NZ624470B2 - Cytotoxic peptides and antibody drug conjugates thereof - Google Patents

Cytotoxic peptides and antibody drug conjugates thereof Download PDF

Info

Publication number
NZ624470B2
NZ624470B2 NZ624470A NZ62447012A NZ624470B2 NZ 624470 B2 NZ624470 B2 NZ 624470B2 NZ 624470 A NZ624470 A NZ 624470A NZ 62447012 A NZ62447012 A NZ 62447012A NZ 624470 B2 NZ624470 B2 NZ 624470B2
Authority
NZ
New Zealand
Prior art keywords
alkyl
aryl
heterocyclyl
haloalkyl
hydrogen
Prior art date
Application number
NZ624470A
Other versions
NZ624470A (en
Inventor
Matthew David Doroski
Russell George Dushin
Edmund Idris Graziani
Andreas Maderna
Donnell Christopher John O
Pavel Strop
Chakrapani Subramanyam
Beth Cooper Vetelino
Original Assignee
Pfizer Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pfizer Inc filed Critical Pfizer Inc
Priority claimed from PCT/IB2012/056224 external-priority patent/WO2013072813A2/en
Publication of NZ624470A publication Critical patent/NZ624470A/en
Publication of NZ624470B2 publication Critical patent/NZ624470B2/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6863—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Abstract

The disclosure relates to cytotoxic pentapeptides of general formula I. The disclosure also relates to antibody drug conjugates and their use in treating cancer. Example compounds include: 2-methylalanyl-N-[(3R,4S,5S)-l-{(2S)-2-[(lR,2R)-3-({(1S)-1-[(7S)-bicyclo[4.2.0]octa-1,3,5-trien-7-yl]-2-methoxy-2-oxoethyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-S-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide 2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(lR,2R)-3-({(1S)-l-[(7R)-bicyclo[4.2.0]octa-1,3,5trien-7-yl]-2-methoxy-2-oxoethyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-S-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide 2-methylalanyl-N-{(3R,4S,5S)-1-[(2S)-2-{(3R,4R,7S,12S)-7-benzyl-14-[3-chloro-4-(propan-2-yloxy)phenyl]-4-methyl-12-[4-(8-methylimidazo[1,2-a]pyridin-2-yl)benzyl]-5,8,14-trioxo-2,9-dioxa-6,13-diazatetradecan-3-yl}pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide xy-2-oxoethyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-S-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide 2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(lR,2R)-3-({(1S)-l-[(7R)-bicyclo[4.2.0]octa-1,3,5trien-7-yl]-2-methoxy-2-oxoethyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-S-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide 2-methylalanyl-N-{(3R,4S,5S)-1-[(2S)-2-{(3R,4R,7S,12S)-7-benzyl-14-[3-chloro-4-(propan-2-yloxy)phenyl]-4-methyl-12-[4-(8-methylimidazo[1,2-a]pyridin-2-yl)benzyl]-5,8,14-trioxo-2,9-dioxa-6,13-diazatetradecan-3-yl}pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide

Description

CYTOTOXIC PEPTIDES AND ANTIBODY DRUG CONJUGATES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefits of US. Provisional Application No. 61/561,255 filed November 17, 2011, and US. Provisional Application No. 61/676,423 filed July 27, 2012, which are both hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION The t invention is directed to novel peptide-based compounds useful as payloads in antibody-drug-conjugates (ADC’s), and payload-linker compounds useful in connection with ADC’s. The present ion r relates to compositions including the aforementioned payloads, d-linkers and ADC’s, and methods for using these payloads, payload-linkers and ADC’s, to treat pathological conditions including cancer.
BACKGROUND Conjugation of drugs to antibodies, either directly or via linkers, involves a consideration of a variety of s, including the identity and location of the chemical group for conjugation of the drug, the mechanism of drug release, the structural elements providing drug e, and the structural modification to the released free drug. In addition, if the drug is to be released after antibody internalization, the mechanism of drug release must be consonant with the intracellular trafficking of the conjugate.
While a number of ent drug classes have been tried for delivery via dies, only 2O a few drug s have proved efficacious as antibody drug conjugates, while having a suitable toxicity profile. One such class is the auristatins, derivatives of the natural product dolastatin 10.
Representative auristatins include (N-methylvaline-valine-dolaisoleuine-dolaproine- edrine) and (N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine). However, there remains a need for additional auristatins with ed properties.
SUMMARY The present ion relates to cytotoxic pentapeptides and antibody drug conjugates thereof represented by formula: or a pharmaceutically acceptable salt or e thereof, wherein, independently for each occurrence, RSA RSB W is R2 0 or R1/N 0 ; R1 is hydrogen C1-C8 alkyl, or C1-C8 haloalkyl or R1 is a linker or a linker-antibody such iKHiHOAOJy Y \LNH z/ z\ )5] Y A or NH2 g ; Y is C2-C20 alkylene-, -C2-C20 heteroalkylene-, C3-C8 carbocyclo-, -arylene-, -C3- rocyclo- -C1-C10alkylene-arylene-, -arylene-C1-C10alkylene-, -C1-C10alkylene (C3- Cgcarbocyclo)- -(C3-Cgcarb0cyclo)-C1-Cloalkyleneg -C1-Cloalkylene-(C3-Cgheter0cyclo) or (C3- Cg heterocyclo)-C1-C10alkylene-; , Lg; W0“? fir“? N O , , O O , O L N N O L N O O O , NH2, NH2, -NH2 or -NHL; G is halogen, -OH, -SH or –S-C1-C6 alkyl; L is an antibody; R2 is en, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are defined as either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; or (ii) R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are defined as either of the following: (i) R4A is en, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; \=/ and C6-C14 aryl optionally tuted , C1-C10 heterocyclyl, C3-C8 carbocycly with l, 2, 3, 4 or 5 groups independently selected from the group ting of -C1-C8 alkyl, -C1- C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl-C(O)OR’ -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR‘, -C(O)N(R')2, -NHC(O)R', -S(O)2R‘, -S(O)R‘, -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of en, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 N/R12 \R12 R12 0/ N/ or R5 is R13 or R13 , , optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-Cg N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’, -Cs alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene- R’, wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-Cgheterocyclyl, C1-C1oalkylene-C3-Cgheterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and R7 is F, Cl, I, Br, NO2, CN and CF3; h is 1, 2, 3, 4 or 5; and X is O.
The present invention relates to cytotoxic pentapeptides and dy drug conjugates thereof represented by formula: or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are defined as either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, l or halogen; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl, halogen or hydrogen; or (ii) R3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R413 are defined as either of the following: (i) R4A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl or l; or (ii) R4A and R413 taken together are C2-C8 alkylene or C1-C8 alkylene; NH-R11 optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group ting of C1-C8 alkyl, -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, ), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; R11 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or R11 is a linker or linker-antibody such as Y O Z Z , or ; Y is C2-C20 alkylene or C2-C20 heteroalkylene; C3-C8 carbocyclo-, -arylene-, -C3- rocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- ocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)-, or - (C3-C8 heterocyclo)-Cl-Cl0alkylene-; Z is , , , , , H H N N N O L N O , O O , O O , O L N NH2, NH2, -NH2 or -NHL; G is halogen, -OH, -SH or –S-C1-C6 alkyl; L is an antibody; R7 is F, Cl, I, Br, NO2, CN and CF3; h is 1, 2, 3, 4 or 5; and X is O.
Another aspect of the invention relates to pharmaceutical compositions including an effective amount of any one of the aforementioned compounds and/or any one of the aforementioned antibody drug ates and a pharmaceutically acceptable carrier or vehicle.
Another aspect of the invention relates to a method of using an effective amount of any one of the aforementioned compounds and/or any one of the aforementioned antibody drug conjugates to treat cancer by administering to a patient in need f an effective amount of said compound and/or conjugate.
Another aspect of the invention relates to a method of ng cancer wherein said cancer includes a tumor, asis, or other disease or er characterized by uncontrolled cell growth wherein said cancer is selected from the group ting of carcinomas of the bladder, breast, cervix, colon, gliomas, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, melanoma, stomach, and testes.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts a graph of umor activity of four conjugates (each administered at 1 mg/kg, Q4dx4) plotted as tumor volume over time.
Figure 2 depicts a graph of anti-tumor activity of six conjugates (each administered at 1 mg/kg, Q4dx4) plotted as drug-treated tumor volume/vehicle-treated tumor volume over time.
Figure 3 depicts the results of the testing of H(C)-#D54 and H(C)-chMAE at 1 mg/kg s Figures 4A, 4B and 4C depict [A] results of testing of H(C)-#D54 and H(K)-MCC-DM1 in a MDA-MBDYT2 mouse xenograft in vivo screening model; [B] s of the testing of H(C)-chMAE and cMMAF in a MDA-MBDYT2 mouse xenograft in vivo screening model; and [C] a comparison of the calculated T/C for all four conjugates. Mice were treated q4dx4, starting on day 1.
Figures 5A, 5B, 5C, SD, SE and SF depict the dose response results of the testing [A] D54, [B] H(C)-chMAE, [C] H(C)-mcMMAF and [D] H(K)-MCC—DM1 in a N87 mouse xenograft in vivo model; [E] a comparison of H(C)-#D54 and H(C)-chMAE; and [F] a comparison of T/C for all four conjugates. Mice were treated q4dx4, starting on day 1.
Figure 6 depicts the dose response results of the testing H(C)-#A115 at 1 mpk, 3 mpk and 10 mpk, in a N87 mouse xenograft in vivo model. Mice were treated q4dx4, starting on day Figure 7 shows data comparing humanized antibody hu08 conjugated to vc-OlOl or mc- 3377, tested in an in vivo xenograft model with PC3MM2 cells, a human prostate cancer cell line that expresses the 0L2 receptor.
Figures 8A h E show [A] the efficacy of rat-human ic otch ADCs dosed at 5mg/kg in HCC2429 lung xenografts; [B and C] the efficacy of rat-human chimeric anti-Notch ADCs dosed at 5mg/kg in MDA-MB-468 breast xenografts; [D and E] the efficacy of rat-human chimeric anti-Notch ADCs dosed at 5mg/kg in N87 gastric xenograft.
DETAILED DESCRIPTION The present invention is directed to cytotoxic pentapeptides, to dy drug conjugates comprising said cytotoxic pentapeptides, and to methods for using the same to treat cancer and other pathological conditions. The invention also s to methods of using such compounds and/ conjugates in vitro, in situ, and in vivo for the detection, diagnosis or treatment of mammalian cells, or associated pathological ions.
Definitions and Abbreviations Unless stated otherwise, the ing terms and phrases as used herein are intended to have the ing meanings. When trade names are used herein, the trade name includes the t formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless ise indicated by context.
The term ”antibody" (or “Ab”) herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e. g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. An intact antibody has primarily two regions: a variable region and a nt region. The variable region binds to and interacts with a target antigen. The variable region es a complementary determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region may be recognized by and interact with the immune system (see, e.g., Janeway et al., 2001, Immuno. Biology, 5th Ed., Garland Publishing, New York). An antibody can be of any type or class (e. g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). The antibody can be derived from any le species. In some embodiments, the antibody is of human or murine origin. An antibody can be, for example, human, humanized or chimeric.
The terms ”specifically binds” and ”specific binding” refer to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity of at least about 1x107 M'l, and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
The term ”monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the dual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier ”monoclonal” indicates the character of the antibody as being ed from a substantially neous population of antibodies, and is not to be ued as requiring production of the antibody by any particular method.
The term ”monoclonal antibodies" specif1cally es ”chimeric” antibodies in which a portion of the heavy and/or light chain is cal to or homologous with the corresponding sequence of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the der of the chain(s) is identical to or homologous with the corresponding sequences of antibodies derived from r species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
As used herein, “H(C)-” refers to trastuzumab (trade name HERCEPTIN®) which is a monoclonal antibody that eres with the HERZ/neu receptor, bound through one of its’ cystine to compound of the invention. As used , “H(K)-” refers to trastuzumab which is a monoclonal antibody that interferes with the HERZ/neu receptor, bound through one of its’ lysines to compound of the invention.
An "intact antibody” is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, CH3 and CH4, as appropriate for the antibody class. The constant domains may be native sequence constant domains (e.g., human native sequence constant s) or amino acid sequence variants An intact antibody may have one or more ”effector functions”, which refers to those biological activities utable to the Fc region (e.g., a native ce Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include complement dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis.
An ”antibody fragmen " comprises a portion of an intact antibody, preferably sing the n-binding or variable region thereof Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecif1c antibody fragments formed from antibody nt(s), a fragment(s) ed by a Fab expression library, or an epitope-binding fragments of any of the above which immuno specif1cally bind to a target antigen (e. g., a cancer cell antigen, a viral antigen or a microbial antigen).
The term ble” in the context of an dy refers to certain portions of the variable domains of the antibody that differ extensively in sequence and are used in the binding and specificity of each particular antibody for its particular antigen. This variability is concentrated in three ts called "hypervariable regions” in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The le domains of native heavy and light chains each comprise four FRs connected by three hypervariable regions.
The term "hypervariable region” when used herein refers to the amino acid residues of an dy which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a ”complementarity determining region” or ”CDR” (e. g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain le domain and 31-35 (H1), 50-65 (H2) and 95-102 (L3) in the heavy chain variable domain; Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a ”hypervariable loop” (e. g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (142) 2O and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). FR residues are those variable domain residues other than the hypervariable region residues as herein defined.
A ”single-chain Fv” or ”scFv” dy fragment comprises the V.sub.H and V.sub.L s of an dy, wherein these domains are present in a single polypeptide chain.
Typically, the Fv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains which enables the scFv to form the d structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabody" refers to small antibody fragments with two n-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of r chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 0 404 097; W0 93/ 1 1 161; and Hollinger et al., 1993, Proc. Natl. Acad.
Sci. USA 90:6444-6448.
”Humanized” forms of non-human (e.g., ) dies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some ces, framework region (FR) residues of the human globulin are ed by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the ent dy or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or ntially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized dy optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
For further details, see Jones et al., 1986, Nature 321 :522-525; Riechmann et al., 1988, Nature 332:323-329; and , 1992, Curr. Op. Struct. Biol. 2:593-596.
As used herein, ”isolated” means separated from other components of (a) a natural source, 2O such as a plant or animal cell or cell culture, or (b) a synthetic organic chemical reaction e.
As used herein, ”purified" means that when isolated, the isolate contains at least 95%, and in another aspect at least 98%, of a compound (e. g., a conjugate) by weight of the e.
An ”isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified ( 1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the dy in situ within recombinant cells since at least one component of the antibody's l nment will not be t. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
An antibody which ”induces apoptosis” is one which induces programmed cell death as determined by g of annexin V, fragmentation of DNA, cell shrinkage, dilation of asmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). The cell is a tumor cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Various s are available for evaluating the cellular events ated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be ted through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells.
The term ”therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; t (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and ably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
To the extent the drug may inhibit the growth of and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or ining the response rate (RR).
The term ”substantial amoun " refers to a ty, i.e. greater than 50% of a population, of a mixture or a sample.
The term ”intracellular metabolite” refers to a compound resulting from a metabolic process or on inside a cell on an antibody-drug ate (ADC). The metabolic process or reaction may be an enzymatic process such as proteolytic ge of a peptide linker of the ADC. Intracellular metabolites include, but are not limited to, antibodies and free drug which have undergone intracellular cleavage after entry, diffusion, uptake or transport into a cell.
The terms ”intracellularly cleaved” and ”intracellular cleavage” refer to a metabolic process or on inside a cell on an ADC or the like, whereby the covalent attachment, e. g., the linker, between the drug moiety and the antibody is broken, resulting in the free drug, or other metabolite of the conjugate dissociated from the antibody inside the cell. The cleaved moieties of the ADC are thus intracellular metabolites.
The term ”bioavailability” refers to the ic bility (i.e., blood/plasma levels) of WO 72813 2012/056224 a given amount of a drug administered to a patient. ilability is an absolute term that indicates measurement of both the time (rate) and total amount t) of drug that s the general circulation from an administered dosage form.
The term ”cytotoxic activity” refers to a cell-killing, a cytostatic or an anti-proliferative effect of a ADC or an ellular metabolite of said ADC. Cytotoxic activity may be expressed as the IC50 value, which is the concentration (molar or mass) per unit volume at which half the cells survive.
A "disorder" is any condition that would benefit from treatment with a drug or antibody- drug conjugate. This includes chronic and acute disorders or diseases including those pathological conditions which predispose a mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include benign and malignant cancers; ia and lymphoid malignancies, neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.
The terms ”cancer” and ”cancerous” refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A ”tumor" comprises one or more cancerous cells.
Examples of a ”patien " include, but are not d to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In an ary embodiment, the patient is a human.
The terms ”treat” or ”treatment,” unless otherwise indicated by context, refer to therapeutic treatment and prophylactic es to prevent relapse, wherein the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, ial or desired clinical results include, but are not limited to, alleviation of symptoms, shment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. ”Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those y having the condition or disorder as well as those prone to have the condition or disorder.
In the context of , the term ing” includes any or all of inhibiting grth of tumor cells, cancer cells, or of a tumor; inhibiting replication of tumor cells or cancer cells, lessening of overall tumor burden or decreasing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.
In the context of an autoimmune disease, the term ”treating” includes any or all of inhibiting replication of cells associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, lessening the mune-antibody burden and ameliorating one or more symptoms of an mune disease.
In the context of an infectious disease, the term ”treating” includes any or all of: inhibiting the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an ious e.
The term ”package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indication(s), usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
As used herein, the terms ”cell,” ”cell line,” and ”cell culture” are used interchangeably and all such designations include progeny. The words ”transformants” and ”transformed cells" include the primary subject cell and cultures or y derived therefrom without regard for the number of transfers. It is also tood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent ons. Mutant progeny that have the same function or biological activity as screened for in the ally ormed cell are included. Where distinct ations are intended, it will be clear from the context.
Unless otherwise ted, the term ”alkyl” by itself or as part of another term refers to a straight chain or branched, saturated hydrocarbon having the indicated number of carbon atoms (e. g., "C1-C3" alkyl refer to an alkyl group having from 1 to 8 carbon atoms). When the number of carbon atoms is not indicated, the alkyl group has from 1 to 8 carbon atoms. Representative straight chain C1-C3 alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl; while branched C1-C8 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tent-butyl, -isopentyl, and methylbutyl; rated C2-C8 alkyls include, but are not limited to, vinyl, allyl, nyl, 2-butenyl, isobutylenyl, l-pentenyl, enyl, 3-methyl-l-butenyl, 2-methyl-2—butenyl, methylbutenyl, l, 2-hexyl, 3- hexyl, acetylenyl, propynyl, l-butynyl, 2-butynyl, l-pentynyl, 2-pentynyl and 3-methyl-l- butynyl.
Unless otherwise indicated, ”alkylene,” by itself of as part of another term, refers to a ted, branched or straight chain or cyclic hydrocarbon radical of the stated number of carbon atoms, typically 1-18 carbon atoms, and having two monovalent radical centers d by the removal oftwo hydrogen atoms from the same or two different carbon atoms of a parent .
Typical alkylene radicals include, but are not limited to: methylene (—CH2-), 1,2-ethylene -CH2CH2-), 1,3-propylene (—CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and the like. A ”C1-C10” straight chain alkylene is a straight chain, saturated hydrocarbon group of the formula -(CH2)1_10-. Examples of a C1-C10 alkylene e methylene, ethylene, propylene, butylene, ene, ne, heptylene, ocytylene, nonylene and decalene.
Unless otherwise indicated, the term ”heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, or combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group ting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may ally be oxidized and the nitrogen atom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. The heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive.
Unless otherwise indicated, the term ”heteroalkylene” by itself or as part of another substituent means a nt group derived from heteroalkyl (as discussed above). For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini.
Unless otherwise indicated, ”aryl,” by itself or an part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of 6-20, ably 6-14, carbon atoms derived by the l of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from e, substituted benzene, naphthalene, anthracene, biphenyl, and the like. A tuted carbocyclic aromatic group (e. g., an aryl group) can be substituted with one or more, preferably 1 to 5, of the following groups: C1-C8 alkyl, -O-(C1-Cg alkyl), -C(O)R', - OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from -H, C1-C8 alkyl and unsubstituted aryl. In some embodiments, a substituted carbocyclic aromatic group can r e one or more of: -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR'.
“Arylene” is the corresponding divalent moiety.
"Substituted alkyl” means an alkyl in which one or more hydrogen atoms are each 2012/056224 independently replaced with a substituent. Typical substituents include, but are not limited to, -X, -R, -O-, -OR, -SR, -S', -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NOz, =N2, -N3, -NRC(=O)R, NR2, -SOg', -SO3H, -S(=O)2R, -OS(=O)ZOR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO32', PO3H2, -AstH2, -C(=O)R, -C(=O)X, -C(=S)R, -COzR, -COz', -C(=S)OR, -C(=O)SR, SR, NR2, -C(=S)NR2, or )NR2, where each X is independently a halogen: -F, -Cl, -Br, or -I; and each R is independently -H, C1-C20 alkyl, C1-C20 heteroalkyl, C6-C20 aryl, C1-C10 heterocyclyl, a protecting group or a prodrug moiety. Aryl, alkylene and heteroalkylene groups as described above may also be similarly substituted.
Unless otherwise indicated, “aralkyl” by itself or part of another term, means an alkyl group, as defined above, tuted with an aryl group, as defined above.
Unless otherwise indicated, ”Cl-Clo heterocyclyl” by itself or as part of r term, refers to a monovalent substituted or unsubstituted aromatic or non-aromatic clic, bicyclic or tricyclic ring system having from 1 to 10, preferably 3 to 8, carbon atoms (also referred to as ring members) and one to four heteroatom ring members ndently selected from N, O, P or S, and derived by removal of one hydrogen atom from a ring atom of a parent ring system. One or more N, C or S atoms in the heterocyclyl can be oxidized. The ring that includes the atom can be aromatic or nonaromatic. Unless otherwise noted, the heterocyclyl is attached to its pendant group at any heteroatom or carbon atom that s in a stable structure. Representative examples of a C1-C10 heterocyclyl include, but are not limited to, tetrahyrofuranyl, oxetanyl, pyranyl, pyrrolidinyl, piperidinyl, piperazinyl, benzofuranyl, benzothiophene, benzothiazolyl, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiopene), furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl including moieties such as 1,2,3,4- tetrshyhro-quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, olyl, epoxide, oxetane and BODIPY (substituted or unsubstituted). A C1-C10 heterocyclyl can be substituted with up to seven groups including, but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, -OR', aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(=O)2R', -S(O)R', halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from -H, C1-C8 alkyl, C1-C8 heteroalkyl and aryl. In some embodiments, a substituted heterocyclyl can also include one or more of: -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR'. “Heterocyclo” “Cl-Clo heterocyclo” is the ponding divalent moiety.
Unless otherwise indicated, oaralkyl” by itself or part of another term, means an alkyl group, as defined above, substituted with an aromatic heterocyclyl group, as defined above.
Heteroaralklo is the corresponding divalent moiety.
Unless otherwise indicated, “C3-C8 carbocyclyl” by itself or as part of r term, is a 3-, 4-, 5-, 6-, 7- or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbocyclic ring derived by the removal of one hydrogen atom from a ring atom of a parent ring system. Representative C3-C3 carbocyclyl e, but are not limited to, cyclopropyl, cyclobutyl, entyl, entadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, eptyl, 1,3-cycloheptadienyl, 1,3,5- cycloheptatrienyl, cyclooctyl, cyclooctadienyl, bicyclo( 1.1.1.)pentane, and bicyclo(2.2.2.)octane.
A C3-C8 carbocyclyl group can be unsubstituted or substituted with up to seven groups including, but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, -OR', aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(=O)2R', -S(=O)R‘, -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; where each R' is independently ed from -H, C1-C8 alkyl, C1-C8 heteroalkyl and aryl. “C3-C8 carbocyclo” is the corresponding nt moiety.
The term ”chiral” refers to molecules which have the property of non-superimposability of the mirror image partner, while the term al” refers to molecules which are superimposable on their mirror image partner.
The term ”stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
”Diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e. g., melting points, boiling , spectral properties, and reactivities. Mixtures of diastereomers may separate under high tion analytical procedures such as electrophoresis and chromatography.
Stereochemical ions and conventions used herein lly follow S. P. , Ed., McGraw-Hill Dictionary of Chemical Terms, McGraw-Hill Book Company, New York ; and Eliel and Wilen, Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York (1994). Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A nd d with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one r. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic e or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms ”racemic e” and ”racemate” refer to an equimolar mixture of two omeric species, devoid of optical activity.
An amino acid ”derivative” includes an amino acid having substitutions or modifications by covalent attachment of a parent amino acid, such as, e. g., by alkylation, glycosylation, acetylation, phosphorylation, and the like. Further included within the definition of ”derivative” is, for example, one or more analogs of an amino acid with substituted linkages, as well as other modifications known in the art.
A ”natural amino acid” refers to arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, e, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and , unless otherwise ted by context.
”Protecting group” refers to a moiety that when attached to a reactive group in a molecule masks, s or prevents that reactivity. Examples of protecting groups can be found in T. W.
Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York, 1999, and Harrison and Harrison et al., dium of Synthetic Organic s, Vols. 1-8 (John Wiley and Sons, 1971- 1996), which are incorporated herein by reference in their entirety. Representative hydroxy protecting groups include acyl groups, benzyl and trityl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. Representative amino protecting groups include, formyl, acetyl, trifluoroacetyl, , benzyloxycarbonyl (CBZ), tert—butoxycarbonyl (Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC), and the like.
Examples of a "hydroxyl protecting group” include, but are not d to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p- methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropyl silyl ether, tbutyldimethyl silyl ether, triphenylmethyl silyl ether, acetate ester, substituted acetate esters, pivaloate, te, methanesulfonate and p-toluenesulfonate.
”Leaving group" refers to a onal group that can be substituted by another functional group. Such leaving groups are well known in the art, and examples include, but are not limited to, a halide (e. g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.
The phrase aceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound. The nd typically contains at least one amino group, and accordingly acid addition salts can be formed with this amino group.
Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, , gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'— methylene-bis-(2-hydroxynaphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically able salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the ceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion. aceutically acceptable solvate” or ”solvate” refer to an association of one or more solvent molecules and a compound or conjugate of the ion. Examples of solvents that form pharmaceutically able solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
The terms “loading” or “drug loading” or “payload loading” represent or refer to the e number of payloads (“payload” and “payloads” are used interchangeable herein with “drug” and “drugs”) per antibody in an ADC le. Drug loading may range from 1 to 20 drugs per antibody. This is sometimes referred to as the DAR, or drug to antibody ratio. itions of the ADCs described herein lly have DAR’s of from 1-20, and in certain embodiments from 1-8, from 2-8, from 2-6, from 2-5 and from 2-4. Typical DAR values are 2, 4, 6 and 8. The average number of drugs per antibody, or DAR value, may be characterized by conventional means such as ible spectroscopy, mass spectrometry, ELISA assay, and HPLC. The quantitative DAR value may also be determined. In some instances, separation, ation, and characterization of homogeneous ADCs having a particular DAR value may be achieved by means such as reverse phase HPLC or electrophoresis. DAR may be limited by the number of attachment sites on the antibody. For e, where the attachment is a cysteine thiol, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a Linker unit may be ed. In some embodiments, the cysteine thiol is a thiol group of a cysteine residue that forms an interchain disulfide bond. In some embodiments, the cysteine thiol is a thiol group of a cysteine residue that does not form an interchain disulfide bond. Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with a linker or linker intermediate.
Only the most reactive lysine groups may react with a reactive linker reagent.
Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug via a linker. Most cysteine thiol residues in the dies exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT). The antibody may be subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. The g (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug- linker relative to the dy, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modif1cation. Where more than one philic group reacts with a inker then the resulting product is a mixture of ADC’s with a distribution of one or more drugs es per antibody. The e number of drugs per antibody may be calculated from the mixture by, for example, dual ELISA antibody assay, specific for antibody and specific for the drug. Individual ADC’s may be identified in the mixture by mass spectroscopy, and separated by HPLC, e. rophobic interaction chromatography.
Below is a list of abbreviations and definitions that may not otherwise be defined or described in this application: DMSO s to dimethyl sulfoxide), HRMS (refers to high resolution mass spectrometry), DAD (refers to diode array ion), TFA (refers to 2,2,2- trifluoroacetic acid or trifluoroacetic acid), TFF (refers to tangential flow flltration), EtOH (refers to ethanol), MW (refers to molecular ), HPLC (refers to high performance liquid chromatography), prep HPLC s to preparative high performance liquid chromatography), etc. (refers to and so forth), trityl (refers l,l',l"-ethane-l,l, l-triyltribenzene), THF s to tetrahydrofuran), NHS (refers to 1-Hydroxy-2,5-pyrrolidinedione), Cbz s to carboxybenzyl), eq. (refers to equivalent), n-BuLi (refers to n-butyllithium), OAc s to acetate), MeOH (refers to methanol), i-Pr (refers to isopropyl or propanyl), NMM (refers to 4- methylmorpholine), and “-“ (in a table refers to no data available at this time). 2012/056224 Compounds and Antibody Drug Conjugates Thereof One aspect of the invention relates to a compound of formula I: 'T O 'l w’N N N N ‘R5 o\ o o\ x or a pharmaceutically able salt or solvate thereof, wherein, independently for each occurrence, RSA R33 R1\N 0 Q R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl, R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl, R3A and R313 are either of the following: (i) R3A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, l or halogen; or (ii) R3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R413 are either of the ing: (i) R4A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and WO 72813 R413 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R413 taken together are C2-C8 ne or C1-C8 heteroalkylene; R5 is and C6-C14 aryl optionally substituted , C1-C10 heterocyclyl, C3-C8 carbocycly with l, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1- C8 alkyl-N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’ -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', R‘, -C(O)N(R')2, -NHC(O)R', -S(O)2R‘, -S(O)R‘, -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; 0 R12 R6 N/ o’ [KEK /R6 \ R12 /R12 0 N or R5 is R13 or R13 , , optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of C1-C8 alkyl, -C1-Cg alkyl-N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’, -Cs alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R‘, -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene- R’, wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-Cgheterocyclyl, C1-C1oalkylene-C3-Cgheterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 cyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-Cg alkenyl, -C2-Cg alkynyl, -C2-Cg alkynyl or -C1-C8 haloalkyl; R12 is en, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O or S; provided that when R3A is hydrogen X is S.
Another aspect of the ion relates to compound of formula 11a: Ha or a pharmaceutically acceptable salt or e thereof, wherein, independently for each occurrence, ELKULHODAJFK Y INH 2/ Z\ 1 R is O or NH2 , ; Y is C2 C20 alkylene-, -C2-C20 heteroalkylene-; -C3-Cg carbocyclo-, ne-, -C3- Cgheterocyclo- -C1-C1oalkylene-arylene-, -arylene-C1-C10alkylene-, -C1-C10alkylene (C3- ocyclo)- -(C3-Cgcarbocyclo)-C1-Cloalkyleneg -C1-Cloalkylene-(C3-Cgheterocyclo)- or (C3- C3 heterocyclo)-C1-C1oalkylene-; O H 37W“? elite NH2 or -NH2; G is halogen, -OH, -SH or —S—C1-C6 alkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R313 are either of the following: (i) 3A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and 3B is C1 C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl or aralkyl or halogen; or (ii) 3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene, R4A and R413 are either of the following: (i) 4A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and 2012/056224 R413 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R413 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; and C6-C14 aryl optionally substituted , C1-C10 cyclyl, C3-C8 carbocycly with l, 2, 3, 4 or 5 groups independently ed from the group consisting of -C1-C8 alkyl, -C1- C8 alkyl-N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’ -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', R', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; 0 R12 R6 / o’ [KEK /R6 N\ R12 /R12 0 N 01‘ R5 is R13 01‘ R13 , , optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-Cg alkyl-N(R’)2, -C1-Cg C(O)R’, -C1-Cg alkyl-C(O)OR’, -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R‘, -S(O)R', -OH, halogen, -N3, 2, -CN, NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene- R’, wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-Cgheterocyclyl, C1-C1oalkylene-C3-Cgheterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-Cg alkenyl, -C2-Cg alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and R7 is ndently selected for each ence from the group consisting of F, Cl, 1, Br, N02, CN and CF3; R10 is hydrogen, -C1-C10alkyl, -C3-Cgcarbocyclyl, -aryl, -C1-C10heteroalkyl, -C3- Cgheterocyclo, -C1-C10alkylene-aryl, -arylene-C1-C10alkyl, -C1-C10alkylene-(C3-Cgcarbocyclo), - (C3-C8 carbocyclo)-C1-C1oalkyl, -C1-C1oalkylene-(C3-Cgheterocyclo), and -(C3-Cs heterocyclo)-C1- Cloalkyl, where aryl on R10 sing aryl is optionally substituted with [R7]h; h is l, 2, 3, 4 or 5; and X is O or S; provided that when R3A is hydrogen X is S.
Another aspect of the invention relates to compound of formula IIIa: 'T O 'l w’N N N MR5 O\ O O\ X 11121 or a pharmaceutically able salt or solvate thereof, wherein, independently for each occurrence, R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl, R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl, R3A and R313 are either of the following: (i) R3A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, n or l; or (ii) R3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R413 are either of the following: (i) R4A is hydrogen, c1—c8 alkyl, c1—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R413 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is R11 0 0 R11 , ’ o R11 , 31>\ f /R11 m N N 11 o N’R11 o o N’R R11 O 0 H a I l N‘R“ ’ Or NH-RH optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C3 alkyl, -O-(C1-Cs alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, 2R' and -SR', wherein each R' is independently selected from the group consisting of en, C1-C8 alkyl and unsubstituted aryl; it);JL0””3’ Y is -C2-C20 ne-, -C2-C20 alkylene-, C3-C8 carbocyclo-, -arylene-, -C3- Cgheterocyclo-, -C1-C10alkylene-arylene-, -arylene-C1-C10alkylene-, -C1-C10alkylene-(C3- Cgcarbocyclo)-, -(C3-Cgcarbocyclo)-C1-Cloalkylene-, -C1-Cloalkylene-(C3-Cgheterocyclo)-, or - (C3-C8 heterocyclo)-C1-C10alkylene-; O H qfimfi white0 NWW/ H NH2 or -NH2; R7 is independently selected for each occurrence from the group consisting of F, Cl, 1, Br, N02, CN and CF3; R10 is hydrogen, -C1-C10alkyl, -C3-Cgcarbocycle, aryl, -C1-C10heteroalkyl, -C3- Cgheterocyclo, -C1-C10alkylene-aryl, -arylene-C1-C10alkyl, -C1-C10alkylene-(C3-Cgcarbocyclo), - (C3-C8 carbocyclo)-C1-C1oalkyl, -C1-C1oalkylene-(C3-Cgheterocyclo), and -(C3-Cs heterocyclo)-C1- Cloalkyl, where aryl on R10 comprising aryl is optionally substituted With [R7]h; h is l, 2, 3, 4 or 5; and X is O or S.
R6 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; and his 1,2, 3,4or5.
Another aspect of the invention s to compound of formula 11b: 11b or a pharmaceutically acceptable salt or solvate thereof, n, independently for each occurrence, R3A R3B Rkl}! Wis R2 0 or R1/N o o H 0% 2\ JL NJLN O \L /Y NH 2 Z‘Yk A R1 is O or NH2 , ; Y is -C2-C20 alkylene-, -C2-C20 alkylene-, -C3-Cg carbocyc10-, -arylene-, -C3- Cgheterocyclo-, -C1-C10alkylene-arylene-, -arylene-C1-C10alkylene-, -C1-C10a1kylene-(C3- Cgcarbocyclo)-, -(C3-Cgcarb0cyc10)-C1-C10a11<ylene-, -C1-Cloalkylene-(C3-Cgheterocyc10)-, or - (C3-C8 cyclo)-C1-C10alkylene-; H O “Y L Z is O O 7 7 a L\”/N mer/O/NYO o o NH2 0r -NHL; L is an antibody; R2 is hydrogen, C1-C3 alkyl or C1-C8 haloalkyl; R3A and R313 are either of the following: (i) R3A is hydrogen, c1—c8 alkyl, c1—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, l or halogen; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, n or aralkyl; or (ii) R3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R413 are either of the following: (i) R4A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413 is hydrogen, C1-C8 alkyl, C1-C8 kyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R413 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; ACES Ages /<(iE§S/\> R5 is , a a 7 R6,oo’, ,‘oo , s N \=/ and C6-C14 aryl ally substituted , C1-C1o cyclyl, C3-C8 carbocycly with l, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1- c8 alkyl-N(R’)2, —c1—c8 alkyl-C(O)R’, —c1—c8 alkyl-C(O)OR’ -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', ', -OH, halogen, -N3, 2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 N/R12 \R12 R12 0/ N/ or R5 is R13 or R13 , , optionally substituted with 1, 2, 3, 4 or 5 groups ndently ed from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’, -Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene- R’, wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-Cgheterocyclyl, alkylene-C3-Cgheterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-Cg alkenyl, -C2-Cg alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O or S; provided that when R3A is hydrogen X is S.
Another aspect of the invention relates to compound of formula IIIb: IIIb or a ceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, 0 Q R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl, R2 is en, C1-C8 alkyl or C1-C8 haloalkyl, R3A and R313 are either of the following: (i) R3A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 cyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R413 are either of the following: (i) R4A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R413 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is R11 o o R11 , ’ o R11 , \ (R11 N N 11 R11 H m /R N’ o o N R11 0 H a [4% (31;?) l ”\RH 31>\ or NH-R11 optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O-(C1-Cg alkyl), -C(O)R', R', R', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, 2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; 0 iQ/THJLHQAOJFKINH R - OANH 1s or 2 Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-Cg yclo-, -arylene-, -C3- Cgheterocyclo-, -C1-C1oalkylene-arylene-, -arylene-C1-C10alkylene-, -C1-C10alkylene-(C3- Cgcarbocyclo)-, -(C3-Cgcarbocyclo)-C1-Cloalkylene-, -C1-Cloalkylene-(C3-Cgheterocyclo)-, or - (C3-C8 heterocyclo)-C1-C10alkylene-; Lfimkw LYVHW/NYO o o NH2 or -NHL; L is an antibody; X is O or S.
Another aspect of the invention relates to compound of formula IIc.
I,'_R3BRSA'NH L Z'-—RL'NI ,/N\ 1-20 or a pharmaceutically able salt or solvate thereof, wherein, independently for each occurrence, ?5va3:“ijDA)5; O \|\NH 1, A R is j‘YJSJ or 0 NH2 ; Y is C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-Cg yclo-, -arylene-, -C3- Cgheterocyclo- -C1-C1oalkylene-arylene-, -arylene-C1-C10alkylene-, -C1-C10alkylene (C3- Cgcarbocyclo)-, -(C3-Cgcarbocyclo)-C1-Croalkylene-, -Cr-Croalkylene-(C3-Cgheterocyclo)-, or - (C3-C8 heterocyclo)-C1-C10alkylene-; Ziffilfmh’hr’ :fif“ frvWONT NH2 or —NH-; L is an antibody; D is —C(R4A,)(R4B,)- or is absent; R2, is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if ””””” ’ ‘~ is t; R3A, and R313, are either of the following: (i) R3A’ is hydrogen, cr—c8 alkyl, cr—c8 haloalkyl, C3-C8 carbocyclyl, Cr—C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R313, is C1-C3 alkyl, C1-C3 haloalkyl, C3-C3 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R33, is C2-C4 alkylene and forms 5-7 member ring as indicated by """"" ' ‘~ ; or (ii) R3A’ and R33, taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A, and R413, are either of the following: (i) R4A’ is hydrogen, cr—c8 alkyl, cr—c8 haloalkyl, C3-C8 yclyl, Cr—C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R43’ is hydrogen, cr—c8 alkyl, cr—c8 haloalkyl, C3-C8 yclyl, Cr-Cro heterocyclyl, aryl, heteroaralkyl or l; or (ii) RAW and R43, taken together are C2-C8 alkylene or C1-C8 heteroalkylene; \=/ and C6-C14 aryl optionally substituted , C1-C10 heterocyclyl, C3-C8 carbocycly with l, 2, 3, 4 or 5 groups ndently ed from the group consisting of -C1-C8 alkyl, -C1- C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl-C(O)OR’ -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', R‘, -C(O)N(R')2, -NHC(O)R', -S(O)2R‘, -S(O)R‘, -OH, halogen, -N3, -N(R')2, -CN, NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, er with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 N/R12 6 \R12 R R 12 0/ N/ or R5 is R13 or R13 , , optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-Cg alkyl-N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’, -O-(C1-Cs alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, 2R', -SR' and arylene- R’, wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-Cgheterocyclyl, C1-C1oalkylene-C3-Cgheterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-Cg alkenyl, -C2-Cg alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O or S; provided that when R3A is hydrogen X is S.
Another aspect of the invention relates to compound of formula IIIc: T O 'F N N\ . .
WI”EN R5_R11_ZI L O\ O O\ X 1-20 or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, 0 Q R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R313 are either of the following: (i) R3A is en, c1—c8 alkyl, c1—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 cyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R413 are either of the following: (i) R4A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R413 taken er are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is R“' o o R“' , ’ o R”, \ ’R11' (31;?) R11- N 11' H IR o N . N’ o R11 o H 9 I ”\RH' ’ or NH_R11' optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O-(C1-Cg , -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, HR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', ', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; Y is -C2-C20 ne-, -C2-C20 heteroalkylene-, -C3-Cg carbocyclo-, -arylene-, -C3- Cgheterocyclo-, -C1-C10alkylene-arylene-, -arylene-C1-C10alkylene-, -C1-C10alkylene-(C3- Cgcarbocyclo)-, -(C3-Cgcarbocyclo)-C1-Cloalkylene-, -C1-C1oalkylene-(C3-Cgheterocyclo)-, or - (C3-C8 heterocyclo)-C1-C10alkylene-; zqffrlfmfi’wiwvgH 0 H HY Eff“ NH2 or —NH-; L is an antibody; XisOorS.
Another aspect of the ion relates to compound of formula IId: ’,_R38R3ANH L [linker]\ I RZ'N or a pharmaceutically acceptable salt or solvate thereof, n, independently for each occurrence, L is an antibody; [linker] is a divalent linker; D is —C(R4A,)(R4B,)- or is absent; R2, is en, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if """"" ' ‘~ is present; R3A, and R313, are either of the following: (i) R3A’ is en, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R313, is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R33, is C2-C4 alkylene and forms 5-7 member ring as indicated by .....~ ' ~ ; or (ii) R3A’ and R33, taken together are C2-C8 ne or C1-C8 heteroalkylene; R4A, and R413, are either of the following: (i) R4A’ is en, ci—c8 alkyl, ci—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413, is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) RAW and R43, taken together are C2-C8 alkylene or C1-C8 heteroalkylene; ACES AEEO:S/\>\ R6 /R12 N\R12 OH R5 is AKKQ , , QQWRB/EGR6,0 0’ o , ,\o , s N \=/ and C6-C14 aryl optionally substituted , C1-C10 heterocyclyl, C3-C8 carbocycly with l, 2, 3, 4 or 5 groups ndently selected from the group consisting of -C1-C8 alkyl, -C1- C8 N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’ -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)N(R')2, )R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group ting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; 0 /R12 /R6 N O \R R6 12 /R12 0/ N or R5 is R13 or R13 , , optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C3 alkyl, -C1-Cg alkyl-N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’, -O-(C1-Cg alkyl), ', -OC(O)R', -C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R‘, -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene- R’, wherein each R' is independently selected from the group ting of hydrogen, C1-C8 alkyl, C1-Cgheterocyclyl, C1-C1oalkylene-C3-Cgheterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-Cg alkenyl, -C2-Cg alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 cyclyl or C6-C14 aryl; R13 is C1-C1o cyclyl; and X is O or S; provided that when R3A is hydrogen X is S.
Another aspect of the invention relates to compound of formula formula IIId: i O ROWJY w’N N N N‘R5' [linker] L 1-20 IIId or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, 0 ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl, R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl, R3A and R313 are either of the following: (i) R3A is hydrogen, cr—c8 alkyl, cr—c8 haloalkyl, C3-C8 carbocyclyl, Cr—Cro heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl, halogen or aralkyl; or (ii) R3A and R313 taken together are C2-C8 ne or C1-C8 heteroalkylene; R4A and R413 are either of the following: (i) R4A is hydrogen, cr—c8 alkyl, cr—c8 kyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R413 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is or NH_R11' ally tuted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; [linker] is a divalent linker; L is an dy; XisOorS.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein the nd is represented by T O T W/N\:)J\N N N /:\ I O\ O O\ X In certain embodiments, the present ion relates to any of the entioned REA R33 RK'}! compounds and attendant definitions, wherein W is R2 0 In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein W is O In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein W is 0 In n embodiments, the present invention relates to any of the entioned compounds and attendant definitions, wherein R1’N R1/N Wis 0 or o In certain embodiments of the invention W is: RSA R33 $1R3A R33 R1 ,N ‘1}1 O R2 R2 R4A R48 Wis 0 O or , , In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl.
In certain ments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R1 is hydrogen.
In certain embodiments, the t invention relates to any of the aforementioned compounds and attendant definitions, wherein R1 is C1-C8 alkyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R1 is methyl.
In certain embodiments, the present invention s to any of the aforementioned compounds and ant ions, wherein R2 is hydrogen, C1-C3 alkyl or C1-C3 haloalkyl.
In certain embodiments, the t invention relates to any of the aforementioned compounds and ant definitions, wherein R2 is hydrogen.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R2 is C1-C8 alkyl.
In certain embodiments, the present invention relates to any of the entioned compounds and attendant definitions, wherein R2 is methyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R1 is hydrogen; and R2 is methyl.
In certain embodiments, the present invention relates to any of the aforementioned 2012/056224 compounds and attendant definitions, wherein R1 is methyl; and R2 is methyl.
In certain embodiments, the present invention relates to any of the entioned nds and attendant definitions, wherein R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3- Cg carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl or aralkyl; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3- Cg carbocyclyl, C1-C10 cyclyl, aryl, heteroaralkyl or aralkyl; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C3 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen.
In certain embodiments, the present invention s to any of the aforementioned compounds and attendant definitions, wherein R3A is halogen.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A is hydrogen.
In certain ments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A is C1-C8 alkyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A is methyl.
In certain embodiments, the t invention relates to any of the aforementioned compounds and attendant definitions, wherein R313 is C1-C8 alkyl.
In certain embodiments, the present invention s to any of the aforementioned compounds and attendant definitions, wherein R313 is methyl.
In certain embodiments, the t invention relates to any of the aforementioned compounds and attendant definitions, wherein R313 is isopropyl.
In certain ments, the t invention relates to any of the aforementioned compounds and attendant definitions, wherein R313 is C3-C8 carbocyclyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R313 is cylohexyl.
In certain embodiments, the t invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A is C1-C3 alkyl, and R313 is C1-C3 alkyl.
In n embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A is methyl; and R313 is methyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A is hydrogen; and R313 is C1-C8 alkyl.
In certain embodiments, the present invention relates to any of the entioned compounds and attendant definitions, wherein R3A is hydrogen; and R313 is isopropyl.
In certain embodiments, the present invention s to any of the entioned compounds and attendant definitions, wherein R3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene.
In certain embodiments, the t invention relates to any of the entioned compounds and attendant definitions, wherein R3A and R313 taken together are C2-C3 alkylene.
In n embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A and R313 taken together are —CH2CH2—.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A and R313 taken er are —CH2CH2CH2—.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant ions, wherein R3A and R313 taken together are — CH2CH2CH2CH2—.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R3A and R313 taken together are C1-C8 heteroalkylene.
In certain ments, the present invention relates to any of the aforementioned nds and attendant definitions, wherein R3A and R313 taken together are —CH20CH2—.
In certain ments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3- Cg carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C3 carbocyclyl, C1-C10 cyclyl, aryl, aralkyl or aralkyl.
In certain embodiments, the present ion relates to any of the aforementioned compounds and attendant definitions, wherein R4A is hydrogen.
In n embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R4A is C1-C8 alkyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R4A is methyl.
In n embodiments, the present invention s to any of the aforementioned compounds and attendant definitions, wherein R413 is hydrogen.
In n embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R413 is C1-C8 alkyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and ant definitions, wherein R413 is methyl.
In certain embodiments, the t invention relates to any of the aforementioned compounds and attendant ions, wherein R4A is C1-C3 alkyl; and R413 is C1-C8 alkyl.
In certain embodiments, the present invention relates to any of the aforementioned nds and attendant definitions, wherein R4A is methyl; and R413 is methyl.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R4A is hydrogen; and R413 is hydrogen.
In certain embodiments, the present invention s to any of the aforementioned compounds and attendant definitions, wherein R4A is en; and R413 is C1-C8 alkyl.
In certain embodiments, the t invention relates to any of the aforementioned compounds and attendant definitions, n R4A and R413 taken together are C2-C8 alkylene or C1-C8 heteroalkylene.
In certain embodiments, the present invention relates to any of the entioned compounds and attendant definitions, wherein R4A and R413 taken together are C2-C3 alkylene.
In certain embodiments, the t invention relates to any of the aforementioned compounds and attendant definitions, wherein R4A and R413 taken together are —CH2CH2—.
In certain embodiments, the present ion relates to any of the aforementioned compounds and attendant definitions, wherein R4A and R413 taken together are —CH2CH2CH2—.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R4A and R413 taken together are — CH2CH2CH2CH2—.
In certain embodiments, the present invention relates to any of the aforementioned compounds and ant definitions, wherein R4A and R413 taken together are C1-C3 heteroalkylene.
In certain embodiments, the t invention s to any of the aforementioned compounds and attendant definitions, wherein R4A and R413 taken er are —CH20CH2—.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R5 is In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R5 is In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R5 is In certain embodiments, the present ion relates to any of the aforementioned MR6 0/ compounds and attendant definitions, wherein R5 is In certain embodiments, the present invention relates to any of the aforementioned nds and attendant definitions, wherein R5 is3 In n embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R5 ism In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R5 is C In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, n R5 is[m In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant ions, wherein R5 is% In certain ments, the present invention relates to any of the aforementioned compounds and ant definitions, wherein R5 is /</\© In certain embodiments, the t invention relates to any of the aforementioned #51ng‘0 compounds and attendant definitions, n R5 is o In certain embodiments, the present invention relates to any of the entioned compounds and attendant definitions, wherein R5 isw.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R5 is R6 In certain ments, the present invention relates to any of the aforementioned compounds and attendant ions, n R5 is 0 0/ In certain embodiments, the present invention relates to any of the entioned In certain embodiments, the present invention relates to any of the aforementioned nds and attendant definitions, wherein R5 is \O 0 In certain embodiments, the present invention relates to any of the aforementioned compounds 3 N and attendant definitions, wherein R5 is \=/ In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, n R6 is hydrogen.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R6 is C1-C8 alkyl.
In certain embodiments, the present invention relates to any of the entioned compounds and attendant definitions, wherein R6 is methyl.
In n embodiments, the present invention relates to any of the aforementioned WO 72813 compounds and attendant definitions, wherein X is O.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant ions, wherein X is S.
In certain embodiments, the present invention relates to any of the aforementioned compounds, or a ceutically able salt or solvate thereof, and attendant def1nitions, wherein the compound is selected from the group consisting of: N-Methyl-L-valyl-N- [(3R,4S,5 S)-3 -methoxy- l - {(2S)[(1R,2R)- l -methoxymethyl-3 - {[(1 S)phenyl- l -(l ,3 -thiazolyl)ethyl] amino} thioxopropyl]pyrrolidin- l -yl} methyl oxoheptanyl] -N-methyl-L-valinamide; NZ- [(1 -Aminocyclopentyl)carbonyl] -N- [(3R,4S,5S)-3 -methoxy- l - {(2 (1R,2R)- l- ymethyl-3 - {[(1 S)phenyl- l -(l ,3 -thiazolyl)ethyl] amino} -3 - thioxopropyl]pyrrolidin- l -yl} methyl- 1 ptanyl] -N-methyl-L-valinamide; 2-Methylalanyl-N—[(3R,4S,SS)methoxy- l - {(2S)[(1R,2R)- l-methoxymethyl {[(1 S)phenyl- l -(l ,3 -thiazolyl)ethyl] amino} thioxopropyl]pyrrolidin- l -yl} methyl oxoheptanyl] -N-methyl-L-valinamide; N-Methyl-L-valyl-N- {(3R,4S,5 S)methoxy- l— [(2S) {(1R,2R)- l -methoxymethyl [(2-phenylethyl)amino] thioxopropyl}pyrrolidin- l -yl] methyl- l -oxoheptanyl} -N-methyl- L-Valinamide; N—Methyl-L-valyl-N—[(3R,4S,SS){(2S)[(1R,2R){[(IS)carboxy 2O phenylethyl] amino} - l -methoxymethyl-3 -thioxopropyl]pyrrolidin- l -yl} -3 -methoxymethyll-oxoheptanyl ] -N-methyl-L-valinamide; N—Methyl-L-valyl-N—[(3R,4S,SS)methoxy- l - {(2S)[(1R,2R)- l -methoxy {[(2S) methoxy- l -oxo-3 -phenylpropanyl] amino} methyl-3 -thioxopropyl]pyrrolidin- l -yl} -5 - oxoheptanyl] -N-methyl-L-valinamide; 2-Methylalanyl-N-[(3R,4S,5 S)- l - {(2 S)[( l R,2R) {[( l S)- l -carboxy phenylethyl] amino} - l -methoxymethyl-3 opropyl]pyrrolidin- l -yl} -3 -methoxymethyll-oxoheptanyl ] -N-methyl-L-valinamide; ylalanyl-N—[(3R,4S,SS)methoxy- l - {(2S)[(1R,2R)- l -methoxy {[(2S) methoxy- l -oxo-3 -phenylpropanyl] amino} methyl-3 -thioxopropyl] pyrrolidin- l -yl} -5 - methyloxoheptanyl] -N-methyl-L-valinamide; NZ- [(1 -Aminocyclopentyl)carbonyl] -N- [(3R,4S,5S)-3 -methoxy- l - {(2 S)[(1R,2R)- l- methoxymethy1-3 -0x0-3 - {[(1 S)pheny1(1,3 -thiazolyl)ethyl] amino } propyl]pyrr01idin- 1-y1} methy10x0heptan—4-yl]-N-methy1—L-valinamide; NZ-[(1-Aminocyclopropyl)carb0nyl]-N-[(3R,4S,5 S)-3 -meth0xy {(2 S)[(1R,2R) methoxymethy1-3 -0x0-3 - {[(1 S)pheny1(1,3 -thiazoly1)ethyl] amino } propy1]pyrr01idin- 1-y1} methy10x0heptan—4-yl]-N-methy1—L-valinamide; 1-Amin0-N—[(2S){[(3R,4S,SS)meth0xy{(2S)[(1R,2R)meth0xymethy1 0x0-3 - {[(1 S)pheny1(1 ,3-thiazolyl) ethyl] amin0}pr0pyl]pyrr01idiny1} methyl 0x0heptanyl] (methyl)amin0} -3 -methy10x0butanyl]cyclohexanecarboxamide; y1alany1-N-[(3R,4S,SS)meth0xy{(2S)[(1R,2R)meth0xymethy1 0x0-3 - {[(1 S)pheny1- 1-(1,3 olyl)ethyl] amino } propy1]pyrr01idiny1} methyl 0x0heptany1]-N-methy1-L-valinamide; 2-Methy1alany1-N— {(3R,4S,5 S)-3 -meth0xy[(2S){(1R,2R)meth0xymethy1—3- 0x0-3 - [(2-pheny1ethyl)amin0]pr0py1}pyrrolidin— 1-y1]—5-methy10x0heptany1}-N-methyl-L- valinamide; 2-Methy1alany1-N-[(3R,4S,SS)meth0xy{(2S)[(1R,2R)meth0xymethy1 0x0-3 - {[(1 -phenylcyclopropy1)methyl] amino } propyl]pyrr01idiny1} methy1- 1 ptan yl] -N-methy1-L-valinamide; 2-Methy1alany1-N— [(3R,4S,5 S) {(2S)[(1R,2R)-3 - {[2-(cyclohepta-2,4,6-trien y1)ethy1] amino} meth0xymethy1-3 -0x0pr0pyl]pyrr01idiny1}-3 -meth0xymethyl 2O 0x0heptany1]-N-methy1-L-valinamide; 2-Methy1alany1-N—[(3R,4S,SS){(2S)[(1R,2R){[(IS)carb0xy phenylethyl] amino} - 1 xymethy1—3 -0x0pr0pyl]pyrr01idiny1} -3 xymethyl 0x0heptany1]-N-methy1-L-valinamide; 2-Methy1alany1-N—[(3R,4S,SS)meth0xy-1 - {(2S)[(1R,2R)meth0xy {[(2S) methoxy0x0-3 -phenylpr0pan—2-yl]amino} methy1-3 -0x0pr0py1]pyrr01idiny1}methy1- eptanyl] -N-methy1—L-valinamide; NZ- [(3 -Amin00xetan-3 -y1)carb0nyl] -N- {(3R,4S,5S)-3 -meth0xy[(2S) R) methoxymethy1-3 -0x0-3 - [(2-phenylethyl)amin0]propyl} pyrrolidin— 1-y1]—5-methy1 0x0heptany1} -N-methy1-L-valinamide; N,2-Dimethy1alany1-N— [(3R,4S,5 S)-3 -meth0xy {(2S)[(1R,2R)meth0xy-3 - {[(2S)- 1-meth0xy0x0-3 -phenylpr0panyl]amino} methy1-3 -thi0x0pr0pyl]pyrr01idiny1} methyl0x0heptanyl]-N-methy1-L-valinamide; N,2-dimethy1alany1-N-[(3R,4S,SS){(2S)[(1R,2R){[(IS)carb0xy phenylethyl] amino} meth0xymethy1—3 -thi0x0pr0pyl]pyrr01idiny1} -3 -meth0xymethy1- 1-0x0heptanyl] -N-methy1—L-valinamide; N,2-Dimethy1alany1-N— [(3R,4S,5 S)meth0xy {(2S)[(1R,2R)meth0xymethy1 {[(1 S)pheny1(1,3 olyl)ethy1] amino} -3 -thi0x0pr0py1]pyrr01idiny1}methy1 0x0heptany1]-N-methy1-L-valinamide; N,2-Dimethy1alany1-N— S,5 S)meth0xy {(2S)[(1R,2R)meth0xymethy10x0-3 - {[(1 heny1(1 ,3-thiazolyl) ethyl] amino } propyl]pyrr01idiny1} hyl 0x0heptany1]-N-methy1-L-valinamide; NZ-(3 -Amin0-2,2-dimethylpropanoyl)-N— [(3R,4S,5 S)-3 -meth0xy {(2S)[(1R,2R) ymethy1-3 -0x0-3 - {[(1 S)pheny1(1,3 -thiazoly1)ethyl] amino } propy1]pyrr01idin- 1-y1} methy10x0heptan—4-yl]-N-methy1—L-valinamide; NZ-(3-Amin0-2,2-dimethylpropan0y1)-N-{(3R,4S,SS)meth0xy[(2S){(1R,2R) methoxymethy1-3 -0x0-3 - [(2-phenylethyl)amin0]propyl} idin— 1-y1]—5-methy1 0x0heptany1} -N-methy1-L-valinamide; 2-Methy1-L-pr01y1-N— [(3R,4S,5 S)-3 -meth0xy 2-[(1R,2R)meth0xymethy1-3 - 0x0-3 - {[(1 S)pheny1- 1-(1,3 -thiazolyl)ethyl] amino } propy1]pyrr01idiny1} methyl 0x0heptanyl] -N-methy1-L-vaLinamide; 2O 2-Methy1alany1-N-[(3R,4S,5 S)- 1- {(2 S) [( 1 R,2R) {[1 -(bicyclo[4.2.0] octa- 1 ,3,5 -trien— 7-y1)meth0xy0xoethyl] amino} meth0xymethy1-3 -0x0pr0pyl]pyrr01idiny1}-3 - methoxy-S-methyl0x0heptan—4-yl] -N-methy1-L-valinamide; 2-Methy1alany1-N-[(3R,4S,SS) {(2S)[(1R,2R) {[bicyclo [4.2.0]octa-1,3,5-trien yl(carb0xy)methyl]amino} meth0xymethy1-3 -0x0pr0pyl]pyrr01idiny1}-3 -meth0xy-5 - methyl0x0heptanyl]-N-methy1-L-valinamide; 2-Methy1alany1-N-[(3R,4S,SS){(2S)[(1R,2R){[(IS,2R)hydroxy phenylpropanyl]amino} meth0xymethy10x0pr0pyl]pyrr01idiny1} -3 -meth0xy-5 - methyl0x0heptanyl]-N-methy1-L-valinamide; methy1alany1—N— {(1 S,2R) {(2S)[(1R,2R) {[(1 S)carb0xy phenylethyl] amino} - 1-meth0xymethy1—3 -0x0pr0pyl]pyrr01idiny1}meth0xy- 1- [(1 S) methylpropyl] 0x0buty1} -N-methy1-L-valinamide; 2-methy1-L-pr01y1—N— [(3 R,4S,SS)meth0xy 2-[(1R,2R)meth0xy-3 - {[(ZS) methoxy0x0-3 -phenylpr0pan—2-yl]amino} methy1-3 0x0pr0py1]pyrr01idin—1-y1}methyl 0x0heptany1]-N-methy1-L-valinamide; 2-methy1-L-pr01y1—N— S,SS)- 1- {(ZS)[(1R,2R) {[(1 S)carb0xy phenylethyl] amino} meth0xymethy1—3 -0x0pr0pyl]pyrr01idiny1}-3 -meth0xymethyl 0x0heptany1]-N-methy1-L-valinamide; 2-methy1alany1-N—[(3R,4S,SS){(ZS)[(1R,2R){[(ZS)tert-but0xy0x0 phenylpropan-Z-yl]amino} meth0xymethy10x0pr0pyl]pyrr01idiny1} -3 -meth0xy-5 - methyl0x0heptanyl]-N-methy1-L-valinamide; Methyl N— [(2R,3R)-3 - {(ZS)[(3R,4S,5 S) {[N—(3 -amin0-2,2—dimethylpr0pan0yl)-L- valyl] (methyl)amino} -3 -meth0xymethylheptan0yl]pyrrolidin-Z-yl} -3 -meth0xy methylpropanoyl] nylalaninate; N,2—dimethy1alany1—N—[(3R,4S,SS){(ZS)[(1R,2R) {[(ZS)tert—but0xy0x0 phenylpropan-Z-yl]amino} meth0xymethy10x0pr0pyl]pyrr01idiny1} -3 xy-5 - methyl0x0heptanyl]-N-methy1-L-valinamide; 2-methy1-D-pr01y1-N—[(3R,4S,5 S)-3 -meth0xy {(ZS)[(1R,2R)meth0xy-3 - {[(ZS) methoxy0x0-3 -phenylpr0pan—2-yl]amino} hy1-3 -0x0pr0py1]pyrr01idiny1}methy1- 1-0x0heptanyl] -N-methy1—L-valinamide; 2-methy1-L-pr01y1-N—[(3R,4S,SS) {(ZS)[(1R,2R) {[(1 1-hydr0xy-1 - phenylpropan-Z-yl]amino} - 1 xymethy10x0pr0pyl]pyrr01idiny1} -3 -meth0xy-5 - methyl0x0heptanyl]-N-methy1-L-valinamide; N,2-dimethy1alany1-N-{(1S,2R){(ZS)[(1R,2R){[(1S)benzy1(methy1amin0)- 2-0xoethyl] amino} meth0xymethy1-3 -0x0pr0py1]pyrr01idin— 1-y1}meth0xy[(l S) methylpropyl] 0x0buty1} -N-methy1-L-valinamide; N,2-dimethy1alany1-N— {(1 S,2R) {(ZS)[(1R,2R) {[(1 S)amin0benzy1 oxoethyl]amin0}meth0xymethy10x0pr0py1]pyrr01idiny1}meth0xy[(IS) methylpropyl] 0x0buty1} -N-methy1-L-valinamide; N,2-dimethy1alany1-N— {(1 S,2R) {(ZS)[(1R,2R) {[(1 S)-1 -benzy10x0 (propylamin0)ethyl] amino} - 1-meth0xymethy1-3 -0x0pr0py1]pyrr01idiny1} meth0xy- 1- [(1 S)methylpr0pyl]0x0buty1}-N-methy1—L-valinamide; N,2-dimethy1a1any1—N-{(1S,2R){(2S)[(1R,2R){[(1S)benzy1(diethy1amin0)- thy1] amino} meth0xymethy1—3 -0x0pr0py1]pyrr01idin— 1-y1}meth0xy[(1 S) methylpropyl] 0x0buty1} -N-methy1—L-va1inamide; N,2-dimethy1a1any1—N— {(1 S,2R) 2-[(1R,2R) {[(1 S)benzy1(tert— butylamin0)0xoethy1] amino} meth0xymethy1—3 -0x0pr0py1]pyrr01idiny1} meth0xy- 1- [(1 S)methy1pr0py1]0x0buty1}-N-methy1—L-valinamide; N,2-dimethy1a1any1—N—[(3R,4S,5S) {(2S)[(1R,2R) {[(1 S,2R)hydr0xy phenylpropany1]amino} meth0xymethy1—3-0x0pr0py1]pyrr01idiny1} -3 -meth0xy-5 - methyl0x0heptany1]-N-methy1—L-valinamide; 3 1—D-isova1y1—N— {(1 S,2R) {(2S)[(1R,2R)-3 - {[(1S)benzy1—2-meth0xy oxoethyl] amino} meth0xymethy10x0pr0py1]pyrr01idiny1} meth0xy [(1 S) methylpropyl] 0x0buty1} -N-methy1—L-va1inamide; 3-methy1-L-isova1y1—N-{(IS,2R){(2S)[(1R,2R){[(1S)benzylmeth0xy oxoethyl] amino} meth0xymethy10x0pr0py1]pyrr01idiny1} meth0xy [(1 S) methylpropyl] 0x0buty1} -N-methy1—L-va1inamide; L-isovalyl-N—{(1S,2R){(2S)[(1R,2R){[(IS)benzy1—2-meth0xy oxoethyl] amino} meth0xymethy10x0pr0py1]pyrr01idiny1} meth0xy [(1 S) methylpropyl] 0x0buty1} -N-methy1—L-va1inamide; D-isovalyl-N— R){(2S)[(1R,2R){[(1S)benzy1—2-meth0xy 2O oxoethyl] amino} meth0xymethy10x0pr0py1]pyrr01idiny1} meth0xy [(1 S) propyl] 0x0buty1} -N-methy1—L-va1inamide; 1,2-dimethy1—L-pr01y1—N- R){(2S)[(1R,2R){[(IS)carb0xy phenylethyl] amino} - 1-meth0xymethy1—3 -0x0pr0py1]pyrr01idiny1}meth0xy- 1- [(1 S) methylpropyl] 0x0buty1} -N-methy1—L-va1inamide; 1,2-dimethy1—D-pr01y1—N— {( 1 S,2R) {(2 S)[( 1 R,2R) {[( 1 S)- 1-carb0xy phenylethyl] amino} - 0xymethy1—3 -0x0pr0py1]pyrr01idiny1}meth0xy- 1- [(1 S) methylpropyl] 0x0buty1} -N-methy1—L-va1inamide; N~2~-[2,2-dimethyl(methy1amin0)pr0pan0y1]-N-{(1S,2R)meth0xy{(2S) [(1R,2R)meth0xymethy1—3-0x0{[(1S)pheny1—1-(1,3-thiazol y1)ethy1] amino } ]pyrr01idiny1} [(1 S)methy1pr0py1]0x0buty1}-N-methy1—L- valinamide; Methyl N— {(2R,3R)[(ZS)- l - {(3R,4S,SS)[ {N— [2,2-dimethyl lamino)propanoyl] -L-Valyl} (methyl)amino] -3 -methoxymethylheptanoyl}pyrrolidin yl] -3 -methoxymethylpropanoyl} nylalaninate; Methyl N— {(2R,3R)-3 -methoxy-3 - [(ZS)- l - {(3R,4S,5 S)-3 -methoxymethyl [methyl(N— { [(2S)methylpiperidinyl] carbonyl} -L-Valyl)amino]heptanoyl } pyrrolidinyl] - 2-methylpropanoyl} -L-phenylalaninate; Methyl N— {(2R,3R)-3 -methoxy-3 - [(ZS)- l - {(3R,4S,5 S)-3 -methoxymethyl l(N— -2—methylpiperidinyl] carbonyl} -L-Valyl)amino] heptanoyl}pyrrolidinyl] - 2-methylpropanoyl} -L-phenylalaninate; N— {(2R,3R)-3 -methoxy [(ZS)- l - {(3R,4S,5 S)-3 -methoxymethyl [methyl(N— - 2-methylpiperidin-2—yl] carbonyl} -L-Valyl)amino] heptanoyl } pyrrolidin—Z-yl] methylpropanoyl} -L-phenylalanine; N— {(2R,3R)-3 -methoxy-3 - [(ZS)- l - {(3R,4S, 5S)methoxymethyl [methyl(N— {[(2R)- 2-methylpiperidin-2—yl] carbonyl} -L-Valyl)amino] heptanoyl } pyrrolidin—Z-yl] methylpropanoyl} -L-phenylalanine; Methyl N-{(2R,3R)[(ZS){(3R,4S,SS)[(N-{[(3R)fluoropyrrolidin yl] carbonyl} -L-Valyl)(methyl)amino] -3 -methoxymethylheptanoyl}pyrrolidin-Z-yl] -3 - methoxy-Z-methylpropanoyl} -L-phenylalaninate; Methyl N-{(2R,3R)[(ZS){(3R,4S,SS)[(N-{[(3R)fluoropyrrolidin yl] carbonyl} -L-Valyl)(methyl)amino] -3 -methoxymethylheptanoyl}pyrrolidin-Z-yl] -3 - methoxy-Z-methylpropanoyl} -L-phenylalaninate; -[(ZS)- l - {[(3R,4S,SS)- l - {(ZS) [(1R,2R) {[2-(cyclohepta-2,4,6-trien— l - yl] amino} - l -methoxymethyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methoxymethyl oxoheptanyl] (methyl)amino} methyl- l-oxobutanyl] methylpiperidinecarboxamide; (2R)-N—[(ZS)- l - {[(3R,4S,SS)- l - {(ZS)[(1R,2R) {[2-(cyclohepta-2,4,6-trien- l - yl)ethyl] amino} - l -methoxymethyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methoxymethyl oxoheptanyl] (methyl)amino} methyl- utanyl] hylpiperidinecarboxamide; 2-methyl-L-prolyl-N- [(3R,4S,5 S)- l - {(ZS)[(1R,2R)-3 - yclohepta-2,4, 6-trien- l - yl)ethyl] amino} - l -methoxymethyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methoxymethyl oxoheptanyl] -N-methyl-L-valinamide; N—[(3R,4S,SS){(ZS)[(1R,2R){[2-(cyclohepta-2,4,6-trieny1)ethy1]amin0} methoxy-Z-methy1-3 -0x0pr0pyl]pyrr01idiny1}-3 -meth0xymethy10x0heptany1]—N~2~- {[(3R)-3 -flu0r0pyrr01idin-3 -yl]carbonyl} -N-methy1-L-valinamide; N—[(3R,4S,SS){(ZS)[(1R,2R){[2-(cyclohepta-2,4,6-trieny1)ethy1]amin0} methoxy-Z-methy1-3 -0x0pr0pyl]pyrr01idiny1}-3 -meth0xymethy10x0heptany1]—N~2~- {[(3 S)-3 -flu0r0pyrr01idin-3 -y1] carbonyl} -N-methy1—L-valinamide; (ZS)-N-[(ZS) 4S,SS)meth0xy{(ZS)[(1R,2R)meth0xymethy10x0 {[(1 heny1(1,3 -thiaz01yl)ethyl]amin0}pr0pyl]pyrr01idiny1}methy1—1- 0x0heptanyl] (methyl)amin0} -3 1—1-0x0butanyl]methylpiperidine-Z-carboxamide; (2R)-N—[(ZS){[(3R,4S,SS)meth0xy{(ZS)[(1R,2R)meth0xymethy10x0 {[(1 S)pheny1(1,3 -thiaz01yl)ethyl]amin0}pr0pyl]pyrr01idiny1}methy1—1- 0x0heptanyl] l)amin0} -3 -methy1—1-0x0butanyl]methylpiperidine-Z-carboxamide; N—2- {[(3R)-3 -flu0r0pyrr01idin-3 rb0ny1} -N- [(3R,4S,5 S)-3 -meth0xy {(ZS) [(1R,2R)meth0xymethy10x0{[(1S)pheny1—1-(1,3-thiazol y1)ethy1] amino } propyl]pyrr01idiny1} methyl- 1-0x0heptany1] -N-methy1—L-valinamide; N—2- {[(3 S)flu0r0pyrr01idiny1]carb0ny1}-N-[(3R,4S,SS)meth0xy{(ZS) [(1R,2R)meth0xymethy10x0{[(1S)pheny1—1-(1,3-thiazol y1)ethy1] amino } propyl]pyrr01idiny1} methyl- 1-0x0heptany1] -N-methy1—L-valinamide; 1,2—dimethy1-D-pr01y1-N—[(3R,4S,SS) {(ZS)[(1R,2R) {[(ZS)(4-amin0phenyl) methoxy- 1 0pan—2-yl] amino} meth0xymethy1—3-0x0pr0py1]pyrr01idiny1} -3 - methoxy-S-methyl0x0heptan—4-yl] -N-methy1-L-valinamide; 1,2—dimethy1-D-pr01y1-N—[(3R,4S,SS) {(ZS)[(1R,2R) {[(ZS)(4-amin0phenyl) methoxy0x0pr0pan—2-yl] amino} meth0xymethy1—3-0x0pr0py1]pyrr01idiny1} -3 - methoxy-S-methyl0x0heptan—4-yl] -N-methy1-L-valinamide; 1,2-dimethy1-L-pr01y1-N- [(3R,4S,5 S)-3 -meth0xy {(ZS)[(1R,2R)meth0xy-3 - {[(ZS)meth0xy-l -0x0-3 -(1,2,3 ,4-tetrahydr0quin01in—6-y1)pr0panyl] amino } hy1—3 - 0x0pr0py1]pyrr01idiny1}methy10x0heptan—4-yl]-N-methy1-L-valinamide; 1,2-dimethy1-L-pr01y1—N- S, SS) {(ZS)[(1R,2R)-3 - {[(ZS)-3 -(4-amin0phenyl) methoxy0x0pr0pan—2-yl] amino} meth0xymethy1—3-0x0pr0py1]pyrr01idiny1} -3 - methoxy-S-methyl0x0heptan—4-yl] -N-methy1-L-valinamide; 1,2-dimethy1—L-pr01y1—N- S,5 S)meth0xy{(ZS)[(1R,2R)meth0xy-3 - {[(ZS)meth0xy-1 -0x0-3 -pheny1pr0pany1] amino} methy1—3 -0x0pr0py1]pyrr01idiny1} methyl0x0heptany1]-N-methy1—L-valinamide; N— {(2R,3R)[(ZS) {(3R,4S,SS)[(N- {[(3R)flu0r0pyrr01idiny1]carb0ny1}-L- valyl)(methyl)amino] -3 -meth0xymethy1heptan0y1} pyrrolidin-Z-yl] -3 -meth0xy methylpropanoyl} -L-pheny1alanine; N— {(2R,3R)[(ZS) {(3R,4S,SS)[(N- {[(3 S)f1u0r0pyrr01idiny1]carb0ny1} -L- valyl)(methyl)amino] -3 -meth0xymethy1heptan0y1} pyrrolidin-Z-yl] -3 -meth0xy methylpropanoyl} -L-pheny1alanine; y1a1any1—N—[(3R,4S,SS) {(ZS)[(1R,2R) {[(2R,4S)carb0xy phenylpentan-Z-yl] amino} meth0xymethy1—3 -0x0pr0py1]pyrr01idiny1}-3 -meth0xy-5 - 0x0heptany1]-N-methy1—L-valinamide; y1a1any1—N-[(3R,4S,SS){(ZS)[(1R,2R)(bicyclo[1.1.1]penty1amin0) methoxy-Z-methy1-3 -0x0pr0py1]pyrr01idiny1}-3 -meth0xymethy10x0heptan—4-y1]—N— methyl-L-valinamide; 2-methy1a1any1—N-[(3R,4S,SS)meth0xy{(ZS)[(1R,2R)meth0xy{[(1R)-2— methoxy-Z-oxo(1 -phenylcyc10pr0py1)ethy1] amino} methy1—3 -0x0pr0py1]pyrr01idiny1} methyl0x0heptany1]-N-methy1—L-valinamide; 2-methy1a1any1—N—[(3R,4S,SS)meth0xy{(ZS)[(1R,2R)meth0xy{[(IS) methoxy-Z-oxo(1 -phenylcyc10pr0py1)ethy1] amino} methy1—3 -0x0pr0py1]pyrr01idiny1} methyl0x0heptany1]-N-methy1—L-valinamide; y1a1any1—N—[(3R,4S,SS){(ZS)[(1R,2R)({(1R)[(7R)-bicyclo[4.2.0]Octa- 1,3,5 -trien—7-y1] meth0xy0xoethy1} meth0xymethy1—3-0x0pr0py1]pyrr01idin yl} meth0xymethy1—1-0x0heptan—4-y1]-N-methy1—L-valinamide; 2-methy1a1any1—N—[(3R,4S,5S){(ZS)[(1R,2R)({(1S)[(7S)-bicyclo[4.2.0]octa- 1,3,5 -trien—7-y1] meth0xy0xoethy1} amino)meth0xymethy1—3-0x0pr0py1]pyrr01idin yl} meth0xymethy1—1-0x0heptan—4-y1]-N-methy1—L-valinamide; N,2-dimethy1a1any1—N— [(3R,4S,5 S)-3 xy {(ZS) [(1 R,2R)meth0xy-3 - {[(ZS)- 1-meth0xy0x0-3 -phenylpr0pany1]amino} methy1—3 -0x0pr0py1]pyrr01idiny1} methyl0x0heptany1]-N-methy1—L-valinamide; 2-methy1alany1-N-[(3R,4S,5S) {(ZS)[(1R,2R)({(1S)[(7R)-bicyclo[4.2.0]octa- 1,3,5 -trien—7-y1] meth0xy0xoethy1} amin0)- 0xymethy1—3-0x0pr0py1]pyrr01idin yl} h0xymethy10x0heptan—4-y1]-N-methy1-L-valinamide; trimethy1alany1-N— [(3R,4S,5 S)-3 -meth0xy {(ZS)[(1R,2R)meth0xy-3 - {[(ZS)meth0xy-l -0x0-3 -phenylpr0panyl] amino} methy1-3 -0x0pr0pyl]pyrr01idiny1} methyl0x0heptanyl]-N-methy1-L-valinamide; N,N,2-trimethylalany1-N—[(3R,4S,SS){(ZS)[(1R,2R){[(IS)carb0xy phenylethyl] amino} meth0xymethy1—3 0pyl]pyrr01idiny1}-3 -meth0xymethyl 0x0heptany1]-N-methy1-L-valinamide; 2-methy1alany1-N— [(3R,4S,5 S) {(ZS)[(1R,2R)-3 -{[(R)-carb0xy(1- phenylcyclopropyl)methy1] amino} meth0xymethy1—3 -0x0pr0py1]pyrr01idin— 1 -y1} -3 - methoxy-S-methyl0x0heptan—4-yl] -N-methy1-L-valinamide; difluoro {2-methy1alany1-N-[(3R,4S,SS) {(ZS)[(3R,4R,7S)benzy1 {2-[(3,5- dimethyl-1H-pyrroly1-kappaN)methylidene]-2H-pyrr01-5 -y1—kappaN} methy1-5 , 8, 13 -tri0x0- 2-0xa-6,9,12-triazapentadecan-3 rr01idiny1}-3 -meth0xymethy1— eptan—4-y1]—N— methyl-L-valinamidato}b0r0n; 2-methy1-D-pr01y1-N—[(3R,4S,5 S)-3 -meth0xy {(ZS)[(1R,2R)meth0xymethy1-3 - 0x0-3 - { [(1 heny1(1 ,3 olyl)ethyl] amino } propy1]pyrr01idiny1} methyl- 1- 0x0heptany1]-N-methy1-L-valinamide; methyl N— {(2R,3R)[(ZS) {(3R,4S,5 S)[ {N— [(3 -amin00xetany1)carb0nyl]-L- valyl} (methyl)amin0] -3 -meth0xymethylheptan0y1}pyrrolidin-Z-yl] meth0xy methylpropanoyl} -L-pheny1alaninate; 2-methy1alany1-N-{(3R,4S,SS)[(ZS) {(3R,4R,7S,12$)benzy1[3 -ch10r0 (propan-Z-yloxy)pheny1]methy1[4-(8-methylimidazo[1,2—a]pyridin-2—yl)benzyl]-5, 8,14- tri0x0-2,9-di0xa-6, 1 3 -diazatetradecan-3 -y1}pyrr01idiny1]—3 -meth0xymethy1— 1-0x0heptan yl} -N-methy1-L-valinamide; 2-methy1a1any1—N—[(3R,4S,SS){(ZS)[(1R,2R){[(ZS){[4-(5-flu0r0-1,3- benzothiazol-Z-yl)methylphenyl] amino} 0x0-3 -phenylpr0pany1] amino} meth0xy methyl-3 -0x0pr0pyl]pyrr01idiny1}-3 -meth0xymethy10x0heptanyl] -N-methy1—L- valinamide; 1 ,2—dimethy1-D-pr01y1-N— [(3R,4S,5 S)-3 -meth0xy {(ZS)[(1R,2R)meth0xy-3 - {[(ZS)meth0xy-l -0x0-3 -phenylpr0panyl] amino} hy1-3 -0x0pr0pyl]pyrr01idiny1} methyl0x0heptanyl]-N-methy1-L-valinamide; N,2-dimethy1alany1-N—[(3R,4S,5S) {(ZS)[(1R,2R) {[(ZS)(1H-ind01y1) methoxy0x0pr0pan—2-yl] amino} meth0xymethy1—3-0x0pr0py1]pyrr01idiny1} -3 - methoxy-S-methyl0x0heptan—4-yl] -N-methy1-L-valinamide; N,2-dimethy1alany1-N— [(3R,4S,5 S)-3 -meth0xy {(ZS)[(1R,2R)meth0xymethy10x0 {[(ZS)0x0-3 -pheny1—1-(pr0peny10xy)pr0panyl]amin0}pr0pyl]pyrr01idin yl} methy1— 1-0x0heptanyl]-N-methy1—L-valinamide; 2-methy1-L-pr01y1—N— [(3 R,4S, SS) {(ZS)[(1R,2R)-3 - {[(ZS)tert-but0xy0x0 phenylpropan-Z-yl]amino} meth0xymethy10x0pr0pyl]pyrr01idiny1} -3 -meth0xy-5 - methyl0x0heptanyl]-N-methy1-L-valinamide; N,2-dimethy1alany1-N— [(3R,4S,5 S)-3 -meth0xy {(ZS)[(1R,2R)meth0xymethy1- 3-0x0({(ZS)-l-0x0pheny1—1-[(1H-1,2,3-triazoly1methy1)amin0]pr0pan yl} amin0)pr0pyl]pyrr01idiny1}methy10x0heptany1]-N-methyl-L-valinamide; N,2-dimethy1alany1-N— [(3R,4S,5 S)-3 -meth0xy 2-[(1R,2R)meth0xymethy1 0x0-3 -pheny1— 1 -(pr0pyny1amin0)pr0panyl] amino } propyl]pyrr01idin yl} methy1— 1-0x0heptanyl]-N-methy1—L-valinamide; N,2-dimethy1alany1—N—[(3R,4S,5S) {(ZS) [(1 R,2R) {[(ZS)(1H-imidaz01—4-y1) y0x0pr0pan—2-yl] amino} meth0xymethy1—3-0x0pr0py1]pyrr01idiny1} -3 - methoxy-S-methyl0x0heptan—4-yl] -N-methy1-L-valinamide; N,2-dimethy1alany1-N— [(3R,4S,5 S) {(2 S)[(1R,2R)-3 - -3 -(4-hydr0xyphenyl) methoxy0x0pr0pan—2-yl] amino} meth0xymethy1—3-0x0pr0py1]pyrr01idiny1} -3 - methoxy-S-methyl0x0heptan—4-yl] -N-methy1-L-valinamide; N,2-dimethy1alany1-N—[(3R,4S,SS) {(ZS)[(1R,2R) {[(1R)carb0xy ethyl] amino} meth0xymethy1—3 -0x0pr0pyl]pyrr01idiny1}-3 -meth0xymethyl tany1]-N-methy1-L-valinamide; 1,2-dimethy1-L-pr01y1—N- S,5 S)meth0xy{(ZS)[(1R,2R)-l-methoxy-Z- methyl-3 -0x0 {[(ZS)0x0-3 -pheny1—1-(piperaziny1)pr0panyl]amin0}pr0pyl]pyrr01idin- 3 O 1 -y1} methy10x0heptan—4-yl]-N-methy1—L-valinamide; 2012/056224 l ,2-dimethyl-L-prolyl-N-[(3R,4S,SS)- l - {(ZS)[(1R,2R) - l -amino phenylpropanyl]amino} - l -methoxymethyloxopropyl]pyrrolidin- l -yl} -3 -methoxy-5 - methyloxoheptanyl] -N-methyl-L-valinamide; and 2-methyl-D-prolyl-N— [(3R,4S,5 S)- l - {(2 S) [( l R,2R)-3 - {[2-(cyclohepta-2,4,6-trien- l - yl)ethyl] amino} - l -methoxymethyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methoxymethyl oxoheptanyl] -N-methyl-L-valinamide.
In certain embodiments, the present invention relates to any of the aforementioned 2/ >9; compounds and attendant definitions, n R1 is .
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R1 is .
In n ments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein R1 is injigw‘iflijfljyINH o)\NH2 In n embodiments, the present invention s to any of the aforementioned compounds and attendant definitions, wherein Y is C2-C20 alkylene.
In certain embodiments, the present invention s to any of the aforementioned compounds and attendant definitions, wherein Y is —(CH2)p—; and p is 1-10.
In n embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein p is 1. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant ions, wherein p is 2. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein p is 3. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein p is 4. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein p is 5. In n embodiments, the present invention s to any of the entioned compounds and attendant def1nitions, wherein p is 6. In certain embodiments, the present invention relates to any of the aforementioned compounds and ant def1nitions, wherein p is 7. In certain ments, the present ion relates to any of the aforementioned compounds and ant tions, wherein p is g. In certain embodiments, the present invention relates to any of the entioned compounds and attendant def1nitions, wherein p is 9. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein p is 10.
In certain embodiments, the present invention relates to any of the aforementioned compounds and ant def1nitions, wherein Y is C2-C20 heteroalkylene.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein Y is H20)qCH2CH2—; and q is 1-10.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein q is 1. In n embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein q is 2. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein q is 3. In certain embodiments, the present invention relates to any of the aforementioned nds and attendant def1nitions, wherein q is 4. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant tions, wherein q is 5. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein q is 6. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein q is 7. In certain embodiments, the present invention relates to any of the aforementioned compounds and ant def1nitions, n q is 8. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein q is 9. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant def1nitions, wherein q is 10.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is 0 In certain embodiments, the present invention relates to any of the aforementioned G/\n/N>3; compounds and attendant definitions, wherein Z is 0 In n ments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein Z isdog; In certain embodiments, the present invention relates to any of the aforementioned compounds and ant definitions, wherein Z is In certain embodiments, the present invention relates to any of the entioned fiENr/O/Nfi0 compounds and attendant definitions, wherein Z is O o .
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is NH2 .
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, n R7 is F or Cl; and h is 4 or 5.
In certain embodiments, the present invention s to any of the aforementioned compounds and ant definitions, n R7 is F; and h is 3, 4 or 5.
In certain embodiments, the present invention relates to any of the aforementioned WO 72813 compounds and attendant definitions, wherein R7 is F; and h is 5.
In certain embodiments, the present invention relates to any of the entioned compounds and attendant definitions, n Z is -NH2.
In certain ments, the t invention relates to any of the aforementioned compounds and attendant definitions, wherein G is Cl. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein G is Br. In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein G is I.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein the compound is selected from the group consisting of the compounds of Table 18B.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein the nd is selected from the group consisting of: HN4»onNvi QMN N o/'\ o\o m \=/ °Qwi “ HZN MV06 N N N u w 0/\ o\o O\ON’s In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is LN},0 In certain ments, the present invention relates to any of the aforementioned L/\n/NF“; compounds and attendant definitions, wherein Z is O In n ments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is .
In certain ments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein Z is -NHL.
In certain embodiments, the present invention s to any of the aforementioned Lri/O/NH Y compounds and attendant definitions, wherein Z is o o .
In certain embodiments, the present invention relates to any of the aforementioned L N compounds and attendant definitions, n Z is NH2.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein L is H(C)-.
In certain embodiments, the present invention relates to any of the aforementioned compounds and attendant definitions, wherein L is an antibody selected from a murine antibody for the treatment of ovarian cancer such as oregovomab (OVAREX® ); a murine Inga antibody for the treatment of ctal cancer such as edrecolomab (PANOREX®) ; an anti-EGFR IgG chimeric antibody for the treatment of mal growth factor positive cancers, such as head and neck cancer, for instance cetuximab (ERBITUX® ); a humanized antibody for the treatment of sarcoma, such as a Humanized Monoclonal Antibody to the Vitronectin Receptor ((XVB3) like Vitaxin®; a humanized IgG1 antibody for the ent of chronic lymphocytic leukemia (CLL) such as alemtuzumab (CAMPATH I/H®); SMART IDlO which is a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; l 3 II Lym—l (ONCOLYM®)which is a abeled murine anti-HLA-DrlO antibody for the treatment of non-Hodgkin's lymphoma; a humanized anti-CD2 mAb for the treatment of n's Disease or dgkin's lymphoma such as ALLOMUNE® ; zumab (CEACIDE®) which is a humanized anti-CEA antibody for the treatment of colorectal cancer; bevacizumab (AVASTIN®) which is a humanized anti- VEGF-A mAb for the treatment of brain, colon, kidney, or lung cancer; Ibritumomab tiuxetan (ZEVALIN®) which is an anti-CD20 monoclonal dy to the treatment of non-Hodgkin’s lymphoma; ofatumumab (ARZERRA®) which is a human anti-CD20 monoclonal antibody for the ent of chronic lymphocytic leukemia; panitumumab (VECTIBIX®) which is a human anti-EGFR monoclonal antibody for the treatment of colon ; rituximab (RITUXAN®) which is an D20 chimeric monoclonal antibody for the treatment of chronic lymphocytic leukemia and non-Hodgkin’s lymphoma; tositumomab R®) which is an anti-CD20 onal antibody for the treatment of non-Hodgkin’s lymphoma; trastuzumab (HERCEPTIN®) which is an anti-HER2 receptor monoclonal antibody for the treatment of breast and stomach cancer; ipilimumab (YERVOY®) which is an anti-CTLA4 human monoclonal antibody for the ent of melanoma; gemtuzumab and inotuzumab ozogamicin.
In another specific embodiment, L includes antibodies selected from anti-I-l3 antibodies, including anti-I- l 3 antibodies used in the treatment of cancer, for instance L-13R0t2 antibodies.
In yet another ic embodiment, L includes antibodies selected from anti-Notch antibodies, including anti-Notch dies used in the treatment of cancer.
In certain embodiments, the antibody L is bound to the linker via a sulfur bond or via a sulfur-sulfur bond.
Another aspect of the invention relates to an antibody drug ate comprising any of the aforementioned nds.
Another aspect of the invention relates to an antibody drug conjugate sing an antibody and any one of the aforementioned compounds.
In certain embodiments, the present invention relates to any of the aforementioned 2012/056224 antibody drug conjugates and attendant definitions, wherein the compound is covalently bound to the antibody.
In certain embodiments, the present invention relates to any of the aforementioned dy drug conjugates and attendant definitions, wherein the compound in said antibody drug conjugate is selected from the group consisting of the compounds of Table 18B, 0 o H2N+/\O+6\)Lu>§rN\i)LNH H N N o A o\ o o\ o N,mS H N N N N 2 VON” o A [i] o\ o o\ o / N S H H N N H o A l o\ o o\ o In certain embodiments, the present invention relates to any of the aforementioned antibody drug conjugates and attendant definitions, wherein the dy drug conjugate ses between 2, 3, 4, 5, 6, 7, 8, 9 or 10 compounds of the invention.
In certain embodiments, the present invention relates to any of the aforementioned antibody drug conjugates and attendant definitions, wherein the antibody drug conjugate comprises 3 or 4 compounds of the invention.
The Antibody Unit (Ab) As noted above, the term ”antibody” (or “Ab”) herein is used in the broadest sense and specifically covers intact onal antibodies, polyclonal dies, monospecific antibodies, multispecific antibodies (e. g., bispecific antibodies), and antibody fragments that t the desired ical activity. In addition, while n aspects of the invention described herein refer to antibody drug conjugates, it is further envisioned that the antibody portion of the 2012/056224 conjugate might be replaced with anything that specifically binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population. For example, instead of containing an antibody a conjugates of the invention could contain a targeting molecule that binds to, complexes with, or reacts with a receptor, antigen or other receptive moiety of a cell population sought to be therapeutically or otherwise biologically modified. Example of such molecules include smaller molecular weight proteins, polypeptide or peptides, lectins, glycoproteins, non-peptides, ns, nt-transport molecules (such as, but not limited to, transferrin), or any other cell binding molecule or substances. In certain aspects, the antibody or other such targeting molecule acts to r a drug to the particular target cell population with which the antibody or other targeting molecule interacts.
Heteroatoms that may be present on an antibody unit include sulfur (in one embodiment, from a sulfliydryl group of an antibody), oxygen (in one embodiment, from a carbonyl, carboxyl or hydroxyl group of an antibody) and nitrogen (in one embodiment, from a primary or secondary amino group of an antibody). These hetero atoms can be present on the dy in the antibody's natural state, for example a naturally-occurring dy, or can be uced into the dy via chemical modification.
In one ment, an antibody unit has a sulfliydryl group and the antibody unit bonds via the sulfhydryl group's sulfur atom.
In another embodiment, the antibody has lysine residues that can react with activated esters (such esters include, but are not d to, N—hydroxysuccinimde, pentafluorophenyl, and p-nitrophenyl esters) and thus form an amide bond ting of the nitrogen atom of the antibody unit and a carbonyl.
In yet another aspect, the antibody unit has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. The reagents that can be used to modify s include, but are not limited to, N—succinimidyl S-acetylthioacetate (SATA) and 2- Iminothiolane hydrochloride (Traut's Reagent).
In another embodiment, the antibody unit can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups.
In yet another embodiment, the antibody unit can have one or more carbohydrate groups that can be oxidized to provide an aldehyde group (see, e. g., Laguzza, et al., 1989, J. Med. Chem. 32(3):548-55). The corresponding de can form a bond with a reactive site such as, for example, hydrazine and ylamine. Other protocols for the modification of proteins for the 2012/056224 attachment or association of drugs are described in Coligan et al., Current ols in Protein Science, vol. 2, John Wiley & Sons (2002) (incorporated herein by reference).
When the conjugates comprise non-immunoreactive protein, polypeptide, or peptide units d of an antibody, useful non-immunoreactive protein, polypeptide, or peptide units include, but are not d to, transferrin, epidermal growth factors (”EGF”), bombesin, gastrin, gastrin- releasing peptide, platelet-derived growth , IL-2, IL-6, transforming grth factors (”TOP”), such as TGF-OL and TGF-B, vaccinia grth factor (”VGF”), insulin and insulin-like growth factors 1 and 11, statin, lectins and apoprotein from low density lipoprotein.
Useful polyclonal antibodies are heterogeneous populations of antibody molecules d from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of dies to a particular antigenic determinant (e. g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by uous cell lines in culture.
Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or ic monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983, 2O Immunology Today 4:72-79; and Olsson et al., 1982, Meth. Enzymol. 92:3-16).
The antibody can also be a bispecif1c antibody. Methods for making bispecif1c dies are known in the art and are discussed infra.
The antibody can be a functionally active nt, derivative or analog of an antibody that immunospecif1cally binds to target cells (e.g., cancer cell antigens, viral antigens, or microbial antigens) or other antibodies that bind to tumor cells or matrix. In this regard, ”functionally active” means that the fragment, derivative or analog is able to elicit anti-anti- idiotype antibodies that recognize the same n that the antibody from which the nt, derivative or analog is derived recognized. cally, in an exemplary embodiment the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C—terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR ces can be used in binding assays with the antigen by any binding assay method known in the art (e. g., the BIA core assay) (for location of the CDR sequences, see, e. g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National ute of Health, Bethesda, Md.; Kabat E et al., 1980, J. logy 125(3):961-969).
Other useful antibodies include fragments of dies such as, but not limited to, F(ab')2 fragments, Fab fragments, Fvs, single chain antibodies, diabodies, triabodies, odies, scFv, scFv-FV, or any other molecule with the same specif1city as the antibody.
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful dies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as for example, those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions.
(See, e.g., U.S. Pat. No. 4,816,567; and U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e. g., U.S. Pat.
No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and zed monoclonal dies can be produced by recombinant DNA ques known in the art, for example using methods described in International Publication No. WO 87/02671; European Patent Publication No. 0 184 187; European Patent Publication No. 0 171 496; European Patent Publication No. 0 173 494; International Publication No. WO 86/01533; U.S.
Pat. No. 4,816,567; European Patent ation No. 012 023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J.
Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; yan et al., 1988, Science 239: 1534; and r et al., 1988, J. Immunol. 141 :4053-4060; each of which is incorporated herein by reference in its entirety.
Completely human antibodies are particularly desirable and can be produced using transgenic mice that are ble of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e. g., all or a n of a polypeptide of the invention. onal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human globulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE dies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93. For a detailed discussion of this technology for producing human dies and human monoclonal antibodies and ols for producing such antibodies, see, e.g., U.S. Pat. Nos. 5,625,126; 425; ,569,825; 5,661,016; 5,545,806; each of which is orated herein by reference in its entirety. Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (now Amgen, Freemont, Calif) and Medarex (Princeton, N.J.).
Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as ”guided selection.” In this approach a selected non-human monoclonal antibody, e. g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (See, e.g., Jespers et al., 1994, Biotechnology 12:899-903). Human antibodies can also be produced using various techniques known in the art, ing phage display libraries (see, e. g., Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581; Quan and Carter, 2002, The rise of monoclonal antibodies as therapeutics, 1n Anti-IgE and Allergic Disease, Jardieu and Pick, eds., Marcel , New York, N.Y., r 20, pp. 427-469).
In other embodiments, the antibody is a fusion n of an antibody, or a functionally active fragment f, for example in which the antibody is fused via a covalent bond (e. g., a peptide bond), at either the N-terminus or the inus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not from an antibody. Preferably, the antibody or fragment thereof is covalently linked to the other protein at the inus of the constant domain. dies include analogs and derivatives that are either modif1ed, i.e., by the covalent attachment of any type of molecule as long as such nt attachment s the antibody to retain its antigen binding immunospecif1city. For example, but not by way of limitation, derivatives and analogs of the antibodies include those that have been further modified, e. g., by ylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the ce of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids.
Antibodies can have modifications (e. g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies can have modifications in amino acid residues identified as involved in the ction between the anti-Fc domain and the FcRn receptor (see, e. g., International Publication No. WO 97/3463 1, which is orated herein by reference in its entirety).
Antibodies immunospecific for a cancer cell antigen can be ed commercially or produced by any method known to one of skill in the art such as, e. g., chemical synthesis or recombinant sion ques. The nucleotide ce encoding antibodies immunospecific for a cancer cell antigen can be obtained, e. g., from the GenBank database or a database like it, literature publications, or by routine g and sequencing.
In a specific embodiment, known antibodies for the treatment of cancer can be used.
Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e. g., inant expression techniques.
The nucleotide sequence encoding antibodies immunospecific for a cancer cell n can be obtained, e. g., from the GenBank database or a database like it, the literature publications, or by routine g and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, OVAREX® which is a murine antibody for the treatment of ovarian cancer; PANOREX® (Glaxo Wellcome, NC) which is a murine Inga dy for the treatment of colorectal cancer; Cetuximab ERBITUX® (Imclone s Inc., NY) which is an anti-EGFR IgG chimeric antibody for the treatment of epidermal grth factor positive s, such as head and neck cancer; Vitaxin® (MedImmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; CAMPATH I/H® (Leukosite, MA) which is a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); SMART IDlO (Protein Design Labs, Inc., CA) which is a humanized anti-HLA-DR antibody for the treatment of non- n's lymphoma; ONCOLYM® (Techniclone, Inc., CA) which is a radiolabeled murine anti-HLA-Drl 0 antibody for the treatment of non-Hodgkin's lymphoma; NE® (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin's ma; CEACIDE® (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer; AVASTIN® (Genentech/Roche, CA) which is a humanized anti-VEGF-A mAb for the treatment of brain, colon, kidney, or lung cancer; ZEVALIN® (Spectrum Pharmaceuticals, NV) which is an anti-CD20 monoclonal antibody to the treatment of non-Hodgkin’s lymphoma; ARZERRA® (GSK, UK) which is a human anti-CD20 monoclonal antibody for the treatment of chronic cytic leukemia; VECTIBIX® , CA) which is a human anti-EGFR monoclonal dy for the treatment of colon cancer; RITUXAN® (Genentech/BioGen, CA) which is an anti-CD20 chimeric monoclonal antibody for the treatment of c lymphocytic leukemia and non-Hodgkin’s lymphoma; BEXXAR® (GSK, UK) which is an D20 monoclonal antibody for the treatment of non-Hodgkin’s lymphoma; HERCEPTIN® (Genentech, CA) which is an anti-HER2 receptor monoclonal antibody for the treatment of breast and stomach cancer; YERVOY® (BMS, NJ) which is an anti-CTLA4 human monoclonal antibody for the ent of melanoma; MYLOTARG® (Wyeth/Pfizer, NY) which is anti-CD33 humanized monoclonal dy conjugated to calicheamicin for the treatment of acute myelogenous leukemia; and, inotuzumab ozogamicin (Wyeth/Pfizer, NY) which is an anti-CD22 humanized monoclonal antibody conjugated to calicheamicin for the treatment of acute lymphocytic leukemia and non-Hodgkin’s lymphoma.
In r specific embodiment, L13 antibodies, including anti-IL13 antibodies used in the treatment of cancer, can be used.
In another specific embodiment, anti-Notch antibodies, including anti-Notch antibodies used in the treatment of , can be used.
In ts to discover ive cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as ed to on one or more normal ncerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the e of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies.
Synthesis of Compounds and Antibody Drug Conjugates Thereof The compounds and conjugates of the invention can be made using the synthetic procedures outlined below in the Exempliflcation.
As described in more detail below, the compounds and conjugates of the invention can be prepared using a section of a linker unit having a reactive site for binding to the compound.
Linker A linker (sometimes referred to as “[linker]” ) is a tional compound which can be used to link a drug and an dy to form an antibody drug conjugate (ADC). Such conjugates are useful, for example, in the formation of imrnunoconjugates directed against tumor associated antigens. Such conjugates allow the selective delivery of cytotoxic drugs to tumor cells.
In one embodiment, the linker has the formula: ignimwy 2W)? A is O N H2 or ; wherein Y is C2-C20 alkylene or C2-C20 heteroalkylene; C3-C8 carbocyclo-, ne-, -C3- Cgheterocyclo-, -C1-C1oalkylene-arylene-, ne-C1-C10alkylene-, -C1-C10alkylene-(C3- Cgcarbocyclo)-, -(C3-Cgcarbocyclo)-C1-Cloalkylene-, -C1-Cloalkylene-(C3-Cgheterocyclo)-, or - (C3-C8 heterocyclo)-C1-C10alkylene-; Zis O NH2 or -NH2; R7 is independently selected for each occurrence from the group consisting of F, Cl, 1, Br, N02, CN and CF3; is hydrogen, -C1-C10alkyl, -C3-Cgcarbocycle, aryl, -C1-C10heteroalkyl, -C3- Cgheterocyclo, -C1-C10alkylene-aryl, -arylene-C1-C10alkyl, -C1-C10alkylene-(C3-Cgcarbocyclo), - (C3-C8 yclo)-C1-C10alkyl, -C1-C10alkylene-(C3-Cgheterocyclo), and -(C3-Cg heterocyclo)-C1- Cloalkyl, where aryl on R10 comprising aryl is optionally substituted with [R7]h; and his 1,2,3,4or5.
In an ADC the linker serves to attach the payload to the antibody.
In one aspect, a second section of the linker unit is introduced which has a second ve site e.g., an ophilic group that is reactive to a nucleophilic group present on an antibody unit (e. g., an antibody). Useful nucleophilic groups on an antibody include but are not d to, sulfhydryl, yl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on a linker unit and forms a covalent bond to a linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for antibody attachment.
In another embodiment, a linker unit has a reactive site which has a nucleophilic group that is reactive to an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a linker unit can react with an electrophilic group on an antibody and form a nt bond to the antibody. Useful nucleophilic groups on a linker unit include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine ylate, and arylhydrazide. The ophilic group on an antibody provides a convenient site for attachment to a linker unit.
Amino functional groups are also useful reactive sites for a linker unit because they can react with carboxylic acid, or activated esters of a compound to form an amide linkage. lly, the peptide-based compounds of the invention can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for e, according to the liquid phase synthesis method (see, e. g., Schroder and Lubke, ”The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
As described in more detail below, the conjugates can be prepared using a section of the linker haVing a reactive site for binding to a compound of the invention and introducing another n of the linker unit haVing a reactive site for an dy. In one , a linker unit has a reactive site which has an electrophilic group that is reactive with a nucleophilic group present on an antibody unit, such as an antibody. The electrophilic group provides a convenient site for antibody attachment. Useful nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the philic group of an antibody is reactive to an electrophilic group on a Linker unit and forms a nt bond to a linker unit.
Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups.
In another embodiment, a linker unit has a reactive site which has a nucleophilic group that is reactive with an electrophilic group t on an antibody unit. The electrophilic group on an dy provides a convenient site for attachment to a linker unit. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a linker unit can react with an electrophilic group on an antibody and form a covalent bond to the antibody. Useful nucleophilic groups on a linker unit include, but are not limited to, hydrazide, oxime, amino, ine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
As used , “mc-” previously known as “MalC—” refers to O As used herein, itPABC—” previously known as “MalCValCitPABC-” refers to O O \L As used herein, “AmPegXC2-” refers to HZNVOWX As used herein, “chalCitPABCAmPegXCZ-“refers to O O 1 O NH2 As used herein, “MalPegXC2ValCitPABC-” refers to O 1N, Br/fifofloW As used herein, “2BrAcPegXC2” refers to 0 g “a As used , “mV-” refers to O 0 As used herein, “mb-” refers to 0 NW As used herein, “me-” refers to O 0 (if s‘ As used herein, “MalC6-” refers to 0 As used herein, “PFPCOPegXCZValCitPABC—” refers Zflifimigwfiw F 0.1 o NH2 As used herein, “PFPCOPegXC2AmPngC2-” refers to F: : :F O O F OWOMHVOM F 0 As used herein, “PFPCOPegXCZAlaAlaAsnPABC-” refers to 2012/056224 :QZLOWOKN HJLN ZIO F {kg H O mom/15R As used herein, “PFPCOPegXC2-” refers to F F o o F oilflobdkf As used herein, “PFPCOPegXCZAmPngCZPABC—” refers to KIWMVMUAV As used , “mcGly-” refers to g/ N\/\/\iNYE 0 0 As used herein, “AzCOC2Ph4AmCOPeg2C2-” refers to VNWGHVMOVIE As used herein, “AzCOC2Ph4AmPeg1C1-“ refers to K?)0i? OVNWG“VWEMI): HZN o As used herein, “AcLysValCitPABC-“ refers to WLNEMNQO o H H 81?; E H o O iH NH2 OJ‘NH2 Conjugation with Transglutaminase In certain embodiments, a compound of the invention may be covalently crosslinked to an Fc-containing 0r Fab-containing polypeptide ered with an acyl donor glutamine-containing WO 72813 tag (e. g., Gln-containing peptide tags or ) or an endogenous glutamine made reactive (i.e., the ability to form a covalent bond as an acyl donor in the presence of an amine and a transglutaminase) by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, mutation, or any combination f on the polypeptide), in the presence of transglutaminase, provided that the compound of the invention comprises an amine donor agent (e. g., small molecule comprising or attached to a reactive amine), thereby forming a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site-specif1cally conjugated to the Fc-containing or Fab-containing polypeptide h the acyl donor glutamine-containing tag or the exposed/accessible/reactive endogenous glutamine. For e, nds of the invention may be conjugated as described in International Patent Application Serial No. , whose entire contents are orated herein by reference. In n embodiments, to facilitate conjugation of the compound ofthe invention to an Fc-containing or Fab-containing polypeptide ered with an acyl donor glutamine-containing tag or an endogenous glutamine made reactive by polypeptide engineering in the presence of transglutaminase, Z is NHZ.
Conjugation to the Human Light Chain Kappa Domain Constant Region In certain embodiments, a compound of the invention may be covalently attached to the side chain of K188 of the human light chain kappa domain constant region (CLK) (full light chain numbering according to Kabat). K188 may also be termed CLK K80, when counting only the human kappa constant region, for example, of SEQ ID NOs: 1, 2, 3 and 4).
For example, compounds of the invention may be conjugated as described in US Patent Application Serial Number 13/180,204, or WO2012/007896 whose entire contents are incorporated herein by reference. In certain ments, to facilitate conjugation to K188 CLK / O R7 | 80), Z is OJSE‘! ; R7 is independently selected for each occurrence from the group consisting of F, Cl, I, Br, N02, CN and CF3; and h is 1, 2, 3, 4 or 5.
In n embodiments, to facilitate conjugation to K188 CLK (CLK-K80), Z is F F The present invention r provides antibody drug conjugates comprising an dy, or antigen binding portions thereof, comprising a constant kappa domain covalently ated to a toxin of the invention, characterized in that at least one toxin of the invention is covalently conjugated to K80 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID N03 or SEQ ID NO: 4 (Table 1).
In some aspects, the number of toxins of the invention covalently conjugated to the at K80 may be a range whose lower limit is ed from the group consisting of about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, and about 2.0, and whose upper limit is selected from the group consisting of about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5. In some aspects, p is about 2.
Conjugation of the toxin with the constant light domain of an antibody is particularly desirable to minimize, or prevent, any interference with binding of the Fc portion of the antibody to Fc receptors (such as FcyR and FcRn) or binding of the antibody to its respective target.
Conversely, conjugation of the respective toxin to the Fc n of an antibody may decrease the antibody half-life in vivo and/or its capacity to interact with the immune system (effector function). Conjugation of the toxin in the variable heavy chain (VH) or variable light chain (VL) region of the antibody carries a risk of diminishing the binding of the antibody to its cognate.
Furthermore, s ation to CLK-K80 is reliable and robust, conjugation to other antibody surface lysines, each of slightly different reactivity and p1 can result in an geneous sample of conjugated antibodies that can release ated molecules at rtune or irregular times, such as during circulation and prior to delivery of the Effector Moiety to the target by antibody recognition.
In addition, the present invention provides for known polymorphisms of the kappa chain V/A at position 45 and A/L at position 83 (giving the 3 identified human constant kappa polymorphisms Km(1):V45/L83 (SEQ ID NO:2), Km(1,2): A45/L83 (SEQ ID N03), and Km(3) A45/V83 (SEQ ID NO:4)). The variability of residues at positions 45 and 83 in SEQ ID NO:1 may be selected so as to only provide for any one, two or all three of the Km( 1), Km( 1,2), and Km(3) rphisms.
Table 1 SEQ 1]) NO DESCRIPTION SEQI. ENCE 1 hLC constant TVAAPSVFIIF PPSD'EQ' .KSG TASWCIJLNN region GENUS KVQW KVDNXLQSGN SQdLSVTdLQDS KDSTYSLSST LTIJS <A DYEK HKXYACEVTH QGL S S PVTKS FNRGEC h LC constant TVAAPSVF: F region Km( 1) FYPREAKVQW polymorphism KDSTYSLSST (V45/L83) QGLSS PVTKS h LC constant region Km(1’2) TVAAPSVFIF POIYmOYPhism FYPREAKVQW A45/L83 KDSTYSLSST QGLSSPVTKS h LC constant ' ' region Km(3) TVAAPSVFIF . TASWC: polymorphism FYPREAKVQW SQ IISVT '- A45/V83 KDSTYSLSST I I HKVYACIT QGLSSPVTKS Wherein x at position 45 is A or V, and x at position 83 is L or V.
In certain ments, the invention es for a composition comprising a compound of the invention covalently conjugated to an antibody (or antigen binding n f), wherein at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the nd of the invention in the composition is conjugated to the antibody or antigen binding portion thereof at K188 CLK.
In certain embodiments, the compounds of the invention F:©:O\n/\/O\/\O/\)J\NJ€(N\-)LNF O O N N\_)\SH Nl/\> H = o o l ' /\ o o o o F F \ \ :II:NMNM,» FfiOMO~0w0~O$OVkN><KNEJLNfi$WMNJOHF F o O O H H o F I o /=\| o\ o O\ o — Dand o o o o Qflofi‘wfikfififir” F OWOWOVL” N?” H O /=\ I /O O F F0 \L \o H iv] F NH H2N O may be ated to the combining site of a catalytic antibody, such as aldolase antibodies, or n g portion thereof. se antibodies contain combining site portions that, when unencumbered (for example by conjugation), catalyze an aldol addition reaction n an aliphatic ketone donor and an aldehyde acceptor. The contents of US Patent Application Publication No. US 2006/205670 are incorporated herein by nce, in particular pages 78-1 18 bing linkers, and paragraphs [0153]-[0233] describing antibodies, useful fragments, variants and modifications thereof, h38C2, combining sites and complimentary determining regions (CDRs), and related antibody technology (Table 2, and exemplary compounds below): O o H H N Hi O?N\g/\/<j/ wowOAATJi/filipgjf% rm“rwwlm*fifi~§i% 33o o H N Hth} 0 O 0 1 o No NH HZNAO H .0 Wam‘lflwfi WVWQEMQ” ”A' g) ; N o o o e 0 \Lw HZN/KO and The term “combining site” includes the CDRs and the adjacent framework residues that are involved in antigen binding.
Table 2 SEQ II) NO DESCRIPTION SEQUENCE h38C2 VL SPSS LSASVGDQVT :TCRSSQSLL HTYGSPYLNW YLQKPGQSP< LLIYKVSNRF SGVPSRFSGS GSGTDFT'IT" SST.QP'T.DFAV YFCSQGTHLP YTFGGGT (V4. K 2012/056224 h38C2ti SGGG LVQPGGSLR; SCAASGFTFS YW SWVRQS WVSfi RHRSDNYAT {YAESVKGRF TISRD SKNT LYLQMNSLRA fiDTG YYCKT YFYSFSYWGQ GTLVTVSS h38C21£j VUQMTQSPSS LSASVGDQVT :TCRSSQSLL {TYGSPYLNW YLQKPGQSP< L'"YKVSNQF SGVPSRFSGS GSGTDFTHT" SSHQPWDFAV YFCSQGTiI YTFGGGT<Vfi {QTVAAPSV EHEPPSDI ' KSGTASVVC; FYPQEAK VQWKVDNAI SGNSQfiSVTZ DSKDSTYSL SSTLTLS YEKHKVYACZ VTiQGLSSPV TKSFNQG h38C2ffl: fiVQ' LVQPGGSLR; FTFS YW SWVRQS PfiKGHfiWVSfi RHRSDNYAT KGQF T13?) SKNT SLQA fiDTG YYC<T YFYSFSYWGQ LVTVSSAS TKGPSVFPLA PSS<STSGGT FPEPVTVSWN SGALTSGVHT YSLSSVVTVP SSSLGTQTY: KVDKRVEPKS CPPC VFLFPP<PKD TUM SQTPfiV DPEVKF WYV DGVEViNA<T YRVVSVHTVH {QDWH GKVY AP fiKT S<A {GQPQEPQVY KNQVSLTCLV (GEYPSD AV NYKTTPPVLD SDGSFFHYSK ' GNVFSCSVMH EALHNHYTQK Conjugation with linkers comprising succinimides, ing ring-opened versions In certain embodiments, the present invention includes a compound of the invention conjugated via a succinimide-based linker or a ring-opened succinimide-based linker. The stability of the succinimide-cysteine linkage has become an area of increasing interest.
Succinimides can be transferred both in vitro and in vivo to exogenous thiol nucleophiles, presumably through a retro-Michael on resulting in a maleimide that is subsequently attacked by a thiol. It is believed that hydrolysis of the ring s in a species that is resistant to the retro-Michael reaction. This renders the resulting conjugate more stable and potentially more efficacious. Conditions may be optimized to forcibly open the succinimide ring on the conjugate. Basic conditions resulted in facile ysis of the ring. For instance, linkers containing a polyethylene glycol (PEG) chain can be hydrolysed at pH 9.2 at 37°C in approximately 12h, and linkers containing an alkyl chain, such as “mc” may require a higher temperature and longer on time in order to drive the ring-opening to completion. 50 mM borate , pH 9.2 a \ iWES o 37 C, 12 h N ‘j’\/Oj\/\n/Payload yload O C02H "MalPengZ" "Mal(H20)Peng2" 50 mM bzgatgbzgffir, pH 9-2 \ / O O WE C02H S/qMPayload SJ;(N\/\/\)J\F’z=lyloadO 0 II II m0 "m(H20)c" Example of forced hydrolysis of ide-based conjugates In order to assess the stability of these conjugates and prioritize samples for in vivo evaluation, an assay was developed that involves the treatment of the maleimide-linked conjugates with excess aqueous glutathione (GSH) or plasma. Aliquots of the reaction e were analyzed at various timepoints to determine the loading of the conjugate, using the methodology bed above. The results (Table 24) te that the drug-antibody linkage is slowly cleaved in a GSH-dependent manner. As expected, the rate of cleavage is highly dependent upon the hydrolysis of the imide ring. Importantly, these results appear to translate to improved PK exposure, as measured by an increase in area-under-curve (AUC) of the conjugate and by an increase in the conjugate/Ab exposure ratio.
Method for assessing the stability of ADCs The ADC sample (30 ug) in PBS is mixed with glutathione (GSH) on to produce final concentration of GSH of 0.5 mM and 3 mg/mL protein concentration. A control sample (without GSH) was likewise prepared from 30 ug ADC diluted to 3 mg/mL in PBS. The GSH- treated ADC sample and the control ADC sample were incubated at 37°C and were sampled at O, 3, and 6 days. Aliquots were reduced with excess TCEP, acidifled by adding 0.1% formic acid solution with 10% acetonitrile and analyzed by for loading by LC/MS as described below.
Sample analysis: Analysis was performed using an Aglient 1100 capillary HPLC coupled with Waters Xevo G2 Q-TOF mass spectrometer. The analytes were loaded onto a Zorbax Poroshell 300SB C8 column (0.5 mm X 75 mm, maintained at 80°C) with 0.1% formic acid, and eluted using a gradient of 20—40% buffer B (80% acetonitrile, 18% 1-propanol, 2% water with 0.1% formic acid) at a flow rate of 20 ul/min over 5.5 minutes. Mass spectrometric detection was carried out in positive, sensitivity mode with capillary e set at 3.3 kV. Data analysis was performed with MaxEnt 1 on in MassLynx and intensities were used for loading calculation based on the previously described formula.
Compositions and Methods of Administration In other embodiments, another aspect of the invention s to pharmaceutical compositions including an effective amount of a compound of the ion and/or antibody drug conjugate thereof and a ceutically acceptable carrier or vehicle. In certain embodiments, the itions are suitable for veterinary or human administration.
The present pharmaceutical compositions can be in any form that allows for the composition to be stered to a patient. For example, the composition can be in the form of a solid or liquid. Typical routes of administration include, without limitation, parenteral, ocular and intra-tumor. Parenteral administration includes subcutaneous injections, intravenous, uscular or intrastemal injection or infusion techniques. In one , the compositions are administered parenterally. In a specific embodiment, the compositions are administered intravenously.
Pharmaceutical compositions can be ated so as to allow a compound of the invention and/or antibody drug conjugate thereof to be bioavailable upon stration of the ition to a patient. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of a compound of the ion and/or antibody drug conjugate thereof in liquid form can hold a plurality of dosage units.
Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors.
Relevant factors include, without tion, the type of animal (e. g., human), the particular form of the a compound of the invention and/or dy drug conjugate thereof the manner of administration, and the composition employed.
The pharmaceutically acceptable carrier or vehicle can be solid or particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid. In addition, the r(s) can be particulate.
The composition can be in the form of a liquid, e. g., a solution, emulsion or suspension.
In a composition for administration by injection, one or more of a tant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
The liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's on, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl l or methyl paraben; idants such as ascorbic acid or sodium bisulf1te; chelating agents such as ethylenediaminetetraacetic acid; buffers such as es, citrates, phosphates or amino acids and agents for the ment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in ampoule, a disposable syringe or a multiple-dose Vial made of glass, plastic or other material. Physiological saline is an exemplary adjuvant. An inj ectable composition is preferably e.
The amount of a compound of the invention and/or dy drug ate thereof that is effective in the treatment of a particular disorder or condition will depend on the nature of the er or condition, and can be determined by standard clinical techniques. In addition, in Vitro or in Vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
The compositions comprise an effective amount of a compound of the invention and/or antibody drug conjugate f such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a nd of the invention and/or antibody drug conjugate thereof by weight of the composition. In an exemplary embodiment, pharmaceutical compositions are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the amount of a compound of the ion and/or antibody drug conjugate thereof.
For intravenous administration, the composition can comprise from about 0.01 to about WO 72813 2012/056224 100 mg of a compound of the invention and/or antibody drug conjugate thereof per kg of the patient's body weight. In one aspect, the composition can include from about 1 to about 100 mg of a compound of the invention and/or antibody drug conjugate thereof per kg of the patient's body weight. In another , the amount administered will be in the range from about 0.1 to about 25 mg/kg of body weight of a compound of the invention and/or antibody drug conjugate thereof.
Generally, the dosage of a compound of the invention and/or antibody drug conjugate thereof stered to a patient is typically about 0.01 mg/kg to about 20 mg/kg of the patient's body weight. In one aspect, the dosage stered to a patient is between about 0.01 mg/kg to about 10 mg/kg of the patient's body weight. In another aspect, the dosage administered to a patient is between about 0.1 mg/kg and about 10 mg/kg of the patient's body weight. In yet another aspect, the dosage administered to a patient is between about 0.1 mg/kg and about 5 mg/kg of the patient's body weight. In yet another aspect the dosage administered is between about 0.1 mg/kg to about 3 mg/kg of the patient's body weight. In yet another aspect, the dosage administered is between about 1 mg/kg to about 3 mg/kg of the patient's body weight.
A compound of the invention and/or antibody drug conjugate thereof can be administered by any convenient route, for example by infusion or bolus injection. Administration can be systemic or local. Various ry systems are known, e. g., encapsulation in liposomes, articles, microcapsules, capsules, etc., and can be used to administer a compound of the invention and/or dy drug conjugate thereof In certain embodiments, more than one compound ofthe invention and/or antibody drug conjugate thereof is administered to a patient.
In specific embodiments, it can be desirable to administer one or more compounds of the invention and/or antibody drug conjugates f locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical ation, e. g., in ction with a wound dressing after surgery; by injection; by means of a catheter; or by means of an t, the implant being of a porous, non-porous, or gelatinous material, ing membranes, such as silastic nes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue. In another embodiment, administration can be by direct injection at the site (or former site) of a manifestation of an autoimmune disease.
In yet another embodiment, the compound of the invention and/or antibody drug conjugate thereof can be delivered in a lled release system, such as but not limited to, a pump or various polymeric materials can be used. In yet another embodiment, a controlled- e system can be placed in proximity of the target of the compound of the invention and/or antibody drug conjugate thereof, e. g., the liver, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in l Applications of Controlled e, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer (Science 249: 1527- 1533 (1990)) can be used.
The term ”carrier” refers to a diluent, adjuvant or excipient, with which a compound or antibody drug conjugate thereof is administered. Such pharmaceutical carriers can be s, such as water and oils, ing those of petroleum, animal, vegetable or synthetic origin. The rs can be saline, and the like. In addition, auxiliary, stabilizing and other agents can be used. In one embodiment, when administered to a patient, the compound or conjugate and pharmaceutically able carriers are sterile. Water is an exemplary carrier when the compound or conjugate are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for inj ectable solutions. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
The present compositions can take the form of solutions, pellets, powders, sustained- release formulations, or any other form suitable for use. Other examples of suitable ceutical carriers are described in ”Remington's Pharmaceutical Sciences” by E. W.
Martin. 2O In an embodiment, the compound of the ion and/or antibody drug conjugate thereof are formulated in accordance with routine procedures as a pharmaceutical composition adapted for enous administration to animals, particularly human beings. Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also e a solubilizing agent. Compositions for intravenous stration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the ion. Generally, the ients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an e or te indicating the quantity of active agent. Where a compound of the invention and/or antibody drug conjugate thereof is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or . Where the compound of the invention and/or antibody drug conjugate thereof is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
The composition can include various materials that modify the physical form of a solid or liquid dosage unit. For e, the composition can include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be encased in a gelatin capsule.
Whether in solid or liquid form, the present compositions can e a cological agent used in the treatment of cancer. eutics Uses of nds and Antibody Drug Conjugates Thereof Another aspect of the invention relates to a method of using the compounds of the invention and antibody drug conjugates thereof for treating .
The compounds of the invention and/or antibody drug conjugates thereof are useful for inhibiting the multiplication of a tumor cell or cancer cell, g apoptosis in a tumor or cancer cell, or for treating cancer in a patient. The compounds of the invention and/or antibody drug conjugates thereof can be used accordingly in a variety of settings for the treatment of animal cancers. Said conjugates can be used to deliver a compound of the ion to a tumor cell or cancer cell. Without being bound by theory, in one embodiment, the antibody of the conjugate binds to or associates with a -cell or a tumor-cell-associated antigen, and the conjugate can be taken up (internalized) inside a tumor cell or cancer cell through receptor-mediated endocytosis or other alization mechanism. The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell.
In certain embodiments, once inside the cell, one or more specific peptide sequences are enzymatically or hydrolytically cleaved by one or more tumor cell or cancer cell-associated proteases, resulting in release of a compound of the invention from the ate. The released compound of the invention is then free to migrate within the cell and induce cytotoxic or cytostatic activities. The conjugate also can be cleaved by an intracellular protease to release a compound ofthe invention. In an alternative embodiment, the nd of the invention is d from conjugate outside the tumor cell or cancer cell, and the compound of the invention subsequently penetrates the cell.
In certain embodiments, the conjugates provide ation-specific tumor or cancer drug targeting, thus reducing general toxicity of the compounds of the ion.
In another embodiment, the antibody unit binds to the tumor cell or cancer cell.
In another embodiment, the antibody unit binds to a tumor cell or cancer cell antigen which is on the surface of the tumor cell or cancer cell.
In r embodiment, the antibody unit binds to a tumor cell or cancer cell antigen which is an extracellular matrix protein associated with the tumor cell or cancer cell.
The specificity of the antibody unit for a particular tumor cell or cancer cell can be important for ining those tumors or cancers that are most effectively treated.
Particular types of cancers that can be treated with a compound of the invention and/or antibody drug conjugate thereof, include but are not limited to, carcinomas of the bladder, breast, cervix, colon, endometrium, , lung, esophagus, ovary, prostate, pancreas, skin, stomach, and testes; and blood born cancers including but not d to leukemias and lymphomas.
Multi-Modalz'ty Therapyfor Cancer. Cancers, including, but not limited to, a tumor, metastasis, or other disease or disorder characterized by rolled cell growth, can be treated or inhibited by administration of a compound of the invention and/or antibody drug conjugate In other embodiments, methods for treating cancer are provided, including administering to a patient in need f an effective amount of a compound of the invention and/or antibody drug ate thereof and a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is that with which the treatment of cancer has been found to be refractory. A nd of the invention and/or dy drug conjugate thereof can be administered to a patient that has also undergone surgery as treatment for the cancer.
In some embodiments, the patient also receives an additional treatment, such as radiation therapy. In a specific embodiment, the nd of the invention and/or antibody drug conjugate thereof is administered concurrently with the chemotherapeutic agent or with ion y. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of a compound of the invention and/or antibody drug conjugate thereof A chemotherapeutic agent can be administered over a series of sessions. Any one or a combination of the chemotherapeutic , such a standard of care chemotherapeutic agent(s), can be administered.
Additionally, s of treatment of cancer with a compound of the invention and/or antibody drug conjugate thereof are provided as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or can prove too toxic, e. g., results in unacceptable or unbearable side effects, for the subject being treated. The patient being d can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, ing on which treatment is found to be acceptable or bearable.
The compounds of the invention and/or antibody drug conjugates f can also be used in an in vitro or ex vivo fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants.
This can e a multi-step process in which the 's autologous hematopoietic stein cells are harvested and purged of all cancer cells, the animal's remaining bone-marrow cell population is then eradicated via the administration of a high dose of a compound of the invention and/or antibody drug conjugate thereof with or without accompanying high dose radiation therapy, and the stem cell graft is infused back into the animal. Supportive care is then provided while bone marrow on is restored and the patient recovers.
Released Species Further embodiments of the invention include the chemical species released, inside or in the vicinity of the cancer cell or tumor cell by what is ed to be enzymatic and/or hydrolytic cleavage by one or more cancer cell or tumor cell-associated proteases. Such compounds include the s described herein, and also include compounds such as those described in the structure : IV or a pharmaceutically acceptable salt or solvate f, wherein, independently for each occurrence, R1 is ”Y Y is C2-C20 ne or C2-C20 heteroalkylene; C3-C8 carbocyclo-, -arylene-, -C3- Cgheterocyclo-, -C1-C10alkylene-arylene-, ne-C1-C10alkylene-, -C1-C10alkylene-(C3- Cgcarbocyclo)-, -(C3-Cgcarbocyclo)-C1-Cloalkyleneg -C1-Cloalkylene-(C3-Cgheterocyclo)-, or - (C3-C8 heterocyclo)-C1-C10alkylene-; 2'"‘%g Zulu/\[r f rugN Z’ ’ is O O , , , Z"\H/N ZYVNW/O/N O o o NH2 or —NHZ” ’; ”71’ co - \\\S' 2 —o C2 e \\ J'I"/\/\N.1$‘/ '02C\|.\‘\\)L§\ +H3N Z”’ is NH3+ + H or NH3 G is halogen, -OH, -SH or —S-C1-C6 alkyl; R2 is hydrogen, C1-C3 alkyl or C1-C8 haloalkyl; R3A and R313 are defined as either of the following: (i) R3A is hydrogen, cl—c8 alkyl, cl—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R313 is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R313 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R413 are defined as either of the following: (i) R4A is hydrogen, c1—c8 alkyl, c1—c8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R413 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 cyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R413 taken together are C2-C8 alkylene or C1-C8 heteroalkylene; and C6-C14 aryl optionally substituted , C1-C10 cyclyl, C3-C8 carbocycly with l, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1- C8 alkyl-N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’ -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR‘, -C(O)N(R')2, )R', -S(O)2R‘, -S(O)R‘, -OH, halogen, -N3, 2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 N/R12 o’ \R R6 12 /R12 0/ N or R 1s R13 or R13 , , optionally substituted with 1, 2, 3, 4 or 5 groups independently ed from the group consisting of C1-C8 alkyl, -C1-Cg alkyl-N(R’)2, -C1-Cg alkyl-C(O)R’, -C1-Cg alkyl-C(O)OR’, -O-(C1-Cg alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, H2, -S(=O)2R', -SR' and arylene- R’, wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-Cgheterocyclyl, alkylene-C3-Cgheterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-Cg alkenyl, -C2-Cg alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and R7 is independently selected for each occurrence from the group consisting of F, Cl, 1, Br, N02, CN and CF3; R10 is hydrogen, -C1-C10alkyl, -C3-Cgcarbocycle, aryl, -C1-C10heteroalkyl, -C3- Cgheterocyclo, -C1-C1oalkylene-aryl, -arylene-C1-C1oalkyl, -C1-C1oalkylene-(C3-Cgcarbocyclo), - (C3-C8 yclo)-C1-C10alkyl, -C1-C10alkylene-(C3-Cgheterocyclo), and -(C3-Cg heterocyclo)-C1- Cloalkyl, where aryl on R10 comprising aryl is optionally substituted with [R7]h; h is l, 2, 3, 4 or 5; and X is O or S.
Of ular interest are nds of formula IV haVing the structures: 0 OH H H N,,, N N W “ R1 T 0 o\ o o\ o O N \ 0 /E\ I 0\ 0 0\ 0 =\© and WO 72813 The invention is further described in the following examples, which are not intended to limit the scope of the invention.
EXEMPLIFICATION Experiments were generally carried out under inert atmosphere gen or argon), particularly in cases where oxygen- or moisture-sensitive reagents or ediates were employed. cial solvents and reagents were generally used without further purification, including anhydrous solvents where appropriate (generally Sure-SealTM products from the Aldrich Chemical Company, Milwaukee, Wisconsin). Products were generally dried under vacuum before being carried on to further reactions or submitted for biological testing. Mass spectrometry data is reported from either liquid chromatography-mass spectrometry (LCMS), atmospheric re al ionization (APCI) or gas chromatography-mass spectrometry (GCMS) instrumentation. Chemical shifts for nuclear ic resonance (NMR) data are expressed in parts per million (ppm, 8) referenced to residual peaks from the deuterated ts employed.
For syntheses referencing procedures in other Examples or Methods, reaction Protocol (length of reaction and temperature) may vary. In general, reactions were followed by thin layer chromatography (TLC) or mass spectrometry, and subjected to p when appropriate.
Purifications may vary between experiments: in general, solvents and the solvent ratios used for eluants/gradients were chosen to provide appropriate R§ or retention times.
Optical rotations were performed on a Perkin-Elmer polarimeter 343 (Serial number 9506).
HRMS were performed on an Agilent 6220 TOP LC/MS. nd names were generated with ACD Labs software.
HPLC and LC-MS Conditions Used for Analysis Protocol A: Column: enex Luna C18 (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 5% B over 1.5 minutes, 5% to 100% B over 8.5 minutes, then 100% B for 1 minute; Flow rate: 0.75 mL/minute. Temperature: 25 °C; Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000 s; Injection volume: 10 uL; Instrument: Agilent 1200 LCMS.
Protocol B: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 50% B over 1.5 minutes, 50% to 100% B over 6.5 minutes, then 100% B over 3 minutes; Flow rate: 0.75 ute. Temperature: 25 OC; Detection: DAD 215 nm; MS (+) range 150-2000 daltons; Injection volume: 10 uL; Instrument: Agilent 1200 LCMS.
Protocol C: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.02% trifluoroacetic acid in water (v/v); Mobile phase B: 0.02% trifluoroacetic acid in methanol (v/v); nt: 50% to 100% B over 10 minutes; Flow rate: 0.75 mL/minute.
Temperature: not controlled; Detection: DAD 215 nm, 254 nm; Injection volume: 10 uL; Instrument: Agilent 1100 HPLC.
Protocol D: : Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.02% trifluoroacetic acid in water (v/v); Mobile phase B: 0.02% trifluoroacetic acid in ol (v/v); Gradient: 5% to 100% B over 8 minutes; Flow rate: 0.75 mL/minute.
Temperature: not controlled; Detection: DAD 215 nm, 254 nm; Injection volume: 10 uL; ment: Agilent 1100 HPLC.
Protocol E: Column: Phenomenex Lux Amylose-2, 250 x 4.6 mm, 5 pm; Mobile phase A: heptane; Mobile phase B: ethanol (denaturated); Gradient: 5% to 100% B over 10 minutes; Flow rate: 1.5 mL/minute. ature: not controlled; Detection: DAD 215 nm, 254 nm; MS (+) range 150-1500 daltons; Injection volume: 10 uL; Instrument: Agilent 1100 LCMS.
Protocol F: Column: Waters Acquity UPLC BEH, C18, 2.1 x 50 mm, 1.7 pm; Mobile phase A: : 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); nt: 5% B over 0.1 minute, 5% to 95% B over 0.7 minute, 95% B over 0.1 minute; Flow rate: 1.25 mL/minute. Temperature: 60 OC; Detection: 0nm; MS (+) range 100-1200 daltons; Injection volume: 5 uL; ment: Waters Acquity.
Protocol G: Column: enex Luna C18 (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.02% trifluoroacetic acid in water (v/v); Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (v/v); Gradient: 0% to 100% B over 8.5 minutes; Flow rate: 1.5 mL/minute.
Temperature: not controlled; Detection: DAD 210 nm; Injection volume: 10 uL; Instrument: Agilent 1100 HPLC.
Protocol H: Column: Phenomenex Gemini-NX, C18, 4.6 x 50 mm, 3pm, 110 A; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 0% to 100% B over 4.10 s, linear then 100% B over 0.4 minute; Flow rate: 1.5 mL/minute. Temperature: 60 °C; Detection: DAD 200-450 nm; MS (+) range 100-2000 daltons; Injection volume: 5 uL; ment: Agilent.
Protocol 1: Column: Atlantis T3, 75 x 3.0 mm, 3 um; Mobile phase A: 0.05% roacetic acid in water (v/v); Mobile phase B: acetonitrile; Gradient: 5% to 95% B over 5.75 minutes; Flow rate: 1.2 mL/minute. Temperature: 45 OC; Detection: DAD 215 nm, 230 nm, 254 nm; MS (+) range: 150-1200 s; Injection volume: 5 uL; Instrument: t 1100 LCMS.
Protocol J: Column: Phenomenex Luna Phenyl-Hexyl, 150 x 3.0 mm, 5 pm; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 5% B over 1.5 minutes, 5% to 100% B over 8.5 minutes, then 100% B over 1 minute; Flow rate: 0.75 mL/minute. Temperature: 25 OC; Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000 daltons; Injection : 10 uL; Instrument: Agilent 1200 LCMS.
Protocol K: : Symmetry-C18, 50 x 2.1 mm, 3.5 um; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in methanol (v/v); Gradient: 10% to 90% B over 6.5 minutes; Flow rate: 0.7 mL/minute. Temperature: room temperature; Detection: DAD 215 nm; MS (+) range 100-1500 daltons; Injection volume: 3 uL; Instrument: Waters 996 PDA.
ProtocolL: Column: XBridge C-18, 150 x 4.6 mm, 3.5 um; Mobile phase A: 5 mM aqueous um acetate solution; Mobile phase B: acetonitrile; Gradient: 10% B over 3 minutes then 10% to 80% B over 14 minutes; Flow rate: 0.7 mL/minute. Temperature: room temperature; ion: DAD 215 nm; MS (+) range 100-1500 daltons; Injection volume: 3 uL; Instrument: Waters 996 PDA.
Protocol M: Column: Phenomenex Luna, 150 x 3.0 mm, 5 um; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in methanol (v/v); Gradient: 50% B over 1.5 minutes, 50% to 80% B over 8.5 minutes, then 80% B over 10 minutes; Flow rate: 0.75 mL/minute. Temperature: 45 OC; Detection: DAD 215 nm, 254 nm; MS (+) range 90-2000 daltons; Injection volume: 10 uL; Instrument: Agilent 1200 LCMS.
Protocol N: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 pm; Mobile phase A: 0.02% trifluoroacetic acid in water (v/v); Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (v/v); Gradient: 0% to 100% B over 23.5 minutes; Flow rate: 1.5 mL/minute.
Temperature: not lled; Detection: DAD 210 nm; Injection Volume: 10 uL; Instrument: Agilent 1100 HPLC Protocol 0: Column: Column: t Poroshell 300SB-C8, 75 x 2.1 mm, 2.6 mm Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); nt: 20% B to 45% B over 4 s; Flow rate: 1.0 mL/minute.
Temperature: 60 OC; Detection: 220 nm; MS (+) range 00Da; ion volume: 10 uL; Instrument: Agilent 1100 LC, Waters MicromassZQ MS. Deconvolution was med using MaxEnt 1.
Protocol P: Column: Column: TSK-gel G3000Sle, 300 x 7.8 mm, 10 um; Mobile phase: Phosphate buffer saline (PBS, 1X), pH 7.4 with 2% acetonitrile; Isocratic; Flow rate: 1 mL/minute. ature: room temperature; Injection Volume: 5 uL; Instrument: Agilent 1100 HPLC.
Protocol Q: Column: Waters Acquity UPLC HSS T3, C18, 2.1 x 50 mm, 1.7 pm; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 5% B over 0.1 minute, 5% to 95% B over 2.5 minutes, 95% B over 0.35 minute; Flow rate: 1.25 mL/minute. Temperature: 60 °C; Detection: 200-450nm; MS (+) range 100-2000 daltons; Injection volume: 5 uL; Instrument: Waters Acquity.
Protocol QI: Column: Waters Acquity UPLC HSS T3, C18, 2.1 x 50 mm, 1.7um; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 5% B over 0.1 minute, 5% to 95% B over 1.5 minute, 95% B over 0.35 minute; Flow rate: 1.25 mL/minute. Temperature: 60 °C; Detection: 200-450nm; MS (+) range 100-2000 daltons; Injection volume: 5 uL; Instrument: Waters Acquity. ol Q2: Column: Xtimate C18, 2.1 x 30 mm, 3 um; Mobile phase A: 0.1% trifluoroacetic acid in water (v/v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v/v); Gradient: 10% to 80% B over 0.9 minutes, 80% B over 0.6 minutes; 100 % B for 0.5 minutes; Flow rate: 1.2 mL/minute. Detection: DAD 220 nM; Temperature: 25 0C; Injection volume: 1 uL; Instrument: Agilent. ol Q3: Column: Xtimate C18, 2.1 x 30 mm, 3 um; Mobile phase A: 0.1% trifluoroacetic acid in water (v/v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v/v); Gradient 0% to 60% B over 0.9 minutes, 60% B over 0.6 minutes; 100% B for 0.5 minutes; Flow rate: 1.2mL/minute. Detection: DAD 220 nM; Temperature: 25 0C; Injection volume: 1 uL; Instrument: Agilent.
Protocol R: Column: enex Luna, 150 x 3.0 mm, 5 um; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in methanol (v/v); nt: 5% B over 1.5 minutes, 5% to 100% B over 8.5 minutes, then 100% B over 1 minute; Flow rate: 0.75 mL/minute. Temperature: 45 OC; Detection: DAD 215 nm, 254 nm; MS (+) range 00 daltons; Injection volume: 10 uL; ment: 305 RP Agilent 1200 LCMS.
Protocol S: Column: Phenomenex Luna, 150 x 3.0 mm, 5 um; Mobile phase A: 0.1% trifluoroacetic acid in water (v/v); Mobile phase B: 0.1% trifluoroacetic in acetonitrile (v/v); Gradient: 5% B over 1.5 minutes, 5% to 95% B over 8.5 minutes, then 100% B over 1 minute; Flow rate: 1.0 mL/minute. Temperature: not lled; Detection: DAD 210 nm; MS (+) range 150-2000 daltons; Injection volume: 10 uL; Instrument: 305 RP Agilent 1100 HPLC.
Protocol T.' Column: is dC18, 50 x 4.6 mm, 5 um; Mobile phase A: 0.05% trifluoroacetic acid in water (v/v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v); nt: 5% to 95% B over 4.0 s; then hold at 95% B over 1 minute; Flow rate: 2 mL/minute. Temperature: room temperature; Detection: DAD 215 nm; MS (+) range 160 -1000 daltons; Injection volume: 3 uL; Instrument: Waters 996 PDA.
Protocol U: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 mm Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 5% B over 1.5 minutes, 5% to 100% B over 8.5 minutes, then 100% B for 1 minute; Flow rate: 0.75 mL/minute. Temperature: 45 OC; Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000 daltons; Injection volume: 10 uL; Instrument: Agilent 1200 LCMS.
Protocol V: Column: HPLC-V Ultimate XB- C18, 50 x 3.0 mm, 3 mm Mobile phase A: 0.225 % trifluoroacetic acid in water (v/v); Mobile phase B: 0.225 % trifluoroacetic acid in acetonitrile (v/v); Gradient: 30% to 90% B over 6 minutes; Flow rate: 1.2 mL/minute.
Temperature: 40 OC; Detection: DAD 220 nm; Injection volume: 1 uL; Instrument: SHIMADZU.
Protocol W: Column: HPLC-V te XB- C18, 50 x 3.0 mm, 3 mm Mobile phase A: 0.1 % roacetic acid in water (v/v); Mobile phase B: 0.1 % trifluoroacetic acid in acetonitrile (v/v); Gradient: 10% to 80% B over 6 minutes; Flow rate: 1.2 mL/minute.
Temperature: 40 OC; Detection: DAD 220 nm; Injection volume: 3 uL; Instrument: SHIMADZU.
Protocol X: Column: YMC-pack ODS-A, 150 x 4.6 mm, 5 um; Mobile phase A: 0.1 % trifluoroacetic acid in water (v/v); Mobile phase B: 0.1 % trifluoroacetic acid in itrile (v/v); Gradient: 10% to 80% B over 6 minutes; Flow rate: 1.2 mL/minute. Detection: DAD 220 nm.
Temperature: 40 OC; Injection volume: 3 uL; Instrument: SHIMADZU.
Protocol Y: Column: YMC-pack ODS-A, 150 x 4.6 mm, 5 um; Mobile Phase A: 0.1% trifluoroacetic acid in water (v/v); Mobile Phase B: 0.1% trifluoroacetic acid in acetonitrile (v/v); Gradient: 0% to 95% B over 10 minutes, then 95% B for 5 minutes; Flow rate: 1.5 mL/minute; Detection: DAD 220 nm; Instrument: Agilent 1100.
Protocol Z: Column: Xtimate C18, 2.1 x 30 pm, 3 um; Mobile Phase A: 0.1% oroacetic acid in water (v/v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v/v); Gradient: 0% to 60% B over 2 minutes; Flow rate: 1.2 . Temperature: 50 °C; Detection: 220 nm, MS (+) range 100 -1000 daltons; Injection : 1 uL; Instrument: ZU.
Protocol AB: Column: Phenomenex Luna C18 (2), 150 x 2.0 mm, 5 mm Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); nt: 5% to 100% B over 10 minutes, then 100% B for 2 minute; Flow rate: 0.5 mL/minute.
Temperature: 25 OC; Detection: DAD 210 nm, 254 nm; MS (+) range 150-2000 daltons; Injection volume: 5 uL; Instrument: Agilent 1100 LCMS.
Protocol BB: Column: Phenomenex Luna C18 (2), 150 x 2.0 mm, 5 mm Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 5% B over 2.0 minutes, 5% to 100% B over 12 minutes, and 100% B for 2 minute, then 100% to 5% B over 1.5 min; Flow rate: 0.75 ute. Temperature: 25 °C; Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000 daltons; Injection : 5 uL; Instrument: Agilent.
Protocol CB: Column: Waters XBridge C18, 4.6 x 50 mm, 5mm Mobile phase A: 0.03% ammonium hydroxide in water (v/v); Mobile phase B: 0.03% ammonium hydroxide in acetonitrile (v/v); Gradient: 5% to 95 % B over 4.0 minutes, then 95% B for 1 ; Flow rate: 2 mL/minute. Temperature: 25 OC; Detection: DAD 215 nm, MS (+) range 160-1000 daltons; Injection volume: 4 uL; ment: Waters ZQ/Alliance 2795 HPLC.
Protocol DB: Column: Waters Atlantis dC18, 4.6 x 50 mm, 5pm; Mobile phase A: 0.05% trifluoroacetic acid in water (v/v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v); Gradient: 5.0% to /95% B over 4.0 minutes, then 95% B for 1 minute. Flow rate: 2 mL/minute. Temperature: 25 °C; Detection: DAD 215 nm, MS (+) range 160-1000 s; Injection volume: 4 uL; Instrument: Waters ZQ/Alliance 2795 HPLC.
Protocol EB: Column: e RP18, 2.1 x 50 mm, 5 um; Mobile phase A: 0.02% ammonium hydroxide in water (v/v); Mobile phase B: 0.02% ammonium hydroxide in acetonitrile (v/v); Gradient 10% to 80% B% over 6 minutes, then 80% for 2 minutes; Flow rate: 1.2 mL/minute. Detection: DAD 220 nm.; Temperature: 50 °C.
Protocol FB: Column: Phenomenex Luna C18 (2), 150 x 2.0 mm, 5 pm; Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Gradient: 5% B over 2.0 minutes, 5% to 100% B over 10 minutes, and 100% B for 2 ; Flow rate: 0.50 mL/minute. Temperature: 25 °C; Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000 daltons; Injection volume: 5 uL; Instrument: Agilent 1200 LCMS.
In some instances some minor alterations to analysis LC-MS and HPLC ions were made such as but not limited a change in gradient or flow rate which is indicated by the symbol HPLC Conditions Used for Purification Method A: Column: Phenomenex Lux Amylose-2, 250 x 21.2 mm, 5 pm; Mobile phase A: Heptane; Mobile phase B: Ethanol (denatured); nt: 5% to 100% B over 6 min; Flow Rate: 27 mL/minute; Detection: DAD 210-360 nm; MS (+) range 150-2000 daltons; Instrument: Waters FractionLynx.
Method B: Column: enex Luna C18(2), 150 x 21.2 mm, 5 pm; Mobile phase A: 0.02% acetic acid in water; Mobile phase B: 0.02% acetic acid in acetonitrile; nt: 5% B over 1.5 s, 5% to 45% B over 8.5 minutes; Flow rate: 27 mL/minute; Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000 daltons; Instrument: Waters FractionLynx.
Method C: Column: Phenomenex Luna C18, 100 x 30 mm, 10 um; Mobile phase A: 0.02% trifluoroacetic acid in water (v/v); Mobile phase B: 0.02% trifluoroacetic acid in ol (v/v); Gradient: 10% to 90% B over 20 minutes; Flow rate: 20 mL/minute.
Temperature: not controlled; Detection: DAD 210 nm, 254 nm; Injection Volume: variable; Instrument: Gilson.
Method D: Column: PhenomeneX Synergi MaX-RP, 150 X 21.2 mm, 4 um; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Gradient: 30% B for 1.5 minutes, 30% to 60% B over 8.5 minutes, 60 to 100% B over 0.5 minutes then 100% B over 2 minutes; Flow rate: 27 mL/ ; Detection: DAD 210-360 nm; MS (+) range 150-2000 daltons; Instrument: Waters FractionLynX.
Method E1 : Column: eneX Luna C18(2), 150 X 21.2 mm, 5 um; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Gradient: 40% B for 1.5 minutes, 40% to 80% B over 8.5 minutes, 80 to 100% B over 0.5 minute then 100% B over 2 minutes; Flow rate: 27 mL/ minute; Detection: Detection: DAD 210-3 60 nm; MS (+) range 150-2000 daltons; Instrument: Waters FractionLynX LCMS.
Method E2: Column: PhenomeneX Luna -hexyl, 150 X 21.2 mm, 5 um. The rest of the Protocols are identical to those described for Method E1.
Method F: Column: PhenomeneX Synergi MaX-RP, 150 X 21.2 mm, 4 um; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in ol; Gradient: 44% B for 1.5 minutes, 44% to 77% B over 8.5 s, then 77% B over 10 minutes; Flow rate: 27 ute; Detection: DAD 210-360 nm; MS (+) range 150-2000 daltons; Instrument: Waters FractionLynX LCMS.
Method G: Column: PrincetonSFC 2-ethylpyridine, 250 X 21.2 mm, 5 pm; Mobile phase A: heptane; Mobile phase B: ethanol (denaturated); Gradient: 1% B for 1.5 minutes, 1% to 50% B over 8.5 minutes; Flow rate: 27 mL/minute; Detection: DAD 0 nm; MS (+) range 150- 2000 daltons; Instrument: Waters FractionLynX LCMS.
Method H: Column: PhenomeneX Luna C18(2), 150 X 21.2 mm, 5 pm; Mobile phase A: 0.02% acetic acid in water; Mobile phase B: 0.02% acetic acid in acetonitrile; Gradient: 20% B over 1.5 minutes, 20% to 60% B over 10.5 minutes; Flow rate: 27 mL/ minute; Detection: DAD 210-360 nm; MS (+) range 150-2000 daltons; ment: Waters FractionLynX LCMS.
Method 1: Column: PhenomeneX Luna C18(2), 150 X 21.2 mm, 5 pm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol; Gradient: 40% B over 1.5 minutes, 40% to 70% B over 8.5 minutes then 70% B over 10 minutes; Flow rate: 27 mL/ minute; ion: DAD 210-360 nm; MS (+) range 150-2000 daltons; Instrument: Waters FractionLynX LCMS.
Method J: Column: PhenomeneX Luna C18, 100 X 30 mm, 5 um; Mobile phase A: 0.02% roacetic acid in water (V/V); Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (V/V); Gradient: 10% to 90% B over 20 minutes; Flow rate: 20 mL/minute. ature: not controlled; Detection: DAD 210 nm, 254 nm; Injection Volume: variable; Instrument: Gilson.
Method K: Column: Phenomenex Luna C18(2), 150 x 21.2 mm, 5 pm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Gradient: 20% B for 1.5 minutes, 20% to 50% B over 8.5 minutes, 50 to 100% B over 0.5 minute then 100% B over 2 minutes; Flow rate: 27 mL/ minute; Detection: Detection: DAD 210-3 60 nm; MS (+) range 150-2000 daltons; Instrument: Waters Fraction Lynx LCMS.
Method L: Column: Phenomenex Luna C18(2), 150 x 21.2 mm, 5 pm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; nt: 30% B for 1.5 minutes, 30% to 50% B over 8.5 minutes, 50 to 100% B over 0.5 minute then 100% B over 2 minutes; Flow rate: 27 mL/ minute; Detection: Detection: DAD 0 nm; MS (+) range 150-2000 s; Instrument: Waters Fraction Lynx LCMS.
Method M: Column: Waters Sunf1re, C18, 19x100 mm, 5 um; Mobile phase A: 0.05% trifluoroacetic acid in water (V/V); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (V/V); Gradient: 0 to 100% over 8.5 s. Flow rate 25 ute. Detection: DAD 215 nm MS (+) range 160-1000 daltons; Instrument: Waters FractionLynx.
Method N: Column: Waters Sunf1re, C18, 19x100 mm, 5 um; Mobile phase A: 0.05% formic acid in water (V/V); Mobile phase B: 0.05% formic acid in acetonitrile (V/V); Gradient: 0 to 100% over 8.5 minutes. Flow rate 25 mL/minute. Detection: DAD 215 nm MS (+) range 160- 1000 daltons; Instrument: Waters onLynx.
Method 0: Column: Phenomenex Luna C18, 21.2 x150 mm, 5 pm; Mobile phase A: 0.1% formic acid in water (V/V) acid in water (V/V); Mobile phase B: 0.1% formic acid in acetonitrile (V/V); nt(V/V); Gradient 20% B over 1.5 minutes, 20% to 40% B over 8.5 s, 40 to 100% B over 0.5 minutes, then hold 100% B for 1.5 minutes. Flow rate: 27 mL/minute. Detection: DAD 210-360 nm; MS (+) range 150-2000 daltons; Instrument: Waters FractionLynx.
Method P: Column: Phenomenex Gemini C18, 21.2><250mm, 5 pm; Mobile phase A: 0.225 % ammonia hydroxide in water (pH 10) (V/V); Mobile phase B: 0.225% ammonia hydroxide in acetonitrile (V/V); Gradient: 45% to 85% B over 10 minutes. Flow rate 35 mL/minute. ion: DAD 220 nm MS (+) range 100-1200 daltons; Instrument: Shimadzu MS Trigger.
Method Q: : Column: Phenomenex i C18, 50x250 mm, 10 um; Mobile phase A: 0.1% trifluoroacetic acid in water (v/v) Mobile phase B: Acetonitrile ; Gradient 10% to 40% B over 25 minutes. Flow rate 100 mL/ minute. Detection: UV/Vis 220 nm; Instrument: Shimadzu LC-8A.
Method R: Column: Phenomenex Luna C18 (2), 250 x 21.2 mm, 5 mm Mobile phase A: 0.1% TFA in water (v/v); Mobile phase B: 0.1% TFA in acetonitrile (v/v); Gradient: 10% to 100% over 30 minutes; Flow rate variable. Temperature: 25 0C; Detection: DAD 215 nm, 254 nm; MS (+) range 150 — 2000 s; Injection volume: 1.8 mL: Instrument: Agilent 1100 Prep HPLC.
In some instances some minor alterations to purification conditions were made such as but not limited to a change in gradient or flow rate which is indicated by the symbol *.
General Procedures General Procedure A: Fmoc removal using diethylamine or piperidine. To a solution of the Fmoc-containing compound in romethane or N,N—dimethylformamide (also referred to as DMF), was added an equal volume of diethylamine or dine. Reaction progress was monitored by LC-MS (or HPLC or TLC). Solvents were removed in vacuo, and in some cases the residue was azeotroped one to four times with heptane. Residue was usually d with dichloromethane and a small amount of methanol before being reduced down onto silica and purified by chromatography on silica gel, eluting with methanol in dichloromethane (or other appropriate mixture of solvents) to afford the desired material (or crude material was used as is). l ure B: Boc removal or t-Bu ester cleavage using trifluoroacetic acid. To a solution of the Boc-containing compound or tert-butyl ester-containing compound in dichloromethane at 0 0C (or at room temperature) was added trifluoroacetic acid, to afford a ratio of 1:4 trifluoroacetic acid:dichloromethane. Reaction progress was monitored by LC-MS (or HPLC or TLC). ts were removed in vacuo. The residue was azeotroped three times with heptane to afford the desired material.
General Procedure C: Boc removal or tert—butyl ester (also refers to t-Bu ester) cleavage using hydrochloric acid in dioxane. To either a solution of Boc-containing compound or tert- butyl ester-containing compound in dioxane (or in some cases no solution, or other relevant solvent) was added a 4 M solution of hydrochloric acid in dioxane. Reaction progress was monitored by LC-MS (or HPLC or TLC). The reaction was concentrated in vacuo and in some cases azeotroped one to four time with heptanes.
General ure D.‘ coupling with 0-(7-azabenzotriazolyl)-N,N,N’,N’- tetramethyluronium hexafiuorophosphate (HATU). To a stirring solution of the amine (1.0 eq.) and acid (1.0-2.0 eq.) in dichloromethane, N,N—dimethylformamide (also referred to as DMF), or a mixture of both, HATU (1.0-2.0 eq.) was added followed by triethylamine (2.0-4.0 eq.) or ropylethylamine (2.0-4.0 eq., also referred to as s base). Reaction progress was monitored by LC-MS (or HPLC or TLC); the reaction was usually completed within three hours.
Solvents were removed in vacuo. The residue was purified by silica gel or reverse phase chromatography or in some cases oped three times with heptanes, diluted with a small amount of ethyl acetate before being reduced down onto silica or C18 bonded silica and purified by silica gel or reverse phase chromatography. l Procedure E.‘ coupling with N—[6-(2,5-dioxo-2,5-dihydro- lH-pyrrol yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-( { [(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]—L- namide (MalcValCitPABC-PNP). To a mixture of the payload amine (1 eq.) and N—[6- ioxo-2,5-dihydro- lH-pyrrolyl)hexanoyl] -L-valyl-N5-carbamoyl-N- [4-( { [(4- nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-omithinamide (MalcValCitPABC-PNP, Eur. Pat.
Appl. (1994), EP624377, 1.0-2.0 eq.) in N,N—dimethylformamide or dimethylacetamide (also referred to as DMA), pyridine (0.0-4.0 eq.), diisopropylethylamine (0.0-4.0 eq.), 2,6- ylpyridine (0.0-4.0 eq., also referred to as 2,6-Luditine) and 1-hydroxybenzotriazole hydrate (0.0l-1.1 eq. also referred to as HOBT) or 3H-[1,2,3]triazolo[4,5-b]pyridin—3-ol (0.01- 1.1 eq., also referred to as HOAT) was added. After stirring at 40 0C-50 0C for 1-48 hours, the reaction mixture was concentrated in vacuo and azeotroped three times with heptane. The crude material was purified by reverse phase chromatography according to the specified method to afford the desired material.
Generalprocedure F.‘ conjugation of cial TIN® antibody with - payload via internal disulfides. Commercially available HERCEPTIN® dy (Genentech Inc) was dialyzed into Dulbecco’s Phosphate Buffered Saline (DPBS, Lonza). The ed antibody was d with addition ofx equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 mM in distilled water) and diluted to 15 mg/mL final antibody concentration using DPBS, 5mM 2,2',2",2"'—(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), pH 7.0-7.4 (Buffer A). The reaction was incubated at 37 0C for 1-2 hours and then cooled to room temperature. Conjugation was performed by addition ofy equivalents of linker-payload (5- 10 mM in dimethylacetamide (DMA)). DMA was added to achieve 10-20% (v/v) total organic solvent component in final reaction mixture, and Buffer A added to achieve 10 mg/mL final antibody concentration. The reaction was incubated for 1-2 hours at room temperature. The reaction mixture was then buffer exchanged into DPBS (pH 7.4) using GE Healthcare Sephadex G-25 M buffer ge columns per manufacturer’s instructions. Crude material was purified by size exclusion chromatography (SEC) using GE AKTA Explorer system with GE Superdex column and PBS (pH 7.4) eluent.
Generalprocedure G: Conjugation reactions were performed in the upper portion of a centrifugal ultraflltration device such as Amicon Ultra 50k Ultracel filters (part #UFC805096, GE). A 132 mM stock solution of L-cysteine was prepared in PBS containing 50 mM EDTA.
This solution (50 uL) was added to a mixture of the respective mutant antibody (5 mg) in 950 uL of PBS containing 50 mM EDTA. The final ne concentration in the reaction mixture was 6.6 mM. After allowing the reaction to stand at room ature (about 23 0C ) for 1.5 hours the reaction tube was centrifuged to concentrate the material to approximately 100 uL. The mixture was diluted to 1 mL with PBS containing 50 mM EDTA. This process was repeated 4 times in order to remove all the cysteine reductant. The resulting material was diluted to 1 mL in PBS containing 50 mM EDTA and treated with 16 uL of a 5 mM solution of the ide linker-payload (from Table 18A in dimethyl acetamide (DMA) ximately 5 equivalents).
After standing at room temperature (about 23 0C ) for 1.5 hours the reaction tube was centrifuged to concentrate the material to approximately 100 uL. The mixture was diluted to 1 mL with PBS.
This process was repeated 2 times in order to remove the excess maleimide reactant. The antibody conjugates were generally purified by size exclusion tography (SEC) using GE AKTA er system with a GE ex200 column and PBS (pH7.4) eluent. The loading of the drug onto the ed site of ation was determined using a variety of methods including mass ometry (MS), reverse phase HPLC, and hydrophobic interaction chromatography (HIC), as has been described elsewhere. The reported value (in Tables 19A and 19B) is generally ed by LC-MS under reducing ions.
Generalprocedure H: A 20 mM TCEP solution (generally 50 to 100 molar equivalents) was added to the antibody (typically 5 mg) such that the final antibody concentration was 5 mg/mL in PBS containing 50 mM EDTA. After allowing the reaction to stand at 37 0C for 1.5 hours, the antibody was buffer exchanged into PBS containing 50 mM EDTA using a 50 kD MW cutoff spin concentration device (3 x 3 mL wash, 10x concentration per cycle). Alternative methods such as TFF or dialysis are also useful in particular circumstances. The resulting antibody was re-suspended in 1 mL of PBS containing 50 mM EDTA and treated with a freshly prepared 50 mM solution of DHA (dehydroascorbate) in 1:1 PBS/EtOH (fmal DHA concentration is typically 1 mM) and allowed to stand at 4 oC overnight. The antibody/DHA mixture was buffer exchanged into PBS containing 50 mM EDTA using a 50 kD MW cutoff spin concentration device (3 x 3 mL wash, 10x concentration per . The resulting antibody was re-suspended in 1 mL of PBS containing 50 mM EDTA and treated with 10 mM maleimide linker-payload in DMA (typically 5-10 equivalents). After standing for 1.5 hours, the material was buffer exchanged (as above) into 1 mL of PBS (3 x 3 mL washes, 10x concentration per cycle). Purification by SEC (as described previously) was med as needed to remove any aggregated material.
Generalprocedure I: The initial conjugation of the linker-payload was performed using the previously described method al Procedure F). The resulting antibody-drug-conjugate was buffer exchanged into a 50 mM borate buffer (pH 9.2) using an ultraflltration device (50 kd MW cutoff). The ing solution was heated to either 37 0C for 24 hours (for the maleimide- Peg linkers) or to 45 0C for 48 hours (for the maleimide-caproyl linkers). The ing on was cooled, buffer-exchanged into PBS, and purified by SEC (as bed previously) in order to remove any aggregated al. LCMS analysis of the material indicated that the succinimide ring had completely opened (90% or more). Note that in es where a methyl ester is present in the payload, the ester is hydrolyzed to the carboxylic acid under the described conditions.
Generalprocedure J: The pentafluorophenyl esters were conjugated to the shown 2O antibody following the procedure previously outlined in “(0201211307896 Al.
Generalprocedure K: The conjugation of amino-alkyl linkers was accomplished via enzyme-mediated ligation as described in WO2012059882 A2.
General Procedure L. N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N— [(3R,4S,5 S) {(2S)[(1R,2R)carboxymethoxypropyl]pyrrolidinyl}-3 -methoxy methyloxoheptanyl]-N-methyl-L-valinamide (prepared in the same manner as #136) was coupled to the relevant amino acid or amine moiety using HATU .0 eq.) in the presence of Hunig’s base (1.-5.0 eq.) in a solution of DMF, dichloromethane, or in some cases a solution of both (or a solution of one or more solvents). Reaction was monitored by LC-MS (or TLC or HPLC). Reaction was concentrated in vacuo and purified y by silica chromatography or by prep HPLC. Fmoc protection was then removed as described in general procedure A followed by concentration in vacuo and purified by silica chromatography or by prep HPLC.
General Procedure M. #151 was coupled to the relevant amine using HATU (1 .0-2.0 eq., or other appropriate coupling t) in the presence of Hunig’s base (1.0-5.0 eq.) in a solution of DMF, dichloromethane, or in some cases a solution of both (or a solution of one or more ts). Reaction was monitored by LC—MS (or TLC or HPLC). Reaction was concentrated in vacuo. Boc de-protection was then performed as described in general procedure B, concentrated in vacuo and purified by silica chromatography or by prep HPLC.
General ure N. 1-(9H-fiuorenyl)oxo-2,7,10,13,16,19,22-heptaoxa azapentacosan—25-oic acid (or other appropriate Fmoc-AmPegXCZ-COOH) was coupled to the relevant cytotoxic pentapeptide (or the cytotoxic pentapeptide containing a protecting group on a reactive moiety other than the N—terminus) using HATU (1 .0-2.0 eq., or other appropriate coupling reagent) in the presence of Hunig’s base (1.0-5 .0 eq. or other appropriate base) in a solution of DMF, dichloromethane, or in some cases a solution of both (or a solution of one or more solvents). Reaction was monitored by LC-MS (or TLC or HPLC). Reaction was concentrated in vacuo. Fmoc de-protection was performed according to general procedure A. In some cases a second de-protection was performed in order to remove a protecting group on a reactive moiety on the cytotoxic pentapeptide using l procedure B (or other relevant procedure known in the literature based on the protecting group). The reaction was concentrated in vacuo and purified by silica chromatography or by prep HPLC.
General ure 0. The appropriate mPegXCZ-COOH is coupled to the relevant cytotoxic eptide (or the cytotoxic pentapeptide containing a protecting group on a reactive moiety other than the N—terminus) and Fmoc de-protection is performed according to general procedure N. The reaction is concentrated in vacuo and then purified by silica chromatography or prep HPLC (or the crude al can be used as is). The riate PABC sequence (such as chalCitPABC, or derivative of) is then installed according to general ure E. In some cases if a protecting group is present on cytotoxic pentapeptide portion of the le de-protection is then performed using general procedure A or general procedure B (or other relevant procedure known in the literature based on the protecting group). The reaction is concentrated in vacuo and purified by silica chromatography or by prep HPLC.
General Procedure P. Followed procedure E replacing chalCitPABC-PNP, with MalPeg3C2ValCitPABC—PNP (prepared in a similar manner to chalCitPABC—PNP).
General Procedure Q. The appropriate Fmoc-AmPegXCZ-COOH is coupled to the relevant xic pentapeptide (or the cytotoxic pentapeptide containing a protecting group on a reactive moiety other than the N—terminus) and Fmoc tection is performed as described in general ure N. The reaction is concentrated in vacuo and then purified by silica chromatography or by prep HPLC (or the crude material can be used as is). To a stirring solution of this residue in DMF at O 0C (or a slightly higher temperature in some cases) bromoacetic acid (1.0-2.0 eq.) was added followed by Hunig’s base (1.0-5.0 eq.) and HATU (1.0-2.0 eq.) The reaction was allowed to warm to room temperature and stir at room temperature while being monitored by LC-MS (or TLC or HPLC). Reaction was concentrated in vacuo and d by prep HPLC.
General Procedure R. Followed procedure E replacing chalCitPABC-PNP, with N—(6- { [(9H-fluorenylmethoxy)carbonyl] amino} hexanoyl)-L-Valyl-N~5~-carbamoyl-N—[4-( { [(4- nitrophenoxy)carbonyl]oxy} methyl)phenyl]-L-omithinamide red in a similar manner to chalCitPABC—PNP). Fmoc de-protection was then performed (general procedure B) followed by prep HPLC purif1cation.
General ure S. To a stirring solution of -dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanal (1.0-3.0 eq.) in methanol the relevant cytotoxic pentapeptide (1.0 eq) was added followed by formic acid. The on was d to stir at room temperature for 1-40 minutes followed by the addition of sodium (cyano-kappaC)(trihydrido)borate(1-) (3.0-6.0 eq., also referred to as sodium cyanborohydride). The reaction was monitored by LC-MS (or TLC or HPLC). In some cases additional 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanal (1.0-3.0 eq.) was added. The reaction was concentrated in vacuo followed by purif1cation using prep HPLC. 2O Generalprocedure T. 4-[3-oxo(2-oxoazetidinyl)propyl]anilinium is prepared as described in the literature (Bioorganic and nal Chemistry Letters. 2012, vol. 22, #13, 4249 — 4253) which is then d to bis(pentafluorophenyl) 3,3'-[ethane-1,2- diylbis(oxy)]dipropanoate using HATU in dichloromethane followed by coupling to the desired cytotoxic pentapeptide. Material is then purified by prep HPLC.
Generalprocedure U. Fmoc-ValCitPABC-PNP is coupled to the desired cytotoxic eptide following general procedure E and then Fmoc is removed following general ure A. This residue is then coupled to [2-oxo({4-[3-oxo(2-oxoazetidin— 1- yl)propyl]phenyl}amino) ethoxy] acetic acid (which is ed by coupling xo(2- oxoazetidin-l-yl)propyl]anilinium with 1,4-dioxane-2,6-dione following general procedure D).
Material is then purified by prep HPLC.
Generalprocedure V. Bis(pentafluorophenyl) 3,3'-[ethane-1,2-diylbis(oxy)]dipropanoate or bis(pentafluorophenyl) 4,7,10,13,16-pentaoxanonadecane-1,19-dioate is coupled to the desired cytotoxic pentapeptide (or in some cases coupled to the desired cytotoxic pentapeptide containing a protecting group on a reactive moiety other than the N—terminus) ing general procedure D. If a protecting group is present, the protecting group is then removed (using relevant procedures described in the literature). Material is then purified by prep HPLC.
Generalprocedure W. 4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl 1-(9H- fluorenyl)oxo-2,7,10-trioxaazatridecanoate is coupled to the desired cytotoxic pentapeptide following l procedure E. Fmoc is removed ing general procedure A.
Bis(pentafluorophenyl) 3,3'—[ethane-1,2-diylbis(oxy)]dipropanoate is coupled to this residue following l procedure D. Material is then purified by prep HPLC.
Generalprocedure XI. N-[(9H-fluorenylmethoxy)carbonyl]-L-alanyl-L-alanyl-N~1~- [4-({[(4-nitrophenoxy)carbonyl]oxy} methyl)phenyl]-N~4~-trityl-L-aspartamide is coupled to the desired cytotoxic pentapeptide ing general procedure E. Fmoc is removed following general procedure A and trityl protecting group is removed following general ure B.
Bis(pentafluorophenyl) 3,3'—[ethane-1,2-diylbis(oxy)]dipropanoate is coupled to this residue following general procedure D. Material is purified by prep HPLC.
Generalprocedure X2. N— {3- [2-(3-ethoxyoxopropoxy)ethoxy]propanoyl} -L-valyl- NNSN-carbamoyl-N- [4-( { [(4-nitrophenoxy)carbonyl] oxy} methyl)phenyl] thinamide is coupled to the desired cytotoxic pentapeptide following l procedure E. Ethyl ester is removed using lithium hydroxide in THF and water. NHS ester is then formed by ng residue with 1-hydroxypyrrolidine-2,5-dione using N,N'—dicyclohexylcarbodiimide in THF.
Material is purified by prep HPLC.
Generalprocedure X3. N-[(9H-fluorenylmethoxy)carbonyl]-D-valyl-N~5~- carbamoyl-N-[4-( { [(2—carboxypropanyl)carbamoyl] oxy}methyl)phenyl] -L-ornithinamide is coupled to #50 following l procedure D in DMSO and acetonitrile. Fmoc is removed following general procedure A ed by coupling with bis(pentafluorophenyl) 3,3'—[ethane- 1,2-diylbis(oxy)]dipropanoate using Hunig’s base in acetonitrile. Material is d by prep HPLC.
Generalprocedure X4. N-[l-(9H-fluorenyl)-3,5,12-trioxo-2,7,10-trioxa azadodecanyl]—2-methylalanine is coupled to #250 following general procedure D in acetonitrile. Fmoc is removed ing general procedure A followed by coupling with ntafluorophenyl) 3,3'—[ethane-1,2-diylbis(oxy)]dipropanoate using Hunig’s base in acetonitrile. Material is purified by prep HPLC.
Generalprocedure X5. L-valyl-N~5~-carbamoyl-N—[4-(hydroxymethyl)phenyl]-L- ornithinamide is coupled to N~2~-acetyl-NN6N-(tert-butoxycarbonyl)-L-lysine following general procedure D. This resulting residue is coupled with bis(4-nitrophenyl)carbonate with Hunig’s base in DMF, followed by coupling with the desired xic pentapeptide following general procedure E. Boc de-protection is then med following general procedure B in acetonitrile.
Residue is purified by prep HPLC.
In some instances minor alterations to reaction conditions were made such as but not limited to order of reagent and reactant addition and or the amount of reagent or reactant which is ted by the symbol *. Furthermore, these l procedures are provided as exemplary only and are non-limiting.
In addition to the General Procedures provided above, relevant dolastatin and auristatin references include the following: Petit er al. J. Am. Chem. Soc. 1989, I I I, 5463; Petit er al. Anti- Cancer Drug Design 1998, 13, 243 and references cited therein; Petit er al. J. Nat. Prod. 2011, 74, 962; WO 12; WO 95/09864; EP 8; WO 07/8848; WO 01/18032; WO 09/48967; WO 09/48967; WO 09/117531; WO 3; US 7,750,116; US 5,985,837; and US 2005/9751; all of which are hereby incorporated by reference in their entireties.
MS Analysis and Sample Preparation Samples were prepped for LC-MS analysis by combining about 20 uL of sample (approximately 1 mg/mL ofADC in PBS) with 20 uL of 20 mM dithiothreitol (DTT). After allowing the mixture to stand at room temperature for 5 minutes, the samples were analyzed according to protocol 0.
The following calculation was performed in order to establish the total g (DAR) of the conjugate: Loading = 2*[LC1/(LC1+LCO)]+2*[HCl/(HCO+HC1+HC2+HC3)]+ 4*[HC2/(HCO+HC1+HC2+HC3)]+6*[HC3/(HCO+HC1+HC2+HC3)] Where the indicated variables are the relative nce of: LCO = unloaded light chain, LCl = single loaded light chain, HCO = unloaded heavy chain, HCl = single loaded heavy chain, HC2 = double loaded heavy chain, and HC3 = triple loaded heavy chain.
LC-MS conditions used are Protocol F for retention time below one minute and Protocol H for the ing experiments unless otherwise indicated.
Preparation of N-[(9H-Flu0renylmeth0xy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S) carboxy—Z-methoxymethylhexanyl] -N-methyl-L-valinamide (#8) DMSO BEN COH \nH 803'PW "'B” L''v''PF?NH THF BH3-THF THF I I tBLIOAc -78, MegoBF4, (CH20I)2 g; \N OtBu 970/ 34% Cbz —° 2 68/0 Cbz Cbz OH 0 CDMT \pfifimu H2, Pd/C NMM ElZNH MBOHI HC', 2MeTHF THF \ orBu OtBu OtBu Cbz 92% N HZNJN OMe o H O O / 'HCI FmocHN—;HFmocHN/\EiLN #@2 Dil Hydrochloride Deprotected\dimer #4 91%0 #5 #5 TFAI CHzClz FmocHNgLfi/YOH 49% /\ O\ O dimer acid CDMT H2N\éj\N NMM 0 TFA 015”2MeTHF NQLNH OtBu M FmocN OH FmOCN “\JLN FmchOH I ected\dimer O /=\ I 0x O 97% I o /=\ I o\ o #6 | TrImer o Trimer acid 97% #7 #8 Step 1. Synthesis of benzyl [(2S,3S)hydroxymethylpentanyl]methylcarbamate (#1). To a solution of N—[(benzyloxy)carbonyl] -N—methyl-L-isoleucine (52.37 g, 187.5 mmol, 1 eq.) in tetrahydrofuran (524 mL, 0.35 M) was added borane-tetrahydrofuran complex (1 M in tetrahydrofuran, 375 mL, 375 mmol, 2 eq.) slowly over 1 hour and the reaction was allowed to stir for 18 hours at room temperature. The on was cooled to 0 °C and water (30 mL) was added over 30 minutes. The reaction mixture was d with 1 M aqueous sodium carbonate solution (100 mL) and tert-butyl methyl ether (250 mL). The aqueous layer was back-extracted with tert-butyl methyl ether (100 mL). The combined organic layers were washed with 1 M aqueous sodium carbonate solution (100 mL), washed with brine (200 mL), dried over magnesium e, filtered, and concentrated in vacuo to provide #1 (48.44 g, 97% yield) as a pale yellow oil, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers: 8 7.26-7.41 (m, 5H), [5 .06 (AB quartet, .9 Hz, 2.8 Hz) and 5.06 (AB quartet, JAB=9.0 Hz, AVAB=9.0 Hz), total 2H], [4.65 (t, J=5.3 Hz) and 4.59 (t, J=5.4 Hz), total 1H], .80 (m, 1H), 3.51-3.60 (m, 1H), 3.41-3.51 (m, 1H), 2.75 and 2.71 (2 s, total 3H), 1.49-1.64 (br m, 1H), 1.24-1.37 (br m, 1H), 0.90-1.02 (br m, 1H), 0.74-0.87 (m, 6H).
Step 2. Synthesis of benzyl methyl[(2S,3S)methyloxopentanyl]carbamate (#2).
To a solution of #1 (8.27 g, 31.2 mmol, 1 eq.) in dimethyl sulfoxide (41.35 mL, 0.75 M), was added triethylamine (8.70 mL, 64.0 mmol, 2.05 eq.) and the mixture was cooled to 0 °C. Sulfur de pyridine complex (10.18 g, 63.96 mmol, 2.05 eq.) was then added portion-wise, while g the internal temperature below 8 °C. The reaction was allowed to reach room temperature and was stirred for 18 hours. The reaction was poured into water (100 mL) and tert- butyl methyl ether (100 mL). The aqueous layer was back-extracted with tert—butyl methyl ether (50 mL) and the combined organic layers were washed with brine (100 mL), dried over magnesium sulfate, filtered, concentrated in vacuo and purified by silica gel chromatography (Gradient: 10% to 60% ethyl acetate in heptane) to provide #2 (7.14 g, 87%) as a colorless oil. 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 9.61 (s, 1H), 7.26-7.42 (m, 5H), .13 (m, 2H), 4.04-4.12 (m, 1H), 2.86 and 2.82 (2 s, total 3H), 1.94-2.11 (br m, 1H), 1.26-1.42 (br m, 1H).
Step 3. Synthesis of tert-butyl (3R,4S,55) {[(benzyloxy)carbonyl](methyl)amino} hydroxymethylheptanoate (#3). Lithium diisopropylamine was prepared by adding n- butyllithium (2.5 M solution in tetrahydrofuran, 35.9 mL, 89.8 mmol, 1.4 eq.) to a solution of diisopropylamine (13.8 mL, 96.3 mmol, 1.5 eq.) in ydrofuran (50 mL, 1.3 M) at -78 0C.
After 1 hour, tert-butyl acetate (15.7 mL, 116 mmol, 1.8 eq.) was added drop-wise and the reaction mixture was stirred for an additional 1.5 hours while being allowed to slowly warm to - OC. The reaction mixture was recooled to -78 OC and a solution of the aldehyde #2 (16.9 g, 64.2 mmol, 1 eq.) in tetrahydrofuran (10 mL) was added. The reaction mixture was stirred for 1.5 hours and then quenched by addition of water (100 mL). After extraction with diethyl ether (2 x 100 mL), the combined c layers were dried over sodium sulfate, d, trated in vacuo and d by silica gel chromatography ent: 0% to 20% acetone in heptane) to provide #3 (8.4 g, 34%) as a colorless oil. LC-MS: m/z 402.4 [M+Na+], retention time = 3.91 s; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers: 8 7.27-7.39 (m, 5H), 5.01-5.12 (m, 2H), [4.93 (d, J=7.2 Hz) and 4.98 (br d, J=7.2 Hz), total 1H], 4.03-4.15 (br m, 1H), 3.68-3.85 (br m, 1H), 2.65 and 2.72 (2 br s, total 3H), 2.28-2.37 (m, 1H), .17 (m, 1H), 1.74-1.90 (br m, 1H), 1.41-1.51 (m, 1H), 1.39 (s, 9H), 0.92-1.01 (m, 1H), 0.77-0.92 (m, 6H).
Step 4. Synthesis of tert-butyl (3R, 4S, 55) {[(benzyloxy)carbonyl](methyl)amino} ymethylheptanoate (#@2). To a solution of #3 (8.4 g, 22 mmol, 1 eq.) in 1,2- dichloroethane (25 mL, 0.88 M) were added molecular sieves (4 A, 0.7 g) and Proton sponge (1,8-bis(dimethylamino)naphthalene) (13.4 g, 59.2 mmol, 2.7 eq.), followed by trimethyloxonium tetrafiuoroborate (9.10 g, 61.6 mmol, 2.8 eq.). After ng overnight, the reaction mixture was filtered through Celite. The filtrate was concentrated in vacuo and the residue was purified by silica gel chromatography (Gradient: 0% to 40% 1:1 acetone:ethyl 2012/056224 e in heptane) to give #@2 (8.7 g, 68%) as a colorless oil. 1H NMR (400 MHz, DMSO-dg), ed to be a mixture ofrotamers: 8 7.28-7.40 (m, 5H), 501-5. 13 (m, 2H), 3.89-4.08 (br m, 1H), 3.70-3.82 (m, 1H), 3.18 and 3.26 (2 s, total 3H), 2.66 and 2.71 (2 br s, total 3H), 2.44-2.53 (m, 1H, assumed; partially obscured by solvent peak), 2.17-2.24 (m, 1H), 1.71-1.86 (br m, 1H), 1.39 and 1.39 (2 s, total 9H), 1.31-1.40 (m, 1H), 0.94-1.08 (m, 1H), 0.76-0.91 (m, 6H).
Step 5. Synthesis of tert-butyl (3R, 4S, 5S)methoxy-5 -methyl (methylamino)heptanoate, hydrochloride salt (#4). To a solution of #@2 (13.37 g, 33.98 mmol, 1 eq.) in methanol (134 mL, 0.1 M) and concentrated hydrochloric acid (3.1 mL, 37.4 mmol, 1.1 eq.) was added 10% palladium on carbon (50% wet) (0.1 wt%; 1.34 g, 3.40 mmol). The e was hydrogenated at 45 psi for 3 hours, then purged with nitrogen, filtered through Celite and concentrated in vacuo to provide #4 (9.20 g, 92%) as a white solid. 1H NMR (400 MHz, CDCl3) 8 9.65 (br s, 1H), 8.97 (br s, 1H), 3.98-4.04 (m, 1H), 3.40 (s, 3H), .13 (br m, 1H), 2.82 (br dd, J=6, 5 Hz, 3H), 2.74-2.80 (m, 1H), 2.68 (dd, half of ABX pattern, J=16.3, 4.2 Hz, 1H), 2.00- 2.10 (br m, 1H), 1.73-1.84 (m, 1H), 1.46 (s, 9H), 1.38-1.45 (m, 1H), 1.13 (d, J=7.0 Hz, 3H), 0.99 (t, J=7.4 Hz, 3H).
Step 6. Synthesis of utyl (3R,4S,5S)[{N-[(9H-fluorenylmethoxy)carbonyl]-L- valyl}(methyl)amino]methoxymethylheptanoate (#5). To a mixture of N—[(9H—fluoren ylmethoxy)carbonyl]-L-valine (18.53 g, 54.60 mmol, 1.3 eq.) and 2-chloro-4,6-dimethoxy-1,3,5- triazine (CDMT) (9.58 g, 54.6 mmol, 1.3 eq.) in 2-methyltetrahydrofuran (118.00 mL, 0.34 M) was added N—methylmorpholine (6.52 mL, 59.1 mmol, 1.5 eq.) followed by #4 (11.80 g, 39.9 mmol, 1 eq.). After 3 hours, the reaction was quenched with water (50 mL) and stirred for 15 minutes. The aqueous layer was separated and back-extracted with 2-methyltetrahydrofuran (50 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution (50 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo to give a colorless oil, which was purified by silica gel chromatography (Gradient: 5% to 40% ethyl acetate in heptane) to give #5 (26.2 g, 91%) as a colorless foam. LC—MS (Protocol 1) m/z 581.3 [M+H+] 604.3 [M+Na+], retention time = 4.993 minutes; 1H NMR (400 MHz, DMSO-dg), possibly a e of rotamers, characteristic major s: 8 7.88 (d, J=7.4 Hz, 2H), 7.71 (d, J=7.4 Hz, 2H), 7.62 (d, J=8.6 Hz, 1H), 7.41 (dd, J=7.4, 7.4 Hz, 2H), 7.27-7.34 (m, 2H), 4.13- 4.32 (m, 4H), .82 (br m, 1H), 3.24 (s, 3H), 2.92 (br s, 3H), 2.54 (dd, , 2.4 Hz, 1H), 2.17 (dd, J=15.4, 9.4 Hz, 1H), 1.95-2.07 (m, 1H), 1.70-1.83 (br m, 1H), 1.40 (s, 9H), 0.83-0.94 (m, 9H), 0.69 (t, J=7.2 Hz, 3H).
Step 7A. Synthesis of tert-butyl (3R, 4S,5S)methoxymethyl[methyl(L- valyl)amino]heptanoate (#6). To a solution of #5 (26 g, 42 mmol, 1 eq.) in tetrahydrofuran (260 mL, 0.16 M) was added diethylamine (22 mL) over 30 minutes. The reaction was stirred for about 6 hours and the suspension was then filtered through Celite and washed with additional tetrahydrofuran (25 mL). The filtrate was concentrated in vacuo to provide a pale yellow oil, which was redissolved in 2-methyltetrahydrofuran (50 mL) and concentrated again to ensure complete removal of diethylamine. The crude oil of #6 (>15.25 g) was taken into the next step without further purification.
Step 73. Synthesis of ,5S)[{N-[(9H-fluorenylmethoxy)carbonyl]-L- valyl}(methyl) amino]methoxymethylheptanoic acid (#@5). According to general procedure B, from #5 (1.62 g, 2.79 mmol, 1 eq.), romethane (10 mL, 0.3 M) and trifluoroacetic acid (3 mL) was synthesized #@5 (1.42 g, 97%) as a solid which was used without further purification. LC—MS m/z 525.3 [M+H+] 547.3 ] retention time = 0.95 minute; 1H NMR (400 MHz, DMSO-dg), characteristic signals: 8 7.89 (d, J=7.6 Hz, 2H), 7.71 (d, J=7.4 Hz, 2H), 7.59 (d, J=8.8 Hz, 1H), 7.41 (dd, J=7.6, 7.4 Hz, 2H), 7.28-7.34 (m, 2H), 4.14- 4.32 (m, 4H), 3.24 (s, 3H), 2.92 (br s, 3H), 2.51-2.57 (m, 1H, assumed; partially obscured by solvent peak), 2.20 (dd, J=15.9, 9.5 Hz, 1H), 1.95-2.06 (m, 1H), 1.70-1.83 (br m, 1H), 1.22-1.36 (br m, 1H), 0.84-0.93 (m, 9H), 0.70 (t, J=7.3 Hz, 3H).
Step 8. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N—methyl-L-valyl-N- S,55)tert—butoxy-3 -methoxymethyloxoheptanyl]-N—methyl-L-valinamide (#7).
To a e of N—[(9H—fluoren—9-ylmethoxy)carbonyl]-N—methyl-L-valine (19.54 g, 55.29 mmol, 1.3 eq.) and #6 (15.25 g, 42.54 mmol, 1 eq.) in yltetrahydrofuran (152 mL, 0.28 M) was added 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (9.71 g, 55.3 mmol, 1.3 eq.). After 10 minutes, N—methylmorpholine (6.6 mL, 60 mmol, 1.4 eq.) was slowly added, while keeping the internal temp below 25 °C. The reaction was stirred for 4 hours and was then quenched by the addition of water (50 mL). After stirring for 15 minutes, the aqueous layer was separated and back-extracted with 2-methyltetrahydrofuran (50 mL). The combined c layers were washed with saturated aqueous sodium bicarbonate solution (100 mL), then were dried over ium e, filtered, and trated in vacuo. The resulting yellow foam was purified by silica gel chromatography (Gradient: 5% to 35% ethyl acetate in heptane) to give #7 (32 g, 97%). 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 7.89 (d, J=7.4 Hz, 2H), 7.62 (d, J=7.4 Hz, 2H), 7.41 (br dd, J=7.4, 7.4 Hz, 2H), 7.29- 7.34 (m, 2H), 4.52-4.69 (br m, 1H), 3.70-3.82 (br m, 1H), 3.22 and 3.25 (2 br s, total 3H), 2.94 and 2.96 (2 br s, total 3H), 2.78 and 2.81 (2 br s, total 3H), 2.11-2.23 (m, 1H), 190-2. 10 (m, 2H), 1.68-1.83 (br m, 1H), 1.40 (s, 9H), 1.21—1.33 (br m, 1H).
Step 9. Synthesis of N-[(9H—fluorenylmethoxy)carbonyl]-N—methyl-L-valyl-N— [(2R,3S,4S)carboxymethoxymethylhexanyl]-N—methyl-L-valinamide (#8). To #7 (32 g, 46 mmol, 1 eq.) in dichloromethane (160 mL, 0.29 M) was added drop-wise over 10 minutes trifluoroacetic acid (17.4 mL, 231 mmol, 5 eq.). After 6 hours, the same amount of trifluoroacetic acid was added and the reaction was continued for 18 hours. The reaction mixture was diluted with toluene (320 mL) and concentrated in vacuo to provide #8 (35.8 g, 97%) as a pinkish oil, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic s: 8 7.90 (d, J=7.0 Hz, 2H), 7.62 (d, J=7.4 Hz, 2H), 7.41 (br dd, J=7.4, 7.0 Hz, 2H), .35 (m, 2H), 4.54-4.68 (br m, 1H), [4.09 (d, J=11 Hz) and 4.22 (d, J=10.9 Hz), total 1H], 3.74-3.84 (br m, 1H), 3.22 and 3.24 (2 br s, total 3H), 2.94 and 2.96 (2 br s, total 3H), 2.78 and 2.80 (2 br s, total 3H), 2.13-2.24 (m, 1H), 1.89- 2.10 (br m, 2H), 1.70-1.81 (br m, 1H).
Preparation of (2R,3R)[(ZS)(tert-But0xycarbonyl)pyrrolidinyl]meth0xy methylpropanoic acid (#11; “Boc-Dap-acid”) (EH 1)OsO4, NMO, tBUOH'Hzo OH N NaH CH3I DMF , [goo Boc BOCH O O H0 970/ 2) NaMnO4, NaIO4, l7- BU4N§gB/EII:H20|2/H20 0 \ CchN, H20 #9 #10 69% (2 steps) #11 Step 1. Synthesis of tert-butyl -[(1R,2S)hydroxymethylbuten yl]pyrrolidinecarboxylate (#9) . To a solution of tert-butyl (2S)formylpyrrolidine carboxylate (10 g, 50 mmol, 1 eq.) in dichloromethane (120 mL, 0.42 M) was added ium (2Z)butenyltrifluoroborate (9.76 g, 60.2 mmol, 1.2 eq.) followed by tetra-nbutylammonium bromide (3.24 g, 5.02 mmol, 0.1 eq.) and water (60 mL). After 13 hours, the reaction was diluted with dichloromethane (150 mL) and water (150 mL). The s layer was separated and back-extracted with dichloromethane (100 mL). The combined organic layers were washed with aqueous sodium chloride solution (5% wt, 200 mL), washed with water (200 mL), and concentrated in vacuo to afford #9 (~13 g) as an orange oil, which was used without further ation. 1H NMR (400 MHz, CDCl3) 8 5.61-5.86 (br m, 1H), 4.97-5.09 (m, 2H), 3.80-3.98 (br m, 2H), 3.45-3.67 (br m, 1H), 3.21-3.29 (m, 1H), 2.14-2.26 (m, 1H), 1.80-2.04 (m, 3H), 1.65-1.76 (m, 1H), 1.47 (s, 9H), 1.12 (d, J=6.6 Hz, 3H).
Step 2. sis of utyl (2S)[(1R,2S)methoxymethylbuten yl]pyrrolidinecarboxylate (#10). Sodium hydride (60% in mineral oil, 3.38 g, 84.4 mmol, 1.1 eq.) was combined with hexane (40 mL), and the mixture was subjected to rapid ical stirring for 5 minutes. The solids were d to settle and the hexane was removed. This procedure was repeated twice to remove mineral oil. N,N—Dimethylformamide (59 mL,1.3 M) was added and the mixture was cooled to 0 OC; methyl iodide (5 mL; 81 mmol, 1.05 eq.) was then added drop-wise, followed by drop-wise addition of a solution of #9 (19.6 g, 76.8 mmol, 1 eq.) in methylformamide (59 mL) over 5 minutes, while keeping the temperature between 0 °C and 5 °C. The reaction was d at 0 0C for 2 hours. The reaction was quenched with saturated aqueous ammonium de on (150 mL), poured into aqueous sodium chloride solution (5% wt, 300 mL), and the mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with 10% s sodium chloride solution (2 x 300 mL), washed with water (200 mL), and concentrated in vacuo. The resulting water-wet oil was reconcentrated from ethyl acetate (150 mL) and d by silica gel chromatography (Gradient: 2% to 10% ethyl acetate in heptane) to afford #10 (15.0 g, 73%) as a colorless oil. 1H NMR (400 MHz, CDCl3), presumed to be a mixture of rotamers: 8 .83 (m, 1H), 4.91-5.06 (m, 2H), 3.81-3.95 (br m, 1H), 3.43 (s, 3H), 3.36-3.61 (m, 2H), 3.19-3.31 (m, 1H), 2.09-2.21 (m, 1H), 1.86-2.02 (br m, 2H), 1.62-1.85 (br m, 2H), 1.47 and 1.49 (2 s, total 9H), 1.09 (d, J=6.6 Hz, 3H).
Step 3. Synthesis of (2R,3R)[(2S)(tert-butoxycarbonyl)pyrrolidinyl]methoxy- 2-methylpropanoic acid (#11). To #10 (25.0 g, 92.8 mmol, 1 eq.) in tert-butanol (100 mL, 0.93 M) was immediately added water (30.00 mL) followed by N—methylmorpholine-N-oxide (25.97 g, 192.1 mmol, 2.07 eq.) and osmium tetroxide (235.93 mg, 928.04 umol, 0.01 eq.) After 12 hours, the mixture was concentrated in vacuo using water (20 mL) to azeotropically remove al tert-butanol. The residue was partitioned between ethyl acetate (500 mL) and water (500 mL) plus brine (150 mL). The s layer was re-extracted with ethyl acetate (250 mL). The combined organic layers were washed with aqueous sodium chloride solution (10 wt%, 200 mL), washed with water (150 mL), and concentrated in vacuo to afford a water-wet pale brown oil that was re-concentrated from ethyl acetate (100 mL) to remove any remaining water. This crude diol (34.76 g) was used without further purification.
To the crude diol (34.76 g, 592.8 mmol, 1 eq.) in acetonitrile (347 mL, 0.1 M) and water (174 mL) was added sodium permanganate (2.03 g, 5.73 mmol, 0.05 eq.). The mixture was cooled to 0 °C and sodium periodate (51.46 g, 240.6 mmol, 2.1 eq.) was added portion-wise over minutes, while keeping the internal temperature below 5 OC. The on was stirred at 0 0C for 4 hours and was then poured into a solution of sodium thiosulfate pentahydrate (65.40 g, 263.5 mmol, 2.3 eq.) in water (100 mL). The mixture was filtered through Celite and the filtrate WO 72813 2012/056224 was concentrated in vacuo. The residue was partitioned between ethyl acetate (200 mL) and water (200 mL). The aqueous layer was back-extracted with ethyl acetate (250 mL), and the combined organic layers were washed with a 10% aqueous citric acid solution. As the desired product was very soluble in water, all the aqueous layers were combined, treated with Celite (100 g) and concentrated in vacuo to yield an off-white paste. Ethyl acetate (150 mL) was added and the mixture was re-concentrated to remove any residual water; this operation was repeated one more time. The paste was treated with ethyl acetate (150 mL) and placed in vacuo at 50 0C for 10 minutes and filtered (repeated twice). These filtrates were combined with the us organic layer (from the citric acid wash), concentrated, d with ethyl acetate (200 mL) and filtered through Celite to remove solids. Finally, this e was concentrated to yield #11 (22.9 g, 69% over two steps) as a yellowish/brown foam. LCMS (Protocol 1): m/z 310.1 [M+Na+], 232.1 [(M - 2-methylpropene)+H+], 188.1 [(M - Boc)+H+], retention time = 3.268 minutes; 1H NMR (400 MHz, DMSO-dg), characteristic signals: 8 3.61-3.85 (br m, 2H), 3.20-3.45 (br m, 4H), 3.03-3. 17 (br m, 1H), 1.59-1.93 (br m, 4H), 1.40 (br s, 9H), 1.02-1.18 (br m, 3H).
Preparation of (2R,3R)Meth0xymethyl-N-[(1S)phenyl(1,3-thiazolyl)ethyl] [(2S)—pyrrolidinyl]propanamide, trifluoroacetic acid salt (#19) and (2R,3R)—3-meth0xy methyl-N-[(1S)—2-phenyl(1,3-thiazolyl)ethyl][(2S)—pyrrolidin yl]pr0panethi0amide, trifluoroacetic acid salt (#18) NMM CICO Et bromoacetaldehyde 0 2 S . d m'6 I t BOCHN\)I\OH NH OH THF BOCHNEJLNHQ Lawessons reagent yace aI 4M HCI BocHN 104C to THF, reflux in dioxane (catalytic) rt —’BOCHN\)LNH2 28;?) —> 5 —> = ' We 82% 77% r: I: acetone reflux #12 #13 #14 1) Lawesson‘s reagent dioxane, 100°C, 5 33% ’\> ” NYgNH $3 2)TFA CHZCI2, 0°Cto rt 0\ S -CF3002H 4 M HCI In donane H2N\/LN “1 DEPC 53 94% #18 D -HC| 97% Et3N, DMF 0°Ctort N\/LN\ \© 81/ }OODOE=\© TFA CH2C|2 (M@ #16 quant. \GCF3002H Step 1. Synthesis of NOI-(tert-butoxycarbonyl)-L-phenylalaninamide (#12). To a on of Boc-Phe-OH (30.1 g, 113 mmol, 1 eq.) in ydrofuran (378 mL, 0.3 M) cooled to -10 0C were added N—methylmorpholine (13.6 mL, 124 mmol, 1.09 eq.), and ethyl chloroformate (11.8 mL, 124 mmol, 1.09 eq.). After 20 minutes, a 30% s ammonium hydroxide solution (45 mL, 350 mmol, 3.1 eq.) was added. The mixture was stirred at room temperature for 18 hours before being concentrated in vacuo. The e was diluted with ethyl acetate and washed sequentially with 1 N aqueous potassium ate solution, water and brine. The organic layer was then dried over sodium sulfate, filtered, and concentrated in vacuo. The white solid was dissolved (this required heating with stirring) in ethyl acetate (about 400 mL); the on was then allowed to cool to room temperature before adding hexane (~1000 mL). After a few minutes, a white material started to precipitate from the reaction mixture. The solid was collected by filtration, washed with heptane (2 x ~150 mL), and dried under vacuum for 18 hours to give #12 (24.50 g, 82%) as a solid. LC-MS: m/Z 263.2 [M-H+], 309.2 [M+HC02'], retention time = 1.85 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, major rotamer: 8 7.35 (br s, 1H), 7.22-7.30 (m, 5H), 7.00 (br s, 1H), 6.78 (d, J=8.6 Hz, 1H), 4.09 (ddd, J=10, 9, 4.5 Hz, 1H), 2.95 (dd, J=13.8, 4.4 Hz, 1H), 2.72 (dd, J=13.7, 10.1 Hz, 1H), 1.30 (s, 9H).
Step 2. Synthesis of tert-butyl [(25)aminophenylthioxopropanyl]carbamate (#13). To a on of #12 (14.060 g, 53.192 mmol, 1 eq.) in tetrahydrofuran (180 mL, 0.296 M), was added s(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-dithione (Lawesson’s reagent) (12.70 g, 31.40 mmol, 0.59 eq.) and the reaction was refluxed for 90 minutes. The reaction was cooled to room temperature and quenched by addition of saturated aqueous sodium bicarbonate solution. The mixture was extracted twice with ethyl acetate and the combined organic layers were dried over sodium sulfate, filtered, and trated in vacuo. The residue was dissolved in ethyl acetate, concentrated in vacuo onto silica and purified by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane), affording #13 (11.50 g, 77%) as a white solid. LC—MS: m/Z 279.4 [M-HT], 225.2 [(M — 2-methylpropene)+H+], 181.2 [(M — Boc)+H+]; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, major rotamer: 8 9.60 (br s, 1H), 9.19 (br s, 1H), 7.23-7.32 (m, 5H), 6.82 (d, J=8.8 Hz, 1H), 4.44 (ddd, J=9.4, 9.1, 4.4 Hz, 1H), 3.00 (dd, J=13.7, 4.5 Hz, 1H), 2.79 (dd, J=13.6, 9.9 Hz, 1H), 1.29 (s, 9H).
Step 3. Synthesis of utyl 2-phenyl(1,3-thiazolyl)ethyl]carbamate (#14).
To a mixture of #13 (5.65 g, 20.2 mmol, 1 eq.) in acetone (101 mL, 0.2 M) was added bromoacetaldehyde diethyl acetal (8.76 mL, 58.2 mmol, 2.89 eq.) and 2 drops of 4 M hloric acid in dioxane. The e was degassed with en three times before being heated to reflux. After 2 hours, the reaction was cooled to room temperature and concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with saturated aqueous sodium bicarbonate solution and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting crude orange oil was d with ethyl acetate WO 72813 before being concentrated in vacuo onto silica and purified by silica gel chromatography (Gradient: 0% to 35% ethyl acetate in heptane) and then by reverse phase chromatography (Method A) to give #14 (625 mg, 10%); HPLC (Protocol E): m/z 304.5 [M+H+], 248.9 [(M - 2- methylprop-l-ene)+H+], retention time = 7.416 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, major rotamer: 8 7.75 (d, J=3.3 Hz, 1H), 7.75 (br d, J=8.6 Hz, 1H), 7.61 (br d, J=3.1 Hz, 1H), 7.25-7.30 (m, 5H), 4.99 =10.5, 8.9, 4.5 Hz, 1H), 3.29- 3.36 (m, 1H, assumed; partially obscured by water signal), 2.98 (dd, J=13.8, 10.6 Hz, 1H), 1.31 (s, 9H).
Step 4. Synthesis of (lS)phenyl(1,3-thiazolyl)ethanamine, hydrochloride salt (#15). According to general procedure C, from #14 (1.010 g, 3.318 mmol, 1 eq.), dioxane (10 mL, 0.33 M) and a 4 M solution of hydrochloric acid in dioxane (20 mL, 80 mmol, 20 eq.) was synthesized #15 (775 mg, 97%). 1H NMR (400 MHz, DMSO-dg) 8 8.95-9.07 (br m, 3H), 7.86 (d, J=3.2 Hz, 1H), 7.73 (d, J=3.2 Hz, 1H), 7.18-7.28 (m, 3H), 7.10-7.15 (m, 2H), .07 (m, 1H), 3.49 (dd, J=13.3, 4.9 Hz, 1H), 3.18 (dd, J=13.4, 10.2 Hz, 1H).
Step 5. Synthesis of tert-butyl (2S)[(1R,2R)methoxymethyloxo{[(1S) phenyl(1,3-thiazolyl)ethyl]amino}propyl]pyrrolidinecarboxylate (#16). To a solution of #11 (280 mg, 0.974 mmol, 1 eq.) and #15 (460 mg, 1.44 mmol, 1.48 eq.) in MN- dimethylformamide (3 mL, 0.32 M) at 0 0C was added lphosphoryl cyanide (DEPC) (93% purity, 212 ”L, 1.30 mmol, 1.34 eq.), followed by triethylamine (367 uL, 2.63 mmol, 2.7 eq.).
After 2 hours at 0 0C, the reaction mixture was warmed to room ature for 18 hours. The reaction mixture was then diluted with ethyl acetate:toluene (2:1, 30 mL) and was washed successively with 1 M aqueous sodium bisulfate solution (35 mL) and 50% saturated aqueous sodium onate solution (4 x 25 mL). The organic layer was dried over sodium sulfate, ed, concentrated in vacuo, and purified by silica gel chromatography (12% to 100% ethyl e in heptane) to give #16 as a light amber oil (374 mg, 81%). LC—MS: m/z 474.4 [M+H+], 374.4 [(M - 2-methylpropene)+H+] retention time = 3.63 minutes; 1H NMR (400 MHz, DMSO-dg), characteristic s: 8 8.66 (d, J=8.5 Hz, 1H), 7.78 (d, J=3.3 Hz, 1H), 7.64 (d, J=3.3 Hz, 1H), 7.21-7.31 (m, 4H), 7.14-7.20 (m, 1H), 5.40 (ddd, J=11.4, 8.5, 4.0 Hz, 1H), 3.23 (br s, 3H), 2.18 (dq, J=9.7, 6.7 Hz, 1H), 1.06 (d, J=6.6 Hz, 3H).
Step 6A. Synthesis of tert-butyl (2S)[(1R,2R)methoxymethyl{[(1S)phenyl- 1-(1,3-thiazolyl)ethyl]amino}thioxopropyl]pyrrolidinecarboxylate (#17). A mixture of #16 (350 mg, 0.739 mmol, 1 eq.) and 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4- dithione (Lawesson’s reagent) (324 mg, 0.776 mmol, 1.05 eq.) in toluene (6 mL, 0.1 M) was warmed to 100 0C. After 10 minutes, the mixture was cooled to room temperature. Insoluble material was removed by filtration and the filtrate was concentrated in vacuo. The e was purified by silica gel chromatography ent: 12% to 80% ethyl acetate in heptane) and then by reverse phase tography (Method E2) to give #17 (120 mg, 33%); HPLC (Protocol J): m/Z 490.2 , retention time = 10.069 minutes; [0thD — 110 (c 0.24, MeOH); 1H NMR (400 MHz, CD3OD), characteristic signals: 8 7.78 (d, J=3.3 Hz, 1H), 7.51 (d, J=3.3 Hz, 1H), 7.32- 7.37 (m, 2H), 7.24-7.30 (m, 2H), 7.17-7.23 (m, 1H), 6.52-6.61 (br m, 1H), 3.62 (br dd, J=15, 4 Hz, 1H), 3.37 (s, 3H), 2.98-3.09 (br m, 1H), 2.53-2.64 (br m, 1H), 1.60-1.78 (m, 2H), 1.49 (s, 9H), 1.27 (d, J=6.5 Hz, 3H).
Step 63. sis of (2R, 3R)methoxymethyl-N-[(1S)phenyl(1,3-thiazol yl)ethyl][(2S)-pyrrolidinyl]propanethioamide, trifluoroacetic acid salt (#18). According to general ure B, from #17 (198 mg, 0.404 mmol, 1 eq.), dichloromethane (6 mL, 0.07 M) and trifluoroacetic acid (2 mL) was synthesized #18 (185 mg, 91%), which was used without further purification. LC-MS: m/z 390.1 [M+H+], retention time = 0.57 minutes; 1H NMR (400 MHz, g) 8 10.91 (d, J=8.2 Hz, 1H), .20 (br m, 1H), 7.86-8.00 (br m, 1H), 7.83 (d, J=3.2 Hz, 1H), 7.69 (d, J=3.3 Hz, 1H), 7.27-7.36 (m, 4H), 7.21-7.26 (m, 1H), 6.33 (ddd, J=11.3, 8.3, 4.4 Hz, 1H), 3.76-3.82 (m, 1H), 3.56 (dd, J=14.6, 4.3 Hz, 1H), 3.45 (s, 3H), 3.28 (dd, J=14.6, 11.3 Hz, 1H), 3.02-3.12 (br m, 1H), 2.89-3.00 (br m, 1H), 2.72-2.89 (m, 2H), 1.69-1.83 (br m, 1H), 1.43-1.58 (m, 2H), 1.20-1.33 (m, 1H), 1.22 (d, J=6.6 Hz, 3H).
Step 7. Synthesis of (2R,3R)methoxymethyl-N-[(lS)phenyl(1,3-thiazol yl)ethyl][(25)-pyrrolidinyl]propanamide, trifluoroacetic acid salt (#19). According to general procedure B, from #16 (607 mg, 1.28 mmol, 1 eq.), dichloromethane (10 mL, 0.13 M) and trifluoroacetic acid (2 mL) was synthesized #19 (640 mg, quantitative), which was used in the next step without further ation. 1H NMR (400 MHz, DMSO-dg) 8 8.96-9.07 (br m, 1H), 8.89 (d, J=8.8 Hz, 1H), 7.87-8.00 (br m, 1H), 7.80 (d, J=3.2 Hz, 1H), 7.66 (d, J=3.3 Hz, 1H), 7.28-7.34 (m, 4H), 7.20-7.27 (m, 1H), 5.43 (ddd, J=11.3, 8.6, 4.2 Hz, 1H), 3.42-3.50 (m, 2H), 3.36 (s, 3H), 3.04-3.14 (br m, 1H), 2.99 (dd, J=14.2, 11.5 Hz, 1H), 2.92-3.02 (m, 1H), 2.78- 2.88 (br m, 1H), 2.34-2.42 (m, 1H), 1.73-1.84 (br m, 1H), 1.55-1.68 (m, 1H), 1.38-1.53 (m, 2H), 1.15 (d, J=6.9 Hz, 3H).
Preparation of (2R,3R)Meth0xymethyl-N-(Z-phenylethyl)[(2S)—pyrrolidin yl]pr0panethi0amide, hydrochloride salt (#23) and (2R,3R)meth0xymethyl-N-(2- phenylethyl)[(2S)—pyrrolidinyl]propanamide, hydrochloride salt (#24) Gt:IES~|ES"© BocN 4 M HCI in CHsgCN 100CS' #21 dioxane microwave BocN H2N 44% HATU, IPerEt. BocN + CH2CI2 o 100% o \ NH \O OH HClindioxane, MeOH #11 #20 16h, quant. .HCI \ NH Dapephenethyl Step IA. Synthesis of tert-butyl (2S){(1R,2R)methoxymethyloxo[(2- ethyl) propyl}pyrrolidinecarboxylate (#20). To #11 (22 g, 77 mmol, 1 eq.) in dichloromethane (383 mL, 0.2 M) and N,N—dimethylformamide (30 mL) were added diisopropylethylamine (26.9 mL, 153 mmol, 2 eq.), 2-phenylethylamine (11.6 mL, 91.9 mmol, 1.2 eq.) and HATU (39.0 g, 99.5 mmol, 1.3 eq.). The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (700 mL) and washed tially with 1 M aqueous hydrochloric acid on (2 x 200 mL) and brine. The organic layer was dried over sodium sulfate, filtered and evaporated in vacuo. The crude material was taken up in dichloromethane and filtered. The filtrate was purified by silica gel chromatography ent: 0% to 100% ethyl acetate in heptane) to give #20 (24 g, 80%) as an off-white solid.
LC-MS: m/Z 392.2 [M+2H+], 291.1 [(M = 0.88 minutes;1H NMR (400 — Boc)+H+], retention time MHz, DMSO-dg), presumed to be a mixture of rotamers: 8 7.80-7.89 (br m, 1H), 7.23-7.29 (m, 2H), 7.15-7.23 (m, 3H), 3.72-3.82 and 3.55-3.62 (2 br m, total 1H), .55 (br m, 1H), 3.31- 3.44 (br m, 2H), 3.29 (s, 3H), 3.12-3.25 (br m, 1H), 2.98-3.12 (br m, 1H), 2.71 (t, J=7.1 Hz, 2H), 2.09-2.19 (m, 1H), 1.71-1.83 (br m, 2H), 1.60-1.70 (br m, 1H), 1.49-1.60 (br m, 1H), 1.41 (s, 9H), 1.03 (d, J=6.8 Hz, 3H).
Step 13. Synthesis of dipyridiniumylpentathiodiphosphonate (#21). orous ulfide (4.45 g, 2.19 mL, 20 mmol, 1 eq.) was added to pyridine (56 mL, 0.36 M) at 80 OC and the mixture was heated at reflux (115 °C) for 1 hour. The mixture was cooled to room temperature and the product was collected by filtration to give #21 as a yellow solid (4.57 g, 60%); mp: 165-167 °C (decomposition); 1H NMR (400 MHz, DMSO-dg) 8 8.78-8.84 (m, 4H), 8.22-8.30 (m, 2H), 7.76-7.83 (m, 4H).
Step 2A. Synthesis of tert-butyl (2S) {(1R,2R)methoxymethyl[(2- phenylethyl)amino]—3-thioxopropyl}pyrrolidinecarboxylate (#22). A mixture of #20 (1.200 g, 3.073 mmol, 1 eq.) and #21 (1.40 g, 3.69 mmol, 1.2 eq.) in acetonitrile (15 mL, 0.20 M) was subjected to microwave radiation at 100 °C for 30 minutes. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (150 mL), and washed sequentially with 0.5 M aqueous hydrochloric acid solution (100 mL) and brine (2 x 50 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 20% to 80% ethyl acetate in heptane) to give #22 (670 mg, 54%) as a white wax-like solid; mp: 107-109 0C; LC—MS: m/Z 407.4 [M+H+], 351.3 [(M - 2- methylprop-l-ene)+H+], 307.3 [(M - Boc)+H+], retention time = 0.99 minutes; 1H NMR (400 MHz, CD3CN), presumed to be a mixture mers: 8 8.28 (br s, 1H), 7.19-7.33 (m, 5H), 3.81- 4.05 (br m, 2H), 3.60-3.81 (br m, 2H), 3.38-3.51 (br m, 1H), 3.36 (s, 3H), 3.02-3.17 (br m, 1H), 2.89-3.02 (m, 2H), .62 (br m, 1H), 1.71-1.85 (br m, 2H), 1.53-1.66 (br m, 2H), 1.45 (br s, 9H), 1.23 (d, J=6.7 Hz, 3H).
Step 3. Synthesis of (2R,3R)methoxymethyl-N—(2-phenylethyl)[(2S)-pyrrolidin yl]propanethioamide, hydrochloride salt (#23). According to procedure C, from #22 (325 mg, 0.799 mmol, 1 eq.), e (5 mL, 0.2 M) and a 4 M hydrochloric acid solution in dioxane (4 mL, 16 mmol, 20 eq.) was sized #23 (274 mg, quantitative) as a white foam; LC-MS: 308.2 [M+H+], retention time = 0.55 minutes.
Step 23. Synthesis of (2R, 3R)methoxymethyl-N-(2-phenylethyl)[(2S)-pyrrolidin- 2-yl] propanamide, hydrochloride salt (#24). To #20 (7.00 g, 17.9 mmol, 1 eq.) in dioxane (50 mL, 0.36 M) and methanol (2 mL) was added a 4 M on of hydrochloric acid in dioxane (20 mL, 80 mmol, 4.4 eq.). After ng for 18 hours, the mixture was concentrated to afford #24 (5.86 g, quantitative) as a gum, which was used without further purification; LC-MS: 292.2 [M+H+], retention time = 0.47 minutes. ation of N-Methyl-L-valyl-N-[(3R,4S,5S)—3-methoxy{(2S)—2-[(1R,2R)—1-meth0xy- 2-methyl{[(1S)—2-phenyl(1,3-thiazolyl)ethyl]amin0}thi0x0pr0pyl]pyrrolidin-l- yl}methyl0x0heptanyl]-N-methyl-L-valinamide (#26) Fmoo’iIrNiL'ipYOH0 s H H \ HATU, Et3N,CHZCIZ,DMF + (MIR/g? 75% a FmOCBIi/NgfiIEHA/NR O /\ O\ O o\ . C 3002F H s \ Trimer acid #18 : #25 _\\(s\ / Et2N CHQCIZ ”HQ“um% 6N #26 S Step 1. Synthesis of N— [(9H—fluorenylmethoxy)carbonyl] -N—methyl-L-valyl-N— [(3R,4S,55)methoxy {(2S)[(1R,2R)methoxymethyl {[(lS)phenyl(1,3- thiazolyl)ethyl] amino} -3 opropyl]pyrrolidinyl}methyloxoheptanyl]—N— methyl-L-valinamide (#25) According to general procedure D, from #8 (480 mg, 0.753 mmol, 1 eq.), dichloromethane (10 mL, 0.07 M), N,N—dimethylformamide (2 mL), the amine #18 (401 mg, 0.941 mmol, 1.25 eq.), HATU (372 mg, 0.979 mmol, 1.3 eq.) and triethylamine (367 ”L, 2.64 mmol, 3.5 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 30% acetone in heptane) to afford #25 (711 mg, 75%) as a solid. LC-MS: m/z 1009.7 [M+H+], retention time = 1.15 s; HPLC (Protocol B): m/z 505.3 ]/2, retention time = 10.138 minutes; 1H NMR (400 MHz, DMSO-dg), ed to be a e of rotamers, characteristic signals: 8 [10.54 (br (1, J=8 Hz) and 10.81 (br (1, J=8 Hz), total 1H], 7.89 (br d, J=7 Hz, 2H), [7.80 (d, J=3.3 Hz) and 7.83 (d, J=3.2 Hz), total 1H], [7.64 (d, J=3.2 Hz) and 7.69 (d, J=3.2 Hz), total 1H], 7.62 (br d, J=7 Hz, 2H), 7.37-7.44 (m, 2H), 7.28-7.35 (m, 4H), 7.20-7.27 (m, 2H), 7.12-7.18 (m, 1H), 6.27-6.35 and 6.40-6.48 (2 m, total 1H), [1.14 (d, J=6.4 Hz) and 1.17 (d, J=6.3 Hz), total 3H].
Step 2. Synthesis of N—methyl-L-valyl-N—[(3R, 4S,55)methoxy {(2S)[( 1R, 2R) methoxymethyl {[( lS)phenyl( 1 ,3-thiazolyl)ethyl] amino} thioxopropyl]pyrrolidinyl} methyloxoheptanyl]-N—methyl-L-valinamide (#26).
According to general procedure A, from #25 (701 mg, 0.694 mmol) in dichloromethane (10 mL, 0.07 M) and diethylamine (10 mL) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give a glass-like solid. Diethyl ether and heptane were added and the mixture was concentrated in vacuo, producing #26 (501 mg, 92%) as a white solid. HPLC (Protocol A): m/z 787.4 , retention time = 7.229 minutes, (purity > 97%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.54 (br (1, J=8 Hz) and 10.81 (br (1, J=8. Hz), total 1H], [7.99 (br d, J=9 Hz) and 8.00 (br d, J=9 Hz), total 1H], [7.80 (d, J=3.3 Hz) and 7.83 (d, J=3.3 Hz), total 1H], [7.65 (d, J=3.2 Hz) and 7.69 (d, J=3.3 Hz), total 1H], 7.29-7.34 (m, 2H), 7.19-7.28 (m, 2H), .19 (m, 1H), [6.31 (ddd, J=11, 8, 4.5 Hz) and 6.45 (ddd, J=11.5, 8, 4.5 Hz), total 1H], [4.57 (dd, J=8.9, 8.7 Hz) and 4.63 (dd, J=8.7, 8.7 Hz), total 1H], 3.16, 3.21, 3.24 and 3.25 (4 s, total 6H), 2.96 and 3.03 (2 br s, total 3H), [1.14 (d, J=6.6 Hz) and 1.17 (d, J=6.4 Hz), total 3H].
Preparation of N2-[(1-Amin0cyclopentyl)carbonyl]-N-[(3R,4S,5S)—3-meth0xy{(2S)—2- [(1R,2R)—1-meth0xymethyl{ [(1 S)—2-phenyl(1 ,3-thiazolyl)ethyl] amin0} thioxopropyl]pyrrolidin-l-yl}methyl0x0heptanyl]-N-methyl-L-valinamide (#30) 1-(Fmoc-amino)cyclo O H N\)OJ\ O BU H 2 f pentanecarboxylic acid [i1 OtBu L>CHCI TFA N OH i —’FmocHNQgNgN FmocHN /\ O quant. \ 0 HATU CHQCIZ iPrgNEt O /\9LT O\ 0 #18 HATU CH2CI2 Et—3_NDMF, gag#RN% Et2NH CH20I2 67% 61% QgN/{OL#:an41% NH NH \\«SN/ Step 1. Synthesis of tert-butyl (3R, 4S, 5S)[{N—[(1-{[(9H—fluoren ylmethoxy)carbonyl] amino} cyclopentyl)carbonyl]-L-valyl} (methyl)amino] -3 xy-5 - methylheptanoate (#27). To #6 (287 mg, 0.801 mmol, 1 eq.) in dichloromethane (4 mL, 0.2 M) were added 1- fluorenylmethoxy)carbonyl]amino}cyclopentanecarboxylic acid (309 mg, 0.879 mmol, 1.1 eq.), diisopropylethylamine (281 uL, 1.60 mmol, 2 eq.) and HATU (376 mg, 0.960 mmol, 1.2 eq.). The mixture was stirred for 18 hours and diluted with ethyl e (15 mL). The reaction mixture was washed with 1 M aqueous hydrochloric acid solution (2 x 5 mL) and with brine (5 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (Gradient: 0% to 60% ethyl acetate in heptane) to provide #27 (502 mg, 91%) as a white foam. LC—MS: m/z 692.3 [M+H+], 714.3 [M+Na+], 636.3 [(M - 2-methylpropene)+H+], ion time = 1.13 minutes; 1H NMR (400 MHz, DMSO-dg), characteristic signals: 8 7.89 (br d, J=7.4 Hz, 2H), 7.67-7.75 (m, 2H), 7.60 (br s, 1H), 7.38-7.44 (m, 2H), 7.30-7.36 (m, 2H), 7.21 (br d, J=8.8 Hz, 1H), 4.44-4.59 (m, 2H), .27 (m, 3H), 3.68-3.78 (br m, 1H), 3.21 (s, 3H), 2.88 (br s, 3H), 2.09-2.20 (m, 2H), 1.39 (s, 9H).
Step 2. Synthesis of (3R,4S, 55)[{N—[( 1- {[(9H-fluorenylmethoxy)carbonyl]amino} cyclopentyl)carbonyl]-L-valyl}(methyl)amino]methoxymethylheptanoic acid (#28). To a solution of #27 (500 mg, 0.723 mmol) in romethane (7 mL, 0.1 M) was added trifluoroacetic acid (3 mL). The reaction mixture initially became orange, then ed over time. After stirring for 18 hours, the solvent was removed in vacuo to give #28 (460 mg, quantitative) as a dark brown glass, which was used without further purification. LC-MS: m/Z 636.3 [M+H+].
Step 3. Synthesis ofNZ-[(1-{[(9H-fluoren ylmethoxy)carbonyl] amino} cyclopentyl)carbonyl] -N—[(3R, 4S, 55)-3 -methoxy {(2S) [( 1R, 2R)methoxymethyl {[( lS)phenyl- 1-( 1 ,3-thiazolyl)ethyl] amino} thioxopropyl]pyrrolidinyl} methyloxoheptanyl]-N—methyl-L-valinamide (#29).
According to general procedure D, from #28 (50 mg, 0.079 mmol, 1 eq.) dichloromethane (3 mL, 0.03 M), N,N—dimethylformamide (0.5 mL), amine #18 (44 mg, 0.087 mmol, 1.1 eq.), ylamine (33.0 uL, 0.237 mmol, 3 eq.) and HATU (36 mg, 0.95 mmol, 1.2 eq.) was sized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 30% acetone in heptane) to give #29 (59 mg, 67%) as a solid. LC-MS: m/z 1007.5 [M+H+], ion time = 1.11 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.54 (br d, J=8 Hz) and 10.80 (br d, J=8 Hz), total 1H], 7.89 (br d, J=7 Hz, 2H), [7.80 (d, J=3.3 Hz) and 7.82 (d, J=3.1 Hz), total 1H], 7.68- 7.75 (m, 2H), [7.64 (d, J=3.2 Hz) and 7.68 (d, J=3.2 Hz), total 1H], .44 (m, 2H), 7.27-7.36 (m, 4H), 7.12-7.25 (m, 4H), [6.30 (ddd, J=11, 8, 4.5 Hz) and 6.39-6.48 (m), total 1H], [4.50 (br dd, J=8, 8 Hz) and 4.54-4.59 (m), total 1H], 4.17-4.29 (m, 3H), 2.89 and 2.96 (2 br s, total 3H), [1.13 (d, J=6.5 Hz) and 1.16 (d, J=6.4 Hz), total 3H].
Step 4. Synthesis of N2-[( ocyclopentyl)carbonyl] R,4S, 55)methoxy {(2S)[(1R,2R)methoxymethyl {[(lS)phenyl(1 ,3-thiazolyl)ethyl] amino} thioxopropyl] pyrrolidinyl}methyloxoheptanyl]-N—methyl-L-valinamide (#30).
According to general procedure A, from #29 (54 mg, 0.054 mmol) in dichloromethane (6 mL, 0.9 mM) and diethylamine (4 mL) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give e #30 (26 mg, 61%) as a solid. HPLC (Protocol A): retention time = 7.233 minutes, m/z 785.4 [M+H+], (purity > 72%). 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.54 (br d, J=8 Hz) and 10.82 (br d, J=8 Hz), total 1H], 8.19-8.27 (m, 1H), [7.80 (d, J=3.2 Hz) and 7.83 (d, J=3.2 Hz), total 1H], [7.65 (d, J=3.3 Hz) and 7.69 (d, J=3.3 Hz), total 1H], 7.28-7.33 (m, 2H), 7.20-7.27 (m, 2H), 7.14-7. 19 (m, 1H), [6.31 (ddd, J=11, 8, 4.5 Hz) and 6.44 (ddd, J=11, 8, 4 Hz), total 1H], [4.53 (dd, J=9, 8 Hz) and 4.60 (dd, J=9, 7.5 Hz), total 1H], 3.24 and 3.25 (2 s, total 3H), 3.17 and 3.21 (2 s, total 3H), 2.93 and 3.00 (2 br s, total 3H), [1.14 (d, J=6.5 Hz) and 1.17 (d, J=6.5 Hz), total 3H].
Preparation of 2-Methylalanyl-N-[(3R,4S,5S)—3-meth0xy{(2S)—2-[(1R,2R)—1-meth0xy methyl{ [(1 S)—2-phenyl(1,3-thiazolyl)ethyl] amino}thi0x0pr0pyl] pyrrolidin-l-yl}- -methyl0x0heptanyl]-N-methyl-L-valinamide (#34) N-Fmoc—ot—aminoisbut ' ' O HQNQLN ync acnd, O OtBu HATU,iPr2NEt,CHZC|2 “\A TFA,CH2C|2 “ OH OtBu —’FmocHN N —> FmocHN N = l .
/\ O\ O Deprotected dimer #6 #18, HATU, Eth, CH2C|2, DMF, KfndL EthH, CH20I2 KrNH N —.H2N N FmocHN , 69% g 60% o I o o o A o\ 0 /\ \ o o \ \ NH #33 NH 8 S #34 ‘\(_/78 ° ‘\\(S\ / Step 1. Synthesis of -fluorenylmethoxy)carbonyl]methylalanyl-N- [(3R,4S,55)tert—butoxy-3 -methoxymethyloxoheptanyl]-N—methyl-L-valinamide (#3 1).
To a solution of #6 (70% pure, 3.13 g, 6.1 mmol, 1 eq.) in dichloromethane (40 mL, 0.15 M) were added N—[(9H—fluorenylmethoxy)carbonyl]methylalanine (1.99 g, 6.12 mmol, 1 eq.), diisopropylethylamine (2.67 mL, 15.3 mmol, 2.5 eq.) and HATU (2.79 g, 7.35 mmol, 1.2 eq.).
The reaction mixture was stirred for 18 hours, diluted with ethyl acetate, washed with 1 M aqueous hydrochloric acid solution and washed with brine. The c layer was dried over sodium sulfate, filtered, and concentrated in vacuo onto silica. The material was then purified by silica gel chromatography (Gradient: 0% to 45% ethyl acetate in heptane) to provide #31 (3.65 g, 90%) as a solid. LC—MS: m/Z 665.5 [M+H+], 688.5 [M+Na+], 610.5 [(M - 2-methylprop ene)+H+]; HPLC (Protocol C): retention time = 9.455 (purity > 94%); 1H NMR (400 MHz, DMSO-dg), characteristic signals: 8 7.89 (d, J=7.4 Hz, 2H), 7.67-7.74 (m, 2H), 7.39-7.48 (m, 3H), 7.31-7.36 (m, 2H), 7.29 (br d, J=8.8 Hz, 1H), 4.47-4.60 (br m, 1H), 4.47 (dd, J=8.6, 8.0 Hz, 1H), 4.18-4.28 (m, 3H), 3.69-3.79 (br m, 1H), 3.21 (s, 3H), 2.88 (br s, 3H), 2.15 (dd, J=15.5, 9.3 Hz, 1H), 1.91-2.01 (m, 1H), 1.67-1.81 (br m, 1H), 1.39 (s, 9H), 1.36 (br s, 3H), 1.30 (s, 3H), 0.75 (d, J=6.6 Hz, 3H), 0.66-0.73 (br m, 3H).
Step 2. sis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N- [(2R, 3S, 4S)carboxymethoxymethylhexanyl] -N—methyl-L-valinamide (#32).
According to general procedure B, from #31 (500 mg, 0.751 mmol) in romethane (7 mL, 0.1 M) and trifluoroacetic acid (3 mL) was sized #32 as a glass (458 mg, quantitative), which was used in the next step without r purification. LC-MS: m/z 611.4 ], 632.2 [M+Na+], retention time = 0.94 minute.
Step 3. Synthesis of N—[(9H—fluorenylmethoxy)carbonyl] methylalanyl-N- [(3R, 4S, 55)-3 xy {(2S)[(1R,2R)methoxymethyl {[(lS)phenyl(1,3 - thiazolyl)ethyl] amino} -3 -thioxopropyl]pyrrolidinyl}methyloxoheptanyl]—N- methyl-L-valinamide (#33). According to l procedure D, from #32 (53.0 mg, $0.083 mmol, 1 eq.), dichloromethane (4 mL, 0.02 M), N,N—dimethylformamide (1 mL), amine #18 (43.8 mg, 0.0870 mmol, 1 eq.), triethylamine (36 uL, 0.26 mmol, 3 eq.) and HATU (39.5 mg, 0.104 mmol, 1.2 eq.) was synthesized the crude desired material, which was d by silica gel chromatography (Gradient: 0% to 30% acetone in heptane) to give #33 (60 mg, 69% over two steps). LC-MS: m/Z 981.4 [M+H+], ion time = 1.090 minutes; 1H NMR (400 MHz, DMSO- d6), presumed to be a mixture of rotamers, characteristic signals: 8 [10.54 (br d, J=8 Hz) and .80 (br d, J=8 Hz), total 1H], 7.86-7.91 (m, 2H), [7.80 (d, J=3.3 Hz) and 7.82 (d, J=3.3 Hz), total 1H], 7.68-7.74 (m, 2H), [7.64 (d, J=3.2 Hz) and 7.68 (d, J=3.3 Hz), total 1H], 7.38-7.44 (m, 2H), .36 (m, 6H), 7.12-7.17 (m, 1H), 6.27-6.34 and 6.40-6.47 (2 m, total 1H), 3.22 and 3.24 (2 s, total 3H), 3.14 and 3.18 (2 s, total 3H), 2.90 and 2.97 (2 br s, total 3H), 1.37 (br s, 3H), 1.31 (2 br s, total 3H), [1.13 (d, J=6.6 Hz) and 1.16 (d, J=6.5 Hz), total 3H].
Step 4. Synthesis of 2-methylalanyl-N-[(3R, 4S, 55)methoxy{(2S)[(1R, 2R) methoxymethyl {[( lS)phenyl( 1 ,3-thiazolyl)ethyl] amino} thioxopropyl]pyrrolidinyl} methyloxoheptanyl]-N—methyl-L-valinamide (#34).
According to general ure A, from #33 (55 mg, 0.055 mmol, 1 eq.) in dichloromethane (6 mL, 0.009 M) and diethylamine (4 mL) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 5% methanol in dichloromethane) to give #34 (25 mg, 60%) as a solid. HPLC (Protocol A): m/z 759.4 [M+H+], retention time = 7.088 minutes, y > 75%). 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.54 (br d, J=8 Hz) and 10.81 (br d, J=8 Hz), total 1H], 8.01-8.08 (m, 1H), [7.80 (d, J=3.1 Hz) and 7.83 (d, J=3.3 Hz), total 1H], [7.65 (d, J=3.2 Hz) and 7.69 (d, J=3.2 Hz), total 1H], 7.29-7.33 (m, 2H), 7.20-7.27 (m, 2H), 7.13-7.19 (m, 1H), 6.27-6.35 and 6.40-6.48 (2 m, total 1H), [4.49 (dd, J=9, 8 Hz) and 4.56 (dd, J=9, 8 Hz), total 1H], 3.24 and 3.25 (2 s, total 3H), 3.17 and 3.21 (2 s, total 3H), 2.92 and 2.99 (2 br s, total 3H), 1.20 and 1.21 (2 s, total 3H), 1.12 and 1.13 (2 s, total 3H), 0.75-0.81 (m, 3H). ation of N-methyl-L-valyl-N-{(3R,4S,5S)meth0xy[(2S)—2-{(1R,2R)—1-meth0xy- 2-methyl[(2-phenylethyl)amino]thioxopr0pyl}pyrrolidin-l-yl]methyl0X0heptan- 4-yl}-N-methyl-L-valinamide (#36) HN 'CI O O H H acid#8 HATU iPr2NEt, Cchlz, DMF Fmocrxx/Ngirpfir'“ m \fiE/Nfixf‘pfi/N o\ —- l 0 88% NH 66% 8 /=\|O\Oo o/'\‘o\oo b \ \ NH #35 NH 3 #36 S b a Step 1. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N—methyl-L-valyl-N— {(3R,4S,55)methoxy[(2S) {(1R,2R)methoxymethyl-3 -[(2-phenylethyl)amino] thioxopropyl}pyrrolidinyl]methyloxoheptanyl}-N—methyl-L-valinamide (#35). To a mixture of #23 (337 mg, 0.983 mmol, 1 eq.) in dichloromethane (8 mL, 0.1 M) and MN- dimethylformamide (1 mL) were added #8 (564 mg, 0.885 mmol, 0.9 eq.), diisopropylethylamine (383 mg, 2.95 mmol, 3 eq.) and HATU (472 mg, 1.18 mmol, 1.2 eq.). After 2 hours, the mixture was diluted with dichloromethane, washed sequentially with 0.1 M aqueous hloric acid and with brine, dried over magnesium e, filtered, and concentrated in vacuo. The residue was purified by reverse phase chromatography (Method G) to give #35 (600 mg, 66%); LC-MS: m/z 926.6 [M+H+], ion time = 1.16 minutes; 1H NMR (400 MHz, DMSO-dg), ed to be a mixture of rotamers, characteristic signals: 8 [9.94 (br t, J=5 Hz) and 10.16-10.23 (br m), total 1H], 7.90 (d, J=7.2 Hz, 2H), [7.71 (br d, J=7 Hz) and 8.06 (br d, J=8 Hz), total 1H], 7.60- 7.65 (m, 2H), 7.41 (br dd, J=7, 7 Hz, 2H), 7.15-7.36 (m, 7H), 3.29 (s, 3H), 1.16-1.22 (m, 3H).
Step 2. Synthesis of N—methyl-L-valyl-N— {(3R,4S,55)methoxy[(2S) {( 1R,2R) methoxymethyl-3 - [(2-phenylethyl)amino] -3 opropyl}pyrrolidinyl]methyl tanyl}-N—methyl-L-valinamide (#36). According to general procedure A, from #35 (465 mg, 0.502 mmol, 1 eq.) in dichloromethane (5 mL, 0.1 M) and diethylamine (5 mL) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give #36 (310 mg, 88%) as a solid. LC- MS: m/z 704.6 [M+H+], retention time = 0.74 minutes; HRMS: m/z calculated for C38H66N5058: 704.4779, found: 704.477 [M+H+]; 1H NMR (400 MHZ, CD3OD), presumed to be a mixture of rotamers, characteristic signals: 8 7.23-7.30 (m, 4H), 7.15-7.22 (m, 1H), [4.68 (d, J=8.6 Hz) and 4.74 (d, J=8.0 Hz), total 1H], 3.39 and 3.40 (2 s, total 3H), 3.12 and 3.22 (2 br s, total 3H), [2.82 (d, J=6.0 Hz) and 2.84 (d, J=6.0 Hz), total 1H], 2.29 and 2.30 (2 s, total 3H), [1.27 (d, J=6.8 Hz) and 1.29 (d, J=6.6 Hz), total 3H], [0.84 (t, J=7.4 Hz) and 0.87 (t, J=7.4 Hz), total 3H].
Preparation of N-methyl-L-valyl-N—[(3R,4S,5S){(2S)—2-[(1R,2R)—3-{[(1S)—1-carb0xy phenylethyl]amin0}meth0xymethylthi0x0propyl]pyrrolidin-l-yl}meth0xy methyl0x0heptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#41) and N- 2012/056224 Methyl-L-valyl-N-[(3R,4S,5S)methoxy{(2S)—2-[(1R,2R)meth0xy{ [(2S)—1- y-l-0X0phenylpr0panyl] amino}methylthi0x0propyl] pyrrolidin-l-yl} methyl0x0heptanyl]-N-methyl-L-valinamide (#42) H2N Hg HATU iPerEt BocN 3% #21 itrile 800% 4 M HCI H3; BOON CH2C'2 DMF microwave 100 °C in dioxane HCI + O O 65% 43% \ NH \ OH b 0 .\\( #39 ? 0\ #110\ HN NJN LiOH,THF,H20 I | o /=-\ -CF3C02H o\ o % #8, HATU, Et3N, CHZCIZ, DMF FmocN 90% (2 steps) 0\ EtZNH CHZCIZ 80% ON/éfiiLN #42 3 0 Step 1. Synthesis of methyl N—{(2R, 3R)[(25)(tert-butoxycarbonyl)pyrrolidin—2-yl]- 3-methoxymethylpropanoyl}-L-phenylalaninate (#37). To a mixture of #11 (2.7 g, 9.4 mmol, 1 eq.) in dichloromethane (30 mL, 0.3 M) and N,N—dimethylformamide (3 mL) were added ropylethylamine (3.30 mL, 18.8 mmol, 2 eq.), L-phenylalanine methyl ester hydrochloride (2.03 g, 9.40 mmol, 1.2 eq.) and HATU (4.79 g, 12.2 mmol, 1.3 eq.). The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (100 mL) and washed sequentially with 1 M hydrochloric acid (2 x 50 mL) and brine. The organic layer was dried over sodium sulfate, filtered and evaporated in vacuo. The crude al was taken up in dichloromethane and filtered. The filtrate was purified by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) to give #37 (2.76 g, 65%) as an off-white solid. LC-MS: m/Z 449.3 [M+H+], 349.2 [(M = 0.88 minutes; 1H NMR (400 MHz, - Boc)+H+] retention time DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 8.28 (d, J=8.2 Hz, 1H), 7.14-7.29 (m, 5H), 4.50 (ddd, J=10.9, 8.1, 4.4 Hz, 1H), 3.64 (s, 3H), 3.23 (s, 3H), 2.15-2.24 (m, 1H), 1.56-1.76 (m, 2H), .55 (m, 11H),1.02(d, J=6.6 Hz, 3H).
Step 2. Synthesis of methyl N— {(2R,3R)[(25)(tert—butoxycarbonyl)pyrrolidinyl]- 3-methoxymethylpropanethioyl} -L-phenylalaninate (#38). A mixture of #37 (1.52 g, 3.39 mmol, 1 eq.) and #21 (1.68 g, 4.41 mmol, 1.3 eq.) in acetonitrile (12 mL, 0.28 M) was subjected to microwave radiation at 100 0C for 1 hour. The mixture was ioned between water and ethyl acetate. The aqueous layer was back-extracted with ethyl acetate. The combined organic layers were washed with 10% aqueous citric acid solution and with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The material was dissolved in a small amount of ethyl acetate and concentrated onto silica in vacuo. Purification by silica gel tography (Gradient: 0% to 30% ethyl acetate in heptane) provided #38 (680 mg, 43%); LC—MS: m/z 465.2 [M+H+], 487.3 ], 365.2 [(M - Boc)+H+], retention time = 0.97 s; HPLC (Protocol B): 465 .2 [M+H+], 487.2 [M+Na+], 365 .2 [(M - Boc)+H+], retention time = 7.444 minutes (purity > 98%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 10.23 (br d, J=7.5 Hz, 1H), 7.17-7.28 (m, 5H), 5.24 (ddd,J=11, 7.5, 4.5 Hz, 1H), 3.66 (s, 3H), 3.28 (s, 3H), 3.21 (dd, , 4.4 Hz, 1H), 3.07 (dd, J=14.2, 11.2 Hz, 1H), 2.65-2.74 (m, 1H), 1.54-1.71 (m, 2H), 1.37 (s, 9H), 1.17 (d, J=6.4 Hz, 3H).
Step 3. Synthesis of methyl N— {(2R,3R)methoxymethyl[(2S)-pyrrolidin yl]propanethioyl}-L-phenylalaninate, hydrochloride salt (#39). According to general procedure C, at 0 0C from #38 (660 mg, 1.42 mmol, 1 eq.), dioxane (10 mL, 0.14 M) and 4 M hydrochloric acid solution in dioxane (20 mL, 80 mmol, 60 eq.) was sized #39 (590 mg) as an off-white solid, which was used in the next step without further purification. LC—MS: m/z 365 .2 [M+H+], retention time = 0.58 minutes; 1H NMR (400 MHz, g) 8 10.67 (d, J=7.7 Hz, 1H), 9.42- 9.54 (br m, 1H), 8.21-8.33 (br m, 1H), 7.20-7.35 (m, 5H), 5.25 (ddd, J=11.1, 7.6, 4.4 Hz, 1H), 3.76 (dd, J=8.9, 3.0 Hz, 1H), 3.68 (s, 3H), 3.39 (s, 3H), 3.24 (dd, J=14.2, 4.5 Hz, 1H), 3.13 (dd, J=14.3, 11.0 Hz, 1H), 2.93-3.09 (m, 3H), 2.85-2.93 (m, 1H), 1.72-1.84 (m, 1H), 1.36-1.60 (m, 3H), 1.22 (d, J=6.6 Hz, 3H).
Step 4. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N—methyl-L-valyl-N- [(3R,4S,55)methoxy {(2S)[(1R,2R)methoxy {[(2S)methoxyoxo phenylpropanyl]amino} methyl-3 -thioxopropyl]pyrrolidin— 1-yl}methyloxoheptan yl]-N—methyl-L-valinamide (#40). According to general procedure D, from #8 (247 mg, 0.387 mmol, 1 eq.), #39 (186 mg, 50.450 mmol, 1.2 eq.), romethane (10 mL, 0.04 M), N,N— dimethylformamide (2 mL), HATU (176 mg, 0.464 mmol, 1.2 eq.) and triethylamine (189 ”L, 1.35 mmol, 3.5 eq.) was synthesized the crude d al, which was purified by silica gel chromatography (Gradient: 0% to 25% acetone in heptane) to give #40 (410 mg, 90% over 2 steps) as an off-white solid. LC—MS: m/Z 984.7 [M+H+], 1006.7 [M+Na+], retention time = 1.15 minutes; HPLC (Protocol C): retention time = 9.683 minutes (purity > 99%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.19 (br d, J=7 Hz) and 10.49 (br d, J=8 Hz), total 1H], 7.90 (d, J=7.5 Hz, 2H), 7.60-7.65 (m, 2H), 7.38- 7.45 (m, 2H), 7.29-7.35 (m, 2H), 7.14-7.28 (m, 5H), [5.20 (ddd, J=11, 7, 4 Hz) and 5.35-5.43 (m), total 1H], 3.65 and 3.69 (2 s, total 3H), [1.15 (d, J=6.5 Hz) and 1.18 (d, J=6.4 Hz), total 3H].
Step 5A. Synthesis of N—methyl-L-valyl-N—[(3R, 4S, 55) {(2S)[( 1R, {[( 15) carboxyphenylethyl]amino} methoxymethyl-3 -thioxopropyl]pyrrolidinyl} -3 - methoxymethyloxoheptan—4-yl]-N—methyl-L-valinamide, trifluoroacetic acid salt (#41). To a solution 0f#40 (401 mg, 0.407 mmol, 1 eq.) in tetrahydrofuran (10 mL, 0.03 M) was added a solution of m ide (24.4 mg, 1.02 mmol, 2.5 eq.) in water (5 mL). After 4 hours, the reaction was concentrated in vacuo and then azeotroped three times with e. The crude material was dissolved in dimethyl sulfoxide (7 mL) and purified by reverse phase chromatography (Method C, 7 injections of 1 mL). The appropriate fractions were concentrated (Genevac) before being diluted with a small amount of methanol in dichloromethane. The mixture was concentrated in vacuo to a glass-like solid. Diethyl ether was then added, followed by heptane, and the mixture was concentrated in vacuo to afford #41 (180 mg, 59%) as a white solid. LC-MS: m/z 748.6 [M+H+], retention time = 0.68 minutes; HPLC (Protocol A): 748.4 [M+H+], retention time = 6.922 s; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 12.9 and 13.1 (2 V br s, total 1H), [10.12 (br d, J=8 Hz) and 10.45 (br d, J=8 Hz), total 1H], 8.75-8.90 (m, 2H), 8.62-8.73 (br m, 1H), 7.13-7.29 (m, 5H), [5.20 (ddd, J=11, 7.5, 4 Hz) and 5.40 (ddd, J=11.5, 8, 4 Hz), total 1H], 4.55-4.73 (m, 2H), 3.23 and 3.25 (2 s, total 3H), 3.16 and 3.18 (2 s, total 3H), 2.97 and 3.01 (2 br s, total 3H), 1.13- 1.20 (m, 3H), 0.73-0.81 (m, 3H).
Step 58. Synthesis of N—methyl-L-valyl-N—[(3R,4S, 55)methoxy{(2S)[(1R,2R) methoxy{[(2S)methoxyoxophenylpropanyl]amino}methyl thioxopropyl]pyrrolidinyl} methyloxoheptanyl]-N—methyl-L-valinamide (#42).
According to general procedure A, from #40 (561 mg, 0.570 mmol, 1 eq.), dichloromethane (10 mL, 0.057 M) and diethylamine (10 mL) was sized #42 (348 mg, 80%) as a white solid after silica gel chromatography (Gradient: 0% to 10% methanol in romethane). LC—MS: m/Z 762.7 [M+H+], retention time = 0.74 minutes; HPLC (Protocol A): 762.4 , retention time = 7.315 minutes y > 95%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.20 (br d, J=7.5 Hz) and 10.50 (br d, J=8 Hz), total 1H], 7.95-8.03 (m, 1H), 7.15-7.29 (m, 5H), [5.20 (ddd, J=11, 7.5, 5 Hz) and 5.39 (ddd, J=11, 7.5, 4 WO 72813 Hz, total 1H], [4.57 (dd, J=8.8, 8.7 Hz) and 4.61 (dd, J=8.7, 8.6 Hz), total 1H], 3.65 and 3.69 (2 , total 3H), 3.24 and 3.25 (2 5, total 3H), 3.16 and 3.17 (2 5, total 3H), 2.96 and 2.99 (2 br 5, total 3H), 2.69-2.79 (m, 1H), 2.62-2.68 (m, 1H), 2.14 and 2.15 (2 br 5, total 3H), [1.15 (d, J=6.6 Hz) and 1.18 (d, J=6.5 Hz), total 3H], [0.75 (t, J=7.4 Hz) and 0.76 (t, J=7.3 Hz), total 3H].
Preparation of ylalanyl-N-[(3R,4S,5S){(2S)[(1R,2R){[(1S)—1-carboxy-2— phenylethyl]amino}methoxy-2—methylthioxopropyl]pyrrolidin-l-yl}methoxy methyloxoheptanyl]-N-methyl—L-valinamide, hydrochloride salt (#44) and 2- methylalanyl-N—[(3R,4S,5S)methoxy{(2S)—2—[(1R,2R)—1-methoxy{[(2S)—1-methoxy- 1-oxophenylpropan-Z-yl]amino}methylthioxopropyl] idin-l-yl}methyl oxoheptanyl]-N-methyl-L-valinamide, hydrochloride salt (#45) MHQE'HC' HATU iPr2NEt N / H g + CHZCIZ DMF S FmocHNfifiN/ifii'fl 0\ U 68% H HC N .
LiOH, THF, H O = “\JCJ: 2 O /\ O\ O N \ FmocHN N s #44 s O /=\ I o\ o O N\H\(O \ OH #43 3 ”9 O\ 0 Et NH, CH2 2 Cl2 N .HC 70% H2N><fl/N\e/U\T o A 0\ O #45 NH 3 [\(O f 0\ Step 1. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N— [(3R,4S,55)methoxy {(2S)[(1R,2R)methoxy {[(2S)methoxyoxo phenylpropanyl]amino} methyl-3 -thioxopropyl]pyrrolidin— 1-yl}methyloxoheptan yl]-N—methyl-L-valinamide (#43). To a solution of #32 (321 mg, 0.881 mmol, 1 eq.) in dichloromethane (5 mL, 0.1 M) and methylformamide (1 mL) were added #39 (484 mg, 50.769 mmol, 0.9 eq.), HATU (353 mg, 0.881 mmol, 1 eq.) and diisopropylethylamine (463 uL, 2.64 mmol, 3 eq.). After stirring for 18 hours, the mixture was diluted with dichloromethane, washed with water and with brine, dried over magnesium sulfate, filtered, and concentrated onto silica in vacuo. The residue was purified by silica gel chromatography (Gradient: 0% to 30% acetone in heptane) to give #43 (574 mg, 68% over two steps) as a white solid. LC-MS: m/z 956.6 [M+H+], ion time = 4.49 minutes; 1H NMR (400 MHz, CD3OD), presumed to be a mixture of rotamers, characteristic signals: 8 7.80 (d, J=7.5 Hz, 2H), 7.64-7.72 (m, 2H), 7.16- 7.35 (m, 7H), [5.43 (dd, J=11, 4.5 Hz) and 5.58 (dd, J=1 1.5, 4 Hz), total 1H], 3.72 and 3.75 (2 s, total 3H), 3.34 and 3.35 (2 s, total 3H), 3.26 and 3.29 (2 s, total 3H), 3.05 and 3.11 (2 br s, total 3H), 1.39 and 1.40 (2 s, total 3H), [1.24 (d, J=6.7 Hz) and 1.29 (d, J=6.4 Hz), total 3H].
Step 2A. Synthesis of 2-methylalanyl-N—[(3R,4S,55){(2S)[(1R,2R)-3 - {[(lS) carboxyphenylethyl]amino} methoxymethyl-3 -thioxopropyl]pyrrolidinyl} -3 - methoxymethyloxoheptan—4-yl]-N—methyl-L-valinamide, hydrochloride salt (#44). To a solution of #43 (100 mg, 0.105 mmol, 1 eq.) in tetrahydrofuran (5 mL, 0.02 M) was added a solution of lithium ide (10 mg, 0.417 mmol, 3 eq.) in water (3 mL). After 3 hours, the on was concentrated in vacuo and purified by reverse phase tography (Method C) to give a trifluoroacetic acid salt, which was dissolved in methanol, treated with a 4 M hydrochloric acid solution in dioxane, and concentrated in vacuo to give #44 (56 mg, 71%) as a white solid.
LC-MS: m/Z 720.6 [M+H+], retention time = 0.67 minutes; HPLC (Protocol D): retention time = 8.851 minutes; 1H NMR (400 MHz, CD3OD), presumed to be a mixture of rotamers, characteristic signals: 8 7.17-7.31 (m, 5H), 3.34 and 3.35 (2 s, total 3H), 3.10 and 3.16 (2 br s, total 3H), 1.62 and 1.64 (2 s, total 3H), 1.53 and 1.55 (2 s, total 3H), [1.26 (d, J=6.5 Hz) and 1.30 (d, J=6.5 Hz), total 3H], 0.84-0.91 (m, 3H).
Step 23. Synthesis of ylalanyl-N—[(3R,4S,55)methoxy {(2S)[(1R,2R) methoxy{[(2S)methoxyoxophenylpropan—2-yl]amino}methyl thioxopropyl]pyrrolidinyl} methyloxoheptanyl]-N—methyl-L-valinamide, hydrochloride salt (#45). According to general procedure A, from #43 (176 mg, 0.184 mmol, 1 eq.), dichloromethane (4 mL, 0.05 M) and diethylamine (4 mL) was sized the crude desired al, which was purified by reverse phase chromatography (Method C). The resulting trifluoroacetic acid salt was dissolved in methanol, treated with a 4 M solution of hloric acid in dioxane, and concentrated in vacuo to give #45 (100 mg, 70%) as a white solid. LC—MS: m/z 734.6 [M+H+], retention time = 0.72 minutes; 1H NMR (400 MHz, , presumed to be a mixture ofrotamers, characteristic signals: 8 7.18-7.31 (m, 5H), 5.41-5.47 and .62 (2 m, total 1H), 3.73 and 3.76 (2 s, total 3H), 3.35 and 3.36 (2 s, total 3H), 3.10 and 3.15 (2 br s, total 3H), 1.62 and 1.64 (2 s, total 3H), 1.53 and 1.55 (2 s, total 3H), [1.25 (d, J=6.6 Hz) and 1.29 (d, J=6.5 Hz), total 3H], 0.84-0.91 (m, 3H).
Preparation of N2-[(1-Amin0cyclopentyl)carb0nyl]-N-[(3R,4S,5S)—3-meth0xy{(2S)—2- [(1R,2R)—1-meth0xymethyl0x0{ [(1S)—2-phenyl(1,3-thiazol yl)ethyl] amin0}pr0pyl] pyrrolidin-l-yl}methyl0x0heptanyl]-N-methyl-L-valinamide (#47) #19 HATU CH2C|2 DMF t FmocHN FmocHNQgNgLA:j$\erH—> éi 69% Ni“6%!“Chg/{bi Z|\//m Step 1. Synthesis ofN-[( 1-{[(9H—fluorenylmethoxy)carbonyl]amino}cyclopentyl)- carbonyl] -N—[(3R,4S,55)methoxy {(2S)[(1R,2R)methoxymethyloxo phenyl(1,3 -thiazolyl)ethyl]amino}propyl]pyrrolidinyl}methyloxoheptan—4-yl]-N— methyl-L-valinamide (#46). To a solution of #19 (353 mg, 0.944 mmol, 1 eq.) in dichloromethane (10 mL, 0.094 M) were added #28 (600 mg, 0.944 mmol, 0.9 eq.), diisopropylethylamine (498 uL, 2.83 mmol, 3 eq.) and HATU (444 mg, 1.13 mmol, 1.2 eq.).
After stirring for two days, the mixture was concentrated in vacuo and the residue was d with ethyl acetate (60 mL), washed with 1 M aqueous hydrochloric acid solution and with brine, dried over sodium sulfate, filtered, and trated in vacuo. The residue was diluted with dichloromethane and filtered. The filtrate was concentrated under reduced pressure onto silica and purified by silica gel chromatography (Gradient: 40% to 100% ethyl acetate in heptane) to give #46 (644 mg, 69%) as a white solid. LC-MS: m/z 991.8 [M+H+], retention time = 1.07 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a e of rotamers, characteristic signals: 8 7.89 (br d, J=7.4 Hz, 2H), [7.77 (d, J=3.3 Hz) and 7.79 (d, J=3.3 Hz), total 1H], 7.66- 7.76 (m, 2H), [7.62 (d, J=3.3 Hz) and 7.65 (d, J=3.3 Hz), total 1H], 7.37-7.44 (m, 2H), 7.11-7.36 (m, 7H), [5.38 (ddd, J=11, 8, 4 Hz) and 5.48-5.57 (m), total 1H], 3.13, 3.17, 3.18 and 3.24 (4 s, total 6H), 2.90 and 3.00 (2 br s, total 3H), [1.05 (d, J=6.6 Hz) and 1.09 (d, J=6.8 Hz), total 3H].
Step 2. Synthesis of N2-[( 1-aminocyclopentyl)carbonyl] -N—[(3R,4S, 55)methoxy {(2S)[(1R,2R)methoxymethyloxo{[(lS)phenyl(1,3-thiazol yl)ethyl] amino } ]pyrrolidinyl} methyl- eptanyl] -N—methyl-L-valinamide (#47). To a mixture of #46 (500 mg, 0.504 mmol, 1 eq.) in tetrahydrofuran (8 mL, 0.06 M) was added diethylamine (4 mL). After stirring for 18 hours, the reaction mixture was concentrated in vacuo and the residue was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give #47 (374 mg, 96%) as a white solid. LC-MS: m/z 769.6 [M+H+], retention time = 0.70 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.64 (br d, J:84 Hz) and 8.87 (br d, J=8.6 Hz), total 1H], [8.22 (br d, J=9.4 Hz) and 8.26 (br d, J=9.4 Hz), total 1H], [7.77 (d, J=3.3 Hz) and 7.80 (d, J=3.3 Hz), total 1H], [7.63 (d, J=3.1 Hz) and 7.66 (d, J=3.3 Hz), total 1H], .31 (m, 5H), [5.39 (ddd, J=11.1, 8.5, 4.2 Hz) and 5.54 (ddd, J=11.7, 8.8, 4.1 Hz), total 1H], [4.53 (dd, J=9.2, 7.6 Hz) and 4.64 (dd, J=9.2, 6.6 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.93 and 3.03 (2 br s, total 3H), [1.05 (d, J=6.8 Hz) and 1.10 (d, J=6.6 Hz), total 3H], 0.73-0.80 (m, 3H).
Preparation of N2-[(1-Aminocyclopropyl)carbonyl]-N-[(3R,4S,5S)methoxy{(ZS) [(1R,2R)—1-meth0xymethyl0X0{[(1S)—2-phenyl(1,3-thiazol yl)ethyl]amin0}pr0pyl]pyrrolidin-l-yl}methyl0X0heptanyl]-N-methyl-L-valinamide (#51) and 1-amin0-N-[(2S){[(3R,4S,5S)methoxy{(2S)—2-[(1R,2R)—1-meth0xy methyl0X0{ [(1 S)—2-phenyl(1 ,3-thiazolyl)ethyl] amino} propyl] pyrrolidin-l-yl}-5— methyl0x0heptanyl](methyl)amin0}methyl0X0butan yl]cyclohexanecarboxamide (#52) BocN CF3002H FmocHNEJLT o\l< TFA CH2CI2 /'\ /O O O\ O FmocHN\)LT/ #5 Sj t3N, H2N51THN DME Et2NH CH2CI2 g I]! 45% (2 steps) FmocHNdkm 30% /\ /O O 0\ NH HzNyb,OH O H2N: Brop iPr2NEt HQN¢L£WN :er/\#5N\£)OLN /O o CH2CI2 34% /=\ I /O O \ NH #50 0 s"\\(sN—l7\ OH H2N 0 N N H2N N = I Brop,iPr2NEt, /0 o CH2C|2, 35% \ NH Step 1. sis of (2R,3R)methoxymethy1-N—[(lS)pheny1(1,3-thiazol yl)ethyl][(2S)-pyrrolidinyl]propanamide, trifluoroacetic acid salt and (3R, 4S, SS)[ {N- uorenylmethoxy)carbonyl] -L-valyl} (methyl)amino] -3 -methoxymethylheptanoic acid (#48). To a solution of #16 (1.0 g, 2.11 mmol, 1 eq.) and #5 (1.22 g, 2.11 mmol, 1 eq.) in dichloromethane (20 mL, 0.1 M) at 0 0C was added trifluoroacetic acid (6 mL). After 3 hours, the mixture was concentrated in vacuo to give the mixture #48 (1.8 g), which was used in the next step without further purification; LC—MS (Protocol K): m/z 374.2 [M+H+], retention time = 2.093 minutes, 525.2 [M+H+], retention time = 4.875 minutes.
Step 2. Synthesis of N2-[(9H—fluoren—9-ylmethoxy)carbonyl] R, 4S, 5S)methoxy 2-[(1R,2R)methoxymethyloxo{[(lS)phenyl(1,3-thiazol yl)ethyl] amino } propyl]pyrrolidinyl} methyl- 1-oxoheptanyl] -N—methyl-L-valinamide (#49). To a solution of #48 (1.8 g, 52.1 mmol, 1 eq.) and diethyl cyanophosphonate (DEPC) (0.51 g, 3.2 mmol, 1.5 eq.) in 1,2-dimethoxyethane (30 mL, 0.07 M) at 0 0C was added triethylamine (1.47 mL, 10.6 mmol, 5 eq.). After stirring at room temperature for 2 hours, the mixture was concentrated in vacuo and the residue was d by silica gel chromatography (10% to 50% ethyl acetate in petroleum ether) to give #49 (0.8 g, 45%). R106 (10% ol in dichloromethane); LC-MS (Protocol K): m/z 881.3 [M+H+], 903.3 [M+Na+], retention time = 4.837 minutes.
Step 3. Synthesis of N—[(3R, 4S, 55)methoxy{(2S)[(1R,2R)methoxymethyl- 3-oxo {[(lS)phenyl- -thiazolyl)ethyl]amino}propyl]pyrrolidin— 1-yl}methyl oxoheptanyl]-N—methyl-L-valinamide (#50). ing to general procedure A, from #49 (0.70 g, 0.79 mmol, 1 eq.), dichloromethane (15 mL, 0.05 M) and diethylamine (10 mL) was synthesized #50 (160 mg, 30%) after purification by silica gel chromatography (Gradient: 0% to % ol in dichloromethane). Rf 0.4 (10% methanol in romethane); LC-MS (Protocol K): m/z 658.3 [M+H+], 680.3 [M+Na+], retention time = 2.760 minutes.
Step 4A. Synthesis ofN2-[(1-aminocyclopropyl)carbonyl]-N—[(3R, 4S, 5S)methoxy {(2S)[(1R,2R)methoxymethyloxo{[(lS)phenyl(1,3-thiazol yl)ethyl] amino } propyl] pyrrolidin— 1-yl} methyloxoheptanyl] hyl-L-valinamide (#51). To a solution of #50 (100 mg, 0.15 mmol, 1 eq.), bromotris(dimethylamino)phosphonium hexafluorophosphate (Brop, 70 mg, 0.18 mmol, 1.2 eq.) and diisopropylethylamine (0.08 mL, 0.45 mmol, 3 eq.) in dichloromethane ( 15 mL, 0.01 M) at 0 0C was added 1- aminocyclopropanecarboxylic acid (18 mg, 0.18 mmol, 1.2 eq.). After 2 hours, the mixture was quenched with water and extracted twice with ethyl acetate. The combined c layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 0% to 5% methanol in dichloromethane) to give #51 (45 mg, 34%). Rf 0.5 (10% methanol in dichloromethane). LC-MS col L): m/z 741.44 [M+H+]; 1H NMR (300 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.64 (br d, J=8 Hz) and 8.88 (br d, J=8 Hz), total 1H], [8.16 (br d, J=9 Hz) and 8.22 (br d, J=10 Hz), total 1H], [7.77 (d, J=3.5 Hz) and 7.79 (d, J=3.5 Hz), total 1H], [7.63 (d, J=3.5 Hz) and 7.65 (d, J=3 Hz), total 1H], .32 (m, 5H), .60 (m, 1H), 3.16, 3.20, 3.21 and 3.26 (4 s, total 6H), 2.93 and 3.02 (2 br s, total 3H), [1.05 (d, J=6.3 Hz) and 1.10 (d, J=6.3 Hz), total 3H].
Step 48. Synthesis of 1-amino-N—[(2S){[(3R,4S,5S)methoxy{(2S)[(1R,2R) methoxymethyl-3 -oxo-3 -{[(1S)phenyl(1,3 -thiazolyl)ethyl] amino } propyl]pyrrolidin- 1-yl} methyloxoheptan—4-yl](methyl)amino}-3 -methyloxobutan yl]cyclohexanecarboxamide (#52). To a solution of #50 (120 mg, 0.18 mmol, 1 eq.), Brop (84 mg, 0.21 mmol, 1.2 eq.) and diisopropylethylamine (0.1 mL, 0.54 mmol, 3 eq.) in dichloromethane (15 mL, 0.009 M) at 0 0C was added 1-aminocyclohexanecarboxylic acid (31 mg, 0.21 mmol, 1.2 eq.). After 2 hours, the mixture was quenched with water and extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 0% to % methanol in dichloromethane) to give #52 (50 mg, 35%). Rf 0.6 (10% methanol in dichloromethane). LC—MS (Protocol K): m/z 783.79 [M+H+]; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.64 (br d, J=8 Hz) and 8.87 (br d, J=9 Hz), total 1H], .28 (m, 1H), [7.77 (d, J=3.5 Hz) and 7.80 (d, J=3.3 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d, J=3.3 Hz), total 1H], 7.12-7.31 (m, 5H), 5.35-5.43 and 5.49- .57 (2 m, total 1H), [4.51 (dd, J=9, 8 Hz), and 4.61 (dd, J=9, 7 Hz), total 1H], 3.16, 3.19, 3.21 and 3.25 (4 s, total 6H), 2.93 and 3.02 (2 br s, total 3H), [1.05 (d, J=6.8 Hz) and 1.10 (d, J=6.8 Hz), total 3H]. ation of 2-Methylalanyl-N-[(3R,4S,5S)—3-meth0xy{(2S)—2-[(1R,2R)—1-meth0xy methyl0x0{ [(1 S)—2-phenyl(1 ,3-thiazolyl)ethyl] amino} propyl] idin-l-yl}-5— methyl0x0heptanyl]-N-methyl-L-valinamide (#54) #19, HATU, Et3N, , DMF FmocHN FmocHNKr 74% ON/E\\)OJ\N .\\(S\ / Et2NH, CH2CI2 75% #H2N>§(H/_; NH Step 1. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N- [(3R, 4S, 55)methoxy {(2S)[(1R, 2R)methoxymethyloxo {[( lS)phenyl (1,3 -thiazolyl)ethyl] amino } propyl]pyrrolidinyl} methyl- 1 ptanyl] -N—methyl-L- valinamide (#53). ing to general procedure D, from #32 (2.05 g, 2.83 mmol, 1 eq.) in dichloromethane (20 mL, 0.1 M) and N,N-dimethylformamide (3 mL), the amine #19 (2.5 g, 3.4 mmol, 1.2 eq.), HATU (1.29 g, 3.38 mmol, 1.2 eq.) and triethylamine (1.57 mL, 11.3 mmol, 4 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 55% acetone in heptane), producing #53 (2.42 g, 74%) as a solid. LC—MS: m/z 965.7 [M+H+], 987.6 [M+Na+], retention time = 1.04 minutes; HPLC (Protocol A): m/Z 965.4 [M+H+], retention time = 11.344 minutes (purity > 97%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, teristic signals: 8 7.86-7.91 (m, 2H), [7.77 (d, J=3.3 Hz) and 7.79 (d, J=3.2 Hz), total 1H], .74 (m, 2H), [7.63 (d, J=3.2 Hz) and 7.65 (d, J=3.2 Hz), total 1H], 7.38-7.44 (m, 2H), 7.30-7.36 (m, 2H), .30 (m, 5H), [5.39 (ddd, J=11.4, 8.4, 4.1 Hz) and 5.52 (ddd, J=11.7, 8.8, 4.2 Hz), total 1H], [4.49 (dd, J=8.6, 7.6 Hz) and 4.59 (dd, J=8.6, 6.8 Hz), total 1H], 3.13, 3.17, 3.18 and 3.24 (4 s, total 6H), 2.90 and 3.00 (2 br s, total 3H), 1.31 and 1.36 (2 br s, total 6H), [1.05 (d, J=6.7 Hz) and 1.09 (d, J=6.7 Hz), total 3H].
Step 2. Synthesis of 2-methylalanyl-N-[(3R, 4S, 55)methoxy {(2S)[(1R, 2R) methoxymethyl-3 -oxo-3 - {[(lS)phenyl(1,3 -thiazolyl)ethyl] amino}propyl]pyrrolidin- 1-yl} methyloxoheptan—4-yl]-N—methyl-L-valinamide (#54). According to general procedure A, from #53 (701 mg, 0.726 mmol) in dichloromethane (10 mL, 0.07 M) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane). The residue was d with diethyl ether and heptane and was trated in vacuo to afford #54 (406 mg, 75%) as a white solid. LC- MS: m/z 743.6 [M+H+], retention time = 0.70 minutes; HPLC (Protocol A): m/z 743.4 [M+H+], retention time = 6.903 minutes, (purity > 97%); 1H NMR (400 MHz, DMSO-dg), presumed to be a e of rotamers, characteristic signals: 8 [8.64 (br d, J:85 Hz) and 8.86 (br d, J:87 Hz), total 1H], [8.04 (br d, J=9.3 Hz) and 8.08 (br d, J=9.3 Hz), total 1H], [7.77 (d, J=3.3 Hz) and 7.80 (d, J=3.2 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d, J=3.2 Hz), total 1H], 7.13-7.31 (m, 5H), [5.39 (ddd, J=11, 8.5, 4 Hz) and 5.53 (ddd, J=12, 9, 4 Hz), total 1H], [4.49 (dd, J=9, 8 Hz) and 4.60 (dd, J=9, 7 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.93 and 3.02 (2 br s, total 3H), 1.21 (s, 3H), 1.13 and 1.13 (2 s, total 3H), [1.05 (d, J=6.7 Hz) and 1.10 (d, J=6.7 Hz), total 3H], 0.73-0.80 (m, 3H).
Preparation of 2-Methylalanyl-N-{(3R,4S,5S)methoxy[(2S)—2-{(1R,2R)—1-methoxy—2- methyloxo[(2-phenylethyl)amino]propyl} pyrrolidin-l-yl]methyloxoheptanyl}- N-methyl-L-valinamide, acetic acid salt (#56) #24, HATU, O I2_‘%Pr NEt CH CI2 FmOCHN><Ir JNjgfl/N N FmocHN>§TONQLN 69% E o /\ l o\ o #32 \ NH Et2NH CH2C|2 H2N><WN$I;W% 49% -OAcOH Step 1. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N- {(3R, 4S, 55)methoxy[(2S) {(1R,2R)methoxymethyl-3 - [(2- phenylethyl)amino]propyl}pyrrolidinyl]—5-methyloxoheptanyl}-N—methyl-L-valinamide (#55). To a solution of #24 (104 mg, 0.256 mmol, 1 eq.) in dichloromethane (10 mL, 0.094 M) were added #32 (156 mg, 0.256 mmol, 0.9 eq.), diisopropylethylamine (135 uL, 0.768 mmol, 3 eq.) and HATU (120 mg, 0.307 mmol, 1.2 eq.). After stirring for 18 hours, the mixture was concentrated in vacuo and the residue was diluted with ethyl acetate (10 mL), washed with 1 M aqueous hydrochloric acid solution (2 x 5 mL) and with brine, dried over sodium e, filtered, and trated in vacuo. The residue was diluted with dichloromethane and filtered. The filtrate was concentrated under reduced pressure onto silica and purified by silica gel tography (Gradient: 0% to 100% ethyl acetate in heptane) to give #55 (44 mg, 19%) as a white solid. LC—MS: m/Z 884.5 [M+2H+], retention time = 1.04 minutes.
Step 2. Synthesis of 2-methylalanyl-N- {(3R, 4S, 55)methoxy- 1- 2- {( 1R, 2R) methoxymethyl-3 -oxo-3 - [(2-phenylethyl)amino]propyl} pyrrolidin— 1-yl] methyl oxoheptanyl}-N—methyl-L-valinamide, acetic acid salt (#56). To a mixture of #55 (44 mg, 0.050 mmol, 1 eq.) in tetrahydrofuran (1 mL, 0.05 M) was added lamine (0.5 mL). After stirring for 18 hours, the reaction mixture was concentrated in vacuo and the residue was purified by reverse phase chromatography (Method B) to give #56 (16.2 mg, 49%) as a solid. LC—MS: m/z 660.8 [M+H+], retention time = 2.23 minutes; HPLC (Protocol A): m/z 660.5 [M+H+], 682.4 [M+Na+], retention time = 6.865 minutes.
Preparation of 2-Methylalanyl-N-[(3R,4S,5S)meth0xy—1-{(2S)[(1R,2R)meth0xy methyl0x0 { [(l-phenylcyclopropyl)methyl] amino} propyl] pyrrolidin—l-yl}methyl tanyl]-N-methyl-L-valinamide (#60) BocN HN #32, HATU, Et3N, #11 HATU Et3N NC LiAIH4, THF CHZCIZ DMF 4 M HCIIn dioxane CH2C|2, DMF O .HCI 72% 68% 97% \ NH 69% #5764 #58 64 >§rN\)Lj:;w H EtzNH CH2C|2 H2N><er\£)LNj/fi:w o /=\ l 83% o\ o #60 \o NéH Step 1. Synthesis of henylcyclopropyl)methanamine #@1. To a solution of 1- cyclopropanecarbonitrile (50 g, 0.34 mol, 1 eq.) in tetrahydrofuran (500 mL, 0.7 M) at 0 0C was added lithium aluminum hydride (23 g, 0.35 mol, 1.03 eq.). The reaction mixture was stirred at 0 0C for one hour and then at reflux for one hour. The reaction e was then cooled down and quenched with water (23 mL) and a 15% aqueous sodium hydroxide solution (69 mL).
The mixture was filtered and concentrated in vacuo to afford #@1 (36 g, 72%). LC-MS: m/z 148.1 , retention time = 0.86 minutes; 1H NMR (400 MHz, CDCl3) 8 7.2-7.4 (m, 5H), 2.78 (s, 2H), 1.19 (br s, 2H), 0.72-0.84 (m, 4H).
Step 2. sis of tert-butyl (2S)[(1R,2R)methoxymethyloxo{[(1- phenylcyclo propyl)methyl]amino}propyl]pyrrolidinecarboxylate (#57). According to general procedure D, from #11 (2.15 g, 7.48 mmol, 1.1 eq.) in dichloromethane (20 mL, 0.3 M) and MN- dimethylformamide (4 mL), 1-(1-phenylcyclopropyl)methanamine #@1 (1.001 g, 6.799 mmol, 1 eq.), HATU (3.10 g, 8.16 mmol, 1.2 eq.) and triethylamine (2.84 mL, 20.4 mmol, 3 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane), producing #57 (1.93 g, 68%) as a solid. HPLC (Protocol A at 45 0C): m/z 417.3 [M+H+], retention time = 10.575 minutes; 1H NMR (400 MHz, g), presumed to be a mixture of rotamers: 8 .81 (m, 1H), 7.20-7.27 (m, 4H), 7.12- 7.19 (m, 1H), 3.33-3.62 and 3.71-3.80 (br multiplets, total 4H), 3.28 (s, 3H), 2.97-3.17 (br m, 2H), 2.14-2.24 (m, 1H), 1.67-1.80 (br m, 2H), 1.45-1.65 (m, 2H), 1.41 (s, 9H), 1.00 (d, J=6.6 Hz, 3H), 0.67-0.93 (m, 4H).
Step 3. Synthesis of (2R,3R)methoxymethyl-N-[(1-phenylcyclopropyl)methyl] pyrrolidinyl]propanamide, hydrochloride salt (#58). ing to general procedure C, from #57 (566 mg, 1.36 mmol, 1 eq.) in e (4 mL, 0.3 M) and 4 M hloric acid solution in dioxane (4 mL, 16 mmol, 11.7 eq.) was synthesized #58 (466 mg, 97%); LC—MS: m/z 318.2 [M+H+], 339.2 [M+Na+], retention time = 0.56 minute; 1H NMR (400 MHz, DMSO-dg) 8 9.53 (br s, 1H), 8.48 (br s, 1H), 8.11 (br dd, J=5.7, 5.6 Hz, 1H), 7.23-7.30 (m, 4H), 7.14-7.21 (m, 1H), 3.58 (dd, J=7.5, 3.9 Hz, 1H), 3.50 (dd, J=13.7, 6.3 Hz, 1H), 3.34 (s, 3H), 3.21-3.29 (br m, 1H), 3.18 (dd, J=13.8, 5.0 Hz, 1H), 3.04-3.13 (br m, 2H), 2.42-2.50 (m, 1H), 1.56-1.89 (m, 4H), 1.04 (d, J=6.9 Hz, 3H), 0.71-0.91 (m, 4H).
Step 4. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N- [(3R, 4S, methoxy {(2S)[(1R, 2R)methoxymethyloxo {[( 1 - phenylcyclopropyl)methyl] amino} propyl]pyrrolidinyl} methyloxoheptanyl] -N— methyl-L-valinamide (#59). According to l procedure D, from #32 (550 mg, 0.902 mmol, 1 eq.), #58 (350 mg, 0.992 mmol, 1.1 eq.) dichloromethane (10 mL, 0.08 M) and MN- dimethylformamide (2 mL), HATU (446 mg, 1.17 mmol, 1.3 eq.) and triethylamine (0.503 mL, 3.61 mmol, 4 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 30% acetone in heptane), producing #59 (618 mg, 69%) as an off-white solid. LC-MS: m/z 908.7 [M+H+], 930.7 ], retention time = 1.07 minutes; HPLC (Protocol B at 45 0C): m/z 908.5 [M+H+], retention time = 8.721 minutes (purity > 97%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 7.89 (d, J=7.5 Hz, 2H), 7.38-7.44 (m, 2H), 7.30-7.36 (m, 2H), [4.49 (dd, J=8.5, 7.8 Hz) and 4.59 (dd, J=8.7, 6.9 Hz), total 1H], 4.18-4.26 (m, 3H), 3.93-4.01 (br m, 1H), 3.23 and 3.26 (2 s, total 3H), 3.16 and 3.16 (2 s, total 3H), 2.91 and 3.05 (2 br s, total 3H), 1.36 and 1.37 (2 br s, total 3H), 1.30 and 1.32 (2 br s, total 3H), [1.00 (d, J=6.7 Hz) and 1.02 (d, J=6.6 Hz), total 3H], 0.67- 0.78 (m, 7H).
Step 5. Synthesis of 2-methylalanyl-N-[(3R,4S, SS)methoxy{(2S)[(1R,2R) methoxymethyl-3 -oxo-3 - { [(1 -phenylcyclopropyl)methyl] amino } propyl]pyrrolidinyl} -5 - methyloxoheptanyl]-N—methyl-L-valinamide (#60). According to general procedure A, from #59 (605 mg, 0.666 mmol, 1 eq.) dichloromethane (10 mL, 0.067 M) and diethylamine (10 mL) was synthesized #60 (379 mg, 83%); HPLC (Protocol A at 45 0C) m/z 685.5 [M+H+], retention time = 7.072 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.03 (br d, J=9.6 Hz) and 8.07 (br d, J=9.4 Hz), total 1H], [7.74 (br dd, J=7, 4 Hz) and 7.99 (br dd, J=5.9, 5.7 Hz), total 1H], 7.20-7.27 (m, 4H), 7.11-7.17 (m, 1H), [4.49 (dd, J=9, 7 Hz) and 4.58 (dd, J=9, 7.5 Hz), total 1H], 3.96-4.04 (br m, 1H), 3.24 and 3.27 (2 s, total 3H), 3.18 and 3.19 (2 s, total 3H), 2.93 and 3.07 (2 br s, total 3H), 1.20 and 1.21 (2 s, total 3H), 1.12 and 1.14 (2 s, total 3H), [1.00 (d, J=6.7 Hz) and 1.03 (d, J=6.7 Hz), total 3H].
Preparation of 2-Methylalanyl-N-[(3R,4S,5S){(2S)[(1R,2R)—3-{[2-(cyclohepta-2,4,6- trien-l-yl)ethyl]amino}meth0xymethyl0X0pr0pyl]pyrrolidinyl}meth0xy methyl0x0heptanyl]-N-methyl-L-valinamide (#66) BocN e HNZ @ BF4 LDA THF CH3CN , EtZO #11, HATU o0c to rt Et3l\l, , DMF HC'In dioxane .73c NH —’ % 73% 59% quant #62 Oabm 65% O /=\ | s—’5% O\ O ‘0 Mb Step 1. Synthesis of cyclohepta-2,4,6-trienylacetonitrile (#61). To a solution of anhydrous itrile (3.12 mL, 56.2 mmol, 1 eq.) in tetrahydrofuran (281 mL, 0.2 M) was added lithium diisopropylamine (1.8 M in e/ ethylbenzene/ ydrofuran, 31.2 mL, 56.2 mmol, 1 eq.) at -78 0C. After 20 minutes at -78 0C, tropylium tetrafluoroborate (10 g, 56 mmol, 1 eq.) was added. After 10 minutes, the reaction was concentrated in vacuo and the residue was diluted with ethyl acetate and washed with water. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to provide a brown oil, which was purified by silica gel chromatography (Gradient: 0% to 10% ethyl acetate in heptane) to provide #61 (1.88 g, %) as a yellow oil. 1H NMR (400 MHz, CDCl3) 8 6.69-6.71 (m, 2H), 6.27-6.32 (m, 2H), 5.28- .33 (m, 2H), 2.61 (d, J=7.2 Hz, 2H), 2.26-2.34 (m, 1H).
Step 2. Synthesis of 2-(cyclohepta-2,4,6-trienyl)ethanamine (#62). To a suspension of lithium aluminum e (911 mg, 24.0 mmol, 1.4 eq.) in anhydrous diethyl ether (75 mL, 0.23 M) at 0 0C was slowly added, drop-wise over 15 minutes, a solution of #61 (2.25 g, 17.2 mmol, 1 eq.) in l ether (15 mL). The reaction was warmed to room temperature. After 5 hours, the reaction was cooled to 0 0C and ed by addition of water (1 mL), then filtered through a small pad of Celite and washed with methanol. The e was dried over sodium sulfate, d, and concentrated in vacuo to provide #62 (1.683 g, 73%) as a golden oil. LC-MS: m/z 136.1 [M+H+], retention time = 0.23 minutes; 1H NMR (400 MHz, CDCl3) 8 6.64-6.67 (m, 2H), .21 (m, 2H), 5.16-5.21 (m, 2H), 2.84-2.89 (m, 2H), 1.86-1.92 (m, 2H), 1.62-1.70 (m, 1H).
Step 3. Synthesis of tert-butyl (2S)[(1R,2R) {[2-(cyclohepta-2,4,6-trien yl)ethyl] amino} - 1 xymethyl-3 -oxopropyl]pyrrolidinecarboxylate (#63). To a solution of #11 (3.57 g, 12.4 mmol, 1 eq.) in dichloromethane (100 mL, 0.1 M) and MN- dimethylformamide (4 mL) was added HATU (5.36 g, 13.7 mmol, 1.1 eq.). After 20 s, triethylamine (5.20 mL, 37.3 mmol, 3 eq.) was added, followed by #62 (1.68 g, 12.4 mmol, 1 eq.), and the mixture was stirred for 18 hours. The on was concentrated in vacuo and the residue was taken up in ethyl acetate and washed with water (50 mL). The aqueous layer was back-extracted with ethyl e (3 times) and the combined c layers were dried, filtered, and concentrated in vacuo to provide a brown oil, which was purified by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) to provide #63 (2.95 g, 59% yield) as a viscous oil. LC—MS: m/z 405.4 [M+H+], 427.4 [M+Na+], retention time = 0.75 minutes; 1H NMR (400 MHz, CDCl3), presumed to be a mixture of rotamers: 8 6.63-6.68 (m, 2H), 6.16-6.23 (m, 2H), 5.19 (br dd, J=9.0, 5.8 Hz, 2H), 3.51-3.63 and 3.71-3.90 (2 br multiplets, total 3H), 3.42 (s, 3H), 3.18-3.29 and 3.34-3.47 (2 br multiplets, total 3H), 2.27-2.45 (br m, 1H), 1.6-2.00 (m, 7H), 1.47 and 1.50 (2 br s, total 9H), 1.16-1.29 (br m, 3H).
Step 4. Synthesis of (2R,3R)-N—[2-(cyclohepta-2,4,6-trienyl)ethyl]—3-methoxy methyl[(25)-pyrrolidinyl]propanamide, hydrochloride salt (#64) Intermediate #63 (400 mg, 0.989 mmol, 1 eq.) was treated with a 4 M solution of hydrochloric acid in dioxane (10 mL, 40 mmol, 40 eq.). After 1 hour, the reaction mixture was concentrated in vacuo and the residue was taken up in romethane and washed with 1 M sodium hydroxide solution. The aqueous layer was xtracted with dichloromethane and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to provide #64 (301 mg, quantitative) as a brown oil, which slowly began to solidify upon standing. LC—MS: m/z 305.3 [M+H+], retention time = 0.54 minutes; HPLC (Protocol G): retention time = 4.848 minutes; 1H NMR (400 MHz, CDCl3), characteristic signals: 8 6.64-6.67 (m, 2H), 6.16-6.22 (m, 2H), 6.08-6.14 (br m, 1H), 5.16-5.22 (m, 2H), 3.44 (s, 3H), 3.31 (dd, J=6.3, 4.5 Hz, 1H), 2.98-3.04 (m, 1H), 2.94 (ddd, J=10.5, 7.2, 5.6 Hz, 1H), 2.81 (ddd, J=10.5, 7.7, 6.7 Hz, 1H), 2.57 (qd, J=7.1, 4.5 Hz, 1H), 1.90-1.97 (m, 2H), 1.49-1.55 (m, 1H), 1.18 (d, WO 72813 J=7.1 Hz, 3H).
Step 5. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N- [(3R, 4S, 55) 1 - {(25) [(1R, 2R) yclohepta-2,4,6-trienyl)ethyl] amino} methoxy methyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxymethyloxoheptanyl]-N—methyl-L- valinamide (#65). According to general procedure D, from #32 (678 mg, 0.937 mmol, 1 eq.) in dichloromethane (9.37 mL, 0.1 M), the amine #64 (300 mg, 0.985 mmol, 1.1 eq.), HATU (427 mg, 1.12 mmol, 1.2 eq.) and diisopropylethylamine (494 uL, 2.81 mmol, 3 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 50% acetone in heptane), ing #65 (546 mg, 65%) as a solid. LC—MS: m/z 896.7 [M+H+], 918.7 [M+Na+], retention time = 1.06 minutes.
Step 6. Synthesis of 2-methylalanyl-N-[(3R, 4S, 55) {(2S)[(1R,2R) {[2-(cyclohepta- 2,4,6-trienyl) ethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxy-5 - methyloxoheptanyl]-N—methyl-L-valinamide (#66). To a solution of #65 (540 mg, 0.603 mmol, 1 eq.) in dichloromethane (10 mL, 0.06 M) was added ylamine (10 mL) and the reaction mixture was stirred for 2 hours. The mixture was concentrated in vacuo and the residue was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give #66 (347 mg, 85%) as a colorless solid. HPLC (Protocol A at 45 0C): m/z 674.5 , retention time = 7.015 minutes. 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rs, characteristic s: 8 [8.03 (br d, J=9 Hz) and 8.05 (br d, J=9 Hz), total 1H], [7.77 (br dd, J=5.5, 5.5 Hz) and 7.98 (br dd, J=5.5, 5.5 Hz), total 1H], 6.54-6.65 (m, 2H), 6.10-6.19 (m, 2H), 5.11-5.19 (m, 2H), [4.48 (dd, J=9, 8 Hz) and 4.54 (dd, J=9, 7.5 Hz), total 1H], 3.94- 4.04 (br m, 1H), 3.26 and 3.29 (2 s, total 3H), 3.17 and 3.19 (2 s, total 3H), 2.93 and 3.06 (2 br s, total 3H), 1.20 and 1.21 (2 s, total 3H), 1.12 and 1.13 (2 s, total 3H), [1.04 (d, J=6.8 Hz) and 1.07 (d, J=6.7 Hz), total 3H].
Preparation of 2-Methylalanyl-N-[(3R,4S,5S){(2S)—2-[(1R,2R){[(1S)—1-carb0xy-2— phenylethyl]amino}meth0xymethyl0x0pr0pyl]pyrrolidin-l-yl}meth0xy methyl0x0heptanyl]-N-methyl-L-valinamide (#69) and 2-methylalanyl-N-[(3R,4S,5S)- 3-meth0xy{(2S)—2-[(1R,2R)—1-methoxy{[(2S)—1-meth0xy0x0phenylpropan yl]amino}methyl0x0pr0pyl]pyrrolidinyl}methyl0x0heptanyl]-N-methyl-L- valinamide (#70) BocN 4 M HCI dloxane_ #32, HATU, iPerEt, CHZCIZ, DMF —> —> O .HCI 88% (2 steps) O \ NH 0 (\(0 V #37 bo\ #67 ©O\ LiOH, THF, H20 H2N><WN\=)LN\J;:\n/% 'CF3C02o /=\ 1 o\ o #59 N H 52% o i: O\ EtZNH,CH2C|2 o N¥JLTH N O /\ O\ O 65% o \ NH Step 1. Synthesis of methyl N—{(2R, 3R)methoxymethyl[(2S)-pyrrolidin yl]propanoyl}-L-phenylalaninate, hydrochloride salt (#67). ing to general procedure C, from #37 (2.39 g, 5.33 mmol, 1 eq.), dioxane (10 mL, 0.53 M) and a 4 M hydrochloric acid solution in dioxane (10 mL, 40 mmol, 7.5 eq.) was synthesized #67 (2.21 g) as a white solid, which was used in the next step without further purification. LC-MS: m/z 349.2 [M+H+], retention time = 0.53 minutes; 1H NMR (400 MHZ, DMSO-dg) 8 .58 (br m, 1H), 8.63 (d, J=8.1 Hz, 1H), 8.51-8.62 (br m, 1H), 7.25-7.33 (m, 4H), 7.18-7.25 (m, 1H), 4.50 (ddd, J=10.8, 8.1, 4.5 Hz, 1H), 3.65 (s, 3H), 3.54 (dd, J=6.8, 4.5 Hz, 1H), 3.20 (s, 3H), 3.11 (dd, J=13.8, 4.5 Hz, 1H), 2.99-3.14 (br m, 3H), 2.89 (dd, , 10.9 Hz, 1H), .50 (m, 1H, assumed; partially ed by solvent peak), 1.77-1.89 (m, 1H), 1.60-1.73 (m, 2H), .57 (m, 1H), 1.05 (d, J=6.8 Hz, 3H).
Step 2. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N- [(3R,4S,55)methoxy {(2S)[(1R,2R)methoxy {[(2S)methoxyoxo phenylpropanyl]amino} methyl-3 -oxopropyl]pyrrolidinyl}methyloxoheptanyl]— N—methyl-L-valinamide (#68). According to general procedure D, from #32 (353 mg, 0.488 mmol, 1 eq.) in dichloromethane (10 mL, 0.04 M), amine #67 (271 mg, 50.5 88 mmol, 1.3 eq.), HATU (223 mg, 0.586 mmol, 1.2 eq.) and diisopropylethylamine (238 “L, 1.71 mmol, 3.5 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 40% acetone in heptane), affording #68 (404 mg, 88% over two steps) as a solid. LC-MS: m/Z 940.7 [M+H+], 962.7 [M+Na+], retention time = 1.04 minutes; HPLC 2012/056224 (Protocol C): retention time = 9.022 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.25 (br d, J=8 Hz) and 8.48 (br d, J=8 Hz), total 1H], 7.89 (d, J=7.4 Hz, 2H), 7.67-7.75 (m, 2H), 7.38-7.44 (m, 2H), .36 (m, 2H), 7.14-7.24 (m, 5H), 4.43-4.69 (m, 3H), 4.17-4.26 (m, 3H), 3.91-3.99 (br m, 1H), 3.63 and 3.65 (2 s, total 3H), 3.19 and 3.24 (2 s, total 3H), 3.14 and 3.15 (2 s, total 3H), 2.90 and 2.99 (2 br s, total 3H), 1.36 and 1.37 (2 br s, total 3H), 1.30 and 1.32 (2 s, total 3H), [1.02 (d, J=6.8 Hz) and 1.06 (d, J=6.6 Hz), total 3H].
Step 3A. Synthesis of 2-methylalanyl-N-[(3R,4S,55) {(2S)[(1R,2R) {[(lS) carboxyphenylethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxy-5 - methyloxoheptanyl]-N—methyl-L-valinamide, trifluoroacetic acid salt (#69). To a on of #68 (143 mg, 0.152 mmol, 1 eq.) in ydrofuran (5 mL, 0.02 M) was added a solution of lithium ide (9.10 mg, 0.378 mmol, 2.5 eq.) in water (3 mL). After 5 hours, the reaction was concentrated in vacuo, azeotroped three times with heptane, dissolved in dimethyl sulfoxide (2.2 mL) and d by reverse phase chromatography (Method C) to give #69 (56 mg, 52%).
HPLC (Protocol A at 45 0C): 704.4 [M+H+], retention time = 6.623 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 8.08-8.22 and 8.37- 8.49 (2 m, total 5H), 7.12-7.28 (m, 5H), 3.18, 3.20 and 3.24 (3 s, total 6H), 2.95 and 3.04 (2 br s, total 3H), 1.52 and 1.53 (2 s, total 3H), 1.39 and 1.41 (2 s, total 3H), [1.02 (d, J=6.8 Hz) and 1.05 (d, J=6.6 Hz), total 3H], 0.74-0.81 (m, 3H).
Step 33. Synthesis of 2-methylalanyl-N-[(3R,4S,55)methoxy {(2S)[(1R,2R) methoxy{[(2S)methoxyoxophenylpropanyl]amino}methyl oxopropyl]pyrrolidinyl}methyloxoheptanyl]-N—methyl-L-valinamide (#70). ing to general procedure A, from #68 (240 mg, 0.255 mmol, 1 eq.), dichloromethane (10 mL, 0.026 M) and diethylamine (10 mL) was synthesized #70 (120 mg, 65%) as a white solid/glass mix after silica gel tography (Gradient: 0% to 10% methanol in dichloromethane). HPLC (Protocol A at 45 0C): m/Z 762.7 [M+H+], 740.4 [M+Na+], retention time = 6.903 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.26 (d, J=8.1 Hz) and 8.49 (d, J=8.3 Hz), total 1H], [8.03 (d, J=9.5 Hz) and 8.07 (d, J=9.5 Hz), total 1H], 7.14-7.27 (m, 5H), 3.63 and 3.67 (2 s, total 3H), 3.16, 3.18, 3.20 and 3.25 (4 s, total 6H), 2.92 and 3.01 (2 br s, total 3H), 1.20 and 1.22 (2 s, total 3H), 1.12 and 1.13 (2 s, total 3H), [1.02 (d, J=6.8 Hz) and 1.06 (d, J=6.7 Hz), total 3H], 0.74-0.80 (m, 3H).
Preparation of N2-[(3-Amin00xetanyl)carbonyl]-N-{(3R,4S,5S)—3-methoxy[(2S)—2- {(1R,2R)—1-meth0xymethyl0x0[(2-phenylethyl)amin0]propyl}pyrrolidinyl] methyl0X0heptanyl}-N-methyl-L-valinamide, acetic acid salt (#75) Boc20,aq .NaO,H O O H2Ng%( dioxane OH 38"/° BocHNgZKOH FmocHN\)LmOH #24 HATU iPr2NEt CH2CI2 DMF 81 % FmocHN/\:CJ)\n‘m(13%;thTHF H2N\)LmN%N\Hb650/ Dimer acid Fmoc:71;amer #@5 \b #75 0 “lb Step 1. Synthesis of 3-[(tert-butoxycarbonyl)amino]oxetanecarboxylic acid (#71). To 1-aminooxetanecarboxylic acid (1.00 g, 8.54 mmol, 1 eq.) in dioxane (15 mL, 0.5 M) was added a solution of sodium hydroxide (1.55 g, 38.7 mmol, 4.5 eq.) in water ( 15 mL) followed by t-butyl dicarbonate (2.09 g, 9.29 mmol, 1.1 eq.) A white solid formed. The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl e and washed with l M aqueous hloric acid solution and with brine. The c layer was dried over sodium sulfate, filtered, and concentrated in vacuo to give #71 (633 mg, 38%) as a white solid. 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers: 8 12.93 (br s, 1H), 7.59 and 7.93 (2 br s, total 1H), 4.71-4.78 (m, 2H), 4.47 (d, J=6.4 Hz, 2H), 1.30 and 1.38 (2 s, total 9H).
Step 2. Synthesis of N2-[(9H—fiuoren—9-ylmethoxy)carbonyl] -N—{(3R, methoxy [(2S) {(1R,2R)methoxymethyloxo-3 -[(2-phenylethyl)amino]propyl}pyrrolidinyl]— 5-methyloxoheptanyl}-N—methyl-L-valinamide (#72). To #@5 (9.47 g, 18.0 mmol, 1 eq.) and #24 (5.90 g, 18.0 mmol, 1 eq.) in romethane (250 mL, 0.072 M) were added diisopropylethylamine (9.52 mL, 54.2 mmol, 3 eq.) and HATU (8.49 g, 21.7 mmol, 1.2 eq.). The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (300 mL) and was washed with 1 M aqueous hydrochloric acid solution (2 x 100 mL) and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was taken up in dichloromethane (250 mL) and filtered. The filtrate was concentrated in vacuo onto silica and purified by silica gel chromatography ent: 0% to 50% acetone in heptane) to provide #72 (11.61 g, 81%) as a light yellow solid.
LC-MS: m/Z 797.6 [M+H+], 819.6 [M+Na+], retention time = 1.06 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 3.26 and 3.28 (2 s, total 3H), 3.18 and 3.20 (2 s, total 3H), 2.95 and 3.10 (2 br s, total 3H), 1.01-1.09 (m, 3H), 0.67- 0.78 (m, 3H).
Step 3. sis ofN— {(3R, 4S, methoxy- 1-[(2S) {( 1R, 2R)methoxymethyloxo-3 -[(2-phenylethyl)amino]propyl}pyrrolidinyl]—5-methyloxoheptanyl}-N—methyl- L-valinamide (#73). To #72 (5.16 g, 6.47 mmol, 1 eq.) in tetrahydrofuran (10 mL, 0.65 M) was added diethylamine (10 mL). After 2 hours, the reaction was concentrated in vacuo and the e was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give #73 (2414 mg, 65%). LC-MS: m/Z 576.5 [M+H+], retention time = 0.64 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 .88 and 7.99-8.10 (2 m, total 1H), 7.14-7.31 (m, 5H), 3.17 and 3.18 (2 s, total 3H), 2.87 and 3.03 (2 br s, total 3H), 1.02-1.08 (m, 3H).
Step 4. Synthesis of N2-( {3-[(tert-butoxycarbonyl)amino]oxetan—3-yl}carbonyl)-N- {(3R, 4S, SS)methoxy[(2S) {(1R,2R)methoxymethyl-3 -oxo [(2- phenylethyl)amino]propyl} pyrrolidinyl] methyloxoheptanyl} -N—methyl-L-valinamide (#74). To #73 (100 mg, 0.174 mmol, 1 eq.) in dichloromethane (4 mL, 0.04) and MN- dimethylformamide (0.5 mL) was added #71 (45.2 mg, 0.208 mmol, 1.2 eq.), followed by diisopropylethylamine (92 ”L, 0.521 mmol, 3 eq.) and HATU (102 mg, 0.260 mmol, 1.5 eq.).
After 16 hours, the reaction was trated in vacuo and the residue was taken up in ethyl e (6 mL) and washed with 1 M aqueous hydrochloric acid solution (2 x 2 mL) and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by reverse phase chromatography d C) to give #74 (140 mg), which was used in the next step without further purification. LC-MS: m/z 774.7 [M+H+], 796.6 [M+Na+], retention time = 0.91 minute.
Step 5. Synthesis of N2-[(3-aminooxetanyl)carbonyl]-N—{(3R,4S,5S)methoxy [(2S) {(1R,2R)methoxymethyloxo-3 -[(2-phenylethyl)amino]propyl}pyrrolidinyl]— -methyloxoheptanyl}-N—methyl-L-valinamide, acetic acid salt (#75). To #74 (140 mg, 50.181 mmol, 1 eq.) in romethane (3 mL, 0.06 M) was added trifluoroacetic acid (1 mL).
After 1 hour, the reaction was concentrated in vacuo and the residue was taken up in ethyl acetate (6 mL) and washed with saturated aqueous sodium bicarbonate solution (2 mL) and with brine.
The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Half of the crude material was d by reverse phase chromatography (Method B) to give #75 (16 mg, 26%, over two steps). LC—MS: m/z 674.6 [M+H+], retention time = 0.68 s; HPLC (Protocol A at 45 0C): m/z 674.5 [M+H+], retention time = 7.128 minutes; 1H NMR (400 MHz, DMSO-dg), ed to be a mixture of rotamers, characteristic signals: 8 7.80-7.87 and 8.02- 8.07 (2 m, total 2H), 7.23-7.30 (m, 2H), 7.14-7.22 (m, 3H), 4.28-4.33 (m, 2H), 3.96-4.04 (br m, 1H), 3.17 and 3.19 (2 s, total 3H), 2.96 and 3.10 (2 br s, total 3H), [1.04 (d, J=7.0 Hz) and 1.07 (d, J=6.6 Hz), total 3H].
Preparation of N,2—Dimethylalanyl-N-[(3R,4S,5S)—3-meth0xy—1-{(2S)[(1R,2R)—1- methoxy{[(2S)—1-meth0xy0X0phenylpr0panyl]amin0}methyl thioxopropyl]pyrrolidin-l-yl}methyl-l-oxoheptanyl]-N-methyl-L-valinamide (#79) and N,2-dimethylalanyl-N—[(3R,4S,5S){(2S)—2-[(1R,2R)—3-{[(1S)carb0xy phenylethyl]amin0}meth0xy—2-methylthi0x0propyl]pyrrolidin-l-yl}meth0xy methyl0x0heptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#80) chHNfifiYOH ' HCI HN HATU iPr2,NEl CH2CI2 FmoHN$R E—_I2NHTHF HN 0 Jn‘m 0\ 0 83 / /\ \ NH /:\ 0 o 0 H NH NH EtNH,THF bHNKrVLNpYOH 2 N u. I Fmocl><nxOH O /=\ I O\ O \ NH #79 S HATU iPr2NEIOCH2CI2 V0 —.MIKEHm o\ / (2 setes)p SN\H\(O‘2.
QO\ o . H LIOH, THF, H 0. HwflNx/HI N .CFco2H 58 % a o /\ o\ o Step 1. Synthesis of methyl N-{(2R,3R)[(2S){(3R,4S,55)[{N—[(9H—fluoren ylmethoxy) carbonyl] -L-Valyl} l)amino] -3 xymethylheptanoyl } pyrrolidinyl] - 3-methoxymethylpropanethioyl} -L-phenylalaninate (#76). According to general procedure D, from #@5 (260 mg, 0.648 mmol, 1 eq.), #39 (340 mg, 50.629 mmol, 1 eq.), dichloromethane (10 mL, 0.065 M), HATU (296 mg, 0.778 mmol, 1.2 eq.) and diisopropylethylamine (339 uL, 1.94 mmol, 3 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 40% acetone in e) to give #76 (466 mg, 83% over two steps) as a solid. LC-MS: m/z 871.5 [M+H+], 893.5 ], retention time = 1.10 minutes; HPLC col C): retention time = 9.249 minutes y > 99%); 1H NMR (400 MHz, DMSO- d5), presumed to be a mixture of rotamers, characteristic signals: 8 [10.19 (br d, J=7.4 Hz) and .49 (br d, J=7.8 Hz), total 1H], 7.89 (br d, J=7.4 Hz, 2H), 7.68-7.75 (m, 2H), 7.54-7.60 (m, 1H), 7.41 (br dd, J=7.4, 7.4 Hz, 2H), 7.28-7.36 (m, 2H), 7.15-7.28 (m, 5H), [5.20 (ddd, J=10.9, 7.3, 4.4 Hz) and 5.34-5.43 (m), total 1H], 3.65 and 3.69 (2 s, total 3H), 3.24 and 3.25 (2 s, total 3H), 3.17 (br s, 3H), 2.93 and 2.98 (2 br s, total 3H), [1.15 (d, J=6.6 Hz) and 1.18 (d, J=6.6 Hz), total 3H].
Step 2. Synthesis of methyl N— R)methoxy[(2S) {(3R,4S,55)methoxy methyl [methyl(L-valyl)amino]heptanoyl} pyrrolidinyl] methylpropanethioyl} -L- phenylalaninate (#77). According to l procedure A, from #76 (460 mg, 0.528 mmol, 1 eq.) tetrahydrofuran (8 mL, 0.07 M) and diethylamine (8 mL) was synthesized #77 (399 mg), which was used in the next step without further purification; LC-MS m/z 649.5 [M+H+], retention time = 0.73 s; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic product signals: 8 [10.20 (d, J=7.4 Hz) and 10.49 (d, J=7.4 Hz), total 1H], 7.15- 7.28 (m, 5H), [5.20 (ddd, J=10.9, 7.2, 4.5 Hz) and 5.34-5.42 (m), total 1H], 3.65 and 3.68 (2 s, total 3H), 3.24 and 3.25 (2 s, total 3H), 3.15 and 3.15 (2 s, total 3H), 2.83 and 2.88 (2 br s, total 3H), [1.15 (d, J=6.6 Hz) and 1.18 (d, J=6.6 Hz), total 3H].
Step 3. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N,2-dimethylalanyl-N— [(3R,4S,55)methoxy 2-[(1R,2R)methoxy methoxyoxo phenylpropanyl]amino} methyl-3 opropyl]pyrrolidin— 1-yl}methyloxoheptan yl]-N—methyl-L-valinamide (#78). According to general procedure D, from #77 (399 mg, £0.52 mmol, 1 eq.), N—[(9H—fluorenylmethoxy)carbonyl]-N,2-dimethylalanine (213 mg, 0.628 mmol, 1.2 eq.), dichloromethane (5 mL, 0.1 M), HATU (239 mg, 0.628 mmol, 1.2 eq.) and diisopropylethylamine (282 uL, 1.62 mmol, 3.1 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 50% acetone in heptane), providing #78 (231 mg, 46% over two steps). LC—MS: m/z 970.7 , 992.6 [M+Na+], retention time = 1.11 minutes; HPLC (Protocol C): retention time = 9.260 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.19 (d, J=7.4 Hz) and 10.47 (d, J=7.8 Hz), total 1H], 7.89 (d, J=7.4 Hz, 2H), 7.61-7.67 (m, 2H), 7.41 (br dd, J=7.4, 7.4 Hz, 2H), 7.14-7.36 (m, 8H), [5.20 (ddd, J=11, 7, 5 Hz) and 5.38 (ddd, J=11, 8, 4 Hz), total 1H], [4.41 (dd, J=8.6, 8.4 Hz) and 4.46 (dd, J=8.2, 8.2 Hz), total 1H], 3.65 and 3.68 (2 s, total 3H), 3.23 and 3.24 (2 s, total 3H), 3.13 (br s, 3H), 2.88 and 2.93 (2 br s, total 3H), 2.84 and 2.85 (2 s, total 3H), 1.31 and 1.32 (2 s, total 3H), [1.15 (d, J=6.6 Hz) and 1.18 (d, J=6.4 Hz), total 3H].
Step 4A. Synthesis ofN,2-dimethylalanyl-N—[(3R,4S,55)methoxy{(2S)[(1R,2R)- 1-methoxy-3 - {[(2S)- 1-methoxyoxo-3 -phenylpropanyl] amino} hylthioxopropyl] pyrrolidin-l-yl} methyloxoheptanyl]-N—methyl-L-valinamide (#79). According to general procedure A, from #78 (223 mg, 0.230 mmol, 1 eq.), dichloromethane (6 mL, 0.04 M) and diethylamine (6 mL) was synthesized #79 (146 mg, 85%) as a white solid after silica gel chromatography (Gradient: 0% to 5% methanol in heptane then 0% to 10% methanol in dichloromethane). HPLC (Protocol A at 45 0C): 749.4 [M+H+], retention time = 7.315 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rs, characteristic signals: 8 [10.20 (d, J=7.6 Hz) and 10.50 (d, J=8.0 Hz), total 1H], 7.79-7.88 (m, 1H), 7.15-7.29 (m, 5H), [5.20 (ddd, J=11, 7, 4 Hz) and 5.38 (ddd, J=11, 8, 4 Hz), total 1H], [4.50 (dd, J=8.8, 8.6 Hz) and 4.56 (dd, J=9, 8 Hz), total 1H], 3.65 and 3.69 (2 s, total 3H), 3.24 and 3.25 (2 s, total 3H), 3.16 (br s, 3H), 2.93 and 2.97 (2 br s, total 3H), 2.10 and 2.11 (2 s, total 3H).
Step 43. Synthesis of methylalanyl-N—[(3R,4S,55){(2S)[( 1R,2R){[(lS) carboxyphenylethyl]amino} methoxymethyl-3 -thioxopropyl]pyrrolidinyl} -3 - methoxymethyloxoheptan—4-yl]-N—methyl-L-valinamide, trifluoroacetic acid salt (#80). To a solution of #78 (170 mg, 0.175 mmol, 1 eq.) in tetrahydrofuran (3 mL, 0.04 M) was added a solution of m hydroxide (12.6 mg, 0.525 mmol, 3 eq.) in water (1.5 mL). After stirring overnight, the solvent was d in vacuo. The residue was azeotroped three times with heptane. The residue was then diluted with dimethyl sulfoxide (2.2 mL) and purified by reverse phase chromatography (Method C) to afford #80 (74 mg, 58%) as a solid. LC-MS: m/z 734.6 [M+H+], retention time = 0.69 minutes; HPLC (Protocol A at 45 0C): 734.4 , retention time = 6.903 minutes y > 96%); 1H NMR (400 MHz, DMSO-dg), presumed to be a e of rotamers, characteristic signals: 8 12.9 and 13.1 (2 v br s, total 1H), [10.12 (d, J=7.4 Hz) and .46 (d, J=7.8 Hz), total 1H], 8.77-8.89 (br m, 2H), [8.47 (d, J=8.6 Hz) and 8.51 (d, J=8.6 Hz), total 1H], 7.21-7.29 (m, 4H), 7.14-7.21 (m, 1H), [5.16-5.23 (m) and 5.38 (ddd, J=11.3, 8.2, 3.9 Hz), total 1H], [4.51 (dd, J=9.0, 9.0 Hz) and 4.57 (dd, J=9.4, 8.6 Hz), total 1H], 3.24 and 3.24 (2 s, total 3H), 3.18 and 3.19 (2 s, total 3H), 2.96 and 3.00 (2 br s, total 3H), 1.51 and 1.53 (2 s, total 3H), 1.40 and 1.42 (2 s, total 3H), 1.14-1.19 (m, 3H), 0.74-0.81 (m, 3H).
Preparation of N,2—Dimethylalanyl-N-[(3R,4S,5S)—3-meth0xy—1-{(2S)[(1R,2R)—1- methoxy-Z-methyl{[(1S)—2-phenyl(1,3-thiazolyl)ethyl]amino} thioxopropyl]pyrrolidin-l-yl}methyl0x0heptanyl]-N-methyl-L-valinamide (#84) FmocHNng/OH HATU, g, o DMF,CH2C|2 N EtZNH,DMF + FmocHN\)J\N 58% E O\ O | /\ /\ CK 0 o .CF3COZH o DImer acid' \ #81 \ NH NH S #18 s,.\\(S e.\\(‘ / i I] urjfm o New“ NWLRNOSHFm°° 0 Fmoc i o /\ l $pw o\o E12NH DMF HATU iPrNE; 95% #82 NH #33 NH #84 CHQCOIZ NH Step 1. Synthesis of N2-[(9H—fluoren—9-ylmethoxy)carbonyl] -N—[(3R,4S,55)methoxy {(2S)[(1R,2R)methoxymethyl phenyl(1 azolyl)ethyl] amino} thioxopropyl]pyrrolidinyl} methyloxoheptanyl]-N—methyl-L-valinamide (#81).
According to general procedure D, from #@5 (620 mg, 1.18 mmol, 1 eq.) dichloromethane (10 mL, 0.1 M), amine #18 (604 mg, 1.42 mmol, 1.2 eq.), diisopropylethylamine (618 HL, 3.54 mmol, 3 eq.) and HATU (539 mg, 1.42 mmol, 1.2 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 30% acetone in heptane) to give #81 (737 mg, 58%). HPLC (Protocol C): retention time = 9.235 s; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.54 (br (1, J=8 Hz) and 10.81 (br (1, J=8 Hz), total 1H], 7.89 (d, J=7.6 Hz, 2H), [7.80 (d, J=3.3 Hz) and 7.83 (d, J=3.1 Hz), total 1H], 7.70-7.75 (m, 2H), [7.64 (d, J=3.1 Hz) and 7.68 (d, J=3.3 Hz), total 1H], 7.55-7.60 (m, 1H), 7.38-7.44 (m, 2H), .35 (m, 7H), [6.31 (ddd, J=11, 8, 4.5 Hz) and .48 (m), total 1H], 3.23 and 3.24 (2 s, total 3H), 3.17 and 3.22 (2 s, total 3H), 2.94 and 3.01 (2 br s, total 3H), [1.14 (d, J=6.4 Hz) and 1.17 (d, J=6.2 Hz), total 3H].
Step 2. Synthesis of N—[(3R,4S,55)methoxy {(2S)[(1R,2R)- 1-methoxymethyl {[(lS)phenyl(1,3 -thiazolyl)ethyl] amino} -3 -thioxopropyl]pyrrolidinyl}methyl oxoheptanyl]-N—methyl-L-valinamide (#82). According to general procedure A, from #81 (733 mg, 0.818 mmol, 1 eq.) in dichloromethane (7 mL, 0.1 M) and diethylamine (7 mL) was synthesized #82 (670 mg), which was used in the next step without further purification. LC—MS: m/z 674.5 [M+H+], retention time = 1.29 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic product signals: 8 [10.55 (br d, J=8 Hz) and 10.84 (br d, J=8 Hz), total 1H], [7.64 (d, J=3.1 Hz) and 7.69 (d, J=3.3 Hz), total 1H], .33 (m, 5H), 6.27-6.35 and 6.38-6.47 (2 m, total 1H), 3.23 and 3.25 (2 s, total 3H), 3.15 and 3.19 (2 s, total 3H), 2.84 and 2.91 (2 br s, total 3H).
Step 3. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N,2-dimethylalanyl-N— [(3R,4S,55)methoxy {(2S)[(1R,2R)methoxymethyl phenyl(1,3- thiazolyl)ethyl] amino} -3 -thioxopropyl]pyrrolidinyl}methyl- 1-oxoheptanyl] -N— methyl-L-valinamide (#83). ing to general procedure D, from #82 (670 mg, 50.818 mmol, 1 eq.), dichloromethane (5 mL, 0.16 M), N-[(9H—fluorenylmethoxy)carbonyl]-N,2- dimethylalanine (304 mg, 0.896 mmol, 1.1 eq.), HATU (372 mg, 0.978 mmol, 1.2 eq.) and diisopropylethylamine (440 uL, 2.53 mmol, 3.1 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 30% acetone in heptane) to give #83 (556 mg, 69% over two steps). LC—MS: m/z 994.7 [M+H+], retention time = 0.69 minutes; HPLC (Protocol C): retention time = 9.333 minutes (purity > 98%); 1H NMR (400 MHz, DMSO-dg), ed to be a mixture of rotamers, characteristic signals: 8 [10.53 (br d, J=8 Hz) and 10.80 (br d, J=8 Hz), total 1H], .91 (m, 2H), [7.80 (d, J=3.3 Hz) and 7.82 (d, J=3.2 Hz), total 1H], [7.64 (d, J=3.2 Hz) and 7.68 (d, J=3.2 Hz), total 1H], .66 (m, 2H), 7.38-7.44 (m, 2H), 7.28-7.36 (m, 5H), .26 (m, 2H), 7.12-7.17 (m, 1H), [6.31 (ddd, J=11, 8, 4.5 Hz) and 6.44 (ddd, J=11, 8.5, 4.5 Hz), total 1H], [4.42 (dd, J=9, 8 Hz) and 4.48 (dd, J=8, 8 Hz), total 1H], 3.22 and 3.24 (2 s, total 3H), 3.13 and 3.17 (2 s, total 3H), 2.89 and 2.97 (2 br s, total 3H), 2.84 and 2.85 (2 s, total 3H), [1.13 (d, J=6.4 Hz) and 1.16 (d, J=6.4 Hz), total 3H].
Step 4. Synthesis of N,2-dimethylalanyl-N—[(3R,4S,55)methoxy{(2S)[( methoxymethyl {[( lS)phenyl( 1 ,3-thiazolyl)ethyl] amino} propyl]pyrrolidinyl} methyloxoheptanyl]-N—methyl-L-valinamide (#84).
According to general procedure A, from #83 (552 mg, 0.555 mmol, 1 eq.) in dichloromethane (10 mL, 0.05 M) and diethylamine (10 mL) was sized the crude desired material, which was diluted with methanol, concentrated in vacuo onto silica, and purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give #84 (406 mg, 95%) as a white solid. LC—MS: m/z 772.8 [M+H+], retention time = 1.35 minutes; HPLC (Protocol A): 774.4 [M+H+], retention time = 7.390 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [10.54 (br (1, J=8 Hz) and 10.81 (br (1, J=8 Hz), total 1H], 7.78-7.84 (m, 2H), [7.65 (d, J=3.1 Hz) and 7.69 (d, J=3.3 Hz), total 1H], 7.29-7.34 (m, 2H), 7.20-7.28 (m, 2H), 7.14-7.19 (m, 1H), 6.27-6.35 and 6.40-6.48 (2 m, total 1H), [4.51 (dd, J=9, 8 Hz) and 4.57 (dd, J=9, 8 Hz), total 1H], 3.24 and 3.25 (2 s, total 3H), 3.16 and 3.21 (2 s, total 3H), 2.94 and 3.00 (2 br s, total 3H), 2.09 and 2.10 (2 s, total 3H), 1.08 and 1.09 (2 s, total 3H), 0.73-0.80 (m, 3H).
N,2-Dimethylalanyl—N-[(3R,4S,5S)meth0xy{(2S)—2-[(1R,2R)—1-meth0xymethyl 0X0{[(1S)—2—phenyl(1,3-thiazolyl)ethyl]amin0}propyl]pyrrolidin-l-yl}methyl oxoheptanyl]-N-methyl-L-valinamide (#88) 2012/056224 HATU iPerEt FmocHNE/lOLmOH .CF (:0 H3 2 CH Cl E NH THFt2 2 2 + H%N9—’:I\© FmocHN/E\\)Lmqgwr \=\/N I O 8°56“ —22-TFA, CH CI “db H N N quantitative H'i‘ é o /\ [ll o\ o o\ o m s \N Step 1. Synthesis of N2-[(9H—fluoren—9-ylmethoxy)carbonyl] -N—[(3R,4S,55)methoxy {(2S)[(1R,2R)methoxymethyloxo{[(lS)phenyl(1,3-thiazolyl)ethyl]amino} propyl] pyrrolidinyl}methyloxoheptanyl]-N—methyl-L-valinamide (#85). To a mixture of#@5 (5.48 g, 10.4 mmol, 1 eq.) and #19 (3.90 g, 10.4 mmol, 1 eq.) in dichloromethane (50 mL, 0.2M) was added diisopropylethylamine (5.51 mL, 31.3 mmol, 3 eq.) followed by HATU (4.91 g, 12.5 mmol, 1.2 eq.). After stirring overnight, the reaction mixture was concentrated in vacuo. The residue was taken up in ethyl acetate (100 mL) and washed with 1 M s hydrochloric acid solution (2 x 30 mL) and with brine (30 mL). The organic layer was dried over sodium e, filtered, and concentrated in vacuo. The residue was taken up in dichloromethane and filtered; the filtrate was purified by silica gel chromatography (Gradient; 0% to 50% acetone in e) to afford #85 (7.20 g, 78%) as a solid. LC—MS: m/z 880.6 [M+H+], retention time = 1.07 minutes.
Step 2. Synthesis of N—[(3R,4S,55)methoxy {(2S)[(1R,2R)- 1 xymethyl- 3-oxo {[(lS)phenyl- 1-(1,3-thiazolyl)ethyl]amino}propyl]pyrrolidin— 1-yl}methyl oxoheptanyl]-N—methyl-L-valinamide (#86). According to general procedure A, from #85 (5.00 g, 5.68 mmol, 1 eq.) in tetrahydrofuran (10 mL, 0.56 M) and lamine (3 mL) was synthesized the crude desired al, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give #86 (2.952 g, 79%) as a solid. LC- MS: m/Z 658.5 [M+H+], 680.5 ] retention time = 0.66 minute; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.64 (br d, J=8.4 Hz) and 8.90 (br d, J=8.8 Hz), total 1H], [7.77 (d, J=3.3 Hz) and 7.80 (d, J=3.3 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d, J=3.3 Hz), total 1H], 7.12-7.31 (m, 5H), [5.39 (ddd, J=11.2, 8.4, 4.2 Hz) and 5.54 (ddd, J=11.9, 8.9, 4.0 Hz), total 1H], 3.15, 3.19, 3.20 and 3.26 (4 s, total 6H), 2.86 and 2.98 (2 br s, total 3H), [1.06 (d, J=6.6 Hz) and 1.11 (d, J=6.6 Hz), total 3H].
Step 3. Synthesis of N—(tert-butoxycarbonyl)-N,2-dimethylalanyl-N—[(3R,4S,55) methoxy-l- {(2S)[(1R,2R)methoxymethyloxo {[(lS)phenyl(1,3-thiazol yl)ethyl] amino} propyl]pyrrolidinyl}methyloxoheptanyl]-N—methyl-L-valinamide (#87). To a mixture of #86 (80.3 mg, 0.122 mmol, 1 eq.) in dichloromethane (4 mL, 0.03 M) was added N—(tert—butoxycarbonyl)-N,2-dimethylalanine (29.1 mg, 0.134 mmol, 1.1 eq.) followed by diisopropylethylamine (64 uL, 0.365 mmol, 3 eq.) and HATU (71.7 mg, 0.183 mmol, 1.5 eq.) After stirring overnight, the reaction mixture was concentrated in vacuo. The residue was taken up in ethyl acetate (6 mL) and washed with 1 M s hydrochloric acid solution (2 x 2 mL) and with brine. The organic solvent was dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was taken up in dichloromethane and filtered; the filtrate was concentrated in vacuo onto silica and d by silica gel chromatography (Gradient: 0% to 50% acetone in heptane) to afford #87 (58 mg, 50%) as a white solid. LC-MS: m/z 857.7 [M+H+], 879.7 [M+Na+], retention time = 0.99 minute; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.64 (br d, J=8 Hz) and 8.87 (br d, J=9 Hz), total 1H], [7.77 (d, J=3.3 Hz) and 7.80 (d, J=3.3 Hz), total 1H], [7.63 (d, J=3.2 Hz) and 7.66 (d, J=3.2 Hz), total 1H], 7.13-7.31 (m, 5H), [6.95 (br d, J=8 Hz) and 7.06 (br d, J=8 Hz), total 1H], 5.35-5.42 and 5.51-5.58 (2 m, total 1H), 3.15, 3.19, 3.20 and 3.26 (4 s, total 6H), 2.94 and 3.03 (2 br s, total 3H), 2.83 and 2.84 (2 s, total 3H), [1.05 (d, J=6.7 Hz) and 1.11 (d, J=6.7 Hz), total 3H].
Step 4. sis of N,2-dimethylalanyl-N—[(3R,4S,55)methoxy{(2S)[( 1R,2R) methoxymethyl-3 -oxo-3 - {[(lS)phenyl(1,3 -thiazolyl)ethyl] amino}propyl]pyrrolidin- 1-yl}methyloxoheptan—4-yl]-N—methyl-L-valinamide (#88). To a mixture of #87 (58 mg, 0.068 mmol, 1 eq.) in dichloromethane (8 mL) was added trifluoroacetic acid (2 mL). After stirring overnight, the reaction mixture was concentrated in vacuo. The residue was taken up in ethyl acetate (10 mL), washed with ted s sodium bicarbonate solution, dried over sodium sulfate, filtered, and concentrated in vacuo to give #88 (52 mg, quantitative). LC-MS 757.6 [M+H+], retention time = 0. 69 ; 1H NMR (400 MHz, g), presumed to be a mixture of rs, characteristic signals: 8 [8.64 (br d, J=8.6 Hz) and 8.87 (br d, J:86 Hz), total 1H], 7.80-7.85 (m, 1H), [7.77 (d, J=3.3 Hz) and 7.80 (d, J=3.1 Hz), total 1H], [7.63 (d, J=3.1 Hz) and 7.66 (d, J=3.3 Hz), total 1H], 7.20-7.31 (m, 4H), 7.13-7.19 (m, 1H), [5.39 (ddd, J=11, 8.5, 4 Hz) and 5.49-5.56 (m), total 1H], [4.51 (dd, J=9, 8 Hz) and 4.61 (dd, J=9, 8 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.94 and 3.03 (2 br s, total 3H), 2.10 and 2.10 (2 s, total 3H), 1.16 (br s, 3H), 1.04-1.12 (m, 6H), 0.72-0.80 (m, 3H).
Preparation of Amin0-2,2-dimethylpr0pan0yl)—N-[(3R,4S,5S)meth0xy{(2S)—2- [(1R,2R)—1-meth0xymethyl0x0{[(1S)—2-phenyl(1,3-thiazol yl)ethyl] amin0}pr0pyl] pyrrolidin-l-yl}methyl-l-oxoheptanyl]-N-methyl-L- valinamide, trifluoroacetic acid salt (#95) .HCI o FmocCl. iperEt 0 #86, HATU, iPrgNEt, 0 HzN/XKOHM» H CH Cl DMF , N N 45% />€J\OH 22—17» 60A) Fm°°HN\><n/N\_/lLNi O l O O O O /\ \ \ S \N #93 \=/ 'CF3002H H H 720/° : I 0 /\ o\ o o\ o s \N Step 1. Synthesis of 3-{[(9H-fluorenylmethoxy)carbonyl]amino}-2,2- dimethylpropanoic acid (#93). To 3-amino-2,2-dimethylpropanoic acid, hydrochloride salt (250 mg, 1.63 mmol, 1 eq.) in dichloromethane (4 mL, 0.4 M) was added ropylethylamine (859 uL, 4.88 mmol, 3 eq.) followed by (9H—fluorenylmethoxy)carbonyl chloride (473 mg, 1.79 mmol, 1.1 eq.) The reaction was stirred for 18 hours and then trated in vacuo. The residue was taken up in ethyl acetate (3 mL) and washed with 1 M aqueous hydrochloric acid solution (2 x 1 mL) and with brine. The organic layer was dried over sodium e, filtered, and ed by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) to give #93 (250 mg, 45%) as an oil. 1H NMR (400 MHz, DMSO-dg) 8 12.22 (s, 1H), 7.89 (d, J=7.4 Hz, 2H), 7.72 (d, J=7.4 Hz, 2H), 7.38-7.44 (m, 2H), .35 (m, 3H), 4.18-4.30 (m, 3H), 3.16 (d, J=6.2 Hz, 2H), 1.05 (s, 6H).
Step 2. Synthesis of NZ-(3- {[(9H—fluorenylmethoxy)carbonyl]amino} -2,2- dimethylpropanoyl)-N— [(3R,4S,55)-3 -methoxy {(2S)[(1R,2R)methoxymethyloxo-3 - {[(lS)phenyl(1,3 -thiazolyl)ethyl] amino } propyl]pyrrolidinyl} methyloxoheptan— 4-yl]-N—methyl-L-valinamide (#94). To #86 (100 mg, 0.152 mmol, 1 eq.) in dichloromethane (4 mL, 0.038 M) and N,N—dimethylformamide (0.5 mL) was added #93 (51.6 mg, 0.152 mmol, 1 eq.) followed by diisopropylethylamine (80.0 uL, 0.457 mmol, 3 eq.) and HATU (89.8 mg, 0.229 mmol, 1.5 eq.). The reaction was d for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (6 mL) and was washed with 1 M aqueous hydrochloric acid solution (2 x 2 mL) and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was taken up in dichloromethane (250 mL) and filtered; the filtrate was concentrated in vacuo onto silica and purified by silica gel chromatography (Gradient: 0% to 50% acetone in heptane) to provide #94 (90 mg, 60%) as a white solid. LC-MS: m/Z 979.8 [M+H+], 1002.7 ], retention time = 1.15 minutes; 1H NMR (400 MHz, DMSO- d5), presumed to be a mixture of rotamers, characteristic t signals: 8 [8.64 (br (1, J:86 Hz) and 8.86 (br d, J=8.6 Hz), total 1H], 7.86-7.91 (m, 2H), [7.77 (d, J=3.3 Hz) and 7.79 (d, J=3.3 Hz), total 1H], 7.67-7.73 (m, 2H), [7.63 (d, J=3.3 Hz) and 7.65 (d, J=3.3 Hz), total 1H], 6.87- 6.95 (m, 1H), [5.39 (ddd, J=11, 8, 4 Hz) and 5.52 (ddd, J=11.5, 9, 4 Hz), total 1H], [4.44 (dd, J=8.4, 8.4 Hz) and 4.55 (dd, J=8.4, 8.4 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.96 and 3.06 (2 br s, total 3H), 0.69-0.77 (m, 3H).
Step 3. sis of amino-2,2-dimethylpropanoyl)-N—[(3R,4S,55)methoxy {(2S)[(1R,2R)methoxymethyloxo{[(lS)phenyl(1,3-thiazol yl)ethyl]amino}propyl] pyrrolidin— 1-yl} methyloxoheptanyl] -N—methyl-L-valinamide, trifluoroacetic acid salt (#95). To #94 (86 mg, 0.088 mmol, 1 eq.) in tetrahydrofuran (2 mL, 0.04 M) was added diethylamine (10 mL). After stirring overnight, the reaction was trated in vacuo and the residue was purified by reverse phase chromatography (Method C) to give #95 (55 mg, 72%). LC-MS: m/Z 757.5 [M+H+], retention time = 0.74 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [8.66 (br d, J=8 Hz) and 8.92 (br d, J=9 Hz), total 1H], [7.91 (br d, J=8 Hz) and 7.97 (br d, J=9 Hz), total 1H], [7.78 (d, J=3.3 Hz) and 7.81 (d, J=3.1 Hz), total 1H], 7.65-7.74 (br m, 3H), [7.63 (d, J=3.3 Hz) and 7.67 (d, J=3.3 Hz), total 1H], 7.12-7.31 (m, 5H), [5.35-5.42 (m) and 5.45-5.52 (m), total 1H], [4.44 (dd, J=9, 9 Hz) and 4.55 (dd, J=9, 9 Hz), total 1H], 3.17, 3.20, 3.22 and 3.25 (4 s, total 6H), 2.96 and 3.05 (2 br s, total 3H), 1.25 and 1.25 (2 s, total 3H), 1.14 and 1.15 (2 s, total 3H), [1.06 (d, J=6.6 Hz) and 1.10 (d, J=6.4 Hz), total 3H], 0.72-0.80 (m, 3H).
Preparation of Nz-(3-Amin0-2,2-dimethylpr0pan0yl)—N-{(3R,4S,5S)—3-methoxy[(2S)—2- {(1R,2R)—1-meth0xymethyl0X0[(2-phenylethyl)amin0]propyl}pyrrolidinyl] 0X0heptanyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#97) HATU iPr2NEt o FmocHN/>€L H CH2CI2 DMF N + HZN/EfiN 65% FmocHNQflrNEJLN= | #93 \ NH EtzNH THF H2N 68% MAR 'OCF3C02H Step 1. Synthesis ofN-(3- {[(9H—fluorenylmethoxy)carbonyl]amino}-2,2- dimethylpropanoyl)-N— {(3R,4S,55)-3 -methoxy[(2S) {(1R,2R)methoxymethyl-3 -oxo-3 - [(2-phenylethyl)amino]propyl}pyrrolidinyl] hyl- 1-oxoheptanyl} -N—methyl-L- mide (#96). To #73 (100 mg, 0.174 mmol, 1 eq.) in dichloromethane (4 mL, 0.04 M) and N,N—dimethylformamide (0.5 mL) was added #93 (59.1 mg, 0.174 mmol, 1 eq.), followed by diisopropylethylamine (92 uL, 0.52 mmol, 3 eq.) and HATU (102 mg, 0.260 mmol, 1.5 eq.).
The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (6 mL) and was washed with 1 M aqueous hydrochloric acid solution (2 x 2 mL) and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0% to 50% acetone in e) provided #96 (102 mg, 65%) as a white solid. LC-MS: m/z 896.7 [M+H+], 918.8 [M+Na+], retention time = 1.14 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rs, characteristic t signals: 8 7.88 (d, J=7.4 Hz, 2H), [7.83 (br dd, J=6, 5 Hz) and 8.03 (br dd, J=6, 5 Hz), total 1H], 7.67-7.73 (m, 2H), 7.36-7.48 (m, 3H), 7.22-7.35 (m, 4H), 7.13-7.21 (m, 3H), 6.86-6.96 (m, 1H), [4.44 (dd, J=8.6, 8.6 Hz) and 4.50 (dd, J=8.6, 8.6 Hz), total 1H], 3.18, 3.19, 3.26 and 3.29 (4 s, total 6H), 2.96 and 3.11 (2 br s, total 3H), 0.70-0.77 (m, 3H).
Step 2. Synthesis of NZ-(3-amino-2,2-dimethylpropanoyl)-N— {(3R,4S,55)methoxy [(2S) {(1R,2R)methoxymethyloxo-3 -[(2-phenylethyl)amino]propyl}pyrrolidinyl]— -methyloxoheptanyl}-N—methyl-L-valinamide, trifluoroacetic acid salt (#97). To #96 (98 mg, 0.11 mmol, 1 eq.) in tetrahydrofuran (2 mL, 0.04 M) was added diethylamine (0.5 mL).
After stirring overnight, the reaction was concentrated in vacuo and the residue was purified by reverse phase chromatography (Method C) to give #97 (58 mg, 68%). LC-MS: m/z 674.4 [M+H+], 696.4 [M+Na+], ion time = 0.74 minutes; HPLC (Protocol A): 674.5 [M+H+], retention time = 7.072 minutes; 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [7.92 (br d, J=8 Hz) and 7.97 (br d, J=8 Hz), total 1H], [7.86 (br dd, J=6, 5 Hz) and 8.07 (br dd, J=6, 5 Hz), total 1H], 7.64-7.74 (br m, 3H), 7.15-7.29 (m, 5H), [4.44 (dd, J=9, 9 Hz) and 4.50 (dd, J=9, 9 Hz), total 1H], 3.26 and 3.29 (2 s, total 3H), 3.18 and 3.20 (2 s, total 3H), 2.96 and 3.10 (2 br s, total 3H), 1.24 and 1.25 (2 s, total 3H), 1.14 and 1.16 (2 s, total 3H), 1.02-1.07 (m, 3H), 0.73-0.80 (m, 3H).
Preparation of 2-methyl-L-prolyl-N-[(3R,4S,5S)—3-meth0xy{(2S)[(1R,2R)—1-meth0xy- 2-methyl0X0{ [(1 S)phenyl(1 ,3-thiazolyl)ethyl] amino} ] pyrrolidin-l-yl} 0x0heptanyl]-N-methyl-L-vaLinamide, trifluoroacetic acid salt (#98) N ’1, OH HATU iPerEt CH2C|2, H2N\)LN then TFA -CF3C02HO(kw/iii», o\_/s_\N To a mixture 1-(tert-butoxycarbonyl)methyl-L-proline (65.1 mg, 0.284 mmol, 1.1 eq.) and #86 (170 mg, 0.258 mmol, 1 eq.) in dichloromethane (5 mL, 0.03 M) was added HATU (0.108 mg, 0.284 mmol, 1.1 eq.) followed by diisopropylethylamine (139 uL, 0.800 mmol, 3.1 eq.). After stirring overnight, the reaction mixture was cooled to 0 0C, dichloromethane (3 mL) was added followed by the slow addition of trifluoroacetic acid (2 mL). The reaction mixture was stirred at 0 0C for 5 minutes, allowed to warm to room temperature and then stirred at room temperature for 30 minutes before being concentrated in vacuo. The residue was azeotroped two times with heptane, diluted with a small amount of dichloromethane and methanol before being concentrated in vacuo onto silica The residue was purified by silica gel tography (Gradient: 0% to 10% methanol in romethane) and then by e phase chromatography d C) to afford #98 (128 mg, 56%) as a white solid. LC—MS: m/z 769.4 [M+H+], retention time = 1.28 minutes; HPLC (Protocol A at 45 0C) m/z 769.4 [M+H+], retention time = 7.146 minutes (purity > 98%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 9.03-9.15 (br m, 1H), 8.77-8.86 (br m, 1H), 8.69-8.76 (m, 1H), [8.66 (d, J=8.2 Hz) and 8.92 (d, J=8.6 Hz), total 1H], [7.78 (d, J=3.1 Hz) and 7.80 (d, J=3.5 Hz), total H], [7.63 (d, J=3.1 Hz) and 7.67 (d, J=3.1 Hz), total 1H], 7.12-7.31 (m, 5H), [5.38 (ddd, J=11, 8, 4 Hz) and 5.47 (ddd, J=11, 9, 4 Hz), total 1H], [4.46 (dd, J=9.4, 9.0 Hz) and 4.55 (dd, J=9.0, 8.6 Hz), total 1H], 3.17, 3.20, 3.22 and 3.25 (4 s, total 6H), 2.98 and 3.04 (2 br s, total 3H), [1.06 (d, J=7.0 Hz) and 1.09 (d, J=6.6 Hz), total 3H], 0.73-0.80 (m, 3H).
Preparation of methyl bicyclo[4.2.0]octa—l,3,5-trien-7—yl)acetate, hloride salt (#102) H N H || 0 0 Br HN + Na, ElOH O/\ 1N NaOH MeOH A0 01 I 017%(2 steps) OHC -HC| H2N / 6N HCI SOCI2MeOH 84% (2 steps) #100 #101 #102 Step 1. sis of ethyl (acetylamino)(bicyclo[4.2.0]octa-1,3,5-trienyl)cyanoacetate (#99). Sodium (464 mg, 20.2 mmol, 1.2 eq.) was allowed to react with te ethanol (40 mL, 0.42 M); to the resulting mixture was added ethyl 2-(acetylamino)cyanoacetate (3.44 g, 20.2 mmol, 1.2 eq.). After 20 minutes at 60 0C, 7-bromobicyclo[4.2.0]octa-1,3,5-triene (3.092 g, 16.89 mmol, 1 eq.) was added and the reaction mixture was heated at reflux overnight, then filtered and concentrated in vacuo. The residue was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to give a dark oil, which was d by silica gel chromatography ent: 0% to 50% ethyl acetate in heptane) to give #99 (4.38 g) as a yellow gum. LC-MS: m/z 273.2 [M+H+], retention time = 2.36 minutes.
Step 2. Synthesis of (acetylamino)(bicyclo[4.2.0]octa-1,3,5-trienyl)acetic acid (#100).
To a mixture of #99 (4.38 mg, <16.1 mmol, 1 eq.) in methanol (30 mL, 0.53 M) was added a 1 N aqueous solution of sodium hydroxide (38 mL, 38 mmol, 2.4 eq.). The reaction mixture was heated at reflux overnight, then concentrated in vacuo, diluted with water (40 mL), and ed with a 1 N aqueous solution of hydrochloric acid (40 mL). The aqueous layer was extracted with dichloromethane (3 x 30 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The resulting oil was purified by silica gel chromatography (Solvent A: romethane; Solvent B: 20% methanol in romethane ning 0.02% trifluoroacetic acid; Gradient: 0% to 40% B) then by supercritical fluid chromatography (Column: Chiralpak AD-H, 250 x 21 mm; Eluent: 85: 15 carbon dioxide/methanol; Flow Rate: 65 g/min; Detection: 210 nm; Instrument: Berger minigram preparative SFC system.). The second eluting peak was isolated to give #100 (600 mg, 17% over two steps) as a single enantiomer (retention time = 3.37 minutes, purity >99%). LC-MS: m/z 220.3 [M+H+], retention time = 2.10 minutes; 1H NMR (400 MHz, CD30D) 8 7.14-7.24 (m, 2H), 7.03-7.09 (m, 2H), 4.59 (d, J=8.6 Hz, 1H), 3.87 (ddd, J=8.5, 5.3, 2.4 Hz, 1H), 3.35 (dd, J=14.5, 5.4 Hz, 1H, assumed; partially obscured by solvent peak), 3.10 (dd, J=14.4, 2.4 Hz, 1H), 2.00 (s, 3H). Optical on: [@1325 +70.9°(c 0.67, methanol) Step 3. Synthesis of amino(bicyclo[4.2.0]octa-1,3,5-trienyl)acetic acid, hydrochloride salt . A e of #100 (200 mg, 0.912 mmol, 1 eq.) and 6 N aqueous hydrochloric acid (12.3 mL, 73.8 mmol, 81 eq.) was heated at reflux overnight. The reaction mixture was concentrated in vacuo to give the single enantiomer #101 (195 mg) as an off-yellow solid, which was used in the next step without further purification.
Step 4. Synthesis of methyl amino(bicyclo[4.2.0]octa-1,3,5-trienyl)acetate, hydrochloride salt (#102). To a mixture of #101 ( 195 mg, <0.913 mmol, 1 eq.) in methanol (20 mL, 0.04 M) was added thionyl chloride (0.666 mL, 9.13 mmol, 10 eq.). After two hours at reflux, the reaction e was concentrated in vacuo to give the single enantiomer #102 (175 mg, 84% over two steps) as a light-colored solid. LC-MS: m/z 192.3 [M+H+], retention time = 0.80 minutes; GC—MS: m/Z 192 [M+H+], retention time = 3.206 minutes; 1H NMR (400 MHz, CD3OD) 8 7.24-7.33 (m, 2H), 7.11-7.18 (m, 2H), 4.40 (d, J=6.9 Hz, 1H), 3.99-4.05 (m, 1H), 3.78 (s, 3H), 3.46 (dd, J=14.8, 5.4 Hz, 1H), 3.23 (dd, J=14.8, 2.5 Hz, 1H).
Preparation of (2R,3R)—3-Meth0xymethyl[(2S)-pyrrolidinyl]propanoic acid, hydrochloride salt (#103) methoxycyclo MOH pentane,4N HCI in e OH Boc 31% H o\ o o\ o -HC| #11 #103 To a mixture of #11 (4.09 g, 14.2 mmol, 1 eq.) in cyclopentyl methyl ether (10 mL, 0.14 M) was added a 4 N solution of hydrogen chloride in dioxane (37 mL, 100 mmol, 7 eq.). After three hours, the reaction mixture was trated in vacuo and azeotroped three times with heptane to give #103 (1000 mg, 31%) as a gum, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-dg) 8 992-1006 (br s, 1H), 8.66-8.80 (br s, 1H), 3.89 (dd, J=5.2, 4.9 Hz, 1H), 3.43-3.53 (m, 1H), 3.39 (s, 3H), 3.06-3.17 (m, 2H), 2.66 (qd, J=7.1, 4.6 Hz, 1H), 1.71-2.03 (m, 4H), 1.11 (d, J=7.1 Hz, 3H).
Preparation of 2-Methylalanyl-N—[(3R,4S,5S){(2S)—2-[(1R,2R){[1-(bicyclo[4.2.0]0cta- 1,3,5-trien-7—yl)meth0xy0x0ethyl]amino}meth0xymethyl pyl]pyrrolidin-l-yl}meth0xymethyl0x0heptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#107) and 2-methylalanyl—N—[(3R,4S,5S){(2S)—2-[(1R,2R) {[bicyclo ]0cta-1 ,3,5—trien-7—yl(carb0xy)methyl] amino}meth0xymethyl pyl]pyrrolidin-l-yl}meth0xymethyl0x0heptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#108) #102, HATU, FCT:PYK_.FmocHN><frfiN\/1LF¢YOF DCM_. FmocHtXrNgLN#32 DCM F 0 F #103 ,prgNEt H (MO iPrZNEt,DCM, H o /\ 99% (zsteps) o /=\ | o\ o O\ 0 764’ o\ o F F #104 mos EtZNH DCM H2N>§rNQLWLE 73/0 -CF3C)O2H #107 Step 1. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N— [(3R,4S,5S)-3 -methoxymethyloxo(pentafluorophenoxy)heptanyl]-N—methyl-L- valinamide (#104). To #32 (4.00 g, 6.56 mmol, 1 eq.) in dichloromethane (20 mL, 0.33 M) and pyridine (1.06 mL, 13.1 mmol, 2 eq.) was added drop-wise pentafluorophenyl trifluoroacetate (2.25 mL, 13.1 mmol, 2 eq.). The reaction mixture was stirred for one hour.
To a second flask containing #32 (360 mg, 0.59 mmol) in dichloromethane (0.6 mL, 1 M) and pyridine (0.095 mL, 1.2 mmol, 2 eq.) was added drop-wise pentafluorophenyl trifluoroacetate (0.203 mL, 1.18 mmol). This reaction mixture was stirred for 15 minutes.
The two reaction mixtures were combined, washed twice with 1 N s hydrochloric acid, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting yellow oil was dissolved in ethyl acetate, pre-adsorbed onto silica gel and purified by silica gel chromatography (Gradient: 0% to 40% ethyl acetate in heptane) to give #104 (4.6 g, 83%) as a white foam containing some impurities. LC-MS: m/z 798.3 [M+Na+], retention time = 1.23 minutes.
Step 2. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N— [(3R,4S,55) {(2S)[(1R,2R)carboxymethoxypropyl]pyrrolidinyl}methoxy methyloxoheptanyl]-N—methyl-L-valinamide (#105). To a mixture of #104 (2.00 g, <2.58 mmol, 1 eq.) in dichloromethane (6 mL, 0.4 M) was added a solution of #103 (483 mg, 2.16 mmol, 1 eq.) in dichloromethane (2 mL) followed by diisopropylethylamine (1.35 mL, 7.73 mmol, 3 eq.). The reaction mixture was stirred for 16 hours, then adsorbed onto silica and purified by silica gel tography ent: 0% to 20% methanol in dichloromethane) to give #105 (1.67 g, 83%) as a white foam. Fractions containing the desired t with impurities (0.571 g) were collected separately.
The above reaction and purification were repeated in a similar fashion using #104 (2.60 g, <3.35 mmol, 1 eq.), #103 (750 mg, 3.35 mmol, 1 eq.), romethane (10 mL, 0.3 M) and diisopropylethylamine ( 1.35 mL, 7.73 mmol, 2.3 eq.) to give #105 (2.4 g, 92%) as a tan foam.
Fractions containing impure product (1.7 g) were ed with the previous impure fractions and purified as described above to afford onal #105 (1.30 g, tative yield for both reactions over two steps). LC—MS: m/z 779.3 [M+H+], 802.3 [M+Na+], retention time = 1.05 minutes.
Step 3. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N— [(3R,4S,55) 2-[(1R,2R) {[1-(bicyclo[4.2.0]octa-1,3,5-trienyl)methoxy oxoethyl]amino } - 1-methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxy-5 -methyl oxoheptanyl]-N—methyl-L-valinamide . To a mixture of #105 (225 mg, 0.289 mmol, 1 eq.) in dichloromethane (15 mL, 0.02 M) and methylformamide (1 mL) was added HATU (136 mg, 0.347 mmol, 1.2 eq.). After five minutes, a solution of amine #102 (72.4 mg, 0.318 2012/056224 mmol, 1 eq.) and diisopropylamine (203 uL, 1.16 mmol, 3 eq.) in dichloromethane (5 mL) was added. After 24 hours, the reaction mixture was washed with brine, dried over sodium sulfate, filtered, concentrated in vacuo onto silica gel, and purified by silica gel chromatography (Gradient: 0% to 50% acetone in e) to give the single enantiomer #106 (210 mg, 76%) as a clear oil. LC—MS: m/z 953.1 [M+H+], retention time = 3.99 s; 1H NMR (400 MHz, CDCl3), presumed to be a mixture of rotamers, characteristic signals: 7.76 (d, J=7.5 Hz, 2H), 7.57-7.64 (m, 2H), 7.40 (dd, J=7.5, 7.4 Hz, 2H), 7.28-7.34 (m, 2H), 4.82-4.88 (m, 1H), 3.95-4.01 (m, 1H), 3.76 and 3.82 (2 s, total 3H), 3.30, 3.31, 3.34 and 3.35 (4 s, total 6H), [1.20 (d, J=7.0 Hz) and 1.20 (d, J=7.0 Hz), total 3H].
Step 4A. Synthesis of 2-methylalanyl-N—[(3R,4S,55){(2S)[(1R,2R)-3 - {[1- lo[4.2.0] octa- 1 ,3,5-trienyl)methoxyoxoethyl] amino} - 1-methoxymethyl-3 - oxopropyl]pyrrolidinyl}-3 -methoxymethyloxoheptanyl]-N—methyl-L-valinamide, trifluoroacetic acid salt (#107). According to general procedure A, from #106 (25 mg, 0.026 mmol, 1 eq.) in dichloromethane (10 mL, 0.003 M) and diethylamine (4 mL) was synthesized the crude d material, which was purified by reverse phase chromatography (Method C) to give the single enantiomer #107 (16 mg, 73%) as a solid. LC-MS: m/z 730.8 [M+H+], retention time = 2.13 minutes; HPLC (Protocol N): retention time = 9.889 minutes.
Step 43. Synthesis of 2-methylalanyl-N—[(3R,4S,55) 2-[(1R,2R) {[bicyclo[4.2.0]octa-1,3 ,5-trienyl(carboxy)methyl] amino} - 1-methoxymethyl oxopropyl]pyrrolidinyl}-3 -methoxymethyloxoheptanyl]-N—methyl-L-valinamide, trifluoroacetic acid salt (#108). The single enantiomer #108 (94.5 mg, 57%) was synthesized from #106 (190 mg, 0.200 mmol) according to a ure similar to the one described for synthesis of #41 from #40. LC-MS: m/z 716.8 , retention time = 2.06 minutes; HPLC (Protocol N): retention time = 9.137 minutes.
Preparation of 2-methylalanyl-N-[(3R,4S,5S)—1-{(2S)—2-[(1R,2R){[(1S,2R)hydr0xy phenylpropan-Z-yl]amino}methoxy-Z-methyl0X0propyl]pyrrolidin-l-yl}meth0xy methyl0x0heptanyl]-N-methyl-L-valinamide (#112) HATU Et3N 4M HCIm #32 HATU Eth 500” 20H DCM DMF dioxane DCM DMF + 74% 34% 95% o OH N-HHCF3002 #109 #1100 FmocHN>§rN\:)LNj;/\\g:ooH H EtZNH DCM N \./U\N o | 51% o _ I #111 #112 (‘3 Step 1. Synthesis of tert-butyl (2S)[(1R,2R) {[(1S,2R)hydroxyphenylpropan—2- yl]amino}methoxymethyloxopropyl]pyrrolidinecarboxylate (#109). To a solution of #11 (2.00 g, 6.96 mmol, 1 eq.) in dichloromethane (21 mL, 0.3 M) and N,N—dimethylformamide (3 mL) was added HATU (3270 mg, 8.35 mmol, 1.2 eq.). After two minutes, the amine (1R,2S)- (+)-norephedrine (1.07 mg, 6.96 mmol, 1 eq.) and triethylamine (1.94 mL, 13.9 mmol, 2 eq.) were added. After two hours, the reaction mixture was diluted with ethyl acetate (100 mL), washed with a 1 M aqueous solution of hloric acid and with brine, dried over sodium sulfate, filtered, concentrated in vacuo, and purified by silica gel chromatography (Gradient: 0% to 60% ethyl acetate in e) to provide #109 (2.18 g, 74%) as a white solid. LC-MS: m/z 321.3 [(M = 3.14 minutes; 1H NMR (400 MHz, DMSO-dg), presumed - Boc)+H+], retention time to be a mixture of rs, characteristic signals: 8 7.64 (d, J=8.6 Hz, 1H), 7.24-7.33 (m, 4H), .21 (m, 1H), 5.35 (br (1, J=5 Hz, 1H), 4.45 (br dd, J=5, 5 Hz, 1H), .00 (m, 1H), 3.30- 3.39 (m, 1H), 3.26 (s, 3H), 2.94-3.07 (m, 1H), 2.04-2.14 (m, 1H), 1.46-1.78 (m, 4H), 1.40 (s, 9H), 0.97-1.04 (m, 6H).
Step 2. sis of (2R,3R)-N—[(1S,2R)hydroxyphenylpropanyl]methoxy methyl[(25)-pyrrolidinyl]propanamide, trifluoroacetic acid salt (#110). According to general procedure C, at 0 0C from #109 (414 mg, 0.984 mmol, 1 eq.), dioxane (5 mL, 0.2 M) and a 4 M solution of hydrogen chloride in dioxane (15 mL, 60 mmol, 60 eq.) was synthesized the crude desired compound, which was purified by reverse phase chromatography (Method C) to give #110 (120 mg, 34%) as a viscous . LC-MS: m/z 321.1 [M+H+], retention time = 0.55 minutes; 1H NMR (400 MHz, DMSO-dg), characteristic signals: 8 7.90 (d, J=8.6 Hz, 1H), 7.28- 7.36 (m, 4H), 7.20-7.27 (m, 1H), 4.46 (d, J=6.2 Hz, 1H), 3.48 (dd, J=8.6, 2.3 Hz, 1H), 3.38 (s, 3H), 2.92-3.16 (m, 3H), 2.24-2.35 (m, 1H), 1.49-1.88 (m, 4H), 1.09 (d, J=6.6 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H).
Step 3. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N— [(3R,4S,55) {(2S)[( 1R,2R) {[( 1S,2R)- oxyphenylpropanyl] amino} - 1-methoxy- 2-methyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxymethyloxoheptan—4-yl]-N—methyl-L- valinamide (#111). According to general procedure D, from #32 (140 mg, 0.230 mmol, 1 eq.), #110 (110 mg, 0.253 mmol, 1.1 eq.), dichloromethane (3 mL, 0.08 M), N,N—dimethylformamide (0.5 mL), HATU (96.2 mg, 0.253 mmol, 1.1 eq) and triethylamine (96 uL, 0.69 mmol, 3 eq.) was synthesized the crude desired product, which was purified by silica gel chromatography (Gradient: 0% to 40% acetone in e) to give #111 (220 mg, 95%). LC-MS: m/z 912.4 [M+H+], 935.4 [M+Na+], retention time = 2.15 minutes; HPLC (Protocol B): m/z 912.5 [M+H+], 934.5 [M+Na+], retention time = 10.138 minutes (purity >94%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, teristic signals: 8 7.89 (d, J=7.8 Hz, 2H), 7.66-7.75 (m, 2H), 7.41 (dd, J=7.4, 7.4 Hz, 2H), 7.12-7.20 (m, 1H), [5.33 (d, J=4.7 Hz) and 5.38 (d, J=4.7 Hz), total 1H], 3.15, 3.18, 3.22 and 3.23 (4 s, total 6H), 1.30, 1.33, 1.36 and 1.39 (4 s, total 6H), 0.95-1.06 (m, 6H).
Step 4. Synthesis of 2-methylalanyl-N—[(3R,4S,55){(2S)[(1R,2R) {[(1S,2R) hydroxy- 1-phenylpropanyl] amino} methoxymethyloxopropyl]pyrrolidin— 1-yl} methoxymethyloxoheptan—4-yl]-N—methyl-L-valinamide (#112). According to general procedure A, from #111 (210 mg, 0.230 mmol) in romethane (5 mL, 0.05 M) and diethylamine (5 mL) was synthesized the crude desired al, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give a e of an oil and solid. Diethyl ether and heptane were added and the e was trated in vacuo, ing #112 (81 mg, 51%) as a white solid. LC-MS: m/z 690.4 [M+H+], retention time = 1.10 minutes; HPLC (Protocol A): m/z 690.5 [M+H+], 712.4 [M+Na+], retention time = 7.229 minutes (purity > 90%); 1H NMR (400 MHz, DMSO-dg), presumed to be a mixture of rotamers, characteristic signals: 8 [7.62 (br d, J=8 Hz), 7.88 (br d, J=8 Hz), 8.07 (br d, J=9 Hz) and 8.11 (br d, J=9 Hz), total 2H], 7.15-7.34 (m, 5H), [5.34 (d, J=4 Hz) and 5.41 (d, J=5 Hz), total 1H], 3.18, 3.21, 3.23 and 3.25 (4 s, total 6H), 2.93 and 3.08 (2 br s, total 3H), 1.15, 1.18, 1.21 and 1.25 (4 s, total 6H). ation of N,2-dimethylalanyl-N-{(1S,2R)—4-{(2S)—2-[(1R,2R){[(1S)—1-carb0xy phenylethyl]amin0}meth0xymethylox0propyl]pyrrolidin-l-yl}meth0xy[(1S)— l-methylpropyl]0X0butyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (115).
HATU Hunig'8 base CHZCIZ EtzNH CHZCIZ Fmoc’H/\\)J\N Fmoc dimer acid#5 111113000©067 HzN/él/(liN 1. N- fluoren-y-|methoxy)carbonyl]N2-d—imethylalanine HATU Hunig'8 base CHZCIZ 2 LiOH THF water HNKPKOrN/EiolxN #114 ROIEEEO— #115 Step 1. Synthesis of methyl N-{(2R,3R)[(2S){(3R,4S,5S)[{N- [(9H-fluoren ylmethoxy)carbonyl]-L-Valyl} (methyl)amino]methoxymethylheptanoyl}pyrrolidinyl] methoxymethylpropanoyl}-L-phenylalaninate (#113). To a stirring mixture of dimer acid#5 (12. 1 g, 23.0 mM) and #67 (11.5 g, 23.0 mM) in 75 mL of dichloromethane under nitrogen, HATU (10.8 g, 27.6 mM) was added followed by Hunig’s base (12.1 mL, 69.0 mM). The reaction was d to stir at room temperature for 15 hours. Reaction was concentrated to a smaller volume, taken up with ethyl acetate and washed with 1 N HCl two times. The organic layer was then washed with brine, dried over sodium sulfate, d, and concentrated in vacuo.
Residue was then purified by silica gel tography (Gradient: 0% to 70% e in heptanes), producing #113 (12.3 g, 62%) as a white solid. LC-MS (Protocol Q): m/z 855.3 [M+H+], 877.2 [M+Na+], retention time = 2.32 minutes; HPLC (Protocol R): /Z 855.5 [M+H+], retention time = 9.596 minutes y > 97%).
Step 2. Synthesis of methyl N-{(2R,3R)methoxy[(2S){(3R,4S,5S)methoxy methyl [methyl(L-valyl)amino]heptanoyl} pyrrolidinyl] methylpropanoyl} -L- phenylalaninate (#114). According to general procedure A, from #113 (12 g, 14 mmol, 1 eq.), dichloromethane (60 mL, 0.24 M) and diethylamine (40 mL, 390 mM) was synthesized #114 (5.9 g, 67%) white/slight yellow solid after ation by silica gel chromatography (Gradient: 0% to 25% methanol in dichloromethane). LC-MS (Protocal Q): m/z 633.0 [M+H+], retention time = 1.19 minutes. HPLC (Protocol A): /2 633.5 [M+H+], ion time = 7.142 minutes (purity > 98%).
Step 3. Synthesis ofN,2-dimethylalanyl-N-{(1S,2R){(2S)[(1R,2R){[(1S) carboxyphenylethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy [(1 S)methylpropyl]oxobutyl}-N-methyl-L-valinamide-trifiuoroacetic acid salt (#1 15). To a stirring mixture of N—[(9H-fluorenylmethoxy)carbonyl]-N,2-dimethylalanine (167 mg, 0.493 mM), #114 (260 mg, 0.411 mM), and HATU (188 mg, 0.493 mM) in 10 mL of dichloromethane, Hunig’s base (0.14 mL, 0.82 mM) was added. The reaction was d to stir at room ature for 1 hour and 20 minutes. Reaction was d down. THF (9 mL) was added to crude material and to this stirring e lithium hydroxide (49.2 mg, 2.06 mM) dissolved in 3 mL of water was added. The reaction was allowed to stir at room temperature for 4 hours.
Reaction was concentrated down followed by purification by medium pressure reverse phase C18 chromatography (Gradient: 5% to 45% water in acetonitrile with 0.02% TFA in each phase) #115 (218 mg, 64%) white solid. LC—MS (Protocol Q): m/Z 718.7 [M+H+], 740.6 [M+Na+], retention time = 1.21 minutes. HPLC (Protocol A at 45 oC): m/z 718.4 [M+H+], retention time = 6.903 minutes. 1H NMR (400 MHz, DMSO-dg), 8 8.81-8.95 (m), .50 (m), 8.42 (d), 8.15 (d), 7.14-7.28 (m), 4.71-4.78 (m), 4.57-4.66 (m), 4.49-4.56 (m), 4.41-4.48 (m), 3.94-4.05 (m), 3.72- 3.79 (m), .60 (m), 2.95-3.33 (m), 2.78-2.89 (m), 2.69 (s), 2.43-2.50 (m), 2.08-2.42 (m), 1.60-1.92 (m), 1.20-1.57 (m), 0.84-1.11 (m), 0.74-0.83 (m).
Preparation of 2-methyl-L-prolyl-N-[(3R,4S,5S)—3-meth0xy{(2S)[(1R,2R)—1-meth0xy- 3-{ [(2S)—1-meth0xy0x0phenylpr0panyl] methyl-3 oxopropyl] pyrrolidin-l- yl}methyl0x0heptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#117) and 2-methyl-L-prolyl-N-[(3R,4S,5S)—1-{(2S)—2-[(1R,2R)—3-{[(1S)—1-carb0xy-2— phenylethyl]amino}meth0xymethyl0x0pr0pyl]pyrrolidin-l-yl}meth0xy methyl0x0heptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#118).
..... H TFA,CH20|2 N N NH 5 | o A o\ o 0 89"/ H o mmN #114 HATU, ChN\gklil N NH .CF3002H o Hunlgs base #117 N - o o \ (\(o o 0— \ NH #116 V0 o—— 1. LiOH THF water (:H2e|22. TFA WNEJLN 74% (2 steps) .CF3002H o #118 Step 1. Synthesis of 1-(tert-butoxycarbonyl)methyl-L-prolyl-N—{(1S,2R){(2S) [(1R,2R){[(1S)benzylmethoxyoxoethyl]amino}methoxymethyl pyl]pyrrolidinyl}methoxy[(1S)methylpropyl]4-oxobutyl}-N-methyl-L- valinamide (#116). To a stirring solution of #1 14 (1.02 g, 1.61 mmol, 1.0 eq.) and 1-(tert- butoxycarbonyl)methyl-L-proline (443 mg, 1.93 mmol, 1.2 eq.) in 12 mL of dichloromethane, HATU (735 mg, 1.93 mmol, 1.2 eq.) was added followed by Hunig’s base (1.12 mL, 6.45 mmol, WO 72813 4.0 eq.). The reaction was allowed to stir at room ature for 2 hours. The reaction was reduced down, diluted with ethyl acetate before being washed with 0.5 N HCl and brine.
Organics where then dried over sodium sulfate, reduced to a smaller , and then reduced down on silica. Silica chromatography was then performed (Gradient: 0%-45% acetone in heptanes) producing #116 (1.02 g, 74%) as a white solid. LC-MS col Q): m/z 844.3 , 867.2 [M+Na+], retention time = 2.15 minutes.
Step 2A. Synthesis of 2-methyl-L-prolyl-N— {(1S,2R){(2S)[(1R,2R){[(1S) benzylmethoxyoxoethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl} methoxy[( 1 S)methylpropyl]oxobutyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (#117). To a stirring solution of#116 (450 mg, 0.533 mmol, 1.0 eq.) in 7 mL of dichloromethane at 0 OC, TFA (3 mL, 40 mmol, 70 eq.) was added. The on was allowed to stir at 0 °C for 5 minutes and then allowed to warm to room temperature while stirring for 20 minutes. Reaction was reduced down, diluted with dichloromethane and a small amount of methanol before being reduced down onto silica. Silica chromatography was then performed (Gradient: 0%-20% methanol in ethyl acetate) producing #117 (396 mg, 89%) as a white solid. LC-MS (Protocol Q): m/Z 744.5 [M--H+], 767.2 [M+Na+], retention time = 1.40 minutes; HPLC (Protocol A at 45 oC): m/z 744.5 [M--H+], retention time = 7.149 minutes (purity > 91%). 1H NMR (400 MHz, DMSO- d6), 8 .14 (m), 8.66 (br d), 8.50 (d), 8.22 (d), 7.12-7.25 (m), .74 (m), .63 (m), 3.93-4.00 (m), 3.73 (dd), 3.63 (d), 3.46-3.57 (m), 3.38-3.45 (m), 3.26-3.23 (m), 3.22-3.25 (m), .22 (m), 2.99- 3.05 (m), 2.93-2.97 (m), 2.80-2.89 (m), 2.75-2.78 (m), 2.64- 2.67 (m), 2.46- 2.50 (m), 2.27- 2.43 (m), 2.00-2.26 (m), 1.85- 1.99 (m), 1.70-1.83 (m), 1.52-1.69 (m), 1.33- 1.51(m), 1.18-1.31 (m), 0.98-1.07 (m), .97 (m), 0.82-0.92 (m), 0.71-0.78 (m).
. Synthesis of 2-methyl-L-prolyl-N-[(3R,4S,5S) {(2S)[(1R,2R) {[(1 S) carboxyphenylethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxy-5 - methyloxoheptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#118). To a stirring solution of #116 (435 mg, 0.515 mmol), in 4 mL of THF under nitrogen, LiOH (24.7 mg, 1.03 mmol, 2.0 eq.) dissolved in 2 mL of water was added. The reaction was allowed to stir at room temperature until LC—MS ted saponif1cation of methyl ester. Reaction was concentrated in vacuo and then placed underneath vacuum. Reaction was diluted with dichloromethane and placed underneath nitrogen. To this stirring mixture TFA (3mL, 40.5 mmol, 80 eq.) was added.
Reaction was allowed to stir at room temperature for 30 s. Reaction was then reduced down. Residue was purified by medium pressure reverse phase C18 chromatography (Gradient: % to 60% acetonitrile in water with 0.02% TFA in each phase) #118 (396 mg, 89%) as a white solid. LC-MS (Protocol Q): m/z 730.2 [M+H+], retention time = 1.18 minutes; HPLC (Protocol A at 45 oC): m/z 730.5 [M+H+], retention time = 7.088 minutes (purity > 98%). 1H NMR (400 MHz, DMSO-dg), 8 9.04-9.13 (m), 8.75-8.87 (m), 8.70 (d), 8.38 (d), 8.11(d), 7.10-7.24 (m), 4.66- 4.74 (m), .64 (m), 4.37-4.47 (m), 3.91-3.99 (m), 3.77 (m), 3.47-3.56 (m), 3.33-3.47 (m), 3.08-3.30 (m), 2.93-3.07 (m), .86 (m), .69 (m), 2.45-2.50 (m), 2.28-2.44 (m), 2.03- 2.27 (m), .02 (m), 1.68-1.86 (m), 1.55-1.67 (m), 1.30-1.47 (m), 1.17-1.29 (m), 0.98-1.05 (m), 0.93-0.97 (m), 0.83-0.92 (m), 0.71-0.79 (m).
Preparation of 2-methylalanyl-N-[(3R,4S,5S)—1-{(2S)—2-[(1R,2R){[(2S)—1-tert—but0xy phenylpropanyl]amino}meth0xymethyl0X0propyl]pyrrolidin-l-yl} methoxy-S-methyl-l-0X0heptanyl]-N-methyl-L-valinamide (#123). 1. 4M HCI in dioxane, 2. Fmoo—OSu tert-butyl ylalaninate, hydrochloride salt Fmoc’N diethylamine % N82003 (aq) HATU, Hunig's base CH2C|2 N CHQCIQ, DMF N 0 B00’ —,Fmoc’ \ NH 79% O quant.
O quant. (2 steps) \ \KfO °H 3 OH o o °t #11 #119 #1 20 ”N #32, HATU, Hunig's base diethylamine CHQCIQ, DMF FmocNKorNEJiN CH20I2 0 2 \ ON?” 0 “.\\(O 62% NH NH yo O #123 #122 Step 1. Synthesis of ) {(2S)[(9H-fluorenylmethoxy)carbonyl]pyrrolidin yl} methoxymethylpropanoic acid . To a stirring solution of #11 (2.4 g, 8.4 mmol, 1.0 eq.) in 10 mL of dioxane under nitrogen, 4M HCl in dioxane(20 mL, 80 mM, 10 eq.) was added. The reaction was allowed to stir at room temperature for 3 hours before being concentrated in vacuo and placed underneath high vacuum. Crude al was then dissolved with 30 mL of 10% Na2C03. This solution was then added to a ng solution of 1- {[(9H- fluorenylmethoxy)carbonyl]oxy}pyrrolidine-2,5-dione (2.96 g, 8.77 mmol, 1.05 eq.) in 30 mL of DME. Reaction was allowed to stir at room temperature until TLC (20% methanol/40% ethyl acetate/40% heptanes) indicated the consumption of Boc de-protected starting material. The reaction was concentrated in vacuo to a smaller volume, washed twice with ether, acidified to pH 2 using concentrated HCl and then extracted three times with a solution of 90% dichloromethane %methanol. The organics where washed with saturated sodium bicarbonate and brine before being dried over sodium sulfate, filtered, and concentrated in vacuo to a brown solid #119 (3.4 g, quant.). LC-MS (Protocol Q): m/z 410.0 [M+H+], retention time = 1.81 minutes.
Step 2. Synthesis tert—butyl N—[(2R,3R) {(2S)[(9H-fluoren ylmethoxy)carbonyl]pyrrolidinyl} -3 -methoxymethylpropanoyl] -L-phenylalaninate (#120).
To a stirring solution of utyl L-phenylalaninate, hydrochloride salt (1.67 g, 6.5 mmol, 1.0 eq.) and #119 (5.9 g, 6.5 mmol, 1.0 eq.) in 50 mL of dichloromethane and 5 mL of DMF, HATU (2.9g, 7.9 mmol, 1.2 eq.) was added followed by Hunig’s base (5.6 mL, 32 mmol, 5.0 eq.). The reaction was allowed to stir at room temperature for 45 minutes. on was reduced down, diluted with ethyl acetate, washed with 0.5 N HCl and brine before being concentrated down onto silica. Silica chromatography was then performed (Gradient: 0%-25% e in heptane) ing #120 (3.14 g, 79%) as a white yellow solid. LC—MS (Protocol Q): m/z 613.1 [M+H+] retention time = 2.37 minutes.
Step 3. Synthesis of tert—butyl N— {(2R,3R)methoxymethyl[(2S)-pyrrolidin yl]propanoyl}-L-phenylalaninate (#121). To a stirring solution of #120 (2.87 g, 4.68 mmol, 1.00 eq.) in 20 mL of dichloromethane, diethylamine (10 mL, 95 mM, 20.5 eq.) was added. The reaction was allowed to stir at room temperature for 2 hours. Another (10 mL, 95 mmol, 20.5 eq.) of diethylamine was added and the reaction was allowed to stir at room temperature for 3 more hours. Reaction was concentrated in vacuo and placed underneath high vacuum producing #121 (1.8 g, quant.) yellow white oil solid mix. LC-MS (Protocol Q): m/Z 391.1 [M+H+] retention time = 1.05 minutes.
Step 4. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N- [(3R,4S,5 S)- 1- {(2S)[(1R,2R) {[(2S)tert-butoxyoxophenylpropanyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxymethyloxoheptan—4-yl]—N— methyl-L-valinamide (#122). To a stirring solution of #121 (0.55 g, 1.0 mmol, 1.0 eq.) in 10 mL of dichloromethane and 1 mL of DMF, #32 (0.62 g, 1.0 mmol, 1.0 eq.) was added followed by HATU (0.42 g, 1.1 mmol, 1.1 eq.) and Hunig’s base (0.72 mL, 4.1 mmol, 4.0 eq.). The reaction was allowed to stir at room temperature for approximately 21 hours. Reaction was reduced down, diluted with ethyl acetate, and then washed with 0.5 N HCl and brine. Organic layer was dried over sodium sulfate, filtered, and concentrated to a smaller volume before being concentrated down onto silica. Silica chromatography was then performed (Gradient: 0%-40% acetone in heptane) producing #122 (0.62 g, 62%) as a white solid. LC-MS col Q): m/z 982.3 [M+H+] retention time = 2.44 s.
Step 5. sis 2-methylalanyl-N-[(3R,4S,5S){(2S)[(1R,2R){[(2S)tert 1-oxo-3 -phenylpropanyl] amino} - 1-methoxymethyl-3 -oxopropyl]pyrrolidin— 1 -yl} - 3-methoxymethyloxoheptanyl]-N-methyl-L-valinamide (#123). To a stirring mixture of #122 (600 mg, 0.611 mmol, 1.00 eq.) in 15 mL of dichloromethane, diethylamine (5 mL, 50 mmol, 80 eq.) was added. The reaction was d to stir at room temperature for 3 hours.
Reaction was concentrated in vacuo and e was d by Silica Chromatography (Gradient: 0%-40% methanol in romethane) producing #123 (0.46 g, 99%) as a solid. LC- MS (Protocol Q1): m/z 760.3 [M+H+] ion time = 0.83 minutes. 1H NMR (400 MHz, CD3OD), 8 7.14-7.30 (m), 4.70-4.78 (m), 4.56-4.64 (m), 4.05-4.19 (m), 3.87 (dd), 3.79-3.84 (m), 3.72-3.77 (m), 3.62-3.70 (m), 3.46-3.56 (m), 3.37- 3.45 (m), 3.33-3.36 (m), 3.16-3.24 (m), 3.09- 3.11 (m), 2.98-3.05 (m), 2.95 (d), 2.91 (d), 2.87 (d), 2.83 (d),2.73-2.79 (m), 2.40-2.51(m), 2.29- 2.39 (m), 2.16-2.28 (m), 2.04-2.15 (m), 2.01 (s), 1.73-1.96 (m), 1.50-1.68 (m), 1.47-1.49 (m), 1.46 (s), 1.43 (s), 1.38 (s), 1.35 (d), 1.23-1.32 (m), 1.17-1.22 (m), 1.15 (d), 1.04-1.11(m), 0.94- 1.03 (m), .91 (m).
Preparation of methyl N-[(2R,3R)—3-{(2S)—1-[(3R,4S,5S)—4-{[N-(3-amin0-2,2- dimethylpropanoyl)—L-valyl](methyl)amin0}meth0xy—5-methylheptanoyl]pyrrolidin-Z- yl}meth0xy-2—methylpropanoyl]-L-phenylalaninate (#126).
Fmoc-OSu #114, HATU, % Na2003 DME gangs base2 2 H N OH 2 \><'r —,Fmoc/N\><(OH —, O 98% O #124 Fmoc’N\>€(N\:)J\N N N l 0\ O m H2N\><WN\:)LN O O m o A o\ o cl) 0 0 /\ l O\ O \ 0 56% CI) #125 #126 Step 1. Synthesis of 3- {[(9H-fluorenylmethoxy)carbonyl]amino}-2,2- dimethylpropanoic acid (#124). A solution of 3-amino-2,2-dimethylpropanoic acid hydrochloride (1.0 g, 6.5 mmol, 1.0eq.) in 10 mL of 10% NazCO3 was added to a on of 1- {[(9H-fluorenylmethoxy)carbonyl]oxy}pyrrolidine-2,5-dione (2.3 g, 6.5 mmol, 1.0 eq.) in 10 mL of DME. The reaction was allowed to stir at room temperature overnight. Reaction was concentrated to a smaller volume and then washed two times with ether. The aqueous layer was acidified to pH <2 with concentrated HCl and then extracted three times with a 10% methanol 90% dichloromethane solution. The organics where combined before being washed with 1M HCl and brine. The organic layer was dried over sodium sulfate and concentrated in vacuo ing #124 (2.2 g, 98%) as a white solid. LC—MS (Protocol Q1): m/z 362.0 [M+Na+] retention time = 0.89 minutes.
Step 2. Synthesis of methyl N—[(2R,3R){(2S)[(3R,4S,5S){[N—(3- fluoren- 9-ylmethoxy)carbonyl] amino} -2,2-dimethylpropanoyl)-L-Valyl] l)amino} -3 xy-5 - methylheptanoyl]pyrrolidinyl}methoxymethylpropanoyl]-L-phenylalaninate (#125). To a stirring solution of #114 (200 mg, 0.316 mmol, 1.00 eq.) in 2 mL of dichloromethane, #124 (107 mg, 0.316 mmol, 1.00 eq.) was added ed by Hunig’s base (0.167 mL, 0.948 mmol, 3.00 eq.) and HATU (149 mg, 0.379 mmol, 1.20 eq.). The reaction was allowed to stir at room temperature for ~12 hours. The reaction was concentrated to a smaller volume, taken up in 10 mL of ethyl acetate, and washed two times with 5 mL of 1M HCl, and once with 5 mL of brine.
The organic layer was dried over sodium sulfate and decanted. Organics where concentrated in vacuo and the crude al was taken up in dichloromethane. The precipitate was filtered. The organic layer was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0%-50% acetone in heptane) ing #125 (235 mg, 78%) as a white solid. LC- MS (Protocol Q): m/z 954.2 [M+H+] retention time = 2.28 minutes.
Step 3. sis of methyl N-[(2R,3R){(2S)[(3R,4S,5S){[N-(3-amino-2,2- dimethylpropanoyl)-L-valyl] (methyl)amino} methoxymethylheptanoyl]pyrrolidinyl} -3 - methoxymethylpropanoyl]-L-phenylalaninate (#126). To a stirring on of #125 (235 mg, 0.246 mmol, 1.00 eq.) in 2 mL of THF, (1 mL, 10 mM, 40.6 eq.) of diethylamine was added.
The reaction was allowed to stir at room temperature for 3 hours. Reaction was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0%-30% ol in ethyl acetate) producing #126 (101 mg, 56%) as a white solid. LC—MS (Protocol Q): m/z 732.2 [M+H+] retention time = 1.32 minutes. 1H NMR (400 MHz, g), 8 8.51 (dd), 8.28 (d), 7.15-7.29 (m), 5.77 (s), .77 (m), 4.44-4.54 (m), 3.94-4.10 (m), 3.73-3.79 (m), 3.66 (d), 3.49-3.60 (m), 3.40-3.48 (m), 3.10-3.36 (m), 3.00-3.09 (m), 2.83-2.98 (m), 2.57-2.77 (m), 2.19- 2.46 (m), 1.87- 2.14 (m), 1.61- 1.86 (m), 1.36-1.55 (m), .36 (m), 1.12-1.22 (m), 0.97-1.11 (m), 0.82-0.96 (m), 0.73-0.81 (m). ation of N,2-dimethylalanyl-N-[(3R,4S,5S)—1-{(2S)—2-[(1R,2R)—3-{[(2S)—1-tert—but0xy- 1-0X0phenylpropan-Z-yl] amino}methoxy-2—methyl0X0pr0pyl] pyrrolidin-l-yl} methoxy-S-methyl-l-0X0heptanyl]-N-methyl-L-valinamide (#130). flingAJfiJase #121, HATU TFA Hunig's base \OC)—>\‘/2CHCI Fm°C\NKgNZJOKN 2 CH CI mekN OH CH2CI2 2 2 DMF, Fmoc\NKfNEJOLN O quam III 78% o\ o 62% #127 #128 WM”Ll/grafi 3:28:me ¢Lj$83£Kg; NH 8% #130 #129 Qo\\/ Step I. Synthesis©of N-[(9H-fluorenylmethoxy)carbonyl]],—N2-dimethylalanyl-N- [(3R,4S,5 S)tert-butoxy-3 -methoxymethyloxoheptanyl] -N-methyl-L-valinamide (#127). To a round bottom flask containing #6 (4.7 g, 7.9 mmol, 1.0 eq.) and N-[(9H-fluoren ylmethoxy)carbonyl]-N,2-dimethylalanine (3.2 g, 9.4 mmol, 1.2 eq.) and a stir bar under nitrogen, 50 mL of dichloromethane was added ed by HATU (3.6 g, 9.4 mmol, 1.2 eq.) and s base (5.5 mL, 32 mmol, 4.0 eq.). The reaction was allowed to stir at room temperature for ~12 hours. Reaction was reduced to a smaller volume, taken up in ethyl acetate, before being washed with 1 N HCl, and brine. Organics where then dried over sodium sulfate, filtered and then reduced down onto silica. Residue was purified by Silica Chromatography (Gradient: 0%-30% acetone in e) producing #127 (4.2 g, 78%) as a white solid. LC-MS (Protocol Q): m/z 680.2 [M+H+] retention time = 2.52 minutes.
Step 2. Synthesis N-[(9H-fluorenylmethoxy)carbonyl]-N,2-dimethylalanyl-N— [(2R,3S,4S)carboxymethoxymethylhexan—3-yl]-N-methyl-L-valinamide, . To a stirring solution of #127 (4.2 g, 6.1 mmol, 1.0 eq.) in 21 mL of dichloromethane under nitrogen, (7 mL, 90 mmol, 10 eq.) of TFA was added. The reaction was allowed to stir at room temperature for N4 hours. on was concentrated in vacuo, azeotroped once with heptane, and then placed underneath high vacuum yielding #128 as a white slight yellow solid (3.8 g, quant.). LC-MS (Protocol Q): m/z 624.2 [M+H+] retention time = 2.01 minutes.
Step 3. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N,2-dimethylalanyl-N— [(3R,4S,5 S)- 1- 2-[(1R,2R) {[(2S)tert-butoxyphenylpropanyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxymethyloxoheptan—4-yl]—N— methyl-L-valinamide (#129). To a stirring solution of #128 (1.67 g, 3.1 mmol, 1.0 eq.) in 20 of dichloromethane and 2 mL of DMF, #121 (2.4 g, 3.1 mmol, 1.0 eq.) was added followed by HATU (1.29 g, 3.39 mmol, 1.1 eq.) and then Hunig’s base (2.2 mL, 12.3 mmol, 4.0 eq.). The reaction was allowed to stir at room temperature for N2 hours. Reaction was reduced down, diluted with ethyl acetate before being washed with 0.5 N HCl and brine. Organics where dried over sodium sulfate and then reduced down onto silica. Residue was purified by Silica Chromatography ent: 0%-50% acetone in es) producing #129 (1.9 g, 62%) as a white solid. LC-MS (Protocol Q): m/z 996.3 [M+H+] retention time =2.53 minutes.
Step 4. sis of N,2-dimethylalanyl-N—[(3R,4S,5 S){(2S)[( 1R,2R){[(2S) tert-butoxyoxo-3 -phenylpropanyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidin yl} methoxymethyloxoheptanyl]-N-methyl-L-valinamide (#130). To a stirring solution of #129 (823 mg, 0.826 mmol, 1.00 eq.) in 15 mL of romethane, diethylamine (4 mL, 40 mmol, 50 eq.) was added. The reaction was allowed to stir at room temperature for ~14 1/2 hours. The reaction was concentrated in vacuo and azeotroped once with heptanes. Residue with diluted with dichloromethane and a small amount of methanol before being reduced down onto silica. Residue was d by Silica Chromatography (Gradient: 0%-20% methanol in ethyl acetate) producing #130 (518 mg, 81%) as a white solid. LC-MS (Protocol Q): m/z 774.3 [M+H+] retention time =1.48 minutes. HPLC col A at 25 oC): m/Z 774.5 [M+H+], retention time = 7.733 minutes (purity > 98%). 1H NMR (400 MHZ, DMSO-dg), 8 8.36 (d). 8.14(d), 7.81 (t), 7.14-7.25 (m), 7.01-7.07 (m), 4.87-4.94 (m), 4.78-4.85 (m,), 4.67-4.76 (m), 4.46-4.65 (m), 4.29-4.40 (m), 3.93-4.03 (m), 3.70- ), 3.49- 3.60 (m), .47 (m), 3.29- 3.36 (m), 3.15-3.28 (m), 2.98-3.13 (m), 2.94 (br s), 2.74-2.89 (m), 2.64-2.69 (m), 2.18-2.45 (m), 2.02-2.14 (m), 1.90-2.01 (m), 1.62-1.87 (m), 1.40-1.55 (m), 1.37 (d), 1.20-1.33 (m), 1.16(d), 1.01-1.10(m), 0.90-0.98 (m), 0.82 -0.89 (m), 0.69-0.79 (m).
Preparation of 2—methyl-D-prolyl-N-[(3R,4S,5S)—3-methoxy{(2S)—2-[(1R,2R)meth0xy- 3-{ [(2S)—1-meth0xy0X0phenylpr0panyl] amino}methyl0X0pr0pyl] pyrrolidin-l- yl}methyl-l-oxoheptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#131).
EV 0H #114, HATU, .CF3C02H Hunig's base,CH2CI2 N 11/ 2. TFA,CH2C|21O1/ND/ $I;fil/ | O —> 54% (2 steps) #131 Step 1. Synthesis of 2-methyl-D-prolyl-N—[(3R,4S,5S)methoxy{(2S)[( 1R,2R) methoxy-3 - {[(2S)- 1 xyoxophenylpropanyl] amino} methyl-3 - oxopropyl]pyrrolidinyl} methyl- 1 ptanyl] -N-methyl-L-valinamide trifluoroacetic acid salt (#131). To a stirring on of #114 (164 mg, 0.259 mmol, 1.0 eq.) and 1-(tert- butoxycarbonyl)methyl-D-proline (71.3 mg, 0.311 mmol, 1.2 eq.) in 4 mL of dichloromethane, HATU (118 mg, 0.311 mmol, 1.2 eq.) was added followed by Hunig’s base (0.180 mL, 1.04 mmol, 4 eq.). The reaction was allowed to stir at room temperature for ~30 minutes. Reaction was reduced down. on was taken up in 3.5 mL of dichloromethane and placed under nitrogen. To this stirring on, TFA (1.5 mL, 20 mmol, 76 eq.) was added. The reaction was allowed to stir at room temperature for N] hour. Reaction was reduced down and placed underneath high vacuum. Purif1cation by (Method J*) affords #131 (119 mg, 54%) as a white solid. HPLC (Protocol A at 45 oC): m/z 744.5 [M+H+], ion time = 7.342 minutes (purity > 98%). 1H NMR (400 MHz, DMSO-dg), 8 9.08-9.18 (m), 8.79-8.89 (m), 8.76 (t), 8.54 (d), 8.29 (d), 7.14-7.31 (m), 4.70-4.79 (m), 4.57- 4.66 (m), 4.45-4.55 (m), 3.96- 4.04 (m), 3.74- 3.80, 3.66 (d), .61 (m), 3.40-3.48 (m), 3.09- 3.34 (m), .09 (m), 2.95-3.00 (m), 2.83- 2.93 (m), 2.36- 2.53 (m), 2.21-2.35 (m), 2.10-2.19 (m), 1.99-2.10 (m), 1.61-1.09 (m), 1.36- 1.53(m), 1.21-1.35 (m), 1.02- 1.10 (m), 0.94- 1.0 (m), 0.86- 0.93 (m), 0.73- 0.82 (m). 2012/056224 Preparation of yl-L-prolyl—N—[(3R,4S,5S){(2S)—2-[(1R,2R)—3-{[(1S,2R)hydroxy- 1-phenylpropanyl]amino}methoxy-Z-methyloxopropyl]pyrrolidin-l-yl}methoxy- -methyloxoheptanyl]-N-methyl-L-valinamide, oroacetic acid salt (#134). #110 HATU Hunig'3 base Ni diethylamine N dichloromelhane FmOC OmNOO THF Fm'4ng #@5 #132 1.1-(lert-buloxycarb0nyl)--melhy|-L-proline H N2 HATU Hunig'3 base dichloromelhane 2. 4 M HCI'In dioxane WdLNHO N .CF co H3 2 dioxane 86%((2sleps) #133 #134 Step 1. Synthesis of N~2~-[(9H-fiuorenylmethoxy)carbonyl]-N-[(3R,4S,5S) {(28)- 2-[(1R,2R)-3 - {[(1 S,2R)hydroxyphenylpropanyl] amino} methoxymethyl-3 - oxopropyl]pyrrolidinyl}-3 -methoxymethyloxoheptan—4-yl] -N-methyl-L-valinamide . To a flask containing #@5 (1.14 g, 2.17 mmol, 1.0 eq.), 10 mL of dichloromethane was added followed by Hunig’s base (1.15 mL, 6.52 mmol, 3.0 eq.), HATU (1.02 g, 2.61 mmol, 1.2 eq.). and #110 (0.776 g, 2.17 mmol, 1.0 eq.). Reaction was allowed to stir at room temperature for 30 s and then concentrated in vacuo. Crude material was taken up in 50 mL of ethyl acetate, washed two times with 25 mL of 1 M HCl, and once with 25 mL of brine. Organics where dried over sodium sulfate and decanted. Organics where concentrated in vacuo, taken up in 30 mL of dichloromethane, and the resulting precipitate was filtered off. Organics where concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0%-50% acetone in heptanes) producing #132 ( 1.33 g, 81%) as a solid. LC-MS (Protocol Q): m/z 849.2 [M+Na+] retention time =2.19 minutes.
Step 2. Synthesis ofN-[(3R,4S,5S){(2S)[(1R,2R){[(1S,2R)hydroxy phenylpropanyl]amino} methoxymethyloxopropyl]pyrrolidinyl} -3 -methoxy-5 - methyloxoheptanyl]-N-methyl-L-valinamide (#133). To a stirring solution of #132 (1.33 g, 1.60 mmol, 1.0 eq) in 10 mL of THF diethylamine (5 mL, 50 mM, 31.3 eq) was added. The reaction was allowed to stir at room temperature for 4 hours. Reaction was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0%-30% methanol in ethyl acetate) ing #133 (418 mg, 43%) as a white solid. LC—MS col Q1): m/z 605.2 [M+H+] retention time =1.48 minutes.
Step 3. Synthesis of 2-methyl-L-prolyl-N—[(3R,4S,5S){(2S)[(1R,2R) {[(1S,2R)- 1-hydroxyphenylpropan—2-yl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 - methoxymethyloxoheptan—4-yl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#134).
HATU (151 mg, 0.398 mmol, 1.2 eq), #133 (201 mg, 0.332 mmol, 1.0 eq.) and 1-(tert- butoxycarbonyl)methyl-L-proline (91.3 mg, 0.398 mM, 1.2 eq.) where combined in a round bottom flask containing a stir bar under nitrogen. 5 mL of dichloromethane was added followed by s base (0.231 mL, 1.33 mmol, 4.0 eq.). The on was allowed to stir at room temperature for ~15 hours. Reaction was then concentrated in vacuo and placed underneath high vacuum. 4 mL of dioxane was then added to the residue followed by 4M HCl in dioxane (4 mL, 20 mmol, 50 eq.). Reaction was then allowed to stir at room temperature for 1 hour. Reaction was then concentrated in vacuo and the residue was purified by medium pressure reverse phase C18 chromatography (Gradient: 5% to 90% acetonitrile in water with 0.02% TFA in each phase) #134 (237 mg, 86%) as a white solid. LC—MS (Protocol Q): m/z 716.3 [M+H+], retention time = 1.16 minutes; HPLC (Protocol A at 45 OC): /2 716.5 [M+H+], retention time = 6.930 s (purity > 98%). 1H NMR (400 MHz, DMSO-dg), 9.12-9.21(m), 8.79-8.90 (m), 8.70-8.78 (m), 7.95 (d), 7.64 (d), 7.25-7.36 (m), 7.16- 7.23 (m), 4.74- 4.80 (m), 4.61- 4.69 (m), 4.41- 4.59 (m), 3.91- 4.06 (m), 3.78 (dd), 3.54- 3.64 (m), 3.45- 3.51 (m), 3.17- 3.36 (m), 3.02- 3.15 (m), 3.00 (br s), 2.40-2.48 (m), 2.24- 2.35 (m), 1.91- 2.21 (m),. 1.68- 1.90 (m), 1.61-1.68 (m), 1.48- 1.59 (m), 1.22- 1.35 (m), 0.97- 1.09 (m), 0.84- 0.97 (m), 0.74-0.83 (m).
Preparation of N,2-dimethylalanyl-N-{(1S,2R)—4-{(2S)[(1R,2R)—3-{[(1S)benzyl (methylamino)oxoethyl] methoxy-Z-methyl0X0pr0pyl] idin-l-yl} methoxy-l-[(1S)—1-methylpr0pyl]0X0butyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#140), N,2-dimethylalanyl-N-{(1S,2R)—4-{(2S)—2-[(1R,2R)—3-{[(1S)amin0benzyl oxoethyl]amino}methoxy-Z-methyl—3-0X0propyl]pyrrolidin-l-yl}meth0xy[(1S)—1- methylpropyl]0X0butyl}-N-methyl-L-valinamide, trifluoroacetic acid salt , N,2- ylalanyl-N-{(1S,2R)—4-{(2S)—2-[(1R,2R)—3-{[(1S)—1-benzyl0X0 lamino)ethyl]amino}methoxy-Z-methyl0X0pr0pyl]pyrrolidin-l-yl}meth0xy— 1-[(1S)—1-methylpr0pyl]0X0butyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#142), N,2—dimethylalanyl-N-{(1S,2R){(2S)[(1R,2R)—3-{[(1 S)benzyl (diethylamino)—2-0xoethyl]amino}meth0xymethyl0X0pr0pyl]pyrrolidin-l-yl} methoxy-l-[(1S)—1-methylpr0pyl]0X0butyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#143), and N,2—dimethylalanyl-N-{(1S,2R){(2S)—2-[(1R,2R){[(1S)—1-benzyl(tert— butylamino)—2-0xoethyl]amino}meth0xymethyl0X0pr0pyl]pyrrolidin-l-yl} methoxy-l-[(1S)methylpr0pyl]0X0butyl}-N-methyl-L-valinamide, oroacetic acid salt (#144). #128 F 1. #11, dioxane, Fkg: dichloromethane Hi F 4M HCI in dioxane —>Fmoc\pyridine N O 2. Hunig's base N i N dichloromethane 98% O /\ /O O F —> 92% (2 steps) #135 Fmom'ixr Pentafluorophenyl 3,3,3-trifluoropropanoate O dichloromethane Hdk L-phenylalanine Fmoc\N N N pyridine Hunig's base, DMF _ N l 5 | —> O /\ O O 92% C? 41% O F #136 #137 Pentafluorophenyl 33 3--trifluoropropanoate dichloromethane Fmoc\N GUN pyridine FmOCFNKSNEJDLN #138 #139 QC F #139 05,00H 1M methyl amine In THF, THF N/iiLRN0%WO #140 % HN\ #139 3 2 7M ammonia in ol, THF KgN/Efj:$810.0: COH #141 OESONHZ #139 1M n-propylamine in THF, THF HNXTKgN/EELN .HCFgCOZ #142 51% #139 1M diethylamine in THF, CF C102H THF HN>§TfiN/Efihl 3 % NH #143 N\’\\ #139 0 o H 1M ten-butylamine in THF, H THF HN>§(N\)I\N N EJW .CFgCOzH | i | 2 H o o o /\ o \ 24% /o #144 Step I . Synthesis of N-[(9H-flu0reny1meth0xy)carb0nyl]-N,2-dimethy1alany1-N- [(3R,4S,5 S)-3 -meth0xymethy10x0(pentafluorophenoxy)heptan—4-y1]-N-methy1-L- valinamide (#135). Pentafluorophenyl 3,3,3-trifluoropropanoate (2.44 mL, 13.4 mmol, 2.0 eq.) was added to a solution of #128 (4.18 g, 6.70 mmol, 1.0 eq.) in 50 mL of dichloromethane followed by pyridine (1.61 mL, 20.1 mmol, 3.0 eq.). Reaction was allowed to stir at room temperature for ~12 hours. Reaction was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0%-70% acetone in heptanes) producing #135 (5.2 g, 98%) as a white foam. LC-MS (Protocol Q1): m/z 812.1 ] retention time =1.24 minutes.
Step 2. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N,2-dimethylalanyl-N— S,5S) {(2S)[(1R,2R)carboxymethoxypropyl]pyrrolidinyl}methoxy methyloxoheptanyl]-N-methyl-L-valinamide(#136). To a stirring solution of 4M HCl in dioxane (10 mL, 25 mmol, 3.7 eq.) in 10 mL of dioxane #11 (2.31 g, 8.05 mmol, 1.2 eq.) was added. The reaction was allowed to stir at room temperature for 6 hours. The reaction was concentrated in vacuo producing a yellow gum. A solution of #135 (5.3 g, 6.7 mmol, 1.0 eq.) in mL of dichloromethane was added to the preVious residue ed by Hunig’s base (3.5 mL, mmol, 3 eq.). The on was allowed to stir at room ature for 4 hours. The reaction was diluted with dichloromethane before being washed with a 1% HCl aqueous solution and then brine. The organics layer was dried over sodium sulfate, concentrated in vacuo, and the e was purified by silica chromatography (Gradient: 20%-50% ethyl e in heptanes followed by 93% ethyl acetate 6.6% methanol and 0.4% acetic acid) producing #136 (4.87 g, 92%) as a off white solid. LC—MS (Protocol Q1): m/z 793.3 [M+H+] retention time =1.07 s.
Step 3. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N,2-dimethylalanyl-N— [(3R,4S,5S)methoxy{(2S)[(1R,2R)methoxymethyloxo (pentafluorophenoxy)propyl]pyrrolidin— 1-yl}methyloxoheptan—4-yl]-N-methyl-L- valinamide (#137). Pentafluorophenyl 3,3,3-trifluoropropanoate (1.3 mL, 7.1 mmol, 2.0 eq.) was added to a solution of #136 (2.8 g, 3.5 mmol, 1.0 eq.) in 30 mL of dichloromethane followed by the addition of pyridine (0.85 mL, 10.6 mM). The reaction was allowed to stir at room temperature for 2 hours. The reaction was concentrated in vacuo, and the residue was purified by silica chromatography (Gradient: 0%-70% acetone in e) producing #137 ( 3.1 g, 92%) as a white powder. LC-MS (Protocol Q1): m/z 959.2 [M+H+] retention time =1.28 minutes.
Step 4. Synthesis of N-[(9H-fluorenylmethoxy)carbonyl]-N,2-dimethylalanyl-N— [(3R,4S,5 S) {(2S)[( 1 R,2R) {[( 1 S)carboxyphenylethyl] amino} methoxy methyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxymethyloxoheptanyl]-N-methyl-L- valinamide(#138). To a stirring solution of #137 (493 mg, 0.514 mmol, 1.0 eq.) in 4 mL of DMF, L-phenylalanine (84.9 mg, 0.514 mmol, 1.0 eq) was added ed by Hunig’s base (0.27 mL, 1.54 mmol, 3.0 eq.). The reaction was allowed to stir at room temperature for ~12 hours.
Reaction was concentrated in vacuo and residue was purified by silica chromatography (Gradient: 0%- 100% ethyl acetate in heptane) producing #138 ( 200 mg, 41%) as a white foam.
LC-MS col Q1): m/z 940.3 [M+H+] retention time =1.08 minutes.
Step 5. Synthesis of -fluorenylmethoxy)carbonyl]-N,2-dimethylalanyl-N— [(3R,4S,5S)methoxy{(2S)[(1R,2R)methoxymethyloxo{[(2S)oxo (pentafluorophenoxy)-3 -phenylpropanyl] propyl]pyrrolidinyl} hyl oxoheptanyl]-N-methyl-L-valinamide (#139). To a ng solution of #138 (200 mg, 0.213 mmol, 1.0 eq.) in 5 mL of dichloromethane, uorophenyl 3,3,3-trifiuoropropanoate (126 mg, 0.426 mM, 2.0 eq.) was added followed by pyridine (0.051 mL, 0.64 mmol, 3.0 eq.). The reaction was allowed to stir at room temperature for ~12 hours. Reaction was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0%-100% ethyl acetate in heptanes) producing #139 (174 mg, 74%) as a yellow oil. LC—MS col Q1): m/Z 1128 [M+Na+] retention time =1.23 minutes.
Step 6A. Synthesis of N,2-dimethylalanyl-N— {( 1 S,2R) {(2S)[( 1 R,2R) {[( 1 S)- 1- benzyl(methylamino)oxoethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}- 2-methoxy[(1S)methylpropyl]oxobutyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#140). To a stirred solution of#139 (20 mg, 0.018 mmol, 1.0 eq.) in lmL of THF methylamine (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) was added, and the mixture was stirred at room temperature for 3 hours. The reaction was reduced down, and diluted with dmso, and subjected to purification (Method J*). The fractions were collected and concentrated in vacuo to give #140 (4.0 mg, 30%) as a white solid. LC-MS (Protocol Q1): m/z 731.2 [M+H+], retention time = 0.70 minutes. 1H NMR (400 MHz, ol-d4), 7.30-7.41 (m), 4.71-4.78 (m), 4.58-4.69 (m), 4.04-4.15 (m), 3.86-3.98 (m), 3.73-3.78 (m), .70 (m), 3.50-3.58 (m), 3.32-3.47 (m), 3.23-3.26 (m), 3.17-3.22 (m), 3.07-3.15 (m), 2.95-2.98 (m), 2.76-2.91 (m), 2.68-2.75 (m), 2.63- 2.66 (m), 2.43-2.51 (m), 2.22-2.28 (m), 1.99-2.11 (m), 1.74-1.96 (m), 1.21-1.31 (m), 1.17-1.20 (m), 0.92-1.10 (m), .89 (m).
Step 63. Synthesis ofN,2-dimethylalanyl-N—{(1S,2R){(2S)[(1R,2R){[(1S) aminobenzyloxoethyl] amino} - 1-methoxymethyl-3 -oxopropyl]pyrrolidin— 1-yl} methoxy [(1 S)- 1 lpropyl] oxobutyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (#141). Following the same procedure as #140 using #139 (20 mg, 0.018 mmol, 1.0 eq.), ammonia solution (7M in methanol, 0.026 mL, 0.18 mmol, 10eq.) and purification (Method 1*), #141 (3.0 mg, 20%) was obtained as a white solid. LC—MS (Protocol Q): m/z 717.2 [M+H+], retention time = 0.79 minutes. 1H NMR (400 MHz, methanol-d4), 7.22-7.30 (m), .21 (m), 4.57-4.4.80 (m), 4.02-4.17 (m), 3.92-3.98 (m), 3.84-3.91 (m), 3.32-3.74 (m), 3.17-3.27 (m), 3.06- 3.14 (m), 2.77-3.05 (m), 2.65 (s), 2.43-2.51 (m), 2.21-2.26 (m), 1.98-2.13 (m), 1.70-1.94 (m), 1.32-1.69 (m), .31 (m), 0.89-1.13 (m), 0.80-0.88 (m).
Step 6C. Synthesis of N,2-dimethylalanyl-N—{(1S,2R){(2S)[(1R,2R){[(1S) oxo(propylamino)ethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidin— 1-yl}- 2-methoxy[(1S)methylpropyl]oxobutyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#142). Following the same procedure as #140 using #139 (20 mg, 0.018 mmol, 1.0 eq.) npropylamine (1M in THF, 0.18 mL, 0.18 mmol, 10. eq) and purification (Method 1*), #142 (3.0 mg, 20%) was obtained as a white solid. LC-MS (Protocol Q): m/z 759.2 , retention time = 0.74 minutes. 1H NMR (400 MHz, methanol-d4), 7.15-7.29 (m), 4.71-4.79 (m), 4.52-4.68 (m), 4.04-4.17 (m), 3.87-3.99 (m), 3.73-3.99 (m), 3.73-3.79 (m), 3.50-3.70 (m), 3.34-3.49 (m), 3.06- 3.23 (m), 2.79-2.99 (m), 2.44-2.50 (m), 2.28-2.43 (m), 2.22-2.27 (m), 1.75-2.10 (m), 1.34-1.61 (m), 1.16-1.29 (m), .10 (m), 0.77-0.89 (m).
Step 6D. Synthesis of N,2-dimethylalanyl-N— {( 1 S,2R) {(2S)[( 1 R,2R) {[( 1 S) (diethylamino)oxoethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}- 2-methoxy[(1S)methylpropyl]oxobutyl}-N-methyl-L-valinamide, roacetic acid salt (#143). Following the same procedure as #140 using #139 (20 mg, 0.018 mmol, 1.0 eq.) diethylamine (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) and purification (Method 1*), #143 (4.0 mg, 30%) was ed as a white solid. LC-MS col Q): m/z 773.3 [M+H+], retention time = 0.77 minutes. 1H NMR (400 MHz, methanol-d4), 7.16-7.33 (m), 5.10-5.17 (m), 4.96-5.07 (m), 4.68-4.75 (m), 4.60-4.65 (m), 3.61-4.23 (m), 3.35-3.67 (m), 3.16-3.26 (m), 2.99-3.15 (m), 2.78- 2.94 (m), 2.30-2.52 (m), 2.19-2.28 (m), 1.73-2.13 (m), 1.83-1.45 (m), 1.19-1.31 (m), 0.92-1.18 (m), 0.80-0.89 (m).
Step 6E. Synthesis of N,2-dimethylalanyl-N— {( 1 S,2R) 2-[( 1 R,2R) {[( 1 S)- 1- benzyl(tert-butylamino)oxoethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidin yl} methoxy[(1 ethylpropyl]oxobutyl}-N-methyl-L-valinamide, trifluoroacetic acid salt #144. Following the same procedure as #140 using #139 (20 mg, 0.018 mmol, 1.0eq.) tert-butylamine (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) and purification (Method 1*), #144 (3.4 mg, 24%) was ed as a white solid. LC-MS (Protocol Q1): m/z 773.3 [M+H+], retention time = 0.74 minutes. 1H NMR (400 MHz, DMSO-dg), 8 8.21 (d), 8.03-7.98 (m), 7.92 (d), 7.81-7.62 (m), 7.46-7.16 (m), 4.83-4.69 (m), 4.68-4.56 (m), 4.21-4.07 (m), 3.92-3.86 (m), 3.83-3.80 (m), 3.74-3.65 (m), 3.60-3.48 (m), 3.47-3.36 (m), 3.28-3.13 (m), 3.11-3.01 (m), 2.96- WO 72813 2.82 (m), 2.69-2.62 (m), 2.54—2.43 (m), .12 (m), 2.00-1.76 (m), 1.69-1.161 (m), 1.60-1.53 0n) 152-098(n0,094-086(n0. ation of N,2-dimethylalanyl-N-[(3R,4S,SS){(ZS)[(1R,2R){[(IS,2R) hydroxy-l-phenylpropan-Z-yl]amino}methoxy-Z-methyl0X0propyl]pyrrolidin-l-yl} methoxy-S-methyl-l-0X0heptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#145). . (182R)--2amIno1--pheny|propan 1 o-I Fmoc\N><gNNgri‘jgjylfloo Hunig's base DMF 2. Piperidine L/3N .CFgCOZH #137 F #145 Step 1. Synthesis ofN,2-dimethylalanyl-N—{(1S,2R){(2S)[(1R,2R){[(1R,2S) hydroxymethylphenylethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl} methoxy-l-[(1S)methylpropyl]oxobutyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#145). To a stirring solution of #137 (300 mg, 0.313 mmol, 1.0 eq.) in 3 mL of DMF, (IS,2R)- 2-aminophenylpropan—1-ol (54.8 mg, 0.344 mmol, 1.1 eq.) was added followed by Hunig’s base (0.164 mL, 0.939 mmol, 3.0 eq). The reaction was allowed to stir at room temperature for ~12 hours. Piperidine 20% solution in DMF (1 mL, 2.2 mmol, 7.0 eq.) was then added and the reaction was allowed to stir at room temperature for 2 hours. Purification (Method J*) followed by concentration of appropriate test tubes produced #145 (190 mg, 74%) as a white powder. LC- MS (Protocol Q): m/z 704.3 [M+H+], retention time = 0.67 minutes. 1H NMR (400 MHz, CD3OD), 8 7.97 (d), 7.73 (d), 7.37-7.41 (m), .36 (m), .25 (m), 4.70- 4.75 (m), 4.58- 4.63 (m), 4.49-4.54 (m), 4.14-4.30 (m), 4.04-4.11 (m), 3.87 (dd), 3.63-3.77 (m), 3.51-3.58 (m), 3.46-3.49 (m), 3.38- 3.43 (m), 3.25-3.37 (m), 3.15- 3.23 (m), 3.11- 3.14 (m), 3.01- 3.02 (m), 2.59-2.64 (m), 2.52-2.55 (m), 2.44-2.52 (m), 2.41-2.43 (m), 2.07-2.26 (m), 1.73-2.0 (m), 1.65- 1.73 (m), 1.59- 1.65 (m), 1.51-1.59 (m), 1.32- 1.46 (m), 1.23- 1.26 (m), 1.08-1.21 (m), 0.94- 1.07 (m), 0.83-0.92 (m).
Preparation of yl-D-isovalyl—N—{(IS,2R){(ZS)[(1R,2R){[(IS)benzyl methoxy-Z-oxoethyl]amino}methoxymethyl0X0propyl]pyrrolidin-l-yl}methoxy- 1-[(1S)—1-methylpropyl]0X0butyl}-N-methyl-L-valinamide (#146), 3-methyl-L-isovalyl-N- {(1S,2R)—4-{(ZS)[(1R,2R){[(1S)benzylmethoxy-Z-oxoethyl]amino}methoxy 0X0propyl]pyrrolidinyl}methoxy[(1 S)methylpropyl]0X0butyl}-N- methyl-L-valinamide (#147), L-isovalyl-N-{(IS,2R){(ZS)[(1R,2R){[(IS)benzyl methoxy-Z-oxoethyl]amino}methoxymethyl0X0propyl]pyrrolidin-l-yl}methoxy- 1-[(1S)—1-methylpr0pyl]0X0butyl}-N-methyl-L-valinamide (#148), and D-isovalyl-N- {(1S,2R)—4-{(2S)—2-[(1R,2R){[(1S)benzylmethoxy-Z-oxoethyl]amino}meth0xy methyl0X0pr0pyl]pyrrolidinyl}meth0xy[(1 S)methylpr0pyl]0X0butyl}-N- -L-valinamide . 1. #114, Hunig's base HATU, CHZCIZ \/ 2. diethylamine \/ O H THF Hdb N Fmoc\ OH —> N N m m H N _ N 69% 2 E | 0 o 0/\ o\o 1. #114, Hunig's base HATU, CHZCIZ 2. diethylamine , THF H I O 1, a, H N FmomN4 OH N (M 82% Mg: 9LT O H O O 1. #114, Hunig's base HATU,CHZC|2 Fmoc‘N/éOH —>2.c diethylamine ’1. THF N MNH H Hi O 35% HZN : l O O m o A o\ o \ o o 1. #114, Hunig's base HATU, CHZCIZ / 2. diethylamine (ij M”H Fm‘ >5?” —>THF o 30% H2“ 5 'I“ o\ O m o A o\ o o o #149 Step IA. Synthesis of 3-methyl-D-isovalyl-N—{(1S,2R){(2S)[(1R,2R) {[(1S) methoxyoxoethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl} methoxy[( 1 S)methylpropyl]oxobutyl} hyl-L-valinamide (#146). A solution of #114 (225 mg, 0.356 mmol, 1.0 eq.) in 2 mL of dichloromethane was added to a solution ofN- uorenylmethoxy)carbonyl]methyl-D-isovaline (126 mg, 0.356 mmol, 1.0eq.) in 4 mL of dichloromethane. Hunig’s base (0.188 mL, 1.07 mmol, 3.0 eq.) was added followed by HATU (167 mg, 0.427 mmol, 1.2 eq). The reaction was allowed to stir at room temperature for 12 hours. The reaction was concentrated in vacuo and then taken up in ethyl acetate before being washed two times with 1M HCl and once with brine. The organic layer was dried over sodium sulfate and decanted. The organic solvent was removed in a genevac. THF (4 mL) was added followed diethylamine (2 mL, 19 mmol, 53.4 eq.). The reaction was allowed to stir for ~12 hours. Reaction was concentrated using a genevac followed by silica tography (Gradient: 0%-30% methanol in ethyl e) producing #146 (183 mg, 69%) as a solid. LC-MS (Protocol Q): m/z 746.4 [M+H+] retention time =1.37 minutes. 1H NMR (400 MHZ, DMSO-dg), 8 8.55 (d), 8.26- 8.36 (m), 7.88-8.03 (m), 7.81 (d), 7.41-7.53 (m), 7.13-7.30 (m), 7.01 (s), 4.71-4.79 (m), 4.44—4.70 (m), 3.96-4.04 (m), .80 (m), 3.62-3.69 (m), 3.40-3.61 (m), 2.76-3.35 (m), 2.67- 2.71 (m), 2.56-2.58 (m), 2.06-2.46 (m), 1.61-1.90 (m), 1.14—1.54 (m), 0.72—1.12 (m).
Step 13. Synthesis of 3-methyl-L-isovalyl-N— {(1 S,2R) {(2S)[(1R,2R) {[(1S) benzylmethoxyoxoethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl} methoxy[( 1 S)methylpropyl]oxobutyl} -N-methyl-L-valinamide (#147). A solution of #114 (224 mg, 0.354 mmol, 1.0 eq.) in 2 mL of romethane was added to a solution ofN- [(9H-fluorenylmethoxy)carbonyl]methyl-L-isovaline (125 mg, 0.354 mmol, 1.0 eq.) in 4 mL of dichloromethane. Hunig’s base (0.187 mL, 1.06 mmol, 3.0 eq.) was added followed by HATU (167 mg, 0.425 mmol, 1.2 eq.). The reaction was allowed to stir at room temperature for 12 hours. The reaction was concentrated in vacuo and then taken up in ethyl acetate before being washed two times with 1M HCl and once with brine. The organic layer was dried over sodium sulfate and decanted. The organic solvent was removed in a genevac. THF (4 mL) was added followed diethylamine (2 mL, 19 mmol, 53.7 eq.). The reaction was allowed to stir for ~12 hours. Reaction was concentrated using a genevac followed by silica chromatography (Gradient: 0%-30% methanol in ethyl acetate) producing #147 (216 mg, 82%) as a solid. LC-MS (Protocol Q): m/z 746.6 [M+H+] retention time =1.29 minutes. 1H NMR (400 MHZ, DMSO-dg), 8 8.56 (m), 8.31-8.39 (m), 8.50 (d), 8.30 (br d), 7.87-8.01 (m), 7.80 (d), 7.40-7.53 (m), 7.14-7.30 (m), 4.45-4.78 (m), 3.94-4.04 (m), 3.70-3.79 (m), .69 (m), 3.42-3.59 (m), 2.97-3.37 (m), 2.80- 2.92 (m), .49 (m), .30 (m), .89 (m), 1.37-1.56 (m), 1.14-1.135 (m), 0.70-1.11 (m).
Step I C. Synthesis of L-isovalyl-N—{(1S,2R){(2S)[(1R,2R){[(1S)benzyl methoxyoxoethyl]amino } - 1-methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy [(1S)methylpropyl]oxobutyl}-N-methyl-L-valinamide . A solution of #114 (447 mg, 0.707 mmol, 1.0 eq.) in 2 mL of dichloromethane was added to a solution of N—[(9H-fluoren ylmethoxy)carbonyl]-L-isovaline (240 mg, 0.707 mmol, 1.0eq.) in 4 mL of dichloromethane. s base (0.373 mL, 2.12 mmol, 3.0 eq.) was added followed by HATU (332 mg, 0.425 mmol, 1.2 eq.). The reaction was allowed to stir at room ature for 12 hours. The reaction was concentrated in vacuo and then taken up in ethyl acetate before being washed two times with 1M HCl and once with brine. The organic layer was dried over sodium sulfate and decanted.
The organic t was removed in a genevac. THF (4 mL) was added followed by lamine (2 mL, 19 mmol, 26.9 eq.) . The reaction was allowed to stir for ~12 hours.
Reaction was concentrated using a genevac followed by silica chromatography (Gradient: 0%- % methanol in ethyl acetate) producing #148 (182 mg, 35%) as a solid. LC—MS (Protocol Q1): m/z 732.3 [M+H+] retention time =0.71 minutes. 1H NMR (400 MHz, DMSO-dg), 8 8.56 (d), 8.46-8.52 (m), 8.30 (d), 8.028 15 (m), 7.98 (d), 7.80 (d), 7.40-7.53 (m), 7.15-7.30 (m), .80 (m), 4.44-4.69 (m), 3.96-4.05 (m), 3.70-3.79 (m), 3.62-3.69 (m), .59 (m), 2.99-3.35 (m), 2.31-2.95 (m), 2.67-2.71 (m), 2.55-2.59 (m), 2.32-2.48 (m), 2.20-2.31 (m), 1.97-2.19 (m), 1.61- 1.88 (m), 1.37-1.56 (m), 1.20-1.34 (m), 1.14-1.19 (m), .11 (m), 0.97-1.01 (m), 0.86-0.96 (m), 0.71-0.83 (m).
Step ID. Synthesis of D-isovalyl-N-{(1S,2R){(2S)[(1R,2R){[(1S)benzyl methoxyoxoethyl]amino } - 1-methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy [(1S)methylpropyl]oxobutyl}-N-methyl-L-valinamide (#149). A solution of #114 (447 mg, 0.707 mmol, 1.0 eq.) in 2 mL of romethane was added to a solution of N—[(9H-fluoren ylmethoxy)carbonyl]-D-isovaline (240 mg, 0.707 mmol, 1.0 eq.) in 4 mL of dichloromethane.
Hunig’s base (0.373 mL, 2.12 mmol, 3 eq.) was added followed by HATU (332 mg, 0.425 mmol, 1.2 eq.). The reaction was allowed to stir at room temperature for 12 hours. The reaction was concentrated in vacuo and then taken up in ethyl acetate before being washed two times with 1M HCl and once with brine. The organic layer was dried over sodium e and decanted. The c solvent was removed in a genevac. THF (4 mL) was added followed by diethylamine (2 mL, 19 mmol, 26.9 eq.). The reaction was allowed to stir for ~12 hours. Reaction was concentrated using a genevac followed by silica chromatography (Gradient: 0%-30% methanol in ethyl acetate) producing #149 (154 mg, 30%) as a solid. LC-MS (Protocol Q): m/z 732.0 [M+H+] retention time =1.24 minutes. 1H NMR (400 MHz, DMSO-dg), 8 8.55 (d), 8.38-8.46 (m), 8.29 (d), 8.03-8.14 (m), 7.97 (d), 7.81 (d), 7.40-7.53 (m), 7.14-7.28 (m), 7.02 (s), 4.71-4.79 (m), 4.43-4.69 (m), 3.96-4.05 (m), 3.71-3.80 (m), 3.62-3.70 (m), 3.49-3.60 (m), 3.40-3.48 (m), 3.15-3.34 (m), 310-3. 14 (m), 3.01-3.09 (m), .00 (m), 2.83-2.93 (m), 2.65-2.71 (m), 2.55- 2.59 (m), 2.32-2.48 (m), .31 (m), 1.61-1.89 (m), 1.37-1.52 (m), 1.21-1.35 (m), 1.15-1.20 (m), 1.02-1.10 (m), 0.75-1.01 (m).
Preparation of 1,2-dimethyl-L-prolyl-N-{(1S,2R){(2S)[(1R,2R){[(1S)—1-carb0xy—2- phenylethyl]amin0}meth0xy—2—methyl0X0propyl]pyrrolidin-l-yl}meth0xy[(1S)— l-methylpropyl]0X0butyl}-N-methyl-L-valinamide . 1. #114, HATU armsmmmsn‘er.
(WOH 2. methanol .. OH THF 0 100% N 69% um%NH #1 51 #150 V0 Step 1. Synthesis of 1,2-dimethyl-L-proline (#150). A parr flask containing 2-methyl-L- proline (1.0 g, 7.7 mmol, 1.0 eq.), 40 mL of methanol, dehyde 37 wt. % in water (2.1 mL, 77 mmol, 10 eq.), and Palladium 10 wt. % on Carbon (313 mg, 2.94 mmol, 0.38 eq.) was placed on a parr shaker and allowed to shake under 40 psi of hydrogen for ~12 hours. Hydrogen was removed and the reaction was d through a pad of celite which was rinsed with a solution of 50% methanol 50% dichloromethane. e was concentrated in vacuo yielding #150 ( 1.1 g, 100%) as a white slight black colored solid. LC-MS (Protocol Q): m/z 144.0 [M+H+] retention time =0.17 minutes.
Step 2. Synthesis of 1,2-dimethyl-L-prolyl-N— {(1S,2R) 2-[(1R,2R){[(1S) carboxyphenylethyl]amino} - 1 xymethyl-3 -oxopropyl]pyrrolidinyl}methoxy [(1S)methylpropyl]oxobutyl}-N-methyl-L-valinamide (#151). To a stirring mixture of #114 (125 mg, 0.198 mmol, 1.0 eq), #150 (37 mg, 0.26 mmol, 1.3 eq.), and HATU (98 mg, 0.26 mmol, 1.3 eq.) in 5 mL of dichloromethane, Hunig’s base (0.14 mL, 0.80 mmol, 4.1 eq.) was added. The reaction was allowed to stir at room temperature for 1 hour. Reaction was concentrated in vacuo. THF (6 mL) was added to crude material. To this stirring mixture LiOH (14 mg, 0.59 mmol, 3.0 eq) dissolved in 2 mL of water was added. Reaction was allowed to stir at room temperature for 90 minutes. Reaction was concentrated in vacuo and residue was purified by medium re reverse phase C18 chromatography (Gradient: 5% to 40% acetonitrile in water with 0.02% TFA in each phase) #151 (147 mg, 69%) as a white solid. LC- MS (Protocol Q): m/Z 744.3 [M+H+], retention time = 1.19 minutes; HPLC col A at 45 oC): m/z 744.4 [M+H+], retention time = 6.631 minutes (purity > 98%). 1H NMR (400 MHz, DMSO- d5), 8 9.57-9.71 (m), 8.75 (d), 8.42 (d), 8.15 (d), 7.14-7.29 (m), 4.70-4.79 (m), 4.40-4.68 (m), 3.95-4.03 (m), 3.73-3.80 (m), 3.37-3.61 (m), 2.97-3.31 (m), 2.79-2.88 (m), 2.66-2.76 (m), 2.54- 2.58 (m), 2.31-2.43 (m), 1.94-2.29 (m), 1.57-1.91 (m), .52 (m), 0.85-1.10 (m), 0.74-0.82 (m) Preparation of 1,2-dimethyl—D-prolyl-N-{(1S,2R)—4-{(2S)—2—[(1R,2R)—3-{[(1S)carb0xy phenylethyl]amin0}meth0xy—2—methyl0X0propyl]pyrrolidin-l-yl}meth0xy[(1S)— l-methylpropyl]0X0butyl}-N-methyl-L-valinamide . 1. #114, HATU Formaldehyde 37 wt. % in water Hunig's base, CHZCIZ MOH Palladium 10 wt. % on Carbon 2. LiOH, water methanol 0 OH THF ., NWN NH I NlOl/A 0 N 100% 11/ 78% #152 #153 00110381-0957 00703598-0711 Step 1. Synthesis of 1,2-dimethyl-D-proline (#152). To a parr flask containing 2-methyl- D-proline (432 mg, 3.34 mmol, 1.0 eq.), Formaldehyde 37 wt. % in water (1.0 mL, 37 mM, 11 eq.), 3.5 mL of methanol and 1 mL of water, Palladium 10 wt. % on Carbon (108 mg, 0.304 mmol, 0.304 eq.) was added. The flask was placed on a parr shaker and allowed to shake under psi of hydrogen for ~48 hours. Hydrogen was removed and reaction was washed through a pad of celite, which was subsequently washed with methanol. The organics where concentrated in vacuo and then azeotroped with toluene affording #152 (517 mg, 100%) as a solid. 1H NMR (400 MHz, methanol-d4): 8 [3.61-3.56 (m, 1H), 3.07-2.96 (m, 1H), 2.68 (br s, 3H), 2.34-2.22 (m, 1H), 2.01-1.88 (m, 1H), 1.87-1.73 (m, 1H), 1.40 (br s, 3H)].
Step 2. Synthesis of methyl-D-prolyl-N-{(1S,2R){(2S)[(1R,2R){[(1S) carboxyphenylethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy [(1S)methylpropyl]oxobutyl} -N-methyl-L-valinamide (#153). To a stirring mixture of #114 (240 mg, 0.379 mmol, 1.0 eq.), #152 (71 mg, 0.49 mmol, 1.3 eq.), and HATU (188 mg, 0.49 mmol, 1.3 eq.) in 10 mL of dichloromethane, Hunig’s base (0.27 mL, 4.1 mM, 4.1 eq.) was added. The on was allowed to stir at room temperature for 1 hour. Reaction was concentrated in vacuo. THF (6 mL) was added to crude al. To this stirring mixture LiOH (36 mg, 1.5 mmol, 4 eq.) dissolved in 2 mL of water was added. Reaction was allowed to stir at room temperature for 1 hour. Reaction was concentrated in vacuo and e was purified by medium pressure reverse phase C18 chromatography ent: 5% to 40% acetonitrile in water with 0.02% TFA in each phase) #153 (220 mg, 78%) as a white solid. LC-MS (Protocol Q): m/z 744.8 [M+H+], retention time = 1.16 minutes; HPLC (Protocol A at 45 oC): /z 744.4 [M+H+], retention time = 6.713 minutes (purity > 98%). 1H NMR (400 MHZ, DMSO-dg), 8 9.72-9.85 (m), 8.65 (t), 8.41 (d), 8.14 (d), .28 (m), 4.69-4.79 (m), .53 (m), 3.95-4.04 (m), 3.73- 3.79 (m), 3.37-3.62 (m), .33 (m), 2.95-3.10 (m), 2.79-2.89 (m), 2.67-2.75 (m), 2.00-2.46 (m), 1.61-1.90 (m), .54 (m), 1.02-1.09 (m), 0.95-1.01 (m), 0.85-0.94 (m), 0.75-0.83 (m) Preparation of N~2~-[2,2-dimethyl(methylamin0)propanoyl]-N-{(1S,2R)meth0xy {(2S)-2—[(1R,2R)—1-meth0xymethyl0X0{[(1S)—2-phenyl(1,3-thiazol yl)ethyl] amin0}pr0pyl] pyrrolidin-l-yl} [(1 S)methylpr0pyl] 0X0butyl}-N-methyl-L- valinamide, trifluoroacetic acid salt (#154). 1. #50, Hunig's base dichloromethane 2. 4M HCI in dioxane dioxane O H I —>I H N\KLNS/\> N\)L \ HN OH HN N 41%(28teps) N : o o o O A I /o o \ .CFacOZH \© #154 Step 1. Synthesis of N~2~-[2,2-dimethyl(methylamino)propanoyl]-N- {(1S,2R) methoxy{(2S)[(1R,2R)methoxymethyloxo{[(1S)phenyl(1,3-thiazol yl)ethyl] amino } propyl]pyrrolidinyl} - 1- [(1 S)methylpropyl]oxobutyl}-N-methyl-L- valinamide, trifluoroacetic acid salt (#154). To Vial ning #50 (100 mg, 0.152 mmol, 1.0 eq.) and 1 mL of dichloromethane, 2,2-dimethyl(methylamino)propanoic acid (36 mg, 0.152 mmol, 1.0 eq.) was added followed by Hunig’s base (0.080 mL, 0.456 mmol, 3.0 eq.) and HATU (66 mg, 0.17 mmol, 1.1 eq.). The reaction was allowed to stir at room temperature for 1 hour.
The reaction was concentrated in vacuo and then taken up in ethyl acetate before being washed two times with 1M HCl and once with brine. The c layer was dried over sodium sulfate and decanted. The reaction was concentrated in vacuo. Dioxane (1 ml) was added followed by 4M HCl in dioxane (1.0 mL, 4.0 mmol, 26 eq.). The on was allowed to stir at room temperature for ~12 hours. The reaction was concentrated in vacuo. The crude material was purified by medium pressure reverse phase C18 tography (Gradient: 10% to 100% acetonitrile in water with 0.02% TFA in each phase) yielding #154 (55.8 mg, 41%) as a solid.
LC-MS (Protocol Q): m/z 771.8 [M+H+]. 1H NMR (400 MHz, DMSO-dg), 8 8.70 (d), 8.45 (d), 7.90-8.15 (m), 7.82 (d), 7.75 (d), 7.55 (dd), 7.40 (dd), 6.90-7.10 (m), 5.10-5.30 (m), 4.45-4.55 (b), 4.30-4.45 (m), 4.20-4.30 (m), 3.75-3.90 (m), 3.50-3.60 (m), 3.15-3.40 (m), .15 (m), 2.85-3.05 (m), 2.60-2.85 (m), 2.25-2.40 (m), 1.80-2.25 (m), 1.70-1.80 (m), 1.20-1.60 (m), 0.80- 1.10 (m), 0.05-0.80 (m).
Preparation of methyl N-{(2R,3R)—3-[(2S)—1-{(3R,4S,5S)—4-[{N-[2,2-dimethyl (methylamino)propanoyl]-L-valyl}(methyl)amin0]meth0xy methylheptanoyl}pyrrolidin-Z-yl]meth0xymethylpropanoyl}-L-phenylalaninate, trifluoroacetic acid salt (#155). 1. #114, Hunig's base dichloromethane 2. 4M HCI in e dioxane l 0 I H Hi HN N HN NdLT MN 0 OH é 17 % (2 steps) 5 O O O /\ \ 0 /O O .CF3C02H #155 Step 1. sis of methyl N—{(2R,3R)[(2S){(3R,4S,5S)[{N—[2,2-dimethyl (methylamino)propanoyl]-L-Valyl}(methyl)amino]methoxymethylheptanoyl}pyrrolidin yl]methoxymethylpropanoyl} -L-phenylalaninate, trifluoroacetic acid salt (#155). To Vial containing #114 (96.2 mg, 0.152 mmol, 1.0 eq.) and 1 mL of dichloromethane, 2,2-dimethyl (methylamino)propanoic acid (36.1 mg, 0.152 mmol, 1.0 eq.) was added followed by s base (0.080 mL, 0.456 mmol, 3.0 eq.) and HATU (66 mg, 0.17 mmol, 1.1 eq.). The reaction was 2012/056224 d to stir at room temperature for 1 hour. The reaction was concentrated in vacuo and then taken up in ethyl acetate before being washed two times with 1M HCl and once with brine. The organic layer was dried over sodium sulfate and decanted. The reaction was concentrated in vacuo. Dioxane (1 ml) was added ed by 4M HCl in dioxane (1.0 mL, 4.0 mmol, 26 eq.).
The reaction was allowed to stir at room temperature for ~12 hours. The reaction was concentrated in vacuo. The crude material was purified by medium re reverse phase C18 chromatography (Gradient: 10% to 100% acetonitrile in water with 0.02% TFA in each phase) ng #155 (22.2 mg, 17%). 1H NMR (400 MHz, DMSO-dg), 8 8.55 (d), 8.22 (d), 8.15-8.35 (m), 7.90-8.05 (m) 7.10-7.25 (m) 4.70-4.80 (m), 4.55-4.65 (m), 4.45-4.52 (m), 3.93-4.00 (m), 3.72-3.78 (m), 3.60-3.70 (m), 3.50-3.60 (m), 3.40-3.50 (m), 2.80-3.30 (m), 2.45-2.60 (m), 2.00- 2.45 (m), 1.60-1.80 (m), 1.35-1.50 (m), 1.10-1.35 (m).
Preparation of methyl N-{(2R,3R)—3-meth0xy[(ZS){(3R,4S,SS)methoxymethyl [methyl(N-{ [(ZS)methylpiperidinyl] carbonyl}-L-valyl)amino] oyl}pyrrolidin yl]methylpropan0yl}-L-phenylalaninate, trifluoroacetic acid salt (#158) and methyl N- {(2R,3R)—3-meth0xy[(ZS){(3R,4S,5S)—3-meth0xymethyl[methyl(N-{[(2R)—2- methylpiperidin-Z-yl] carbonyl}-L-valyl)amino] heptanoyl}pyrrolidin-Z-yl] methylpropanoyl}-L-phenylalaninate, trifluoroacetic acid salt (#159) >Ljio 0H OH SP0 >LJOLO “0H O >l\JOkO 0 N Separation N O N #156 #157 1) #156, HATU, iPrNEtg, @(NEJ‘NN o CHZCI2, DMF H 2) TFA, CH20I2 49% #158 \ NH ' CF3COQH 1) #157, HATU, iPrNEt2, CH2CI2,DMF NH o -“ H 2) TFA, CH Cl2 2 N\:)L'Tl N 33% #159 0 wk?0 Step 1. (Synthesis of (ZS)(tert-butoxycarbonyl)methylpiperidinecarboxylic acid (#156) and (2R)(tert—butoxycarbonyl)methylpiperidinecarboxylic acid (#157). t— butoxycarbonyl)methylpiperidinecarboxylic acid (500 mg, 2.06 mmol, 1 eq.) was separated by supercritical fluid chromatography (Column: Chiralcel OJ-H, 250 x 21 mm; Eluent: 90: 10 carbon dioxide/ethanol; Flow Rate: 65 g/min; to give the corresponding enantiomers. The first g peak (retention time = 1.57 minutes) was isolated to give #156 as a gum (140 mg, 28%) (stereochemistry arbitrarily assigned as the S enantiomer). 1H NMR (400 MHz, CDCl3) 8 3.83- 3.90 (m, 1H), 2.93-3.01 (m, 1H), 1.87-1.97 (m, 1H), 1.67-1.77 (m, 3H), 1.48-1.66 (m, 2H), 1.46 (s, 3H), 1.44 (s, 9H). Optical rotation: [0t]D25 -21.70 (c 0.40, form). The second eluting peak (retention time = 2.22 minutes) was isolated to give #157 as an oil (255 mg, 51%) (stereochemistry arbitrarily assigned as the R enantiomer). 1H NMR (400 MHz, CDCl3) 8 3.83- 3.90 (m, 1H), 2.93-3.01 (m, 1H), 1.87-1.97 (m, 1H), 1.67-1.77 (m, 3H), 1.48-1.66 (m, 2H), 1.46 (s, 3H), 1.44 (s, 9H). Optical rotation: [0t]D25 +30.2°(chloroform).
Step 2A. Synthesis of methyl N— {(2R,3R)methoxy[(2S) {(3R,4S,5S)methoxy- -methyl [methyl(N— {[(2S)methylpiperidinyl] carbonyl} -L valyl)amino]heptanoyl}pyrrolidinyl] methylpropanoyl} -L-phenylalaninate, trifluoroacetic acid salt (#158). To a solution of #156 (8.3 mg, 0.034 mmol, 1 eq.) in dichloromethane (0.3 mL) and N,N—dimethylformamide (0.05 mL), was added N,N—diisopropylethylamine (0.018 mL, 0.102 mmol, 3 eq.), followed by HATU (16.1 mg, 0.041 mmol, 1.2 eq.). The reaction was stirred for minutes and #114 (23.4 mg, 0.037 mmol, 1.1 eq.) was added and stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane (2.5 mL) and 10 % citric acid (1.5 mL) was added. The layers were separated using a phase tor cartridge and the aqueous layer extracted with dichloromethane (2 X 2.5 mL) and the combined organic layers were concentrated in vacuo. The e was dissolved in dichloromethane (4 mL) and trifluoroacetic acid (0.5 mL) was added. The reaction was stirred at room temperature for 2 hours, then concentrated in vacuo. Purification by reverse phase chromatography (method M*) afforded #158 (10.6 mg, 49%). HPLC (Protocol T): m/z 758.4 , retention time = 2.53 minutes (purity >99%). 1H NMR (400 MHz, DMSO-d6), 8 8.74-8.90 (m), 8.49-8.55(m), 8.24 (d), 8.08- 8.12 (m), 7.94-8.01 (m), 7.14-7.26 (m), 4.71-4.77 (m), 4.57-4.68 (m), 4.44-4.55(m), .0 (m), 3.73-3.78 (m), 3.40-3.72 (m), 3.16-3.32 (m), 2.98-3.16 (m), .92 (m), 2.47-2.56 (m), 2.38- 2.44 (m), 2.20-2.37 (m), 2.08-2.19 (m) 1.74-1.88 (m), 1.61-1.73 (m), 1.52-1.59 (m), 1.22-1.52 (m), 1.05 (dd), .00 (m), 0.85-0.93 (m), 0.74-0.79 (m).
Step 23. Synthesis of methyl N-{(2R,3R)methoxy[(2S){(3R,4S,5S)methoxy- -methyl l(N— {[(2R)methylpiperidinyl]carbonyl} -L- amino]heptanoyl}pyrrolidinyl] methylpropanoyl} -L-phenylalaninate, roacetic acid salt (#159). To a solution of #157 (7.8 mg, 0.032 mmol, 1 eq.) in dichloromethane (0.3 mL) and N,N—dimethylformamide (0.05 mL), was added N,N—diisopropylethylamine (0.017 mL, 0.096 mmol, 3 eq.) followed by HATU (14.9 mg, 0.038 mmol, 1.2 eq.). The reaction was stirred for minutes and #114 (22.1 mg, 0.035 mmol, 1.1 eq.) was added and stirred at room ature for 3 hours and then concentrated in vacuo. Purification of the e by silica gel chromatography (Gradient: 0 to 80% acetone in heptane) afforded a white solid which was dissolved in dioxane (0.2 mL) and 4N HCl in e (0.2 mL) was added. The reaction was stirred at room temperature for 2 hours and additional 4N HCl in dioxane (0.1 mL) was added.
The reaction was stirred for 2 hours at room temperature, concentrated in vacuo. Purification by reverse phase chromatography (Method M*) afforded #159 (6.6 mg, 33 %). HPLC (Protocol T ): m/z 758.4 [M+H+], retention time = 2.46 minutes (purity = 89%). 1H NMR (400 MHz, DMSO- d6), 8 8.86-8.95 (m), 8.75-8.84(m), 8.48-8.54 (m), 8.33-8.45 (m), .27 (m), 8.17-8.19 (m), 7.99-8.12 (m), 7.83-7.91 (m), 7.13-7.29 (m), 7.04-7.08 (m), 4.69-4.76 (m), 4.55-4.66 (m), 4.45- 4.53 (m), .01 (m), 3.41-3.78 (m), 3.28-3.33 (m), 3.24-3.27 (m), 3.16-3.23 (m), 3.11-3.15 (m), .10 (m), 2.93-3.02 (m), 2.91-2.93 (m), 2.84-2.91 (m), 2.76-2.82 (m), .71(m), 2.60-2.63 (m), 2.53-2.55 (m), 2.47-2.53 (m), 2.40-2.46 (m), 2.30-2.38 (m), 2.20-2.30 (m), 2.06- 2.17 (m), 1.75-1.87 (m), 1.52-1.74 (m), 1.35-1.51 (m), 1.14-1.34 (m), 1.01-1.08 (m), 0.92-1.0 (m), 0.85-0.94 (m), 0.74-0.82 (m).
Preparation of ,3R)meth0xy[(2S)—1-{(3R,4S,5S)meth0xymethyl [methyl(N-{ [(2S)methylpiperidinyl] carbonyl}-L-valyl)amin0] heptanoyl}pyrrolidin yl]methylpr0pan0yl}-L-phenylalanine, trifluoroacetic acid salt (#162) and N-{(2R,3R)—3- methoxy [(2S)—1-{(3R,4S,5S)—3-meth0xymethyl [methyl(N- { [(2R)—2-methylpiperidin- 2-yl] carbonyl}-L-valyl)amin0] oyl}pyrrolidin-Z-yl] methylpr0pan0yl} -L- phenylalanine, trifluoroacetic acid salt. (#163). 011156, HATU, N\)l\j?\n/%Sf:2)TFA, CH2C121) er/THF 0th002H ,pma CH2012, DMF WN0 .—. 39 % qua 111(twos1p 1 i l #160 #162 NH /\ 0\ 0 0/ \ NH *1“ ‘9 K?) 0, 011157, HATU, CF002H 1 L0H / \12,PrNE1 was1111; CH2C12, DMF ZjTFA, CH2012H\)1\NH \ .—/> 68 / (two sslep ) NH NH {0 #163 Step IA. Synthesis of methyl N-{(2R,3R)[(2S){(3R,4S,5S)[(N—{[(2S)(tert- butoxycarbonyl)methylpiperidinyl]carbonyl} -L-Valyl)(methyl)amino] methoxy-5 - methylheptanoyl } pyrrolidinyl] -3 -methoxymethylpropanoyl} -L-phenylalaninate (#1 60). To a solution of #156 (106 mg, 0.436 mmol, 1 eq.) in dichloromethane (3 mL) and MN- dimethylformamide (0.5 mL) was added diisopropylethylamine (0.228 mL, 1.31 mmol, 3 eq.) followed by HATU (205 mg, 0.523 mmol, 1.2 eq.). The on was stirred for 15 s and #114 (276 mg, 0.436 mmol, 1 eq.) was added and stirred at room temperature for 2 hours. The reaction was diluted with dichloromethane (10 mL) and washed with 10% citric acid (3 X 5 mL).
The organic layer was dried over sodium sulfate, filtered and the filtrate concentrated in vacuo.
Purification of the residue by silica gel chromatography (Gradient: 0 to 80% e in e) afforded #160 (145 mg, 39%). LC—MS (protocol Q1): m/z 858.8 [M+H+], retention time = 1.12 minutes.
Step 13. Synthesis ofmethyl N-{(2R,3R)[(2S){(3R,4S,5S)[(N—{[(2R)(tert— butoxycarbonyl)methylpiperidinyl]carbonyl} -L-valyl)(methyl)amino] methoxy-5 - methylheptanoyl}pyrrolidinyl]methoxymethylpropanoyl} -L-phenylalaninate (#161). To a solution of#157 (109 mg, 0.448 mmol, 1 eq.) in dichloromethane (3 mL) and MN- dimethylformamide (0.5 mL), was added diisopropylethylamine (0.234 mL, 1.34 mmol, 3 eq.) followed by HATU (205 mg, 0.538 mmol, 1.2 eq.). The reaction stirred for 15 minutes and #114 (284 mg, 0.448 mmol, 1 eq.) was added. After stirring at room temperature for 2 hours, the mixture was diluted with dichloromethane (10 mL), washed with 10 % citric acid (3 X 5 mL).
The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. Purification of the residue by silica gel chromatography (Gradient: 0 to 100% acetone in heptane) afforded #161 (185 mg, 48%). LC—MS (Protocol Q): m/Z 858.3 [M+H+], retention time = 2.25 minutes.
Step 2A. Synthesis of N—{(2R,3R)methoxy[(2S){(3R,4S,5 S)methoxy methyl [methyl(N— {[(2S)methylpiperidinyl]carbonyl} -L- valyl)amino]heptanoyl}pyrrolidinyl] methylpropanoyl} nylalanine, trifluoroacetic acid salt (#162). To a solution of #160 (145 mg, 0.169 mmol, 1 eq.) in tetrahydrofuran (1.25 mL) was added lithium hydroxide (8 mg, 0.338 mmol, 2 eq.) dissolved in water (0.75 mL). The reaction was d at room temperature for 2 hours and evaporated to dryness in vacuo. The residue was dissolved in dichloromethane (2.5 mL) and roacetic acid (1 mL) was added.
The on was stirred for 30 minutes, concentrated in vacuo and purified by medium pressure e phase C18 chromatography (Gradient: 0% to 100% acetonitrile in water with 0.02% TFA in each phase) to afford the title compound #162 (145 mg, quantitative) as a white solid. HPLC (Protocol U): m/z 744.5 [M+H+], retention time = 7.121 minutes (purity = 98%). 1H NMR (400 MHz, DMSO-d6). 8 8.76-8.96 (m), 8.52-8.58 (m), 8.38-8.43 (m), 8.11- 8.16 (m), 7.27-7.30 (m), .27 (m), .05 (m), 4.71-4.79 (m), 4.48-4.67 (m), 4.39-4.47 (m), 3.79-4.22 (m), 3.71- 3.78 (m), 3.38-3.57 (m), 3.22-3.30 (m), 3.14-3.23 (m), .13 (m), 2.96-3.06 (m), 2.76-2.87 2012/056224 (m), 2.66-2.68 (m), 2.47—2.57 (m), 2.42—2.44 (m), 2.06-2.40 (m), 1.73-1.89 (m), 1.51—1.72 (m), 1.36-1.49 (m), 1.20—1.35 (m), 1.00—1.09 (m), .99 (m), 0.83-0.94 (m), 0.73-0.80 (m).
Step 28. Synthesis of N—{(2R,3R)methoxy[(2S){(3R,4S,5 ethoxy methyl[methyl(N— {[(2R)methylpiperidinyl]carbonyl} -L- valyl)amino] heptanoyl} pyrrolidin—2-yl] methylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt (#163). Compound #161 (185 mg, 0.216 mmol, 1 eq.) was converted to the crude title compound #163, using the procedure described for the preparation of #162. The crude material was purified by medium pressure reverse phase C18 chromatography (Gradient: 0% to 85% acetonitrile in water with 0.02% TFA in each phase) to yield #163 (127 mg, 68%) as a White solid. HPLC (Protocol U): m/z 744.5 [M+H+], ion time = 7.077 minutes (purity = 98%). 1H NMR (400 MHz, DMSO-d6). 8 8.79-8.99 (m), .49 (m), 8.12-8.17 (m), 7.31-7.34 (m), 7.11-7.27 (m), 7.05-7.09 (m), 4.71-4.77 (m), 4.54-4.68 (m), 4.40-4.53 (m), 3.88-4.39 (m), 3.71- 3.77 (m), 3.39-3.58 (m), 3.22-3.32 (m), 3.10-3.22 (m), 3.04-3.09 (m), 2.92-3.03 (m), 2.77-2.88 (m), 2.68-2.71 (m), 2.47-2.57 (m), .45 (m), 2.30-2.42 (m), 2.03-2.29 (m), 1.74-1.88 (m), 1.52-1.73 (m), .51 (m), 1.17-1.37 (m), 1.00-1.07 (m), 0.95-0.99 (m), 0.84-0.93 (m), 0.73- 0.81 (m).
Preparation of methyl N-{(2R,3R)—3-[(2S){(3R,4S,5S)—4-[(N-{[(3R)flu0r0pyrrolidin yl]carbonyl}-L-valyl)(methyl)amin0]methoxy—S-methylheptanoyl}pyrrolidin-Z-yl] methoxy—Z-methylpropanoyl}-L-phenylalaninate, trifluoroacetic acid salt (#172) and methyl N-{(2R,3R)—3-[(2S)—1-{(3R,4S,5S)—4-[(N-{[(3R)flu0r0pyrrolidinyl]carbonyl}-L- (methyl)amin0]meth0xy—5—methylheptanoyl}pyrrolidin—Z-yl]meth0xy methylpropanoyl}-L-phenylalaninate, trifluoroacetic acid salt (#173).
% Pd/C 0 methanol 0 o o F 45 PSI H2 F F F OL'. R 30020 (i OH NaOH aq, MeOH LIOH aq..
NBn —> N300 —> NBoc —> NBoc 74% 99% quant. #164 #166 #1 68 #170 % Pd/C O methanol 0 O O F 45PS|H F F F ’c O 2 7. O 1, OH ’4 OLI.
NBn —> NBoc —> NBoc —> NBoc 63% 99% quant. #165 #167 #1 69 #171 1) #168, HATU iPrNElg, ,DMF F 0 HM H N CFSCOZH 2) TFA, CHZCIZ OngJN o /\ I o\ o 51% (two steps) \ 1) #169, HATU, rPrNEtg, CHZCIZ, DMF 2) TFA, CHZCIZ CFficozH 45%(lwosteps) WW" N ; 'i‘ o o \ o Step 1. Synthesis of methyl (3R)benzylfluoropyrrolidinecarboxylate (#164) and methyl (3 S)benzylfluoropyrrolidinecarboxylate (#165). Known l 1-benzyl fluoropyrrolidinecarboxylate (3900 mg, 16.4 mmol, 1 eq.) was separated by supercritical fluid chromatography (Column: Chiralpak 1C, 250 x 21 mm; Eluent: 95:5 carbon dioxide/propanol; Flow Rate: 65 g/min; to give the corresponding enantiomers. The first eluting peak (retention time = 3.37 minutes) was isolated to afford #164 (1720 mg, 36%) as a single omer (stereochemistry arbitrarily assigned as R enantiomer). 1H NMR (400 MHz, TMS-CDC13; 8 7.17—7.30 (m, 5H), 3.74 (s, 3H), 3.65 (d, J= 12.9 Hz, 1H), 3.63 (d, J= 12.9 Hz, 1H), 2.86—3.03 (m, 3H), 2.61 (q, J: 8.0 Hz, 1H), 2.34—2.46 (m, 1H), 2.13-2.26 (m, 1H). Optical rotation: ]325 +24.7O (chloroform). The second eluting peak (retention time = 3.91 minutes) was isolated to afford #165 (1600 mg, 33%) as a single enantiomer (stereochemistry arbitrarily assigned as S enantiomer). 1H NMR (400 MHZ, CDCl3; (CH3)4Si), 8 7.17—7.30 (m, 5H), 3.74 (s, 3H), 3.65 (d, J=12.9 Hz, 1H), 3.63 (d, J=12.9 Hz, 1H), .03 (m, 3H), 2.61 (q, J= 8.0 Hz, 1H), 2.34—2.46 (m, 1H), 2.13-2.26 (m, 1H). l rotation: [01]]325 - 23.3o oform).
Step 2A. Synthesis of 1-tert—butyl 3-methyl (3R)fluoropyrrolidine-1,3-dicarboxylate . To a solution ning #164 (355mg, 1.50 mmol, 1 eq.) and Di-tert-butyl carbonate (400mg, 1.8 mmol, 1.2 eq.) in methanol (15.5 mL) was added 10% Pd/C (70 mg). The reaction was hydrogenated at 45 psi in a Parr shaker at for 22 hours, filtered over celite, and the filtrate concentrated in vacuo and purified by silica gel chromatography (Gradient: 0 to 30% ethyl e in e) to afford #166 as a clear oil. (272 mg, 74%). 1H NMR (400 MHz, CDCl3), 8 3.87 (s, 3H), 3.85-3.66 (m, 3H), 3.56 (m, 1H), 2.53-2.28 (m, 2H), 1.51 (s, 9H).
Step 28. sis of 1-tert-butyl 3-methyl (3 S)fluoropyrrolidine-1,3-dicarboxylate (#167). Compound #165 (362mg, 1.53 mmol, 1 eq.) was ted to #167 in 63% yield using the method described above for #164. 1H NMR (400 MHz, CDCl3), 8 3.87 (s, 3H), 3.85-3.66 (m, 3H), 3.56 (m, 1H), 2.53-2.28 (m, 2H), 1.51 (s, 9H).
Step 3A. Synthesis of (3R)(tert-butoxycarbonyl)fluoropyrrolidinecarboxylic acid (#168). To a solution of #166 (272mg, 1.10 mmol, 1 eq.) dissolved in methanol (2.96 mL) was added an aqueous solution of sodium hydroxide (2.5 M, 0.88 mL) and the reaction was stirred at room temperature for 3.5 hours. The reaction was quenched with 10% aqueous citric acid (5 mL), ethyl acetate (100 mL) was added, and the layers separated. The organic layer was washed with 10% citric acid, water, and brine, dried over sodium sulfate, filtered and concentrated in vacuo to afford #168 as a white solid. (253mg, 99%). 1H NMR (400 MHz, CDCl3), 8 3.96-3.69 (m, 3H), 3.59 (m, 1H), 2.59-2.33 (m, 2H), 1.51 (s, 9H). LC-MS (Protocol Q1): m/z 232.1 [M- H+], retention time = 0.67 s. Chiral HPLC retention time: 3.39 min (purity = 99%). n: Chiralpak AD-H, 4.6mm x 25cm, mobile phase 5-60% COz/Methanol, flow rate 3.0 mL/min); Optical rotation: [0L]D25 4.8 (c= 0.52, MeOH) Step SB. Synthesis of (3 S)(tert-butoxycarbonyl)fluoropyrrolidinecarboxylic acid (#169). To a solution of #167 (238 mg, 0.963 mmol, 1 eq.) dissolved in ol (2.6 mL) was added an aqueous on of sodium hydroxide (2.5 M, 0.88 mL) and the reaction was stirred at room temperature for 3 hours. The reaction was then quenched with 10% aqueous citric acid (5 mL) and ethyl acetate (100 mL) was added, and the layers were separated. The organic layer was washed with 10% citric acid, water, and brine, then dried over sodium e, filtered and concentrated in vacuo to afford #169 as a white solid (221 mg, 99%). 1H NMR (400 MHz, CDCl3), 8 3.96-3.69 (m, 3H), 3.59 (m, 1H), 2.59-2.33 (m, 2H), 1.51 (s, 9H). LC-MS (Protocol Q1): m/z 232.1 [M-H+], retention time = 0.67 minutes. Chiral HPLC retention time: 3.95 min y = 98%)(Column: Chiralpak AD-H, 4.6mm x 25cm, mobile phase 5-60% COz/Methanol, flow rate 3.0 mL/min); Optical rotation: [0t]D25 -3.6 (c= 0.55, MeOH) Step 4A. Synthesis of lithium (3R)(tert-butoxycarbonyl)fluoropyrrolidine carboxylate (#170). To a solution of #168 ( 50 mg, 0.21 mmol, 1 eq.) in methanol (0.2 mL) was added a solution of lithium hydroxide (9.2 mg, 0.38 mmol, 1.8 eq.) dissolved in water (0.1 mL).
Next, tetrahydrofuran ( 0.3 mL) was added and the reaction was stirred at 45 °C for 18 hours.
The reaction was concentrated in vacuo and the material was azeotroped (3X) with toluene (2 mL) to obtain #170 (51mg, 100%) as a white solid which was used in the next step without further purification.
Step 48. Synthesis of lithium (3 S)(tert—butoxycarbonyl)fluoropyrrolidine carboxylate (#171). To a solution of #169 ( 75 mg, 0.32 mmol, 1 eq.) in methanol (0.3 mL) was added a solution of lithium hydroxide (13.8 mg, 0.572 mmol, 1.8 eq.) in water (0.4 mL). Next, tetrahydrofuran ( 0.45 mL ) was added and the reaction was stirred at 45 0C for 18 hours. The reaction was concentrated in vacuo and the material was azeotroped (3X) with toluene (4 mL) to obtain #171 (77 mg, 100%) as a white solid, which was used in the next step without further purification.
Step 5A. Synthesis ofmethyl N-{(2R,3R)[(2S){(3R,4S,5S)[(N—{[(3R) fluoropyrrolidinyl]carbonyl}-L-valyl)(methyl)amino]methoxy methylheptanoyl } pyrrolidinyl] -3 -methoxymethylpropanoyl} -L-phenylalaninate, trifluoroacetic acid salt (#172) . To a suspension of #168 (36.9 mg, 0.158 mmol, 1 eq.) and #114 (100 mg, 0.15 8 mmol, 1.0 eq.) in N,N—dimethylformamide (0.8 mL) and dichloromethane (3.6 mL) was added MN—diisopropylethylamine (0.083 mL, 0.474 mmol, 3 eq.) followed by HATU (60.7 mg, 0. 15 8 mmol, 1.0 eq.) and the on was stirred at room temperature for 18 hours.
The reaction was diluted with ethyl acetate and was washed successively with water, 10% s citric acid (W/V), and brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to give 220 mg (164% of theory) of a crude intermediate. A portion of this crude ediate (50 mg, 23%) was dissolved in dichloromethane (1.5 mL) and trifluroacetic acid (0.4 mL) was added. The mixture was stirred at room temperature for 2 hours and evaporated to dryness in vacuo. Purification by e phase chromatography (Method M* ) afforded #172 (15.8 mg, 51%) LC—MS (Protocol Q): m/z 748.9 [M+H+] retention time = 1.29 minutes. 1H NMR (DMSO-d6_), 8 9.16-9.43 (m), 8.48-8.53 (m), 8.40-8.44 (m), 8.34-8.39 (m), 8.22-8.30 (m), 8.098 16 (m), 7.87-7.91 (m), 7.77-7.83 (m), 7.12-7.24 (m), 4.56-4.72 (m), 4.41- 4.54 (m), 3.92-4.00 (m), 3.70-3.75 (m), 3.39-3.66 (m), 3.20-3.25 (m), .20 (m), 2.97-3.11 (m), 2.94 (br s), 2.75-2.89 (m), 2.63- 2.69 (m), 2.47-2.54 (m), 2.29- 2.45 (m), 2.15- 2.27 (m), 2.02-2.15 (m), 1.57-1.87 (m), 1.33- 1.51 (m), 1.19-1.30 02 (dd), 0.82-0.97 (m), 0.70-0.79 (In)- Step 53. Synthesis of methyl N— R)[(2S) S,5S)[(N— {[(3R) fluoropyrrolidin-3 -yl]carbonyl} -L-valyl)(methyl)amino] -3 xy-5 - methylheptanoyl } idinyl] -3 -methoxymethylpropanoyl} -L-phenylalaninate, trifluoroacetic acid salt (#173). To a suspension of #169 (36.9 mg, 0.158 mmol, leq. ) and #114 (100.0 mg, 0. 15 8 mmol, 1 eq.) in N,N—dimethylformamide (0.8 mL) and dichloromethane (3.6 mL) was added N,N—diisopropylethylamine (0.083 mL, 0.474 mmol, 3 eq.) was added, followed by HATU (60.7 mg, 0.158 mmol, 1 eq.). The reaction was stirred at room temperature for 14 hours, diluted with ethyl acetate, and washed sively with water, 10% aqueous citric acid (W/V), and brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to give 180 mg (134% of theory) of a crude intermediate. A portion of this crude intermediate (50 mg, 27%) was dissolved in dichloromethane (1.5 mL) and trifluroacetic acid (0.4 mL) was added and the mixture stirred at room temperature for 2 hours and evaporated to s in vacuo.
Purification by reverse phase chromatography (Method M* ) ed #173 (17.6 mg, 45%). LC- MS (Protocol Q): m/z 748.9 [M+H+] retention time =1.29 s. 1H NMR (DMSO-d6) 8 9.35-9.50 (m), 9.22-9.34 (m), 8.47-8.52 (m), 8.39-8.45 (m), 8.30-8.37 (m), 8.22-8.24 (m), 8.09- 8.13 (m), 7.80-7.85 (m), 7.67-7.72 (m), 7.09-7.24 (m), 6.97-6.98 (m), 4.65-4.72 (m), 4.55-4.64 (m), 4.41-4.50 (m), 3.92-3.99 (m), 3.42-3.75 (m), 3.32-3.39 (m), 3.25-3.31(m), 3.21-3.24 (m), .20 (m), .11 (M), .05 (m), 2.95 (br s), 2.83-2.88 (m), 2.76-2.82 (m), 2.65-2.70 (m), 2.44-2.54 (m), 2.15-2.42 (m), 2.02-2.14 (m), 1.57-1.84 (m), 1.33-1.48 (m), 1.19-1.30 (m), 1.02 (dd), 0.92-0.97(m), 0.82-0.91 (m), 0.70-0.77 (m).
Preparation of (2S)—N—[(2S){[(3R,4S,5S){(2S)—2-[(1R,2R)—3-{[2-(cyclohepta-2,4,6-trien- 1-yl)ethyl]amin0}meth0xy—2—methyl0X0pr0pyl]pyrrolidin-l-yl}meth0xymethyl oxoheptanyl](methyl)amin0}methyl0X0butanyl]methylpiperidine-2— carboxamide , hydrochloride salt (#178) and (2R)—N-[(2S)—1-{[(3R,4S,5S){(2S)—2- [(1R,2R)—3-{[2-(cyclohepta—2,4,6-trienyl)ethyl]amino}meth0xymethyl oxopropyl]pyrrolidin-l-yl}meth0xymethyl—1-0X0heptanyl](methyl)amin0} methyl-l-oxobutan-Z-yl]methylpiperidine-Z-carboxamide, hydrochloride salt (#180).
F F O o F F F o F O m Fm“HN o WHNJrNtfigYN; chHN\)L | endme 9L»: IPVZNELCHQClQ /\ /O o N F o ' I a —> o o —> = | /\ / F \ NH /\ o\ o F 92% 58% #@5 #174 Step 1. Synthesis of pentafluorophenyl (3R,4S,5S)[{N—[(9H-fluoren ylmethoxy)carbonyl]-L-Valyl}(methyl)amino]methoxymethylheptanoate . To a solution of #@5 (19.43 g, 37.03 mmol, 1 eq.) in dichloromethane (100 mL) and pyridine (5.86 g, 74.1 mmol, 2 eq.) was added pentafluorophenyl trifluoroacetate (20.7 g, 74.1 mmol, 2 eq.) and the reaction was d at room ature for 1 hour. The reaction was concentrated in vacuo and purified by silica gel chromatography ent: 0 to 52% ethyl e in heptane) to afford #174 (23.58 g, 92%) as a yellow oil. LC—MS (Protocol Q1): m/z 691.2 [M+H+], ion time = 1.23 s.
Step 2. Synthesis of N—[(3R,4S,5S) {(2S)[(1R,2R) {[2-(cyclohepta-2,4,6-trien yl)ethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 xymethyl oxoheptanyl]-N~2~-[(9H-fluorenylmethoxy)carbonyl]-N-methyl-L-valinamide (#175). To a solution of#174 (706 mg, 1.02 mmol, 1 eq.) and #64 (311mg, 1.02 mmol, 1 eq.) in dichloromethane (3 mL) was added MN—diisopropylethylamine (400 mg, 3.07 mmol, 3 eq.).
After 18 hours of stirring at room temperature, the reaction was concentrated in vacuo and purified by silica gel chromatography (Gradient: 0 to 100% ethyl acetate in heptane) to afford #175 (560 mg, 68%) as a white solid. LC-MS (Protocol Q1): m/z 611.8 [M+H+], retention time = 1.15 minutes.
Step 3. Synthesis of N—[(3R,4S,5S) {(2S)[(1R,2R) {[2-(cyclohepta-2,4,6-trien yl)ethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxymethyl oxoheptanyl]-N-methyl-L-valinamide (#176). According to the general procedure A, from #175 (560 mg, 0.690 mmol, 1 eq.) in dichloromethane (9 mL), and N,N—diethylamine (6.0 mL), was synthesized the crude desired compound, which was purified by by silica gel chromatography (Gradient: 0 to 50% methanol in dichloromethane) to afford #176 (351 mg, 87%) as a yellow oil. LC-MS (Protocol Q1): m/z 589.5 [M+H+], ion time = 0.72 minutes.
Step 4A. Synthesis oftert-butyl (2S){[(2S){[(3R,4S,5S){(2S)[(1R,2R){[2- (cyclohepta-2,4,6-trienyl)ethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 - ymethyloxoheptanyl](methyl)amino}-3 -methyloxobutanyl]carbamoyl} methylpiperidinecarboxylate (#177). According to the general procedure D, from #176 (100 mg, 0.170 mmol, 1 eq.), #156 (53.8 mg, 0.221 mmol, 1.3 eq.), dichloromethane (4.5 mL), HATU (84.9 mg, 0.221 mmol, 1.3 eq.) and N,N—diisopropylethylamine (0.123 mL, 0.697 mmol, 4.1 eq.), was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0 to 100% ethyl acetate in heptane) to afford #177 (145 mg, assume WO 72813 quantitative yield) as a white solid. LC—MS (Protocol Q1): m/z 814.7 [M+H+], retention time = 1.14 minutes.
Step 43. Synthesis oftert—butyl (2R){[(2S){[(3R,4S,5S){(2S)[(1R,2R){[2- (cyclohepta-2,4,6-trienyl)ethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 - methoxymethyloxoheptanyl](methyl)amino}-3 -methyloxobutanyl]carbamoyl} methylpiperidinecarboxylate (#179). According to the general procedure D, from #176 (100 mg, 0.170 mmol, 1 eq.), #157 (53.8 mg, 0.221 mmol, 1.3 eq.), dichloromethane (4.5 mL), HATU (84.9 mg, 0.221 mmol, 1.3 eq.) and N,N—diisopropylethylamine (0.123 mL, 0.697 mmol, 4.1 eq.), was synthesized the crude desired al, which was purified by silica gel chromatography (Gradient: 0 to 100% ethyl acetate in heptane) to afford #179 (155 mg, assume quantitative yield) as a white solid. LC—MS (Protocol Q1): m/z 814.7 , retention time = 1.14 minutes.
Step 5A. Synthesis of -[(2S){[(3R,4S,5S){(2S)[(1R,2R){[2- (cyclohepta-2,4,6-trienyl)ethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 - methoxymethyloxoheptanyl](methyl)amino}-3 -methyloxobutanyl]—2- methylpiperidinecarboxamide salt . According to the general procedure , hydrochloride C, from #177 (143 mg, 0.176 mmol, 1 eq.) and 4M solution of hydrochloric acid in dioxane (2.0 mL) was synthesized the desired material as a gum (145 mg). A portion of this crude residue (25 mg) was azeotroped with a mixture of methanol/acetonitrile to afford #178 (20 mg, 89% yield) as a white solid. 1H NMR (400 MHz, DMSO-dg), 8 .07 (m), .96 (m), 8.58 (d), 8.02- 8.08 (m), 7.77-7.83 (m), 7.25-7.31 (m), .24 (m), 6.56-6.67 (m), 6.12-6.21(m), 5.13-5.22 (m), 4.72-4.81 (m), 4.63-4.70 (m), 4.50-4.59 (m), 407-4. 16 (m), 3.98-4.05 (m), 3.80-3.86 (m), 3.55-3.76 (m), 3.46-3.54 (m), 3.38 -3.44 (m), 3.26-3.35 (m), .24 (m), 3.05-3.18 (m), 2.98- 3.04 (m), 2.40-2.55 (m), 2.27-2.35 (m), 2.08-2.27 (m), 1.74-1.98 (m), 1.50-1.74 (m), 1.20-1.46 (m), 0.73-1.16 (m). LC-MS (Protocol Q1): m/z 714.6 [M+H+], retention time = 0.76 s.
HPLC (Protocol U): m/z 714.5 [M+H+] retention time = 7.124 minutes (purity =91%).
Step 53. Synthesis of (2R)-N—[(2S){[(3R,4S,5S){(2S)[(1R,2R){[2- (cyclohepta-2,4,6-trienyl)ethyl] amino} methoxymethyl-3 opyl]pyrrolidinyl}-3 - methoxymethyloxoheptanyl](methyl)amino}-3 -methyloxobutanyl]—2- methylpiperidinecarboxamide, hydrochloride salt (#180). According to the general procedure C, from #179 (162 mg, 0.199 mmol, 1 eq.) and 4M solution of hydrochloric acid in dioxane (2.0 mL) was synthesized the desired material as a gum (155 mg). A portion of this gum (25 mg) was azeotroped with a 1/1 mixture of methanol/acetonitrile to afford #180 (20 mg, 83%) as a solid. 1H NMR (400 MHz, DMSO-d6), 8 9.02-9.13 (m), 8.83-8.93 (m), 8.39-8.46 (m), 8.00-8.06 (m), 7.78 (t), 7.24-7.30 (m), 7.16-7.21 (m), 6.54-6.65 (m), 6.09-6.19 (m), 5.1 1-5. 18 (m), 4.69-4.78 (m), 4.59-4.68 (m), 4.46-4.56 (m), 4.08-4.13 (m), 3.95-4.03 (m), 3.77-3.85 (m), 3.54-3.73 (m), 3.43- 3.53 (m), 3.37-3.42 (m), 3.24-3.33 (m), 3.16-3.22 (m), 3.03- 3.15 (m), 2.99-3.02 (m), .98 (m), 2.65-2.76 (m), 2.41-2.54 (m), 2.15-2.39 (m), 2.072 15 (m), 1.51-1.94 (m), 1.49 (d), 1.38 (t), 1.20-1.32 (m), 1.02-1.09 (m), 0.84-0.97 (m), 0.73-0.81 (m). LC-MS (Protocol Q1): m/z 714.6 [M+H+], retention time =0.76 HPLC col U): m/z 714.4 [M+H+], retention time = 7.409 minutes (purity =90%).
Preparation of 2-methyl-L-prolyl-N-[(3R,4S,5S)—1-{(2S)—2-[(1R,2R)—3-{[2—(cyclohepta-2,4,6- trien-l-yl)ethyl]amino}meth0xymethyl0X0pr0pyl]pyrrolidinyl}meth0xy methyl0x0heptanyl]-N-methyl—L-valinamide, formic acid salt (#182) and N- [(3R,4S,5S)—1-{(2S)—2- [(1R,2R)—3-{ [2-(cyclohepta-2,4,6-trienyl)ethyl] amin0}-1 -meth0xy-2— methyl0X0pr0pyl]pyrrolidin-l-yl}meth0xymethyl—l-oxoheptanyl]-N~2~-{[(3R) fluoropyrrolidinyl]carbonyl}-N-methyl—L-valinamide, trifluoroacetic acid salt (#184) and N-[(3R,4S,SS){(2S)[(1R,2R)—3-{[2-(cyclohepta-2,4,6-trienyl)ethyl]amin0} methoxy-Z-methyl0X0propyl]pyrrolidin-l-yl}meth0xymethyl—1-0X0heptanyl]- N~2-{ [(3S)—3-flu0ropyrrolidinyl] carbonyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#186).
HATUB oxane PrrQNEt CHQCl —> —> 57% (two steps) #131 0 #132 H N\)J\2 N = T /\ o O #170 o HATU \ Pr,2NEt CHQCIQ NEH/2%”! HCl/d llllll NH 0 ______ ”WNW 595“” HNOifH/iliN/o 0%:CFHCOH #176 #183 #134 #171 HATU IPerEt. CH2011 o BocN f “\A HCl/d llllll O —> F H N —.HN » UL : o /\ | a sstep) N lo : | o /\ /o CFHCOH #185 #136 Step IA. Synthesis of 1-(tert-butoxycarbonyl)methyl-L-prolyl-N-[(3R,4S,5S){(2S)- 2-[(1R,2R)-3 - {[2-(cyclohepta-2,4,6-trienyl)ethyl] amino} methoxymethyl-3 - oxopropyl]pyrrolidinyl}-3 -methoxymethyl- 1 ptan—4-yl] -N-methyl-L-valinamide (#181). ing to the general procedure D, from #176 (100 mg, 0.170 mmol, 1 eq.), (S) (tert-butoxycarbonyl)methyl-L-proline (50.7 mg, 0.221 mmol, 1.3 eq.), dichloromethane (4.3 mL), HATU (84.9 mg, 0.221 mmol, 1.3 eq.) and N,N—diisopropylethylamine (0.123 mL, 0.697 mmol, 4.1 eq.), was synthesized the crude desired material, which was purified by silica gel tography (Gradient: 0 to 100% ethyl acetate in heptane) to afford #181 (142 mg, assume quantitative yield). LC-MS (Protocol Q1): m/z 800.6 [M+H+], retention time = 1.11 minutes.
Step 18. Synthesis oftert-butyl (3R){[(2S){[(3R,4S,5S){(2S)[(1R,2R){[2- hepta-2,4,6-trien- 1 hyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 - methoxymethyloxoheptanyl](methyl)amino}-3 -methyloxobutanyl]carbamoyl}-3 - fluoropyrrolidinecarboxylate (#183). To a solution of #170 (18.2 mg, 0.076 mmol, 1 eq.) in dichloromethane (1.8 mL) and N,N—dimethylformamide (0.3 mL) was added N,N— diisopropylethylamine (0.040 mL, 0.228 mmol, 3 eq.) followed by HATU (29.2 mg, 0.076 mmol, 1 eq.). After stirring for 10 minutes at room temperature, #176 (45 mg, 0.076 mmol, 1 eq.) was added. The reaction was stirred at room temperature for 18 hours and additional HATU (29 mg, 0.076 mmol, 1 eq.) was added. After 8 hours the reaction was concentrated in vacuo to provide #183 (61.0 mg, quantitative) which was taken into the next step without further purification. LC-MS (Protocol Q1): m/z 826.6 [M+Na+], retention time = 1.05 minutes.
Step 1C. Synthesis oftert-butyl -{[(2S){[(3R,4S,5S){(2S)[(1R,2R){[2- hepta-2,4,6-trienyl)ethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 - methoxymethyloxoheptanyl](methyl)amino}-3 -methyloxobutanyl]carbamoyl}-3 - fluorocyclopentanecarboxylate (#185). To a on of #171 (24 mg, 0.1 mmol, 1 eq.) in dichloromethane (2.35 mL) and N,N—dimethylformamide (0.33 mL) was added N,N— diisopropylethylamine (0.053 mL, 0.300 mmol, 3 eq.) ed by HATU (38.4 mg, 0.100 mmol, 1 eq.). After stirring at room temperature for 10 minutes, #176 (58.9 mg, 0.1 mmol, 1 eq.) was added. The on was stirred at room temperature for 18 hours and onal quantity of HATU (38.4 mg, 0.100 mmol, 1 eq.) and N,N—dimethylformamide (0.2 mL) was added and stirred for an additional 9 hours. The reaction was concentrated in vacuo to give #185 (80 mg, quantitative), which was taken into the next step t further purification. LC-MS (Protocol Q1): m/z 804.6 [M+H+], retention time = 1.05 minutes.
Step 2A. Synthesis of 2-methyl-L-prolyl-N-[(3R,4S,5S) {(2S)[(1R,2R) {[2- (cyclohepta-2,4,6-trienyl)ethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 - methoxymethyloxoheptan—4-yl]-N-methyl-L-valinamide, formic acid salt (#182).
According to the general procedure C, from #181 (136 mg, 0.170 mmol, 1 eq.) and 4M solution of hydrochloric acid in dioxane (2 mL) was synthesized the desired material as a gum ( 142 mg).
A portion of this crude e (20 mg, 14%) was azeotroped with a 1/1 mixture of methanol/acetonitrile and then purified by reverse phase tography (Method 0) to obtain #182 (10 mg, 57% over two steps) as a solid. HPLC (Protocol U): m/z 700.4 [M+H+], retention time = 7.106 minutes (purity > 90%). 1H NMR (400 MHz, DMSO-dg), 8 8.25-8.39 (m), 8.20- 8.25 (m), 7.96-7.99 (m), 7.74-7.77 (m), 6.55-6.63 (m), 6.10-6.18 (m), 5.11- 5.18 (m), 4.66-4.72 (m), 4.51-4.61 (m), 4.46-4.50 (m), 3.96-4.01 (m), 3.37-3.86 (m), 3.20-3.36 (m), 3.11-3.19 (m), .11 (m), 2.98-3.03 (m), 2.90-2.96 (m), .79 (m), 2.65-2.73 (m), 2.57-2.63 (m), 2.47- 2.56 (m), 2.36-2.46 (m), 2.26-2.32 (m), 2.14-2.25 (m), 2.03-2.10 (m), 1.92-2.03 (m), 1.72-1.92 (m), 1.64-1.72 (m), 1.60-1.64 (m), 1.50-1.59 (m), 1.39-1.48 (m), .32 (m), 1.18-1.22 (m), 1.12-1.14 (m), 1.01-1.09 (m), 0.83-1.00 (m), .83 (m), 0.70- 0.74 (m).
Step 23. Synthesis of N—[(3R,4S,5S){(2S)[(1R,2R){[2-(cyclohepta-2,4,6-trien- 1-yl)ethyl] amino} - 1-methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 -methoxymethyl- 1- oxoheptanyl]-N~2~- {[(3R)-3 -fluoropyrrolidinyl]carbonyl} -N-methyl-L-valinamide trifluoroacetic acid salt (#184). According to the general procedure C, from #183 (61.1 mg, 0.076 mmol, 1 eq.), romethane (0.3 mL), and a 4M on of hydrochloric acid in dioxane (0.9 mL) was synthesized the crude desired material (140 mg). A portion of the crude material (98 mg, 69 %) was purified by reverse phase chromatography (Method M*) to give #184 (7.6 mg, 20% over two steps). HPLC (Protocol T): m/z 704.5 [M+H+], retention time = 2.50 minutes (purity = 84%). 1H NMR (400 MHz, DMSO-dg), 8 8.67-8.71 (m), 8.44-8.48 (m), 8.27-8.33 (m), 8.22-8.27 (m), 7.97-8.03 (m), 7.84-7.89 (m), 7.74-7.81 (m), .48 (m), 7.23- 7.29 (m), 7.17-7.21 (m), .66 (m), 6.10-6.19 (m), 5.10-5.19 (m), 4.66-4.75 (m), 4.51-4.63 (m), 3.96-4.04 (m), 3.78-3.85 (m), 3.65-3.73 (m), 3.46-3.62 (m), 3.37-3.45 (m), 3.24-3.37 (m), 3.21-3.24 (m), .21 (m), 3.02-3.13 (m), 2.95-3.00 (m), 2.80-2.82 (m), 2.66-2.71 (m), 2.47- 2.57 (m), 2.24-2.46 (m), 2.09-2.24 (m), 1.95-2.05 (m), 1.49-1.93 (m), 1.22-1.34 (m), 1.02-1.09 (m), 0.83-1.01 (m), 0.74-0.82 (m).
Step 2C. Synthesis of N—[(3R,4S,5S)- 1- {(2S)[( 1R,2R) yclohepta-2,4,6-trien- 1-yl)ethyl] amino} - 1-methoxymethyl-3 opyl]pyrrolidinyl}-3 -methoxymethyl- 1- oxoheptanyl]-N~2~- {[(3 S)-3 -fiuoropyrrolidin-3 -yl]carbonyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (#186). According to the general procedure C, from #185 (80.3 mg, 0.1mmol, 1 eq.), dichloromethane (0.4 mL), and a 4M solution of hloric acid in dioxane (1.2 mL) was synthesized the crude desired material, in which a portion ( 94 mg, 53%) was purified by reverse phase chromatography (Method M*) to give #186 (11.8 mg, 32% over two steps). 1H NMR (400 MHz, DMSO-d6), 8 .07 (m), 8.29-8.41(m), 7.99-8.04 (m), 7.76-7.84 (m), .68 (m), 6.12-6.21 (m), 5.12-5.21 (m), 4.87-4.99 (m), 4.69-4.79 (m), 4.49-4.67 (m), 3.98-4.06 (m), 3.80-3.87 (m), 3.64-3.76 (m), 3.55-3.64 (m), 3.47-3.54 (m), 3.39-3.46 (m), 3.26- 3.39 (m), 3.22-3.25 (m), 3.18- 3.22 (m), 3.06-3.14 (m), 2.98-3.01 (m), 2.55- 2.57 (m), 2.42-2.49 (m), 2.11-2.38 (m), 2.09 (s), 1.78-1.97 (m), 1.72-1.77 (m), 1.51-1.71 (m), 1.24-1.36 (m), 1.07 (dd), 0.83-1.03 (m), 0.75- 0.82 (m). HPLC (Protocol T ): m/z 704.5 [M+H+], retention time = 2.48 minutes (purity = 100%).
Preparation of (2S)—N—[(2S){[(3R,4S,5S)—3-meth0xy{(2S)-2—[(1R,2R)—1-meth0xy methyl0X0{ [(1 henyl(1 ,3-thiazolyl)ethyl] amino} propyl] pyrrolidin-l-yl}-5— methyl0x0heptanyl](methyl)amin0}methyl—l-oxobutan-Z-yl]methylpiperidine carboxamide, formate salt (#188) and (2R)—N-[(2S)—1-{[(3R,4S,5S)meth0xy{(2S)—2- [(1R,2R)—1-meth0xymethyl0X0{[(1S)—2-phenyl—1-(1,3-thiazol yl)ethyl] amin0}pr0pyl] pyrrolidin-l-yl}methyl0X0heptanyl] l)amin0} methyl-l-oxobutan-Z-yl]methylpiperidine-Z-carboxamide, formate salt (#190) and N~2~- {[(3R)flu0ropyrrolidinyl]carbonyl}-N—[(3R,4S,5S)—3-meth0xy{(2S)[(1R,2R)—1- methoxy-Z-methyl0X0{[(1S)—2-phenyl(1,3-thiazol yl)ethyl]amin0}pr0pyl]pyrrolidin-l-yl}methyl0X0heptanyl]-N-methyl-L-valinamide , trifluoroacetic acid salt. (#192) and (3S)flu0ropyrrolidinyl]carbonyl}-N- [(3R,4S,5S)—3-meth0xy{(2S)—2-[(1R,2R)methoxy-Z-methyl0X0{[(1 S)phenyl (1,3-thiaz01—2—yl)ethyl]amin0}pr0pyl] pyrrolidin-l-yl}methyl-l-oxoheptanyl] -N- methyl-L-valinamide , trifluoroacetic acid salt. (#194). «155 o HATU 1Pr~N E1 o \\ H H . A . .s H H _ N N N CHZClz, ' NdL N N 9'0““ N $11 N N = _.H g I HCOzH soc O o o O = o I \ o | O o O /\ \ /\ 51% s \N \ 5&5 \N 39% \=/ 11187 mm *157 HATU. iPer El. 0 AN HCl H CHZCQ ndL N N HCOZH goo N o o o o —> o /"\ I o\ o o\ o \ \ s \N \_IN o 37% \_/— H N2 \RLN N N 1:189 1:190 /=\ I o\ o o\ o s \N *155 HATU iPrZNE‘B ONEJLTF H 3mmAND HCl F H H #55 CH25‘, cFacozH s \Nrum O\ o\ O O\ o s\=\/N #191 #192 x159 HATU iPrNEI e F H CH012 BecN M:ONEiNH HN ; CFgC02H fl» ON\)kN o S\=\/N \ S\_/_\N 1:19: «1:14 Step IA. Synthesis of tert-butyl (2S) {[(2S) {[(3R,4S,5S)methoxy 2- [(1R,2R)methoxymethyloxo{[(1S)phenyl(1,3-thiazol yl)ethyl] amino } propyl]pyrrolidinyl} methyl- 1-oxoheptanyl] (methyl)amino} -3 -methyl oxobutanyl]carbamoyl}methylpiperidinecarboxylate (#187). ing to the general procedure D, from #86 (280 mg, 0.4 mmol, 1 eq.), #156 (100 mg, 0.4 mmol, 1 eq.), romethane (5mL), HATU (182 mg, 0.48 mmol, 1.2 eq.) and isopropylethylamine (100 mg, 0.8 mmol, 2 eq.) was synthesized the crude desired al, which was purified by silica gel chromatography (Gradient: 0.01 to 0.05% methanol in dichloromethane) to afford #187 (220 mg, 62%) as a white solid. HPLC (Protocol V): m/z 883.57 [M+H+], retention time = 3.23 minutes (purity = 95%).
Step 18. Synthesis of tert-butyl (2R){[(2S){[(3R,4S,5S)methoxy{(2S) [(1R,2R)methoxymethyloxo{[(1S)phenyl(1,3-thiazol yl)ethyl] amino } propyl]pyrrolidinyl} methyl- 1-oxoheptanyl] (methyl)amino} -3 -methyl oxobutanyl]carbamoyl}methylpiperidinecarboxylate (#189). According to the general procedure D, from #86 (400 mg, 0.6 mmol, 1 eq.), #157 (146 mg, 0.6 mmol, 1 eq.), dichloromethane (10 mL), HATU (259 mg, 0.72 mmol, 1.2 eq.) and N,N—diisopropylethylamine (158 mg, 1.2 mmol, 2 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0.01 to 0.05% methanol in dichloromethane) to afford #189 (220 mg, 37%) as a white solid. HPLC (Protocol W): m/z 883.7 , retention time = 4.12 minutes (purity = 95%).
Step I C. Synthesis of tert-butyl (3R)fluoro{[(2S){[(3R,4S,5 S)methoxy {(2S)[(1R,2R)methoxymethyloxo{[(1S)phenyl(1,3-thiazol yl)ethyl] amino } propyl]pyrrolidinyl} methyloxoheptanyl] (methyl)amino } methyl oxobutanyl]carbamoyl}pyrrolidinecarboxylate (#191). According to the l procedure D, from #86 (300 mg, 0.45 mmol, 1 eq.), #168 (106 mg, 0.45 mmol, 1 eq.), dichloromethane (10 mL), HATU (194 mg, 0.54 mmol, 1.2 eq.) and ropylethylamine (117 mg, 0.9 mmol, 2 eq.) was synthesized the crude desired material, which was purified by reverse phase chromatography (Method P) to afford #191 (159 mg, 40%) as a white solid. HPLC col X): m/z 873.4 [M+H+], retention time = 3.32 minutes (purity = 99%).
Step ID. Synthesis of tert—butyl (3 S)fluoro{[(2S){[(3R,4S,5 ethoxy {(2S)[(1R,2R)methoxymethyloxo{[(1S)phenyl(1,3-thiazol yl)ethyl] amino } propyl]pyrrolidinyl} methyl- 1 ptanyl] (methyl)amino} hyl oxobutanyl]carbamoyl}pyrrolidinecarboxylate (#193). According to the general procedure D, from #86 (300 mg, 0.45 mmol, 1 eq.), #169 (106 mg, 0.45 mmol, 1 eq.), dichloromethane (10 mL), HATU (194 mg, 0.54 mmol, 1.2 eq.) and diisopropylethylamine (117 mg, 0.9 mmol, 2 eq.) was synthesized the crude desired material, which was purified by reverse phase chromatography (Method P) to afford #193 (149 mg, 37%) as a white solid. HPLC (Protocol X): m/z 873.4 [M+H+], retention time = 3.34 minutes (purity = 98%).
WO 72813 Step 2A. Synthesis of (2S)-N-[(2S){[(3R,4S,5S)methoxy{(2S)[(1R,2R) methoxymethyl-3 -oxo-3 -{[(1S)phenyl(1,3 olyl)ethyl] amino } propyl]pyrrolidin- 1-yl} methyloxoheptan—4-yl](methyl)amino}-3 -methyloxobutanyl]—2- methylpiperidinecarboxamide, formic acid salt . According to the general procedure C, from #187 (20 mg, 0.023 mmol, 1 eq.), dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M solution of hydrochloric acid in dioxane (0.26 mL) was synthesized the crude desired material, which was d by reverse phase chromatography (Method N*) to obtain #188 (11.6 mg, 61%,); HPLC (Protocol T): m/z 783.8[M+H+], retention time = 2.53 minutes (purity= 96%). 1H NMR (400 MHz, DMSO-dg), 8 8.82-8.87 (m), 8.60-8.63 (m), 8.26-8.29 (m), 7.84-7.90 (m), 7.75- 7.81 (m), 7.60-7.67 (m), 7.21-7.31 (m), 7.14-7.19 (m), 5.49-5.55 (m), 5.37-5.42 (m), 4.69-4.75 (m), 4.59-4.65 (m), 4.50-4.56 (m), 3.95-4.01 (m), 3.77-3.82 (m), 3.54-3.61 (m), 3.47-3.53 (m), 3.24-3.45 (m), 3.14-3.23 (m), 3.03-3.08 (m), .03 (m), 2.78-2.80 (m), 2.64-2.73 (m), 2.60- 2.62 (m), 2.47-2.56 (m), 2.31-2.45 (m), 2.13-2.28 (m), 2.00-2.07 (m), 1.92-1.99 (m), 1.71-1.86 (m), 1.58-1.70 (m), 1.50-1.56 (m), 1.38-1.49 (m), 1.15-1.36 (m), 1.08-1.14 (m), .07 (m), 0.90-1.02 (m), 0.83-0.90 (m), 0.73-0.80 (m), 0.70-0.73 (m).
Step 28. Synthesis of (2R)-N—[(2S){[(3R,4S,5S)methoxy{(2S)[( methoxymethyl-3 -oxo-3 -{[(1S)phenyl(1,3 -thiazolyl)ethyl] amino } propyl]pyrrolidin- 1-yl} hyloxoheptan—4-yl](methyl)amino}-3 -methyloxobutanyl]—2- methylpiperidinecarboxamide, formic acid salt. . According to the general procedure C, from #189 (20 mg, 0.022 mmol, 1 eq.), dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M solution of hydrochloric acid in dioxane (0.26 mL) was synthesized the crude desired material, which was purified by e phase chromatography (Method N*) to obtain #190 (13.2 mg, 73%,) HPLC (Protocol T): m/z 783.7[M+H+], retention time = 2.5 minutes (purity= 100%). 1H NMR (400 MHz, DMSO-dg), 8 8.83-8.86 (m), 8.60-8.62 (m), 8.24-8.27 (m), 7.83-7.89 (m), 7.78-7.80 (m), 7.75-7.77 (m), 7.64-7.66 (m), .63 (m), 7.20-7.31 (m), 7.13-7.19 (m), 5.49- .55 (m), 5.36-5.42 (m), 4.65-4.74 (m), 4.60-4.65 (m), 4.50-4.56 (m), 3.95-4.01 (m), 3.76-3.81 (m), 3.53-3.62 (m), 3.47-3.52 (m), 3.22-3.45 (m), 3.14-3.21 (m), 2.97-3.05 (m), 2.93-2.96 (m), 2.79-2.86 (m), 2.76-2.78 (m), 2.65-2.68 (m), 2.48-2.56 (m), 2.37-2.43 (m), 2.29-2.35 (m), 2.17- 2.27 (m), 2.04-2.11 (m), 1.93-2.00 (m), 1.70-1.85 (m), 1.53-1.69 (m), .48 (m), 1.28-1.36 (m), 1.13-1.26 (m), 1.08-1.12 (m), 0.98-1.07 (m), .97 (m), 0.84-0.91 (m), 0.73-0.78 (m).
Step 2C. Synthesis ofN~2~- {[(3R)fluoropyrrolidinyl]carbonyl}-N-[(3R,4S,5 S) methoxy {(2S)[(1R,2R)methoxymethyl-3 -oxo-3 -{[(1S)phenyl(1,3 -thiazol yl)ethyl] amino } propyl]pyrrolidin— 1-yl} hyloxoheptan—4-yl] -N-methyl-L-valinamide , trifluoroacetic acid salt. (#192). According to the general ure C, from #191 (10 mg, 0.011 mmol, 1 eq.), dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M solution of hydrochloric acid in dioxane (0.13 mL) was synthesized the crude desired material, which was d by reverse phase chromatography d M*) to obtain #192 (5.1mg, 60 %) HPLC (Protocol T): m/Z 773.5[M+H+ ], retention time = 2.43 minutes (purity = 100%).1H NMR (400 MHz, DMSO- d6) 5 9.32-9.44 (m), 9.17-9.21 (m), 8.91 (d), 8.63-8.69 (m), 8.38-8.43 (m), 8.22-8.27 (m), 7.80 (dd), 7.66 (dd), 7.14-7.33 (m), 5.49-5.57 (m), 5.37-5.45 (m), 4.10-4.78 (m), 3.97-4.06 (m), 3.77- 3.83 (m), 3.33-3.65 (m), 3.15-3.29 (m), 2.95-3.09 (m), 2.82-2.83 (m), 2.67-2.71 (m), 2.55-2.57 (m), 2.32-2.54 (m), 2.10-2.31 (m), 2.09 (s), 1.72-1.90(m), 1.57-1.72 (m). 1.38-1.50 (m), 1.15- 1.38 (m), 1.09 (dd), 0.85-1.0 (m), 0.75-0.83 (m).
Step 2D. Synthesis - {[(3 S)fluoropyrrolidinyl]carbonyl} R,4S,5 S) methoxy-l-{(2S)[(1R,2R)methoxymethyloxo{[(1S)phenyl(1,3-thiazol yl)ethyl]amino}propyl]pyrrolidin— 1-yl} methyloxoheptan—4-yl]-N-methyl-L-valinamide , trifluoroacetic acid salt. (#194). According to the general procedure C, from #193 (10 mg, 0.011 mmol, 1 eq.), dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M solution of hydrochloric acid in dioxane (0.13 mL) was synthesized the crude desired material, which was purified by reverse phase tography (Method M* ) to obtain #194 (9 mg, 93%) HPLC col T): m/Z 773.8 [M+H+], retention time = 2.42 minutes (purity = 100%). 1H NMR (400 MHz, DMSO- d6) 8 9.39-9.52 (m), 9.21-9.35 (m), 8.90 (d), 8.63-8.69 (m), 8.42-8.48 (m), 8.29 -8.34 (m), 7.80 (dd), 7.66 (dd), 7.22-7.33 (m), .21 (m), 5.47-5.57 (m), 5.36-5.44 (m), 4.43-4.93 (m), 3.97- 4.05 (m), 3.64-3.83 (m), .61 (m), 3.15-3.29 (m), 3.06-3.09 (m), 2.95-3.05 (m), 2.89-2.95 (m), 2.82-2.84 (m), .72 (m), .56 (m), 2.48-2.53 (m), 2.28-2.48 (m), 2.11-2.28 (m), 2.09 (s), 1.57-1.72 (m), 1.38-1.47 (m), 1.15-1.37 (m), 1.09 (dd), 0.85-0.99 (m), 0.78 (t).
Preparation of 1,2—dimethyl-D-prolyl-N-[(3R,4S,5S)—1-{(2S)—2-[(1R,2R)—3-{[(2S)(4- aminophenyl)—1-meth0xy0X0pr0panyl]amino}meth0xymethyl oxopropyl]pyrrolidin-l-yl}meth0xymethyl0X0heptanyl]-N-methyl-L-valinamide, formate salt (#200) and 1,2-dimethyl-D-prolyl-N-[(3R,4S,5S){(2S)—2-[(1R,2R)—3-{[(2S)—3- (4-amin0phenyl)—1-meth0xy0X0pr0panyl]amin0}meth0xymethyl oxopropyl]pyrrolidin-l-yl}meth0xymethyl0X0heptanyl]-N-methyl-L-valinamide, formate salt (#201).
OmiF o #152 W0N\HN/éii/N W0 HATU iPerEt N\HNglx/N F H NQN2 OtBu TFA FjilFo CH2C|2 ; III CH2C|2 pyridine CHZCIZ If’z“\ ()‘~\ C) OtBu —> O OH 81% (three steps) #6 #195 #196 F F FQOKF W0 W0 NHN/{N/N #103 iPerEt ng0 N\ HN\ijiN/\IN>%:\>kOQFTCH2C'2 MEL] pyridine CH2CI2 #198 #199 #197 #215 IPerEl a DMF/CHZCIZ, O ‘3‘!“ 43 “/6 F CF3CO2H HN N F 0 N\ \ F I; O 0 #200 O ? F O o .IF’IENEL N 'CF3002H DMF/CHZClg. NHN N __ \ i‘ O\ O O\ #201 56% | N Step 1. Synthesis of 1,2-dimethyl-D-prolyl-N-[(3R,4S,SS)tert-butoxymethoxy methyloxoheptan—4-yl]-N-methyl-L-valinamide . According to the general procedure D, from #6 (7.75 g, 21.6 mmol, 1 eq.), #152 (3.88 g, 21.6 mmol, 1 eq.), dichloromethane (100 mL), HATU (9.8 g, 25.9 mmol, 1.2 eq.), and diisopropylethylamine (11.1 g, 86.4 mmol, 4 eq.) was synthesized the crude desired material, which was purified by silica gel tography (Gradient: 20 to 55% ethyl acetate in petroleum ether) to afford #195 (11.1 g, tative yield) as a yellow oil.
Step 2. Synthesis of 1,2-dimethyl-D-prolyl-N—[(2R,3S,4S)carboxymethoxy methylhexanyl]-N-methyl-L-valinamide (#196). According to the general procedure B, from #195 (11.1 g, 21.6 mmol, 1 eq.), dichloromethane (100 mL) and oroacetic acid (40 mL) was synthesized the crude desired material, to obtain #196 (10.1 g, quantitative yield) which was used in the next step without further purification. LC-MS (Protocol Z): m/Z 428.5 , retention time = 0.9 minutes.
Step 3. Synthesis of 1,2-dimethyl-D-prolyl-N—[(3R,4S,5S)methoxymethyloxo (pentafluorophenoxy)heptanyl]-N-methyl-L-valinamide (#197). To a cooled solution (0 °C) of #196 (4.0 g, 9.4 mmol, 1 eq.) in dichloromethane (40 mL) was added dropwise pyridine (2.95 g, 37.6 mmol, 4eq.) followed by a solution of pentafluorophenyl trifluoroacetate (3.9 g, 13.6 mmol, 1.4 eq.) in dichloromethane (5 mL). The mixture was stirred at room temperature for one hour, and the solvent was concentrated in vacuo. The e was purified by silica gel chromatography (Gradient: 1 to 10% methanol in dichloromethane) to afford compound #197 (4.5 g, 81.2% (over three steps) as white solid.
Step 4. sis of 1,2-dimethyl-D-prolyl-N—[(3R,4S,5S){(2S)[(1R,2R) carboxymethoxypropyl]pyrrolidinyl} -3 -methoxymethyloxoheptanyl]-N-methyl-L- valinamide, trifluoroacetic acid salt . To a cooled solution (0 °C) of #197 (4.0 g, 7.4 mmol, 1 eq.) in dichloromethane (25 mL) was added dropwise diisopropylethylamine (3.4 g, 26.3 mmol, 3.5 eq.) followed by a on #103 (2.3 g, 7.6 mmol, 1.02 eq.) in dichloromethane (15 mL). After the addition, the mixture was stirred at room temperature for 16 hours and the solvent was removed in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to % ol in dichloromethane) followed by a second purification by e phase chromatography (Method Q) to give #198 (1.57 g, 57.5%) as white solid HPLC (Protocol X): m/z 597.49 [M+H+] retention time = 8.879 minutes (purity = 98%). Chiral HPLC retention time: 3.328 min (purity = 98%) Column: Column: Chiralcel OJ-H, 250 x 4.6 mm, 5 pm; Mobile phase: methanol (0.05% diethylamine ) in C02 from 5% to 40% over 15 minutes; Flow rate: 2.35 mL/minute.
Step 5. Synthesis of 1,2-dimethyl-D-prolyl-N—[(3R,4S,5S)methoxy{(2S) [(1 R,2R)- 1 xymethyl-3 -oxo-3 -(pentafluorophenoxy)propyl]pyrrolidinyl}-5 -methyl- 1-oxoheptanyl]-N-methyl-L-valinamide (#199). To a solution of #198 (280 mg, 0.394 mmol, 1 eq.) in dichloromethane (2 mL) was added pyridine (75 mg, 0.94 mmol, 2.4 eq.) followed by a solution of pentafluorophenyl trifluoroacetate (268 mg, 0.94 mmol, 2.4 eq.) in dichloromethane (1.5 mL). The mixture was stirred at room ature for 2.5 hours, and the solvent was concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to % methanol in dichloromethane) to afford compound #199 (279 mg, 39%) as white solid. LC- MS (Protocol Q1): m/z 763.5 [M+H+], retention time = 0.93 minutes.
Step 6A. Synthesis of 1,2-dimethyl-D-prolyl-N—[(3R,4S,5S)methoxy{(2S) [(1 R,2R)methoxy-3 - {[(2S)methoxyoxo-3 -(1,2,3,4-tetrahydroquinolinyl)propan—2- yl] amino} methyl-3 -oxopropyl]pyrrolidinyl}methyloxoheptanyl]-N-methyl-L- valinamide, trifluoroacetic acid salt (#200). To a e of #199 (25 mg, 0.033 mmol, 1 eq) and #215 ((7.7 mg, 0.033 mmol, 1 eq.) in dichloromethane (1.5 mL), N,N—diisopropylethylamine (30.2 mg, 2.31 mmol, 7 eq.) was added. The reaction was d for 5 minutes and N,N- dimethylformamide (0.5 mL) was added. After stirring for 2 1/2 hours, additional N,N- diisopropylethylamine (30.2 mg, 2.31 mmol, 7 eq.) was added. After 3 1/2 hours, more N,N- dimethylformamide (0.75 mL) was added and the mixture d for 18 hours. Additional N,N- diisopropylethylamine (15.1 mg, 1.15 mmol, 3.6 eq.) and N,N—dimethylformamide (0.25 mL) were added and reaction stirred at room temperature for 48 hours. The reaction mixture was concentrated in vacuo and the crude product was purified by reverse phase chromatography (Method M*) to give #200 (12.9 mg, 48 %). HPLC (Protocol T ): m/z HPLC (Protocol T): m/z 407.7, double charge [2+], retention time = 1.69 minutes (purity = 100 %). 1H NMR (400 MHz, DMSO-d6) 8 9.72-9.82 (m), 8.61-8.67 (m), 8.42-8.48 (m), 8.19-8.24 (m), .27 (m), 7.12- 7.14 (m), 6.94-7.01 (m), 6.88- 6.94 (m), 6.78- 6.84 (m), 6.67- 6.74 (m), 6.57-6.64 (m), 4.69-4.77 (m), .68 (m), 4.53-4.60 (m), 4.46-4.53 (m), 4.37-4.45 (m), 3.97-4.05 (m), .81 (m), .67 (m), 3.53-3.62 (m), 3.44-3.52 (m), 3.32-3.38 (m), 3.27-3.32 (m), 3.22-3.27 (m), 3.15- 3.22 (m), 3.06-3.14 (m), .01 (m), 2.92-2.96 (m), 2.74-2.83 (m), 2.61-2.74 (m), 2.57-2.61 (m), 2.48-2.56 (m), .46 (m), 2.25-2.36 (m), 2.00-2.20 (m), 1.67-1.91 (m), 1.47-1.60 (m), 1.37-1.47 (m), 1.24-1.35 (m), 1.03-1.10 (m), 0.95-1.00 (m), 0.88-0.94 (m), 0.76-0.83 (m).
Step 63. Synthesis of 1,2-dimethyl-D-prolyl-N—[(3R,4S,5S){(2S)[(1R,2R){[(2S)- 3 -(4-aminophenyl)methoxy-1 -oxopropanyl] amino} methoxymethyl-3 - oxopropyl]pyrrolidinyl} -3 -methoxymethyloxoheptan—4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (#201). To a mixture of #199 (25 mg, 0.033 mmol, leq) and the commercially available methyl 4-amino-L-phenylalaninate (8.8 mg, 0.033 mmol, 1 eq.) in dichloromethane (1.5 mL), N,N—diisopropylethylamine (30.2 mg, 2.31 mmol, 7 eq.) was added.
The reaction was stirred for 5 minutes and N,N—dimethylformamide (0.5 mL) was added. After 4 hours, onal MN—diisopropylethylamine (37.75 mg, 2.88 mmol, 8.25 eq.) was added and the e stirred for 50 minutes. Additional N,N—dimethylformamide (0.75 mL) was added and the reaction was stirred for 66 hours, concentrated in vacuo and the crude t was purified by reverse phase chromatography (Method M* ) to give #201 (14.3 mg, 56 %); HPLC (Protocol T): m/z 387.2, double charge [2+], retention time = 1.50 minutes (purity: 100%).; 1H NMR (400 MHz, DMSO-d6) 8 9.68-9.84 (m), 8.57-8.66 (m), 8.46-8.51 (m), 8.23-8.29 (m), 7.18-7.28 (m), 7.11-7.16 (m), 7.03-7.08 (m), 6.97-7.02 (m), 4.67-4.75 (m), 4.58-4.66 (m), 4.34-4.57 (m), 3.95- 4.03 (m), 3.85-3.90 (m), 3.73-3.81 (m), 3.66-3.72 (m), 3.57-3.66 (m), 3.50-3.57 (m), 3.42-3.49 2012/056224 (m), 3.32-3.38 (m), 3.14—3.30 (m), 2.95—3.11 (m), 2.84-2.94 (m), 2.78-2.81 (m), 2.63-2.74 (m), 2.46-2.57 (m), 2.34—2.45 (m), 2.19—2.34 (m), 1.972. 19 (m), 1.67-1.90 (m), 1.45-1.62 (m), 1.34— 1.41 (m), 1.21—1.34 (m), 1.01—1.10(m),0.82—0.99(m),0.72—0.81 (m).
Preparation of 1,2-dimethyl-L-prolyl-N-[(3R,4S,SS)methoxy{(ZS)[(1R,2R) methoxy { [(ZS)-1 -meth0xy0X0(1 ,2,3,4-tetrahydr0quinolinyl)pr0panyl] amino}- yl—3-0X0pr0pyl]pyrrolidin-l-yl}methyl-l-oxoheptanyl]-N-methyl-L-valinamide, formate salt. (#207) and methyl—L-prolyl-N-[(3R,4S,SS){(ZS)[(1R,2R){[(ZS) (4-amin0phenyl)—1-meth0xy0X0pr0panyl]amin0}meth0xymethyl oxopropyl]pyrrolidin-l-yl}meth0xymethyl0X0heptanyl]-N-methyl-L-valinamide formate salt (#208) and 1,2-dimethyl—L-prolyl-N-[(3R,4S,SS)methoxy{(ZS)[(1R,2R)— 1-meth0xy{[(ZS)meth0xy0X0phenylpr0pan—2—yl]amin0}methyl oxopropyl]pyrrolidin-l-yl}methyl0X0heptanyl]-N-methyl-L-valinamide, trifluoroacetic acid salt (#209) _: o FfioEF #150 W N\HN\')J\N m; Fr; HATU 'PerEt TFA N\ HN\)L H N?“ 01BU : 2 CH2CI2 / pyridine CH2CI2 /_\ CH2C|2 O OlBu —> 82% two steps #202 #203 2 Q—KVL m N:HN:)OJ\N o #103 N HNQL iPr2NE1 \ F E /N F CH2C|2 o\ ne CH2CI2 o O \ F o 97% #204 #205 O OH #206 #215 'Pbiz/lF/CHZCb‘NEt‘ 0::(0 O N\HN‘I)LN 'CF3C02H 44% /‘\’ -0 NH2 0% 0 NH2 g O ‘ o IPr2NEt, QEKVLN\ iI ' DMF/CH2CIZ‘ o N \ O NH2 51 % #203 ,0 O O\ 'o NH2 04 o (DH/Q N\HN\é)L;\J I NEt‘ /\ ‘DIIrAQF/CHZCIZ,P SN 41 % O 0‘ #209 N 2012/056224 Step 1. sis of 1,2-dimethyl-L-prolyl-N—[(3R,4S,5S)tert—butoxymethoxy methyloxoheptanyl]-N-methyl-L-valinamide (#202). According to the general procedure D, from #6 (4.3 g, 12 mmol, 1 eq.), #150 (2.15 g, 12 mmol, 1 eq.), dichloromethane (50 mL), HATU (5.46 g, 14 mmol, 1.2 eq.), and diisopropylethylamine (8.17 mL) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 20 to 55% ethyl acetate in petroleum ether) to afford #202 (5 .2 g, 89 %) as a yellow oil.\ Step 2. Synthesis of methyl-L-prolyl-N—[(2R,3S,4S)carboxymethoxy methylhexanyl]-N-methyl-L-valinamide (#203). According to the general procedure B, from #202 (5.2 g, 10.77 mmol, 1 eq.), dichloromethane (45 mL), and trifluoroacetic acid (20 mL) was synthesized the crude desired material, to obtain #203 (7 g, quantitative yield) which was used in the next step without further purification.
Step 3. Synthesis of methyl-L-prolyl-N—[(3R,4S,5S)methoxymethyloxo (pentafluorophenoxy)heptanyl]-N-methyl-L-valinamide (#204). To a cooled solution (0 °C) of #203 (7.0 g, 10.77 mmol, 1 eq.) in dichloromethane (15 mL) was added dropwise pyridine (3.41 g 43.08 mmol, 4eq.) followed by a solution of pentafluorophenyl roacetate (6.03 g, 21.54 mmol, 2 eq.) in dichloromethane (7 mL). The mixture was stirred at room temperature for one hour, and the solvent was concentrated in vacuo. The e was purified by silica gel chromatography (Gradient: 1 to 10% methanol in dichloromethane) to afford compound #204 (8 g, 82% over two steps) as yellow solid.
Step 4. Synthesis of 1,2-dimethyl-L-prolyl-N—[(3R,4S,5 S) {(2S)[(1R,2R) carboxymethoxypropyl]pyrrolidinyl} -3 -methoxymethyloxoheptanyl]-N-methyl-L- valinamide (#205). To a cooled solution (0 °C) of #204 (8.0 g, 10.77 mmol, 1 eq.) in romethane (25 mL) was added dropwise diisopropylethylamine(5 .6 g, 43.08 mmol, 4 eq.) followed by a solution of #103 (3.22 g, 10.77 mmol, 1 eq.) in dichloromethane ( 15 mL). After the addition, the e was stirred at room temperature for 16 hours and the solvent was removed in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to 10% methanol in dichloromethane) to give #205 (2.2 g, 33 %) as a yellow solid HPLC (Protocol X): m/Z 597.42 [M+H+], retention time = 8.729 minutes (purity > 97%), Chiral HPLC retention time: 2.87 min (purity = 89%) Column: cel OD-3, 150 x 4.6 mm, 3 um; Mobile phase: ethanol (0.05% diethylamine ) in C02 from 5% to 40% over 12 minutes; Flow rate: 2.5 mL/minute. 2012/056224 Step 5. Synthesis of 1,2-dimethyl-L-prolyl-N—[(3R,4S,5S)methoxy{(2S) [(1 R,2R)methoxymethyl-3 -oxo-3 -(pentafluorophenoxy)propyl]pyrrolidinyl}-5 l- 1-oxoheptanyl]-N-methyl-L-valinamide (#206). To a solution of #198 (0.28 g, 0.47 mmol, 1 eq.) in dichloromethane (2 mL) was added pyridine (75 mg, 0.94 mmol, 2 eq.) ed by a solution of pentafluorophenyl trifluoroacetate (268 mg, 0.94 mmol, 2 eq.) in dichloromethane (1.5 mL). The mixture was stirred at room temperature for 2.5 hours, and the solvent was concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to % methanol in dichloromethane) to afford compound #206 (348 mg, 97%) as white solid. LC- MS (protocol Q1): m/z 763.5 [M+H+], retention time = 0.9 minutes.
Step 6A. sis of methyl-L-prolyl-N—[(3R,4S,5S)methoxy{(2S) [(1 R,2R)methoxy-3 - {[(2S)methoxyoxo-3 -(1,2,3,4-tetrahydroquinolinyl)propan—2- yl] amino} methyl-3 -oxopropyl]pyrrolidinyl}methyloxoheptanyl]-N-methyl-L- valinamide, trifluoroacetic acid salt. (#207). The title compound was prepared from #206 (25 mg, 0.033 mmol, leq.) and #215 (7.7 mg, 0.033 mmol, 1 eq) using the method described above for preparation of #200. The crude product was purified by reverse phase chromatography (Method M*) to give #207 (11.7 mg, 44%). HPLC (Protocol T ): m/z 407.6, double charge [2+], retention time = 1.59 minutes (purity = 100%). 1H NMR (DMSO-d6) 8 9.57-9.69 (m), 8.68-8.76 (m), 8.42-8.47 (m), 8.23-8.29 (m), 8.18-8.23 (m), .27 (m), 6.95-7.01 (m), 6.89-6.94 (m), 6.80- 6.86 (m), 6.70-6.78 (m), .67 (m), 4.69-4.77 (m), 4.60-4.68 (m), 4.46-4.60 (m), 4.34-4.46 (m), 3.95-4.03 (m), 3.87-3.91 (m), 3.79-3.85 (m), 3.74-3.79 (m), 3.60-3.66 (m), 3.49-3.60 (m), 3.41-3.49 (m), 3.12-3.35 (m), 3.04-3.12 (m), 2.89-3.04 (m), 2.68-2.83 (m), 2.61-2.67 (m), 2.45- 2.55 (m), 2.34-2.44 (m), 2.08-2.33 (m), 2.05-2.08 (m), 1.92-2.05 (m), 1.75-1.91 (m), 1.65-1.75 (m), 1.59-1.64 (m), 1.34-1.58 (m), 1.20-1.31 (m), 1.01-1.09 (m), .99 (m), 0.84-0.93 (m), .83 (m), 0.72-0.80 (m).
Step 68. Synthesis of 1,2-dimethyl-L-prolyl-N—[(3R,4S,5S){(2S)[(1R,2R){[(2S)(4-aminophenyl)methoxy-1 -oxopropanyl] amino} methoxymethyl-3 - pyl]pyrrolidinyl}-3 -methoxymethyloxoheptan—4-yl] -N-methyl-L-valinamide trifluoroacetic acid salt (#208). To a mixture of #206 (25.0 mg, 0.033 mmol, 1 eq.), and the cially available methyl 4-amino-L-phenylalaninate (8.8 mg, 0.033 mmol, 1 eq.) in dichloromethane (1.5 mL),N,N—diisopropylethylamine (30.2 mg, 2.31 mmol, 7 eq.) was added.
The reaction was stirred for 5 minutes and N,N—dimethylformamide (0.5 mL) was added. After 2 1/2 hours, additional N,N—diisopropylethylamine (30.2 mg, 2.31 mmol, 7 eq.) was added. After stirring for 3 1/2 hours, onal N,N—dimethylformamide (0.75 mL) was added and The WO 72813 reaction was stirred for 66 hours, concentrated in vacuo and the crude product was purified by e phase chromatography (Method M*) to give #208 (13 mg, 51 %); HPLC (Protocol T): m/z 387.2, double charge [2+], retention time = 1.58 s (purity: 100%); 1H NMR (400 MHz, DMSO-d6) 8 9.54-9.69 (m), 8.68-8.75 (m), 8.46-8.50 (m), 8.33-8.37 (m), 8.22-8.31 (m), 8.09-8.14 (m), 7.17-7.27 (m), 7.07-7.16 (m), 6.99-7.05 (m), 6.92-6.99 (m), 4.69-4.75 (m), 4.60- 4.68 (m), 4.42-4.59 (m), 4.34-4.42 (m), 3.95-4.03 (m), 3.85-3.90 (m), 3.74-3.80 (m), .72 (m), 3.62-3.65 (m), 3.42-3.62 (m), 3.31-3.36 (m), 3.24-3.30 (m), 3.11-3.24 (m), 3.03-3.11 (m), 2.96-3.03 (m), 2.81-2.92 (m), 2.65-2.76 (m), 2.43-2.55 (m), 2.34-2.43 (m), 2.06-2.33 (m), 1.93- 2.05 (m), .89 (m), 1.66-1.74 (m), .65 (m), 1.47-1.57 (m), 1.34-1.43 (m), 1.20-1.32 (m), 1.10-1.14 (m), 1.00-1.09 (m), 0.83-0.99 (m), 0.72-0.81 (m).
Step 6C. Synthesis of 1,2-dimethyl-L-prolyl-N—[(3R,4S,5S)methoxy{(2S) [(1 R,2R)methoxy-3 - {[(2S)methoxyoxo-3 -phenylpropanyl] amino} methyl-3 - oxopropyl]pyrrolidinyl} hyloxoheptanyl] -N-methyl-L-valinamide, trifluoroacetic acid salt (#209). The title compound was prepared from #206 (25.0 mg, 0.033 mmol, 1 eq.) and methyl -L-phenylalaninate hydrochloride (7.1 mg, 0.033 mmol, 1 eq.) using the method described above for preparation of #200. The crude product was purified by reverse phase chromatography d M* ) to give #209 (10.3 mg, 41%). HPLC (Protocol T): m/z 758.7 [M+H+], retention time = 1.787 minutes (purity= 100%).1H NMR (DMSO-d6) 8 9.57-9.70 (m), 8.70-8.75 (m), 8.50-8.56 (m), 8.32-8.42 (m), 8.24-8.26 (m), 8.10-8.15 (m), 7.14-7.27 (m), 7.12- 7.13 (m), 6.98-7.00 (m), 4.69-4.77 (m), 4.55-4.67 (m), 4.43-4.53 (m), 3.94-4.02 (m), 3.73-3.78 (m), 3.62-3.68 (m), 3.48-3.60 (m), 3.39-3.48 (m), 3.23-3.33 (m), 3.13-3.22 (m), .13 (m), 3.02-3.08 (m), 2.96-3.01 (m), 2.81-2.94 (m), 2.76-2.80 (m), 2.66-2.75 (m), 2.62-2.66 (m), 2.46- 2.55 (m), 2.31-2.45 (m), 2.09-2.29 (m), 2.05-2.09 (m), 1.93-2.04 (m), .88 (m), .74 (m), 1.59-1.65 (m), 1.36-1.52 (m), 1.21-1.35 (m), 1.01-1.08 (m), 0.94-1.00 (m), 0.83-0.94 (m), 0.73-0.81 (m).
Preparation of methyl (2S)—2-amin0(1,2,3,4-tetrahydroquinolinyl)propanoate O N\ /0 130020 N N \ NBS \ N / TFA/HZO/ / Elan BPO "-5"le THF_ acetonitrile CHZCIZ / Br —> / —> (“KfN —> —> HZN COOMe 32% (2 steps) N /o 74% (2 steps) #210 #211 #212 H H \ N N H2,Pd-C TFA O EtOH CHZCIZ o ” COOMe 80% o ” COOMe 85% HZN COOMe #213 #214 #21 5 Step 1. Synthesis of momethyl)quinoline (#210). A solution of 6-methquuinoline (5 g, 35 mmol, 1 eq.), N—Bromosuccinimide (8.1 g, 45.5 mmol, 1.3 eq.) and benzoyl peroxide (840 mg, 3.5mmol, 0.1 eq.) in carbon tetrachloride (100 mL) was stirred at reflux for 3 hours and then cooled to room temperature. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was dissolved in tetrahydrofuran (100 mL) and filtered. The filtrate was directly used in the next step without further purification Step 2. Synthesis of 6- {[(2S,5R)-3,6-dimethoxy(propanyl)-2,5-dihydropyrazin—2- yl]methyl}quinoline (#211). To a cooled solution (-70 0C) of (2R)-3,6-dimethoxy(propan yl)-2,5-dihydropyrazine (25.8 g, 140 mmol, 2 eq.) in tetrahydrofuran (200 mL) was added dropwise n-butyllithium (2.5 M, 64.4 mL, 161 mmol. 2.3eq.) and then stirred for 30 minutes. A solution of #210 (15.4 g, 70 mmol, 1 eq.) in tetrahydrofuran (150 mL) was added dropwise at - 65°C and then the on was d for 2 hours at this temperature. The reaction was quenched by saturated aqueous ammonium chloride (100 mL) and extracted with ethyl acetate (100 mL).
The organic phase was dried over sodium e and trated in vacuo. The residue was purified by silica column chromatography (Gradient: 10 to 16% ethyl acetate in eum ether) to afford #211 (7.3 g, 32%, over two steps) as yellow solid. LC-MS (Protocol Z): m/z 326.2[M+H+], retention time = 0.88 minutes.
Step 3. Synthesis of methyl (2S)amino(quinolinyl)propanoate (#212). To a 2O solution of #211 (7.3 g, 22.5 mmol, 1 eq.) in water (25 mL) and acetonitrile (80 mL) was added roacetic acid (9 mL) at 0 OC and the on was stirred at 10°C overnight. The organic layer was removed in vacuo and the remaining aqueous layer was basif1ed to pH 9 with sodium carbonate, which was directly used for the next step.
Step 4. Synthesis of methyl -[(tert-butoxycarbonyl)amino](quinolin yl)propanoate (#213). To a solution #212 (5.2 g, 22.5 mmol, 1 eq.) and triethylamine (9.1 g, 90 mmol, 4eq.) in mixed solvent of methanol (30 mL) and water (50 mL) was added di-tert-butyl dicarbonate (17.5 g, 78.75 mmol, 3.5 eq.) at 0 OC and then the solution was stirred at 10 °C overnight. The reaction mixture was filtered and the filter cake was washed with methanol (20 mL X 2). The filtrate was extracted with ethyl acetate (50 mL X 2) and the organic phase was concentrated in vacuo. The residue was purified by silica column chromatography (Gradient: 25 to 50% ethyl acetate in petroleum ether) to afford #213 (5.5 g, 74% over two steps) as yellow oil.
LC-MS (Protocol Z): m/z 331.2[M+H+], retention time = 0.76 minutes.
Step 5. Synthesis of methyl (2S)[(tert-butoxycarbonyl)amino](1,2,3,4- tetrahydroquinolin—6-yl)propanoate (#214) A suspension of #213 (1.5 g, 4.55 mmol, 1 eq.) and palladium on carbon (150 mg) in ethanol (20 mL) was stirred at 50 0C under 30 psi of hydrogen overnight. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated in vacuo. The residue was purified by silica column chromatography (Gradient: 40% ethyl e in petroleum ether) to afford #214 (1.2 g, 80%) as an oil. 1H NMR (400 MHz, CDCl3): 8 6.70 (d, 2H), 6.40 (m, 1H), 4.95 (m, 1H), 4.49 (m, 1H), 3.77 (s, 3H), 3.28 (m, 2H), 2.97 (m, 2H), 2.71 (m, 2H), 1.95 (m, 2H), 1.26 (s, 9H), HPLC (Protocol Y): m/z 357.0 [M+Na+] retention time = 5.304 s (purity >98%). Chiral HPLC retention time: 4.64 min (purity = 98%)(Column: Chiralcel OJ—H, 150 x 4.6 mm, 5 mm Mobile phase: ethanol (0.05% diethylamine ) in C02 from 5% to 40% over 15 minutes; Flow rate: 2.5 mL/minute.
Step 6. sis of methyl (2S)amino(1,2,3,4-tetrahydroquinolinyl)propanoate (#215). To a solution of #214 (750 mg, 2.25 mmol, 1 eq.) in dichloromethane (20 mL) was added dropwise trifluoracetic acid (2 mL) at 0 oC and then the solution was stirred at 20 oC ght.
The reaction mixture was concentrated in vacuo and the residue was dissolved in water (20 mL).
The solution was basified with sodium carbonate and ted with ethyl acetate/tetrahydrofuran (30 mL X 3). The organic phase was dried over sodium e and concentrated in vacuo to afford #215 (450 mg, 85%) as a yellow oil. 1H NMR (400 MHz, CDC13): 8 6.70 (d, 2H), 6.40 (m, 1H), 3.73 (s, 3H), 3.67 (m, 1H), 3.30 (m, 2H), 2.96 (m, 1H), 2.75 (m, 3H), 1.96 (m, 2H), 1.50 (br, 2H), 1.26 (br, 1H)., HPLC (Protocol Y): m/z 235.14 [M+H+] ion time = 4.35 minutes (purity > 96%). Chiral HPLC retention time: 5.71 min (purity = 98%). n: Chiralcel OJ-H, 150 x 4.6 mm, 5 mm Mobile phase: ethanol (0.05% diethylamine ) in C02 from 5% to 40% over minutes; Flow rate: 2.5 mL/minute.
Preparation of N-{(2R,3R)—3-[(2S)—1-{(3R,4S,5S)—4-[(N-{[(3R)flu0r0pyrrolidin bonyl}-L-valyl)(methyl)amin0]methoxy—S-methylheptanoyl}pyrrolidin-Z-yl] methoxy—2-methylpropanoyl}-L-phenylalanine, trifluoroacetic acid salt (#217) and N- {(2R,3R)[(ZS){(3R,4S,SS)[(N-{[(3S)flu0r0pyrrolidinyl]carbonyl}-L- valyl)(methyl)amin0]meth0xy—5-methylheptanoyl}pyrrolidin—2—yl]meth0xy methylpropanoyl}-L-phenylalanine, trifluoroacetic acid salt (#219). #168, HATU, 1) LiOH PrNEtZ, BocN waler/THF FOHEJJLN ”ZN/giggfiviio ,DMF 2) TFA (:H2(:I2 —> Fell/1i“ 86/((lhreesleps) #216 #217 #114 #169, HATU, OOOQO: fPrN EIZ’ 1)LiOH water/THF CH2C'2v DMF 2) TFA CH2CI 0 2 —>BocN ..F HN -F “\i —, N 91/ (three steps) 5 o T \ NH #219 #218 “_.K(0 @011 Step IA. Synthesis ofmethyl N—{(2R,3R)[(2S){(3R,4S,5S)[(N—{[(3R)(tert- butoxycarbonyl)fluoropyrrolidinyl]carbonyl}-L-Valyl)(methyl)amino]methoxy methylheptanoyl}pyrrolidinyl]methoxymethylpropanoyl}-L-phenylalaninate (#216). To a solution of#168 (36.9 mg, 0.158 mmol, 1 eq.) and #114 (100 mg, 0.158 mmol, 1 eq.) in dichloromethane (3.6 mL) and N,N—dimethylformamide (0.8 mL), was added diisopropylethylamine (0.083 mL, 0.474 mmol, 3 eq.) followed by HATU (60.7 mg, 0. 15 8 mmol, 1eq.). The reaction was allowed to stir at room temperature for 18 hours, diluted with ethyl e (25 mL), washed with water (1X), 10% citric acid (IX) and brine (1X). The organic layer was dried over sodium sulfate, filtered, and the filtrate trated in vacuo to give crude #216 (220 mg, 164% of theory) which was used in next step without r purification. HPLC (protocol Q): m/z 848.6 [M+H+], retention time = 2.10 minutes.
Step 13. Synthesis of methyl N— {(2R,3R)[(2S) {(3R,4S,5S)[(N— {[(3 S)(tert- carbonyl)fluoropyrrolidinyl]carbonyl}-L-Valyl)(methyl)amino]methoxy methylheptanoyl}pyrrolidinyl]methoxymethylpropanoyl} -L-phenylalaninate (#21 8). To a solution of#169 (36.9 mg, 0.158 mmol, 1 eq.) and #114 (100 mg, 0.158 mmol, 1 eq.) in dichloromethane (3.6 mL) and N,N—dimethylformamide (0.8 mL), was added diisopropylethylamine (0.083 mL, 0.474 mmol, 3 eq.) followed by HATU (60.7 mg, 0. 15 8 mmol, 1eq.). The reaction was allowed to stir at room temperature for 18 hours, diluted with ethyl acetate (25 mL), washed with water (1 X), 10% citric acid (IX) and brine (1X). The organic layer was dried over sodium sulfate, filtered, and the filtrate concentrated in vacuo to e crude #218 (180 mg, 134% of theory) which was used in next step without further purification.
HPLC (protocol Q): m/z 848.6 [M+H+], retention time = 2.10 minutes.
Step 2A. Synthesis of N—{(2R,3R)[(2S){(3R,4S,5S)[(N—{[(3R) fluoropyrrolidinyl]carbonyl}-L-Valyl)(methyl)amino]methoxy methylheptanoyl } pyrrolidinyl] -3 -methoxymethylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt (#217). To a solution of crude #216 (134 mg) in ydrofuran (4 mL) was added lithium hydroxide (1M, 0.5 mL). The reaction was d at room temperature for 18 hours and concentrated in vacuo. The residue was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) was added. The reaction was stirred for 4 hours and concentrated in vacuo. The crude material was purified by reverse phase chromatography (Method M*) to obtain #217 (60 mg, 86% over two steps) as a gum. LC-MS (protocol Q): m/z 734.93 , ion time = 1.19 minutes. 1H NMR (DMSO-d6) 12.62-12.83 (m), 9.30-9.43 (m), 9.17-9.28 (m), 8.34-8.41 (m), 8.22-8.31 (m), .15 (m), 7.87-7.93 (m), 7.76-7.81 (m), 7.11-7.23 (m), 4.93- 4.99 (m), 4.81-4.88 (m), 4.55-4.71 (m), 4.48-4.54 (m), 4.37-4.45 (m), 3.92-3.99 (m), 3.69-3.75 (m), 3.31-3.65 (m), 3.25-3.30 (m), 3.20-3.24 (m), 312-3. 19 (m), 3.08-3.10 (m), 2.97-3.07 (m), 2.92-2.97 (m), 2.75-2.84 (m), 2.64-2.70 (m), 2.43-2.57 (m), 2.28-2.43 (m), 2.15-2.26 (m), 2.02- 2.15 (m), 1.56-1.87 (m), 1.31-1.48 (m), 1.05-1.30 (m), 0.97-1.06 (m), 0.82-0.97 (m), 0.71-0.79 (In)- Step 23. Synthesis of N—{(2R,3R)[(2S){(3R,4S,5S)[(N—{[(3S) pyrrolidinyl]carbonyl}-L-valyl)(methyl)amino]methoxy methylheptanoyl } pyrrolidinyl] -3 -methoxymethylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt . To a solution of crude #218 (100 mg) in tetrahydrofuran (4 mL) was added 1.0 M lithium hydroxide in water (0.5 mL). The reaction was d at room 2O temperature for 18 hours and then concentrated in vacuo. The crude material was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) was added. The reaction was stirred for 4 hours and then concentrated in vacuo. The crude material was purified by reverse phase chromatography (Method M*) to obtain #219 as a gum (60 mg, 91% over two steps). LC-MS (protocol Q): m/z 734.97 [M+H+], retention time = 1.14 s. 1H NMR (400 MHz, DMSO- d6), 8 12.62-12.85 (m), 9.32-9.43 (m), 9.13-9.26 (m), .46 (m), 8.30-8.39 (m), 8.25-8.29 (m), 8.08-8.13 (m), 7.79-7.85 (m), 7.67-7.72 (m), 7.10-7.23 (m), 4.94-5.01 (m), 4.83-4.89 (m), 4.64-4.73 (m), 4.56-4.63 (m), 4.44-4.50 (m), 4.37-4.44 (m), 3.92-3.99 (m), .74 (m), 3.24- 3.55 (m), 3.11-3.24 (m), 3.07-3.10 (m), 3.02-3.06 (m), 2.98-3.02 (m), 2.93-2.97 (m), 2.75-2.85 (m), 2.68-2.69 (m), .67 (m), 2.45-2.55 (m), 2.26 -2.44 (m), 2.15-2.25 (m), 2.03-2.14 (m), 1.55-1.87 (m), .47 (m), 1.15-1.31(m), 0.98-1.05 (m), 0.91-0.98 (m), O.82-O.91(m), 0.71- 0.78 (m).
Preparation of 2-methylalanyl-N-{(3R,4S,5S)[(2S)—2-{(3R,4R,7S)—7—benzylmethyl [(4S,5R)—5-methyl-Z-oxoimidazolidinyl]-5,8,13-trioxooxa-6,9,12-triazaoctadecan yl}pyrrolidinyl]methoxymethyloxoheptanyl}-N-methyl-L-valinamide . 1. 4M HCI in e 2. HATU, Hunig's base, DMF H H H2N/\/N‘Boc o O HOW/V'ENVO H o .
~ NH , , JNNN‘H H N HATU, Hunlgs base, DMF Fmoc’ \HLOH U H: 35% #253 o 0 FmOC/NgkN/VNWv/I" NF0H H H H piperdine DMF. H N\)L2 N n,_ N i ”N \n/\/\/ E F0 : H 0 . NH \“ NH 84% \© \\ #255 O H H H H ,, #253, #105, HATU. Fmom N\)L (MNEJLNNNW/..
Hunig's base. DMF H : H N>=o E Ii! 0 O O ‘ O /\ /O O \ NH #256 O H H H #256,piperdine,DMF H N\)L /\/N N N N N HZN i = H >‘0 o o \© o .
- T \ c‘ NH o /\ /o 0 50% \ #257 00703673-0529 Step 1. Synthesis of 9H-fluorenylmethyl [(2S)({2-[(tert butoxycarbonyl)amino]ethyl} oxophenylpropanyl]carbamate . Following general procedure D using N-[(9H-fluorenylmethoxy)carbonyl]-L-phenylalanine (500 mg, 1.29 mmol, 1.0 eq), tert-butyl (2-aminoethyl)carbamate (207 mg, 1.29 mmol, 1.0 eq.), HATU (620 mg, 1.55 mmol, 1.2 eq.) and Hunig’s base (0.452 mL, 2.58 mmol, 2.0 eq) in 6 mL of DMF #253 was yielded as a white solid (620 mg, 91%) following concentration of solvent and re- crystallization using ethyl acetate. LC-MS (Protocol Q1): m/z 552.3 [M+Na+], retention time = 1.01 minutes.
Step 2. Synthesis of N—alpha-[(9H-fluorenylmethoxy)carbonyl]-N-[2-({6-[(4S,5R) methyloxoimidazolidinyl]hexanoyl}amino)ethyl]-L-phenylalaninamide (#254). Boc protection was removed using general procedure C using #251 (88 mg, 0.17 mmol, 1.0 eq.) and 4M HCl (2.0 mL, 8.0 mmol, 48 eq.) followed by concentration in vacuo. ng reaction was then performed following general procedure D using crude residue, 6-[(4S,5R)methyl oxoimidazolidin—4-yl]hexanoic acid (35.6 mg, 0.166 mmol, 1.0 eq.), HATU (73.2 mg, 0.18 mmol, 1.1 eq.), and Hunig’s base (0.087 mL, 0.50 mmol, 3.0 eq.) in 2 mL of DMF followed by WO 72813 purification (Method J) yielding #254 (35 mg, 34%) as a white solid. LC-MS (Protocol Q1): m/z 626.3 [M+H+], retention time = 0.86 minutes.
Step 3. Synthesis of {6-[(4S,5R)methyloxoimidazolidin—4- yl]hexanoyl} amino)ethyl]-L-phenylalaninamide (#255). Following general procedure A using #254 (35 mg, 0.056 mmol, 1.0 eq.), piperidine (0.10 mL, 1.0 mmol, 20 eq.) in 0.5 mL of DMF followed by purification using silica chromatography (0-30% methanol in dichloromethane) affords #253 (19 mg, 84%). LC—MS (Protocol Q1): m/z 404.2 [M+H+], retention time = 0.48 minutes.
Step 4. sis of N-[(9H-fluorenylmethoxy)carbonyl]methylalanyl-N— S,5S)[(2S){(3R,4R,7S)benzylmethyl[(4S,5R)methyl oxoimidazolidinyl]-5,8, 13 -trioxooxa-6,9,12-triazaoctadecan—3-yl}pyrrolidinyl]—3 - methoxymethyloxoheptan—4-yl}-N-methyl-L-valinamide (#256). Following l procedure D using #105 (36.6 mg, 0.047 mmol, 1.0 eq.), #255 (19 mg, 0.047 mmol, 1.0 eq.), HATU (22.4 mg, 0.056 mmol, 1.2 eq.) and Hunig’s base (0.025 mL, 0.141 mmol) in 1.5 mL of DMF following by purification (Method J) yielded #256 (15 mg, 27%) as a white solid. LC-MS (Protocol Q1): m/z 1164.8 [M+H+], retention time = 0.99 s.
Step 5. Synthesis of 2-methylalanyl-N— {(3R,4S,5S)- 1-[(2S) {(3R,4R,7S)benzyl methyl[(4S,5R)methyloxoimidazolidinyl]-5,8,13-trioxooxa-6,9,12- triazaoctadecan-3 -yl}pyrrolidinyl]—3 -methoxymethyloxoheptanyl}-N-methyl-L- valinamide (#257). Following general procedure A using #256 (5mg, 0.004 mmol, 1.0 eq.) and piperidine (0.02 mL, 0.2 mmol, 50 eq.) in 0.7 mL of DMF followed by purification (Method J) afforded #257 (2 mg, 50%) as a colorless glass. LC-MS (Protocol Q1): m/z 1164.8 [M+H+], retention time = 0.99 minutes. 1H NMR (400 MHZ, DMSO-dg), 8 8.44-8.52 (m), 8.06-8.20 (m), 7.96-8.01 (m), 7.69-7.83 (m), 7.20-7.28 (m), 7.11-7.19 (m), .83 (m), 3.19-3.26 (m), 3.03- 3.12 (m), 2.98 (s), 2.91 (s), 2.75 (s), 2.65-2.70 (m), 2.01-2.36 (m), 1.65-1.87 (m), 1.39-1.57 (m), 1.13-1.37 (m), 1.04-1.08 (m), 0.74-1.01 (m).
Preparation of N-[5-(2,5-di0X0-2,5-dihydro-lH-pyrrol-l-yl)pentan0yl]-N,2-dimethylalanyl— N-{(1S,2R)—4-{(ZS)[(1R,2R)—3-{ [(1 arboxy-Z-phenylethyl] methoxy methyl0X0propyl]pyrrolidinyl}meth0xy[(1 S)—1-methylpr0pyl]0X0butyl}-N- methyl-L-valinamide (mv#1 1 5). #115, 0 «A HATU, Hunig's base O O DMF.CH2C|2 “\i QN N N N N OH \ I ' l o o\ o 3.3% O O 0 \ NH Step I . ation of N—[5-(2,5-dioxo-2,5-dihydro- 1 H-pyrrolyl)pentanoyl] -N,2- dimethylalanyl-N—{(1 S,2R){(2S)[(1R,2R){[(1S)carboxyphenylethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy[(1S)methylpropyl] oxobutyl}-N-methyl-L-valinamide (mv#115). To a stirring solution of 5-(2,5-dioxo-2,5-dihydro- 1H-pyrrolyl)pentanoic acid (12 mg, 0.061 mM) in 0.4 mL of dichloromethane, and 0.1 mL of DMF, HATU (23.2 mg, 0.061 mM) was added followed by Hunig's base (0.033 mL, 0.188 mM).
The reaction was allowed to stir for 5 minutes before #115 (39 mg, 0.047 mM) was added as a on in 0.4 mL of dichloromethane, and 0.1 mL of DMF. The on was allowed to stir at room temperature for 3 hours and 15 minutes before being quenched through the addition of water containing a small amount of TFA. Reaction was then reduced down. Crude material was dissolved with DMSO and purified by reverse phase chromatography (Method J). The appropriate fractions were concentrated then (Genevac). Material was then further purified by reverse phase chromatography (Method K) with the appropriate fractions being concentrated ac). Material was then transferred to a small vial using dichloromethane and methanol before being reduced down (Genevac) to afford mv#115 (1.4 mg, 3.3%) oil/solid mix. HPLC (Protocol A at 45 OC): m/z 897.5 [M+H+], retention time = 9.149 minutes (purity > 97%).
Preparation of N-[6-(2,5-di0X0-2,S-dihydro-lH-pyrrol-l-yl)hexan0yl]-N,2—dimethylalanyl- N-{(1S,2R)—4-{(2S)—2-[(1R,2R)—3-{ [(1 arboxy-Z-phenylethyl] methoxy 0X0pr0pyl]pyrrolidinyl}meth0xy[(1 S)—1-methylpr0pyl]0X0butyl}-N- methyl-L-valinamide (mc#1 1 5) o Oxalyl de 0 triethylamine O CHZCIZ O / o N\/\/\)J\ / o CHZCIZ / 1 )J\ OH —> N\/\/\)J\ KrOH OH CI 100% O HT 16% O [I] O O #248 0 #249 lithium hydroxide THF water HZNjLRN0%boz #113 —> /=\ #250 #249 HATU, Hunig's base #250 DMF, CHZCIZ ng|>§fNEjiRN(9%ng mc#115 Step 1. Synthesis of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl chloride (#248).
To a stirring solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoic acid (3.15 g, 14.9 mM) in 15 mL of dichloromethane, oxalyl chloride (1.61 mL, 17.9 mM) was added followed by one drop of DMF. The on was allowed to stir at room temperature for three hours. The reaction was concentrated in vacuo. The residue was dissolved in a one to one solution of heptane and dichloromethane and then concentrated in vacuo. This process was repeated two more times producing a solid #248 (3.43 g, 100%). . 1H NMR (400 MHz, DMSO-dg): 8 [7.02 (s, 2H), 3.43 (m, 2H), 2.53 (m, 1H), CH2.18 (m, 1H), 1.54 (m, 4H), 1.26 (m, 2H).] Step 2. Synthesis of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]—N,2- dimethylalanine (#249). To a stirring solution of #248 (600 mg, 2.61 mM) in 10 mL of dichloromethane, N,2-dimethylalanine (306 mg, 2.61 mM) was added ed by ylamine (1.09 mL, 7.84 mM). The reaction was allowed to stir at room temperature for three hours. romethane was added to the reaction and the organic layer was washed three times with water and two times with brine. The organic layer was separated and then dried over sodium sulfate before being concentrated in vacuo. The crude residue was purified by silica chromatography (0-30% methanol in dichloromethane) on silica which had been previously neutralized with ylamine yielding a white solid #249 (127 mg, 16%). LC-MS (Protocol Q): m/z 309.0 [M-H'], retention time = 0.96 minutes.
Step 3. Synthesis of N—{(2R,3R)methoxy[(2S) {(3R,4S,5S)methoxymethyl— 4- [methyl(L-valyl)amino]heptanoyl } pyrrolidin—2-yl] methylpropanoyl} -L-phenylalanine (#250). To a stirring solution of #113 (2.10 g, 2.46 mM) in 10 mL of THF, lithium hydroxide (228 mg, 5.16 mM) was added followed by 3 mL of water. The reaction was allowed to stir at room temperature for 2 hours. The reaction was acidified though the addition of 1 M HCl and then concentrated in vacuo. The resulting white solid was taken up in 20 mL of acetonitrile and mL of water. The aqueous layer removed and the organic layer was washed once with water.
The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Ethyl acetate (20 mL) was then added and the crude solid was allowed to stir for 30 minutes, before being filtered to yield a white solid #250 (1.42 g, 94%). LC-MS (Protocol Q): m/z 619.5 [M+H+], retention time = 1.10 minutes. HPLC (Protocol A at 45 oC) m/z 619.4 [M+H+], retention time = 6.732 minutes.
Step 4. Synthesis of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]—N,2- ylalanyl-N- {(1 4- {(2S)[(1R,2R) {[(1 S)- 1-carboxyphenylethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy[(IS)methylpropyl] oxobutyl}-N-methyl-L-valinamide 5). To a stirring solution of #249 (382 mg, 1.23 mM) in 5 mL of romethane, HATU (482 mg, 1.23 mM) was added followed by triethylamine (0.52 mL, 1.23 mM). The reaction was d to stir for 1 hour at room temperature ed by the addition of #250 (762 mg, 1.23 mM). The reaction was allowed to stir for 3 hours. on was concentrated in vacuo. Reverse phase purification (MethodL ) ed by lyophilization yielded a white solid mc#120 (124 mg, 11%). HPLC (Protocol A at 45 oC;) m/z 2O 911.5 [M+H+], retention time = 9.676 minutes.
Preparation of N- [4-(2,5-di0X0-2,5-dihydr0-1H-pyrrol-l-yl)butan0yl] -N,2-dimethylalanyl— N-{(1S,2R)—4-{(ZS)[(1R,2R)—3-{ [(1 S)—l-carboxy-Z-phenylethyl] amin0}methoxy methyl0X0pr0pyl]pyrrolidinyl}meth0xy[(1 ethylpr0pyl]0X0butyl}-N- methyl-L-valinamide (mb#115).
O O O #115 \ “\JL ég/Vfiol/ N OH HATU, Hunig's base _ N N DMF, CH CI N/\/\n,N 5 2 2 \ | O O /\ o\ 0 Ci NH Step I . Synthesis of N—[4-(2,5-dioxo-2,5-dihydro- 1 H-pyrrolyl)butanoyl] -N,2- dimethylalanyl-N- {(1 S,2R) {(2 S)[(1R,2R)-3 - {[(1 S)- 1-carboxyphenylethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy[(IS)methylpropyl] WO 72813 oxobutyl}-N-methyl-L-valinamide (mb#115). A stirring solution of 4-(2,5-dioxo-2,5-dihydro- 1H-pyrrolyl)butanoic acid (1.2 equivalents), HATU ( 1.2 lents), and Hunig’s base (3 equivalents) in DMF and dichloromethane is d to stir for 30 minutes. Compound #115 (1 lent) is then added as a solution in dichloromethane and DMF. Reaction is monitored by LC-MS. Reaction is concentrated down and purification is completed by Isco medium pressure reverse phase chromatography (Gradient: 5%-100% water in acetonitrile).
Preparation of N-[7-(2,5-di0X0-2,5-dihydro-lH-pyrrol-l-yl)heptan0yl]-N,2—dimethylalanyl— N-{(1S,2R)—4-{(ZS)[(1R,2R)—3-{ [(1 S)—l-carboxy-Z-phenylethyl] amin0}methoxy methyl0X0pr0pyl]pyrrolidinyl}meth0xy[(1 S)—1-methylpr0pyl]0X0butyl}-N- methyl-L-valinamide (me#1 1 5).
O #115 O qWVYOI—I O HATU, Hunig's base / N\/W\[FN>€(N\=)LT N DMF, CHZCIZ O /\ O\ o O O O O O \ NH me#115 ~ Step 1. sis of N-[7-(2,5-dioxo-2,5-dihydro-lH-pyrrolyl)heptanoyl]—N,2- dimethylalanyl-N—{(1 S,2R){(2S)[(1R,2R){[(1S)carboxyphenylethyl]amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy[(IS)methylpropyl] oxobutyl}-N-methyl-L-valinamide (me#115). A stirring solution 7-(2,5-dioxo-2,5-dihydro-1H- pyrrol-l-yl)heptanoic acid (1.2 equivalents), HATU (1.2 equivalents), and s base (3 equivalents) in DMF and dichloromethane is allowed to stir for 30 minutes. Compound #115 (1 equivalent) is then added as a on in dichloromethane and DMF. Reaction is monitored by LC-MS. Reaction is concentrated down and purification is completed by Isco medium pressure 2O reverse phase tography (Gradient: 5%-100% water in acetonitrile).
Preparation of N-[6-(2,5—di0X0-2,5-dihydr0-1H-pyrrol—l-yl)hexan0yl]-L-valyl-N~5~- carbamoyl-N-(4-{(SS,118,12R)—12-(2-{(ZS)[(1R,2R){[(IS)carboxy phenylethyl]amin0}meth0xy-2—methyl0X0propyl]pyrrolidin-l-yl}oxoethyl) isopropyl-4,5,5,10-tetramethyl[(1S)methylpr0pyl]-3,6,9-tri0X0-2,13-di0xa-4,7,10- triazatetradec-l-yl}phenyl)-L-0rnithinamide (chalCitPABC#115). chaICitPABC-PNP OJLTfiHN\aJLN Hunig's base 0 ' H o A O 2,6-Lutidine / N\)\N Ci HOAt,DMA N N a N” H 0 H - O O - #115 —> O \L chalCitPABC#115 6 1 OH NH Step 1. Synthesis of N—[6-(2,5-dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl]—L-valyl- N~5~-carbamoyl-N—(4- {(8S,1 IS, 12R)- 1 2-(2- {(2S)[(1R,2R) {[(1 S)carboxy phenylethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}oxoethyl)isopropyl- 4,5 ,5 , 1 O-tetramethyl- 1 1-[(1S)methylpropyl]—3 ,6,9-trioxo-2, 13 -dioxa-4, 7,10-triazatetradec yl}phenyl)-L-ornithinamide (chalCitPABC#115). A solution of mcCValCitPABC (Linker # D, 1 equivalent) and #115 (1 equivalent) in DMF is prepared. Hunig’s base (4 equivalents), 2,6- Luditine (4 equivalents), and HOAT (0.2 equivalents) is added. Reaction is monitored by LC- MS. Reaction is concentrated down and purification is completed by Isco medium pressure e phase chromatography ent: 5%-100% water in acetonitrile).
Preparation of N-(21-amin0-4,7,10,13,16,19-hexaoxahenicosan0yl)—N,2—dimethylalanyl- N-{(1S,2R)—4-{(ZS)[(1R,2R)—3-{[(1S)-l-carboxy-Z-phenylethyl]amin0}methoxy methyl0X0propyl]pyrrolidinyl}meth0xy[(1 S)—1-methylpr0pyl]0X0butyl}-N- methyl-L-valinamide (AmPeg6C2#115). 1. #115 HATU, s base DMF, CHZCIZ 0 O O H N 2. piperdine N Fmoc/ O OH HZNVOMNfiNJN 6 I E | O /\ O O 6 \ 3 NH O 5Y0 AmPegGC2#115 ) OH Step I . Synthesis ofN—(21-amino-4,7,10,13,16,19-hexaoxahenicosan—1-oyl)-N,2- ylalanyl-N-{(1S,2R){(2 S)[(1R,2R)-3 - {[(1 S)- 1-carboxyphenylethyl] amino} methoxymethyl-3 -oxopropyl]pyrrolidinyl}methoxy[(IS)methylpropyl]—4- oxobutyl}-N-methyl-L-valinamide (AmPeg6C2#115). A solution of 1-(9H-fluorenyl)oxo- 2,7,10,13,16,19,22-heptaoxaazapentacosanoic acid (1 equivalent), HATU (1 equivalent), and s base (3 lents) is allowed to stir for 30 minutes. Compound #115 is added as a solution in DMF. Reaction is monitored by LC-MS. When ng reaction is near tion, piperidine (5 equivalents) is added. Fmoc de-protection is monitored by LC-MS. Reaction is 2012/056224 trated down and purification is completed by Isco medium pressure reverse phase chromatography (Gradient: 5%-100% water in acetonitrile).
Preparation of 1,2-dimethyl—D-prolyl-N—[(3R,4S,5S){(2S)[(1R,2R)({(2S)[4-({N-[6- (2,5-di0x0-2,5-dihydr0-1H—pyrrolyl)hexan0yl] glycyl}amin0)phenyl]meth0xy oxopropan-Z-yl}amino)methoxymethyl0x0propyl]pyrrolidin-l-yl}meth0xy methyl0x0heptanyl]-N-methyl-L-valinamide mcGly#201. \i/ O O H \l/ H O 0 “\Jko/ 2 OTVLO/H HOJK’N N nor TFA/DCM HN\)I\o/ o , —.= O 0 _ 98% 0 O DCC/DMF o o \©\ N/U\/N UNJK/NWNH H 320/ N ° H NH2 H Y\/\/\ o / o / #251 o #252 #198 H \ (j/ N N H O HATU,D|PEA,DMF N N \n/\ V IN ”'1’ J'nw H o O 16.2% O/‘\ O\ 0 I N O\ mcGly#201 Step1 : Synthesis of methyl N—(tert-butoxycarbonyl)( {N—[6-(2,5-dioxo-2,5-dihydro- 1H yl)hexanoyl]glycyl}amino)-L-phenylalaninate (#251): To a solution of methyl 4-amino- N—(tert-butoxycarbonyl)-L-phenylalaninate (4.1 g, 15.3 mmol, leq.) in dry N,N— dimethylformamide (70 mL) was added N,N’-Dicyclohexylcarbodiimide (2.9 g, 15.3 mmol, leq.) at 0 0C. The mixture was stirred at 0 0C for 30 minutes. A solution of 2-(6-(2,5-dioxo-2,5- dihydro-lH-pyrrolyl)hexanamido)acetic acid (3 g, 10.2 mmol, 0.66 eq.) in dry N,N— dimethylformamide (20 mL) was added at 0 0C. The mixture was stirred at room ature for 3 days. The mixture was filtered. The filtrate was poured into ice water (200 mL) and extracted with EtOAc (200 mLX3). The extract was washed with brine (200 mL), dried over Na2S04 and concentrated in vacuo to afford #251 (1.8 g, 32.3% yield) as a light yellow solid. HPLC (Protocol Q2) [M+Na+] 567.3, retention time = 1.02 min Step 2: sis of methyl 4-({N—[6-(2,5-dioxo-2,5-dihydro- lH-pyrrol- 1- yl)hexanoyl]glycyl} amino)-L-phenylalaninate : To a on of #251 (800 mg, 1.47 mmol, 1 eq.) in dichloromethane (16 mL) was added TFA (4.8 mL) at 0 0C. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated in vacuo. The residue was dissolved in water and filtered. The filtrate was lyophilized to afford #252 (800 mg, 97.5%) as a white solid. HPLC (Protocol Q3) [M+H+] 445.4, retention time = 0.90 min.
Step 3: Synthesis of 1,2-dimethyl-D-prolyl-N—[(3R,4S,5S){(2S)[(1R,2R)({(2S)[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]glycyl}amino)phenyl]—1-methoxy- 1-oxopropanyl} - 1-methoxymethyl-3 -oxopropyl]pyrrolidin— 1-yl} -3 -methoxy-5 - methyloxoheptanyl]-N-methyl-L-valinamide (mcGly#201). To a solution of #198 (94 mg, 0.13mmol, 1 eq.) and #252 (60.3 mg, 0.18 mmol, 1.4 eq.) in N,N—dimethylformamide (2 mL) was added HATU (64.2 mg, 0.13 mmol, 1 eq.) followed by N,N— diisopropylethylamine (66 mg, 0.52 mmol). The solution was stirred at room temperature for 1 hour. The reaction mixture was neutralized with aq. critic acid and concentrated to give crude product, which was purified by silica gel chromotography (eluted with MeOH/DCM from 1% to 7%), then purified again by preparative TLC (Methanol:dichloromethane: =1: 10) to give 201 (25 mg, 16.2%) as a white solid : ESI-MS : m/z 1023.59 [M+H+], HPLC (ProtocolEB) retention time = 4.0 minutes (Purity = 96%). 1H NMR (DMSO-d6) 9.88 (d, 1H), 8.48 (d, 0.5H), 8.24 (d, 0.5H), 8.11 (m, 1H), 7.82 (m, 1H), 7.47 (d, 2H), 7.15 (m, 2H), 7.01 (s, 2H), 4.67 (m, 3H), 3.96 (m, 4H), 3.65 (m, 4H), 3.40 (m, 4H), 3.27 (m, 7H), 3.16 (m, 5H), 2.24 (m, 8H), 1.50 (m, 11H), 1.19 (m, 21H). ation of 1,2—dimethyl-L-prolyl-N-[(3R,4S,5S)—1-{(2S)—2-[(1R,2R)—3-{[(2S)—1-{[6-(2,5- di0X0-2,5-dihydr0-1H-pyrrol-l-yl)hexyl]amino}0X0phenylpr0panyl]amin0} methoxy-Z-methyl0X0pr0pyl]pyrrolidin-l-yl}meth0xy-5—methyl0X0heptanyl]-N- methyl-L-valinamide (MalC6Am#151).
HATU 6m Hunig's base gahéoromethane $3“hagyflmo [:ONONNHJ—Ff 86% 0 Step I . Synthesis of 1,2-dimethyl-L-prolyl-N—[(3R,4S,5S){(2S)[(1R,2R){[(2S)- 1-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexyl]amino}oxophenylpropanyl]amino}- 1-methoxymethyl-3 -oxopropyl]pyrrolidinyl}-3 xymethyloxoheptan—4-yl]—N— methyl-L-valinamide (MalC6Am#151). ing general procedure D using #151 (20 mg, 0.023 mmol, 1.0 eq.), 1-(6-aminohexyl)-1H-pyrrole-2,5-dione (7.0 mg, 0.030 mmol, 1.3 eq.), HATU (11.4 mg, 0.030 mmol, 1.3 eq.), and s base (0.016 mL, 0.092 mmol, 1.3 eq.) in 2 mL of dichloromethane, and 0.2 mL of DMF followed by purification using medium pressure reverse phase C18 chromatography (Gradient: 5% to 80% acetonitrile in water with 0.02% TFA in each phase) d MalC6Am#151 (18.4 mg, 86%) as a clear oil/solid mix. LC-MS (Protocol Q): m/z 922.3 [M+H+], retention time = 1.43 minutes; HPLC (Protocol A at 45 oC): m/z 922.4 [M+H+], retention time = 7.203 minutes.
Preparation of 2,5—dioxo-2,5-dihydro-1H-pyrrol-l-yl)hexanoyl]-L-valyl-N-(4- {(6S,9R,10R)—6-benzyl[(ZS){(3R,4S,SS)[(1,2—dimethyl-L-prolyl-L- valyl)(methyl)amino]methoxymethylheptanoyl}pyrrolidin—Z-yl]methyl-3,8-dioxo- 2,11-dioxa-4,7-diazadodecyl}phenyl)-N~5~-carbamoyl-L-ornithinamide (chalCitPABC#246) chaICilPABC-PNP Hunig'5 base 26-Lulidine HOAI DMA o HJkOHMQ VJKA/VNO \ NH — N7?)' N\ /\ N #246 chaICilPABC#246 H2N*0 Step 1. Synthesis of N—[6-(2,5-dioxo-2,5-dihydro- lH-pyrrolyl)hexanoyl]—L-valyl-N— (4-{(6S,9R,10R)benzyl[(2S){(3R,4S,5S)[(1,2-dimethyl-L-prolyl-L- valyl)(methyl)amino] -3 -methoxymethylheptanoyl}pyrrolidinyl] methyl-3 ,8-dioxo-2,1 1- dioxa-4,7-diazadodec- 1-yl}phenyl)-N~5~-carbamoyl-L-ornithinamide (chalCitPABC#246).
Following general procedure E using #246 (29.2 mg, 0.035 mmol, 1.0 eq.), tPABC-PNP (28.8 mg, 0.039 mmol, 1.1 eq.), 2,6-Luditine (0.016 mL, 0.14 mmol, 4.0 eq.), s base (0.025 mL, 0.14 mmol, 4.0 rq.), and HOAT (4.8 mg, 0.035 mmol, 1.0 eq.) in 2.0 mL of DMA ed by purification using medium pressure reverse phase C18 chromatography (Gradient: 5% to 50% acetonitrile in water with 0.02% TFA in each phase) yielded chalCitPABC#246 (21 mg, 45%) as a clear oil/solid mix. LC-MS (Protocol Q): m/z 1327.9 , retention time = 1.36 minutes.
TIN® in vitro and in vivo Studies It is noted that for the following studies HERCEPTIN® in the e of ated cytotoxic agents shows no significant in vitro potency or in vivo efflcacy at equivalent antibody concentrations.
In vitro Cell Assay Procedure Target expressing (BT474 (breast cancer), N87 (gastric cancer), HCC1954 (breast cancer), DYT2 (breast cancer)) or non-expressing (MDA-MB-468) cells were seeded in 96-well cell culture plates for 24 hours before treatment. Cells were treated with 3-fold serially diluted antibody-drug conjugates or free compounds (i.e., no antibody conjugated to the drug) in ate at 10 concentrations. Cell viability was determined by CellTiter 96® AQueous One Solution Cell Proliferation MTS Assay (Promega, Madison WI) 96 hours after treatment.
Relative cell viability was determined as percentage of untreated control. 1C50 values were calculated using a four parameter logistic model #203 with XLfit V4.2 (IDB S, Guildford, Surry, UK). Results are shown in Tables 20, 21A and 21B.
In vivo MDAMB-361 DYT2 Tumor Xenograft Model In vivo efficacy studies of antibody-drug conjugates were performed with the Her2+ MDAMB-361 DYT2 cell line. For efficacy studies, 10 million tumor cells in 50% matrigel were implanted subcutaneously into 6-8 week old irradiated nude mice. When the tumor sizes reached between 0 mm3 drugs or vehicle were administered h bolus tail vein injection. Mice were injected with 1 mg/kg of antibody drug conjugates treated four times every four days ). Tumor volume is measured twice a week for the first 50 days and once weekly thereafter by a Caliper device and calculated with the following formula: Tumor volume = (length x widthz) / 2. Results were compared across studies by normalizing the tumor regression of the drug-treated mice by ng the tumor volume by the e-treated tumor volume (T/C).
Six compounds were tested in the three different DYT2 xenograft studies to determine their anti-tumor ty. The results of a representative study with four of the compounds demonstrates cant tumor regression from the vehicle-treated mice over the 50 day ation period (Figure 1). To compare the results of nds in the three studies, anti-tumor activity was normalized by dividing the reated tumor volume by the vehicle- treated tumor volume (T/C). A plot of the six T/C values (Figure 2) demonstrates that each of the six compounds causes complete (or almost complete) tumor regression over the observation period which was up to 107 days for one of the studies.
Results of the testing of H(C)-#D54, H(C)-chMAE, H(C)-mcMMAF and H(K)-MCC- DMl in the MDA-MBDYT2 xenograft studies are shown in Figure 4. Tumor volume in treatment group over control group (T/C) plot allows comparison between conjugates (see Figure 5C). These results demonstrate that H(C)-#D54 displays equivalent y to TIN® conjugates with H(C)-chMAE, H(C)-mcMMAF and is superior to H(K)-MCC-DM1 in this model.
In vivo N87 Tumor Xenograft Model (HERCEPTIN®) In vivo efficacy studies of antibody-drug conjugates were performed with target- expressing xenograft models using the N87 cell lines. For efficacy study, 7.5 million tumor cells in 50% matrigel are implanted subcutaneously into 6-8 weeks old nude mice until the tumor sizes reach between 250 and 350 mm3. Dosing is done through bolus tail vein injection. Depending on the tumor response to treatment, animals are injected with 1-10 mg/kg of antibody drug conjugates d four times every four days. All experimental animals are monitored for body weight changes weekly. Tumor volume is ed twice a week for the first 50 days and once weekly thereafter by a Caliper device and calculated with the ing formula: Tumor volume = (length x widthz) / 2. Animals are humanely sacrif1ced before their tumor volumes reach 2500 mm3. The tumor size is observed to decrease after the first week of treatment. Animals may be monitored continuously for tumor re-growth after the treatment has discontinued.
Results of the testing of H(C)-#D54, H(C)-chMAE, H(C)-mcMMAF and H(K)-MCC- DM1 in the N87 mouse xenograft in vivo screening model are shown in Figures 3 and 5. These results demonstrate that H(C)-#D54 is superior/similar to the H(C)-chMAE ate and is more potent than the H(C)-mcMMAF and H(K)-MCC-DM1 conjugates in this model.
Pharmacokinetics and Toxicokinetics Mouse pharmacokinetics and rat toxicokinetics were determined from single dose mouse pharmacokinetic and rat toxicology studies (see Tables 22 and 23). Mouse cokinetics and rat toxicokinetics were determined from single dose mouse pharmacokinetic and rat toxicology studies. Mouse cokinetics were determined from samples collected from nude mice that were administered a single 3 mg/kg dose. Samples were collected for up to 336 h. Rat toxicokinetics were determined in rats (Sprague-Dawley (Crl:CD (SD))) that were administered a single administration of H(C)-vc-MMAE or H(C)-#D54 at doses of 3, 10, and 30 mg/kg, or administered H(C)-mc-MMAD or H(C)-mc-MMAF at 10, 30, and 100 mg/kg. Samples were collected for up to 336 hours. Circulating concentrations of total dy and ADC were measured using ELISA assays. Area under the curve (AUC) was calculated for the total dy and ADC for each ADC. ADC to dy AUC ratios were also calculated.
Exposure of H(C)-#D54 total antibody and ADC were greater than that observed for H(C)-vc-MMAE in mice at 3 mg/kg and at all doses ted in rats. The ADC to Ab AUC ratio for H(C)-#D54 was also greater than that observed for c-MMAE. These results suggest that H(C)-#D54 has greater exposure and that the ADC and/or linker payload are potentially more stable than H(C)-vc-MMAE.
Toxicity The target ndent toxicity of #D54 and comparator linker-payloads itPABC-MMAD and chalCitPABC-MMAE) conjugated to a non-cross reactive monoclonal antibody (IgGl) were assessed in a single-dose rat toxicity study with a two-week WO 72813 observation period. The doses of the antibody drug conjugates (ADCs) were 0, 3, 10 and 30 mg/kg with an n is 5 males/group and the linker-payload loading was similar among the conjugates (3.8, 3.2 and 4, respectively). These studies included at least daily clinical observations, weekly body weights, clinical pathology (end of in-life) and necropsy (Day 15-17) with microscopic examination of 9 or more tissues and any gross lesions.
Mortality with related body weight changes and signs of morbidity were observed at the mg/kg dose for all conjugates and at the 10 mg/kg dose for the MMAD conjugate. There were no clinical observations or body weight changes in the surviving groups.
The target organs of the conjugates identified by microscopic examination in the ing dose groups were as follows. The conjugate at 10 mg/kg had debris in the lumen of the epididymis (5/5, l to mild), inflammation at the base of the heart (1/5 rats, minimal) and increased mitosis in the cornea (1/5 rats, minimal). There were no histological findings for the conjugate at 3 m/kg. For the MMAD conjugate in the surviving dose group at 3 mg/kg, there were changes in and related to the bone marrow and in the testis and ymidis. For the MMAE conjugate at 10 mg/kg, there were changes in the bone , kidney, liver and epididymis. At the 3 mg/kg dose for this conjugate, there were kidney changes and increased mitosis in the liver. Thus, in studies of r design and in the surviving , the conjugate did not have the bone marrow f1ndings seen with the comparator conjugates and also did not have the liver or kidney findings seen with one of the comparators.
In summary, the maximum tolerated dose (MTD) of the conjugate and the MMAE ate was 10 mg/kg and the MTD of the MMAD conjugate was 3 mg/kg. The no observed adverse effect level (NOAEL) of the conjugate was 3 mg/kg whereas the NOAEL of the comparator linker-payload conjugates was less than 3 mg/kg. This study demonstrates how the toxicological profile for #D54 compares to certain compounds described in the art.
Anti-IL-13Ra2 ADC In Vitro and In Vivo Studies Anti-IL-13Ra2 Antibodies and ADCs The zed antibody hu08 specifically binds to the IL-13R0t2 receptor. The amino acid and the nucleotide sequences for hu08 are shown in Table 3. Kabat CDRs are underlined.
Table 3. Amino acid and nucleotide sequences of humanized antibody hu08.
SEQ II) NO PTION SEQUENCE hu08 heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVR variable region QAPGKGLEWVATVSSGGSYIYYADSVKGRFTISRDNAK amino acid sequence NSLYLQMNSLRAEDTAVYYCARQGTTALATRFFDVWG (CDRs underlined). QGTLVTVSS hu08 light chain DIQMTQSPSSLSASVGDRVTITCKASE2DVGTAVAWYQQ variable region KPGKAPKLLIYSASYRSTGVPSRFSGSGSGTDFTLTISSLQ amino acid sequence PEDFATYYCS ZHHYSAPWTFGGGTKVEIK (CDRs underlined). hu08 heavy chain SGGGLVQPGGSLRLSCAASGFTFSRNGMSWVR amino acid sequence QAPGKGLEWVATVSSGGSYIYYADSVKGRFTISRDNAK (CDRs ined). NSLYLQMNSLRAEDTAVYYCARS2GTTALATRFFDVWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW SCSVMHEALHNHYTQKSLSCSPGK hu08 light chain DIQMTQSPSSLSASVGDRVTITCKASS2DVGTAVAWYQQ amino acid sequence KPGKAPKLLIYSASYRSTGVPSRFSGSGSGTDFTLTISSLQ (CDRs underlined). PEDFATYYCS ZHHYSAPWTFGGGTKVEIK TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC hu08-heavy chain GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGC nucleotide sequence AGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCTCC GGCTTCACCTTCAGTAGGAATGGCATGTCTTGGGTGA GGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGGCCA CCGTTAGTAGTGGTGGTAGTTACATCTACTATGCAGA CAGTGTGAAGGGGCGGTTCACCATCTCCAGGGACAAC GCCAAGAACTCCCTGTACCTCCAGATGAACTCCCTGA GGGCCGAGGATACCGCCGTGTACTACTGTGCCAGACA AGGGACTACGGCACTAGCTACGAGGTTCTTCGATGTC TGGGGCCAGGGCACCCTGGTGACCGTGTCCTCTGCGT CGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC GAGCACCTCTGGGGGCACAGCGGCCCTGGGC TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG GGAACTCAGGCGCCCTGACCAGCGGCGTGCA CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT GGGCACCCAGACCTACATCTGCAACGTGAATCACAAG CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCC AAATCTTGTGACAAAACTCACACATGCCCACCGTGCC CAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT CGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CGTGGAGTGGGAGAGCAATGGGCAGCCGGA GAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT GCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAA hu08- light chain GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGC tide sequence CTCTGTGGGCGACAGAGTGACCATCACCTGTAAGGCC AGTCAGGATGTAGGTACTGCTGTAGCCTGGTATCAGC AGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTC GGCATCCTACCGGTCCACTGGCGTGCCTTCCAGATTCT CCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATC TCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTG CCAGCACCATTATAGTGCTCCGTGGACGTTTGGCGGC GGAACAAAGGTGGAGATCAAGACTGTGGCTGCACCA TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGG ATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT GCAGGACAGCAAGGACAGCACCTACAGCCT CAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGA GAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGT Humanized anti-IL-13R0t2 antibody hu08 was ated to various linker-payload combinations of the present invention, as provided in Table 4. The antibody drug conjugates were prepared ing to the methods of the present invention.
Table 4 L-13R0t2 ADCs.
ADC Linker-Payload # Corresponding ADC lature IL13Ra2-AB08-v1010-hG1-(C)_chalCitPABC-#54 c-0 1 01 IL13Ra2-AB08-v1010-hG1-(C)_mc-#115 hu08-mc-3377 2-AB08-v1010-hG1-(C)_mc-0#118 hu08-mc-0131 IL13Ra2-AB08-v1010-hG1-(C)_MalPeg6C2-#117 hu08-Malpeg-6121 IL13Ra2-AB08-v1010-hG1-(C)_mc-#117 hu08-mc-6121 IL13Ra2-AB08-v1010-hG1-(C)_chalCitPABC-#226 hu08-vc-3906 IL1 3Ra2-AB08-v1010-hG1-(C)_chalCitPABC-# 1 12 c-6780 2-AB08-v1010-hG1-(C)_mc-#69 hu08-mc-8261 IL13Ra2-AB08-v1010-hG1-(C)_mc-#226 c-3906 hngG8.84-chalCitPABC-#54 hngG8.84-v00101 hngG8.84-mc-#115 hngG8.84-mc3377 In Vitro Cytotoxicity Assay with anti-IL-13Ra2 ADCs Cell lines expressing the IL-13R0t2 antigen and a negative control cell line, were cultured with increasing concentrations of anti-IL-13R0t2 ADCs comprising the hu08 antibody conjugated to various linker payloads of the present invention. After four days, viability of each culture was assessed. IC50 values were calculated by logistic non-linear regression and are presented as ng Ab/mL.
The data demonstrates that the anti-IL-13R0t2 antibody huO8v1.0/1.0 conjugated to six different auristatin payloads is effective against both of the IL-13R0t2 positive cell lines tested (PC3MM2), having an IC50 ranging from 1.1 to 4.9 ng Ab/mL or 7.3-32.7 pM (Table 5). All ADCs were not active t the IL-13R0t2 negative cell line, H460, and the non— IL-13R02 binding control ADCs, hngG8.84-vc0101 and hngG8.84-mc3377, were not active against any of the cell lines tested.
Table 5. IC50 (ng Ab/mL) values of humanized anti-IL-13R0t2 ADCs. 1C50 (ng Ab/mL) H460 PC3MM2 hu08—ve0101 . 2.5 _ >400000 hu08—mc3377 4.3 1.2 2.2 >400000 hu08—mc—0131 3.2 1.3 2.1 >400000 hu08-Mal e_-6121 3.4 >400000 al e _-0131' 4.9 >400000 hu08—mc—6121 2.4 >400000 hu08—vc—3906 2.9 >400000 hu08 vc-6780 1.2 2.2 0 hngG8.84-vc0101 >400000 >400000 >400000 84-mc3377 >400000 >400000 >400000 In Vivo Subcutaneous Xenograft Models with anti-IL13Ra2 ADCs The humanized antibody hu08 specifically binds to the lL-13Ra2 receptor. hu08 ADCs with eleven ent linker-payload combinations were tested in an in vivo xenograft model.
Female, athymic (nude) mice were injected subcutaneous with PC3MM2. Mice with staged tumors, approximately 0.1 to 0.3 g (n = 8 to 10 mice/treatment group), were administered intravenously q4d x 4 with normal saline (vehicle), hu08v1 .0/ 1.0 ADCs with linker-payloads vc- 0101, 0, vc-3906, mc-8261, mc-0131, mc-6121, mc-3377, MalPeg-8261, MalPeg-0131, MalPeg-6121, or MalPeg-3906, and a non-binding Ab (hulgG8.84) ated with vc-0101 or mc-3377, at a dose of 2 or 3 mg Ab/kg. The ADCs were dosed based on Ab content. Tumors were measured at least once a week and their size is calculated as mm3 = 0.5 x (tumor widthz) x (tumor length).
The in vivo efficacy s listed in Table 6 show a range of anti-tumor ty with the various ADCs tested. The relative order of potency is hu08-vc-0101 > hu08-vc-6780 > hu08-mc- 0131 > hu08-mc-6121 > hu08-mc-3906 > hu08-MalPeg-0131 >hu08-MalPeg-6121 > hu08- MalPeg-3906 > > hu08-mc-8261. At the 3 mg/kg dose level, both hu08-vc-0101 and hu08-mc- 3377 demonstrated antitumor activity, whereas the non-binding Ab (hulgG8.84) conjugated with vc-0101 or mc-3377 had no activity and were r to the vehicle control.
Table 6. Efficacy of anti-lL-13Ra2 ADCs in PC3MM2 xenografts.
PC3MM2 xenograft, tumor volume (mm +/- SEM) Day Day Dgy Day Day Day Day Day -1 17:07 16 20 30 42 52 638 l 1349 Vehicle 0 GT 27 8211133 GT GT GT GT GT GT GT GT GT GT hu08—mc— 0131 hu08- MalPeg— 61 2 1 hu08- MalPeg— 2 01 3 1 hu08—Vc- 01 01 hu08—Vc- 6780 hu08—mc— 6121 hu08- mc3377 hngG8.84- vc0101 hngG8.84- mc3377 GT= group ated due to large tumor size otch ADC In Vitro and In Vivo Studies Anti-Notch Antibodies and ADCs Humanized antibodies, th8 and hu75, and rat-human chimeric antibodies, ch28 and ch75 bind to the Notch receptor. The amino acid and nucleotide sequences for hu28 , specifically and hu75 are provided in Table 7. Kabat CDRs are underlined.
Table 7. Amino acid and nucleotide sequences of humanized anti-Notch antibodies.
DESCRIPTION SEQUENCES hu28 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFTFRDYGMTWVRQAPGKGL Variable Region EWVAYISSGSNYIYYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDT amino acid ce AVYYCARRGPFVLDAWGQGTLVTVSS (CDRs underlined). hu28 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFTFRDYGMTWVRQAPGKGL amino acid sequence EWVAYISSGSNYIYYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDT (CDRs underlined). AVYYCARRGPFVLDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS Human IgGl Constant VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA Region PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG hu28 Heavy Chain GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGG nucleotide sequence GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAG GGACTATGGAATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTGGCCTATATTAGTAGTGGTAGCAATTACATCTATT ATGCAGAAGCGGTGAAGGGCCGATTCACCATCTCCAGAGACAAC GCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGA GGACACGGCTGTGTATTACTGTGCGAGACGAGGCCCGTTTGTTTT GGATGCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCGTC GACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAG CACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGAC TCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG GCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAAC ACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACA ACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG TGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT CCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGA GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCC CGGGT hu28 Light Chain DIQMTQSPSSLSASVGDRVTITCKASQSINRYLHWYQQKPGKAPKLL Variable Region IYNANGLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNTWP amino acid sequence mFGGGTKVEIK (CDRs ined). hu28 Light Chain DIQMTQSPSSLSASVGDRVTITCKASQSINRYLHWYQQKPGKAPKLL amino acid sequence IYNANGLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNTWP (CDRs underlined). mFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH Human kappa KVYACEVTHQGLSSPVTKSFNRGEC Constant Region hu28 Light Chain GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA nucleotide sequence GGAGACAGAGTCACCATCACTTGCAAAGCAAGTCAGAGTATTAA CAGGTACTTACACTGGTATCAGCAGAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATAATGCAAACGGTTTGCAAACGGGGGTCCCAT CAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCA GTCTGCAACCTGAAGATTTTGCAACTTACTACTGTTTGC AGCATAATACGTGGCCGGACACGTTTGGCGGAGGGACCAAGGTG AAACGGACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCC CCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCTCTGTGGTGTGT CTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAA GGTGGACAACGCTCTGCAGTCCGGCAACTCCCAGGAGTCTGTGA CCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCTACCC TGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCC TGTGAGGTGACCCACCAGGGCCTGTCCTCTCCTGTGACCAAGTCC TTCAACCGGGGCGAGTGC hu75 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGYAFTDYWMTWVRQAPGKGL Variable Region EWVAEISPNSGGTNFNEKFKGRFTISVDNAKNSLYLQMNSLRAEDT amino acid sequence AVYYCARGEIRYNWFAYWGQGTLVTVSS (CDRs underlined). hu75 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGYAFTDYWMTWVRQAPGKGL amino acid sequence EWVAEISPNSGGTNFNEKFKGRFTISVDNAKNSLYLQMNSLRAEDT (CDRs underlined). AVYYCARGEIRYNWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS Human IgGl Constant LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP region CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG hu75 Heavy Chain GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGG tide sequence GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGTTATGCATTCAC TGACTACTGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTGGCCGAAATTTCTCCTAACAGTGGTGGTACTAACT TCAATGAAAAGTTCAAGGGCCGATTCACCATCTCCGTTGACAACG CCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAG GCTGTGTATTACTGTGCGAGAGGGGAAATCCGTTACAA TTGGTTTGCTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC AGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCC CAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTG ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGC CAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCCCCGGGT hu75 Light Chain DIQMTQSPSSLSASVGDRVTITCKASQNVGNNIAWYQQKPGKAPKL Variable Region LIYYASNRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQRLYNS amino acid sequence MFGGGTKVEIK (CDRs underlined). hu75 Light Chain DIQMTQSPSSLSASVGDRVTITCKASQNVGNNIAWYQQKPGKAPKL amino acid sequence LIYYASNRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQRLYNS (CDRs underlined). EFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR Human kappa EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK nt Region HKVYACEVTHQGLSSPVTKSFNRGEC hu75 Light Chain GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA nucleotide sequence GGAGACAGAGTCACCATCACTTGCAAGGCCAGTCAGAATGTGGG TAATAATATAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATTATGCATCTAACCGGTACACTGGGGTCCCAT CAAGGTTCAGTGGCAGTGGATATGGGACAGATTTCACTCTCACCA TCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGC GTCTTTACAATTCTCCATTCACGTTCGGCGGAGGGACCAAGGTGG AGATCAAACGGACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCCC CTTCCGACGAGCAGCTGAAGTCTGGCACCGCCTCTGTGGTGTGTC ACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAG GTGGACAACGCTCTGCAGTCCGGCAACTCCCAGGAGTCTGTGACC GAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCTACCCTG ACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTG TGAGGTGACCCACCAGGGCCTGTCCTCTCCTGTGACCAAGTCCTT CAACCGGGGCGAGTGC zed otch antibodies, hu28 and hu75, and rat-human chimeric anti-Notch antibodies, ch28 and ch75, were conjugated to various linker-payload ations of the present invention, as provided in Table 8. The antibody drug conjugates were prepared according to the methods of the present invention.
Table 8. Anti-Notch ADCs.
Corresponding ADC ADC Linker-Payload # Nomenclature \ otchv1010-hG1-(C)_chalCitPABC-#54 hu28-v00101 \ otchv1010-hG1-(C)_chalCitPABC-#1 12 hu28-vc6780 \ otchv1913-hG1-(C)_chalCitPABC-#54 hu75-vc0101 \ otchv1913-hG1-(C)_chalCitPABC-#1 12 hu75-vc6780 \ otchcG1-(C)_chalCitPABC-#54 ch28-v00101 \ otch-2 8-cG 1 -(C)_chalCitPABC-# 1 12 ch28-vc6780 \ otch-2 8-cG1-(C)_mc-#54 ch28-ch 101 \ otchcG1-(C)_mc-0#1 18 ch28-ch 1 31 \ otch-2 (C)_mc-#1 15 ch28-mc3 377 \ otch-2 8-cG1-(C)_mc-#69 ch28-mc8261 \ otch-2 8-cG1-(C)_MalPeg6C2-0#1 18 ch28-MalPeg6C2-0131 \ otchcG1-(C)_MalPeg6C2-#69 ch28-MalPeg6C2-8261 \ otchcG1-(C)_me-0#1 18 ch28-me0131 \ otchcG1-(C)_m(H20)c-0#1 18 (H20)c-0131 \ otchcG1-(C)_chalCitPABC-#54 ch75-vc0101 \ otch-7 5-cG 1 -(C)_chalCitPABC-# 1 12 ch75-vc6780 \ otchcG1-(C)_mc-0#1 18 ch75-ch 1 31 \ otchcG1-(C)_mc-#1 15 ch75-mc3 377 \ otch-7 5-cG1-(C)_MalPeg6C2-0#1 18 ch75-MalPegC2-0131 \ otchcG1-(C)_MalPeg6C2-#69 ch75-MalPeg6C2-8261 \ 5-cG1-(C)_me-0#1 18 ch75-me0131 \ otchcG1-(C)_m(H20)c-0#1 18 ch75-m(H20)c-0131 huNeg8.8-(C)_chalCitPABC-#54 huNeg8.8-v00101 huNeg8.8-(C)_chalCitPABC-#1 12 huNeg8.8-vc6780 huNeg8.8-(C)_mc-0#118 huNeg8.8-ch 1 31 .8-(C)_mc-#115 .8-mc3377 huNeg8.8-(C)_me-0#118 huNeg8.8-me0131 huNeg8.8-(C)_MalPeg6C2-#69 huNeg8.8-MalPeg6C2-8261 ch2H6-(C)_mc-#69 ch2H6-mc8261 In Vitro Cytotoxicity Assays with Anti-Notch ADCs The effects of anti-Notch ADCs were assessed on 1) cell lines endogenously expressing Notch protein: HCC2429 (lung cancer), OVCAR3 (ovarian cancer) and MDA-MB-468 (breast cancer), 2) cell lines engineered to over-express Notch n: MDA-MB-468/hNotch and U2OS/hNotch, and 3) a negative control cell line (SW900) using an MTS cellular viability indicator (Promega, Madison, WI). These cell lines were cultured with increasing concentrations of anti-Notch ADCs comprising humanized anti-Notch antibodies, hu28 and hu75, and rat- human chimeric anti-Notch antibodies, ch28 and ch75, conjugated to various -payload combinations of the present invention. As a specificity control for the anti-Notch-ADCs, non- targeted control-ADCs (huNeg8.8-ADCs or ch2H6-ADCs) were also tested on the same cell lines. After four days, ity of each culture was assessed. IC50 values were calculated by logistic non-linear sion and ted as ng Ab/mL. The drug antibody ratio (DAR) is also provided.
Table 9 shows IC50 (ng Ab/mL) values of the zed anti-Notch ADC treatments.
HCC2429 and MDA-MB-468/hNotch cell lines had two individual repeats. The data demonstrates that the zed anti-Notch ADCs with s linker-payloads were active and induced cell death in the Notch expressing and over-expressing cancer cell lines HCC2429, OVCAR3, MDA-MB-468, MDA-MB-468/hNotch, U2OS/hNotch, but not in the negative control cell line SW9OO lacking Notch expression. The non-targeted control-ADCs either lacked potency (LP) and therefore IC50 values were not generated as indicated, or were lly active 2O at the highest doses tested. Anti-Notch ADCs having IC50 values equal to or higher than IC50 values for control-ADCs were considered to lack potency in vitro and indicted as LP.
Table 9.1C50 (ng Ab/mL) values of humanized anti-Notch ADCs. 1C50 (ng Ab/mL) . .
MDA- MDA-MB-468/ UZOS/ HM” OVCARS SW90" M3468 hu28-vc0101 3.9 473 2940 6545 1330 —--—- —-—- huNeg8.8-vc6780 4.2 LP Table 10 shows IC50 (ng Ab/mL) values of the rat-human chimeric anti-Notch ADC treatments. For experiments with 2-4 individual s, average IC50 values were calculated along with standard error of the mean (S.E.M.). The data demonstrates that the rat-human chimeric anti-Notch ADCs with s linker-payloads were active and induced cell death in the Notch expressing and over-expressing cancer cell lines HCC2429, , MDA-MB-468, MDA-MB-468/hNotch, UZOS/hNotch. The non-targeted control-ADCs either lacked potency (LP) and therefore 1C50 values were not generated as indicated, or were lly active at the t doses tested. Anti-Notch ADCs having 1C50 values equal to or higher than 1C50 values for control-ADCs were considered to lack potency in vitro and ed as LP.
Table 10.1C50 (ng Ab/mL) values of rat-human ic anti-Notch ADCs (nd= not determined) —.—-—--ICSO (ng Ab/mL) :: S.E.M. ch28-mc8261 3.7 LP nd 12147::4806.4 nd nd ———- ———- huNeg8.8-MalPeg6C2-8261 4. 1 ch28-mc0131 3. 4 251::77. 5 6::1.0-3:0.5 ch75-mc0131 3. 3 671::406. 5 8202:2773.0 huNeg8.8-me0131 ch28-mc3377 -7 ch75-mc3377 huNeg8.8-mc3377 I ch28MalPeg6CZ0131 ——_—_ 3 ~ chm-wow- ch75-vc6780 4- 1004:1710 _—_ LP huNeg8.8-vc6780 —-——- In vivo Human Tumor Xenograft Models with Anti-Notch ADCs Humanized anti-Notch antibodies, th8 and hu75, and rat-human chimeric anti-Notch antibodies, ch28 and ch75, were conjugated to various linker-payload combinations and tested in 1 non-small cell lung cancer (NSCLC), HCC2429 lung cancer, MDA-MB-468 breast cancer and N87 gastric cancer xenograft models. For each model described below the first dose was given on Day 0. The tumors were measured at least once a week and their volume was calculated with the formula: tumor volume (mm3) = 0.5 x (tumor )(tumor length). The mean tumor volumes (z: S.E.M.) for each treatment group were calculated having a maximum of animals and a minimum of 6 animals to be included.
A. 1 NSCLC Xenografts The effects of anti-Notch ADCs were examined in immunodeficient mice on the in vivo growth of human tumor xenografts that were established from fragments of freshly resected 37622A1 NSCLC tumors obtained in accordance with riate consent procedures (Asterand). The 37622A1 NSCLC patient-derived xenografts were subcutaneously passaged in vivo as fragments from animal to animal in nude (Nu/Nu) female mice. When the tumors reached a volume of 150 to 300 mm3, they were staged to ensure uniformity of the tumor size among various treatment groups. The 37622A1 NSCLC model was dosed intraveneously four times every four days (Q4dx4) with PBS vehicle, humanized anti-Notch ADCs, l huNeg-8.8 ADCs and cisplatin at the doses provided in Table 11.
Cisplatin is a platinum-based anti-cancer agent used in the treatment of cancer and considered a standard-of—care therapy. Cisplatin cross-links DNA thereby inducing sis and cell grth inhibition. The data demonstrates that anti-Notch ADCs hu28-vc0101, hu28-vc6780, hu75-vc0101 and hu75-vc6780 inhibited growth of 37622A1 NSCLC xenografts. Further, the data shows that anti-Notch ADCs inhibited tumor grth more ly than control huNeg8.8- ADCs. Furthermore, the data shows that anti-Notch ADCs inhibited tumor growth more potently than cisplatin indicating a r potency than a platinum-based standard-of—care herapeutic drug.
Table 11. Efficacy of otch ADCs in 37622A1 NSCLC aftsEM) 37622A1 NSCLC xenografts, tumor volume (mm3, mhu£2%é%8_ Cisplatin 5 182 185 :17 : 11 226 226 ::26 ::15 265 280 : 28 :Z29 246 301 :30 : 34 277 345 : 41 :47 219 309 : 31 :Z37 218 373 ::42 :Z50 264 401 :52 : 58 DAY 28 246 446 , , :53 :51 :64 DAY 32 42 72 332 482 :99 :13 :15 :52 : :77 :62 :104 :1 1 :21 :79 :59 :79 :94 :80 :117 :26 :31 :103 :78 :114 :83 DAY 42 75 151 495 612 :34 :37 :128 :92 DAY 46 92 172 610 723 :44 :47 :165 :119 :120 :63 :62 :135 :202 :139 :158 :67 :93 :195 :251 :193 :185 :51 :100 :251 :184 :231 :220 :85 :138 :162 :232 :273 :193 :111 :160 :191 :147 :227 :254 :152 :272 :202 :173 :251 :260 :162 :235 :209 :203 :305 0 ' ' :264 B. HCC2429 Lung Xenografts Similar in vivo experiments were performed with the HCC2429 lung cancer cell line as described above. To generate xenografts, nude (Nu/Nu) female mice were implanted subcutaneously with 3.5xlo6 HCC2429 cells in 50% Matrigel (BD Biosciences). When the tumors reached a volume of 200 to 400 mm3, the tumors were staged to ensure uniformity of the tumor mass among various treatment groups. The HCC2429 lung model was dosed intraveneously Q4dx4 with PBS vehicle, zed anti-Notch ADCs and control huNeg-8.8 ADCs at the doses provided in Tables 12 and 13. The data demonstrates that anti-Notch ADCs hu28-vc0101, hu28-vc6780, hu75-vc0101 and hu75-vc6780 ted grth of HCC2429 lung xenografts in a ependent manner. Further, the data shows that anti-Notch ADCs inhibited tumor growth more potently than control huNeg8.8-ADCs at the l and 3 mg/kg doses for anti- Notch ADCs with chlOl linker-payloads and at the 3 and 10 mg/kg doses for otch ADCs with vc6780 linker-payloads. Furthermore, the data demonstrates that a 3mg/kg dose of hu28- chlOl was more potent than a 10 mg/kg dose ofhu28-vc6780.
WO 72813 Table 12. y of anti-N0tch—vc0101 ADCs in HCC2429 lung xenografts.
HCC2429 Lung xenografts, tumor volume (mm3 +/- SEM) PBS hu28-vc0101 hu75-vc0101 huNeg-8.8-vc0101 0 s 1 :24 :23 :26 :30 28 23 29 30 33 27 :52 :52 :36 :50 39 37 66 59 72 50 :73 :78 :68 :78 44 93 74 91 97 73 :120 :101 :119 : 146 74 132 100 100 DAY 56 704 U) 00 Table 13. Efficacy of anti-N0tch—vc6780 ADCs in 9 lung xenografts.
HCC2429 Lung xenografts, tumor volume (mm3 1 SEM) hu28-vc6780 hu75-vc6780 huNeg-8.8-vc6780 DAY -1 245 244 245 245 244 246 245 244 244 245 :28 :22 19 30 16 22 26 20 DAY 1 398 369 427 402 49 53 DAY 3 689 655 : 83 97 :140 : 33 98 97 101 114 117 :200 : 24 115 131 154 136 172 The HCC2429 lung model was also dosed intravenously Q4dx4 with PBS e, rat- human chimeric anti-Notch ADCs and control huNeg-8.8 ADCs, at a dose of 5mg/kg as provided in Figure 8A. The data demonstrates that anti-Notch ADCs with non-cleavable (mc) and cleavable (vc) s and various payload combinations inhibited growth of HCC2429 lung xenografts. Further, the data shows that rat-human chimeric anti-Notch ADCs ted tumor growth more potently than control huNeg8.8-ADCs. Furthermore, the data trates that rat- human chimeric anti-Notch ADCs with ch 1 01 linker-payloads were more potent than the other anti-Notch ADCs tested.
C. MDA-MB-468 Breast Xenografts Similar in vivo experiments were performed with the -468 breast cancer cell line as described above. MDA-MB-468 cells are classified as a triple-negative breast cancer (TNBC) basal-like subtype since they lack expression of the estrogen receptor, progesterone receptor and human mal growth factor or 2 (HERZ) (Lehmann, BD, et al, J Clin Invest. 2011;121(7):2750—2767). To generate xenografts, female SCID Hairless Outbred (SHO) mice were orthotopically implanted with 10x106 MDA-MB-468 cells containing 50% Matrigel (BD Biosciences) in the mammary fat pad. When the tumors reached a volume of 250 to 450 mm3, the tumors were staged to ensure uniformity of the tumor mass among various treatment groups. The MDA-MB-468 breast model was dosed intraveneously Q4dx4 with PBS e, humanized anti-Notch ADCs and control huNeg-8.8 ADCs at the doses provided in Tables 14 and 15. The data trates that anti-Notch ADCs hu28-chlOl, hu28-vc6780, hu75-vc0101 and hu75-vc6780 inhibited growth of MDA-MB-468 breast xenografts in a dose-dependent manner. Further, the data shows that anti-Notch ADCs inhibited tumor growth more potently than control huNeg8.8-ADCs at the l and 3 mg/kg doses for ADCs with chlOl linker-payloads and l, 3 and 10 mg/kg doses for ADC with vc6780 linker-payloads. Furthermore, the data demonstrates that a 1 mg/kg dose of anti-Notch ADCs with chlOl linker-payloads were more potent than a 3 mg/kg dose of anti-Notch ADCs with vc6780 linker-payloads.
Table 14. Efficacy of anti-Notch—chlOl ADCs in MDA-MB-468 breast xenografts.
MDA-MB-468 Breast afts, tumor volume (mm3 : PBS hu28-vc0101 c0101 huNeg-8.8-vc0101 Dose mg/kg DAY 0 343 347 348 336 347 347 348 334 346 12 15 :22 :19 K)O :22 :21 23 H o 1.1 0 DAY 4 441 359 439 403 4;4; 4; 410 439 424 447 442 k) 00 m N 1.1 0 32 27 :25 K)O 26 NO NU) DAY 11 495 227 496 28 27 N U) 37 DAY 14 581 147 560 20 U) 0\ O\ \leH-h 0N©\l 578 43 10 :12 U) 0\ DAY 21 63 8 60 O\ ::46 :8 1;; o loWM O\] -DAY 26 671 DAY 29 238 :ZSO 67 H HM oxox\]J> ::79 73 VI 00 DAY 39 892 310 :Z84 88 a4; ”“42 ::104 95 HH u: u: 4;4;N ._. ._. 4; u: ::134 “—123 HH O\0 HH\l 00 DAY 50 n—118 :14; 0000 04> ‘10\] DAY 53 4; u: N 127 [Ioo DAY 56 UI U) UI 128 U:U) O\OU) 140 U:00 DAY 63 DAY 67 611 DAY 70 0 0 Table 15. Efficacy of anti-Notch—vc6780 ADCs in MDA-MB-468 breast xenografts.
MDA-MB-468 Breast xenografts, tumor volume (mm3 :: SEM) hu28-vc6780 hu75-vc6780 huNeg-8.8-vc6780 mg/kg :9 9 1 8 :16 DAY 4 394 DAY 7 DAY 19 DAY 22 DAY 25 DAY 28 DAY 32 DAY 35 DAY 40 DAY 43 DAY 46 DAY 49 DAY 53 DAY 56 DAY 60 DAY 63 The MDA-MB-468 breast model was also dosed eneously Q4dx4 with PBS vehicle, rat-human chimeric anti-Notch ADCs and control huNeg-8.8 ADCs, at a dose of 5mg/kg as provided in Figures 8B and 8C. The data demonstrates that man chimeric anti-Notch ADCs with non-cleavable (mc) and cleavable (vc) linkers and various payload combinations inhibited growth of MDA-MB-468 breast xenografts. Further, the data shows that rat-human chimeric otch ADCs inhibited tumor growth more potently than l huNeg8.8-ADCs. rmore, the data demonstrates that rat-human chimeric anti-Notch ADCs with ch 1 01 linker-payloads were more potent than the other rat-human chimeric anti-Notch ADCs tested.
D. N87 Gastric Xenografts Similar in vivo experiments were performed with the N87 gastric cancer cell line as described above. To generate xenografts, nude (Nu/Nu) female mice were ted aneously with 7.5 x106 N87 cells in 50% Matrigel (BD Biosciences). When the tumors reached a volume of 250 to 450 mm3, the tumors were staged to ensure uniformity of the tumor mass among various treatment groups. The N87 gastric model was dosed intraveneously Q4dx4 with PBS vehicle, humanized anti-Notch ADCs, control huNeg-8.8 ADCs and cisplatin at the doses provided in Tables 16 and 17. The data demonstrates that anti-Notch ADCs hu28- chlOl, hu28-vc6780, hu75-vc0101 and hu75-vc6780 inhibited grth ofN87 gastric xenografts in a dose-dependent manner. r, the data shows that anti-Notch ADCs inhibited tumor growth more potently than control huNeg8.8-ADCs at the 1, 3, 5 mg/kg doses for ADCs with vc0101 linker-payloads and 3 and 10 mg/kg doses for ADCs with vc6780 linker-payloads.
Furthermore, the data demonstrates that ADCs with chlOl linker-payloads were in general more potent than cisplatin standard-of—care therapy and ADCs with vc6780 linker-payloads.
Table 16. y of otch—chlOl ADCs in N87 gastric xenografts.
N87 Gastric xenografts, tumor volume (mm3 :: SEM) hu28-vc0101 hu75-vc0101 huNeg—8.8-vc0101 Cisplatin s s 321 326 321 321 324 321 21 13 8 9 l9 16 369 393 344 392 362 ll 14 15 U) UI 7 25 Np—A l4 13 21 ._. 4; l3 l7 >— O 7 ll 17 v—A 9 23 16 16 24 llov—A 99 50 211 104 1 4 13 37 p—A O\ 106 43 251 91 17 14 53 18 22 16 59 18 23 14 72 16 149 34 314 15 73 334 Table 17. Efficacy of anti-Notch—vc6780 ADCs in N87 gastric xenografts.
N87 Gastric xenografts, tumor volume (mm3 :: SEM) ------_—3 mg/kg ::14 ::14 ::10 ::13 ::8 :ZZO -------::16 ::24 ::24 ::26 ::18 ::3 7 379 545 592 351 DAY 25 DAY 28 DAY 33 733 1290 : ::195 ::128 DAY 36 817 1265 - :Z222 ::11 1 DAY 39 880 1429 - :247 ::121 DAY 42 - :244 DAY 63 DAY 70 DAY 77 The N87 gastric model was also dosed intraveneously Q4dx4 with PBS e, rat- human chimeric anti-Notch ADCs and control huNeg-8.8 ADCs, at a dose of 5mg/kg as provided in Figure 8D. The data demonstrates that rat-human ic anti-Notch ADCs with non- cleavable (mc) and cleavable (vc) linkers and various payload combinations inhibited growth of N87 gastric xenografts. Further, the data shows that rat-human chimeric anti-Notch ADCs inhibited tumor growth more potently than control huNeg8.8-ADCs. Furthermore, the data demonstrates that man chimeric anti-Notch ADCs with chlOl linker-payloads were more potent than the other anti-Notch ADCs .
The N87 gastric model was also dosed intravenously Q4dx4 with PBS vehicle and rat- human chimeric anti-Notch ADCs ch28-mc0131, ch75-mc0131, ch28-m(H20)c-0131 and ch75 - m(H20)c-0131 at a dose of 5mg/kg as provided in Figure 8E. The data demonstrates that rat- human chimeric anti-Notch ADCs having mc0131 and m(H20)c-0131 linker-payloads inhibited growth ofN87 gastric xenografts. Further, the data demonstrates that rat-human chimeric anti- Notch ADCs having m(H20)c-0131 linker-payloads were more potent than rat-human chimeric anti-Notch ADCs having mc0131 linker-payloads.
Table 18A - ed compounds oxic es with linkers) of the invention Quantity Preparation Lmker-Payload # ation method in mg method (Yield) . General chalCitPABC #34 4.7 (12 A)0 _ procedure E General MalPeg3C2-#4l 36 (28%) procedure D 125 (88%) .9 (25%) (28%) 1.8 (6%) General MalValCitPABC-#44 VIethod F 4 (10%) procedure E 12.8 (53 %) MalPeg3C2-#45 7.5 (28%) procedure D General MalPeg6C2-#45 Method E 1 * 13.6 (45%) procedure D General chalCitPABC-#45 Method C & Method E1 5.8 (15%) procedure E General chalCitPABC-#54 Method D 33 (36%) procedure E General mc-#69 Method C 30.2 (24%) procedure D General MalPeg6C2-#69 Method C 3.6 (13 h)0 ure D General chalCitPABC-#69 Method I 51 (9%) procedure E General chalCitPABC-#70 Method D 6.9 (12%) procedure E General chalCitPABC-#75 5.3 (14%) procedure E General mc-#79 5.6 (19 A1)0 ure D .5 (10%) 9.5 (26%) General chalCitPABC-#112 Method C 11.8 (21%) procedure E General mV-#115 Method J 1.4 (3.3 A1)0 procedure D me—#115 — L 124 (11%) General General chalCitPABC-#115 Method K 4.9 (12%) procedure E procedure D procedure D procedure D General chalCitPABC#47 Method E1 3. 3 (20%) procedure E chalCitPABC#26 Method E1 2. 3 (20%) procedure E General mc-#26 Method E1 5.4 (10%) ure D General chalCitPABC#42 Method E1 10.8 (38%) procedure E General chalCitPABC#36 Method E1 12. 6 (32%) procedure E mc-#42 Nixie: D Method E1 7.1 (83%) General AmPeg6C2#54 Method J 44 (67%) ure N General MalPeg3C2-#54 Method J 19 (69%) procedure D General tPABCAmPeg6C2#54 Method J 12 (42%) procedure 0 General chalCitPABCAmPeg3C2#54 Method J 12. 4 (30%) procedure 0 General MalPeg3C2#47 Method 1* 19 (62%) procedure D General AmPeg6C2#47 Method J 50 (77%) procedure N General chalCitPABCAmPeg3C2#47 6.4 (18%) procedure 0 General chalCitPABCAmPeg6C2#47 18 (50%) procedure 0 General MalPeg3C2#42 Method J 22 (70%) procedure D General AmPeg6C2#42 Method J 53 (75%) procedure N General chalCitPABCAmPeg6C2#42 Method J 15. 4 (43%) procedure 0 2012/056224 General chalCitPABCAmPeg3C2-#42 Method J* 12 (26%) procedure 0 General MalPeg3C2-#26 Method J* 13.8 (51 At)0 procedure D General Inc-#41 Method J* 9.6 (38%) ure D General AmPeg6C2-#26 Method J 59 (87 At)0 procedure N General chalCitPABCAmPeg3C2-#26 Method J 23.4 (45%) procedure 0 General MalPeg3C2ValC1tPABC #26 Method J 16 (42%) procedure P General chalCitPABCAmPeg6C2 #26 Method J 15 (38%) procedure 0 General mC-#36 Method J* 26 (80%) procedure D General MalPeg6C2-#54 Method J 27 (67 At)0 procedure D General MalPeg3C2ValCitPABC-#47 Method J 17 (49%) procedure P General 3C2-#36 Method J* 9.2 (33 At)0 procedure D MalPeg6C2-#47 Method J* 24 (78%) ure D General MalPeg6C2-#26 Method J* 29 (75%) procedure D General MalPeg6C2-#36 Vlethod J 18 (58%) procedure D General chalCitPABCAmPeg3C2-#36 d J 16 (51%) procedure 0 General AmPeg6C2-#36 Vlethod J 51 (78 At)0 procedure N General chalCitPABC-#60 Vlethod J 1.9 (4.3%) procedure E General chalCitPABCAmPeg6C2-#36 Vlethod J 1 1.6 (35%) procedure 0 General chalCitPABCAmPeg3C2-#4l Vlethod J 5.7 (26%) procedure 0 General MalPeg6C2-#60 Vlethod J 24 (75%) procedure D AmPeg6C2 #60 Nixie:N Vlethod J 31 (80%) 3C2-#60 prg‘xfi D Vlethod J 17.3 (58%) General Method J With AcOH as MalPeg6C2-#4l 1 1 (28 4)0 modifier AmPeg6C2-#66 prg‘xfiN Method J 5 (10%) chalCitPABCAmPeg6C2-#60 Method J 12 (34%) procedure 0 General eg6C2-#66 Method J 8 (60%) procedure Q General Method J With AcOH as mc-#66 8.2 (32%) procedure D modifier General chalCitPABC-#88 Method J 7.9 (45%) procedure E General chalCitPABC-#88 Method 1 7.5 (39%) procedure E General mc-#92 Method F 15 (66 A1)0 ure D General chalCitPABC-#44 Method F 1.6 (4%) procedure E General Method H Without chalCitPABC-#108 Method F 6.1 (14%) ure E General NHSCOPegZCZValCitPABC-#66 Method F* 6.8 (32%) procedure X2 General chalCitPABC-#98 Method 1* 4.8 (1 1%) procedure E chalCitPABC-#95 Method 1* 13 (28%) procedure E General MalPeg3C2-#69 Method K 12.8 (35%) procedure D General AmPeg6C2-#69 Method J* 71 (69 A1)0 procedure N General chalCitPABC-#84 Method 1* 4.9 (1 1%) procedure E General AmCapValCitPABC-#54 Method K* 97 (53%) orocedure R 7.1 (16%) .8 (36%) 7.4 (22%) 11.7 (29%) 3.8 (12%) 11 (4.5%) 2.8 (8%) 13.6 (29%) General mc-#226 Slllca chromatography. . 290 (40 A1)0 procedure D 2012/056224 THC-#1 18 Method 1* 16.2 (42%) procedure D General mC-#131 Method 1* 16.3 (51%) procedure D General mb-#1 18 Method 1* 7.9 (23%) procedure D General chalCitPABC-#134 Method 1* 17 (33%) procedure E General mC-#145 Method K 6 (20%) procedure D General 6C2-#126 Method 1* 16.4 (26%) procedure D General mC-#126 Method K* 16.3 (32%) procedure D General HIV-#118 Method 1* 1 1.7 (34%) procedure D General mc-#172 Method 1* 10 (56%) procedure D General MalPeg6C2-#226 Method K* 15 (10%) procedure D General MalPeg6C2-#145 Method K 1.8 (3.7 A1)0 General Method H* Without 11164162 1.2 )0 modifier mc-#163 Method K* 9.9 (26%) procedure D General chalCitPABC #231 Method 1* 0.2 (4%) procedure E General MalPeg6C2-#238 s111ca chromatography. 240 (77 At)0 General medium pressure C18 MalPeg6C2 #239 0 - 104 (39%,) chromatography me #123 11:£:D silica chromatography 345 (quant.) MalC6-#54 magi: 8 Method 1* 16.3 (30%) mC-#231 Nita? D Method 1* 10 (60%) MalC6-#1 18 magi: 8 Method 1* 5.3 (10%) chalCitPABC-#123 prOGCZIEIa; E silica chromatography 179 (60%) mc-#237 p533: D Method 1* 12.6 (47%) mc-#158 p533: D Method 1* 7.1 (28%) MalC6Am-#151 Nixie: D Method 1* 18.4 (86%) General MethOd J .ACOH as* PFPCOPegZCZ ValCitPABC-#54 70 (68%) procedure X3 r General medium pressure C18 chalCitPABC-#1 54 10 (19%) ure E chromatography MalC6Am-#153 wildfire: D Method K* 18.7 (47%) PFPCOPegZCZAmPeg2C2-#69 proiilgjgm Method R* 40 (64%) General medium pressure C18 chalCitPABC-#246 21 (45%) procedure E chromatography General PFPCOPeg2C2AlaAlaAsnPABC-#54 Method 12* 16.8 (54%) procedure X1 General PFPCOPeg2C2-#54 Method R* 4.1 (56%) procedure V PFPCOPeg2C2AmPeg2C2PABC-#54 M00040* -0.0004)0 General AmPeg6C2-#115 MMM* -(004000 General eg5C2-#115 * -0 —mcGly-#201 —silica chromatography —25 (16.2%) AzCOC2Ph4AmCOPeg2C2-#54 AzCOCZPh4AmPeg1C1ValCitPABC- Ph4AmPeg 1C 1ValCitPABC- AzCOC2Ph4AmCOPeg2C2-#69 AzCOC2Ph4AmCOPeg2C2-#1 15 AcLysValCitPABC-#54 Table 18B - Selected compounds (cytotoxic peptides with linkers) of the ion Mass spectrum: LC-MS 0r HPLC linker with payload # observed Hill and IUPAC Name retention time in minutes: ESI-MS N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— l-N-{4-[(8S,118,12R)[(2S)-butanyl]—12-(2- HPLC (Protocol 2-[(1R,2R)methoxymethyl {[(1S) M) : 1380.6 phenyl(1,3 -thiazolyl)ethyl] amino} -3 - chalCitPABC-#34 [M+Na+], (12.899 thioxopropyl]pyrrolidinyl}oxoethyl)-5 ,5 ,10- minutes) trimethyl- 3,6, 9-trioxo(propanyl)-2,13 -dioxa- 4,7,10-triazatetradecyl]phenyl}-N5-carbamoyl-L- ornithinamide LC-MS: 1031.7 N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-l- [M+}P], 1054.8 yl)ethoxy]ethoxy} ethoxy)propanoyl] -N-methyl-L-Valyl- ] (0.88 N—[(3R,4S,SS){(2S)[(1R,2R) {[(1S)carboxy- 3C2-#41 minutes); HPLC 2-phenylethyl]amino} methoxymethyl (Protocol D) thioxopropyl]pyrrolidinyl}-3 -methoxymethyl : 10.559 minutes oxoheptan—4-yl] hyl-L-Valinamide LC-MS: 1178.2 N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)oxo- [M+H+], 1197.4 3,6,9,12,15,18-hexaoxahenicosan—21-yl]-N-methyl-L- [M+Na+] (3.50 valyl-N-[(3R,4S,5 S)methoxy{(2S)[(1R,2R) MalPeg6C2-#42 minutes); HPLC methoxy- 3- {[(2S)methoxyoxo-3 -phenylpropan (Protocol Q) yl]amino} methyl-3 -thioxopropyl]pyrrolidinyl}-5 - : 25.235 minutes methyl- 1 ptanyl] -N-methyl-L-Valinamide LC-MS: 913.7 [M+1F] (0.85 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— 2-methylalanyl-N—[(3R,4S,5S){(ZS)[(1R,2R) minutes); { [(1 S)carboxyphenylethyl]amino} methoxy HRMS: Calc: methyl-3 -thioxopropyl]pyrrolidiNyl} - 3-methoxy-5 - 913.5103 [M+H+], methyl oxoheptaN—4-yl] -N-methyl-L-Valinamide Obsd: 913.5103.
LC-MS: 1003.8 [M+}P](0.82 N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol oxy]ethoxy} ethoxy)propanoyl] mcthylalanyl-N- minutes); HPLC [(3R,4S,SS){(2S)[(1R,2R){[(1S)carboxy Machg3C2-#44 (Protocol A) phenylethyl]amino} mcthoxy-2 -methyl-3 - : 1003.5 [M+H+], thioxopropyl]pyrrolidinyl}-3 -mcthoxymethyl 1026.4 [M+Na+] oxohcptan—4-yl] -N-methyl-L-Valinamidc (9.095 minutes) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)oxo- 3,6,9,12,15,18-hcxaoxahenicosanyl] LC-MS: 1135.8 methylalanyl-N-[(3R,4S,SS){(2S)[(1R,2R) Machg6C2-#44 [M+}P] (0.83 {[(1 S)carboxyphenylethyl]amino} methoxy minutes) methyl-3 -thioxopropyl]pyrrolidinyl}-3 -mcthoxy-5 - methyloxohcptanyl] -N-methyl-L-Valinamidc N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— LC-MS: 1318.9 [M+}P] (0.89 l-N-{4-[(8S,118,12R)[(2S)-butaN—2-yl]—12- (2-{(2S)[(1R,2R) carboxy minutes); HPLC phenylethyl]amino} mcthoxy-2 -methyl-3 - MalValCitPABC-#44 (Protocol A) thioxopropyl]pyrrolidiN— 1-yl} oxoethyl)-5 ,5 ,10- : 1319.6 [M+}P] , trimethyl-3,6,9-trioxo(propaN—2-yl)-2,13-dioxa- 1342.6 [M+Na+] 4,7, 10-tr1azatetradecyl]phenyl} rbamoyl-L- (9.132 minutes) ornithinamidc LC-MS: 927.7 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— [M+H*] (0.92 2-mcthylalanyl-N— [(3R,4S,5 S) -3 -mcthoxy{(2S) s); [(1R,2R)mcthoxy-3 -{[(2S)mcthoxyoxo HRMS: Calc: phenylpropaN—2-yl]amino} methyl-3 - 927.5260 [M+H+] thioxopropyl]pyrrolidiN— 1-yl}methyloxoheptaN—4- Obsd: 927.5259. yl] -N-methyl-L-Valinamidc N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol yl)ethoxy]ethoxy} ethoxy)propanoyl] mcthylalanyl-N- LC-MS: 1017.8 MalPeg3C2-#45 [M+}F] (0.90 [(3R,4S,5 ethoxy {(2 S)[(1R,2R)methoxy{[(2S)mcthoxyoxo-3 -phcnylpropan minutes); yl]amino} mcthyl-3 -thioxopropyl]pyrrolidin—1-yl}-5 - oxohcptanyl] -N-methyl-L-Valinamidc LC-MS: 1149.9 [M+H+] (0.90 N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)oxo- 3,6,9,12,15,18-hcxaoxahenicosanyl] minutes); HPLC methylalanyl-N— [(3R,4S,5 S)-3 -methoxy{(2S) (Protocol A at 45 MalPeg6C2-#45 R)mcthoxy-3 -{[(2S)mcthoxyoxo 0C) : 1150.5 1M+H+], phenylpropanyl]amino} methyl 1171.5[M+Na+] thioxopropyl]pyrrolidinyl}-5 -methyloxohcptan yl] -N-methyl-L-Valinamidc (9.788 minutes) N— [6-(2,5 -dioxo-2, 5-dihydro-1H-pyrrolyl)hexanoyl]— LC-MS: 1332.8 [M+H+] (1.86 L-Valyl-N—{4-[(8S,118,12R)[(2S)-butanyl]—12-(2- {(2 S)[(1R,2R)methoxy- 3 )mcthoxy minutes); HPLC oxo-3 -phenylpropanyl]amino} mcthyl-3 - chalCitPABC-#45 (Protocol A at 45 propyl]pyrrolidinyl}oxoethyl)-5 ,5 ,10- 0C) : 1333.6 [M+H*] (9737 trimethyl-3 rioxo(propan—2-yl)-2, 1 3 -dioxa- 4,7, 10-triazatctradec-1 -yl]phenyl} -N5-carbamoyl-L- minutes) ornithinamidc N— [6-(2,5 -dioxo-2, 5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N—{4-[(8S,118,12R)[(2S)-butanyl]—12-(2- HPLC (Protocol A {(2 (1R,2R)methoxymcthyl oxo-3 - { [( 1 S)- at 45 0C) : 1342.6 2-phenyl(1,3 -thiazol chalCitPABC-#54 [M+H+] (9.114 yl)ethyl]amino } propyl]pyrrolidin— 1-yl} oxocthyl)- minutes). 5 ,5 1 0-tr1mcthyl- 3,6,9-tr1oxo(propanyl)-2,1 3- dioxa-4,7, 10-triazatetradec- 1 enyl} -N5-carbamoyl- L-ornithinamidc LC—MS: 897.7 N—[6-(2,5-dioxo-2,5-dihydro- I H-pyrrol- I -yl)hexanoyl]— [M+}P], 919.7 2-methy1alanyl-N-[(3R,4S,5S)- I - {(2S)[(IR,2R) (0.81 s); { [(1 S)- I -carboxyphenylethyl]amino} - I -mcthoxy HPLC (Protocol A methyl-3 -oxopropyl]pyrrolidin- I -yl} -3 -mcthoxy-5 - at 45 0C) : 897. 5 methyloxohcptanyl]-N-mcthyl-L-Valinamide [M+H*] (9.058 N—[1-(2,5-dioxo-2,5-dihydro-IH-pyrrol-I-yl)-2I-oxo- HPLC (Protocol A 12, I 5, I 8-hexaoxahcnicosan-2 I-yl] at 45 0C) : 1120.6 alanyl-N-[(3R,4S,5S)- I - {(2S) R) Machg6C2-#69 [M-I-PP] , I 142 .5 { [(1 S)- I -carboxyphenylethyl]amino} - I -mcthoxy (9.076 minutes) -3 opyl]pyrrolidin- I -yl} -3 -mcthoxy-5 - methyloxohcptanyl] -N-methyl-L-Valinamidc 2- {2-[2-(2,5-dioxo-2,5-dihydro- I H-pyrrol- I - yl)ethoxy]cthoxy} ethoxy)propanoyl] -N-mcthyl-L-Valyl- HPLC (ProtocolM) N—[(3R,4S,5S)- I - {(2S)[( IR,2R) {[( I S)- I -carboxy- chalCitPABC-#69 : 1326.6 [M+Na+] 2-phcnylcthyl]amino} - I -mcthoxymcthyl (11.962 s) oxopropyl]pyrrolidiN— I -yl} -3 -mcthoxy-5 -mcthyl- I - oxohcptaN—4-yl] -N-methyl-L-Valinamidc N—[6-(2,5-dioxo-2,5-dihydro- I H-pyrrol- I -yl)hexanoyl]— L-Valyl-N- {4-[(8S,I IS, I2R)-I I-[(2S)-butaN—2-yl] - I 2- HPLC (Protocol A S)[(1R,2R)- I-mcthoxy {[(2S)mcthoxy- I - at 45 0C) : 1317.6 oxophcnylpropaN—2-yl]amino} mcthyl chalCitPABC-#70 [M+H+] (9.282 oxopropyl]pyrrolidiN-I-yl}oxocthyl)-5,5,10- minutcs) trimethyl-3 ,6,9-trioxo(propaN—2-yl)-2,13-dioxa- 4,7,10-triazatctradcc-I-yl]phcnyl}-N5-carbamoyl-L- ornithinamidc N—[6-(2,5-dioxo-2,5-dihydro- I H-pyrrol- I -yl)hexanoyl]— LC-MS: 1273.9 L-Valyl-NS-carbamoyl-N-{4-[({[3-({(2S) [M+H+] (0.82 [{(3R,4S,5S)methoxy- I-[(2S) {( I R,2R)- I - minutes); HPLC methoxymethyloxo[(2- chalCitPABC-#75 (Protocol A at 45 cthyl)amino]propyl } pyrrolidin- I -yl] -5 -mcthyl- I - 0C): 1273.6 [M+}P], (8.814 oxohcptan—4-yl} (methyl)amino] -3 -mcthyl- I -oxobutan- 2-yl} carbamoyl) oxctan-3 - minutes) yl]carbamoyl} oxy)mcthyl]phcnyl} -L-ornithinamidc N—[6-(2,5-dioxo-2,5-dihydro- I H-pyrrol- I -yl)hexanoyl]— HPLC (Protocol A N,2-dimethylalanyl-N-[(3R,4S,5 S)-3 -methoxy- I - {(2S)- at 45 °C) ; 941.5 [M+H+], 963.4 2-[(1R,2R)-I-mcthoxy{[(2S)-I-mcthoxy-I-oxo ] (10.444 phenylpropaN—2-yl]amino} methyl-3 - thioxopropyl]pyrrolidiN— I-yl} mcthyl- I -oxoheptaN—4- minutes) yl] -N-methyl-L-Valinamidc N—[6-(2,5-dioxo-2,5-dihydro- I ol- I -yl)hexanoyl]— L-Valyl-N— {4-[(8S,I IS, I 2R)-I I-[(2S)-butany1]— I 2-(2- {(2S)[(IR,2R)-I-methoxy{[(2S)-I-mcthoxy HPLC (Protocol A) oxo-3 -phcnylpropanyl]amino } mcthyl chalCitPABC-#79 : 1346.6 [M+}P], thioxopropyl]pyrrolidin- I -yl } oxoethyl)-4,5 ,5 ,10- (9.807 minutes) tctramethyl-3 ,6,9-trioxo(propanyl)-2, 13 -dioxa- 4,7,10-triazatctradcc-I-yl]phcnyl}-N5-carbamoyl-L- ornithinamidc LC-MS: 1282.6 N—[6-(2,5-dioxo-2,5-dihydro- I H-pyrrol- I -yl)hexanoyl]— [M+}P] (0.79 L-Valyl-N— {4-[(8S,I IS, I 2R)-I I-[(2S)-butany1]— I 2-(2- minutes); HPLC {(2S)[(1R,2R)-I-methoxymcthyloxo chalCitPABC-#92 col A at 45 (quinolinylamino)propyl]pyrrolidin- I -yl} 0C): 1282.6 oxoethyl)-5,5, 10-trimcthyl-3,6,9-trioxo(propanyl)- [M+H+] (7 .95 3 2,13-dioxa-4,7,10-triazatctradcc-I-yl]phcnyl}-N5- minutes) carbamoyl-L-ornithinamidc WO 72813 N—[6-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol- 1 xanoyl]— L-Valyl-N—{4-[(SS,118,12R)[(ZS)-butanyl](2- HPLC col {(ZS)[(1R,2R){[(1S)carboxy M) : 1288.6 chalCitPABC-# 1 12 [M+}P] phenylethyl]amino} - 1 xy-2 -methyl-3 - 1310.6 [M+Na+] (11.757 0X0p1‘0pyl]py1‘r0lidinyl} oxoethyl)-5 ,5,10- trimethyl- 3,6, 9-trioxo(propanyl)-2,13 -dioxaminutes 4,7, lO-trlazatetradec- 1 enyl} -N5-carbamoyl-L- ornithinamide N—[ 5 -(2,5 -dioxo-2,5 -dihydro- 1 H-pyrrol- 1 ntanoyl] - HPLC col A N,2-dimcthylalanyl-N— {(1 S,2R)-4— {(ZS)[(1R,2R) at 45 oC): m/z {[(1 S)carboxyphenylethyl]amino} methoxy 897.5 [M+H+], methyl-3 -oxopropyl]pyrrolidinyl} methoxy (9.149 minutes) [( 1 S)methylpropyl] oxobutyl} -N—methyl-L- valinamidc N—[6-(2,5-dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl]— HPLC (Protocol A N,2-dimcthylalanyl-N— {(1 S,2R)-4— {(ZS)[(1R,2R) at 45 °C;) m/z {[(1 S)carboxyphenylethyl]amino} methoxy 911.5 [M+H+], methyl-3 -oxopropyl]pyrrolidinyl} methoxy (9.676 minutes) [( 1 S)methylpropyl] oxobutyl} -N—methyl-L- valinamidc N—[4-(2,5-dioxo-2,5-dihydro- 1 H-pyrrolyl)butanoyl] - mcthylalanyl-N— {(1 S,2R)-4— {(ZS)[(1R,2R) {[(1 S)- 1 xyphenylethyl]amino} methoxy methyl-3 -oxopropyl]pyrrolidinyl} methoxy [( 1 S)methylpropyl] oxobutyl} -N—methyl-L- valinamidc N—[7-(2,5 -dioxo-2,5 -dihydro- 1 H-pyrrolyl)heptanoyl] - N,2-dimcthylalanyl-N— {(1 S,2R)-4— {(ZS)[(1R,2R) {[(1 S)carboxyphenylethyl]amino} methoxy methyl-3 opyl]pyrrolidinyl} methoxy [( 1 S)methylpropyl] oxobutyl} -N—methyl-L- valinamidc N—[6-(2,5-dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl]— L-Valyl-N—{4-[(SS,118,12R)[(ZS)-butanyl](2- HPLC (Protocol {(ZS)[(1R,2R){[(1S)carboxy M): m/z 1317.7 phenylethyl]amino} methoxy-2 -methyl-3 - chalCitPABC-#1 15 [M+H+] (12.261 oxopropyl]pyrro minutes) lidinyl} oxoethyl)-4,5,5, lO-tetramethyl-3,6,9- trioxo- 8-(propanyl)-2, 1 3 -dioxa-4,7, 1 zatetradecyl]phenyl} -N~5~-carbamoyl-L-ornithinamide N~2~-[(1- {[6-(2,5-dioxo-2,5-dihydro- lH-pyrrol- 1- yl)hexanoyl] amino} cyclopropyl)carbonyl] -N- HPLC (Protocol [(3R,4S,5S)methoxy {(2 S)[(1R,2R)methoxy- M): m/z 934.5 [M+}P] 2-methyloxo { [(1 henyl(1 ,3-thiazol (11.94 yl)ethyl] minutes) amino } ]pyrrolidin— 1 -yl} methyl- 1 ptan yl] -N-methyl-L-Valinamide N~2~-[(1- {[6-(2,5-dioxo-2,5-dihydro- lH-pyrrol yl)hexanoyl]amino } cyclopentyl)carbonyl] -N- HPLC (Protocol [(3R,4S,5S)methoxy {(2 S)[(1R,2R)methoxy- M): m/z 962.5 [M+H+] 2-methyloxo { [(1 S)phenyl(1 ,3-thiazol (13.014 yl)ethyl] minutes) amino } ]pyrrolidin— 1 -yl} hyloxoheptan yl] -N-methyl-L-Valinamide N—[6-(2,5-dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl]— HPLC (Protocol 2-methylalanyl-N— [(3R,4S,5 S) -3 -methoxy {(ZS) M): m/z 936.5 [(1R,2R)methoxymethyloxo{[(1S)phenyl- mc-#54 [M+H+] (9.22 1-(1,3 -thiazolyl)ethyl]amino } propyl]pyrroli minutes) dinyl} - 5-methyloxoheptanyl] -N-methyl-L- valinamidc N— [6-(2,5 -dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl] - L-Valyl-N~5~-carbamoyl-N—[4-({[(1-{[(ZS)-l- {[(3R,4S,SS)methoxy{(ZS)[(1R,2R) HPLC (Protocol methoxy-Z-methyl-3 - oxo-3 - { [(1 S)phenyl(1 ,3 - M): m/z 1368.6 thiazol-Z-yl chalCitPABC-#47 [M+H+] (13.157 )ethyl]amino } propyl]pyrrolidinyl} - 5-methyl minutes) oxoheptan—4-yl](methyl)amino} -3 -methyloxobutar1— yl] carbamoyl} cyclopentyl)carbamoyl] oxy} methyl)phen yl] -L-ornithinamide N— [6-(2,5 -dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl] - L-Valyl-N— {4-[(5 S,8S ,118,12R)[(2S)-butan—2-yl]— HPLC (Protocol 12-(2-{(ZS)[(1R,2R)methoxymcthyl-3 - { [( 1 S) - M): m/z 1386.6 2-phenyl- 1-(1,3 -thiazolyl)ethyl] amino } - 3-t chalCitPABC-#26 [M+}P] (16.21 hioxopropyl]pyrrolidinyl} oxoethyl)-4,10- minutes) dimethyl- 3,6,9-trioxo-5 ,8-di(propanyl)-2, 1 3 -dioxa- 4,7,10-triazatetradec-l-yl]phenyl}-N~5~-carbamoyl-L- ornithinamide N— [6-(2,5 -dioxo-2,5-dihydro- 1 ol- 1 xanoyl] - HPLC (Protocol N-methyl-L-Valyl-N— S,SS)-3 -methoxy {(28) A*): m/z 980.5 [( 1 R,2R)methoxymethyl { [( 1 S)phenyl- 1- [M+H+] (10.628 (1,3 -thiazolyl)ethyl] amino} -3 -thioxopropyl]py minutes) rrolidinyl} -5 -methyloxoheptanyl]-N-methyl-L- valinamidc N—[6-(2,5-dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl]— L-Valyl-N—{4-[(SS,8S,118,12R)[(ZS)-butanyl]- 12-(2-{(2S)[(1R,2R)methoxy{[(ZS) HPLC (Protocol methoxy oxo-3 -phenylpropanyl]amino} methyl- A*): m/z 1361.7 chalCitPABC-#42 3 _ [M+H+] (9.831 thioxopropyl]pyrrolidinyl} oxoethyl)-4,10- dimethyl-3,6,9-trioxo-5,8-di(propanyl)-2,13-dioxa- 4,7,10-triazatetradec-l-yl]phenyl}-N~5~-carbamoyl-L- ornithinamide N— 5 -dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl] - L-Valyl-N— {4-[(5 S,8S ,118,12R)[(2S)-butan—2-yl]— HPLC (Protocol 12- {2-[(2S){(1R,2R)mcthoxymcthyl- 3- [(2- A*): m/z 1324.6 chalCitPABC-#36 [M+Na+23] phenylethyl)amino] -3 -thioxopropyl} pyrrolidiny (9.987 l]oxoethyl}-4,10-dimethyl-3 ,6,9-trioxo-5 ,8- minutes) panyl)-2, 1 3 -dioxa—4,7, 1 O-triazatetradec yl]phenyl} -N~5~-carbamoyl-L- ornithinamide N— [6-(2,5 -dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl] - HPLC col yl-L-Valyl-N— [(3R,4S,SS)-3 xy {(28) A*): m/z 955.5 [( 1 R,2R)methoxy-3 - { [(ZS)methoxyoxo-3 - [M+H+] (10.679 phenylpropan—Z-yl]amino} methyl- 3-thioxopropyl]p minutes) dinyl} - 5-methyloxoheptanyl]-N-methyl- L-Valinamidc N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-l-oyl)- LC-MS (Protocol 2-methylalanyl-N— [(3R,4S,5 S) -3 -methoxy {(ZS) H): m/z 1078.7 [(1R,2R)methoxy-2 -methyl-3 -oxo-3 - { [( 1 S)phenyl- AmPeg6C2-#54 [M+H*] (2.56 1-( 1 ,3-thiaz olyl)ethyl]amino } propyl]pyrrolid minutes) inyl} -5 -methyloxoheptan—4-yl]-N-methyl-L- valinamidc N-[3 -(2- {2- [2-(2,5-dioxo-2,5 -dihydro- 1 H-pyrrol- l - LC-MS (Protocol yl)ethoxy]ethoxy} ethoxy)propanoyl] methylalanyl-NH ): m/z 1026.6 [(3R,4S,5 S)methoxy {(2 S)[(1R,2R)-1 -methoxy- 3C2-#54 [M+H+] (3.54 2-methyl-3 -oxo-3 - { [(1 S)phenyl(1 ,3 ol- minutes) 2-yl)ethyl]amino } propyl]pyrrolidin— 1 -yl} methyl oxoheptan—4-yl] -N-methyl-L-Valinamide N— [6-(2,5 -dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N— {4-[(3OS,3 3S, 34R) -3 3 - [(ZS)-butanyl] (2-{(ZS)[(1R,2R)methoxymethyl-3 -oxo-3 - LC-MS (Protocol {[(1S)phenyl(1,3 -thiazolyl)ethyl] amino} chalCitPABCAmPeg6C2- H): m/z 1677.9 [M+H+] propyl]pyrrolidinyl}oxoethyl)-27,27,3 2-trimethyl- #54 (3.48 3 ,25 ,2 8, 31-tetraoxo-3 0-(propan—2-yl)- minutes) 2,7,10,13,16,19,22,35-octaoxa-4,26,29,32- tetraazahexatriacontyl]phenyl} -N~5~-carbamoyl-L- ornithinamide N— [6-(2,5 -dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N— {4-[(218,24S,25 R)-24— [(ZS)-butanyl] LC-MS (Protocol (2-{(ZS)[(1R,2R)methoxymethyl-3 -oxo-3 - chalCitPABCAmPeg3C2- H): m/z 1545.8 {[(1S)phenyl(1,3 olyl)ethyl] amino} #54 [M+H+] (3.48 ]pyrrolidinyl}oxoethyl)-18,18,2 3-trimethyl- minutes) 3 ,16,19,22-tetraoxo(propan—2-yl)-2,7,10,13 ,26- pentaoxa—4, 17,20,2 3-tetraazaheptacosyl]phenyl} - N~5~-carbamoyl-L-ornithinamide N~2~-[(1- {[3-(2- (2,5-dioxo-2,5-dihydro-1H- pyrrol LC-MS (Protocol yl)ethoxy]ethoxy} )propanoyl]amino } cyclopentyl) Q1): m/z 1052.7 carbonyl] -N- [(3R,4S,5 S)-3 -methoxy {(ZS) 3C2-#47 [M+H+] (0.88 [(1R,2R)methoxymethyl-3 -oxo- S)ph minutes) enyl- 1-(1,3 -thiazolyl)ethyl]amino } propyl]pyrrolidin- 1-yl} - 5-methyloxoheptanyl]-N-methyl-L- midc 1-amino-N-(1-{[(ZS){[(3R,4S,5S)methoxy {(ZS)[(1R,2R)methoxymethyloxo {[(1S)- LC-MS (Protocol 2 l(1,3 -thiazol H): m/21104.88 AmPeg6C2-#47 [M+H+] yl)ethyl]amino } propyl]pyrrolidinyl} methyl (2.65 oxoheptanyl](methyl)ami minutes) no} - 3-methyloxobutan—2-yl]carbamoyl}cyclopentyl)- 3 ,6,9,12,15 ,18-hexaoxahenicosanamide N— [6-(2,5 -dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-valyl-N~5~-carbamoyl-N-(4-{16-[(1-{[(ZS) {[(3R,4S,5S)methoxy{(ZS)[(1R,2R) methoxy-Z-methyl-3 - oxo-3 -{[(1S)phenyl(1,3 - LC-MS (Protocol thiazol-Z- chalCitPABCAmPeg3C2- H): m/z 1571.8 #47 [M+H+] yl)ethyl]amino } propyl]pyrrolidinyl} - 5-methyl (3.56 oxoheptan—4-yl](methyl)amino} -3 -methyloxobutar1— minutes) 2-yl]carbamoyl} cyclopentyl)amino] -3 ,16-dioxo- ,13 -tetraoxaazahexadecyl}phenyl)-L- ornithin amide N— [6-(2,5 -dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N~5~-carbamoyl-N—(4- {25 -[(1-{[(ZS) {[(3R,4S,SS)methoxy{(ZS)[(1R,2R) y-Z-methyl-3 - oxo-3 -{[(1S)phenyl(1,3 - HPLC col l-Z- chalCitPABCAmPeg6C2- H): m/z 1703.8 #47 [M+H+] yl)ethyl]amino } propyl]pyrrolidinyl} - 5-methyl (3.57 oxoheptan—4-yl](methyl)amino} -3 -methyloxobutar1— minutes) 2-yl]carbamoyl} cyclopentyl)amino] -3 ,25 -dioxo- 2,7,10,13,16,19,22-heptaoxaazapentacos yl} pheny1)- L-ornithinamide N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol yl)cthoxy]cthoxy} )propanoyl] -N-mcthyl-L-Valyl- LCfMS (”“0001 N-[(3R,4S,5S)mcthoxy{(ZS)[(1R,2R) H). m/z 1045 .7 C2-#42 methoxy- 3- mcthoxyoxo-3 -phcnylpropan [M+H+] (3 .92 yl]a minutes) mino} mcthyl-3 opropyl]pyrrolidin—1-yl}-5 - methyloxohcptanyl] -N-mcthyl-L-Valinamidc N-(21-amino-4,7,10,13,16,19-hcxaoxahcnicosan—1-oyl)- LC-MS (Protocol N-mcthyl-L-Valyl-N— [(3R,4S,5S)-3 xy {(2S) H): m/z 1097.7 [(1R,2R)mcthoxy-3 -{[(2S)mcthoxyoxo Amch6C2-#42 [M+H+] (2.80 phenylpropanyl]amino } mcthyl- 3-thioxopropyl]py s) rrolidinyl} -5 -mcthyloxohcptanyl]-N-mcthyl-L- valinamidc N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hcxanoyl]— L-Valyl-N— {4-[(27S,3OS,33S,34R)-3 3-[(2S)-butanyl] - 34-(2-{(2S)[(1R,2R)mcthoxy{[(2S) LC-MS col methoxy oxo-3 -phcnylpropanyl]amino} mcthyl- chalCitPABCAmch6C2- H): m/z 1696.8 3 -thioxopropyl]pyrrolidin—1-yl}oxocthyl)-26,3 2- #42 [M+H+] (3 .73 dimcthyl- 3,25 ,28,31-tctraoxo-27,3 0-di(propanyl)- minutes) 2,7,10,13,16,19,22,35-octaoxa-4,26,29,32- tctraazahcxatriacontyl]phcnyl} -N~5~-carbamoyl-L- ornithi namidc N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hcxanoyl]— L-Valyl-N—{4—[(18S,21S,24S,25R)-24—[(2S)-butanyl]- LC-MS (Protocol 25-(2-{(2S)[(1R,2R)mcthoxy{[(2S) chalCitPABCAmch3C2- H): m/z 1564. 8 methoxy oxo-3 -phcnylpropanyl]amino} mcthyl- #42 [M+H+] (3 .70 3 -thioxopropyl]pyrrolidin— 1 -yl} oxocthyl)- 17,2 3- minutes) dimcthyl- 3,16,19,22-tctraoxo- 18,2 1-di(propanyl)- 2,7,10,13,26-pcntaoxa-4,17,20,23 azahcptacos yl]phenyl} -N~5~-carbamoyl-L- inamidc N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol LC-MS (Protocol yl)cthoxy]cthoxy} cthoxy)propanoyl] -N-mcthyl-L-Valyl- H): m/z 1070.6 N—[(3R,4S,SS)mcthoxy {(2S)[(1R,2R) Machg3C2-#26 [M+H*] (3.94 methoxymcthyl{[(1S)phcnyl(1,3-thiazolyl minutes) )cthyl]amino} - 3-thioxopropyl]pyrrolidin—1-yl} methyloxohcptanyl] -N-mcthyl-L-Valinamidc N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hcxanoyl]— HPLC (Protocol N-mcthyl-L-Valyl-N—[(3R,4S,5S){(2S)[(1R,2R) A): m/z 941.5 { [(1 S)- 1 xyphcnylcthyl]amino} mcthoxy [M+Hj (9.883 -3 -thioxopropyl]pyrrolidinyl}-3 -mcth minutes) oxy-5 -mcthyl oxohcptanyl] -N-mcthyl-L- valinamidc N-(21-amino-4,7,10,13,16,19-hcxaoxahcnicosanoyl)- LC-MS (Protocol N-mcthyl-L-Valyl-N— [(3R,4S,5S)-3 -mcthoxy {(2S) H): m/z 1122.6 [(1R,2R)mcthoxymcthyl{[(1S)phcnyl Amch6C2-#26 [M+H*] (2.76 (1,3 -thiazolyl)cthyl]amino} thioxopropyl]pyr minutes) rolidinyl} -5 -mcthyloxohcptanyl]-N-mcthyl-L- valinamidc N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hcxanoyl]— L-Valyl-N—{4—[(18S,21S,24S,25R)-24—[(2S)-butanyl] LC-MS (Protocol {(2S)[(1R,2R)mcthoxymcthyl{[(1S)- chalCitPABCAmch3C2- H): m/z 1588.0 2-phcr1yl(1,3-thiazolyl)cthyl]amino}-3 #26 [M+H+] (3 .74 -thioxopropyl]pyrrolidinyl} oxocthyl)- 17,23 - minutes) dimcthyl- 3,16,19,22-tctraoxo- 18,2 1-di(propanyl)- 2,7,10,13,26-pcntaoxa-4,17,20,23 -tctraazahcptacos nyl} -N~5~-carbamoyl-L- ornithinamidc N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol yl)ethoxy]ethoxy} ethoxy)propanoyl] yl-N— {4- LC-MS (Protocol [(5S,8S,118,12R)[(2S)-butanyl]—12-(2-{(2S) H): m/z 1476.8 [(1R,2R)methoxymethyl{[(1S)phenyl(1, MalPeg3C2ValCitPABC-#26 [M+H+] (3.81 3 -thiazolyl)ethyl]amino} -3 opropyl]pyrrolidin— minutes) 1-yl}oxoethyl)-4,10-dimethyl-3,6,9-trioxo-5,8- di(propanyl)-2,13 -dioxa—4,7, 1 O-triazatetradec yl]phenyl} -N~5~-carbamoyl-L- ornithinamide N-[6-(2,5 -dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N—{4-[(27S,3OS,33S,34R)[(ZS)-butanyl]- {(ZS)[(1R,2R)methoxymethyl{[(1S)- LC-MS (Protocol 2-pher1yl(1,3 -thiazolyl)ethyl]amino} -3 chalCitPABCAmPeg6C2- H): m/z 1721.9 -thioxopropyl]pyrrolidinyl}oxoethyl)-26,32- #26 [M+H+] (3.75 dimethyl- 3,25 -tetraoxo-27,3 0-di(propanyl)- minutes) 2,7,10,13,16,19,22,35-octaoxa-4,26,29,32- tetraazahexatriacontyl]phenyl} -N~5~-carbamoyl-L- ornithin amide N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— LC-MS (Protocol N-methyl-L-Valyl-N— {(3R,4S,5 S)-3 xy [(2S) Q1): m/z 897.7 R)methoxymethyl[(2- [M+H+] (1.00 phenylethyl)amino] -3 -thioxopropyl} pyrrolidinyl] -5 - minutes) methyl oxoheptan—4-yl} -N-methyl-L-Valinamide 2,5-dioxo-2,5-dihydro-1H-pyrrolyl)oxo- 3,6,9,12,15,18-hexaoxahenicosanyl] LC-MS col methylalanyl-N—[(3R,4S,5S)methoxy{(2S) H): m/z 1158.7 MalPeg6C2-#54 [M+H+] [(1R,2R)methoxymethyloxo{[(1S)phenyl- (3.55 1 -( 1 ,3 -th minutes) iazolyl)ethyl] amino } propyl]pyrrolidinyl} -5 - methyloxoheptanyl] -N-methyl-L-Valinamide N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol yl)ethoxy]ethoxy} ethoxy)propanoyl] -L-Valyl-N~5~- carbamoyl-N-[4-({[(1-{[(ZS){[(3R,4S,SS) methoxy {(2S)[(1R,2R)methoxymethyl LC-MS (Protocol oxo { [(1 H): m/z 1458.7 MalPeg3C2ValCitPABC-#47 [M+H+] S)pher1yl(1 ,3-thiazol (3.56 yl)ethyl]amino } propyl]pyrrolidinyl} methyl minutes) oxoheptan—4-yl](methyl)amino} -3 -methyloxobutar1— yl] carbamoyl} cyclopentyl)carbamoyl] oxy} methyl)phen yl] -L-ornithinamide N-[3 -(2- {2- [2-(2,5-dioxo-2,5 -dihydro-1H-pyrrol yl)ethoxy]ethoxy} ethoxy)propanoyl] -N-methyl-L-Valyl- LC-MS (Protocol N-{(3R,4S,SS)methoxy[(2S){(1R,2R) H): m/z 987.7 MalPeg3C2-#36 [M+H+] methoxymethyl-3 - enylethyl)amino] -3 - (3.97 thioxoprop minutes) yl}pyrrolidinyl]—5 -methyl- 1-oxoheptanyl} -N- methyl-L-Valinamide 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)-N-(1-{[(2S)- 1-{[(3R,4S,SS)methoxy{(ZS)[(1R,2R) LC-MS (Protocol methoxymethyloxo{[(1S)phenyl(1,3- H): m/z 1184.7 MalPeg6C2-#47 [M+H+] thiazolyl)ethyl] amino } propyl]pyrrolidinyl} -5 -me (3.67 thyl oxoheptanyl](methyl)amino} -3 -methyl anyl]carbamoyl} cyclopentyl)-3 ,6,9, 12,15 ,18- hexaoxahenicosan—2 1 -amide WO 72813 2012/056224 N-[l-(2,5-dioxo-2,5-dihydro- lH-pyrrol-l-yl)oxo- LC-MS (Protocol 3,6,9,12,15,l8-hexaoxahenicosan—2l-yl]—N-methyl-LH ): m/z 1202.7 valyl-N-[(3R,4S,5 S)methoxy{(ZS)[(1R,2R) MalPeg6C2-#26 [M+H+] (3.93 methoxy-Z-methyl- 3- {[(1 S)-2 -pher1yl- l-( l , 3 -thiazo minutes) lyl)ethyl]amino} -3 -thioxopropyl]pyrrolidin- l -yl} oxoheptanyl] -N-methyl-L-Valinamide N-[ l dioxo-2,5 -dihydro- lH-pyrrol-l-yl)oxo- LC-MS col 3 ,6,9, 12,1 5 l 8-hexaoxahenicosan—2 l -yl] -N-methyl-L- H): m/z 1118.8 valyl-N- S,5 S)-3 -methoxy[(ZS){(1R,2R) MalPeg6C2-#36 [M-H] (3.96 y-Z-methyl-3 - enylethyl)amino] -3 -thio minutes) xopropyl } pyrrolidin- l -yl] -5 -methyl oxoheptanyl} - N—methyl-L-Valinamide N— [6-(2,5 -dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl] - L-Valyl-N—{4-[(18S,21S,24S,25R)[(ZS)-butanyl]- LC-MS (Protocol 25- {2-[(ZS){(1R,2R)mcthoxymcthyl- 3- [(2- chalCitPABCAmPeg3C2- H): m/z 1506.8 ethyl)amino] - 3-thioxopropyl} pyrrolidin- l #36 [M+H+] (3.76 -yl]oxoethyl} - 17,23 -dimethyl-3 ,16, l 9,22-tetraoxo- minutes) 18,21-di(propanyl)-2,7,10,l3 ,26-pentaoxa- 4,17,20,23 -tetraazaheptacos- l -yl]phenyl} -N~5~- oyl-L-ornithinamide N-(21-amino-4,7,10, l3,l6,l 9-hexaoxahenicosan-l-oyl)- LC-MS (Protocol N-methyl-L-Valyl-N— {(3R,4S,5 S)-3 -methoxy- l - [(2S) H): m/z 1039.7 {(1R,2R)- l -methoxymethyl-3 -[(2- AmPeg6C2-#36 [M+H+] (2.68 phenylethyl)amino] -3 -thioxopropyl} pyrrolidin- l -yl] -5 - minutes) methylo xoheptanyl} -N-methyl-L-Valinamide N— 5 -dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl] - L-Valyl-N—{4-[(8S,118,12R)[(ZS)-butanyl](2 HPLC (Protocol {(ZS)[(1R,2R)-l -methoxymethyl-3 -oxo {[(1- M): m/z 1307.6 chalCitPABC-#60 [M+H+] phenylcyclopropyl)methyl]amino } propyl]pyrrol (12.696 idin- l -yl} ethyl)- 5,5 ,10-trimethyl-3 ,6,9-trioxo minutes) (propan-Z-yl)-2, l 3 -dioxa-4,7, l O-triazatetradec- l - yl]phenyl} -N~5~-carbamoyl-L- ornithinamide N— [6-(2,5 -dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl] - L-Valyl-N— {4- 3 08,3 3 S,34R)-3 3- [(ZS)-butanyl] LC-MS (Protocol 34- {2-[(ZS){(1R,2R)mcthoxymcthyl- 3- [(2- chalCitPABCAmPeg6C2- H): m/z 1638.0 phenylethyl)amino] - 3-thioxopropyl} idin- l #36 [M+H+] (3.77 -yl] oxoethyl} -26,32-dimethyl-3 ,25 ,28, 3 l -tetraoxo- minutes) 27,30-di(propanyl)-2,7,10,13 , 16,1 9,22,3 5 -octaoxa- 4,26,29, 32-tetraazahexatriacont- l -yl]phenyl} -N~5~- carbamoyl-L-ornithinamide N— [6-(2,5 -dioxo-2,5-dihydro- l H-pyrrol- l xanoyl] - L-Valyl-N—{4-[(18S,21S,24S,25R)[(ZS)-butanyl] LC-MS (Protocol 25 -(2- {(ZS)[(1R,2R)-3 -{[(1S)carboxy chalCitPABCAmPeg3C2- H): m/z 1550.9 phenylethyl]amino} - l -methoxymethyl- 3-thioxoprop #41 [M+H+] (3.53 yl]pyrrolidin- l -yl} oxoethyl)- 17,23 -dimethyl- minutes) 3, 16,1 9,22-tetraoxo- l 8,2 l-di(propanyl)-2,7, 10,1 3 ,26 pentaoxa—4, l 7,20,2 3-tetraazaheptacos- l -yl]phenyl} - N~5~-carbamoyl-L-ornithinamide N-[ l -(2,5 -dioxo-2,5 -dihydro-lH-pyrrol-l-yl)oxo- 3 ,6, 9,12,15 l8-hexaoxahenicosan-2 l -yl] LC-MS (Protocol , methylalanyl-N— [(3R,4S,5 S)-3 -methoxy- l- {(ZS) H): m/z 1101.8 MalPeg6C2-#60 [M+H+] [(1R,2R)- l -methoxy-2 -methyl-3 -oxo-3 - {[(1 - (3.66 phenylcyclopropyl)mc minutes) thyl]amino} propyl]pyrrolidin— l -yl} -5 -methyl tan—4-yl] -N-methyl-L-Valinamide N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-l-oyl)- LC-MS (Protocol 2-methylalanyl-N— [(3R,4S,5 S) -3 -methoxy {(2S) H): m/z 1021.7 [(1R,2R)methoxymethyloxo{[(1- AmPeg6C2-#60 [M+H+] (2.57 phenylcyclopropyl)methyl]amino } propyl]pyrrolidin minutes) yl} -5 -m ethyloxoheptanyl] -N-methyl-L-Valinamide N-[3 -(2- {2- [2-(2,5-dioxo-2,5 -dihydro- 1 H-pyrrol- l - LC-MS (Protocol yl)ethoxy]ethoxy} ethoxy)propanoyl] methylalanyl-NH ): m/z 969.7 [(3R,4S,5 S)methoxy {(2 S)[(1R,2R)-1 -methoxy- MalPeg3C2-#60 [M+H+] (3.65 2-methyl-3 -oxo-3 -{[(1-phenylcyclopropyl)methyl]a minutes) mino } propyl]pyrrolidinyl} - yl oxoheptan—4- yl] -N-methyl-L-Valinamide N-[ 1-(2,5 -dioxo-2,5 -dihydro- 1 olyl)-2 1 -0X0- LC-MS col 3 ,6,9,12,15 ,18-hexaoxahenicosan-2l-yl]—N-methyl-L- H): m/z 1163.0 valyl-N-[(3R,4S,5S){(2S)[(1R,2R) {[(1S) MalPeg6C2-#4l [M-H] (3.70 carboxy-Z-phenylethyl]amino} methoxymethyl-3 s) -thioxopropyl]pyrrolidin— 1 -yl} -3 -methoxy-5 -methyl oxoheptan—4-yl] -N-methyl-L-Valinamide N-(21-amino-4,7,10,13 -hexaoxahenicosan—l-oyl)- LC-MS (Protocol 2-methylalanyl-N— [(3R,4S,5 S) {(2S) [( 1 R,2R)-3 - Q1): m/z 1009.8 {[2-(cyclohepta—2,4,6-trienyl)ethyl]amino} AmPeg6C2-#66 [M+H+] (0.72 methoxy-Z-methyl-3 -oxopropyl]pyrrolidjnyl} me minutes) thoxy-5 -methyloxoheptanyl] -N-methyl-L- valinamidc N— [6-(2,5 -dioxo-2,5-dihydro- 1 H-pyrrolyl)hexanoyl] - L-Valyl-N— {4-[(30S,3 3S, 34R)-3 3- [(2S)-butanyl] LC-MS (Protocol (2- {(2S)[(1R,2R)methoxymethyl-3 -oxo-3 - {[(1- chalCitPABCAmPeg6C2- H): m/z 1621.0 phenylcyclopropyl)methyl]amino } propyl]pyrro #60 [M+H+] (3.55 lidinyl} oxoethyl)-27,27,32-trimethyl-3 ,25,28,31- minutes) tetraoxo-3 0-(propan—2-yl)-2,7,10,l 3,16,19,22,3 5- octaoxa-4,26,29,32-tetraazahexatriacontyl]phenyl} - N~5~-carbamoyl-L-ornithinamide N— 5 -2,5-dihydro- 1 H-pyrrolyl)hexanoyl] - HPLC (Protocol 2-methylalanyl-N— [(3R,4S,5 S) -3 -methoxy {(2S) M): m/z 911.5 [( 1 R,2R)- 1 xy-3 - { [(2S)methoxyoxo-3 - [M+H+] (11.847 phenylpropan-Z-yl]amino } methyl- 3- pyl]pyrrol minutes) idinyl} methyloxoheptanyl]-N-methyl-L- midc N-(24-bromooxo-4,7,10,13,16,19-hexaoxa—22- azatetracosanoyl)methylalanyl-N— [(3R,4S,5 S) LC-MS (Protocol {(2S)[(1R,2R){[2-(cyclohcpta-2,4,6-tricn Q1): m/z 1129.8 2AcAmPeg6C2-#66 [M+H+] yl)ethyl]amino} methoxymethyl-3 - (0.85 oxopropyl]pyrrol minutes) idinyl} -3 -methoxy-5 -methyloxoheptanyl] -N- methyl-L-Valinamide N— [6-(2,5 -dioxo-2,5-dihydro- 1 olyl)hexanoyl] - LC-MS (Protocol 2-methylalanyl-N— [(3R,4S,5 S) {(2S) [( 1 R,2R)-3 - Q1): m/z 867.7 {[2-(cyclohepta—2,4,6-trienyl)ethyl]amino} [M+H+] (0.90 y-Z-methyl-3 opyl]pyrrolidinyl} m minutes) ethoxy- 5 loxoheptanyl] -N-methyl-L- valinamidc N— [6-(2,5 -dioxo-2,5-dihydro- 1 H-pyrrol- 1 xanoyl] - L-Valyl-N—{4-[(8S,11S,12R)[(2S)-butanyl]—12-(2- LC-MS (Protocol {(2S)[(1R,2R)methoxymethyl-3 -oxo {[(1S)- Q1): m/z 1355.9 2-phenyl( 1 ,3-thiazolyl)ethyl]amino} p chalCitPABC-#88 [M+H+] (0.87 ropyl]pyrrolidin— 1 -yl} yl)-4,5,5 ,10-tetramethylminutes ) 3,6,9-trioxo- 8-(propanyl)-2,l3-dioxa-4,7,10- triazatetradecyl]phenyl} -N~5~-carbamoyl-L- ornithinamide N— {6- [(bromoacetyl)amino]hexanoyl} -L-Valyl-N— {4- [(8S,11S,12R)[(2S)-butanyl]—12-(2-{(2S) LC-MS col [( l R,2R) { [2-(cyclohepta-2,4,6-trier1— l - Q1): m/z 1314.9 yl)ethyl]amino} - l-methoxy-Z-methyl-3 - chalCitPABC-#88 [M+H+] (0.91 oxopropyl]py1rolidinyl minutes) }oxoethyl)-5,5, l 0-trimethyl-3,6,9-trioxo(propan yl)-2, l 3 -dioxa- 4,7, azatetradec- l enyl} -N~5~- carbamoyl-L-ornithinamide N—[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— LC-MS (Protocol Z-methylalanyl-N- [(3R,4S,5 S) -3 xy- l - 2- Q1): m/z 876.7 [M+H+] [(lR,2R)- l -methoxymethyloxo(quinolin (0.75 ylamino)propyl]pyrrolidin- l -yl} -5 -methyloxohept anyl] -N-methyl-L-Valinamide 2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— L-Valyl-N—{4-[(8S,11S,12R)[(2S)-butanyl]—12-(2- HPLC (Protocol {(2S)[(1R,2R){[(1S)carboxy A): m/z 1318.6 phenylethyl]amino} - l -methoxy-2 -methyl-3 - tPABC-#44 [M+}P] (9.174 thioxopropyl]py minutes) rrolidin- l -yl} oxoethyl)-5,5, l 0-trimethyl-3,6,9-trioxo- 8-(propanyl)-2, l 3 -dioxa-4,7, l 0-triazatetradec- l - yl]phenyl} -N~5~-carbamoyl-L- ornithinamide N—[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— Z-methylalanyl-N-[(3R,4S,5S){(2S)[(1R,2R) HPLC (Protocol {[(R)-(7S)-bicyclo[4.2.0]octa-1,3,5-trien A): m/z 909.5 [M+H+] yl(carboxy)methyl]amino} - l -methoxy-Z-methyl-3 - (9.063 oxoprop minutes) yl]pyrrolidin- l -yl} -3 -methoxymethyl- l -oxoheptan—4- -N-meth l-L-Valinamide N—[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— L-Valyl-N-{4-[(8S,11S,12R)(2-{(2S)[(1R,2R) {[(R)-(7S)-bicyclo[4.2.0]octa-1,3,5-trien HPLC (Protocol yl(carboxy)methyl]amino} - l -methoxy-Z-methyl-3 - M): m/z 1315.7 chalCitPABC-#108 [M+}P] oxopr (11.89 opyl]pyrrolidin- l -yl} oxoethyl)-l l-[(2S)-butanyl] - minutes) ,5, l 0-trimethyl-3,6,9-trioxo(propanyl)-2,l 3- dioxa-4,7, l O-triazatetradec- l -yl]phenyl} -N~5~- carbamoyl-L-ornithinamide N-[3-(2- {3-[(2,5-dioxopyrrolidjn— l -yl)oxy]—3- oxopropoxy} ethoxy)propanoyl] -L-Valyl-N— {4- [(8S,11S,12R)[(2S)-butanyl]—12-(2-{(2S) HPLC (Protocol [( l R,2R) { [2-(cyclohepta-2,4,6-trier1— l - NHSCOPegZCZValCitPABC- M): m/z 683.3 yl)ethyl] amino} - l -metho #66 [M+H*2] (10.03 ethyl-3 opyl]pyrrolidin- l -yl} oxoethyl)- minutes) ,5, l 0-trimethyl-3,6,9-trioxo(propanyl)-2,l 3- dioxa-4,7, l zatetradec- l -yl]phenyl} -N~5~- carbamoyl-L-ornithinamide 2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— L-Valyl-N~5~-carbamoyl-N— {4-[( { [(2S) { [(2S) 4S,5S)methoxy{(2S)[(1R,2R) HPLC (Protocol methoxy-Z-methyloxo { [(l S)phenyl- l -(l ,3- M): m/z 1368.6 chalCitPABC-#98 thiazol [M+H+] (12.504 yl)ethyl]amino } propyl]pyrrolidin- l -yl} -5 -methyl- 1- minutes) oxoheptan—4-yl](methyl)amino} -3 -methyloxobutar1— 2-yl]carbamoyl } methylpyrrolidin- l - yl]carbonyl} oxy)methyl]phenyl} -L-ornithinamide WO 72813 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N—{4-[(9S,1ZS,13R)[(ZS)-butanyl](2- LC-MS (Protocol {(ZS)[(1R,2R)methoxymethyloxo {[(1S)- Q): m/z 1356.5 yl( 1 ,3-thiazolyl)ethyl]amino} p chalCitPABC-#95 [M+H+] (1.79 ropyl]pyrrolidin— 1 -yl} oxoethyl)-6,6,1 1-tr1methyl- minutes) 3,7,10-trioxo(propanyl)-2,14-dioxa-4,8,11- pentadecyl]phenyl} -N~5~-carbamoyl-L- ornithinamide N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol HPLC (Protocol yl)ethoxy]ethoxy} ethoxy)propanoyl] methylalanyl-N- M): m/z 987.5 [(3R,4S,SS){(ZS)[(1R,2R){[(1S)carboxy MalPeg3C2-#69 [M+H+] (10.702 phenylethyl] amino} methoxymethyl-3 -oxoprop minutes) yl]pyrrolidinyl}-3 -methoxymethyloxoheptan—4- -N-meth l-L-Valinamide N-(21-amino-4,7,10,13,16,19-hexaoxahenicosanoyl)- LC-MS (Protocol 2-methylalanyl-N—[(3R,4S,5S){(ZS)[(1R,2R) H): m/z 1040.1 AmPeg6C2-#69 [M+H+] {[(1 S)carboxyphenylethyl]amino} methoxy (2.12 -3 -oxopropyl]pyrrolidinyl}-3 -methoxy minutes) methyloxoheptanyl] -N-methyl-L-Valinamide 2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N—{4-[(8S,118,12R)[(ZS)-butanyl](2- LC-MS (Protocol {(ZS)[(1R,2R)methoxymcthyl {[(1S) Q): m/z 1371.4 phenyl-l-(1,3 olyl)ethyl]amino} -3 -thio chalCitPABC-#84 [M+H*] (1.89 xopropyl]pyrrolidinyl} yl)-4,5 ,5 ,10- minutes) ethyl-3 rioxo(propanyl)-2, 13 -dioxa- 4,7,10-triazatetradecyl]phenyl}-N~5~-carbamoyl-L- ornithinamide N-(6-aminohexanoyl)-L-Valyl-N— {4-[(8S,1 1 S, 12R)- 1 1- [(ZS)-butanyl](2- {(ZS)[(1R,2R)methoxy LC-MS (Protocol methyloxo{[(1S)phenyl(1,3-thiazol H): m/z 1262.3 AmCapValCitPABC-#54 [M+H*] yl)ethyl]amino } propyl]pyrrolidin— 1-yl} oxoethyl (2.35 )-5,5,10-trimethyl-3,6,9-trioxo(propanyl)-2,13- minutes) dioxa-4,7,10-triazatetradecyl]phenyl}-N~5~- carbamoyl-L-ornithinamide N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N—{4-[(8S,118,12R)[(ZS)-butanyl](2- LC-MS (Protocol {(ZS)[(1R,2R)methoxy{[(ZS)methoxy Q): m/z 1330.9 oxo-3 -phenylpropanyl]amino} methyl-3 -oxo chalCitPABC-#226 [M+H+] (1.77 ]pyrrolidin—1-yl}oxoethyl)-4,5,5,10- minutes) ethyl-3 ,6,9-trioxo(propanyl)-2, 13 -dioxa- 4,7,10-triazatetradecyl]phenyl}-N~5~-carbamoyl-L- ornithinamide N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hexanoyl]— L-Valyl-N~5~-carbamoyl-N— {4-[( { [(ZS) { [(ZS) {[(3R,4S,SS)methoxy{(ZS)[(1R,2R) LC-MS (Protocol methoxy- 3- {[(ZS)methoxyoxo-3 -phenylpropan Q): m/z 1342.6 chalCitPABC-# 1 17 [M+H+] yl]am (1.80 1110} methyl oxopropyl]pyrrolidinyl} methyl- minutes) 1 - oxoheptan—4-yl] (methyl)amino} -3 -methyl oxobutan—Z-yl] carbamoyl} hylpyrrolidin yl]carbonyl} oxy)methyl]phenyl} -L-ornithinamide 1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)oxo- HPLC (Protocol 3,6,9,12,15,18-hexaoxahenicosanyl]methyl-LM ): m/z 1185.6 prolyl-N—[(3R,4S,5S)methoxy {(ZS)[(1R,2R) MalPeg6C2-#98 [M+H+] (11.985 methoxy-Z-methyloxo{[(1S)phenyl(1,3 minutes) -thiazolyl)ethyl] amino} propyl]pyrrolidin— 1 -yl} -5 - methyloxoheptanyl] -N-methyl-L-Valinamide N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hcxanoyl]— L-Valyl-N~5~-carbamoyl-N— {4-[( { [(2S) { [(2S) LC-MS (Protocol {[(3R,4S,SS){(2S)[(1R,2R){[(1S)carboxy Q): m/z 1328 .6 phenylcthyl]amino} mcthoxymcthyl-3 -oxopro chalCitPABC-#1 18 [M+H+] (1 .68 pyl]pyrrolidinyl}-3 -mcthoxy-5 -mcthyl oxohcptan- minutes) 4-yl](methyl)amino } -3 -mcthyl oxobutan yl]carbamoyl}mcthylpyrrolidin yl]carbonyl} oxy)mcthyl]phcnyl} -L-ornithinamidc N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hcxanoyl]— L-Valyl-N—{4-[(8S,118,12R)[(2S)-butanyl]—12-(2- HPLC (Protocol {(2S)[(1R,2R){[(1S)carboxy M): m/z 1 3 53 .6 phenylcthyl]amino} - 1 xy-2 -mcthyl-3 - chalCitPABC-#80 [M+Na+] (12 .751 thioxopropyl]py minutes) rrolidinyl}oxocthyl)-4,5 ,5 tramcthyl-3 ,6,9- trioxo- 8-(propanyl)-2,13 -dioxa-4,7,10-triazatctradccyl]phenyl} -N~5~-carbamoyl-L-ornithinamidc 1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)oxo- LC-MS (Protocol 3,6,9,12,15,18-hcxaoxahcnicosanyl]mcthyl-L- Q): m/z 1145 .6 prolyl-N-[(3R,4S,5S){(2S)[(1R,2R) {[(1S) C2-#1 18 [M+H+] (1 .66 yphcnylcthyl]amino } mcthoxymcthyl- s) 3- oxopropyl]pyrrolidin— 1 -yl} -3 -mcthoxy-5 -mcthyl oxohcptan—4-yl] -N-mcthyl-L-Valinamidc N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)oxo- 3,6,9,12,15,18-hcxaoxahcnicosanyl] HPLC (”“0001 methylalanyl-N-[(3R,4S,5S){(2S)[(1R,2R)—3- M): m/z 1146.6 C2-#230 {[(R)-carboxy( 1 -phenylcyclopropyl)mcthyl]ammo} . + [M+H ] (12.071 minutes) methox.y-.2- mcthyl-3 -oxopropyl]pyrrolld1nyl}-3 -mcthoxy methyloxohcptanyl] -N-mcthyl-L-Valinamidc N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hcxanoyl]— L-Valyl-N~5~-carbamoyl-N-{4-[({[(2R){[(2S) 4S,SS)mcthoxy{(2S)[(1R,2R) LC-MS (Protocol Q): m/z 1367.3 methoxymcthyloxo{[(1S)phcnyl(1,3- chalCitPABC-#232 thlaZOI [M+H+] (1.81 yl)cthyl]ammo } ]pyrrolldmyl} -5 -mcthyl- 1-. . . minutes) oxohcptan—4-yl](methyl)am1no} -3 -mcthyloxobutar1— 2-yl]carbamoyl}mcthylpyrrolidin yl]carbonyl} oxy)mcthyl]phcnyl} -L-ornithinamidc 1-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)hcxanoyl]— LC-MS (Protocol 2-mcthyl-L-prolyl-N—[(3R,4S,5S)mcthoxy{(2S) Q): m/z 937.4 [(1R,2R)mcthoxy-3 -{[(2S)mcthoxyoxo [M+H+] (1 .91 phenylpropanyl]amino} mcthyl-3 -oxopropyl]pyr minutes) rolidinyl} -5 loxohcptanyl] -N-mcthyl-L- valinamidc 1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl)oxo- HPLC (Protocol 3,6,9,12,15,18-hcxaoxahcnicosanyl]mcthyl-L- M): m/z 1161.6 prolyl-N-[(3R,4S,5S)mcthoxy{(2S)[(1R,2R) Machg6C2-#1 17 [M+H+] (12.1 15 methoxy{[(2S)mcthoxyoxophcnylpropan minutes) yl]amino} mcthyl-3 -oxopropyl]pyrrolidinyl}-5 - methyloxohcptanyl] -N-mcthyl-L-Valinamidc N—[ 5 -(2,5 -dioxo-2,5 -dihydro-1H-pyrrolyl)pcntanoyl]— LC'MS (”010001 2-mcthylalanyl-N—[(3R,4S,SS){(2S)[(1R,2R) Q): m/z 8 83 .3 . + {[(1 S)carboxyphcnylcthyl]ammo} mcthoxy [M+H ] (1 .57 methyl-3 -oxopropyl]pyrrolld1nyl}-3 -mcthoxy-. . -mcthyl- 1 ptan—4-yl] -N-mcthyl-L-Valmamldc N— [4-(2,5 -dioxo-2,5 -dihydro-1H-pyrrolyl)butanoyl]— HPLC (Protocol ylalanyl-N—[(3R,4S,5S){(2S)—2-[(1R,2R) M): m/z 869 5 mb-#69 {[(1S)carboxyphcnylcthyl]amino}mcthoxy [M+.H+] (10 874 methyl-3 -oxopropyl]pyrrolidinyl} -3 -mcthoxy-5 minute 5). -mcthyl oxohcptan—4-yl] -N-mcthyl-L-Valinamidc N-(21-amino-4,7,10, l3,l6,l 9-hexaoxahenicosan-l-oyl)- 2-methylalanyl-N- {(3R,4S,5 S)[(ZS) LC-MS (Protocol {(3R,4R,7S,lZS)benzyl[3 -chloro(propan—2- Q1): m/z 1514.3 yloxy)phenyl] methyl- l 2- [4-(8-methylimidazo[ l ,2- AmPeg6C2-#234 [M+H+] (0.76 a]pyridin minutes) yl)benzyl] -5 ,8, l 4-trioxo-2, 9-dioxa-6, l 3- diazatetradecan-3 -yl} pyrrolidin- l -yl] hoxy-5 - methyl oxoheptanyl} -N-methyl-L-Valinamide N-(21-amino-4,7,10,l3 , 16,1 9-hexaoxahenicosan— l-oyl)- 2-methylalanyl-N— [(3R,4S,5 S) {(ZS) [( 1 R,2R)-3 - LC-MS (Protocol - l - { [4-(5 - l ,3 -benzothiazolyl) Q1): m/z 1280.2 AmPeg6C2-#235 [M+H*] methylphenyl]amino} - l -oxo -3 -phenylpropan—2-yl]amin (0.87 o} - l -methoxy-2 -methyl-3 -oxopropyl]pyrrolidin- l -yl} -3 - minutes) methoxy-S-methyloxoheptanyl] -N-methyl-L- valinamidc l-[6-(2,5-dioxo-2,5-dihydro- l ol- l -yl)hexanoyl]— LC-MS (Protocol 2-methyl-L-prolyl-N—[(3R,4S,SS){(ZS)[(1R,2R) Q): m/z 923.3 [M+H*] {[(l S)- l -carboxyphenylethyl]amino} - l -methoxy (1.73 methyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methox minutes) y-5 -methyloxoheptan—4-yl] -N-methyl-L-Valinamide N-[l-(2,5-dioxo-2,5-dihydro- lH-pyrrol-l-yl)oxo- 3,6,9,12,15, lS-hexaoxahenicosan-Zl-yl]—2- LC-MS (Protocol methylalanyl-N-[(3R,4S,SS){(ZS)[(1R,2R) Q1): m/z 1175.3 MalPeg6C2-#123 [M+H*] - l -tert-butoxy- l -oxophenylpropan—2- (0.99 yl] amino} - l -meth minutes) oxy-Z-methyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methoxy- loxoheptan—4-yl] -N-methyl-L-Valinamide N—[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— LC-MS (Protocol N,2-dimethylalanyl-N-[(3R,4S,5 S)-3 -methoxy- l - {(28)- Q): m/z 925.7 2-[(1R,2R)- l -methoxy { [(ZS)- l-methoxy- l -oxo [M+H*] (1.85 phenylpropan-Z-yl]amino} methyl-3 -oxopropyl]py minutes) rrolidin- l -yl} -5 -methyloxoheptanyl] -N-methyl-L- midc l-[7-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)heptanoyl] - LC-MS (Protocol 2-methyl-L-prolyl-N—[(3R,4S,SS){(ZS)[(1R,2R) Q): m/z 937.7 [M+H+] {[(l S)- l -carboxyphenylethyl]amino} - l -methoxy (1.80 methyl-3 opyl]pyrrolidin— l -yl} - 3-metho minutes) xy- 5-methyl- l ptanyl] hyl-L-Valinamide l-[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— LC-MS (Protocol Z-methyl-D-prolyl-N-[(3R,4S,SS)methoxy- l - {(ZS) Q): m/z 937.3 R)- l -methoxy-3 - { [(ZS)- l-methoxy- l -oxo [M+H+] (1.88 phenylpropan-Z-yl]amino} methyl-3 opyl]pyr minutes) rolidin- l -yl} -5 -methyloxoheptanyl] -N-methyl-L- valinamidc l-[4-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)butanoyl] - LC-MS (Protocol 2-methyl-L-prolyl-N—[(3R,4S,SS){(ZS)[(1R,2R) Q): m/z 895.3 mb-#118 [M+H+] {[(l S)- l -carboxyphenylethyl]amino} - l -methoxy (1.63 methyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methox y-5 -methyloxoheptan—4-yl] -N-methyl-L-Valinamide N—[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— L-Valyl-N~5~-carbamoyl-N— {4-[( { [(ZS) { [(ZS)- l - {[(3R,4S,SS){(ZS)[(1R,2R){[(lS,2R) LC-MS (Protocol hydroxy- l lpropanyl]amino} - l -methoxy Q): m/z 1314.3 chalCitPABC-#134 [M+H*] methyl (1.67 -3 -oxopropyl]pyrrolidin- l -yl} hoxy-5 -methyl minutes) oxoheptan—4-yl](methyl)amino} -3 -methyloxobuta11— 2-yl]carbamoyl } methylpyrrolidin- l - yl]carbonyl} oxy)methyl]phenyl} -L-ornithinamide 2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— LC-MS (Protocol N,2-dimethylalanyl-N-[(3R,4S,5S){(ZS)[(1R,2R)- Q1): m/z 897.34 3 - {[(l S,2R)hydroxy- l -phenylpropanyl] amino} - l - [M+H*] (0.90 methoxymethyl-3 -oxopropyl]pyrrolidjn— l -yl minutes) } -3 -methoxy-5 -methyloxoheptanyl] -N-methyl-L- valinamide methyl N- {(2R,3R)- 3-[(ZS){(3R,4S,5 S)[{N-[1- HPLC (Protocol (2,5 -dioxo-2,5 ro- lH-pyrrol- l 4,24-dimethyl- M): m/z 1169.6 21,25 -dioxo-3 2, l 5 ,18-hexaoxaazapentacosan- MalPeg6C2-#126 [M+Na+] (12.583 25 -yl] -L-Valyl} (methyl)amino] -3 -methoxy-5 -met minutes) hylheptanoyl} pyrrolidin—Z-yl] -3 -methoxy methylpropanoyl} -L-phenylalaninate methyl N- [(2R, 3R)-3 -{(ZS)[(3R,4S,5 S) { [N-(3 - HPLC (Protocol {[6-(2,5 -dioxo-2,5 -dihydro- l H-pyrrol- l - M): m/z 925.5 yl)hexanoyl]amino} -2,2-dimethylpropanoyl)-L- [M+H+] (12.994 yalyl](methyl)amino} -3 -methoxy minutes) methylheptanoyl]pyrrolidin—2-yl} -3 - methoxymethylpropanoyl] -L-phenylalaninate l- [5 -(2,5 -dioxo-2,5 ro- l H-pyrrol- l -yl)pentanoyl] - LC-MS (Protocol 2-methyl-L-prolyl-N- [(3R,4S,5 S){(ZS)[(1R,2R)-3 - Q): m/z 909.2 [M+H+] { [(1 S)- l -carboxyphenylethyl]amino} - l -methoxy (1.68 methyl-3 -oxopropyl]pyrrolidin— l -yl} - 3-metho minutes) xy- 5-methyl- l -oxoheptanyl] -N-methyl-L-Valinamide methyl N— [(2R,3R)-3 [(3R,4S,5 S) { [N- ( {(3 S)- l - [6-(2,5 -dioxo-2,5 -dihydro- l H-pyrrol- l - LC-MS (Protocol yl)hexanoyl] -3 pyrrolidin—3 -yl} yl)-L- Q1): m/z 941.3 [M+H*] yalyl](methyl)amino} -3 -methoxy (0.96 methylheptanoyl]pyrrolidi nyl} methoxymethylpropanoyl] -L- alaninate N-[ l -(2,5-dioxo-2,5 -dihydro- lH-pyrrol-l-yl)oxo- 3,6,9,12, l 5, l 8-hexaoxahenicosan-2 l -yl]-N,2- LC-MS (Protocol dimethylalanyl-N— [(3R,4S, 5S) -3 -methoxy- l- {(ZS) Q): m/z 1147.3 MalPeg6C2-#226 [M+H+] [(1R,2R)-l-methoxy-3 - { [(ZS)- l-methoxy- l -oxo-3 - (1.76 phenylpropa minutes) nyl]amino} hyloxopropyl]pyrrolidin- l -yl} methyl- 1 ptan—4-yl] -N-methyl-L-Valinamide N-[ l -(2,5 -dioxo-2,5 -dihydro-lH-pyrrol-l-yl)oxo- 3,6,9,12, l 5, l 8-hexaoxahenicosan-2 l -yl]-N,2- LC-MS (Protocol dimethylalanyl-N— [(3R,4S, SS) {(ZS) [( 1 R,2R)-3 - Q1): m/z 1141.3 MalPeg6C2-#l 45 [M+Na*] {[( l S,2R)hydroxy- l -phenylpropanyl]amino} - l - (0.87 methoxy minutes) hyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methoxy-5 - methyloxoheptanyl] hyl-L-Valinamide N—[(2R,3R){(2S)[(3R,4S,SS){[N-({(ZS)[6- LC-MS (Protocol (2,5 -dioxo-2,5 -dihydro- lH-pyrrol- l -yl)hexanoyl] Q): m/z 937.3 methylpiperidin-Z-yl} carbonyl)-L- [M+H+] (1.50 yalyl](methyl)amino} -3 -methoxy s) methylheptanoyl]pyrrolidin—Z-yl} - 3-methoxymethylpropanoyl] -L-phenylalanine N—[(2R,3R)-3 -{(ZS)[(3R,4S,5 S){[N-({(2R)[6- HPLC (Protocol (2,5 -dioxo-2,5 -dihydro- lH-pyrrol- l -yl)hexanoyl] A): m/z 937.5 methylpiperidin-Z-yl} yl)-L- [M+Ht] yalyl](methyl)amino} -3 -methoxy (7.855minutes) methylheptanoyl]pyrrolidin—Z-yl} - 3-methoxymethylpropanoyl] -L-phenylalanine (N-[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l-yl)hexanoyl] - L-Valyl-N—{4-[(8S,11S,12R)(2-{(2S) [(3R,4R,7S)benzyl- l 5- {2-[(3,5-dimethyl- l H-pyrrol- LC-MS (Protocol 2-yl-kappaN)methylidene] -2H-pyrrol- 5-yl-kappaN} Q1): m/z 1640.4 met tPABC-#23 1 [M+Na+23] (0.94 hyl- 5 ,8, l 3 -trioxooxa-6,9, l 2-triazapentadecan-3 - minutes) rolidin— l -yl} oxoethyl)-l l- [(2S)-butanyl]— ,5, l 0-trimethyl-3,6,9-trioxo(propanyl)-2,l 3- dioxa-4,7, l O-triazatetradec- l -yl]phenyl} -N~5~- carbamoyl-L-ornithinamidato)(difluoro)boron N-[ l -(2,5-dioxo-2,5 -dihydro- lH-pyrrol-l-yl)oxo- 3,6,9,12, l 5, l 8-hexaoxahenicosan-2 l -yl]-N,2- LC-MS (Protocol dimethylalanyl-N— [(3R,4S, 5S) -3 -methoxy- l- {(2S) Q1): m/z 1173.3 [( l R,2R)- l -methoxy-2 -methyl-3 -oxo- 3- { [(2S)oxo-3 - MalPeg6C2-#238 [M+H*] (0.96 phenyl minutes) - l -(propen- l -yloxy)propan no } propyl]pyrrolidin- l -yl} -5 -methyl oxoheptan—4-yl] -N-methyl-L-Valinamide l-[ l -(2,5 -dioxo-2,5 -dihydro-lH-pyrrol-l-yl)oxo- 3,6,9,12,l 5,l8-hexaoxahenicosanyl]methyl-L- LC-MS (Protocol prolyl-N-[(3R,4S, 5 S){(2S)[(1R,2R) {[(2S) Q): m/z 1201.3 MalPeg6C2-#Z39 [M+H+] tcrt-butoxy- l-oxophenylpropanyl]amino} - l -m (2.02 ethoxy-Z-methyl-3 opyl]pyrrolidin- l -yl} - 3- minutes) methoxymethyloxoheptanyl] -N-methyl-L- valinamidc N— [6-(2,5 -dioxo-2,5-dihydro- l H-pyrrol- l xanoyl] - 2-methylalanyl-N— [(3R,4S,5 S) {(2S) [( 1 3 - LC-MS (Protocol {[(2S)- 1 butoxy- l - oxo-3 -phenylpropan Q1): m/z 953.3 [M+H+] yl]amino} - l -methoxymethyl-3 -oxopropyl]pyrrolidin— (1.04 minutes) -yl} -3 -methoxy-5 -methyloxoheptan—4-yl] -N-methyl- L-Valinamidc N-[6-(2,5 -dioxo-2,5 -dihydro- lH-pyrrol- l -yl)hexyl] LC-MS (Protocol methylalanyl-N— [(3R,4S,5 S)-3 -methoxy- l- {(2S) Q): m/z 922.3 [( l R,2R)- l -methoxy-2 -methyl-3 -oxo-3 - { [( l S)phenyl- MalC6-#54 [M+H*] (1.50 l -(l ,3 -thiazolyl)ethyl] amino } propyl]pyrrolidin minutes) - l -yl} methyl- l -oxoheptan—4-yl] -N-methyl-L- valinamidc {N-[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l - yl)hexanoyl] methylalanyl-N-[(3R,4S,5 S)- l- {(2S) LC-MS (Protocol [(3R,4R,7S)benzyl- l 5- {2-[(3,5-dimethyl- l ol- Q1): m/z 1213.3 2-yl-kappaN)methylidene] -2H-pyrrol- 5-yl-kappaN} [M+H+] (0.98 methy minutes) l-5,8, l xooxa-6,9, l zapentadecan yl]pyrrolidin- l -yl} -3 -methoxymethyl- l -oxoheptan—4- yl] hyl-L-Valinamidato} (difluoro)boron l- [6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexyl] LC-MS (Protocol methyl-L-prolyl-N—[(3R,4S,5S){(2S)[(1R,2R) Q1): m/z 909.3 MalC6-#1 18 [M+H*] {[(l S)- l -carboxyphenylethyl]amino} - l -methoxy (0.76 methyl-3 -oxopropyl]pyrrolidin- l -yl} -3 -methoxy-5 minutes) -methyl - l -oxoheptan—4-yl] -N-methyl-L-Valinamide N—[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l xanoyl]— L-Valyl-N—{4-[(8S,11S,12R)[(2S)-butanyl]—12-(2- LC-MS col {(2S)[(1R,2R){[(2S)tert-butoxyoxo Q1): m/z 1358.3 phenylpropan—Z-yl] amino} - l -methoxy-Z-methyl-3 chalCitPABC-#123 [M+H+] (0.97 -oxopropyl]py1rolidinyl}oxoethyl)-5,5,10- minutes) trimethyl- 3,6, 9-trioxo(propanyl)-2, l 3 -dioxa- 4,7,10-triazatetradec-l-yl]phenyl}-N~5~-carbamoyl-L- ornithinamide N— [6-(2,5 -dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl] - LC-MS (Protocol N,2-dimcthylalanyl-N-[(3R,4S,5 S){(2S)[(1R,2R)- Q1): m/z 964.4 3 - { 3-( l H-indol-3 -yl)- l -methoxy- l -oxopropan [M+H*] (0.96 yl]amino} - l -methoxymethyl-3 -oxopropyl]pyr minutes) rolidin- l -yl } - 3-methoxymethyloxoheptanyl] -N- methyl-L-Valinamide methyl N— [(2R,3R)-3 -{(2S)[(3R,4S,5 S) { [N- ( {(2 S)- l dioxo-2,5 -dihydro- l H-pyrrol- l - HPLC (Protocol yl)hexanoyl] methylpiperidin—2-yl} carbonyl)-LM ): m/z 951.4 [M+H+] valyl](methyl)amino} -3 xy (12.839 methylheptanoyl]pyrrolidin minutes) yl} -3 -methoxymethylpropanoyl] -L- phenylalaninate 1,2-dimethyl-L-prolyl-N-[(3R,4S, SS) {(2 S) [(1R,2R)-3 ){[6-(2,5 -dioxo-2,5 -dihydro-1H- LC-MS (Protocol pyrrol- l -yl)hexyl] amino} - l -oxo- 3-phenylpropan—2- Q): m/z 922.3 MalC6Am-#151 [M+H+] yl]amino} - l -methoxymethyl-3 -oxopropyl]pyrrolidin— (1.43 l -yl minutes) } -3 -methoxy-5 -methyloxoheptanyl] -N-methyl-L- valinamidc N-(3- {2-[3-oxo (pentafluorophenoxy)propoxy]ethoxy} oyl)-L- N- {4- [(SS,1 IS ,12R)[(2S)-butanyl]—12-(2- LC-MS (Protocol 2-[(1R,2R)- l -methoxymethyl-3 -oxo {[( l S)- PFPCOPeg2C2ValCitPABC- Q): m/z 1502.8 [M+H+] 2-pher1yl-l-(l,3 -thiazolyl)eth #54 (1.98 yl]amino} propyl]pyrrolidin- l -yl} oxoethyl)-5 ,5 ,10- minutes) trimethyl- 3,6, 9-trioxo(propanyl)-2, l 3 -dioxa- 4,7,10-triazatetradec-l-yl]phenyl}-N~5~-carbamoyl-L- ornithinamide N—[6-(2,5-dioxo-2,5-dihydro- l H-pyrrol- l -yl)hexanoyl]— L-Valyl-N—{4-[(9S,12S,13R)[(2S)-butanyl]—13-(2- LC-MS (Protocol {(ZS)[(1R,2R)- l -methoxymethyloxo {[( l S)- Q1): m/z 1370.2 2-phenyl- l -( l ,3-thiazolyl)ethyl]amino} p chalCitPABC-#154 [M+H*] (0.93 ropyl]pyrrolidin- l -yl} oxoethyl)-4,6,6,l l-tetramethyl- s) 3,7, l 0-trioxo(propanyl)-2, l 4-dioxa-4,8,l l- triazapentadec- l -yl]phenyl} -N~5~-carbamoyl-L- ornithinamide 1,2-dimethyl-D-prolyl-N— [(3R,4S,5 S){(ZS) [(1R,2R)-3 -{[(ZS){[6-(2,5 -dioxo-2,5 -dihydro-1H- HPLC (Protocol pyrrol- l -yl)hexyl] amino} - l -oxo- 3-phenylpropan—2- A): m/z 922.5 MalC6Am-# 15 3 [M+}P] yl]amino} - l -methoxymethyl-3 -oxopropyl]pyrrolidin— (7.352 l -yl minutes) } -3 xy-5 -methyloxoheptanyl] -N-methyl-L- valinamidc N—[ l l,20-dioxo(pentafluorophenoxy)-4,7,14,17- tetraoxa- 10-azaicosan- l - oyl] methylalanyl-N- HPLC (Protocol [(3R,4S,5 S) {(ZS) [( 1 R,2R)-3 -{[(1S)carboxy BB): m/z 1217.6 PFPCOPeg2C2AmPeg2C2-#69 [M+H+] phenylethyl]amino} - l xy-2 -methyl-3 - (12.936 OXOPmpyllpyr minutes) rolidin- l -yl } - 3-methoxymethyloxoheptanyl] -N- -L-Valinamide N— [6-(2,5 -dioxo-2,5-dihydro- l ol- l -yl)hexanoyl] - L-Valyl-N-(4-{(6S,9R,10R)benzyl[(2S) LC-MS col {(3 R,4S,5 S)-4—[(1,2-dimethyl-L-prolyl-LQ ): m/z 1327.9 chalCitPABC-#246 [M+H+] valyl)(methyl)amino] -3 -methoxy-5 - (1.36 methylheptanoyl} pyrrolidin-2 -yl] - 9-methyl-3 ,8-dioxo-2,l l-dioxa-4,7-diazadodcc- l - yl} phenyl) -N~5~-carbamoyl-L-ornithinamide N-(3- {2-[3-oxo (pentafluorophenoxy)propoxy]ethoxy} propanoyl)-L- alanyl-L-alanyl-N~1~-{4-[(8S,118,12R)[(ZS)- HPLC (Protocol 2-yl]— 12-(2- {(2S)[(1R,2R)-1 -methoxy PFPCOPeg2C2AlaAlaAsnPAB AB): m/z 1503.6 [M+H+] methyl-3 -oxo-3 - { [( 1 S)pher1yl( 1 ,3-thi C-#54 (8.06 azolyl)ethyl]amino } propyl]pyrrolidinyl} minutes) oxoethyl)-5 ,5 ,10-trimethyl-3,6,9-trioxo(propanyl)- 2,13 -dioxa-4,7, 1 O-triazatetradecyl]phenyl} -L- aspartamidc 2-methyl-N—(3- {2- [3 -oxo-3 - (pcntafluorophenoxy)propoxy]ethoxy} propanoyl)alanyl- HPLC (Protocol N-[(3R,4S,5 S)-3 -methoxy{(2S)[(1R,2R) AB): m/z 1098.4 eg2C2-#54 [M+H+] methoxymethyl-3 - oxo-3 - { [(1 S)phenyl(1 ,3 - (8.44 thiazolyl)ethyl]amino } prop minutes) yl]pyrrolidinyl} -5 -methyl oxoheptanyl] -N- methyl-L-Valinamide N- { [(4- { [1 1,20-dioxo(pentafluorophen0xy)- 4,7,14,17-tetraoxaazaicosan-l- HPLC (Protocol oyl] amino } benzyl) oxy]carbonyl} methylalanyl-NPFPCOPeg2C2AmPeg2C2PAB AB): m/zl428.2 [(3R,4S,5 S)methoxy {(2 S)[(1R,2R)-1 -methoxy- C-#54 [M+Na+] (10.32 2-methyloxo{[(l S minutes) )phenyl(1 , 3-thiazol yl)ethyl]amino } propyl]pyrrolidinyl} - 5 l oxoheptan—4-yl] -N-methyl-L-Valinamide N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-l-oyl)- HPLC (Protocol N,2-dimethylalanyl-N-[(3R,4S,5S){(2S)[(1R,2R)- A): m/z 1053.5 AmPeg6C2-#115 3 - { [(1 S)carboxyphenylethyl]amino} methoxy [M+H*] (7.35 methyl-3 -oxopropyl]pyrrolidinyl} -3 -methox minutes) y-5 -methyloxoheptan—4-yl] hyl-L-Valinamide N,2-dimethyl-N-[ 1 9- 0x0- 1 9-(pentafluorophenoxy)- ,13 ,l6-pentaoxanonadecan-l-oyl]alar1yl-N— LC-MS (Protocol [(3R,4S,5 S) 2- [( 1 R,2R)-3 -{[(1S)carboxy Q): m/z 1205.1 PFPCOch5C2-#115 [M+H*] phenylethyl]amino} methoxy-2 l-3 - (1.99 oxopropyl]pyrro minutes) lidinyl} -3 -methoxy-5 -methyl oxoheptanyl] -N- methyl-L-Valinamide 1,2-dimethyl-D-prolyl-N- [(3R,4S,5 S){(2S) HPLC (Protocol [(1R,2R)-3 -( 3- [4-( {N— [6-(2,5-dioxo-2, 5-dihydro- 1 H-pyrrolyl)hexanoyl] glycyl } amino)phenyl] EB): (4.0 minutes) mcGly-#201 methoxy oxopropan—2-yl} amino)methoxy : ESI-MS m/z methyl- 3-o 1023.59 [M+H*] yl]pyrrolidinyl} methoxy- 5-methyl tan—4-yl] -N-methyl-L-Valinamide 2-methyl-N—(3 - {2- [3 -oxo- 3-( {4- [3 -oxo-3 -(2- tidin HPLC (Protocol pyl]phenyl} amino)propoxy]ethoxy} propanoyl)ala AzCOC2Ph4AmCOch2C2- FB): m/z 1132.4 #54 [M+}P] nyl-N—[(3R,4S ,5 S)methoxy{(2S)[(1R,2R) (10.18 methoxymethyl-3 - oxo-3 - { [(1 S)phenyl(1 ,3 - minutes) lyl)ethyl] amino } propyl]pyrrolidinyl} -5 - oxoheptanyl] -N-methyl-L-Valinamide N- { [2- oxo( { 4- [3 -oxo- 3-(2- oxoazetidin yl)propyl]phenyl} amino)ethoxy]acetyl} -L-Valyl-N— {4- HPLC (Protocol [(8S,118,12R)[(2S)-butanyl]—12-(2-{(2S) AzCOC2Ph4AmPeg1C1ValCit FB): m/z 1465.8 [(1R,2R)methoxy-2 -methyl-3 -oxo-3 - { [( 1 S)phenyl- PABC-#54 [M+}P] (10.97 1-(1,3 -thiazolyl)ethyl]amino } propyl]pyrrolidinyl} - minutes) 2-oxoethyl) -5 ,5 ,10-trimethyl- 3,6,9-triox o- 8-(propan yl)-2,13 -dioxa-4,7, lO-triazatetradecyl]phenyl} -N~5~- carbamoyl-L-ornithinamide N- { [2-oxo( { 4- [3 -oxo- 3-(2- oxoazetidin yl)propyl]phenyl} amino)ethoxy]acetyl} -L-Valyl-N~5~- carbamoyl-N-[4-({[(1-{[(ZS){[(3R,4S,5 S)-3 - HPLC (Protocol methoxy{(ZS)[(1R,2R)methoxymethyl-3 - AZCOCZPh4AmPeg1C1ValCit FB): m/z 1491.8 oxo{[(1S)phenyl(1,3-thiazol PABC-#30 [Mi-PF] (10.56 yl)ethyl]amino}propyl]pyrrolidinyl}methyl s) oxoheptanyl](methyl)amino} -3 loxobutan- yl] carbamoyl} cyclopentyl)carbamoyl] oxy} methyl)phen yl] -L-ornithinamide 2-methyl-N—(3 - {2- [3 -oxo- 3-( {4- [3 -oxo-3 -(2- oxoazetidin HPLC col yl)propyl]phenyl} amino)propoxy]ethoxy} propanoyl)ala AZCOCZPh4AmCOPegZC2_ AB): m/z 1065.3 #69 [M+H+] (1202 nyl-N-[(3R,4S,SS){(ZS)[(1R,2R){[(IS) carboxy-Z-phenylethyl]amino } - 1 xymethyl minutes) oxopropyl]py1rolidinyl}-3 -methoxy-5 -methyl oxoheptan—4-yl] -N-methyl-L-Valinamide N,2-dimethyl-N-(3- {2-[3-oxo({4-[3-oxo(2- oxoazetidin HPLC (Protocol yl)propyl]phenyl} amino)propoxy]ethoxy} propanoyl)ala AzCOC2Ph4AmCOPeg2C2- AA): m/21078.6 nyl-N-[(3R,4S,SS){(ZS)[(1R,2R){[(IS) #115 [M+}F] (12.02 carboxy-Z-phenylethyl]amino } - 1 xymethyl minutes) oxopropyl]py1rolidinyl}-3 -methoxy-5 -methyl oxoheptan—4-yl] -N-methyl-L-Valinamide N~2~-acetyl-L-lysyl-L-Valyl-N- {4-[(8$,1 1s,1 2R)- 1 1- [(ZS)-butanyl](2- {(ZS)[(1R,2R)methoxy LC-MS (Protocol . Q): m/Z 1319.3 methyloxo{[(1S)phenyl(1,3-th1azol AcLysValCitPABC—#54 yl)ethyl]am1no}propyl]pyrrolid1nyl}oxoethyl)- [M+H+2] (1 34' ,5,10-trimethyl-3,6,9-trioxo-8—(propanyl)-2,13- dioxa-4,7,10-triazatetradecyl]phenyl}-N~5~- carbamoyl-L-omithinamide, trifluoroacetic acid salt Table 19A - Selected conjugates of the invention Theoretical A Amount of "- mass 01‘ phosphanetriyltripropanoic ADC-Linker-Payload # payload/linker nker-Payload 0r TCEP/ PL (x/y) General MalPeg3C2-#41 procedure F H-(C)_MalPeg6C2-#42 procedure F General H-(C)_mc-#44 2.3/7.5 913 procedure F General H-(C)_MalPeg3C2-#44 2.2/7 1003 procedure F General H-(C)_MalPeg6C2-#44 2.0/7 1 135 procedure F General H-(C)_chalCitPABC-#44 2.5/7.5 1319 procedure F General H-(C)_Mal-PEG3C2-#45 23/75 1017 procedure F General H-(C)_Mal-PEG6C2-#45 2.05/10 1 149 procedure F General cha1CitPABC-#45 2.5/10 1342 procedure F General H-(C)_mc-#54 22/75 897 procedure F General H-(C)_Mal-PEG6C2-#69 1119 procedure F General H-(C)_chalCitPABC-#69 1303 procedure F General H-(C)_chalCitPABC-#70 1317 procedure F General H-(C)_mc-#79 941 procedure F General H-(C)_chalCitPABC-#79 1345 procedure F General H-(C)_mc-#1 15 911 procedure F General H-Al 14C-(C1 -#51 934.21 procedure G General H-Al 14C-(C1 -#47 962.27 procedure G General H-Al 14C-(C1 14)_mc-#54 936.2 procedure G H-Al 14C-(C1 14)_chalCitPABC- General 50/10 1367.72 #47 procedure H H-Al 14C-(C1 14)_chalCitPABC- General 100/10 8 #54 procedure H H-Al 14C-(C1 14)_chalCitPABC- General 50/10 1385.8 #26 ure H General H-Al 14C-(C1 14)_mc-#26 50/10 980.35 procedure H H-Al 14C-(C1 14)_chalCitPABC- l 50/10 1302.69 #36 procedure H H-Al 14C-(C1 14)_chalCitPABC- General 100/7 1360.73 #42 procedure H General H-Al 1 14)_mc-#42 50/10 955.27 procedure H General H-(C)-chalCitPABC-#54 2.5/10 1342 procedure F H-(C)_chalCitPABCAmPeg3C2- General 2.5/9 1544 #54 procedure F H-(C)_chalCitPABCAmPeg6C2- General 2.6/10 1677 #54 procedure F General H-(C)_mc-#47 1.9/10 962.27 procedure F General H-(C)_MalPeg3C2-#54 1.9/10 1026 procedure F H-(C)_mc-#54 2.07/10 936.2 procedure F H-(C)_chalCitPABCAmPeg3C2- General 2.3/7.5 1569.96 #47 procedure F General H-(C)_MalPeg3C2-#47 23/75 1052 ure F H-(C)_chalCitPABCAmPeg3C2- General /75 1562.87 #42 procedure F H-(C)_mc-#41 2.5/10 941.24 procedure F H-(C)_chalCitPABCAmPeg3C2- General 2.5/10 1589.04 #26 procedure F H-(C)_chalCitPABCAmPeg6C2- General 2.4/7 1701.9 #47 procedure F General MalPeg3C2-#42 2.3/7 1044.58 procedure F WO 72813 H-(C)_chalCitPABCAmPeg6C2- General #26 procedure F chalCitPABCAmPeg6C2- General #42 ure F General MalPeg6C2-#54 procedure F General H-(C)_MalPeg6C2-#47 procedure F General H-(C)_MalPeg6C2-#26 procedure F General H-(C)-MalPeg6C2-#42 procedure F General mc-#36 00 \O0\ procedure F General H-(C)_chalCitPABC-#60 1284.61 procedure F General H-(C)_MalPeg3C2-#26 1070.42 procedure F H-(C)_chalCitPABCAmPeg3C2- General 1505.93 #36 procedure F H-A 1 14C- General (C 1 1 4)_chalCitPABCAmPeg3C2- 1505.93 procedure H General H-Al 14C-(C1 14)_MalPeg6C2-#54 1158.5 procedure H General H-(C)_MalPeg3C2-#60 969.23 procedure F General H-(C)_MalPeg6C2-#60 1101.4 procedure F General H-(C)_MalPeg6C2-#41 1163.5 procedure F General H-(C)-mc-#69 2.2/7.5 00 \O\1 procedure F General H-(C)_MalPeg3C2-#36 2.15/10 987.31 procedure F H-(C)_chalCitPABCAmPeg6C2- General 2.25/10 1636 #36 procedure F General H-(C)_MalPeg6C2-#36 2.15/10 1119.5 procedure F H-(C)_chalCitPABCAmPeg3C2- General 2.5/10 1549.94 #41 procedure F H-(C)-MalPeg3C2-#41 2.3/7.5 1031 procedure F H-(C)_chalCitPABCAmPeg6C2- l 2.5/10 1620 #60 procedure F General H-Al 14C-(C1 14)_mc-#66 50/7 866.5 procedure H H-L398C+L443C- General 50/7 1341.68 (C398+C443)_chalCitPABC-#54 procedure H H-K392C+L443C- General 100/10 1341 (C392+C443)_chalCitPABC-#54 procedure H H-L443C-(C443)_chalCitPABC- General 100/10 1341 #54 procedure H C+V422C- General 50/7 1341.68 C398+C422 chalCitPABC-#54 orocedure H General H-(C)-mc-#44 23/75 913 procedure F General Mal-PEG3C2-#45 23/75 1017 procedure F General H-(C)_2AcAmPeg6C2-#66 2.4/10 procedure F General H-(C)-Mal-PEG6C2-#45 2.05/10 procedure F H-(C)-mc-#79 procedure F MalPeg3C2-#44 procedure F General chalCitPABC-#70 procedure F General H-(C)-MalPeg6C2-#44 procedure F H-Al 14C-(C1 14)_chalCitPABC- General #69 procedure H General H-(C)-chalCitPABC-#79 procedure F H-Al 1 14)_chalCitPABC- General 100/7.5 #79 procedure H General H-(C)-chalCitPABC-#44 2.5/7.5 procedure F H-Al 14C-(C1 14)_chalCitPABC- General #88 procedure H General H-(C)-chalCitPABC-#69 ure F General 2AcAmCapValCitPABC-#66 procedure F H-Al 14C-(C1 14)_chalCitPABC- General #45 procedure H H-Al 14C-(C1 14)_chalCitPABC- General #34 procedure H General H-Al 14C-(C1 l4)_mc-#45 procedure H General H-Al 14C-(C1 14)_mc-#70 procedure H General H-(C)_chalCitPABC-#1 12 procedure F General Mal-PEG6C2-#69 procedure F H-Q347C-(C347)_chalCitPABC- General #69 procedure H H-Y373C-(C373)_chalCitPABC- General #69 procedure H H-E388C-(C388)_chalCitPABC- General #69 procedure H H-N42 1C-(C421)_chalCitPABC- General #69 procedure H H-L443C-(C443)_chalCitPABC- General #69 procedure H C-(C443)_chalCitPABC- General #79 procedure H H-Al 14C-(C1 14)_chalCitPABC- General #95 ure H H-Al 14C-(C1 14)_chalCitPABC- General #98 procedure H General H-Al 14C-(C1 l4)_MalPeg3C2-#69 procedure H General H-N297Q-(Q)_AmPeg6C2-#42 NA procedure K WO 72813 General H-N297Q-(Q)_AmPeg6C2-#54 procedure K General H-N297Q-(Q)_AmPeg6C2-#47 procedure K General H-N297Q-(Q)_AmPeg6C2-#36 procedure K General H-N297Q-(Q)_AmPeg6C2-#26 procedure K General H-N297Q-(Q)_AmPeg6C2-#66 procedure K General H-L443C-(C443)_MalPeg6C2-#69 100/10 procedure H H-Q347C-(C347)_MalPeg6C2-#69 100/10 1119 procedure H General C-(C388)_MalPeg6C2-#69 100/10 1119 procedure H General H-N421C-(C421)_MalPeg6C2-#69 100/10 1119 procedure H General H-E380C-(C380)_MalPeg6C2-#69 100/10 1119 procedure H H-L398C+L443C- General 1119 (C3 98+C443)_MalPeg6C2-#69 procedure H H-K392C+L443C- General 1119 C392+C443 MalPe_6C2-#69 ure H General H-kAl C111)_MalPeg6C2-#69 1119 procedure H General H-kKl83C-(kC183)_MalPeg6C2-#69 1119 procedure H General H-kK207C-(kC207)_MalPeg6C2-#69 1119 procedure H H-Al 14C-(C1 14)_chalCitPABC- General 1314.59 #108 procedure H H-Al 14C-(C1 alCitPABC- General 1371 #84 procedure H H-Al 14C-(C1 14)_chalCitPABC- General 1330 #226 procedure H General H-A114C-(C114)_mc-#108 909.12 procedure H H-Al 14C-(C1 14)_chalCitPABC- General 1342 #1 17 ure H H-Al 1 14)_chalCitPABC- General 1316 #1 15 procedure H General H-Al 14C-(C1 14)_MalPeg6C2-#98 1184 procedure H IL13Ra2-AB08-V1010-hG1- General 1341.68 (C)_chalCitPABC-#54 procedure F IL13Ra2-AB08-V1010-hG1-(C)_mc- General 897.12 #69 procedure F IL13Ra2-AB08-V1010-hG1- General 1119 (C)_MalPeg6C2-#69 ure F IL13Ra2-AB08-V1010-hG1- General 1302 (C)_chalCitPABC-#69 procedure F General H-A114C-(C114)_MalPeg6C2-0#118 1145 ure H H-Al 14C-(C1 14)_chalCitPABC- General 1328 0# 1 1 8 procedure H H-Al 14C-(C1 14)_chalCitPABC- General 100/15 1332 #80 procedure H General C-(C114)_mc-#117 100/15 procedure H H-Al 14C-(C1 14)_chalCitPABC- General 100/15 #232 procedure H H-Al14C-(C114)_MalPeg6C2-#230 H-Al14C-(C114)_MalPeg6C2-#117 H-A114C-(C114)_mc-#115 H-A114C-(C114)_mV-#115 C-(C114)_mb-#69 H-Al 14C-(C1 14)_mV-#69 General H-A114C-(C114)_mc-0#118 100/ 15 923 procedure H General H-(C)_mc-#1 17 2.0/6.5 937 ure F General H-(C)_MalPeg6C2-#1 17 procedure F General H-(C)_mc-0#1 18 orocedure F General H-(C)_MalPeg6C2-0#1 18 procedure F IL13Ra2-AB08-V1010-hG1-(C)_mc- General 0#1 18 procedure F IL13Ra2-AB08-V1010-hG1-(C)_mc- General #226 procedure F 2-AB08-V1010-hG1-(C)_mc- General #1 17 procedure F IL13Ra2-AB08-V1010-hG1- General (C)_MalPeg6C2-#1 17 procedure F IL13Ra2-AB08-V1010-hG1- (C)_MalPeg6C2-0#1 18 C-(C114)_MalPeg6C2-#226 H-A114C-(C114)_mc-#172 H-A114C-(C1 14)_mb-0#118 H-A114C-(C114)_me-0#118 H-Al 14C-(C1 14)_chalCitPABC- #134 H-A114C-(C114)_mc-#131 H-Al14C-(C114)_MalPeg6C2-#126 C-(C114)_MalPeg6C2-#123 H-A114C-(C114)_mc-#126 H-A114C-(C1 14)_mV—0#1 18 General H-(C)_MalPeg6C2-#226 2.4/7 1 147 procedure F General H-(C)_mc-#226 2.4/7 procedure F IL13Ra2-AB08-V1010-hG1- General 3.0/10 (C)_MalPeg6C2-#226 ure F General NotchcG1-(C)_mc-0#1 18 procedure F General NotchcG1-(C)_mc-#115 procedure F General NotchcG1-(C)_MalPeg6C2-0#1 18 procedure F General NotchcG1-(C)_me-0#1 18 937 procedure F General NotchcG1-(C)_mc-0#1 18 923 procedure F IL13Ra2-19F9-cG1- General 1341.68 (C)_chalCitPABC-#54 procedure F IL13Ra2-19F9-cG1- General 2.5/7 1288 alCitPABC-#1 12 procedure F NotchcG1-(C)_chalCitPABC- General 2.3/7 1288 # 1 1 2 procedure F General 28-cG1-(C)_MalPeg6C2-#69 procedure F General NotchcG1-(C)_MalPeg6C2-#69 orocedure F General H-(C)_m(H20)c-0#1 18 procedure I General H-(C)_Mal(H20)Peg6C2-0#1 18 procedure I General H-(C)_Mal(H20)Peg6C2-#69 procedure I General H-(C)_m(H20)c-#69 procedure I General H-(C)_me-0#1 18 procedure F General H-(C)_mV-0#1 18 procedure F General H-(C)_mb-0#1 18 procedure F General H-A114C-(C114)_MalC6-#54 procedure H General H-A114C-(C114)_mc-#231 ure H H-Al14C-(C114)_MalC6-0#118 procedure H General Mal(H20)Peg6C2-#1 15 procedure I General H-A114C-(C114)_mc-#158 procedure H H-Al 14C-(C1 14)_chalCitPABC- General #23 1 procedure H General m(H20)c-#1 15 ure I General NotchcG1-(C)_mc-#115 procedure F General NotchcG1-(C)_me-0#1 18 procedure F General NotchcG1-(C)_MalPeg6C2-0#1 18 3.0/7.0 procedure F General H-A114C-(C114)_mc-#237 100/10 963 procedure H General H-A114C-(C114)_mc-#145 100/10 897 ure H General H-Al14C-(C114)_MalPeg6C2-#145 procedure H General H-A114C-(C114)_mc-#162 procedure H General H-Al 14C-(C1 14)_MalC6Am-#151 procedure H General NotchcG1-(C)_m(H20)c-0#1 18 procedure I General NotchcG1-(C)_m(H20)c-0#1 18 procedure I 88)_COPeg2C2ValCitPABC- General #54 procedure J— IL13Ra2-AB08-V1010-hG1- General 2.3/7 (C)_Mal(H20)Peg6C2-0#1 18 procedure F IL13Ra2-AB08-V1010-hG1- General 2.3/7 (C)_Mal(H20)Peg6C2-#1 15 procedure F 2-AB08-V1010-hG1-(C)_mc- General 2.35/7 #115 procedure F IL13Ra2-AB08-V1010-hG1- General 2.8/7 C m H20 c-0#118 orocedure F General H-(C)_chalCitPABC-0#1 18 2.2/7 procedure F IL13Ra2-AB08-V1010-hG1- General 2.35/7 H20)c-#1 15 procedure F H-Al 14C-(C1 14)_chalCitPABC- General 100/5 #154 procedure H General H-Al 14C-(C1 14)_MalC6Am-#153 100/10 procedure H IL13Ra2-AB08-V1010- General Q347C+kK183C-hG1- 100/10 procedure H (C347+kC183)_chalCitPABC-#54 1L13Ra2-AB08-V1010-Q347C-hG1- General 100/10 _chalCitPABC-#54 procedure H IL13Ra2-AB08-V1010-hG1- General (kKl88)_COPeg2C2AmPeg2C2-#69 procedure J IL13Ra2-AB08-V1010-hG1- General (kKl88)_COPeg2C2ValCitPABC-#54 procedure J IL13Ra2-AB08-V1010-L443C-hG1- General 100/10 1341.68 (C443)_chalCitPABC-#54 procedure H 2-AB08-V1010- General L443C-hG1- 100/10 1341.68 procedure H (C392+C443)_chalCitPABC-#54 IL13Ra2-AB08-V1010- General kK183C-hG1- 100/10 1341.68 procedure H (C443+kC183)_chalCitPABC-#54 General H-(C)_chalCitPABC-#98 2.2/7 1367 procedure F H-Al 14C-(C1 alCitPABC- General 100/10 1327 #246 procedure H H-H435A-(C)_chalCitPABC-#54 2.2/8 1341.7 procedure F H-M428L+N434S- General 2.2/8 1316 (C)_chalCitPABC-#70 procedure F H-M428L+N434S- General 2.2/8 1341.7 (C)_chalCitPABC-#54 procedure F H-E388C+N421C- General 100/10 1341 (C388+C421)_chalCitPABC-#54 procedure H H-Q347C+K392C- General 100/10 1341 (C347+C392)_chalCitPABC-#54 procedure H H-L443C+kK183C- General 1341 (C443+kC183)_chalCitPABC-#54 procedure H H-Q347C+kK183C- General 1341 kC183)_chalCitPABC-#54 ure H H-Q347C-(C347)_chalCitPABC- General 1341 #54 procedure H H-K392C+L443C-(C392+C443)_mc- General #1 15 procedure H H-E388C+N421C-(C388+C421)_mc- General #1 15 procedure H C+K392C-(C347+C392)_mc- General #1 15 procedure H H-L443C+kK183C- General 100/10 911 (C443+kC183)_mc-#115 procedure H H-Q347C+kK183C- General 100/10 911 kC183)_mc-#115 procedure H General H-Q347C-(C347)_mc-#115 911 procedure H H-kK183C-(kC183)_chalCitPABC- General 1341 #54 orocedure H H-E388C-(C388)_chalCitPABC- General 1341 #54 procedure H General H-kK183C-(kC183)_mc-#115 911 procedure H General H-E388C-(C388)_mc-#1 15 911 procedure H General H-L443C-(C443)_mc-#1 15 911 procedure H H-N421C-(C421)_chalCitPABC- l 1341 #54 procedure H General H-N421C-(C421)_mc-#115 911 procedure H General H-Al 14C-(C1 14)_mcGly-#201 1023 procedure G Table 19B - Selected conjugates of the invention Mass spectra: HPLC-SEC retention time and HPLC A mass Loading 0r Drug ADC-Linker-Payload # for the Heavy Chain (HC) portion per dy (up to 6 Da difference with theoretical A ratio (DAR) SEC (Protocol 0): 7.317 minutes; HPLC H'(C)—Malpeg3cz'#4l :P U.) (Protocol P): HC A mass = 1032 SEC (Protocol 0): 7.177 minutes; HPLC H-(C)_MalPeg6C2-#42 U.) \0 (Protocol P): HC A mass = 1180 .
SEC (Protocol 0): 7.195 s; HPLC H-(C)_mc-#44 :P .1; (Protocol P): HC A mass = 915 SEC col 0): 7.247 minutes; HPLC H-(C)_MalPeg3C2-#44 U.) . .1; (Protocol P): HC A mass = 1005 SEC (Protocol 0): 7.237 minutes; HPLC H-(C)_MalPeg6C2-#44 5” 4; (Protocol P): HC A mass = 1135 . SEC (Protocol 0): 7.351 minutes; HPLC H'(C)—mcva1C“PABC'#44 7"N col P): HC A mass = 1321 SEC (Protocol 0): 7.364 minutes; HPLC H-(C)_Mal-PEG3C2-#45 7" U.) (Protocol P): HC A mass = 1017 SEC (Protocol 0): 7.419 minutes; HPLC H-(C)_Mal-PEG6C2-#45 U.) \0 (Protocol P): HC A mass = 1154 .
. SEC Protocol 0 : 7.159 minutes; HPLC H-(C)_chalCltPABC-#45 (Protocol P):)HC A mass = 1343 SEC (Protocol 0): 7.192 minutes; HPLC H-(C)_mc-#54 (Protocol P): HC A mass = 899 SEC (Protocol 0): 7.350 minutes; HPLC H-(C)_Mal-PEG6C2-#69 (Protocol P): HC A mass = 1122 SEC (Protocol 0): 7.254 minutes; HPLC H-(C)_chalC1tPABC-#69. 4.5 (Protocol P): HC A mass = 1305 mcva1C1tPABC'#70.
SEC (Protocol 0): 7.466 s; HPLC (Protocol P): HC A mass = 1318 SEC (Protocol 0): 7.478 minutes; HPLC H_(C) mc-#79 (Protocol P): HC A mass = 946 . SEC (Protocol 0): 7.635 minutes; HPLC H_(C) chalCltPABC-#79 (Protocol P): HC A mass = 1349 SEC col 0): 7.510 s; HPLC H (C)_mc-#115 3'54 col P): HC A mass = 912 H-A114C (C114)_mc-#51 — H-Al 14C (C1 14)_mc-#47 — H-A114C (C114)_mc-#54 — NN WU.) H-A114C-(C1 14)_chalCitPABC-#47 2 H-A114C-(C1 14)_chalCitPABC-#54 1.9 —1.9 SEC (Protocol P): 7.681 minutes; HPLC H-A114C-(C114)_chalCltPABC-#42 .H u.
(Protocol 0): HC A mass = 1378 H-Al 14C-(C1 14)_mc-#42 - No SEC (Protocol P): 7.159 minutes; HPLC H'(C)'mcva1C1tPABC'#54 .1; ,_.
(Protocol 0); HC A mass = 1343 .
H-(C)_chalCitPABCAmPeg3C2-#54 H-(C)_chalCitPABCAmPeg6C2-#54 H-(C)_mc-#47 AA-p No.0 SEC (Protocol P): 7.179 minutes; HPLC H'(C)—Malpeg3cz'#54 w \1 (Protocol 0); HC A mass = 1028 .
H-(C) m0 #54 — 4 H-(C)_chalCitPABCAmPeg3C2 #47— 3.7 H-(C)_chalCitPABCAmch6C2-#47 SEC col P): 7.142 s; HPLC H-(C)_Machg3C2-#42 (Protocol 0): HC A mass = 1050 H-(C)_chalCitPABCAmch6C2-#26 chalCitPABCAmch6C2-#42 SEC (Protocol P): 7.254 minutes; HPLC H (C) Machg6C2-#54 7P.
(Protocol 0); HC A mass = 1161 SEC (Protocol P): 7.303 minutes; HPLC H (C) MalPeg6C2 #47 (Protocol 0); HC A mass = 1182 H (C) Machg6C2 #26 SEC (Protocol P): 7.177 minutes; HPLC H (C) MalPeg6C2 #42 (Protocol 0); HC A mass = 1180 H (C) mc #36 H (C) chalCltPABC #60 H-(C)_chalC1tPABCAmPcg3C2 #36 H-A 1 1 4C- \omoooowon-b-b H-A114C-(C114) Machg6C2-#54 H (C) Machg3C2-#60 H (C) Machg6C2-#60 H (C) Machg6C2-#41 (111000103211Protocol P : 7.192 minutes; HPLC SEC H-(C)_chalC1tPABCAmch6C2 #36 SEC (Protocol P): 7.317 minutes; HPLC (Protocol 0); HC A mass = 1032 ' H (C) Machg3C2 #41 H-(C)_chalC1tPABCAmch6C2 #60— H-Al 14C-(Cl -#66 — H-P-PrbmrbwrbgrprpNE—‘P’P’P’PP’7P7PHN OOt—w-FNNUI U.) 00 (C398+C443)_cha1CitPABC-#54 H-K392C+L443C- (C392+C443)_cha1CitPABC-#54 SEC (Protocol P): 8.827 minutes; HPLC H-L443C-(C443)_chalCitPABC-#54 col 0V : HC A mass = 1344 H-L398C+V422C- IINU.) (C398+C422)_cha1CitPABC-#54 SEC (Protocol P): 7.195 s; HPLC H (C) mc-#44 :P 4; (Protocol 0): HC A mass = 915 SEC (Protocol P): 7.364 minutes; HPLC H'(C) Mal PEG3C2'#45 7" w (Protocol 0): HC A mass = 1017 H-(C)_2AcAmch6C2-#66 SEfilfsssszfgi?1331911121131?ch U.) \0 SECoizizzzllé’filré? 5315;352:133?3317211311315“ w 4; SECiéféféé’Sf’o‘i?#3261322??? U.) \] SEC (Protocol P): 7.237 minutes; HPLC H'(C)'Malpeg6cz'#44 3.4 (Protocol 0); HC A mass = 1135 H-A114C-(Cl14)_chaICitPABC-#69— 2 SEC (Protocol P): 7.635 s; HPLC H (C)'mcva1C1tPABC #79 col 0): HC A mass = 1349 H-A114C (C114) chalCltPABC #79 - . SEC (Protocol P): 7.351 s; HPLC H (C) movalCItPABC #44 (Protocol 0); HC A mass = 1321 H-A114C (C114)_chaICIth’%BC #88— . SEC (Protocol P): 7.254 minutes; HPLC H'(C)'mcva1C1tPABC #69 (Protocol 0); HC A mass = 1305 H-(C)_2AcAmCapValC1tPABC-#66 . SEC (Protocol P): 7.083 minutes; HPLC H'(C) movalCItPABC #112 (Protocol 0): HC A mass = 1291 SEC (Protocol P): 7.350 mlnutcs; HPLC H-(C)-Mal-PEG6C2-#69 (Protocol 0): HC A mass = 1122 H-Q347C-(C347)_cha1CitPABC-#69 C-(C373)_cha1CitPABC-#69 H E388C _chalCitPABC #69 H-N421C (C421)_cha1CitPABC #69- -— H L443C (C443)_cha1CitPABC-#69- - — H—L44sc—<c443>_cha1chABc-#79— H—A114c-<c114> chaICttPABc-tws— H-A114C (Cl 14):cha1C1tPABC #98 H A114C (C114) Machg3C2-#69- - — H-N297Q (Q) AmPeg6C2 #42_ _ — H-N297Q-(Q)_Amch6C2-#47 H-N297Q-(Q) Amch6C2-#36 .
H-N297Q-(Q)_Amch6C2-#66 2.8 SEC(ErotocorohmwmomProtocol P : 7.012 minutes; HPLC C-(C388)_MalPeg6CZ-#69—— H-N4ZIC-(C421)_MalPeg6CZ-#69—— H-E380C-(C380)_Ma1Peg6C2-#69 1.8 (C3 98+C443)_MalPeg6C2-#69 ‘ ——_(C392+C443)_Ma1ch6C2-#69 ‘ H-A114C-(C114)_mc-#108 1.9 H-A114C-(Cl 14)_chaICitPABC-#115—— H-Al 14C-(Cl 14)_MalPeg6CZ-#98—— IL13Ra2-AB08-V1010-hG1- (C)_cha1CitPABC-#54 ~ IL13Ra2-AB08-V1010-hG1-(C)_mc-#69 3.5 IL13Ra2-AB08-V1010-hG1- (C)_Ma1ch6C2-#69 ‘ IL13Ra2-AB08-V1010-hG1- (C)_chalCitPABC-#69 H-A114C-(C114)_Ma1Peg6C2-0#1 18 H-Al 14C-(C1 alCitPABC- 0# 1 1 8 NNNNNAHAHAHAccooxoooooxo H-A114C-(C114)_mV-#115 H-Al 14C-(C1 14)_mb-#69 H-Al 14C-(C1 14)_mV-#69 H-Al 14C-(C1 l4)_mc-0#l 18 SEC (Protocol P): 7.797 s; HPLC H-(C)_mc-#117 U.) U.
(Protocol 0): HC A mass = 937 SEC (Protocol P): 8.005 minutes; HPLC H'(C)—Malpeg6cz'#1 17 (Protocol 0); HC A mass = 1163 H-(C)_mc-0#1 18 - SEC (Protocol P): NA; PlPLC col 0): HC H-(C)_MalPeg6C2-0#118 (”A \01— A mass — 1148 IL13Ra2-AB08-V1010-hG1-(C)_mc- 0#118 IL13Ra2-AB08-V1010-hG1-(C)_mc- .4; o #226 IL13Ra2-AB08-V1010-hG1-(C)_mc- U.) U.) #1 17 IL13Ra2-AB08-V1010-hG1- U.) U.) (C)_MalPeg6C2-#1 17 IL13Ra2-AB08-V1010-hG1- (C)_MalPeg6C2-0#1 18 _ ¥O H-A114C-(C1 14)_MalPeg6C2-#126 1— C-(C114)_mc-#126 H-Al 14C-(C1 —0#1 18 SEC (Protocol P): 7.501 minutes; HPLC H'(C)—Malpeg6cz'#226 A u. col 0); HC A mass = 1150 SEC (Protocol P): 7.418 minutes; HPLC H-(C)_mc-#226 A (Protocol 0): HC A mass = 927 IL13Ra2-AB08-V1010-hG1- (C)_MalPeg6C2-#226 NotchcG1-(C)_mc-0#1 18 A SEC (Protocol P): 7.015 minutes; HPLC NOtCh'28'0G1'(C)—mc'#1 15 w (Protocol 0): HC A mass = 911 NotchcG1-(C)_MalPeg6C2-0#1 18 - A SEC (Protocol P): 7.182 minutes; HPLC NOtCh 28 0G1 (C)_mc 0#118_ _ _ _ w (Protocol 0): HC A mass = 937 NotchcG1-(C)_mc-0#1 18 w IL13Ra2-19F9-cG1- (C)_chalCitPABC-#54 IL13Ra2-19F9-cG1- (C)_chalCitPABC-#1 12 NotchcG1-(C)_chalCitPABC- #112 NotchcG1-(C)_Machg6C2-#69 4.3 NotchcG1-(C) Machg6C2-#69 - 3.8 SEC (Protocol P): 7.010 minutes; HPLC H'(C)—m(HZO)°'0#1 18 4‘1 (Protocol 0): HC A mass = 942 SEC (Protocol P): 6.964 minutes; HPLC H'(C)—Mal(HZO)Peg6C2'0#1 18 (Protocol 0); HC A mass = 1166 H-(C)_Mal(H20)ch6C2-#69 H-(C)_m(H20)c-#69 H-(C)_me-0#1 18 — H-(C)_mV-0#1 18 — SEC (Protocol P): 7.032 minutes; HPLC H (CD—“lb 01“ 18_ _ (Protocol 0): HC A mass = 896 H-A114C-(Cll4)_MaIC6-#54— H-Al 14C-(C1 14)_mc-#231 — —H-Al l 14)_MaIC6-0#1 18— SEC col P): 6.936 minutes; HPLC H'(C)—Mal(HZO)Peg6C2'#1 15 (Protocol 0); HC A mass = 1152 H-A114C-(C114)_mc-#158 — —H-A114C-(Cl 14)_chaICitPABC-#231— SEC (Protocol P): 6.928 minutes; HPLC H'(C)—m(HZO)C'#1 15 (Protocol 0): HC A mass = 930 —NotchcG1-(C)_mc-#115 — —Notch0G1-(C)_me-0#1 18— —NotchcG1-(C)_Ma1Peg6C2-0#1 18 4C-(C114)_mc-#237 —H-A114C-(C114)_mc-#145 H-A114C-(C1 14)_Machg6C2-#145 H-A114C-(C114)_mc-#162 —H-Al 14C-(C1 14)_Ma1C6Am-#151 —NotchcG1-(C)_m(H20)c-0#1 18 —NotchcG1-(C)_m(H20)c-0#1 18 H-(kKl88)_COPeg2C2ValCitPABC- (C)_Mal(H20)ch6C2-0#1 18 (Protocol 0): HC A mass = 1164 IL13Ra2-AB08-V1010-hG1- (C)_Ma1(H20)Peg6CZ-#115 — 2-AB08-V1010-hG1-(C)_mc- SEC (Protocol P): 7.813 minutes; HPLC IL13Ra2-AB08-V1010-hG1- H-(C)_chalCitPABC-0#1 18 (C)_m(H20)c-#115 (Protocol 0): HC A mass = 930 H-A114C-(Cl 14)_chaICitPABC-#154— H-Al 14C-(Cl 14)_MaIC6Am-#153— 2-AB08-V1010- Q347C+kK183C-hG1- kC183)_chalCitPABC-#54 IL13Ra2-AB08-V1010-Q347C-hG1- (C347)_cha1CitPABC-#54 IL13Ra2-AB08-V1010-hG1- (kKl88)_COPeg2C2AmPeg2C2-#69 IL13Ra2-AB08-V1010-hG1- (kKl88)_COPeg2C2ValCitPABC-#54 IL13Ra2-AB08-V1010-L443C-hG1- (C443)_cha1CitPABC-#54 WO 72813 IL13Ra2-AB08-V1010-K392C+L443C- hG1-(C392+C443)_chalCitPABC-#54 IL13Ra2-AB08-V1010-L443C+kK183C- hG1 -(C443+kC 183)_chalCitPABC- . SEC69Protocol P : 7.232 minutes; HPLC H-H435A-(C)_cha1CitPABC-#54 H-M428L+N434S-(C)_cha1CitPABC- H-M428L+N434S-(C)_chalCitPABC-— ——H-E388C+N421C- ——(C347+C392)_cha1CitPABC-#54 ——(C443+kC183)_cha1CitPABC-#54 —H-Q347C-(C347)_chaICitPABC-#54— H-K392C+L443C-(C392+C443)_mc- #1 15 #1 15 H-Q347C+K392C-(C347+C392)_mc- #1 15 H-L443C+kK183C-(C443+kC183)_mc- #1 15 C+kK183C"C347+kCI83””— #1 15 H-Q347C-(C347)_mc-#115 — —H-E38SC-(C388)_chaICitPABC-#54— —H-kK183C-(k0183)_mc-#115— SEC (Protocol P): 7.364 minutes; HPLC H'E388C'(C388)—mc'#1 15 (Protocol 0): HC A mass = 914 H-L443C-(C443)_mc-#1 15 - C-(C421) chalCitPABC-#54 H-N421C-(C421)_mc-#115 H-Al 14C-(C1 14)_mcGly-#201 Table 20 - ICso values for ed compounds (cytotoxic peptides) of the invention MDA-MB- (3‘:ng N87GMEAN 361-DYT2 Example # ICso (nM) GMEAN 1C50 [C50 (nM) (11M) 0.368 0.543 1.045 0.682 6.709 1.853 0.211 1.119 0.499 1.205 1.111 #51__— #56 0.316 1 256 0.766 #117 0.096 0.103 0.118 #118 0 100.000 100.000 #123 0.125 0.089 0.129 #126 0.315 0.375 0.454 #130 0.050 0.076 0.039 #131 0.072 0.185 0.081 #134 0.108 0.115 0.134 #141 3.367 3.018 _ #142 0.279 0.259 #143 _ _ #144 0.172 0.182 0.174 #145 0.185 0.167 0.229 #146 0.435 0.195 0.387 #148 0.429 0.219 0.502 #162 31.448 21.610 27.824 #173 0.088 0.099 0.067 #178 0.968 1.262 0.911 #192 #194 #200 #201 #207 #208 #209 #217 —— #219 —— #220 35.163 0 100.000 #221 32.402 87.857 65.401 #222 0158 #223 7589 #224 0383 #225 3449 10524 7575 #226 0.118 0.478 0.106 #227 11.008 12.899 #228 0.105 0.078 #229 18.372 10.218 #230 100.000 70.236 #231 3.706 15.127 22.855 #232 0071 0194 0095 #233 1074 8413 5042 #234 0684 0756 2004 #235 0852 1320 1278 #236 _0020 0023 0010 #237 0162 0217 0278 #238 0139 0077 0084 #239 _ #240 _11.710 19.930 23.480 #241 _0364 0388 0494 #242 34-529 #243 _1252 1301 1284 #244 73.123 100.000 100.000 #245 11.793 33.037 31.856 #246 - 3159 10828 5430 #247 _1007 2061 1334 #257 _ WO 72813 Table 21A” - IC50 values for selected conjugates of the invention BT474 HCC1954 N87 IC50 0f IC50 0f IC50 0f 1—10 (II O IC ADC Linker-Payload # Antibody 50 IC dy 50 Antibody (HM) (HM) (HM) (n_/mL (n_/mL (n_/mL H-(C)_Ma1Peg3C2-#41 0.725 25.592 0.465 16.617 4.02 175.448 H-(C) MalPeg6C2-#42 0.502 19.855 0.604 24.783 >14.090 >6681.150 H-(C)_mc-#44 3.553 121.414 14.464 493.077 >841.360 >294195.31 H-(C)_Ma1Peg3C2-#44 2.603 114.847 5.113 225.594 >440.881 >263246'53 Ma1Peg6C2-#44 1.318 58.155 1.466 64.663 98.174 4873.851 H-(C)_cha1CitPABC-#44 0.188 6.717 0.155 5.329 0.781 28.146 1.274 1.423 9072.131 0.258 0.204 16.737 0.436 0.992 138.026 1.938 0.356 12.995 5.743 2427.639 H-(C) tPABC-#69 0.18 7.17 0.073 2.878 <0.185 <8.946 0.133 0.078 8.722 0.483 0.654 297.254 0.152 0.127 21.134 0.272 — 0.109 41.768 — 3.269 — - 8.216 4.294 - 7.195 H-A114C- -0. 3 1 0.696 H-A114C- 0174 (C1 a1CitPABC-#54 ‘ 0 17 H-A114C- 2.548 28.2 (C114) chalCitPABC-#26 HA114C-(C114)_mc-#26 >60.648 — >1000.00 - >980.026 2007 26.18 - (C1 14)_cha1CitPABC-#36 ' 13.579 H-A114C 0‘16 - 0.524 (C114)_cha1CitPABC-#42 H-A114C-(C114)_mc-#42 0.81 — 1.54 — 44.164 H-(C)-cha1CitPABC #54 0.292 0.27 — 0.345 (C)_cha1CitPABCAmPeg3C2- 15. 134 14.33 (C)_cha1CitPABCAmPeg6C2- 1 .898 .4 H-(C)_mc-#47 4.429 — 3.52 - 20.007 H-(C)_Ma1Peg3C2-#54 2.181 — 1.54 - >41.711 H-(C)_mc-#54 3.565 — 6.28 - 48.566 (C)_cha1CitPABCAmPeg3C2- 5 .228 >1000.00 - >543.852 H-(C)_Ma1Peg3C2-#47 1.467 — 1.29 - 16.856 >1000.00 (C)_cha1CitPABCAmPeg3C2- 1.587 4 95 _ )_mc-#41 0.506 0.68 - 7.543 >1000.00 (C)_cha1CitPABCAmPeg3C2- 11.211 >1000.00 _ (C)_cha1CitPABCAmPeg6C2- 0.935 2.46 — 14.283 IIH-(C)_MalPeg3C2-#42 0.517 0 5 1 - 5.479 >1000.00 (C)_cha1CitPABCAmPeg6C2- 10.992 >1000.00 _ (C)_cha1CitPABCAmPeg6C2- 1.819 1. 97 - 75.643 H-(C)_MalPeg6C2-#54 1.02 H-(C)_MalPeg6C2-#47 1.42 MalPeg6C2-#26 9.55 H-(C)-Ma1Peg6C2-#42 0.55 >1000.00 H-(C)_mc-#36 >1000.000 >1000.00 _ H-(C)_cha1CitPABC-#60 0.835 6.45 - 14.917 >1000.00 Ma1Peg3C2-#26 11.506 9.43 _ >1000.00 (C)_cha1CitPABCAmPeg3C2- >1000.000 >1000.00 _ H-A1 1 4C- (C 1 1 1CitPABCAmPeg3 >1000.000 >1000.00 — >325.714 C2-#36 H-A114C-(C114)_Ma1Peg6C2- 1.228 201 — 133.426 H-(C)_Ma1Peg3C2-#60 >1000.000 >1000.00 _ >1000.00 H-(C)_MalPeg6C2-#60 >1000.000 >1000.00 _ H-(C)_Ma1Peg6C2-#41 1.166 0.36 — 5.882 H-(C)-mc-#69 0.427 0.47 >1000.00 H-(C)_Ma1Peg3C2-#36 720.826 >1000.00 _ >1000.00 (C)_cha1CitPABCAmPeg6C2- >1000.000 >1000.00 >1000.00 H-(C)_MalPeg6C2-#36 878.903 159.1 (C)_cha1CitPABCAmPeg3C2- 2.28 H-(C)-Ma1Peg3C2-#41 0.725 0.54 4.004 (C)_cha1CitPABCAmPeg6C2- 979. 982 00 392.905 >1000.00 H-A114C-(C114)_mc-#66 17.235 - >1000.00 _ H-L398C+L443C- (C398+C443)_cha1CitPABC- 0.249 0.27 - 0.678 H-K392C+L443C- (C392+C443)_cha1CitPABC- <0.195 0.42 - <0.254 H-L443C- <0'130 0.32 - <0.267 (C443)_cha1CitPABC-#54 H-L398C+V422C- (C398+C422)_cha1CitPABC- 0.387 0.27 - 0.504 ——3553 >507.23 - >878.489 2.886 68.41 - >834.717 H-(C)_2AcAmPeg6C2-#66 419 >1000.00 — >100)0.00 H-(C)-Ma1-PEG6C2-#45 ——1.274 2.74 - >268.047 H-(C)-mc-#79 ——0.483 0.65 - 7.576 Ma1Peg3C2-#44 2.603 5.11 - >440.881 0.09 - <0.179 Ma1Peg6C2-#44 -- l.3l8 1.47 - 98.174 - 0.207 (Cl 14)_cha1CitPABC-#69 ' H-(C)-cha1CitPABC-#79 0.152 0.15 - 0.469 H-A114C- (Cl 14)_cha1CitPABC-#79 -- 0 124 0.12 ‘ 0.386 H-(C)-cha1CitPABC-#44 0.252 .0 5 - 0.732 H-Al 14C- 8 127 >1000.00 - ' 62.825 (C 1 14)_cha1CitPABC-#88 H-(C)-cha1CitPABC-#69 0.13 3 p H - 0.249 H-(C)_2AcAm#C6a6pVa1C1tPABC---0.436 H-A114C- -- 0 217 (C 1 14)_cha1CitPABC-#45 ' 0.2 H-Al 1 4C- 3724 >1000.00 (C1 14)_cha1CitPABC-#34 ——6.431 00 ——0.349 ——0.226 0.453 H-Q347C- 0 368 (C347)_cha1CitPABC-#69 ' 9.0.0.0 oUINO\ oxA-pw H-Y373C- 0 359 (C373)_cha1CitPABC-#69 .-' H-E388C- H-N421C- (C443)_cha1CitPABC-#69 ‘ (C443)_cha1CitPABC-#79 ‘ 0.36 - ‘ 0 852 (Cl 14)_cha1CitPABC-#95 ‘ 0.24 0 258 (Cl 14)_cha1CitPABC-#98 H-Al14C-(C1fi1643_MalPeg3C2- 0.221 0.58 - 1.589 H-N297Q-(Q)_AmPeg6C2-#42 0.466 0.36 5.42 ——0557 037 H-N297Q-(Q)_AmPeg6C2-#47 0.346 0.43 — 4.337 H-N297Q-(Q)_AmPeg6C2-#36 3.003 00 — 284.267 H-N297Q-(Q)_AmPeg6C2-#26 0.991 1.07 — 35.331 >1000.00 H-N297Q-(Q)_AmPeg6C2-#66 13.812 >1000.00 H-L443C-(C443)_MalPeg6C2- 0.251 0.25 - 1.989 H-Q347C-(C3Q673_MalPeg6CZ' 0.267 3 H-13388C—(C388)_MalPeg6CZ- 0.382 H-N421C-(C421)_Ma1Peg6C2- 0.35 4s 0C-(C32E;_MalPeg6CZ- 0.482 H-L398C+L443C- 0 226 03 (C398+C443)_Ma1Peg6C2-#69 ' H-K392C+L443C- 0 268 (C392+C443)_MalPeg6C2-#69 ' H-kAl 1 1C 1.635 (kCl 1 1)_Ma1Peg6C2-#69 H-kK183C- 0257 .0 u. 2. 3 (kC183)_Ma1Peg6C2-#69 (kC207)_Ma1Peg6C2-#69 ‘ C114 chalCitPABC-#108 ' H-A114C- H-A114C- o. N .0 .— H-A114C-(C114)_mc-#108 >1000.000 >1000.00— H-Al 14C° 3 - 0239 (C1 14)_cha1CitPABC-#1 17 H-Al 14C- 0202 O N. 0211 (C1 14)_cha1CitPABC-#1 15 H-Al 14C-(C 1#194§_Ma1Peg6C2- 0.576 1.46 H-Al 14C-(C1 14)_Ma1Peg6C2- 0.257 0.505 0#1 18 (C1 14)_cha1CitPABC-0#1 18 ‘ O U.) _ (C 1 14)_cha1CitPABC-#80 ' H-A114C-(C114)_mc-#117 0.197 H-Al 14C- 0 376' ’—‘ U.) _ (C1 a1CitPABC-#232 H-Al 14C-(C1 14)_Ma1Peg6C2- 0.504 0.85 #230 H_A114c-(C;11411)7_MalPeg6C2- 0.335 0.21 0 792 0.243 0.23 0.21 0.15 0.256 0.43 0.215 0.27 0.151 .0 H 0.162 0283 0.07 O ~ _. 00 <0~10 0269 0.15 H-Al 14C-(C1 14)_Ma1Peg6C2- 0 28 0.22 - ' 0.685 #226 H-A114C-(C114)_mc-#172 0.296 0.41 — 0.694 H-A114C-(C1 14)_mb-0#118 0.318 0.33 — 0.709 C-(C114)_me-0#118 0.256 0.33 H-A114C- 0 301 - ' 0.34 0.501 (C1 14)_cha1CitPABC-#134 H-A114C-(C114)_mc-#131 0.357 0.76 - 1.614 H-Al14C-(C114)_Ma1Peg6C2- 0.284 0.36 - 1.377 #126 H-Al14C-(C114)_Ma1Peg6C2- 0.362 w 4> - 1.867 #123 H-A114C-(C114)_mc-#126 0.319 H-A114C-(C114)_mV-0#118 0.209 H-(C)_Ma1Peg6C2-#226 0.575 0.0.0.0. NN-b ch H-(C)_mc-#226 0.359 0.18 m H-(C)_m(H20)c-0#1 18 0.26 0.11 H-(C)_Ma1(H20)Peg6C2-0#1 18 0.482 0.19 —u_ H-(C)_Ma1(H20)Peg6C2-#69 0.832 0.51 H-(C)_m(H20)c-#69 0.418 0.28 me-0#1 18 0.186 0.11 H-(C)_mV-0#1 18 0.201 0.14 H-(C)_mb-0#1 18 0.222 0.13 H-A114C-(C1 14)_Ma1C6-#54 0.662 H-A114C-(C114)_mc-#231 000 >1000.00 - >1000 H-Al14C-(C114)_Ma1C6-0#118 0.976 113 H-(C)_Ma1(H20)Peg6C2-#1 15 1.06 0.28 - 3.439 H-A114C-(C114)_mc-#158 0.247 0.35 - 0.739 H-A114C- 1.178 24.41 - 13.447 (C1 14)_cha1CitPABC-#231 m(H20)c-#1 15 0.393 0.17 - 0.498 H-A114C-(C114)_mc-#237 0.97 0.68 - 27.907 H-A114C-(C114)_mc-#145 4.681 585.59 - 643.391 H-Al14C-(C114)_Ma1Peg6C2- 12.856 190.59 - 89.125 #145 H-A114C-(C114) mc-#162 0.377 0.15 - 1.144 C-(C1 14)_Ma1C6Am- .04;N o ;_. - 0.694 #151 (kKl 88)_COPeg2C2ValCitPAB C-#54 H-(C)_cha1CitPABC-0#1 18 0.227 .0 ’— 4; - 0.182 H-A114C- 0.323 .0 w N - 0.363 (C1 14)_cha1CitPABC-#154 H-A114C-(C1 14)_Ma1C6Am- 0.377 .0N\1 #153 H-(C)_cha1CitPABC-#98 0.21 1 0.14 - 0.162 H-A114C- 0 357 .0o u. - ' 3.197 (C1 14)_cha1CitPABC-#246 0.17 - 0.237 H-M428L+N434S- 0 322' 0.1 _ (C)_cha1CitPABC-#70 H-M428L+N434S- 0 354 (C)_cha1CitPABC-#54 ' H-E388C+N421C- (C388+C421)_cha1CitPABC- 1.38 — 0.855 H-Q347C+K392C- (C347+C392)_cha1CitPABC- 0.276 - 0.29 - 0.147 - H-L443C+kK183C- (C443+kC183)_cha1CitPABC <0.129 0.37 - <01 1 1 -#54 H-Q347C+kK183C- kC183)_cha1CitPABC 0.146 0.25 - 0 08 -#54 (05153213111301.1215 (05113361513219.5115 H-kK183C- kC183 mc-#115 — . 0.269 H-E388C-(C388)_mc-#115 0.302 — . 0.301 H-L443C-(C443)_mc-#115 0.222 — . 0.259 H-N421C- <0‘05 1 <0‘05 1 (C421)_cha1CitPABC-#54 H-N421C-(C421)_mc-#115 0.312 0 306 H-Al14C-(C114)_mcG1y-#201 0.321 _ Table 21B - IC50 values for selected conjugates of the invention —DYT2 MDA-MB-468 IC50 of IC50 0f IC IC ADC-Linker-Payload # (111510) Antibody (111510) Antibody (ng/mL) (ng/mL) Ma1Peg3C2-#41 >69 685 .581 — >35714 286 H-(C)_Ma1Peg6C2-#42 33.396 5455.61 >629.281 >25857 971 mc-#44 >1000 000 >34090.909 >1000.000 >34090 909 H-(C)_Ma1Peg3C2-#44 >1000 000 >441 17.647 >1000.000 >441 17.647 H-(C)_Ma1Peg6C2-#44 >1000 000 >441 17.647 >1000.000 >441 17.647 H-(C)_cha1CitPABC-#44 0.203 7.246 >1000.000 >35714.286 H-(C)_Ma1—PEG3C2-#45 >1000.000 >34883.721 >1000.000 >34883 721 Ma1—PEG6C2-#45 >1000.000 >38461.5 38 >1000.000 >38461 538 ————— ————— ————— H-A114C-(C114)_mc-#54 >1000.000 — —>1000.000 H-A114C-(C114)_cha1CitPABC-#47 383.667 445.014 - ————— ————— ———— —H-Al 14C-(C114)_chalCitPABC-#36 ——— H-Al 14C-(C1 14)_chalCitPABC-#42 -- H-A114C-(C114)_mc-#42 >727.245 567.735 H-(C)-chalCitPABC-#54 0.275 — 471.905 —H—(C)_cha1CitPABCAmPeg3CZ-#54 ——— —H—(C)_cha1CitPABCAmPeg6CZ-#54 ——— W "- H-C MalPe_3C2-#54 >1651.007 >1651.007 ———— ———— _I-_——— ———— ———— -I__——— ———— ———— -I__——— ———— ———— ———— _I-_——— ———— ———— _I———— ———— ———— H-Al 14C- >1000'000 >1000‘000 (C1 14)_chalCitPABCAmPeg3C2-#36 H-A114C-(C1 14)_MalPeg6C2-#54 000 000 H-(C)_MalPeg3C2-#60 000 — >1000.000 H-(C)_MalPeg6C2-#60 >1000.000 >1000.000 H-(C)_MalPeg6C2-#41 >1000.000 000 H-(C)-mc-#69 >71.831 — 49 H-(C)_MalPeg3C2-#36 000 >1000.000 H-(C)_chalCitPABCAmPeg6C2-#36 >1000.000 - >1000.000 H-(C)_MalPeg6C2-#36 >1000.000 >1000.000 H-(C)_chalCitPABCAmPeg3C2-#41 >1000.000 >1000.000 H-(C)-MalPeg3C2-#41 >69.685 - >1000.000 H-(C)_chalCitPABCAmPeg6C2-#60 >1000.000 >1000.000 H-L398C+L443C- 0463 801354 (C398+C443)_chalCitPABC-#54 H-K392C+L443C- <0'171 - 565'01 (C392+C443)_chalCitPABC-#54 H-L443C-(C443)_chalCitPABC-#54 0.371 — 500.958 H-L398C+V422C- 0'48 610'884 (C398+C422) chalCitPABC-#54 H- C -mc-#44 >1000.000 >1000.000 ———— ———— _I———— ———— ———— _I———— ———— H—Al14C—(Cl14)_cha1CitPABC-#69 ———— H—(C)—chaICitPABC-#79 ———— H-Al 14C-(C1 alCitPABC-#79 ——— H—(C)-chalCitPABC-#44 ——— H-Al 14C-(C1 14)_chalCitPABC-#88 -- H-(C)-chalCitPABC-#69 0.098 >1000.000 H-(C)_2AcAmCapVaICitPABC-#66 ——— H-Al 14C-(C1 14)_chalCitPABC-#45 ——— H-Al 14C-(C1 14)_chalCitPABC-#34 ——— C-C114 mC-#70 >1000.000 >1000.000 H-(C)_chaICitPABC-#112——— H-(C)-Mal-PEG6CZ-#69 ——— H-Q347C- C347 chalCitPABC-#69 ——— H-Y373C-(C373)_chalCitPABC-#69 ——— H-E388C-(C388)_chalCitPABC-#69 ——— H-N421C- C421 chalCitPABC-#69 ——— H-L443C-(C443)_chalCitPABC-#69 ——— H-L443C-(C443)_chalCitPABC-#79 ——— H-A114C- C114 chaICitPABC-#95 ——— H-Al 14C-(C1 14)_chalCitPABC-#98 ——— H—A114C-(Cl14)_MalP6g302-#69 ——— H-N297Q-(Q)_AmPeg6CZ-#42 ——— H-N297Q-Q Amps-6024154 ——— H-N297Q-(Q)_AmPeg6CZ-#47 ——— H-N297Q-(Q)_AmPeg6CZ-#36 ——— Q-Q Amps-602426 ——— Q-(Q)_AmPeg6CZ-#66 ——— H—L443C-(C443)_MalPeg6CZ-#69 ——— H-Q347C-C347 Ma1P6-6cz-#69 ——— H—E3880-(0388)_M61P6g602-#69 ——— H—N4210-(C421)_MaIP6g6CZ-#69——— H-L398C+L443C- ”27 846418 C443)_MalPeg6C2-#69 —--—H-K392C+L443C- ———— H-kK183C-(kCI83)_MalP6g6CZ-#69 ——— H-kK207C-(kczo7)_MalP6g6cz-#69 ——— H-Al 14C-(C1 14)_chalCitPABC-#108 ——— H-Al 14C-(C1 14)_chalCitPABC-#84 ——— H-Al 14C-(C1 14)_chalCitPABC-#226 ——— H-A114C-(Cll4)_mc-#108 ——— H-Al 14C-(C1 14)_chalCitPABC-#1 17 ——— H-Al 14C-(C1 14)_chalCitPABC-#1 15 ——— C-(Cl14)_MaIP6g6CZ-#98 ——— H-Al 14C-(C1 14)_MalPeg6C2-0#1 18 -- H-A114C-(C1 14)_chalCitPABC-0#1 18 0.215 >1000.000 H-Al 14C-(C1 14)_chalCitPABC-#80 ——— C-(Cll4)_mc-#117 ——— H-Al 14C-(C1 14)_chalCitPABC-#232 ——— H—Al140-10114)_MalP6g6CZ-#230 ——— H—Al140-10114)_MalP6g6CZ-#117——— ———— ———— H-A114C-(C114)_mb-#69 >1000.000 — >1000.000 H-A114C-(C114)_mV-#69 000 — >1000.000 —432.816 —194.684 —361.061 —541.542 H-(C) MalPeg6C2 0#118 465.455 H A114C-(C114) MalPeg6C2-#226 574.794 H A114C (C114)_mc #172 —500.864 H A114C (C114) mb 0#118 —506.604 H A114C (C114)_me 0#118 —903.571 H A114C C114 Inc #131 --480.901 HA114C C114 Inc #126 —— H-A114C (C114)_mV-0#118 —— H (C) MalPeg6C2-#226 —— HiC Inc #226 —— H (C) m(H20)c 0#118 —— H-(C) Ma1(H20)Peg6C2 0#118 —— H C Mal H20 Pe_6C2 #69 —— H-(C) m(H20)c #69 —— H (C):mV-0#118 —— H-C 18 —— H (C114) Ma1C6 #54 —— H-A114C (C114) Inc #231 —— H-A114C C114 Ma1C6 0#118 —— H-(C)_Ma1(H20)Peg6C2-#1 15 —— H A114C (C114) Inc #158 — H-A114C- C114 chalCltPABC #231 — H (C) m(H20)c #115 — H-A114C-(C114) MalPeg6C2 #145 -->1000.000 H-A114C-(C114) MalC6Am #153 —— chalC1tPABC-#98 —— —380.393 H-E388C+N421C- 826.243 (C3 88+C421)_cha1CitPABC-#54 H-Q347C+K392C- 390.7 (C347+C392)_cha1CitPABC-#54 H-L443C+kK183C- 395.707 384.028 (C347+kC1 83)_cha1C1tPABC-#54 —393.412 _ —— H E388C+N421C C421)_mc #115 0.227 — >1000.000 H-Q347C+K392C-(C347+C392)_mc #115 0.068 — 934.867 H-L443C+kK183C-(C443+kC183)_mc- 0.071 757.604 #1 1 5 H-Q347C+kK183C-(C347+kC183)_mc- 0.073 741.434 #1 1 5 H-Q347C-(C347)_mc-#115 0.098 — 888.128 H-kKl83C-(kC183)_cha1CitPABC-#54 1.329 — 160.012 H-E388C-(C388)_cha1CitPABC-#54 0.658 — 287.88 H-N421C-(C421)_mc-#115 0.108 668.857 H-Al 14C-(C1 14)_mcGly-#201 0.073 Table 22 - Selected cokinetic values in rats for conjugates of the invention and ed pharmacokinetic values in rats for conjugates comprising MMAD, MMAE or MMAF. AUCs were calculated at a O-last of 0-336 h except where noted.
AUC (0-last) - ADC Dose ADC Ab ADC/.Ab Ratlo 3 33901 45601 74 110-1054 ——-1_ ——m_ 100 146000 212000 69 4‘§;ZE§6C2' H<c>-mc-MMAD H<c>-vc-MMAE _-—-— H<c>-mc-MMAF Ha<>-Mcc-DM1 39100 49600 79 1 denotes a O-last of 0-3 12 hours 2 denotes a O-last of 0-168 hours 3 denotes a O-last of 0-96 hours Table 23 - Selected pharmacokinetic values in mice for conjugates of the invention and for conjugates comprising MMAD, MMAE or MMAF. AUCs were calculated at a O-last of 0-336 h except where noted.
AUC (0-last) ( _*H0urs/mL) Ratlo H(C) #D44 3 10701 27201 H(C)-#D7O 2240 4890 H(C)-#D69 2490 4770 52 3594 H c -MalPEG6C2-<>MMAD H(C)-mc-MMAD 3580 4970 72 c-MMAE 1600 3290 9 H(C)-mc-MMAF U.) 3080 4800 64 1 denotes a O-last of 0-168 hours Table 24 — Data showing stability of conjugates prepared using pened versus ring closed imide-based linkers.
Herceptin ADC GSH stability (6d) Mouse ADC Mouse PK (%loading remaining AUC ADC/Ab on da 6 (u_*h/mL mc-#1 18 ring-closed 65% 2160 55% ring-opened 87% 3490 65% MalPeg6C2-#1 18 ring-closed 82% 2010 70% ring-opened 100% 3000 77% mc-#8261 ring-closed 51% 3590 52% __———MalPeg6C2-#8261 ring-closed ———— Table 25A - Selected payloads and their methods of synthesis Prepared in the Same Manner as or ation Quantity in mg Example Preparation Method (Yield) Method example #107 Vlethod M 10.5 mg (43%) silica example #146 354 mg (78 A)0 chromatography l procedure L Method 1* 19.4 mg (77%) example #131 Method E1* 30 mg (51%) #229 example #151 Method J* 16 mg (61%) General ure L Method J* 69 mg (42%) General procedure Method 1* 4.2 mg (44%) GeneralIprocedure #235 51h” example #145 38.6 mg (93%) chromatography silica example #145 419 mg (81 A)0 chromatography silica #239 example #130 315 mg (48 A)0 chromatography #240 example #142 Method E1* 6 mg (20%) example #142 Method E 1 * 6 mg (20%) example #145 Method J* 8 mg (10%) #243 example #145 Method J* 12 mg (22%) #244 example #145 Method J* 9.6 mg (20%) #245 General procedure M medluglrgressue 38 mg (55%) #246 example #130 medluglgressue 78 mg (80%) #247 example #178 Method M* 10.5 mg (57%) Table 25B - Selected payloads and their IUPAC name and characterization data Mass spectrum: LC-MS or HPLC observed Hill and Example IUPACNAME retention time in minutes: 1H NMR (400 MHz, DMSO-dg unless ted otherwise 2-methylalanyl-N—[(3R,4S,5 S) {(ZS)[(1R,2R){[(2R,4S) carboxyphenylpentan HPLC (Protocol 0311;113:534651 [M+}P]. (1.57 #220 yl]amino} methoxymethyl oxopropyl]pyrrolidinyl} - 3- methoxymethyloxoheptan yl] -N-methyl-L-Valinamide 2-methylalanyl-N—[(3R,4S,5 S) 2-[(1R,2R) lo[1.1.1]pentylamino) HPLC (Protocol DB): m/z 622.42 [Mi-PF] (1.57. #221 methoxymethyl m1nutes) oxopropyl]pyrrohdrnyl} - 3-. . y-5 -methyloxoheptan yl] -N-methyl-L-Valinamide LC-MS (Protocol H): m/z 744.9 [M+H+] (2.19 minutes). me$§§$11811§¥12$§3€5$2§£ 1H NMR (400 MHz, CDC13) 5 7.16-7.22 (m), 6.99-7.08 (m), 6.42-6.51 (m), 6.10-6.17 (m), .96 (m), 4.65- methoxy{[(1R)methoxy 4.79 (m), 4.27-4.36 (m), 4.04-4.27 (m), 3.95-4.02 (m), oxo(1- 3.87-3.93 (m), 3.64-3.84 (m), 3.44-3.57 (m), 3.22-3.42 #222 phenylcyclopropyl)ethyl]amino}- (m), 308-3. 17 (m), 2.98-3.07 (m), 2.90-2.93 (m), 2.85- 2-methyloxopropyl]pyrrolidin- 2.89 (m), 2.53-2.57 (m), 2.35-2.51 (m), 2.19-2.27 (m), 1-yl } - yl tanyl] - 2.02-2.16 (m), 1.93-2.00 (m), 1.77-1.90 (m), 1.57-1.70 N-meth (m),1.35-1.52(m),1.26-1.33(m),1.19-1.25(m),1.11- yl'L'Vahnamlde 1.16 (m), 1.03—1.11 (m), 0.83-1.02 (m), 0.79-0.88 (m).
LC-MS (Protocol H): m/z 744.4 [M+H+] (2.17 minutes). 2-methylalanyl-N—[(3R,4S,5 S) 1H NMR (400 MHZ, CD3OD) 5 8.19-8.24 (m), 7.87- methoxy{(ZS)[(1R,2R) 7.92 (m), 7.20-7.38 (m), 4.71-5.04 (m), 4.61-4.71 (m), methoxy{[(IS)methoxy 4.47-4.52 (m), 4.38-4.44 (m), 405-4. 13 (m), 3.99-4.04 oxo-l-(l- (m), 3.90-3.98 (m), 3.64-3.73 (m), 3.52-3.60 (m), 3.46- #223 phenylcyclopropyl)ethyl]amino}- 3.52 (m), 3.37-3.46 (m), 3.35-3.37 (m), .35 (m), 2-methyloxopropyl]pyrrolidin- .28 (m), .19 (m), 308-3. 14 (m), 3.01-3.06 -methyloxoheptanyl]— (m), 2.84-2.87 (m), 2.43-2.63 (m), 1.96-2.20 (m), 1.68- N—meth 1.95 (m), 1.60-1.66 (m), 1.52-1.57 (m), 1.33-1.44 (m), yl-L-Valinamide 1.27-1.32 (m), 1.23-1.27 (m), 1.12-1.17 (m), 1.04-1.10 (m), 0.96-1.03 (m), 0.90-0.96 (m), 0.82-0.90 (m).
LC-MS (Protocol H): m/z 730.8 [M+H+] (2.15 minutes). 2-mcthylalanyl-N- S,5 S) 1HNMR (400 MHz, CD3OD) 5 7.09-7.18 (m), 6.95- {(2S)[(1R,2R)({(1R) 7.08 (m), 4.88-4.93 (m), 4.75-4.85 (m), .74 (m), [(7R)-bicyclo[4.2.0]octa-1,3 ,5 - 4.62-4.70 (m), 4.50-4.59 (m), 409-4. 16 (m), 3.96-4.06 tricnyl] methoxy (m), .90 (m), 3.67-3.76 (m), 3.58-3.67 (m), 3.58- oxocthyl } amino)- 1-mcthoxy 3.67 (m), 3.45-3.54 (m), 3.33-3.44 (m), .44 (m), methyl-3 - oxopropyl]pyrrolidin 3.28-3.33 (m), 3.10-3.27 (m), 300-3. 10 (m), 2.93-3.00 yl} -3 -mcthoxy-5 lo (m), 2.75-2.78 (m), 2.56-2.65 (m), 2.36-2.45 (m), 2.17- an—4-yl] -N-methyl-L- 2.35 (m), 1.94-2.16 (m), 1.67-1.94 (m), 1.48-1.67 (m), valinamidc 1.27-1.33(m),1.23-1.27(m), 1.17-1.26 (m), 1.08-1.17 (m), 0.98-1.07 (m), 0.86-0.98 (m), 0.77-0.84 (m). 2-mcthylalanyl-N- [(3R,4S,5 S) {(ZS)[(1R,2R)({(1S)-11(75)- bicyclo[4.2.0]octa-1,3 ,5 yl] mcthoxy oxocthyl} amino)mcthoxymcthyl-3 - LC-MS (Protocol H): m/z 730.9 [M-l-PP] (2.19 minutes) oxopropyl]py1rolidinyl}- 3- methoxymcthylo an—4-yl] -N-methyl-L- valinamidc N,2-dimcthylalanyl-N- LC-MS (Protocol Q): m/z 732.4 [M+H+] (1.24 minutes). [(3R,4S,5 S)-3 -methoxy{(2S) 1HNMR 5 8.47-8.53 (m), 8.24-8.29 (m), 7.81-7.91 (m), [(1R,2R)methoxy{[(2S) 7.14-7.27 (m), 4.54-4.75 (m), 4.44-4.54 (m), 3.94-4.02 methoxyoxo-3 -phenylpropan (m), 3.72-3.78 (m), 3.61-3.69 (m), .36 (m), 3.14- yl]amino} methyl-3 - 3.28 (m), 2.99-3.08 (m), 2.81-2.97 (m), 2.29-2.57 (m), oxopropyl]py1rolidinyl}- 5- 2.16-2.29(m),1.91-2.16(m), 1.60-1.87(m),1.35-1.53 mcthyl- cptanyl] -N- (m), 0.99-1.33 (m), 0.80-0.99 (m), 0.71-0.80 (m). methyl-L-Valinamidc 2-methylalanyl-N-[(3R,4S,5 S) {(2S)[(1R,2R)({(1S) bicyclo[4.2.0]octa-1,3 ,5 - tricnyl] methoxy oxocthyl } amino)- 1-mcthoxy LC-MS (Protocol Q): m/z 730.4 [M-l-PP] (1.29 minutes) methyl-3 - oxopropyl]pyrrolidin yl} -3 -mcthoxy-5 -mcthylo xohcptan—4-yl] -N-methyl-L- valinamidc N,N,2-trimcthylalanyl-N- [(3R,4S,5 S)-3 -methoxy{(2S) [(1R,2R)methoxy{[(2S) methoxyoxo-3 -phenylpropan HPLC (Protocol A*): m/z 746.5 [M-l-PP] (7.103 yl]amino} methyl-3 - minutes) oxopropyl]py1rolidinyl}- 5- mcthyl- 1-oxohcptanyl] -N- methyl -L-Valinamidc N,N,2-trimcthylalanyl-N- [(3R,4S,5 S){(2S)[(1R,2R)-3 - { [(1 S)- 1 xy cthyl]amino } - 1-mcthoxy LC-MS (Protocol Q1): m/z 732.3 [M+H+] (0.70 methyl- 3- pyl]pyrrolidin minutes) yl} -3 -mcthoxy-5 -methyl oxohcptanyl] -N-mcthyl-L- valinamid 2-mcthylalanyl-N— [(3R,4S,5 S) 2-[(1R,2R) {[(R)- carboxy( 1 - phenylcyclopropyl)methyl]amino} - HPLC (Protocol G): m/z 730.4 [M+H+] (1.25 minutes) 1 -mcthoxymcthyl-3 - oxopropyl]py1rolidinyl}- 3- methoxy-5 -mcthyloxoheptan yl] -N-methyl-L-Valinamide LC-MS (Protocol Q1): m/z 1020.6 [M+H+] (0.83 difluoro {2-mcthylalanyl-N- s). 1H NMR (400 MHz, CD30D) 5 8.19-8.23 [(3R,4S,5 S){(2S) (m), 7.99-8.07 (m), 7.93-7.98 (m), 7.41-7.45 (m), 7.23- [(3R,4R,7 S)benzyl-15 - {2-[(3,5 - 7.31 (m), 7.17-7.22 (m), 7.00-7.04 (m), 6.32-6.37 (m), dimcthyl- 1H-pyrrolyl- 6.20-6.24 (m), 4.72-4.93 (m), 4.61-4.69 (m), 4.05-4.17 kappaN)methylidenc] -2H-pyrrol-5 - (m), 3.88-3.93 (m), 3.72-3.81 (m), 3.63-3.70 (m), 3.56- yl-kappaN} mcthyl-5 ,8, 13 - 3.62 (m), 3.48-3.56 (m), 3.25-3.44 (m), 3.16-3.25 (m), trioxooxa-6,9,12- 3.09-3.14 (m), 2.98-3.09 (m), 2.81-2.90 (m), 2.54-2.67 triazapcntadccan (m), 2.39-2.53 (m), 2.09-2.32 (m), .97 (m), 1.60- -3 -yl]pyrrolidinyl} -3 -methoxy- 1.69 (m), 1.52-1.59 (m), 1.32-1.44 (m), 1.28-1.32 (m), -mcthyloxohcptanyl] -N- 1.16-1.21 (m), 0.98-1.09 (m), 0.86-0.98 (m), 0.79-0.90 methyl-L-Valinamidato }boron (m).
LC-MS (Protocol Q): m/z 769.3 [M+H+] (1.34 minutes). 1HNMR 5 9.04-9.17 (m), 8.88-8.94 (m), 8.70- 8.86 2-methyl-D-prolyl-N— [(3R,4S,5 S)- (m), .67 (m), 7.79-7.84 (m), 7.76-7.79 (m), 7.65- 3 xy{(2S)[(1R,2R) 7.69 (m), 7.61-7.64 (m), 7.20-7.31 (m), 7.12-7.20 (m), methoxymcthyl-3 -oxo { [(1 S)- 5.44-5.52 (m), 5.34-5.46 (m), 4.70-4.78 (m), 4.56-4.67 2-phcnyl(1,3 -thiazol (m), 4.47-4.54 (m), 3.94-4.04 (m), 3.76-3.83 (m), 3.52- yl)ethyl]amino } propyl]pyrrolidin- 3.61 (m), .52 (m), 3.28-3.35 (m), 3.10-3.27 (m), 1-yl } - 5-mcthyl oxohcptanyl] - 2.93-3.08 (m), 2.77-2.80 (m), 2.64-2.70 (m), 2.35-2.54 N—mcthyl-L-Valinamidc (m), 2.09-2.34 (m), 1.96-2.09 (m), 1.54-1.88 (m), 1.38- 1.52(m),1.18-1.36(m),1.03-1.13(m), 0.81-1.01(m), .81 (m).
LC-MS (Protocol Q): m/z 732.2 [M+H+] (1.28 minutes). methyl N-{(2R,3R)[(2S) 1HNMR 5 8.48- 8.53 (m), 8.22-8.28 (m), 7.80-7.92 {(3R,4S,5 S)[ {N— [(3 - (m), 7.14-7.28 (m), 4.74-4.79 (m), 4.54-4.72 (m), 4.43- aminooxetan— 3-yl)carbonyl] -L- 4.52 (m), 4.24-4.35 (m), 4.07-4.12 (m), 3.94-4.02 (m), valyl} (methyl)amino] mcthoxy- 3.72-3.78 (m), 3.61-3.69 (m), 3.48-3.58 (m), 3.40-3.48 lheptanoyl} pyrrolidin-2 - (m), 3.11-3.35 (m), 2.98-3.11 (m), 2.75-2.97 (m), 2.64- yl] hoxy 2.69 (m), 2.30-2.55 (m), 2.17-2.28 (m), 2.03-2.14 (m), methylpropanoyl} -L- 1.92-2.02(m),1.59-1.87(m), .54(m),1.21-1.33 alaninatc (m), 1.112-1.20 (m), 1.00-1.09 (m), 0. 70-098 (m).
LC-MS (Protocol H): m/z 589.9 [M+H+ ] (2.29 ylalanyl-N-{(3R,4S,5S) minutes). 1H NMR (400 MHz, CD30D) 5 8.55-8.61 [(2S){(3R,4R,7S,12S)benzyl- (m), 8.40- 8.45 (m), .39 (m), 8.23-8.28 (m), 8.14- 14-[ 3 -chloro(propan—2- 8.19 (m), 7.84-7.95 (m), 7.79-7.84 (m), 7.71-7.77 (m), yloxy)phenyl]—4-mcthyl[4-(8- 7.61-7.68 (m), 7.46-7.52 (m), 7.34-7.40 (m), 7.09-7.27 methylimidazo[ 1 ,2-a]pyridin (m), 7.03-7.09 (m), 4.77-4.90 (m), 4.58-4.77 (m), 4.43- yl)benzyl] -5 ,8,14-trioxo-2,9-dioxa- 4.55 (m), 4.17-4.33 (m), .16 (m), 4.00-4.07 (m), 6, 1 3 -diazatetr 3.79-3.85 (m), 3.58-3.70 (m), 3.44-3.52 (m), 3.12-3.40 adecan-3 -yl}pyrrolidin—1-yl]—3 - (m), 2.80-3.12 (m), 2.64-2.71 (m), .64 (m), 2.38- methoxymcthyloxoheptan 2.47 (m), 2.00-2.33 (m), .00 (m), 1.46-1.63 (m), yl} -N-mcthyl-L-Valinamidc 1.29-1.44 (m), .16 (m), 0.91-1.07 (m), 0.79-0.87 (m)- LC-MS (Protocol Q1): m/z 944.3 [M+H+] (0.84 minutes). 1H NMR (400 MHZ, CD3OD) 5 8.54-8.59 2-methylalanyl-N—[(3R,4S,5S) (m), 8.29- 8.33 (m), 7.87-8.02 (m), 7.80-7.87 (m), 7.68- {(2S)[(1R,2R){[(2S){[4- 7.74 (m), 7.62-7.67 (m), 7.20-7.38 (m), 4.98-5.06 (m), (5-fluoro-1,3-benzothiazolyl) 4.84-4.97 (m), 4.66-4.79 (m), 4.61-4.66 (m), 4.13-4.19 methylphcnyl]amino}oxo (m), 3.98-4.04 (m), 3.91-3.96 (m), 3.79-3.85 (m), 3.64- phenylpropan—2-yl]amino} 3.73 (m), 3.38-3.56 (m), 3.34-3.38 (m), 3.28-3.34 (m), methoxymcthyl 3.17-3.27 (m), 3.12-3.16 (m), 3.03-3.11 (m), 2.99-3.03 oxopropyl]pyrrolidinyl}-3 (m), .87 (m), 2.80-2.82 (m), 2.69-2.71 (m), 2.31- -mcthoxymcthyloxohcptan- 2.54 (m), 2.27-2.31 (m), 2.06-2.27 (m), 1.88-2.00 (m), 4-yl]-N-methyl-L-Valinamidc 1.74-1.88 (m), 1.64-1.74 (m), 1.59-1.64 (m), .59 27-1.48(m),1.19-1.26(m),1.11-1.16(m),1.06- 1.11 (m), 0.96-1.05 (m), 0.86-0.94 (m), 0.77-0.83 (m). 1 ,2-dimethyl-D-prolyl-N— [(3R,4S,SS)methoxy{(2S) [(1R,2R)methoxy{[(2S) methoxyoxo-3 -phenylpropan LC-MS (Protocol Q1): m/z 758.3 [M+H+] (0.74 no} mcthyl minutes) oxopropyl]py1rolidinyl}- 5- mcthyl- 1-oxohcptanyl] -N- methyl -L-Valinamidc LC-MS (Protocol Q1): m/z 771.2 [M+H+] (0.67 . minutes). 1H NMR (400 MHz, CD30D) 5 7.95-7.96 (m), 7.48-7.55 (m), .48 (m), 7.26-7.31 (m), 6.94- [(3R 4§§éillfit駧13n&ll§'2R)_3_ {,[(2,S)(1H-indolylS 7.18 (m), .49 (m), 5.19-5.22 (m), 5.11-5.17 (m), 4.97-5.00 (m), 4.78-4.87 (m), 4.68-4.77 (m), 4.59-4.64 methoxy oxopropan (m), 4.27-4.34 (m), 3.99-4.16 (m), 3.84-3.92 (m), 3.78- yl]amino}methoxymethyl 3.82 (m), 3.62-3.78 (m), 3.49-3.59 (m), .49 (m), oxopropyl]py1rolidinyl} 3.20-3.41 (m), 2.99-3.20 (m), 2.95-2.98 (m), 2.82-2.86 methoxy-S-methyloxoheptan (m), 2.77-2.79 (m), 2.62-2.68 (m), 2.28-2.49 (m), 2.19- yl]-N- 2.27 (m), 1.98-2.16 (m), 1.56-1.91(m), 1.31-1.49 (m), methyl-L-Valinamidc 1.19-1.30(m),1.15-1.19(m),1.06-1.13(m), 0.88-1.03 (m), .87 (m).
LC-MS (Protocol Q1): m/z 758.84 [M+H+] (0.71 mcthylalanyl-N- minutes). 1H NMR (400 MHZ, CD3OD) 5 .32 S,5S)methoxy{(ZS) (m), 5.86-6.00 (m), 5.28-5.40 (m), 5.17-5.27 (m), 4.97- [(1R,2R)methoxymcthyl 5.04 (m), 4.69-4.91 (m), 4.57-4.69 (m), 4.05-4.21 (m), oxo{[(2S)oxophcnyl .96 (m), 3.79-3.88 (m), 3.71-3.78 (m), 3.62-3.70 (propcn—1-yloxy)propan (m), 3.25-3.56 (m), 3.15-3.24 (m), 3.08-3.14 (m), 2.90- yl]amino}propyl]pyrrolidinyl}- 3.02 (m), 2.79-2.87 (m), 2.42-2.52 (m), .38 (m), -methyloxohcptanyl]—N- 2.12-2.20 (m), 2.03-2.12 (m), 2.00-2.03 (m), .1.96 methyl-L-Valinamidc (m), 1.33-1.70 (m), 1.23-1.32 (m), 1.17-1.23 (m), 1.12- 1.17 (m), 1.05-1.10 (m), .05 (m), 0.82-0.89 (m).
LC-MS (Protocol Q): m/z 786.6 [M+H+] (1.46 minutes). 2-methyl-L-prolyl-N—[(3R,4S,SS)- 1H NMR 5 8.35-8.42 (m), 8.21- 8.31 (m), 8.14-8.20 1-{(2S)[(1R,2R){[(2S) (m), 7.15-7.29 (m), 4.66-4.76 (m), 4.53-4.65 (m), 4.46- tcrt-butoxyoxophcnylpropan- 4.53 (m), 4.32-4.42 (m), 4.07-4.15 (m), 3.96-4.04 (m), 2-yl]amino}methoxymcthyl- 3.76-3.82 (m), 3.41-3.61 (m), 3.30-3.38 (m), 3.16-3.30 3-oxopropyl]pyrrolidinyl} (m), 3.08-3.15 (m) 2.99-3.08 (m), 2.92-2.96 (m), 2.78- mcthoxymcthyloxohcptan 2.90 (m), 2.63-2.78 (m), 2.37-2.58 (m), 2.18-2.36 (m), yl]-N-methyl 2.03-2.13 (m), 1.89-2.01 (m), 1.64-1.88 (m), 1.35-1.62 -L-Valinamide (m), 1.31-1.35 (m), .31 (m), 1.03-1.14 (m), 0.70- 1.01 (m). 2012/056224 [(3R41;E‘sii?::¥11fi:§?1§(_28)~2 LC-MS(ProtocolQ1): m/z 798.2 [M+H+] (0.66 [(1R,21,2)methoxymethyl minutes). 1H NMR (400 MHz, CD30D) 5 8.43-8.49 oxoj3-({(2S)oxophcnyl (m), 7.50—7.53 (m), 7.42-7.48 (m), 7.06-7.20 (m), 4.21— 4.83 (m), 3.95—4.13 (m), 3.76-3.88 (m), 3.53-3.67 (m), [(1H_1 2 3-triazol ylmethyl);11;in0]pmpan_2_ .47 (m), 3.08-3.15 (m) 3.003 16 (m), .90 (m), 2.70—2.73 (m), .69 (m), 2.45-2.58 (m), 2.34— y1}amin0)pr0pyl]pm0hdinyl}- 2.41 (m),2.21-2.29(m),2.12-2.21 (m), 1.55—2.09 (m), 1_oxoheptan_4_y1]¥N_methyl_L_5-meth 1_ .54 (m), 1.16-1.36 (m), 1.04-1.14(m),0.85-0.99 vahnamide (m), 0.73-0.80 (m), .02 (m).
LC-Ms (Protocol Q1): m/z 755.1 [M+H+] (0.69 [(312,4151isclu31fi11fiigy112S)2. minutes). 1H NMR (400 MHz, CDgOD) 5 8.36-8.67 (m), 7.26-7.50 (m), 7.10-7.26 (m), 5.13—5.17(m),4.95— [(1R92R)_1_methoxy_2_methy1 4.99 (m), 4.67-4.84 (m), 4.61-4.66 (m), 4.50-4.60 (m), OXO_3_ {[(2S)oxophenyl .12(m), 3.69-3.75 (m), 3.56-3.66 (m), 3.44—3.54 (pr0p_2_yn_1_ylamm0)pmpan_2_ (m), 3.19-3.44(m) 3.12—3.19(m), 3.03—3.12(m),2.74— ylhmindmowlbmohdiny1}- 2.94 (m), 2.37-2.60 (m), 2.14-2.36 (m), 1.60-2.13 (m), -mcthyloxohcptany 1.47—1.59 (m), 1.19—1.40 (m), 1.11-1.16 (m), 0.88-1.11 l]-N-methyl-L-Valinamide (m), 0.75-0.84 (m), 0.02-0.06 (m).
LC-MS (Protocol Q1): m/z 722.95 [M-l-PP] (0.52 minutes) 1H NMR (400 MHZ, CD30D) 5 8.78-8.86 (m), N,2-dimcthylalanyl-N- 8.71-8.73 (m), 7.96-8.00 (m), 7.34—7.40 (m), 4.74—4.91 [(31%4S,SS) {(2S)[(1R,2R) (m), 4.67-4.71 (m), 4.55-4.63 (m), 4.13—4.22 (m), 4.04— {[(ZS)(1H-imidaz01_4_y1)_1_ 4.10 (m), 3.97—4.01 (m), 3.84-3.92 (m), 3.66-3.82 (m), methoxy_1_oxopmpan_2_ 3.42-3.64 (m), 3.26-3.42 (m) 3.11—3.21 (m), 2.90—2.92 yl]amino}mcthoxymcthyl (m), .84 (m), 2.59-2.64 (m), 2.48-2.56 (m), 2.32— oxopropyl]pymhdin_1_y1}_3_ 2.41 (m), 2.09—2.24 (m), 1.99-2.08 (m), 1.68-1.95 (m), methoxy-S-mcthyloxoheptan 1.59-1.66(m),1.51-1.58(m), 1.35—1.45 (m). 1.22-1.26 (m), 1.17—1.21 (m). 0.95—1.12(m),0.83—0.89(m). _N_methy1_L_Vahnamide LC-MS (Protocol Q1): m/z 748.2 [M+H+] (0.52 minutes) 1H NMR (400 MHZ, CD3OD), 5 8.91-8.99 (m), 8.42-8.46 (m), 8.15-8.20 (m), .01 (m),7.00-7.10 N,2-dimethylalanyl-N- (m), 6.64-6.74 (m), .26 (m), 5.06-5.09 (m), 4.79- [(31%4S,SS) {(ZS)[(1R,2R) 4.95 (m), 4.65-4.79 (m), 4.59-4.65 (m),4.12-4.21 (m), {[(ZS)(4-hydroxyphcnyl) 4.05—4.12(m), 3.91—3.99 (m), 3.84-3.90 (m), 3.67-3.79 methoxy_1_oxopr0pan_2_ (m), 3.60-3.66 (m), 3.39—3.57 (m), 3.34—3.39 (m) 3.29— yl]amino}mcthoxymcthyl 3.34 (m), 3.12—3.27 (m), 2.98-3.00 (m), 2.78-2.88 (m), oxopropyl]pmohdm_1_yl}_3_ 2.61-2.65 (m), 2.55—2.57 (m), 2.46-2.53 (m), 2.10-2.36 methoxy_5_methyl_1_0xoheptan_4_ VI} (in33 1611916911“3211611613 (1133151132 (in33 1‘3? N'methyl'L'Valmamlde~~ .m,.-. m,.-. m,.-. m, 0.98-1.08 (m), 0.84-0.92 (m).
LC-MS (Protocol Q1): m/z 718.4 [M+H+] (0.66 minutes) 1H NMR (400 MHz, CD3OD), 5 .92 N 2-dimethylalanyl-N- (m), 7.71-7.76 46-7.53 (m), 7.40-7.46 (m), 7.19- 7.33 (m), 4.81-4.96 (m), .77 (m), 4.60-4.65 (m), [(3R 4S,SS){(ZS)[(1R 2R)_3_’ {,[(1R)carboxy, 4.47—4.53 (m),4.01-4.17(m), 3.94-3.98 (m), .86 (m), 3.68-3.76 (m), 3.56-3.64 (m), 3.40—3.50 (m), 3.36- phenylethyl]amin0}_1_methoxy_2_ 3.40 (m) 3.26-3.35 (m), 3.23-3.26 (m), 3.16-3.22 (m), methYL3_Oxopmpyl]pmohdm_1_ 3.12-3.16 (m), 2.94-3.06 (m), 2.91—2.93 (m), 2.86-2.88 yl}methoxymethyl (m),2.41-2.66(m),2.32-2.41 (m), 1.97—2.23 (m), 1.85- tanyl]-N-methyl-L- 1.97 (m), 1.71-1.85 (m), 1.62-1.68 (m), 1.50-1.61 (m), Vahnamide .46 (m), .14 (m), 0.85-0.92 (m).
LC-MS (Protocol Q): m/z 835.0 [M+Na+] (0.87 minutes) 1H NMR 5 9.58-9.69 (m), 8.84-9.16 (m), 8.69- 1,2-dimcthyl-L-prolyl-N— 8.77 (m), 8.54-8.60 (m), .50 (m), 8.32-8.42 (m), S,5 S)-3 -methoxy{(2S) .30 (m), .31 (m), 7.00-7.01 (m), 4.97-5.06 [(1R,2R)methoxymcthyl-3 - oxo-3 -{[(2S)oxo-3 -phcnyl (m), 4.88-4.97 (m), 4.57-4.75 (m), 4.45-4.57 (m), 3.84- 4.45 (m), 3.62-3.84 (m), 3.40-3.62 (m), 3.13-3.33 (m), (piperazinyl)propan 2.77-3.10 (m), 2.67-2.75 (m), 2.47-2.57 (m), 2.38-2.45 yl]amino } propyl]pyrrolidinyl} - (m), .35 (m), 1.58-1.88 (m), 1.37-1.55 (m), 1.22- -mcthyl oxohcptanyl] 1.32 (m), 0.97-1.06 (m), 0.84-0.97 (m), 0.73-0.81 (m).
-N-methyl-L-Valinamidc LC-MS (Protocol Q): m/z 366.2[M+H+ ] (0.91 minutes) 1,2-dimethyl-L-prolyl-N- 1H NMR 5 9.56-9.65 (m), 8.70-8.76 (m), 8.05-8.09 (m), [(3R,4S,SS){(ZS)[(1R,2R) 7.77-7.92 (m), 7.14—7.30 (m), 4.60-4.72 (m), 4.46-4.57 {[(2S)aminophenylpropan (m), 3.61-4.39 (m), 3.41-3.61 (m), .33 (m), 2.97- yl]amino} hoxymethyl 3.09 (m), 2.79-2.94 (m), .74 (m), 2.3 8-2.56 (m), oxopropyl]pyrrolidinyl} 2.13-2.37 (m), 1 .93-2. 13 (m), 1.45- 1.89 (m), 1 .2 1- 1.32 methoxy-S-methyloxohcptan (m), 1.09-1.14 (m), 1.03-1.08 (m), 084-095 (In), 0.73- yl] -N-mcthyl-L-Valin 0.80 (m). amide 2-methyl-D-prolyl-N—[(3R,4S,5S) {(2S)[(1R,2R) {[2- (cyclohepta-2,4,6-trien yl)cthyl]amino}methoxy HPLC (Protocol DB): m/z 700.5 1 [M-l-PP] (2.56 methyl- 3- oxopropyl]pyrrolidin minutes) yl} -3 -mcthoxy-5 -methyl oxohcptanyl] -N-mcthyl-L-Valin amide

Claims (63)

What is claimed is:
1. A nd of formula I: or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each ence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A isC1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; or (ii) R3A and R3B taken er are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 carbocycly and C6-C14 aryl optionally tuted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 C(O)R’, -C1-C8 alkyl- C(O)OR’ -O-(C1-C8 alkyl), -C(O)R', R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R12 O R6 N O R12 R6 R12 O N or R5 is , R13 , or R13 optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’, -O-(C1-C8 , -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, 2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene-R’, wherein each R' is independently selected from thegroup consisting of hydrogen, C1-C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 l or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O.
2. A compound of formula IIa: or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; Y O Z Z R1 is , or ; Y is -C2-C20 alkylene-, 0 heteroalkylene-; -C3-C8 carbocyclo-, -arylene-, -C3- rocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene-, 0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)- or - (C3-C8 heterocyclo)-Cl-Cl0alkylene-; R10 N O Z is , , , O NH2 or -NH2; G is halogen, -OH, -SH or –S-C 1-C6 alkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl or halogen; or (ii) R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 carbocycly and C6-C14 aryl optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’ -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - ', -C(O)N(R')2, )R', -S(O)2R', -S(O)R', -OH, halogen, -N3, 2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, er with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R12 O R6 N O R12 R6 R12 O N or R5 is , R13 , or R13 optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene-R’, wherein each R' is independently selected from thegroup consisting of hydrogen, C1-C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is en, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 l or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 cyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and R7 is independently selected for each occurrence from the group consisting of F, Cl, I, Br, NO2, CN and CF3; R10 is hydrogen, -Cl-Cl0alkyl, -C3-C8carbocyclyl, -aryl, -Cl-C10heteroalkyl, -C3- C8heterocyclo, -Cl-Cl0alkylene-aryl, -arylene-Cl-Cl0alkyl, -Cl-Cl0alkylene-(C3-C8carbocyclo), -(C3-C8 carbocyclo)-Cl-Cl0alkyl, -Cl-Cl0alkylene-(C3-C8heterocyclo), and -(C3-C8 heterocyclo)-Cl-Cl0alkyl, where aryl on R10 comprising aryl is optionally tuted with [R7]h; h is 1, 2, 3, 4 or 5; and X is O.
3. A compound of formula IIIa: IIIa or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, l or n; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 ne or C1-C8 heteroalkylene; R5 is R11 O , O R11 , , O R11 , N N R11 R11 R11 O O N O O N R11 O , H , , , , H R11 or NH-R11 optionally substituted with 1, 2, 3, 4 or 5 groups independently ed from the group consisting of C1-C8 alkyl, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently edfrom the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; R11 is , or Y is 0 alkylene-, -C2-C20 heteroalkylene-, C3-C8 carbocyclo-, -arylene-, - C3-C8heterocyclo-, 0alkylene-arylene-, -arylene-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)-, or -(C3-C8 heterocyclo)-Cl-Cl0alkylene-; N O Z is , ,, , O , NH2 or -NH2; G is halogen, -OH, -SH, or –S-C1-C6alkyl R7 is independently selected for each occurrence from the group consisting of F, Cl, I, Br, NO2, CN and CF3; h is 1, 2, 3, 4 or 5; and X is O.
4. A compound of formula IIb: or a ceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; Y O Z Z R1 is , or ; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3- C8heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- ocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)-, or -(C3-C8 heterocyclo)-Cl-Cl0alkylene-; Z is , , , L N L N O O O , NH2 or -NHL; L is an antibody; R2 is hydrogen, C1-C8 alkyl or C1-C8 kyl; R3A and R3B are either of the following: (i) R3A isC1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R3B taken er are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the ing: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is en, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 carbocycly and C6-C14 aryl optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’ -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, n, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of en, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O R12 O R6 N O R12 R6 R12 O N or R5 is , R13 , or R13 optionally substituted with 1, 2, 3, 4 or 5 groups independently ed from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and e-R’, n each R' is independently selected from thegroup consisting of hydrogen, C1-C8 alkyl, C1-C8heterocyclyl, alkylene-C3-C8heterocyclyl and aryl, or two R’ can, er with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O.
5. A compound of formula IIIb: IIIb or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is en, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl or aralkyl; or (ii) R4A and R4B taken er are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is R11 O , O R11 , , O R11 , N N R11 R11 R11 O O N O O N R11 O , H , , , , H R11 or NH-R11 optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of C1-C8 alkyl, -O-(C1-C8 alkyl), -C(O)R', R', - C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, H2, -S(=O)2R' and -SR', wherein each R' is independently selectedfrom the group consisting of en, C1-C8 alkyl and unsubstituted aryl; R11 is , or Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3- C8heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)-, or -(C3-C8 heterocyclo)-Cl-Cl0alkylene-; Z is , , , L N L N O O O , NH2 or -NHL; L is an antibody; X is O.
6. A compound of formula IIc: R3B' R3A' H O H L Z' R1' N N N N D N R5 O O O O X 1-20 or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each ence, O O O Y N N H H R1’ is or O NH2 ; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3- C8heterocyclo-, -Cl-C10alkylene-arylene-, ne-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)-, or -(C3-C8 heterocyclo)-Cl-Cl0alkylene-; H O N N Z’ is O , O , , N O O O , NH2 , or –NH-; L is an antibody; D is –C(R4A’)(R4B’)- or is absent; R2’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if is present; R3A’ and R3B’ are either of the following: (i) R3A’ isC1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B’ is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 cyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R3B’ is C2-C4 alkylene and forms 5-7 member ring as ted by ; or (ii) R3A’ and R3B’ taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A’ and R4B’ are either of the following: (i) R4A’ is hydrogen, C1-C8 alkyl, C1-C8 kyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A’ and R4B’ taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 carbocycly and C6-C14 aryl optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’ -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - ', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O R12 R6 N O R12 R6 R12 O N or R5 is , R13 , or R13 optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 C(O)R’, -C1-C8 alkyl- C(O)OR’, -C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, )R', -S(O)2R', -S(O)R', -OH, n, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene-R’, wherein each R' is independently selected from thegroup consisting of en, C1-C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O.
7. A compound of formula IIIc: IIIc or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each ence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A isC1-C8 alkyl, C1-C8 kyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, l or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group ting of C1-C8 alkyl, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selectedfrom the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; O O O Y N N H H R11’ is or O NH2 ; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3- C8heterocyclo-, 0alkylene-arylene-, ne-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)-, or -(C3-C8 heterocyclo)-Cl-Cl0alkylene-; H O N N Z’ is O , O , , N O O O , NH2 , or –NH-; L is an dy; X is O.
8. A compound of formula IId: R3B' R3A' H O H L [linker] N N N N D N R5 O O O O X 1-20 or a ceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, L is an antibody; [linker] is a divalent linker; D is –C(R4A’)(R4B’)- or is absent; R2’ is en, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if is present; R3A’ and R3B’ are either of the following: (i) R3A’ is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B’ is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R3B’ is C2-C4 alkylene and forms 5-7 member ring as indicated by ; or (ii) R3A’ and R3B’ taken together are C2-C8 ne or C1-C8 heteroalkylene; R4A’ and R4B’ are either of the following: (i) R4A’ is en, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A’ and R4B’ taken together are C2-C8 ne or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 ycly and C6-C14 aryl optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’ -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O R12 R6 N O R12 R6 R12 O N or R5 is , R13 , or R13 optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’, -O-(C1-C8 alkyl), ', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene-R’, wherein each R' is independently selected from thegroup consisting of hydrogen, C1-C8 alkyl, eterocyclyl, alkylene-C3-C8heterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 l, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O.
9. A compound of formula IIId: IIId or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A isC1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or n; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or l; or (ii) R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken er are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O-(C1-C8 , -C(O)R', -OC(O)R', - C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is ndently selectedfrom the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; [linker] is a divalent linker; L is an dy; X is O.
10. The compound, salt or solvate of any one of claims 1-9, wherein W is .
11. The compound, salt or solvate of any one of claims 1-9, wherein W is
12. The compound, salt or solvate of any one of claims 1-9, n R2 is hydrogen, or C1-C8 alkyl.
13. The compound, salt or solvate of any one of claims 1-9, n R3A isC1-C8 alkyl.
14. The compound, salt or solvate of claim 13, wherein R3A is methyl.
15. The compound, salt or solvate of any one of claims 1-5, 7 or 9, wherein R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene.
16. The compound, salt or solvate of any one of claims 1, 2, 4, 6 or 8, wherein R5 is , , , or .
17. The compound, salt or solvate of any one of claims 5-9 wherein antibody is ed from trastuzumab, oregovomab, edrecolomab, cetuximab, a zed monoclonal antibody to the vitronectin receptor (αvβ3); alemtuzumab; a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma;131I Lym-1; a murine anti-HLA-Dr10 dy for the treatment of non-Hodgkin's lymphoma; a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin's lymphoma; labetuzumab; bevacizumab; ibritumomab tiuxetan; ofatumumab; mumab; mab; tositumomab; ipilimumab; gemtuzumab; an anti-IL13 antibody; and an anti-Notch antibody.
18. A compound, or a pharmaceutically acceptable salt or solvate thereof, selected from: O S N N NH O O O O O N N O S O H O S H H N N NH N N N N N N NH H2N N H2N N O O O O O , H2N O O O O O O O O O O , S N , O H N N H N H2N N O H H N N O O O N N H2N N N O O O H2N N O O O NH O O O O O , , , H N N N O H2N H N O O N O N O O H2N O H NH O O H N O O O O N N NH H2N N O O O O OH O O O , O , , O H N N H N H2N N O H N N H N HN N O O O H2N N N O O N O O O S N O O O O O O S N , O N , O H H N O H H N N N H N H2N N N N NH N O O S N O O O H2N O H O O O O O N N O O O O O , , , O H H N N N N N H N NH O H2N O O H N O O O N N O O O H2N N OH NH O O NH O O O O O OH O O , , O O H H N N N N O H N H2N N H N NH H2N N N O O O O O O N O O O O O O O O O O O NH NH O O O , , , H O N N H O HN N H N N N N N N N N O O O H O O O O O O H O O O NH O OH O NH O O O NH , , , N N O HN N H O N N H O O O HN N N N O O HN N H O O O N O O O O O O N O H NH O NH2 NH O , HN , , O O H H N N O O H N N HN N H N HN N N N HN N N O O O O H O O O O H O O O O O O N OH N NH O O , , , O H H N O H O H N N H N H N N N N N N H2N O O H2N N H2N N O O O O O , O O O O O O O O O , O O O O O , O H H N N O H N N N H N N N N N O O O O O O O O O H2N N O O O H H O O O O O N N OH O OH , , , S H O O H O O H H N N N HN N N H N N N HN N N O N O O O O O O O O O O O O O O , , N O H O , NH NH H O H O N NH N N O N H N N N N O O O O N O O O O O O O O N , N O O H O O H O , N O H O , F H O O HN HN F H N N O N N H H N N N N N N N O O O O O O O O H O O O O O O N , N O H O O H O , , O O H H H H N N N N N N F O H H N N N N HN N N H H N O O O O O O O O O O , O O O O O , , F O H H N N HN N N O H H O H N H N N S N N N N N S O N N N N O O O O H H O O O O O O O O O O , , , N H F O H H N N HN N N N S F O H H O O N HN N N N S O N N O O O O HN N O O O O O N , O , O O O H O , O NH2 H O O N O O O NH2 HN N N N N HN N N O HN N O N N O O O O O O O O N O O H O , N O N H O , O H O , F O H H H O N N HN N N N F H H N N N HN N N N N O O O O O O O O O O OH , O O O O O O O OH , N O H O , N N O HN N H O N N H O O O H2N N N N O O H2N N O O O NH O O O O O OH O N OH O H O HN , O , , O H O H O H H N H N H N N N N N N N H2N N H2N N H2N N O O O O O O O O O O O , O O O O O , O O O O O , N N HN N O O O O H O O O H H N N N NH H N O N N H2N N O O O H2N N O O O O O O O , O O O O O , , H O N N N N H O N O H O N O O N N O N H N N O O O O H2N N OH O O N O O O O O OH O H O , N O H O , , O O O H H H H N N N N N N N+ N N H2N N H O O O B- NH O O N S O O O F O O H F N N , , O H H O H N N N N N N H2N N H2N N O O O O O O O O O O H O O O N Cl O N O H , O O H H H N O O N N O O N O H O O N N H H O N N N N N H2N O O HN N O O O N O O O O O O O S N O H O N F, , H , O O N O O H N H O H N N H H N N N O O H N N N N H O N HN O N O O O N HN N N O H O O O O O O O O NH O O , , , O O O O O H H O H H N N N N H N N HN N N O N N N HN H HN N O N O O O O O O O O O O O O O O O , NH N , OH, O H H H H N N N N N N H2N N H O O O O O O O NH
19. A compound, or a pharmaceutically able salt or solvate thereof, selected from: 344344 345345
20. A payload-linker or an antibody-drug-conjugate comprising a radical of a compound of claim 18 or 19.
21. An antibody drug conjugate comprising a radical of a compound of any one of claims 1-3.
22. A nd, or a pharmaceutically acceptable salt or solvate thereof, selected from: O O H H O N N N O H O O N N N N H O O O O O N N H H S N O O O NH2 , O O O H N N N N N H O O O O O H O N O OH O O O H N O O O N N N O O O N O H O O O O H N OH O O O H N N N O N O O H O O O H O O N NH O N N O H O OH O NH2 , O H N N N O N O H O O O O H O O O N NH N N O H O H O O NH2 , O O O H H N N N O O O H N N N N H O O O O O N N O H H O NH2 , O H N N N O N O H O O O O H O O N N N N O NH H OH H O O NH2 , O O O N O O N N N N O H N O O O N N N O O O O O O NH O NH O O O O OH OH , , O O N N O N N O O O H O O O N O N N H NH H O O O NH OH O NH2 , N N S O O O S O O H O H NH N N NH N N N N N N N O H O O O O O H O O O O O O , , O O H H N N N N N N O H O O O O O S N O O S O H O O O N N N NH N N H N N N O O O O O O H O H O NH2 , N N H2N O O O HN N O O O O O O O N O H S , O O O H N N O N N N H O O O O O N O N O H S , O O O N N O O O NH O O O HN N H O O O N N O O O N N O O N O H H N O O H S H2N O , O H N N O O O HN N O O O N O H O O O H O N N N O N N N O H H O S O H H2N O , O O H H N N O N N O O N N H O O O O O O S N , N N H2N O O O HN N O O O O O O N O O O H S , O N N O O O HN N O O O N O H O O O H N N N O O N N N O H H O O H S H2N O , O O O N N O O O NH O O O HN N H O O O N N O O O N N O O N O H H O N O H S H2N O O O O O O O N N N O O O N N O O O O O N O H S , O O O N N O O O N N H H N O N O O O O O N N O N O H H O N O H S H2N O , H O O N H O O O N O O H N O O N N O O O O S N O , O O O N N O O O N N H H N N O O O NH N N O O H H O O NH2 , O O O O O O H N N N O O O N N N O O O O O O O H N N O O N N O H O NH O , , , , O O O H O O O H N N O O O HN N N O O O O O O O O , O O O O O O N N N O O O N N H OH O O O O O O , , , , , , N N N O N H O O O O O N O N N H O N N N N N N N H O O O O O O N H O , H O O H H N N O N N O N N S O O O O O O O O , O O H H N N N O O N N S H H H N N O O O O O O N N H H N O O O O O NH H2N O , O O H H N N N O O N N S H H H N N O O O O O O N N H H N O O O O O NH H2N O , H O H O H N N O N N O N N OH N O O O O O O O O , H O H O H N O N N N O N N OH O O O N O O O O O and
23. An antibody-drug-conjugate comprising a radical of a compound of claim 22.
24. A compound of Claim 1, or a pharmaceutically acceptable salt or solvate thereof, which is
25. A compound of Claim 1, or a ceutically acceptable salt or solvate thereof, which is
26. A compound of Claim 1, or a pharmaceutically acceptable salt or solvate thereof, which is
27. A compound of Claim 1, or a pharmaceutically acceptable salt or e thereof, which is
28. A compound of Claim 1, or a pharmaceutically acceptable salt or solvate thereof, which is
29. A compound of Claim 1, or a pharmaceutically acceptable salt or solvate thereof, which is
30. A compound of Claim 1, or a pharmaceutically acceptable salt or solvate thereof, which is
31. A nd of Claim 1, or a pharmaceutically acceptable salt or solvate thereof, which is
32. A pharmaceutical composition sing a compound of any one of claims 1-31, or a pharmaceutically acceptable salt or solvate thereof, and pharmaceutically acceptable excipient.
33. Use of a therapeutically effective amount of a compound of any one of claims 1-31 or a pharmaceutical composition of claim 32 in the manufacture of a ment for treating cancer.
34. Use of a therapeutically effective amount of a compound of formula I: or a pharmaceutically acceptable salt or e thereof, wherein, independently for each occurrence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; (ii) R3A and R3B taken er are C2-C8 ne or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 carbocycly or C6-C14 aryl, optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’ -C8 alkyl), -C(O)R', -OC(O)R', - ', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consist ing of hydrogen, C1-C8 alkyl and tituted aryl, or two R’ can, together with the nitrogen to which they are attached, form O O R6 N O R6 R12 a C1-C10 heterocyclyl; or R5 is , R13 , or R13 optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl-C(O)OR’, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', R', -S(O)R', -OH, n, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene-R’, wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R’ can, er with the nitrogen to which they are ed, form a C1-C10 heterocyclyl; R6 is hydrogen, - C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O; in the manufacture of a medicament for treating cancer.
35. The use of claim 34, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
36. Use of a therapeutically effective amount of a compound of formula IIb: or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; R1 is , or ; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3- C8heterocyclo-, 0alkylene-arylene-, ne-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)-, or -(C3-C8 heterocyclo)-Cl-Cl0alkylene-; Z is , , , L N L N O O O , NH2 or -NHL; L is an dy; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; (ii) R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 kyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 carbocycly or C6-C14 aryl, optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’ -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', ', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consist ing of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O R12 R6 N O R12 R6 R12 O N or R5 is , R13 , or R13 ally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’, -C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and e-R’, wherein each R' is ndently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 cyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O; in the cture of a ment for treating cancer, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
37. Use of a therapeutically effective amount of a compound of formula IIIb: IIIb or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each ence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, [Annotation] fip C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl, halogen or aralkyl; (ii) R3A and R3B taken er are C2-C8 alkylene or C1-C8 alkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, aralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is R11 O , O R11 , , O R11 , N N R11 R11 R11 O O N O O N R11 O , H , , , , H R11 or NH-R11 optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; R11 is , or Y is 0 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3- C8heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3-C8heterocyclo)-, or 8 heterocyclo)-Cl-Cl0alkylene-; Z is , , , L N L N O O O , NH2 or -NHL; L is an antibody; and X is O; in the manufacture of a medicament for treating cancer.
38. The use of claim 37, wherein said cancer is selected from the group consisting of carcinomas of the r, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, ry gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
39. Use of a therapeutically effective amount of a compound of formula IIc: R3B' R3A' H O H L Z' R1' N N N N D N R5 O O O O X 1-20 or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, O O O Y N N H H R1’ is or O NH2 ; Y is 0 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3- C8heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene-, 0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, -Cl-Cl0alkylene-(C3- C8heterocyclo)-, or -(C3-C8 heterocyclo)-Cl-Cl0alkylene-; H O N N Z’ is O , O , , N O O O , NH2 , or –NH-; L is an antibody; D is –C(R4A’)(R4B’)- or is absent; R2’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if is present; R3A’ and R3B’ are either of the following: (i) R3A’ is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B’ is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 cyclyl, aryl, heteroaralkyl, halogen or l, or R3B’ is C2-C4 alkylene and forms 5-7 member ring as indicated by ; or (ii) R3A’ and R3B’ taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A’ and R4B’ are either of the following: (i) R4A’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A’ and R4B’ taken er are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 carbocycly or C6-C14 aryl, optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’ -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', (R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, 2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consist ing of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R12 O R6 N O R12 R6 R12 O N or R5 is , R13 , or R13 optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’, -O-(C1-C8 alkyl), -C(O)R', R', - ', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene-R’, wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8heterocyclyl, alkylene-C3-C8heterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O; in the manufacture of a medicament for treating cancer.
40. The use of claim 39, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, , colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, ry gland, thyroid gland, skin, stomach, and testes; and blood born s selected from the group consisting of leukemias and lymphomas.
41. Use of a eutically effective amount of a compound of formula IIIc: IIIc or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 yclyl, C1-C10 heterocyclyl, aryl, aralkyl, halogen or aralkyl; (ii) R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or l; and R4B is en, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is O , O , , O , N N O O N O O N O , H , , , , NH- . optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; O O O Y N N H H R11’ is or O NH2 ; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3- C8heterocyclo-, -Cl-C10alkylene-arylene-, ne-Cl-Cl0alkylene-, -Cl-Cl0alkylene-(C3- C8carbocyclo)-, -(C3-C8carbocyclo)-Cl-C10alkylene-, 0alkylene-(C3-C8heterocyclo)-, or 8 heterocyclo)-Cl-Cl0alkylene-; H O N N Z’ is O , O , , N O O O , NH2 , or –NH-; L is an antibody; and X is O; in the manufacture of a medicament for treating cancer.
42. The use of claim 41, wherein said cancer is selected from the group consisting of carcinomas of the bladder, , cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
43. Use of a therapeutically effective amount of a compound of formula IId: R3B' R3A' H O H L [linker] N N N N D N R5 O O O O X 1-20 or a pharmaceutically acceptable salt or e thereof, wherein, independently for each ence, L is an antibody; [linker] is a divalent linker; D is –C(R4A’)(R4B’)- or is absent; R2’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if is present; R3A’ and R3B’ are either of the following: (i) R3A’ is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B’ is C1-C8 alkyl, C1-C8 kyl, C3-C8 carbocyclyl, C1-C10 cyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R3B’ is C2-C4 alkylene and forms 5-7 member ring as indicated by ; or (ii) R3A’ and R3B’ taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R4A’ and R4B’ are either of the following: (i) R4A’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B’ is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or l; or (ii) R4A’ and R4B’ taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is , , , , , , , , , , , , , , C1-C10 heterocyclyl, C3-C8 carbocycly or C6-C14 aryl optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’ -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, n, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, 2R' and -SR', wherein each R' is independently selected from the group consist ing of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 cyclyl; R12 O R6 N O R12 R6 R12 O N or R5 is , R13 , or R13 optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N(R’)2, -C1-C8 alkyl-C(O)R’, -C1-C8 alkyl- C(O)OR’, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', -SR' and arylene-R’, wherein each R' is independently selected from the group consisting of en, C1-C8 alkyl, C1-C8heterocyclyl, alkylene-C3-C8heterocyclyl and aryl, or two R’ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O; in the manufacture of a ment for the treatment of cancer.
44. The use of claim 43, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, trium, kidney, lung, esophagus, ovary, prostate, pancreas, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group ting of leukemias and lymphomas.
45. Use of a therapeutically effective amount of a compound of formula IIId: IIId or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, W is , O , or O ; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are either of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 cyclyl, aryl, heteroaralkyl, halogen or aralkyl; (ii) R3A and R3B taken er are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are either of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is O , O , , O , N N O O N O O N O , H , , , , NH- . optionally substituted with 1, 2, 3, 4 or 5 groups ndently selected from the group consisting of C1-C8 alkyl, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', - C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, -CN, -NHC(=NH)NH2, -NHCONH2, -S(=O)2R' and -SR', wherein each R' is independently selected from the group ting of en, C1-C8 alkyl and unsubstituted aryl; [linker] is a divalent linker; L is an antibody; and X is O; in the manufacture of a medicament for the ent of cancer.
46. The use of claim 45, wherein said cancer is ed from the group consisting of carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, ry gland, thyroid gland, skin, stomach, and ; and blood born cancers selected from the group consisting of leukemias and lymphomas.
47. Use of a therapeutically effective amount of the nd: or a ceutically acceptable salt or solvate thereof in the manufacture of a ment for the treatment of cancer.
48. The use of claim 47, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, as, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
49. Use of a eutically effective amount of the compound: or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating cancer.
50. The use of claim 49, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, salivary gland, thyroid gland, skin, stomach, and ; and blood born cancers ed from the group consisting of ias and lymphomas.
51. Use of a therapeutically effective amount of the compound: or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating cancer.
52. The use of claim 51, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, trium, kidney, lung, esophagus, ovary, prostate, as, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
53. Use of a therapeutically effective amount of the compound: or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating cancer.
54. The use of claim 53, wherein said cancer is selected from the group ting of carcinomas of the bladder, breast, , colon, endometrium, kidney, lung, gus, ovary, prostate, as, salivary gland, d gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
55. Use of a therapeutically ive amount of the compound: or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating cancer.
56. The use of claim 55, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
57. Use of a therapeutically effective amount of the compound: or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating cancer.
58. The use of claim 57, wherein said cancer is selected from the group consisting of carcinomas of the bladder, breast, cervix, colon, trium, kidney, lung, esophagus, ovary, prostate, pancreas, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
59. Use of a eutically effective amount of the compound: or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating cancer.
60. The use of claim 59, wherein said cancer is selected from the group consisting of carcinomas of the r, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, te, pancreas, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of ias and lymphomas.
61. Use of a therapeutically effective amount of the compound: or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a ment for treating cancer.
62. The use of claim 61, wherein said cancer is selected from the group consisting of omas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, salivary gland, thyroid gland, skin, stomach, and testes; and blood born cancers selected from the group consisting of leukemias and lymphomas.
63. The compound of any one of claims 1-9, 18, 19 or 22, substantially as herein bed with reference to any one of the Examples and/or
NZ624470A 2011-11-17 2012-11-07 Cytotoxic peptides and antibody drug conjugates thereof NZ624470B2 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201161561255P 2011-11-17 2011-11-17
US61/561,255 2011-11-17
US201261676423P 2012-07-27 2012-07-27
US61/676,423 2012-07-27
PCT/IB2012/056224 WO2013072813A2 (en) 2011-11-17 2012-11-07 Cytotoxic peptides and antibody drug conjugates thereof

Publications (2)

Publication Number Publication Date
NZ624470A NZ624470A (en) 2015-10-30
NZ624470B2 true NZ624470B2 (en) 2016-02-02

Family

ID=

Similar Documents

Publication Publication Date Title
US9249186B2 (en) Cytotoxic peptides and antibody drug conjugates thereof
AU2021290341A1 (en) Antibody drug conjugates (ADCs) and antibody prodrug conjugates (APDCs) with enzymatically cleavable groups
JP6560681B2 (en) Sulfonamide-containing linkage systems for drug conjugates
CA2934617A1 (en) Antibody drug conjugates (adcs) with kinesin spindle protein (ksp)
CA2990408A1 (en) Antibody drug conjugates of kinesin spindel protein (ksp) inhibitors with anti-b7h3-antibodies
CA2990398A1 (en) Antibody drug conjugates of kinesin spindel protein (ksp) inhibitors with anti-cd123-antibodies
WO2016096610A1 (en) Antibody-drug conjugates (adcs) of ksp inhibitors with aglycosylated anti-tweakr antibodies
CA2990394A1 (en) Antibody drug conjugates of kinesin spindel protein (ksp) inhibitors with anti-tweakr-antibodies
EP4110402A1 (en) Camptothecin derivatives and conjugates thereof
JP2019507126A (en) Tubulicin analogues and methods for their preparation
JP2018123122A (en) Calicheamicin derivatives and antibody-drug conjugate thereof
AU2015264844B2 (en) Cytotoxic peptides and antibody drug conjugates thereof
NZ624470B2 (en) Cytotoxic peptides and antibody drug conjugates thereof
BR112018008490B1 (en) CRYPTOPHYCIN COMPOUNDS AND CONJUGATES, PROCESSES FOR THE PREPARATION THEREOF, MEDICINE AND PHARMACEUTICAL COMPOSITION