OA20532A - Integrin ligands and uses thereof. - Google Patents

Integrin ligands and uses thereof. Download PDF

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Publication number
OA20532A
OA20532A OA1202000151 OA20532A OA 20532 A OA20532 A OA 20532A OA 1202000151 OA1202000151 OA 1202000151 OA 20532 A OA20532 A OA 20532A
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Prior art keywords
mmol
equiv
cargo
ανβό integrin
integrin ligand
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OA1202000151
Inventor
Zhen Li
Xiaokai Ll
Erik W. BUSH
Rui ZHU
Dongxu Shu
Jonathan Benson
Patrick Shao
Matthew FOWLER-WATTERS
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Arrowhead Pharmaceuticals, Inc.
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Publication of OA20532A publication Critical patent/OA20532A/en

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Abstract

Synthetic αvβ6 integrin ligands of Formula I having serum stability and affinity for integrin αvβ6, which is a receptor expressed in a variety of cell types, are described. The described ligands are usful for delivering cargo molecules, such as RNAi agents or other oligonucleotide-based compounds, to cells that express integrin αvβ6, and thereby facilitating the uptake of the cargo molecules into these cells. Compositions that include αvβ6 integrin ligands and methods of use are also described.

Description

INTEGRIN LIGANDS AND USES THEREOF
Cross Reference To Related Applications
[0001] This application daims priority to United States Provisional Patent Application Serial No. 62/580,398, filed on November 1, 2017, United States Provisional Patent Application Serial No. 62/646,739, filed on March 22, 2018, and United States Provisional Patent Application Serial No. 62/679,549, filed on June 1,2018, the contents of each of which are incorporated herein by reference in their entirety.
Background
[0002] Integrin alpha-v beta-6 (ανβό), which is expressed in varions cell types including épithélial cells, isa receptor for the latency-associated peptide (LAP) of TGF-β and for the extracellular matrix (ECM) proteins fibronectin, vitronectin, and tenascin. Although barely détectable in normal healthy adult epithelîa, ανβό integrin is upregulated during wound healing and in different cancers (e.g., colon, ovarîan, endométrial, and gastric cancer), and often associâtes with poor cancer prognosis. It has been shown that ανβό integrin can promote cell invasion and migration in metastasis, and inhibit apoptosis. ανβό integrin may also regulate expression of matrix métalloprotéases (MMPs) and activate TGF-βΙ. There is increasing evidence, prîmarîly from in vitro studies, that suggestthat ανβό integrin may promote carcinoma progression. Thus, integrin ανβό is attractive as a tumor biomarker and potential therapeutic target in view of, among other things, its rôle in expression of matrix métalloprotéases (MMPs) and activation of TGF-βΙ.
[0003] The in vivo delivery of therapeutically effective compounds, such as drug compounds, to the desired cells and/or tissues, continues to be a general challenge for the development of drug products. There continues to exist a need for stable and effective targeting ligands that are able to selectively target cells or tissues, which can be employed to facilitate the targeted delivery of cargo molécules (e.g., a therapeutically active compound or ingrédient) to spécifie cells or tissues.
Indeed, there is a general need for targeting ligands that can be conjugated to one or more cargo molécules of choice, such as one or more drug products or other payloads, to facilitate the delivery of the cargo molécules to desired cells or tissues in vivo. Moreover, there exists a need for compounds that target integrin alpha-v beta-6, which are suitable to be conjugated to cargo molécules, to deliver the cargo molécules to cells expressing integrin alpha-v beta-6, in vivo. With respect to spécifie cargo molécules, such as therapeutic oligonucleotide-based compounds (e.g., an antisense oligonucleotides or an RNAi agents), there exists a need for targeting ligands that are able to target integrin alpha-v beta-6 that can be conjugated to oligonucleotide-based compounds to deliver the therapeutic to cells and/or tissues expressing integrin alpha-v beta-6, and facilitate the entry of the therapeutic into the cell through receptor-mediated endocytosîs, pinocytosis, or by other means.
SüMMARY
[0004] Described herein are novel, synthetic ανβό integrin ligands (also referred to herein as ανβό ligands), The ανβό integrin ligands disclosed herein are stable in sérum and hâve affinîty for, and can bind with specificity to, ανβό integrins. The ανβό integrin ligands can be conjugated to cargo molécules to facilitate the delivery of the cargo molécule to desired cells or tissues that express ανβό integrin. such as to épithélial cells.
[0005] Also disclosed herein are methods of delivery of a cargo molécule to a tissue and/or cell expressing ανβό integrin in vivo, wherein the methods încluding administering to a subject one or more ανβό integrin ligands disclosed herein that hâve been conjugated to one or more cargo molécules. Further disclosed are methods of treatment of a subject having a disease, symptom, or disorder for which the delivery of a therapeutic cargo molécule (e.g., an active pharmaceutical ingrédient) to a cell expressing ανβό integrin is capable of treating the subject, wherein the methods include administering to a subject one or more ανβό integrin ligands disclosed herein that hâve been conjugated to one or more therapeutic cargo molécules.
[0006] In some embodiments, described herein are methods of inhibîting expression of a target gene in a cell, wherein the methods include administering to the cell an effective amount of one or more ανβό integrin ligands that hâve been conjugated to one or more oligonucleotide-based compounds (e.g., an oligonucleotide-based therapeutic) capable of inhibîting expression of a target gene in a cell, such as an RNAi agent. In some embodiments, described herein are methods of inhibîting expression of a target gene in a cell of a subject, wherein the subject is administered an effective amount of one or more ανβό integrin ligands that hâve been conjugated to one or more oligonucleotide-based compounds capable of inhibîting expression of a target gene in a cell, such as an RNAi agent.
[0007] Further described herein are compositions that include ανβό integrin ligands, The compositions described herein can be pharmaceutical compositions that include one or more ανβό integrin ligands disclosed herein conjugated to one or more therapeutic substances, such as an RNAi agent or other cargo molécule.
[0008] In some embodiments, described herein are methods of treatment of a subject having a disease or disorder mediated at least in part by expression of a target gene, wherein the methods including administering to a subject in need thereof an effective amount of a pharmaceutical composition, — wherein the pharmaceutical composition includes one or more ανβό integrin ligands disclosed herein conjugated to one or more oligonucleotide-based compounds, such as an RNAi agent.
[0009] In a first aspect, this dîsclosure provides synthetic ανβ6 întegrîn ligands.
[0010] In some embodiments, an ανβό integrin ligand disclosed herein includes the structure of the
S or a pharmaceutically acceptable sait thereof, wherein, n is an înteger from 0 to 7;
J isC-H or N;
Z is OR13, N(R13)2 or SR13;
R' is H, optionally substîtuted Cj-Cé alkyl, OH, COOH, CON(R5)2, OR6, or R1 comprises a cargo molécule, wherein each R5 is independently H or Ci-Cô alkyl, and R6is H or Ci-Ce alkyl;
R2, RP1 and Rp2 are each independently H, halo, optionally substîtuted cycloalkylene, optionally substîtuted arylene, optionally substîtuted heterocycloalkylene, or optionally substîtuted heteroarylene, or R2, Rpl and RP2 may comprise a cargo molécule;
Rw is H or optionally substîtuted alkyl;
R is H or optionally substîtuted alkyl, or R11 and R' together with the atoms to which they are attached form an optionally substîtuted heterocycle;
R12 is H or optionally substîtuted alkyl;
each R13 is independently H, optionally substîtuted alkyl, or R13 comprises a cargo molecuie;
R14 is optionally substîtuted alkyl; and wherein at least one of R1, R2, R13, RPI and Rp2 comprises a cargo molecuie.
[0011] In some embodiments, an ανβ6 integrin ligand disclosed herein can be conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 25 to 10, 10 to 30, 10 to 25, 10to20, 10 to 15, I5to30, 15to25, I5to20, 20 to 30, 20 to 25, or 25 to 30) cargo molécules (e.g., any of the cargo molécules described herein or known in the art).
[0012] In some embodiments, more than one ανβ6 integrin ligand disclosed herein (e.g., 2, 3, 4, 5,
6, 7, 8, or 1 to 8, I to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 ανβό integrin ligands) can be 30 conjugated to one cargo molecuie (e.g., any of the cargo molécules described herein or known in the art).
[0013] In another aspect, this dîsclosure provides compositions that include one or more ofthe ανβό integrin ligands described herein. For example, in some embodiments, compositions comprising one or more ανβό integrin ligands disclosed herein include one or more oligonucleotide-based compound(s), such as one or more RNAi agent(s), to be delivered to a cell in vivo. In some embodiments, described herein are compositions for delivering an RNAi agent to a cell in vivo, wherein the RNAi agent is linked to one or more ανβό integrin ligands.
[0014] Compositions that include one or more ανβό integrin ligands are described. In some embodiments, a composition comprises a pharmaceutically acceptable excipient. In some embodiments, a composition that încludes one or more ανβό integrin ligands comprises one or more other pharmaceutical substances or pharmaceutically active ingrédients or compounds. In some embodiments, médicaments that include one or more ανβό integrin ligands are described herein.
[0015] Compositions that include one or more ανβό integrin ligands disclosed herein conjugated to one or more cargo molécules can facilitate the delivery of the cargo molecuele in vivo or in vitro to cells that express integrin ανβό. For example, compositions that include one or more ανβό integrin ligands disclosed herein can deliver cargo molécules, such as oligonucleotide-based compounds, in vivo or in vitro, to type I and II alveolar épithélial cells, goblet cells, secretory épithélial cells, cilîated épithélial cells, corneal and conjunctival épithélial cells, dermal épithélial cells, cholangiocytes, enterocytes, ductal épithélial cells, glandular épithélial cells, and épithélial tumors (carcinomas).
[0016] In another aspect, the présent dîsclosure provides methods comprising the use of one or more ανβό integrin ligands and/or compositions as described herein and, if desired, bringing the disclosed ανβό integrin ligands and/or compositions into a form suitable for administration as a pharmaceutical product. In other embodiments, the dîsclosure provides methods for the manufacture of the ligands and compositions, e.g., médicaments, described herein.
[0017] Compositions that include one or more ανβό integrin ligands can be administered to subjects in vivo using routes of administration known in the art to be suitable for such administration in view of the cargo molécule sought to be administered, including, for example, inhaled (aérosol or dry powder formulations), intranasal, subcutaneous, intravenous, intraperitoneal, intradennal, transdermal, oral, sublingual, topical, or intratumoral administration. In some embodiments, the compositions that include one or more ανβό integrin ligands may be administered for systemic delivery, for example, by intravenous or subcutaneous administration. In some embodiments, the compositions that include one or more ανβό integrin ligands may be administered for localized delivery, for example, by inhaled delivery via dry powder inhaler or nebulîzer. In some embodiments, the compositions that include one or more ανβό integrin ligands may be administered for localized delivery by topical administration.
[0018} In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a type I alveolar épithélial ce 11 in vivo, wherein the methods include admimstering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0019[ In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a type Π alveolar épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0020] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a goblet cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0021] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a secretory épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0022] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a ciliated épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0023] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a corneal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0024] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a conjunctival épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0025] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a dermal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0026) In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a cholangiocyte in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0027] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to an enterocyte in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0028] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a ductal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
{0029] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a glandular épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécule.
[0030] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to an épithélial tumor (carcinoma) in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more cargo molécules.
[0031] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to a type I alveolar épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a type I alveolar épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of atarget gene in a type I alveolar épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affînity for ανβό integrin.
[0032] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to a type II alveolar épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a type I] alveolar épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of înhibitîng the expression of a target gene in a type II alveolar épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affînity for ανβό integrin.
[0033] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to a goblet cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a goblet cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in a goblet cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affînity for ανβό integrin.
[00341 In some embodiments, disclosed herein are methods of delivering an oligonucieotide-based compound to a secretory épithélial cell in vivo, wherein the methods include admmistenng to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucieotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a secretory épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAÎ agents. In some embodiments, disclosed herein are methods of înhîbiting the expression of a target gene in a secretory épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affïnity for ανβό integrin.
[0035] In some embodiments, disclosed herein are methods of delivering an oligonucieotide-based compound to a cîliated épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucieotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a ciliated épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in a ciliated épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affïnity for ανβό integrin.
[0036] In some embodiments, disclosed herein are methods of delivering an oligonucieotide-based compound to a corneal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucieotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a corneal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in a corneal épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affïnity for ανβό integrin.
[0037] In some embodiments, disclosed herein are methods of delivering an oligonucieotide-based compound to a conjunctival épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucieotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a conjunctival épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in a conjunctival épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having afhnity for ανβό integrin.
[0038] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to a dermal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a dermal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in a dermal épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affinity for ανβό integrin.
[0039] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to a cholangiocyte in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a cholangiocyte in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in a cholangiocyte in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affinity for ανβό integrin.
(0040] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to an enterocyte in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to an enterocyte in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in an enterocyte in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affinity for ανβό integrin.
[0041] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to a ductal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods ofdelivering an RNAi agent to aductal épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in a ductal épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands havîng affinity for ανβό integrin.
[0042] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to a glandular épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to a glandular épithélial cell in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in a glandular épithélial cell in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affinity for ανβό integrin.
[0043] In some embodiments, disclosed herein are methods of delivering an oligonucleotide-based compound to an épithélial tumor (carcinoma) in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods of delivering an RNAi agent to an épithélial tumor (carcinoma) in vivo, wherein the methods include administering to the subject one or more ανβό integrin ligands conjugated to the one or more RNAi agents. In some embodiments, disclosed herein are methods of inhibiting the expression of a target gene in an épithélial tumor (carcinoma) in vivo, wherein the methods include administering to the subject an RNAi agent conjugated to one or more ligands having affinity for ανβό integrin.
|0044] Unless otherwise defmed, ail technical and scientific terms used herein hâve the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or équivalent to those described herein can be used in the practice or testing of the présent invention, suitable methods and materials are described below. AH publications, patent applications, patents, and other référencés mentioned herein are incorporated by reference in their entirety. In case of conflict, the présent spécification, including définitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0045] Other objects, features, aspects, and advantages of the invention will be apparent from the following detailed description and from the daims.
Detailed Description ανβό Integrin Ligands.
[0046] Described herein are synthetic ανβό integrin ligands having sérum stability and affïnity for integrin ανβό. The ανβό integrin ligands can be used to target cells that express integrin ανβό in vitro, in situ, ex vivo, and/or in vivo. In some embodiments, the ανβό integrin ligands disclosed herein can be conjugated to one or more cargo molécules to preferentially direct and target the cargo molécules to cells that express integrin ανβό in vitro, in situ, ex vivo, and/or in vivo. In some embodiments, the cargo molécules include or consist of pharmaceutically active compounds. In some embodiments, the cargo molécules include or consist of oligonucleotide-based compounds, such as RNAi agents. In some embodiments, the ανβό integrin ligands disclosed herein are conjugated to cargo molécules to direct the cargo molécules to épithélial cells in vivo.
[0047] In a First aspect, this disclosure provides synthetic ανβό integrin ligands.
[0048] In some embodiments, an ανβό integrin ligand disclosed herein includes the structure of the following formula:
or a pharmaceutically acceptable sait thereof, wherein, n is an integer from 0 to 7;
J isC-H or N;
Z is OR13, N(R,3)2 or SR13;
R1 is H, optionally substituted Ci-C& alkyl, OH, COOH, CON(R5)?, OR6, or R1 comprises a cargo molécule, wherein each R5 is independently H or Ci-Cô alkyl, and R6 is H or Ci-Cô alkyl;
R2, RPI and RP2 are each independently H, halo, optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene, or R2, RP1 and RP2 may comprise a cargo molécule;
R10 is H or optionally substituted alkyl;
R11 is H or optionally substituted alkyl, or R11 and R1 together with the atoms to which they are attached form an optionally substituted heterocycle;
R12 is H or optionally substituted alkyl;
each R13 is independently H, optionally substituted alkyl, or R13 comprises a cargo molécule;
R14 is optionally substituted alkyl; and wherein at least one of R1, R. ,R13,RP1 and RP2 comprises a cargo moiecule.
[0049] In some embodiments, n=3 in Formula I. In some embodiments, n=4 in Formula I.
[0050] In some embodiments of Formula I, R2 is napthylene. In some embodiments of Formula I, R2 is substituted napthylene and R2 also comprises a cargo moiecule.
[0051] In some embodiments an ανβό întegrîn ligand disclosed herein includes the structure of the following formula:
or a pharmaceutîcally acceptable sait thereof, wherein, n is an integer from 0 to 7 (Le., n is 0, 1,2, 3, 4, 5, 6, or 7);
J is C-H or N;
R1 îs H, Ci-Cè alkyl, CH(R3)(R4), OH, COOH, CH2CH2CH2NH2, CONHR5, OR6, wherein R3 is H or Ci-Cè alkyl, R4 is H, Ci-Ce alkyl, R5 is H or Ci-Cè alkyl, and R6 îs H or Ci-Ct, alkyl;
R2 is optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene,
R10 is H or optionally substituted alkyl;
R11 is H or optionally substituted alkyl, or R11 and R1 together with the atoms to which they are attached form an optionally substituted heterocycle;
R12 is H or optionally substituted alkyl;
R13 is H or optionally substituted alkyl;
R14 is optionally substituted alkyl;
wherein at least one of R1 or R2 includes a cargo moiecule.
[0052] In some embodiments, n=3 in Formula IL In some embodiments, n=4 in Formula 11.
[0053] In some embodiments, an ανβό integrin ligand disclosed herein includes the structure of the following formula:
(Formula III),
or a pharmaceutically acceptable sait thereof, wherein, n is an înteger from 1 to 7 (Le., n is 1,2,3, 4, 5, 6, or 7);
R7 includes one or more cargo molécules; and
Rs is one or more optionally substituted divalent cyclic moieties having 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, such as cycioalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl), cycloalkenyl (e.g., cyclopentenyi, cyclobutenyl, cyclopentenyl, cyciohexenyl, or cycloheptenyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, pyrîmidinyl, pyrîdazinyl, pyrrole, pyrazole, imidazole, thiophene, benzothiophene, thiazoie, benzothiazole, furan, oxazole, isoxazole, 10 benzofuran, îndole, indazole, benzimidazole, oxadiazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, quinolinyl, isoquinolinyl, or quinoxalinyl), or heterocyclyl (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidîne, dioxane, or dioxolane).
[00541 In some embodiments, n=3 in Formula III. In some embodiments, n=4 in Formula III.
[0055] In some embodiments, an ανβό integrin ligand disclosed herein includes the structure of the 15 following formula:
or a pharmaceutically acceptable sait thereof, wherein, n is an integer from I to 7 (i.e., n is 1,2, 3, 4, 5, 6, or 7); and
R9 includes one or more cargo molécules.
]0056] In some embodiments, n=3 in Formula IV. In some embodiments, n=4 in Formula IV.
[0057] In another aspect, the invention provides integrin targeting ligand precursor of the structure:
or a pharmaceutically acceptable sait thereof, wherein, n is an integer from 0 to 7;
J is C-H or N;
Z is OR13, N(RI3)2 or SR’3;
R1 îs H, optionally substituted Ci-Cô alkyl, OH, COOH, CON(R5)2, OR6, or R1 comprises a linking group conjugated to a reactive group, wherein each R3 is independently H or Ci-Cè alkyl, and R6 is H or Ci-Câ alkyl;
R2, Rpl and Rp2 are each independently H, halo, optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene, or R2, RP1 and RP2 may comprise a linking group conjugated to a reactive group;
R10 is H or optionally substituted alkyl;
R11 is H or optionally substituted alkyl, or R11 and R1 together with the atoms to which they are attached form an optionally substituted heterocycle;
R12 is H or optionally substituted alkyl;
each R13 is independently H, optionally substituted alkyl, or R13 comprises a linking group conjugated to a reactive group;
R14 is optionally substituted alkyl; and wherein at least one of R1, R2, R13, RPI and RP2 comprises a linking group conjugated to a reactive group.
[0058] In some embodiments, an ανβό integrin ligand disclosed herein can be conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 30, 10to25, I0to20, 10 to 15, 15 to 30, 15 to 25, 15 to 20, 20 to 30, 20 to 25, or 25 to 30) cargo molécules (e.g., any of the cargo molécules described herein or known in the art).
[0059] In some embodiments, more than one ανβό integrin ligand disclosed herein (e.g., 2, 3, 4, 5, 6, 7, 8, or 1 to 8, l to 7, 1 to 6, I to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 ανβό integrin ligands) can be conjugated to one cargo molécule (e.g., any of the cargo molécules described herein or known in the art).
[0060] In some embodiments, the ανβό integrin ligands disclosed herein are optionally conjugated to one or more cargo molécules via a linking group, such as, for example, a polyethylene glycol (PEG) group.
[0061] In some embodiments, the ανβό integrin ligands disclosed herein are optionally conjugated to one or more cargo molécules via a scaffold that includes at least one attachaient point for each ligand and at least one attachment point for each cargo molécule. In some embodiments, the ανβό integrin ligands comprise, consist of, or consist essentîally of, one cargo molécule. In some embodiments, the ανβό integrin ligands comprise, consist of, or consist essentîally of, more than one cargo molécule.
[0062] In some embodiments, the ανβό integrin ligand comprises, consists of, or consists essentîally of, any of Structure 1, Structure 2, Structure 5, Structure 5.1, Structure 5.2, Structure 6, Structure 6.1, Structure 6.2, Structure 6.3, Structure 6.4, Structure 7, Structure 8, Structure 9, Structure 10, Structure 1 1, Structure 12, Structure 13, Structure 14, Structure 15, Structure 16, Structure 17, Structure 18, Structure 19, Structure 20, Structure 22, Structure 23, Structure 24, Structure 25, Structure 27, Structure 29, Structure 30, Structure 31, Structure 32, Structure 33, Structure 34, Structure 35, Structure 36, or Structure 37, each as disclosed herein.
[0063] Any ofthe ανβ6 integrin ligands disclosed herein can be linked to a cargo molécule, a réactivé group, and/or a protected reactive group. A reactive group can be used to facilitate conjugation ofthe ανβό integrin ligand to a cargo molécule. The ανβό integrin ligands disclosed herein can increase targeting of a cargo molécule to an ανβό integrin or to a cell expressing an ανβό integrin. A cargo molécule can be, but is not limîted to, a pharmaceutically active ingrédient or compound, a prodrug, or another substance with known therapeutic or diagnostic benefit. In some embodiments, a cargo molécule can be, but is not lîmited to, a small molécule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified oligonucieotide-based compound (e.g., an antisense oligonucleotide or an RNAi agent), a natural or modified nucleic acid, a peptide, an aptamer, a polymer, a polyamine, a protein, a toxin, a vitamin, a polyethylene glycol, a hapten, a digoxigenin, a biotin, a radioactive atom or molécule, or a fluorophore. In some embodiments, a cargo molécule includes a pharmaceutically active ingrédient or a prodrug. In some embodiments, a cargo molécule includes an oligonucleotide-based compound as a pharmaceutically active ingrédient. In some embodiments, a cargo molécule includes an RNAi agent as a pharmaceutically active ingrédient.
[0064] As used herein, the terni “alkyl” refers to a saturaied aliphatic hydrocarbon group, straight chain or branched, having from 1 to 10 carbon atoms unless otherwise specîfied. For example, “CiCô alky 1” includes alkyl groups having 1,2, 3, 4, 5, or 6 carbons in a linear or branched arrangement. Non-limiting examples of alkyl groups include methyl, ethyl, κρ-propyl, to'Abutyl, tt-hexyl. As used herein, the term “aminoalkyl” refers to an alkyl group as defined above, substituted at any position with one or more amino groups as permitted by normal valency. The amino groups may be unsubstituted, monosubstituted, or di-substituted. Non-limiting examples of aminoalkyl groups include aminomethyl, dimethylaminomethyl, and 2-aminoprop-l-yl.
[00651 As used herein, the term “cycloalkyl” means a saturated or unsaturated nonaromatic hydrocarbon ring group having from 3 to 14 carbon atoms, unless otherwise specified. Non-limiting examples of cycloalkyl groups include, but are not Iimited to, cyclopropyl, methyl-cyclopropyl, 2,2dimethyl-cyclobutyl, 2-ethyl-cyclopentyi, and cyclohexyl. Cycloalkyls may include multiple spiroor fused rings. Cycloalkyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted on any position as permitted by normal valency.
[00661 As used herein, the term “cycloalkylene” refers to a divalent radical of a cycloalkyl group as described herein. Cycloalkylene is a subset of cycloalkyl, referringto the same resîdues as cycloalkyl, but having two points of substitution. Examples of cycloalkylene include cyclopropylene, ,
1,4-cyclohexylene, Γ , and 1,5-cyclooxylene /. Cycloalkylene groups are optionally mono-, di-, tri-, tetra-, or penta-substituted on any position as permitted by normal valency. Cycloalkylene groups may mono-, di-, or tri-cyclic.
[0067| As used herein, the term “alkenyl” refers to a non-aromatic hydrocarbon radical, straight, or branched, containing at least one carbon-carbon double bond, and having from 2 to 10 carbon atoms unless otherwise specified. Up to five carbon-carbon double bonds may be présent in such groups. For example, “C2-C6” alkenyl is defmed as an alkenyl radical having from 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight, branched, or cyclic portion of the alkenyl group may contain double bonds and is optionally mono-, di-, tri-, tetra-, or penta-substituted on any position as permitted by normal valency. The term “cycloalkenyl” means a monocyclîc hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.
[0068| As used herein, the term “alkynyl” refers to a hydrocarbon radical, straight or branched, containing from 2 to 10 carbon atoms, unless otherwise specified, and containing at least one carboncarbon triple bond. Up to 5 carbon-carbon triple bonds may be présent. Thus, “C2-C6 alkynyl” means an alkynyl radical having from 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight or branched portion ofthe alkynyl group may be optionally mono-, di-, tri-, tetra-, or penta-substituted on any position as permitted by normal valency.
[0069] As used herein, “alkoxy!” or alkoxy” refers to -O-alkyl radical having the indicated number of carbon atoms. For example, Cj-s alkoxy is intended to include Ci, C2, C3, C4, C5, and CT alkoxy groups. For example, Ci-s alkoxy, is intended to include Ci, C2, C3, C4, C5, Cô, C7, and Cg alkoxy groups. Examples ofalkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i—propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n^octoxy.
[0070] As used herein, “keto” refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyi, heteroaryl, or aryl group as defîned herein attachcd through a carbonyl bridge. Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, or hexanoyl), alkenoyl (e.g., acryloyl) alkynoyl (e.g., ethynoyl, propynoyl, butynoyl, pentynoyl, or hexynoyl), aryloyl (e.g., benzoyl), heteroaryloyl (e.g., pyrroloyl, imîdazoloyl, quinolînoyl, or pyridinoyl).
[0071] As used herein, “alkoxycarbonyl” refers to any alkoxy group as defîned above attachcd through a carbonyl bridge (i.e., -C(O)O-alkyi). Examples ofalkoxy carbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, iso-propoxy carbonyl, n-propoxycarbonyl, tbutoxy carbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl,
[0072] As used herein, “aryloxycarbonyl” refers to any aryl group as defîned herein attached through an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.
|0073] As used herein, “heteroaryloxycarbonyl” refers to any heteroaryl group as defîned herein attached through an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.
[0074] As used herein, “aryl” or “aromatic” means any stable monocyclic or polycyclic carbon ring of up to 6 atoms in each ring, wherein at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyL In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring. Aryl groups are optionally mono-, di-, tri-, tetra-, or pentasubstituted on any position as permitted by normal valency.
[0075| As used herein, the term “arylene” refers to a divalent radical of an aryl group as described herein. Arylene is a subset of aryl, referring to the same residues as aryl, but having two points of substitution. Examples of arylene include phenylene, which refers to a divalent phenyl group. Arylene groups are optionally mono-, di-, tri-, tetra-, orpenta-substituted on any position as permitted by normal valency.
[0076] As used herein, the term “halo” refers to a halogen radical. For instance, “halo” may refer to a fluorine (F), chlorine (Cl), bromine (Br), or an iodine (I) radical.
[0077] As used herein, the term “heteroaryl” represents a stable monocyclic or polycyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from I to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not hnmted to, acndmyl, carbazolyl, cinnohnyl, quinoxahnyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. “Heteroaryl” is also understood to include the N-oxide dérivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring. Heteroaryl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted on any position as permitted by normal valency.
[0078] As used herein, the terni “heteroarylene” refers to a divalent radical of a heteroaryl group as described herein. Heteroarylene is a subset of heteroaryl, referring to the same residues as heteroaryl, but having two points of substitution. Examples of heteroaryl include pyrîdînylene, pyrimidinylene, and pyrrolylene. Heteroarylene groups are optionally mono-, di-, tri-, tetra-, or penta-substituted on any position as permitted by normal valency.
[0079] As used herein, the term “heterocycle,” “heterocyclic,” or “heterocyclyl” means a 3- to 14membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N, and S, încluding polycyclic groups. As used herein, the term “heterocyclic” is also considered to be synonymous with the terms “heterocycle” and “heterocyclyl” and is understood as also having the same définitions set forth herein. “Heterocyclyl” includes the above mentîoned heteroaryls, as well as dîhydro and tetrahydro analogs thereof. Exampies of heterocyclyl groups include, but are not limited to, azetidinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolînyl, furanyl, imidazolyl, indolînyl, indolyl, indolazinyl, indazolyl, isobenzofuranyi, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazolîne, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl, pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tctrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidînyl, pyrîdin-2-onyl, pyrrolidinyl, morphoiinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothîophenyl, dihydrobenzoxazolyl, dîhydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dîhydro pyrazinyl, dîhydropyrazolyi, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyi, dihydroquinolinyl, dihydrotetrazoly I, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorphohnyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom. Heterocyclyl groups are optîonally mono-, dî-, tri-, tetra-, or penta-substituted on any position as pcrmitted by normal valency.
[0080] As used herein, the term “heterocycloalkyl” means a 3- to 14-membered nonaromatic hcterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N, and S, including polycyclic groups. Examples of heterocyclyl groups include, but are not limited to, azctidinyl, oxopiperazînyl, oxopyrrolidînyl, oxomorpholinyl, oxetanyl, pyranyl, pyridinonyl, pyrimidinonyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyi, dihydrofuranyl, dihydroimidazolyl, dîhydroisooxazolyl, dihydroisothiazolyl, dîhydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazoiyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyi, dihydrotetrazoly 1, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dioxidothiomorpholinyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocycloalkyl substituent can occur via a carbon atom or via a heteroatom. Heterocyclyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted on any position as pcrmitted by normal valency.
|0081] As used herein, the term “heterocycloalkylene” refers to a divalent radical of a heterocycloalkyl group as described herein. Heteroycloalkylene is a subset of heterocycloalkyl, referring to the same residues as heterocycloalkyl, but havîng two points of substitution. Examples of heterocycloalkylene include piperidinylene, azetidinylene, and tetrahydrofuranylene. Heterocycloalkylene groups are optionally mono-, di-, tri-, tetra-, or penta-substituted on any position as permitted by normal valency.
[0082] As used herein, the tenns “treat,” “treatment,” and the like, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and/or frequency of one or more symptoms of a disease in a subject. As used herein, “treat” and “treatment” may include the prévention, management, prophylactic treatment, and/or inhibition of the number, severity, and/or frequency of one or more symptoms of a disease in a subject.
[0083) As used herein, the phrase “introducing into a cell,” when referring to an RNAi agent, means functionally delîvering the RNAi agent into a cell. The phrase “functîonal delivery,” means that delivering the RNAi agent to the cell in a manner that enables the RNAi agent to hâve the expected biological activity, e.g., sequence-speciftc inhibition of gene expression.
(0084] Unless stated otherwise, use of the symbol as used herein means that any group or groups may be linked thereto that is in accordance with the scope of the inventions described herein. (0085] As uscd herein, the terni “isomers” refers to compounds that hâve identical molecular formulae, but that differ in the nature or the sequence ofbonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stéréoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is termed a “chiral center.” When the compounds described herein contain olefinîc double bonds or other centers of géométrie asymmetry for which the isomeric structure is not specifically defined, it is intended that the compounds can include both E and Z géométrie isomers individually or in a mixture. The compounds of Formula I or their pharmaceutically acceptable salts, for example, are meant to include al! possible isomers, as well as their racmeic and optically pure forms. Likewise, unless expressiy stated otherwise, ail tautomeric forms are also intended to be included.
[0086] As used herein, a linking group is one or more atoms that connects one molécule or portion of a molécule to another to second molécule or second portion of a molécule. In the art, the terms linking group and spacers are sometimes used interchangeably. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with a linking group. In some embodiments, a linking group can include a peptide-cleavable linking group. In some embodiments, a linking group can include or consist of the peptide phenylalanine-citrulline-phenylalanine-proline. In some embodiments, a linking group can include or consist of a PEG group.
[0087] As used herein, the term “linked” when referring to the connection between two molécules means that two molécules are joined by a covalent bond or that two molécules are associated via noncovalent bonds (e.g., hydrogen bonds or ionic bonds). In some examples, where the term “linked” refers to the association between two molécules via noncovalent bonds, the association between the two different molécules has a Kd of less than 1 x ΙΟ'4 M (e.g., less than I x I0'5 M, less than 1x10' 6 M, or less than 1 x ΙΟ'7 M) in physiologically acceptable buffer (e.g., phosphate buffered saline). Unless stated, the term linked as used herein may refer to the connection between a first compound and a second compound either with or without any intervening atoms or groups of atoms.
[0088] The person of ordinary skill in the art would readily understand and appreciate that the compounds and compositions disclosed herein may hâve certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated State, depending upon the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their State of protonation based on the pH ofthe environment, as would be readily understood by the person of ordînary skill in the art.
[0089] Structures may be depicted as having a bond “floating” over a ring structure to indicate binding to any carbon or heteroatom on the ring as permitted by valency. For example, the structure
R indicates that R may replace any hydrogen atom at any of the five available positions on the ring. “Floating’ bonds may also be used in bicyclic structures to indicate a bond to any position on either ring of the bicycle as permitted by valency. In the case of bicycles, the bond will be shown R “floating” over both rings, for example, indicates that R may replace any hydrogen atom at any of the seven available positions on the ring.
[0090] As used in a claim herein, the phrase “consisting of ’ excludes any élément, step, or ingrédient not specified in the claim. hen used in a claim herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic 15 and novel characteristic(s) of the claimed invention.
[0091] Described herein is the use of the described ανβό integrin ligands to target and deliver a cargo molécule to a cell that expresses ανβό integrin. The cargo molécule can be delivered to a cell in vitro, in situ, ex vivo, or in vivo.
[0092] In some embodiments of Formula Ib, the lînking group is a PEG group containing 2-20 20 ethylene glycol units.
[0093] In some embodiments of Formula Ib, the reactive group is an azide.
[0094] In some embodiments, the ανό integrin ligand hâve structures that include, consist of, or consist essentially of any of the structures represented by the following:
(Structure 8);
(Structure il);
(Structure 30);
moiety comprising a cargo molécule.
[0095] In some embodiments, an ανβό integrin ligand disclosed herein can be conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27,
28, 29, or 30; or 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10to30, 10to25, 10 to 20, 10 to 15, 15 to 30, 15to25, 15 to 20, 20 to 30, 20 to 25, or 25 to 10 30) cargo molécules (e.g., any of the cargo molécules described herein or known in the art).
[0096] In some embodiments, more than one ανβό integrin ligand disclosed herein (e.g., 2, 3, 4, 5,
6, 7, 8, or 1 to 8, 1 to 7, 1 to 6, ! to 5, 1 to 4, ! to 3, 1 to 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 ανβό integrin ligands) can be 31 conjugated to one cargo molécule (e.g., any of the cargo molécules described herein or known in the art).
[0097] In some embodiments, the ανβό integrin ligands disclosed herein are optionally conjugated to one or more cargo molécules via a linking group, such as, for example, a polyethylene glycol 5 (PEG) group.
[0098] In some embodiments, the ανβό integrin ligands disclosed herein are optionally conjugated to one or more cargo molécules via a scaffold that includes at least one attachment point for each ligand and at least one attachment point for each cargo molécule. In some embodiments, the ανβό integrin ligands comprise, consist of, or consist essentially of, one cargo molécule. In some 10 embodiments, the ανβό integrin ligands comprise, consist of, or consist essentially of, more than one cargo molécule.
[0099] In some embodiments, the ανβό integrin ligand comprises, consists of, or consists essentially of, any of Structure 1, Structure 2, Structure 5, Structure 5.1, Structure 5.2, Structure 6, Structure 6.1, Structure 6.2, Structure 6.3, Structure 6.4, Structure 7, Structure 8, Structure 9, Structure 10, Structure 15 11, Structure 12, Structure 13, Structure 14, Structure 15, Structure 16, Structure 17, Structure 18,
Structure 19, Structure 20, Structure 22, Structure 23, Structure 24, Structure 25, Structure 27, Structure 29, Structure 30, Structure 31, Structure 32, Structure 33, Structure 34, Structure 35, Structure 36, or Structure 37, each as disclosed herein.
[0100] Any ofthe ανβό integrin ligands disclosed herein can be linked to a cargo molécule, a reactive 20 group, and/or a protected reactive group. A reactive group can be used to facilitate conjugation of the ανβό integrin ligand to a cargo molécule. The ανβό integrin ligands disclosed herein can increase targeting of a cargo molécule to an ανβό integrin or to a cell expressing an ανβό integrin. A cargo molécule can be, but is not limited to, a pharmaceutically active ingrédient or compound, a prodrug, or another substance with known therapeutic benefït. In some embodiments, a cargo molécule can 25 be, but is not limited to, a small molécule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified oligonucleotide-based compound (e.g., an antisense oligonucleotide or an RNAi agent), a natural or modified nucleic acid, a peptide, an aptamer, a polymer, a polyamîne, a protein, a toxin, a vitamin, a polyethylene glycol, a hapten, a digoxigenin, a biotin, a radioactive atom or molécule, or a fluorophore. In some 30 embodiments, a cargo molécule includes a pharmaceutically active ingrédient or a prodrug. In some embodiments, a cargo molécule includes an oligonucleotide-based compound as a pharmaceutically active ingrédient. In some embodiments, a cargo molécule includes an RNAi agent as a pharmaceutically active ingrédient.
[0101] In one aspect, the invention provides for a structure comprising an ανβό integrin ligand as described herein, a linking group, and a scaffold, wherein the scaffoid is bound to a cargo molécule.
In some embodiments, the structure may comprise the ligand in monodentate form. In some embodiments, the structure may comprise the ligand in bidentate form. In some embodiments, the structure may comprise the ligand in tridentate form. In some embodiments, the structure may comprise the ligand in tetradentate form.
[0102] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0103] In some embodiments, the ανβό integrin ligand of Structure 1 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0104] In some embodiments, an ανβό integrin ligand can be synthesizedto include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0105] In some embodiments, an ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
Ο
N H
[0106] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0107] In some embodiments, the ανβό integrin ligand of Structure 2 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0108] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0109] In some embodiments, the ανβό integrin ligand can be synthesîzed to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
(Structure 2b).
(0110] In some embodiments, the ανβό integrin ligand can be synthesîzed to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[OUI] In some embodiments, the ανβό integrin ligand of Structure 5 is lînked to one or more cargo molécules (e.g., RNAi agent(s)).
[0112] In some embodiments, the ανβό integrin ligand can be synthesîzed to include a reactive group, 10 a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0113] In some embodiments, the ανβό integrin ligand can be synthesîzed to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0114] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 5.1).
[0115] In the embodiments, the length of the PEG in the PEG-azide réactivé group may be varied.
In some embodiments, the ανβό integrin ligands of Structure 5.1 can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and comprises the following structure:
(Structure 5.1b).
[0116] A reactive group (or protected reactive group) can be used to facîlitate the conjugation of the ανβό integrin ligand to a moiecule of interest, e.g., to a cargo moiecule (either directly or via one or more scaffolds and/or linker).
[0117] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 5.2)
[0118] In some embodiments, the ανβό integrin ligand of Structure 5.2 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0119] In some embodiments, a PEG-azide reactive group may be replaced with an alkyl-azide reactive group. In some embodiments, the ανβό integrin ligand can be synthesized to include an alkyl-azide reactive group and comprises the following structure:
[0120] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 6)
[0121] In some embodiments, the ανβό integrin ligand of Structure 6 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0122] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a réactivé group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0123} In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene 5 glycol (PEG)-azide reactive group, and comprises the following structure:
(0124] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0125] In some embodiments, the ανβό integrin ligand of Structure 6.1 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0126] In the embodiments, the length of the PEG in the PEG-azide reactive group may be varied. In some embodiments, the ανβό integrin ligands can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
N3 ° ° (Structure 6.1b)
[0127] A reactive group (or protected reactive group) can be used to facilitate the conjugation of the ανβό integrin ligand to a molecuie of interest, e.g., to a cargo molecuie (either directiy or via one or more scaffolds and/or linker).
[0128] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
-b- (Structure 6.2)
[0129] In some embodiments, the ανβό integrin ligand of Structure 6.2 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0130] in some embodiments, a PEG-azide reactive group may be replaced with an alkyl-azide reactive group. In some embodiments, the ανβό integrin ligand can be synthesized to include an 15 alkyl-azide reactive group, and comprises the following structure:
[0131] A reactive group (or protected reactive group) can be used to facîlitate the conjugation of the ανβό integrin ligand to a molécule of interest. e.g., to a cargo molécule (either directly or via one or more scaffolds and/or linker).
[0132] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0133] In some embodiments, the ανβό integrin ligand of Structure 6.3 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0134] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule. In some embodiments, the ανβό integrin ligands can be synthesized to include an azide reactive group and comprises the following structure:
(Structure 6.3b)
[0135] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0135] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 6.4) |0136] In some embodiments, the ανβό integrin ligand of Structure 6.4 is linked to one or more
S cargo molécules (e.g., RNAi agent(s)).
[0137] In some embodiments, the ανβό integrin ligand can be synthesized to include an azide reactive group and comprises the following structure:
(Structure 6.4b)
[0138] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 7)
[0139] in some embodiments, the ανβό integrin ligand of Structure 7 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0140] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X încludes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0141] In some embodiments, the ανβό integrin ligand can be synthesized to include a PEG-azîde reactive group, and comprises the following structure:
[0142] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0143] In some embodiments, the ανβό integrin ligand of Structure 8 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0144] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
(Structure 8a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0145] In some embodiments, the ανβό integrin ligand can be synthesized to include a PEG-azide reactive group, and comprises the following structure:
[0146] in some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 9)
[0147] In some embodiments, the ανβό integrin ligand of Structure 9 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0148] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
Η Ν
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0149] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
N3
Ο
Ν Η
(Structure 9b)
[0150] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 10)
[0151] In some embodiments, the ανβό integrin ligand of Structure 10 is linked to one or more cargo molécules (e.g., R1XIAi agent(s)).
[0152] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 1 Oa), wherein X includes a reactive group, a protected reactive group, or a cargo moiecule (e.g., an RNAi agent).
[0153] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0154] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0155] In some embodiments, the ανβό integrin ligand of Structure 11 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0156] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo moiecule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0157] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene 5 glycol (PEG)-azide reactive group, and comprises the following structure:
[0158] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0159] In some embodiments, the ανβό integrin ligand of Structure 12 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0160] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 12a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0161] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
(Structure 12b)
[0162] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0163] In some embodiments, the ανβό integrin ligand of Structure 13 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0164] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 13a), wherein X includes a reactive group, a protected réactivé group, or a cargo molécule (e.g., an RNAi agent).
[0165] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
(Structure 13b)
[0166] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure :
(Structure 14)
[0167] In some embodiments, the ανβό integrin ligand of Structure 14 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[01681 In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0169] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0170] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0171] In some embodiments, the ανβό integrin ligand of Structure 15 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0172] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0173] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
(Structure 15b)
[0174] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 16)
[0175] In some embodiments, the ανβό integrin ligand of Structure 16 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0176] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0177] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
N
(Structure 16b)
[0178] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 17)
[0179] In some embodiments, the ανβό integrin ligand of Structure 17 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0180] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected réactivé group, or a cargo molécule, and comprises the following structure:
wherein X includes a réactivé group, a protected réactivé group, or a cargo molécule (e.g., an RNAi agent).
[0181J In some embodiments, the ανβό integrin ligand can be synthesîzed to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0182] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0183] In some embodiments, the ανβό integrin ligand of Structure 18 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0184] In some embodiments, the ανβό integrin ligand can be synthesîzed to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 18a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0185] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
Ns 0 0 (Structure 18b)
[0186] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 19)
[0187] In some embodiments, the ανβό integrin ligand of Structure 19 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0188] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a réactivé group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0189] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0190] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0191] In some embodiments, the ανβό integrin ligand of Structure 20 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0192] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a réactivé group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0193] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0194] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 22)
[0195] In some embodiments, the ανβό integrin ligand of Structure 22 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0196] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 22a), wherein X includes a réactivé group, a protected réactivé group, or a cargo moiecule (e.g., an RNAi agent).
[0197] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
/^-./0//^-- /^-./-0/^-- /\/o
N3 O O (Structure 22b) (0198] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 23)
[0199] In some embodiments, the ανβό integrin ligand of Structure 23 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0200] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo moiecule, and comprises the following structure:
X (Structure 23a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0201] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
(Structure 23b)
[0202] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 24)
[0203] In some embodiments, the ανβό integrin ligand of Structure 24 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0204] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 24a), whereîn X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0205] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0206] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following
[0207] In some embodiments, the ανβ6 integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAÎ agent(s)).
[0208] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecuie, and comprises the following structure:
(Structure 25a), wherein X includes a reactive group, a protected reactive group, or a cargo molecuie (e.g., an RNAi agent).
[0209] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0210] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 27)
[0211] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g,, RNAi agent(s)).
[0212] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, 5 a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 27a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0213] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0214] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0215] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[02161 1η some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0217] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, 5 a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0218] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azidc reactive group, and comprises the following structure:
[0219] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0220] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0221] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, 5 a protected reactive group, or a cargo molécule, and comprises the following structure:
(Structure 30a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0222] In some embodiments, the ανβό integrin ligand can be synthesized to include a poiyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
(Structure 30b)
[0223] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 31)
[0224] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0225] In some embodiments, the ανβό integrin ligand can be synthesîzed to include a reactive group, 5 a protected reactive group, or a cargo molécule, and comprises the following structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0226] In some embodiments, the ανβό integrin ligand can be synthesîzed to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
(Structure 31b)
[0227] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0228] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0229] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, 5 a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 32a), wherein X includes a reactive group, a protected réactivé group, or a cargo molécule (e.g., an RNAi agent).
[0230] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0231] In some embodiments, an ανβό integrin ligand disclosed herein comprises the foliowing structure:
(Structure 33)
[0232] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0233] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group.
a protected reactive group, or a cargo molécule, and comprises the foliowing structure:
wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi 10 agent).
[0234] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the foliowing structure:
N
(Structure 33b).
[0235] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 34)
[0236] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0237] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 34a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0238] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
N3 0 0 (Structure 34 b)
[0239] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
(Structure 35)
[0240] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0241] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, 10 a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 35a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0242] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
(Structure 35b)
[0243] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0244] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0245] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, 10 a protected reactive group, or a cargo molécule, and comprises the following structure:
X (Structure 36a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
|0246] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0247] In some embodiments, an ανβό integrin ligand disclosed herein comprises the following structure:
[0248] In some embodiments, the ανβό integrin ligand of Structure 25 is linked to one or more cargo molécules (e.g., RNAi agent(s)).
[0249] In some embodiments, the ανβό integrin ligand can be synthesized to include a reactive group, 10 a protected reactive group, or a cargo molécule, and comprises the following structure:
Z (Structure 37a), wherein X includes a reactive group, a protected reactive group, or a cargo molécule (e.g., an RNAi agent).
[0250] In some embodiments, the ανβό integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, and comprises the following structure:
[0251] The reactive group as disclosed in any of Structure la, Structure Ib, Structure 2a, Structure 2b, Structure 5a, Structure 5b, Structure 6a, Structure 6b, Structure 7a, Structure 7b, Structure 8a, Structure 8b, Structure 9a, Structure 9b, Structure 10a, Structure 10b, Structure 1 la, Structure 1 1 b, Structure 12a, Structure 12b, Structure I3a, Structure 13b, Structure 14a, Structure 14b, Structure 15a, Structure 15b, Structure 16a, Structure 16b, Structure 17a, Structure 17b, Structure 18a, Structure 18b, Structure 19a, Structure 19b, Structure 20a, Structure 20b, Structure 22a, Structure 22b, Structure 23a, Structure 23b, Structure 24a, Structure 24b, Structure 25a, Structure 25b, Structure 27a, Structure 27b, Structure 29a, Structure 29b, Structure 30a, Structure 30b, Structure 31a, Structure 31b, Structure 32a, Structure 32b, Structure 33a, Structure 33b, Structure 34a, Structure 34b, Structure 35a, Structure 35b, Structure 36a, Structure 36b, Structure 37a, or Structure 37b can be used to attach the ανβό integrin ligand to a molecuie of interest, i.e., to a cargo molecuie such as an RNAi agent. The cargo molecuie can be any molecuie that is desired to be targeted to an ανβό integrin-expressing cell.
Multidentate ανβό Integrin Ligands and Scaffolds
[0252] As disclosed herein, in some embodiments, one or more ανβό integrin ligands may be linked to one or more cargo molécules. In some embodiments, only one ανβό integrin ligand is conjugated to a cargo molecuie (referred to herein as a “monodentate” or “monovalent” ligand). In some embodiments, two ανβό integrin ligands are conjugated to a cargo molecuie (referred to herein as a “bidentate” or “divalent” ligand), in some embodiments, three ανβό integrin ligands are conjugated to a cargo molecuie (referred to herein as a “tridentate” or “trivalent” ligand). In some embodiments, four ανβό integrin ligands are conjugated to a cargo molecuie (referred to herein as a “tetradentate” or “tetravalent” ligand). In some embodiments, more than four ανβό integrin ligands are conjugated to a cargo molecuie.
[0253] In some embodiments, where only one ανβό integrin ligand is conjugated to a cargo molécule (referred to herein as a “monodentate” ligand), the ανβό integrin ligand may be conjugated directly to the cargo molécule. In some embodiments, an ανβό integrin ligand disclosed herein can be conjugated to a cargo molécule via a scaffold or other linker structure.
[0254] In some embodiments, the ανβό integrin ligands disclosed herein include one or more scaffolds. Scaffolds, also sometimes refrred to in the art as linking groups or linkers, can be used to facilitate the linkage of one or more cargo molécules to one or more ανβό integrin ligands disclosed herein. Useful scaffolds compatible with the ligands disclosed herein are generally known in the art. Non-limiting examples of scaffolds that can be used with the ανβό integrin ligands disclosed herein include, but are not limited to polymers and polyamino acids (e.g., bis-glutamic acid, poly-L-lysine, etc.). In some embodiments, scaffolds may include cysteine linkers or groups, DBCO-PEG1-24-NHS, Propargyl-PEGi-24-NHS, and/or multidentate DBCO and/or propargyl moîetîes.
|0255] In some embodiments, the scaffold used for linking one or more ανβό integrin ligands disclosed herein to one or more cargo molécules has the following structure:
[0256] The use of Scaffold 1, for example, facilitâtes efficient conjugation with both the ανβό integrin ligand monomers and the one or more cargo molécules. Scaffold 1 includes an amine reactive /?-nitrophenol (also called 4-nitrophenol) ester, an amide linkage, and three of PEG2 units, as well as terminal alkynes. The 4-nitrophenol ester can be conjugated with the primary amine on a cargo molécule, such as the primary amine on an RNA trigger formulated with a terminal amine group (e.g., NHz-Ce), through amide formation. The terminal alkyne can be conjugated with azido modified ligands (both peptides and small molécules) through copper-catalyzed click chemistry.
[0257] In some embodiments, the cargo molécule is an RNAi agent. In some embodiments, Scaffold 1 may be attached to the terminal end of an RNAi agent, such as to the 5’ terminal end of the sense strand of an RNAi agent. For example, the 5’ terminal end of the sense strand of an RNAi agent may be modîfied to include a Cé amine (-Cô-NFh) attached to the 5’ end of the 5’ terminal nucléotide of the RNAi agent. An RNAi agent having such a Co amine modification (or another other modification resulting in a terminal amine) may be readily conjugated to Scaffold 1, as shown in by the représentation in the following structure:
(Structure 380), wherein indicates an RNAi agent.
[0258] The alkyne groups of Structure 380, above, may then be conjugated to the ανβό integrin ligands disclosed herein to form tridentate ανβό integrin ligands.
[0259] In some embodiments, a scaffold may be synthesized using DBCO (dibenzocyclooctyne), which can be represented by the following structure:
indicates attachment to a reactive group or a moiety comprising cargo molécule.
[0260} In some embodiments, triazole groups are formed between the RNAi agent and the ανβό integrin ligands disclosed herem, as shown in the following general structure:
(Structure 390), wherein indicates any suitable scaffold or linking groups that can be used to link a ligand to an RNAi agent, and indicates an RNAi agent.
[0261] In some embodiments, a scaffold may be synthesîzed as a phosphoramidite compound, which can allow for a tridentate ligand to be readily coupled to the 5’ terminal end of the sense strand of an
RNAi agent through phosphoramidite synthesis, as shown in the following structure:
(Structure 400).
[0262] After synthesizing to attach the compound of Structure 400 to the 5’ terminal end of the sense strand of the RNAi agent, the terminal alkynes can then be linked to the ανβό integrin ligands disclosed herein.
[0263] In some embodiments, an ανβό integrin ligand disclosed herein comprises Structure 1, Structure 2, Structure 5, Structure 5.1, Structure 5.2, Structure 6, Structure 6.1, Structure 6.2,
Structure 6.3, Structure 6.4, Structure 7, Structure 8, Structure 9, Structure 10, Structure 11, Structure
12, Structure 13, Structure 14, Structure 15, Structure 16, Structure 17, Structure 18, Structure 19, Structure 20, Structure 22, Structure 23, Structure 24, Structure 25, Structure 27, Structure 29,
Structure 30, Structure 31, Structure 32, Structure 33, Structure 34, Structure 35, Structure 36,
Structure 37, wherein the ανβό integrin ligand is a tridentate ligand, linked via a scaffold.
[0264] In some embodiments, an ανβό integrin ligand disclosed herein comprises Structure 2 in a tridentate form, and can be represented by the following structure:
(Structure 700)
[0265] In some embodiments, an ανβό integrin ligand disclosed herein comprises Structure 6.1 in a tridentate form, and can be represented by the following structure:
[0266] [n some embodiments, an ανβό integrin ligand disclosed herein comprises Structure 6.1 in a tridentate form, and can be represented by the following structure:
(Structure 701a)
[0267] In some embodiments, an ανβό integrin ligand disclosed herein comprises Structure 6.1 in a tridentate form that includes a glutaric linker, and can be represented by the following structure:
(Structure 701b)
[0268] In some embodiments, an ανβό integrin ligand disclosed herein comprises Structure 6.1 in a tridentate form conjugated to an RNAi agent, and may be represented by the following structure:
HO
(Structure 7Ûlc), wherein
indicates an RNAi agent.
[0269] In some embodiments, an ανβό integrin ligand disclosed herein comprises Structure 6.1 in a tridentate form, and may be represented by the following structure:
[0270] In some embodiments, an ανβό integrin ligand disclosed herein comprises Structure 6.1 in a tridentate form conjugated to an RNAi agent, and may be represented by the following structure:
(Structure 701 e), wherein indicates any suitable scaffoid that can be used to link a ligand and a RNAi agent, and indicates a RNAi agent.
Reactive groups and protected réactivé groups.
[0271] Reactive groups are well known in the art and provide for formation of covalent linkages between two molécules or reactants. Suitable reactive groups for use in the scope of the inventions herein include, but are not limited to: amino groups, amide groups, carboxyiic acid groups, azides, alkynes, propargyl groups, BCN(biclclo[6.l .0]nonyne, DBCO(dibenzocyclooctyne) thiols, maleimîde groups, aminooxy groups, N-hydroxysuccinimide (NHS) or other activated ester (for example, PNP, TFP, PFP), bromo groups, aldéhydes, carbonates, tosylates, tetrazînes, transcyclooctene (TCO), hydrazides, hydroxyl groups, disulfides, and orthopyridyl disulfide groups.
[0272] Incorporation of reactive groups can facilitate conjugation of an ανβό integrin ligand disclosed herein to a cargo molécule. Conjugation reactions are well known in the art and provide for formation of covalent linkages between two molécules or reactants. Suitable conjugation reactions for use in the scope ofthe inventions herein include, but are not limited to, amide coupling reaction, Michael addition reaction, hydrazone formation reaction and click chemistry cycloaddition reaction.
[0273] In some embodiments, the ανβό integrin targeting ligands disclosed herein are synthesized as a tetrafluorophenyl (TFP) ester, which can be displaced by a reactive amino group to attach a cargo molécule. In some embodiments, the integrin targeting ligands disclosed herein are synthesized as an azide, which can be conjugated to a propargyl or DBCO group, for example, via click chemistry cycloaddition reaction, to attach a cargo molécule.
[0274] Protected reactive groups are also commonly used in the art. A protecting group provides temporary Chemical transformation of a reactive group into a group that does not react under conditions where the non-protected group reacts, e.g, to provide chemo-selectivity in a subséquent Chemical reaction. Suitable protected reactive groups for use in the scope of the inventions herein include, but are not limited to, BOC groups (t-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), carboxybenzyl (CBZ) groups, benzyl esters, and PBF (2,2,4,6,7-pentamethyldihydrobenzofuran-5sulfonyl).
Cargo Molécules (including RNAi agents)
[0275] A cargo molécule is any molécule which, when detached from the ανβό integrin ligands described herein, would hâve a désirable effect on a cell comprising an ανβό integrin receptor. A cargo molécule can be, but is not limited to, a pharmaceutical ingrédient, a drug product, a prodrug, a substance with a known therapeutic benefit, a small molécule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid or polynucleotide, a peptide, a polymer, a polyamine, a protein, an aptamer, a toxin, a vitamin, a PEG, a hapten, a digoxigenin, a biotin, a radioactive atom or molécule, or a fluorophore. In some embodiments, one or more cargo molécules (e.g., the same or different cargo molécules) are linked to one or more ανβό integrin ligands to target the cargo molécules to a cell expressing an ανβό integrin.
[0276] In some embodiments, the one or more cargo molécules is a pharmaceutical ingrédient or pharmaceutical composition. In some embodiments, the one or more cargo molécules is an oligonucleotide-based compound. As used herein, an “oligonucleotide-based compound” is a nucléotide sequence containing about 10-50 (e.g., 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10to26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 50, 12 to 48, 12 to 46, 12to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, I2to34, 12to32, 12to30, 12to28, 12to26, 12to24, 12to22, 12to20, 12 to 18, 12 to 16, 12 to 14, 14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16to38, 16 to 36, 16 to 34, 16 to 32, 16to 30, 16to28, 16 to 26, 16 to 24, 16to22, 16 to 20, 16 to 18, 18 to 50, 18 to 48, 18 to 46, 18 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 38, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 50, 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, to 28 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32, 32 to 50, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40, 40 to 50, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50, 44 to 48, 44 to 46, 46 to 50, 46 to 48, or 48 to 50) nucléotides or nucléotide base pairs. In some embodiments, an oligonucleotidebased compound has a nucleobase sequence that is at least partially complementary to a coding sequence in an expressed target nucleîc acid or target gene within a cell. In some embodiments, the oligonucleotide-based compounds, upon delivery to a cell expressing a gene, are able to inhibit the expression of the underlying gene, and are referred to herein as “expression-inhibiting oligonucleotide-based compounds.” The gene expression can be inhibited in vitro or in vivo.
[0277] “Oligonucleotide-based compounds” include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), ribozymes, interfering RNA molécules, and dicer substrates. In some embodiments, an oligonucleotide-based 5 compound is a single-stranded oligonucleotide, such as an antisense oligonucleotide. In some embodiments, an oligonucleotide-based compound is a double-stranded oligonucleotide. In some embodiments, an oligonucleotide-based compound is a double-stranded oligonucleotide that is an RNAi agent.
[0278] In some embodiments, the one or more cargo molécules is/are an ‘RNAi agent,” which as 10 defined herein is a composition that contaîns an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molécule that is capable of degrading or înhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence spécifie manner. As used herein, RNAi agents may operate through the RNA interférence mechanism (i.e., înducing RNA interférence through interaction with the RNA interférence pathway machinery (RNA-înduced silencing complex or 15 RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). Whîle it is believed that RNAi agents, as that tenn is used herein, operate primarily through the RNA interférence mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short (or small) interfering RNAs (siRNAs), double-strand 20 RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. The antisense strand ofthe RNAi agents described herein is at least partially complementary to the mRNA being targeted. RNAi agents can include one or more modified nucléotides and/or one or more nonphosphodiester linkages.
[0279] Typically, RNAi agents can be comprised of at least a sense strand (also referred to as a 25 passenger strand) that includes a first sequence, and an antisense strand (also referred to as a guide strand) that includes a second sequence. The length of an RNAi agent sense and antisense strands can each be 16 to 49 nucléotides in length. In some embodiments, the sense and antisense strands of an RNAi agent are independently 17 to 26 nucléotides in length. In some embodiments, the sense and antisense strands are independently 19 to 26 nucléotides in length. In some embodiments, the 30 sense and antisense strands are independently 21 to 26 nucléotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucléotides in length. The sense and antisense strands can be either the same length or different lengths. The RNAi agents include an antisense strand sequence that is at least partially complementary to a sequence in the target gene, and upon delivery to a cell expressing the target, an RNAi agent may inhibit the expression of one or more target genes in vivo or in vitro.
(0280] Oligonucleotide-based compounds generally, and RNAi agents specifically, may be comprised of modified nucléotides and/or one or more non-phosphodiester linkages. As used herein, 5 a “modified nucléotide” is a nucléotide other than a ribonucleotide (2'-hydroxyl nucléotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucléotides are modified nucléotides. As used herein, modified nucléotides include, but are not limited to, deoxyribonucleotides, nucléotide mimics, abasic nucléotides, 2'-modified nucléotides, 3' to 3' linkages (inverted) nucléotides, 10 non-natural base-comprising nucléotides, bridged nucléotides, peptide nucleic acids, 2',3'-seco nucléotide mimics (unlocked nucleobase analogues, locked nucléotides, 3'-O-methoxy (2' intemucieoside linked) nucléotides, 2'-F-Arabino nucléotides, 5'-Me, 2'-tluoro nucléotide, morpholino nucléotides, vînyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucléotides, and cyclopropyl phosphonate containing nucléotides. 2-modified nucléotides (i.e. a 15 nucléotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucléotides, 2'-deoxy-2'-fluoro nucléotides, 2'deoxy nucléotides, 2'-methoxyethyl (2-O-2-methoxylethyl) nucléotides, 2'-amino nucléotides, and 2'-alkyl nucléotides.
[0281] Moreover, one or more nucléotides of an olîgonucleotide-based compound, such as an RNAi 20 agent, may be linked by non-standard linkages or backbones (i.e., modified intemucieoside linkages or modified backbones). A modified intemucieoside lînkage may be a non-phosphate-containîng covalent intemucieoside linkage. Modified intemucieoside linkages or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., 25 methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkylphosphorami dates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkyl phosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5f linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are 30 linked 3'-5' to 5'-3' or 2'-5r to 5'-2'.
[0282] It is not necessary for ail positions in a given compound to be uniformly modified. Conversely, more than one modification may be incorporated in a single oligonucleotîde-based compound or even in a single nucléotide thereof.
[0283] In some embodiments, the cargo moiecule is an RNAi agent for inhibiting alpha ENaC gene expression. The cargo moiecule may be an RNAi agent described in International Patent Application No. PCT/US18/40874, which is herein incorporated by reference in its entirety.
[0284] The RNAi agent sense strands and antisense strands may be synthesized and/or modified by 5 methods known in the art. For example, the disclosure of RNAi agents directed to the inhibition of alpha-ENaC expression may be found, for example, in International Patent Application Publication No. WO 2008/152131, which is incorporated by reference herein in its entirety. Additîonal dîsclosures related to RNAi agents may be found, for example, in the disclosure of modifications may be found, for example, in International Patent Application No. PCT/IJS2017/0455446 to 10 Arrowhead Pharmaceuticals, Inc., which also is incorporated by reference herein in its entirety.
In some embodiments, the one or more cargo molecule(s) can include or consist of a PEG moiety that can acts as a pharmacokinetic (PK) modulator. In some embodiments, the one or more cargo molécules can include a PEG moiety having about 20-900 ethylene oxide (CH2-CH2-O) units (e.g., 20 to 850, 20 to 800, 20 to 750, 20 to 700, 20 to 650, 20 to 600, 20 to 550, 20 to 500, 20 to 450, 20 15 to 400, 20 to 350, 20 to 300, 20 to 250, 20 to 200, 20 to 150, 20 to 100, 20 to 75, 20 to 50, 100 to
850, lOOto 800, 100 to 750, 100 to 700, lOOto 650, lOOto 600, 100 to 550, lOOto 500, 100 to 450, 100 to 400, 100 to 350, lOOto 300, 100 to 250, 100 to 200, lOOto 150, 200 to 850, 200 to 800, 200 to 750, 200 to 700, 200 to 650, 200 to 600, 200 to 550, 200 to 500, 200 to 450, 200 to 400, 200 to 350, 200 to 300, 200 to 250, 250 to 900, 250 to 850, 250 to 800, 250 to 750, 250 to 700, 250 to 650, 20 250 to 600, 250 to 550, 250 to 500, 250 to 450, 250 to 400, 250 to 350, 250 to 300, 300 to 900, 300 to 850, 300 to 800, 300 to 750, 300 to 700, 300 to 650, 300 to 600, 300 to 550, 300 to 500, 300 to 450, 300 to 400, 300 to 350, 350 to 900, 350 to 850, 350 to 800, 350 to 750, 350 to 700, 350 to 650, 350 to 600, 350 to 550, 350 to 500, 350 to 450, 350 to 400, 400 to 900, 400 to 850, 400 to 800, 400 to 750, 400 to 700, 400 to 650, 400 to 600, 400 to 550, 400 to 500, 400 to 450, 450 to 900, 450 to 25 850, 450 to 800, 450 to 750, 450 to 700, 450 to 650, 450 to 600, 450 to 550, 450 to 500, 500 to 900,
500 to 850, 500 to 800, 500 to 750, 500 to 700, 500 to 650, 500 to 600, 500 to 550, 550 to 900, 550 to 850, 550 to 800, 550 to 750, 550 to 700, 550 to 650, 550 to 600, 600 to 900, 600 to 850, 600 to 800, 600 to 750, 600 to 700, 600 to 650, 650 to 900, 650 to 850, 650 to 800, 650 to 750, 650 to 700, 700 to 900, 700 to 850, 700 to 800, 700 to 750, 750 to 900, 750 to 850, 750 to 800, 800 to 900, 850 30 to 900, or 850 to 900 ethylene oxide units). In some embodiments, the one or more cargo molecule(s) consist of a PEG moiety having approximateiy 455 ethylene oxide units (about 20 kilodalton (kDa) molecular weight). In some embodiments, a PEG moiety has a molecular weight of about 2 kilodaltons. In some embodiments, a PEG moiety has a molecular weight of about 20 kilodaltons. In some embodiments, a PEG moiety has a molecular weight of about 40 kilodaltons. The PEG moieties described herein may be linear or branched. The PEG moieties may be discrète (monodîspersed) or non-discrete (polydispersed). PEG moieties for use as a PK enhancing cargo molécule may be purchase commercially. In some embodiments, the one or more cargo molecule(s) include a PEG moiety that can act as a PK modulator or enhancer, as well as a different cargo 5 molécule, such as a pharmaceutically active ingrédient or compound.
[0285] The described ανβό integrin ligands include salts or solvatés thereof. Solvatés of an ανβό integrin ligand is taken to mean adductions of inert solvent molécules onto the ανβό integrin ligand which form owing to theîr mutual attractive force. Solvatés are, for example, mono- or dihydrates or addition compounds with alcohols, such as, for example, with methanol or éthanol.
[0286] Free amino groups or free hydroxyl groups can be provided as substituents of ανβό integrin ligands with corresponding protecting groups.
[0287] The ανβό integrin ligands also include, e.g., dérivatives, i.e., ανβό integrin ligands modified with, for example, alkyl or acyl groups, sugars or oligopeptides, which are cleaved either in vitro or in an organism.
[0288] In some embodiments, an ανβό integrin ligand disclosed herein facilitâtes the delivery of a cargo molécule into the cytosol of a cell presenting an ανβό integrin on its surface, either through ligand-mediated endocytosis, pinocytosis, or by other means. In some embodiments, an ανβό integrin ligand disclosed herein facilitâtes the delivery of a cargo molécule to the plasma membrane of a cell presenting an ανβό integrin.
Pharmaceutical Compositions
[0289] In some embodiments, the présent disclosure provides pharmaceutical compositions that include, consist of, or consist essentially of, one or more of the ανβό integrin ligands disclosed herein. [0290] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective 25 amount of an Active Pharmaceutical Ingrédient (API), and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingrédient (API, therapeutic product) that are intentionally inciuded in the drug delivery System. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery System during 30 manufacture, b) protect, support or enhance stability, bîoavailabilîty or patient acceptability of the
API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.
«I
[0291 ] Excipients include, but are not limited to: absorption enhancers, anti-adhérents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delîvery polymers, dextran, dextrose, diluents, dis intégrants, emulsifiers, extenders, fi llers, flavors, glidants, humectants, lubricants, oils, polymers, préservâtives, saline, salts, solvents, sugars, 5 suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
[0292] The pharmaceutical compositions described herein can contain other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional component îs a pharmaceutically-active materîal. Pharmaceutically-active materials include, but are not limited 10 to: anti-prurîtics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.), small molécule drug, antibody, antibody fragment, aptamers, and/or vaccine. [0293] The pharmaceutical compositions may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents, or antioxidants. They may also contain other agent with 15 a known therapeutic benefit.
[0294] The pharmaceutical compositions can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Adrninistration can be made by any way commonly known in the art, such as, but not limited to, topical (e.g., by a transdermal patch), pulmonary (e.g., by inhalation or insufflation of powders or aérosols, including 20 by nebulizer, întratracheal, intranasal), epidermal, transdermal, oral or parentéral. Parentéral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscuîar injection or infusion; subdermal (e.g., via an implanted device), intracranial, intraparenchymal, intrathecal, and intraventricular, administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection. The 25 pharmaceutical compositions may be administered orally, for example in the form of tablets, coated tablets, dragées, hard or soft gélatine capsules, solutions, émulsions or suspensions. Administration can also be carried out rectally, for example using suppositories; locally or percutaneously, for example using ointments, creams, gels, or solutions; or parentérally, for example using injectable solutions.
[0295] Pharmaceutical compositions suitable for injectable use include stérile aqueous solutions (where water soluble) or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of mîcroorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, éthanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maîntained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be préférable to include isotonie agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0296] Stérile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingrédients enumerated above, as required, followed by fîlter sterilization. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle which contains a basic dispersion medium and the required other ingrédients from those enumerated above. In the case of stérile powders for the préparation of stérile injectable solutions, methods of préparation include vacuum drying and freeze-drying which yields a powder of the active ingrédient plus any additional desired ingrédient from a previously sterile-filtered solution thereof.
[0297] Formulations suitable for intra-articular administration can be in the form of a stérile aqueous préparation of any ofthe ligands described herein that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodégradable polymer Systems can also be used to présent any of the ligands described herein for both intraarticular and ophthalmic administration.
[0298] The active compounds can be prepared with carriers that will protect the compound against rapid élimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery Systems. Biodégradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactîc acid. Methods for préparation of such formulations will be apparent to those ski lied in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
[0299] A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics, or anti-in fl ammatory agents (e.g., antihistamine, diphenhydramine, etc.). As used herein, “pharmacologically effective amount,” Nherapeutically effective amount,” or simply “effective amount” refers to that amount of an the pharmaceutical ly active agent to produce a pharmacological, therapeutic or préventive resuit.
[0300] Médicaments containing an ανβό integrin ligand are also an object ofthe présent invention, as are processes for the manufacture of such médicaments, which processes comprise bringing one 5 or more compounds containing a ανβό integrin ligand, and, if desired, one or more other substances with a known therapeutic benefit, into a pharmaceutically acceptable form.
[0301] The descrîbed ανβό integrin ligands and pharmaceutical compositions comprising ανβό integrin ligands disclosed herein may be packaged or included in a kit, container, pack, or dispenser. The ανβό integrin ligands and pharmaceutical compositions comprising the ανβό integrin ligands 10 may be packaged in pre-ftlled syringes or vials.
Cells, Tissues, and Non-Human Organisms
[0302] Cells, tissues, and non-human organisms that include at least one of the ανβό integrin ligands described herein is contemplated. The cell, tissue, or non-human organism is made by delivering the 15 ανβό integrin ligand to the cell, tissue, or non-human organism by any means available in the art. In some embodiments, the cell is a mammalian cell, including, but not limited to, a human cell.
Targeting Groups, Linking Groups, Pharmacokinetic (PK) Modulators, and Delivery Vehicles
[0303] In some embodiments, an ανβό ligand is conjugated to one or more non-nucleotide groups including, but not limited to, a linking group, a pharmacokinetic (PK) modulator, a delivery polymer, or a delivery vehicle. The non-nucieotide group can enhance targeting, delivery, or attachment of the cargo molécule. Examples of targeting groups and linking groups are provided in Table 6. The nonnucleotide group can be covalently linked to the 3' and/or 5' end of either the sense strand and/or the antisense strand. In embodiments where the cargo molécule is an RNAi agent, the RNAi agent contaîns a non-nucleotide group linked to the 3' and/or 5' end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5' end of an RNAi agent sense strand. An ανβό ligand can be linked directly or îndirectly to the cargo molécule via a lînker/linking group. In some embodiments, a ανβό ligand is linked to the cargo molécule via a labile, cleavable, or réversible bond 30 or linker.
[0304] In some embodiments, a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, a nonnucléotide group enhances endocytosis of the RNAi agent.
[0305] Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a cargo molécule to which they are attached to improve cell-specific (including, in some 5 cases, organ spécifie) distribution and cell-specific (or organ spécifie) uptake of the cargo molécule.
In some embodiments, a targeting group may comprise an ανβό ligand as described herein. In some embodiments, a targeting group comprises a linker. In some embodiments, a targeting group comprises a PK modulator. In some embodiments, an ανβό ligand is linked to a cargo molécule using a linker, such as a PEG linker or one, two, or three abasic and/or ribitol (abasic ribose) residues, 10 which in some instances can serve as linkers.
[0306] Cargo molécules can be synthesized having a reactive group, such as an amino group (also referred to herein as an amine). In embodiments where the cargo molécule is an RNAi agent, the reactive group may be linked at the 5f-terminus and/or the 3'-terminus. The reactive group can be used subsequently to attach an ανβό ligand using methods typical in the art.
[0307] For example, in some embodiments, an RNAi agent is synthesized having an NHs-Cé group at the 5'-tenninus of the sense strand of the RNAi agent. The terminal amino group subsequently can be reacted to form a conjugate with, for example, a group that includes an ανβό integrin targeting ligand. In some embodiments, an RNAi agent is synthesized having one or more alkyne groups at the 5'-terminus of the sense strand of the RNAi agent. The terminal alkyne group(s) can subsequently be reacted to form a conjugate with, for example, a group that includes an ανβό integrin targeting ligand.
[0308] In some embodiments, a linking group is conjugated to the ανβό ligand. The linking group facilitâtes covalent linkage of the ανβό ligand to a cargo molécule, pharmacokinetic modulator, delivery polymer, or delivery vehicle. Examples of linking groups, include, but are not limited to: 25 Alk-SMPT-C6, Alk-SS-C6, DBCO-TEG, Me-Alk-SS-C6, and C6-SS-Alk-Me, reactive groups such a primary amines and alkynes, alkyl groups, abasic residues/nucleotides, amino acids, tri-alkyne functionalized groups, ribitol, and/or PEG groups.
[0309] A linker or linking group is a connection between two atoms that links one Chemical group (such as an RNAi agent) or segment of interest to another Chemical group (such as an ανβό ligand, 30 pharmacokinetic modulator, or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage can optionally include a spacerthat increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the linkage. Spacers include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucléotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description.
[0310] In some embodiments, ανβό ligands are linked to cargo molécules without the use of an additional linker. In some embodiments, the ανβό ligand is designed having a linker readily présent 5 to facilitate the linkage to a cargo molécule. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents can be linked to their respective targeting groups using the same linkers. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents are linked to their respective targeting groups using different linkers.
[0311] Examples of certain linking groups are provided in Table A.
100
[0312] Alternatively, other linking groups known in the art may be used.
[0313] The above provided embodiments and items are now illustrated with the following, nonlimiting examples.
Examples
[0314] The following examples are not lîmiting and are intended to illustrate certain embodiments disclosed herein.
Example 1. Synthesis of ανβό integrin ligands
[0315] Some of the abbreviations used in the following experimental details of the synthesis of the examples are defined as follows: h or hr = hour(s); min = minute(s); mol = mole(s); mmol = millimole(s); M = molar; μΜ = micromolar; g = gram(s); pg = microgram(s); rt or RT = room température; L= liter(s); mL = milliliter(s); wt = weight; Et2O = diethyl ether; THF = tetrahydrofuran; DM S O = dimethyl sulfoxîde; EtOAc = ethy 1 acetate; EtjN or TE A = triethy lamine;
APr2NEt or DI PEA or DIEA = diisopropylethy lamine; CH2C12 or DCM = methylene chloride; CHCh = chloroform; CDCk = deuterated chloroform; CCh = carbon tetrachloride; MeOH = methanol; EtOH = éthanol; DM F = dimethyl formamide; B OC = /-butoxycarbonyl; CBZ = benzyloxycarbonyl; TBS = t— butyldimethylsilyl; TBSC1 or TBDMSCl = t—butyldîmethylsilyl chloride; TFA = trifluoroacetic acid; DMAP = 4-dimethylaminopyridine; NaNa = sodium azide;
Na2SO4 = sodium sulfate; NaHCO2 = sodium bicarbonate; NaOH = sodium hydroxide; MgSOi = magnésium sulfate; K2COj = potassium carbonate; KO H = potassium hydroxide; NFLOH =
102 ammonium hydroxide; NHæl = ammonium chloride; S1O2 = silica; Pd-C = palladium on carbon;
HCl = hydrogen chloride or hydrochloric acid; NMM = W-methyhnorphohne; H2 = hydrogen gas; KF = potassium fluoride; EDC-HC1 = N-tS-Dimethylaminopropylj-N-ethylcarbodiimide hydrochloride; MTBE = methyL/cri-butyl ether; Ar = argon; N2 = nitrogen; Rt = rétention tîme.
[0316] Chemical names for structures 1-37 were automatically generated using ChemDraw® software.
Synthesis of Structure 1b ((14Sy17S)-l-aï.ido-14-(5-((4-methylpyridin-2-yl)amino)pentanamido)17~(4-(naphth alen-l-yl)phenyl)-l S-oxo-3,6,9,12-tetraoxa-16-azanoHadecan-19-oic acid).
1. TFA, i-Pr3SiH CH2CI2
2. Fmoc-OSu NaHCO3
3. Column
[0317] Compound 1 (Methyl (S)-(-)-1-trîtylazîridine-2-carboxylate (4.204 g, 12.24 mmol, 1.0 equiv.) and triisopropylsilane (3.877 g, 5.02 mL, 24.48 mmol, 2 equiv.) were dîssolved in DCM (40 mL), the solution was cooled to 0 °C, and then TFA (8.5 eq) was added dropwise. The solution remained for 1 hour at 0 °C. The reaction was monitored by TLC Hexane : Elhyi Acetate (8 : 2). The solution was dried to yield a mixture of white precipîtate and light yellow oil. Hexanes (40 mL) were added and heated gently over heat gun until ail white precipîtate dîssolved. The addition of hexanes resulted in two layers, a clear upper layer and an oil layer. The hexane layer was poured off and the oil layer was retained. The hexanes addition was repeated and once again poured off. The oil was allowed to dry. The aziridine (1.06 g, 10.5 mmol) was dîssolved in THF / H2O (2 / 1) 60 mL total. Fmoc-OSu (5.312 g, 15.75 mmol, 1.5 eq) and NaHCOj (2.646 g, 31.5 mmol, 3 eq to keep pH = 8.5) were added to the mixture at room temp and allowed to react overnight. The reaction was monitored by TLC, Hexane:Ethyl Acetate 8:2. The mixture was concentrated until ail the THF was removed, then diluted with ethyl acetate (350 mL) and H2O (25 mL). The layers were separated, and the organics washed with FhO (40 mL). The organîcs were then washed with pH 3-4 water (2 x 40 mL), then H2O (40 mL), then saturated aq. NaCl solution (40 mL). The organic phase was dried over Na2SÛ4, filtered, and concentrated. The product was purîfted on silica column 10%-20% ethyl acetate in hexanes.
103
hrs RT
HO-PEG4-N3
BF3 Et2O
CH2CI2
Set at 0°C
N H
O
[0318] Compound 2 (Fmoc-aziridine) (L46 g, 4.52 mmol) and HO-PEG4-N3 (1.983 g, 9.04 mmol, 2 eq) were dissolved in DCM. The mixture was cooled to 0 °C. Boron trifluoride diethyl etherate (12 drops) was added dropwise. The mixture was stirred at RT for 48 hours. The reaction was monitored by TLC, DCM with 5% MeOH. The reaction was quenched with NH4CI saturated solution (5 mL), diluted with DCM (60 mL) and washed with H2O (3 x 20 mL), saturated aq. NaCI solution (20 mL), dried over NaîSO4, filtered, and concentrated. The product was purified on a siIica column, 40%-60% ethyl acetate in hexanes.
[0319] Compound 3 was dissolved in a solution of 20% triethylamine in DMF. The reaction was monitored by TLC. The product was concentrated.
[0320] Compound 5 (tert-Butyl(4-methylpyridin-2-yl)carbamate) (0.501 g, 2.406 mmol, 1.0 equiv.) 15 was dissolved in DMF (1 7 mL). To the mixture was added NaFI (0.116 mg, 3.01 mmol, 1.25 eq, 60 % dispersion in minerai oîl) at room température. The mixture stirred for 10 min, then ethyl 5bromovalerate (0.798 g, 3.82 mmol, 0.604 mL) was added. After 3 hours the reaction was quenched with éthanol (18 mL) and concentrated. The product was dissolved in DCM (50 mL) and washed with saturated aq. NaCI solution (50 mL), dried overNa2SO4, filtered and concentrated.
The product was purified on silica column, gradient 0-5% methanol in DCM.
104
[0321] Compound 7 (0.80 g, 2.378 mmol) was dissolved in 100 mL of acetone :0.l M NaOH (1:1), and the reaction was monitored by TLC (5% ethyl acetate in hexane). The organics were concentrated, and the mixture was acidified to pH 3-4 with 0.3 M citric acid (40 mL). The product 5 was extracted with DCM (3 x 75 mL). The organics were pooled, dried over NazSCL, filtered and concentrated. The product was used without further purification.
TBTU (1.8 eq), DIPEA (2 eq) DMF
Work-up Column
[0322] Compound 4 was dissolved (0.340g, 1.104 mmol) in DMF (10 mL). To the solution was added TBTU (0.531g, 1.655 mmol) and diisopropylethylamine (0.320 mL, 1.839 mmol). Then compound 8 was added (0.295g, 0.9197 mmol). The reaction was monitored by LC-MS and TLC (DCM with 5% MeOH). The reaction was complété in 2 hours. The product was concentrated and dissolved in ethyl acetate (150 mL), and washed with pH 3-4 H2O (2 x 12 mL). Then the product was washed with H2O (2x12 mL), saturated aq. NaHCOs solution (12 mL), then saturated aq. NaCl solution (12 mL). The organic phase was dried overNajSCU, filtered and concentrated. The 15 product was purified on silica column, hexanes 20% in ethyl acetate to 100% ethyl acetate.
105
[0323] Compound 9 was dissolved (0.330g, 0.540 mmol) in 10 mL of MeOH:dioxane [1:1 J and 1 M LiOH solution (10 ntL) The mixture was stirred at rt for 2 hr, monîtored by LC-MS and TLC (EtOAc). The organics were concentrated away, and the mixture was diluted with H2O (5 mL) and acidifled to pH 4. The product was extracted with ethyl acetate (2 x 50 mL). The organics were pooled, washed with saturated aq. NaCl solution (10 mL), dried over NaiSCU, filtered and concentrated. The product was used without further purification.
[0324] Compound 11 ((S)-3-(4-Bromopheny!)-3-((tert-butoxycarbonyl)amino)-propionic acid) (2.0g, 5.81 mmol) was dissolved in DM F (40 mL). To the mixture was added K2CO3 (1.2 g, 8.72 mmol). Then iodomethane (1.65 g, 11.62 mmol, 0.72 mL) was added. The reaction was monîtored by TLC (hexane : ethyl acetate (7 :3)). Upon completion, the mixture was cooled to 0 °C and H2O (20 mL) and MTBE (40 mL) were added. The product was extracted with MTBE (4 x 40 mL). The
106 combined organic phase was washed with saturated aq. NaHCOs (40 mL) then HîO (4 x 40 mL). The mixture was dried over Na2SÛ4, filtered and concentrated.
[0325] To dried product compound 12 (1.0 g, 2.7915 mmol) was added compound 13(1Naphthalene Boronic Acid (0.960 g, 5.583 mmol, 2 eq)). To the mixture was added [ 1,1 '5 Bis(diphenylphosphino)ferrocene]dichloropalladium(II) or Pd(dppf)Ch (0.0817 g, 0.1117 mmol, 0.4 eq) along with NaiCOs (0.888 g, 8.375 mmol, 3 eq). Next, 1,4-dioxane (5 mL) and HjO (0.2 mL) were added, and the mixture was stirred at 100 °C for 4 hr. The reaction was monitored by TLC (hexane : ethyl acetate (7 :3)). The product was purifïed by si lie a chromatography, gradient 0% to 50% ethyl acetate in hexanes.
[0326] Compound 14 (0.200 g, 0.493 mmol) was dissolved in DCM (2.5 mL), then TFA (0.45 mL) was added. The reaction was monitored by TLC, (DCM : methanol (9 : 1)). Upon completion, the reaction mixture was concentrated. The residuewas dissolved in DCM (4 mL) and washed with saturated aq. NaHCCh solution (2x2 mL) then saturated aq. NaCl solution (2x2 mL). The organic phase was dried over NasSOi, filtered and concentrated. The product w'as used without further purification.
[0327] Compound 10 (0.3224g, 0.54 mmol) was dissolved in DMF (7 mL). To the mixture was added TBTU (0.236 g, 0.735 mmol) and diisopropylethylamine (0.170 mL, 0.98 mmol). Then compound 15 was added (0.1496g, 0.49 mmol). The reaction was stirred at RT for 2 hours. The
107 réaction was monitored by LC-MS. The mixture was concentrated, and the residue was disseIved in ethyl acetate (90 mL), and washed with pH 3-4 H2O (3x10 mL). The product was washed with H2O (2x10 mL), saturated aq. NaHCOs solution (10 mL), and then saturated aq. NaCl solution (1 x 10 mL). The organîc phase was dried over Na2SO4, fiItered and concentrated. The product was purified by silîca chromatography using DCM, gradient to 5% MeOH.
[0328] A Compound 16 was dissolved (0.250g, 0.2828 mmol) in MeOH : dioxane [1:1] (4 mL) and 1 M LiOH (4 mL) The mixture was stirred at RT for 2 hr. The organics were concentrated away, and the residue was diluted with H2O (3 mL) and acidified to pH 4. The product was extracted with ethyl acetate (3 x 20 mL). The organics were pooled and washed with saturated aq. NaCl solution (10 mL). The product was dried over Na2SO4. The product was dissolved (0.200 g, 0.2299 mmol) in 2 mL DCM : TFA [25:75] and stirred at RT for 2 hours. To the mixture was added toluene (4 mL). The mixture was concentrated, then coevaporated with acetonitrile (2 x 4mL). The product was purified by HPLC, gradient 35% ACN to 50% over 30 minutes, 0.1% TFA buffer. => [M+H]+ calculated for C4iH5iN7O8: 769.90, found: 770.45; Ή NMR (400 MHz, DMSO) δ 8.64 (d, 1H), 8.07 (d, 1H), 8.00 (d, 1H), 7.95 (d, 1H), 7.78 (t, 2H), 7.60-7.40 (m, 8H), 6.80 (s, 1H), 6.67 (d, 1 H), 5.31 (q, 1 H), 4.55 (m, 1H), 3.62-3.45 (m, 18H), 3.40 (t, 2H), 3.25 (m, 2H), 2.80 (dd, 2H), 2.30 (s, 3H), 2.20 (t, 2H), 1.55 (m, 4H).
0 Synthesis of Structure 2b ((14S,17S)-l-azido-14-(4-((4-methylpyridin-2-yl)andno)butanamido)l7-(4fnaphthalen-l-yl)phenyl)-15~oxo-3t6,9,12-tetraoxa-16-azanonadecan-l9-oic acid).
[0329] Compound 5 (tert-Butyl(4-methylpyridin-2-yl)carbamate) (0.501 g, 2.406 mmol, 1 equiv.) was dissolved in DM F (17 mL). To the mixture was added NaH (0.116 mg, 3.01 mmol, 1.25 eq, 60 % dispersion in oil) The mixture stirred for 10 min before adding Compound 20 (Ethyl 4Bromobutyrate (0.745 g, 3.82 mmol, 0.547 mL)) (Sigma 167118). After 3 hours the reaction was
108 quenched with éthanol (18 mL) and concentrated. The concentrate was dissolved in DCM (50 mL) and washed with saturated aq. NaCl solution (i x 50 mL), dried overNazSCh, filtered and concentrated. The product was purified on silica column, gradient 0-5% Methanol in DCM.
[0330] Compound 21 was dissolved (0.80 g, 2.378 mmol) in 100 mL of Acetone : 0.1 M NaOH
[1:1]. The reaction was monitored by TLC (5% ethyl acetate in hexane). The organics were concentrated away, and the residue was acidified to pH 3-4 with 0.3 M Citrîc Acid (40 mL). The product was extracted with DCM (3 x 75 mL). The organics were pooled, dried over Na2SC>4, filtered and concentrated. The product was used without further purification
[0331] Compound 22 was dissolved (0.340g, 1.104 mmol) in DMF (10 mL). To the mixture was added TBTU (0.531g, 1.655 mmol) and dîisopropylethylamine (0.320 mL, 1.839 mmol). Then Compound 10 (0.295g, 0.9197 mmol) was added. The reaction was monitored by LC-MS and TLC (DCM with 5% MeOH). The reaction was complété in 2 hr. The mixture was concentrated,
IS dissolved in ethyl acetate (150 mL), and washed with pH 3-4 HjO (2 x 12 mL). The mixture was then washed with HsO (2x12 mL), saturated aq. NaHCOj solution (12 mL), then saturated aq. NaCl solution (12 mL). The organic phase was dried over NazSCU, filtered and concentrated. The
109
product was purified on silica column, Hexanes 20% in ethyl acetate to 100% ethyl acetate.
[0332] Compound 23 was dissolved (0.330g, 0.540 mmol) in 10 mL of MeOH : Dioxane [1:1] and 1 M LiOH (10 mL) The mixture was stirred at room température for 2 hours and monitored by LC5 MS and TLC (100% EtOAc). The organics were concentrated, and the residue was diluted with
H?O (5 mL), and acidified to pH 4. The product was extracted with ethyl acetate (2x50 mL). The combined organic phase was washed with saturated aq. NaCl solution (1x10 mL). The organic phase was dried over NazSCL, fiItered and concentrated. The product was used without further purification.
[0333] Compound 24 was dissolved (0.3224g, 0.54 mmol) in DMF (7 mL). To the mixture was added TBTU (0.236 g, 0.735 mmol) and diisopropylethy lamine (0.170 mL, 0.98 mmol).
Compound 15 was then added (0.1496g, 0.49 mmol). The mixture was stirred at room température for 2 hours. The reaction was monitored by LC-MS. The mixture was concentrated, and the residue was dissolved in ethyl acetate (90 mL) and washed with pH 3-4 FfO (3x10 mL). The concentrate
110 was washed with H2O (2x10 mL), saturated aq. NaHCOj solution (10 mL), and then saturated aq. NaCl solution (10 mL). The organic phase was dried overNasSCL, filtered and concentrated. The product was purified on silica column, DCM, gradient to 5% MeOH.
[0334] Compound 25 was dissolved (0.250g, 0.2828 mmol) in MeOH : Dioxane [1:1] (4 mL) and 1
M LiOH (4 mL). The mixture was stirred at room température for 2 hr, monîtored by LC-MS. The organics were concentrated, and the residue was diluted with H2O (3 mL) and acidifîed to pH 4. The product was extracted with ethyl acetate (3 x 20 mL). The organics were pooled and washed with saturated aq. NaCl solution (1x10 mL). The organic phase was dried overNasSCL and concentrated. The residue was dissolved (0.200 g, 0.2299 mmol) in 2 mL DCM / TFA (25 / 75) and stirred at RT for 2 hours while monîtored by LC-MS. Toluene (4 mL) was added, and the mixture was concentrated. Then acetonitrile (2x4 mL) was added, and the mixture was concentrated. The product was purified on HP LC, gradient 35% ACN to 50% over 30 minutes, 0.1% TFA buffer. [M+H]+ calculated for C^^NyOs: 755.87, found: 756.32; Ή NMR (400 MHz, DMSO) δ 8.64 (t,
1H), 8.17-8.10 (m, 1H), 8.00 (d, 1H), 7.95 (d, 1H), 7.80 (d, 1H), 7.75 (m, 1 H), 7.60-7.40 (m, 8H),
6.8 (s, 1H), 6.67 (d, 1H), 5.31 (q, 1 H), 4.55 (m, IH), 3.62-3.45 (m, 18H), 3.40 (t, 2H), 3.25 (m, 2H), 2.80 (dd, 2H), 2.30 (s, 3H), 2.26 (t, 2H), 1.80 (m, 2H).
Synthesis of Structure 5b, 5.1b, and 5.2b.
Structure 5b (3-(4-(2-(2-(2-azidoethoxv)ethoxv)ethoxv)-3,5-dichloronhenvlÎ-3-(2-(5-((4methvlpvridiii-2-vl)amino)pentanamido)acetamido)propanoic acid)
[0335] To a solution of Compound 5 (0.98 g, 4.70 mmol, 1 equiv.) in dry DMF (10 mL) was added NaH (0.226 g, 5.647 mmol, 1.2 equiv., 60% oil dispersion) portion-wise at 0 °C underNs atmosphère. The reaction mixture was kept at 0 °C for 30 min followed by the addition of compound 6(1.18 mL, 5.647 mmol, 1.2 equiv.) at the same température. After additional stirring at
111 °C for 30 min the mixture was allowed to warm to room température. A fier stirring at room température for 1 hour, the reaction was quenched by saturated NH4CI aqueous solution. The aqueous phase was extracted with ethyl acetate (3 x 20 mL) and the organic layer was combined, dried over NazSCU, and concentrated. The product was separated by CombiFlash® using si!ica gel as the stationary phase. LC-MS: [M+H]+ 337.20, found 337.39.
[0336] To a solution of compound 7 (1.347 g, 4.00 mmol, 1 equîv.) in THF (5 mL) and H2O (5 mL) was added lithium hydroxide monohydrate (0.505 g, 12.01 mmol, 5 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for I h, the reaction mixture was acidified by HCI (6 N) to pH 4.0. The aqueous phase was extracted with ethyl acetate (3 x 20 mL) and the organic layer was combined, dried over NaiSCU, and concentrated. LCMS: [M+H]+ 309.17, found 309.39.
[0337] To a solution of Compound 8 (1.163 g, 3.77 mmol, 1 equiv.), Compound 45 (568 mg, 4.52 mmol, 1.2 equîv.), and TBTU (1.453 g, 4.52 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (1.97 mL, 11.31 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred 3 hours. The reaction was quenched by saturated NaHCOj solution (20 mL). The aqueous layer was extracted with ethyl acetate (3x10 mL), and the organic phase was combined, dried over anhydrous Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: calculated [M+H]+ 380.21, found 380.51.
112
[0338] To a solution of compound 47 (1.0 g, 5.23 mmol, 1 equiv.) and malonic acid (1.09 g, 10.47 mmol, 2 equiv.) m éthanol (10 mL) was added ammonium acetate (0.807 mg, 10.47 mmol, 2.0 equiv.) at room température. The reaction mixture was stirred at reflux overnight. The solîd was fiItered and washed with cold éthanol. The product was used directly for further steps without further purification.
LC-MS: calculated [M+H]+ 250.00, found 250.16.
[0339] To a solution of compound 46 (1.412 g, 3.72 mmol, 1 equiv.) in THF (5 mL) and HzO (5 mL) was added lithium hydroxide monohydrate (0.469 g, 11.16 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for 3 hours, the reaction mixture was acidified by HCl (6 N) to pH 4.0. The aqueous phase was extracted with ethyl acetate (3 x 20 mL) and the organic layer was combined, dried over Na2SO4, and concentrated. LC-MS: calculated [M+H]+ 366.20, found 366.46.
[0340] To a suspension of compound 49 (0.531 g, 2.12 mmol, 1 equiv.) in anhydrous methanol (10 mL) was added thionyl chloride (308 uL, 4.24 mmol, 2.0 equiv.) on ice bath. The reaction was warmed to room température and stirred overnight. The solvent was removed under reduced pressure and the product was directly used without further purification. LC-MS: calculated [M+H]+ 264.01, found 264.20.
51 OH
[0341] To a solution of compound 50 (150 mg, 0.410 mmol, 1 equiv.), compound 51 (148 mg, 0.492 mmol, 1.2 equiv.), and TBTU (158 mg, 0.492 mmol, 1.2 equiv.) in anhydrous DMF (5 mL) was added diisopropylethylamine (0.214 mL, 1.23 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred 3 hours. The reaction was quenched by saturated NaHCOj
113 aqueous solution (10 mL) and the product was extracted with ethyl acetate (3 x 20 mL). The organîc phase was combined, dned over Na2SO4, and concentrated. The product was purified by
CombiFlash® using silica gel as the stationary phase and was eluted with 2-4% methanol in DCM.
[0342] To a solution of compound 52 (80 mg, 0.130 mmol, 1 equiv.) and azido-PEGj-OTs (86 mg,
0.262 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added K2CO4 (36 mg, 0.262 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred for 1 hr at 80 °C. The solvent was removed by rotary evaporalor. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 2-4% methanol in DCM. LC-MS: calculated [M+H]+ 768.28, found 769.
Structure 5b
[0343] To a solution of compound 53 (58 mg, 0.0755 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide monohydrate (10 mg, 0.226 mmol, 3.0 equiv.) at room température, The mixture was stirred at room température for another 2 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried overNa2SO4, and concentrated. TFA (0.25 mL) and DCM (0.75 mL) was added into the residue and the mixture was stirred at room température for another 1 hour. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 654.21, found 655.
Structure 5.1b (3-(4-((14-azido-3,6,9,12-tetraoxatetradecvl)oxv)-3,5-dichlorophenyl)-3-(2-(5((4-niethvlpvridin-2-yl)aminoÎpentanamido)acctamido)propanoic acid)
114
[0344] To a solution of compound 52 (100 mg, 0.163 mmol, 1 equiv.) and azido-PEGs-OTs (205 mg, 0.491 mmol, 3 equiv.) in anhydrous DMF (2 mL) was added K2CO3 (68 mg, 0.491 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred for 1 hour at 80 °C. The solvent was removed by rotary evaporator. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 2-3% methanol in DCM. LC-MS: calculated [M+H]+ 856.33, found 857.07.
*tfa
[0345] To a solution of compound 55 (119 mg, 0.139 mmol, 1.0 equiv.) in THF (4 mL) and water(4 mL) was added lithium hydroxide ( 10 mg, 0.417 mmol, 3.0 equiv.) at room température. The mixture 10 was stirred at room température for another 1 hr. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over NazSiX and concentrated. TFA (2 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hours. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 742.27, found 743.02,
Structure 5.2b (3-(4-(ï8-azidooctyl)oxv)-3,5-dichlorophenvl)-3-(2-(5-((4-tnethvlpyridin-2vl)amino)pentanamido)acetamido)propanoic acid)
115 ” [0346] To a solution of compound 52 (89 mg, 0.14 mmol, 1 equiv.) and 1,8-dibromooctane (80 uL,
0.436 mmol, 3 equiv.) in acetone (2 mL) was added K2CO3 (60 mg, 0.436 mmol, 3 equiv.) at room température. The reaction mixture was stirred for 6 hours at 55 °C. The reaction was quenched by saturated NaHCOj solution and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. LC-MS: calculated [M+H]+ 801.23, found 801.98.
[0347] To a solution of compound 57 (97 mg, 0.114 mmol, 1 equiv.) in anhydrous DMF (2 mL) was added sodium azide (15 mg, 0.229 mmol, 2 equiv.) at room température. The reaction mixture was 10 stirred for 2 hours at 80 °C. The reaction was quenched by water and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over NajSOi, and concentrated. Theproductwas used directly without further purification. LC-MS: calculated [M+H]+ 764.32, found 765.07.
[0348] To a solution of compound 58 (78 mg, 0.101 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (7 mg, 0.304 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hr. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (2 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LCMS: calculated [M+H]+ 650.25, found 650.83.
Synthesis of Structure 6b, 6.1b, 6.2b, 6.3b, and 6.4b,
116
Structure 6b ((S)-3-(4-(4-(2-(2-(2-azidoethoxv)ethoxv)ethoxv)naphthalen-l-yl)phenvl)-3-(2-(4((4-methvlpvridiii-2-vl)amino)butanamitio)acetamido)propanoic acid)
[0349] To a solution of Compound 22 (1.1 g, 3.95 mmol, 1 equiv.), Compound 45 (595 mg, 4.74 mmol, 1.2 equiv.), and TBTU (1.52 g, 4.74 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (2.06 mL, 11.85 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stîrred 3 hours. The reaction was quenched by saturated NaHCOs solution (10 mL). The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic phase was combined, dried over anhydrous Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: calculated [M+H]+ 366.20, found 367.
[0350] To a solution of compound 61 (2 g, 8.96 mmol, 1 equiv.), and compound 62 (2.13 mL, 17.93 mmol, 2 equiv.) in anhydrous DMF (10 mL) was added K2CO3 (2.48 g, 17.93 mmol, 2 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred overnight. The reaction was quenched by water (10 mL). The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic phase was combined, dried over anhydrous Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase.
[0351] To a solution of compound 60 (1.77 g, 4.84 mmol, I equiv.) in THF (5 mL) and H2O (5 mL) was added lithium hydroxide monohydrate (0.61 g, 14.53 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for 3 hours,
117 the reaction mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3 x 20 mL) and the organic layer was combined, drîed overNa2SÜ4, and concentrated. LC-MS: calculated [M+H]+ 352.18, found 352.
[0352] To a solution of compound 63 (1.88 g, 6.0 mmol, 1.0 equiv.) in anhydrous THF (20 mL) was added n-BuLi in hexane (3.6 mL, 9.0 mmol, 1.5 equiv.) drop-wise at -78 °C. The reaction was kept at -78 °C for another I hour. Triisopropylborate (2.08 mL, 9.0 mmol, 1.5 equiv.) was then added into the mixture at -78 °C. The reaction was then warmed up to room température and stirred for another 1 hour. The reaction was quenched by saturated NH4CI solution (20 mL) and the pH was adjusted to 10 3. The aqueous phase was extracted with EtOAc (3 x 20 mL) and the organic phase was combined, dried over Na2SÛ4, and concentrated.
[0353] Compound 12 (300 mg, 0.837 mmol, 1.0 equiv.), Compound 65 (349 mg, 1.256 mmol, 1.5 equiv.), XPhos Pd G2 (13 mg, 0.0167 mmol, 0.02 equiv.), and K3PO4 (355 mg, l.675mmol, 2.0 15 equiv.) were mixed in a round-bottom flask. The Dask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 times). Then, THF (8 mL) and water (2 mL) were added via syringe. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at room température for ovemight. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 χ 10 mL). The organic phase was 20 dried over Na2SÛ4, concentrated, and purified via CombiFlash® using silica gel as the stationary phase and was eluted with 15% EtOAc in hexane. LC-MS: calculated [M+H]+ 512.24, found 512.56.
118
[0354] Compound 66 (858 mg, 1,677 mmol, 1.0 equiv.) was cooled by ice bath. HCl in dioxane (8,4 mL, 33.54 mmol, 20 equiv.) was added into the flask. The reaction was warmed to room température and stirred for another 1 hr. The solvent was removed by rotary evaporator and the product was directly used wîthout further purification. LC-MS: calculated [M+H]+ 412.18, found 412.46.
[0355] To a solution of compound 64 (500 mg, 1.423 mmol, 1 equiv.), compound 67 (669 mg, 1.494 mmol, 1.05 equiv.), and TBTLJ (548 mg, 0.492 mmol, 1.2 equiv.) in anhydrous DMF (15 mL) was added diisopropylethylamine (0.744 mL, 4.268 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched by saturated NaHCOs aqueous solution (10 mL) and the product was extracted with ethyl acetate (3 x 20 mL). The organic phase was combined, dried over NajSOi, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. The yield was 96.23%. LC-MS: calculated [M+H]+ 745.35, found 746.08.
119
[0356] To a solution of compound 68 (1.02 g, 1.369 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd/C (0.15 g, 50% H2O) at room température. The reaction mixture was warmed to room température and the reaction was monitored by LC-MS. The reaction was kept at room température overnight. The solids were filtered through Celite® and the solvent was removed by rotary evaporator. The product was directly used without further purification. LC-MS: [M+H]+ 655.31, found 655.87.
[0357] To a solution of compound 69 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEGs-OTs (100 10 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added K2CO3 (42 mg, 0.305 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred for 6 hours at 80 °C. The reaction was quenched by saturated Nal-ICOj solution and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over NajSCL, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: calculated [M+H]+ 812.39, found 15 813.14.
120
[0358] To a solution of compound 70 (77 mg, 0.0948 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (7 mg, 0.284 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 2 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over NasSOi, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) was added înto the residue and the mixture was stirred at room température for another 3 hours. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 698.32, found 698.81.
Structure 6.1b ((SÎ-3-(4-(4-((14-azi(lo-3,6,9,12-tetraoxatetradecvl)oxv]naphthalen-l-
[0359] To a solution of compound 69 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEGs-OTs (128 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added K2CO3 (42 mg, 0.305 mmol, 2 15 equiv.) at 0 °C. The reaction mixture was stirred for 6 hours at 80 °C. The reaction was quenched by saturated NaHCOj solution and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over NaïSOi, and concentrated. LC-MS: calculated [M+H]+ 900.40, found 901.46.
[0360] To a solution of compound 72 (59 mg, 0.0656 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (5 mg, 0.197 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hr. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SÛ4, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) was added into the resîdue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 786.37, found 786.95.
Structure 6.2b ((S)-3-(4-(4-((8-azidooctvl)oxv)naphthaIen-l-vl)phenvl)-3-(2-(4-((4methvlpvridin-2-yl)amino)butanamido)acetamido)propanoic acid)
[0361] To a solution of compound 69 (150 mg, 0.229 mmol, 1 equiv.) and 1,8-dibromooctane (127 uL, 0.687 mmol, 3 equiv.) in acetone (2 mL) was added K2CO3 (95 mg, 0.687 mmol, 3 equiv.) at 15 room température. The reaction mixture was stirred for overnight at 55 °C. The reaction was quenched by saturated NaHCOî solution and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried overNasSOi, and concentrated. LC-MS: calculated [M+HJ+ 845.34, found 845.91.
[0362] To a solution of compound 74 (97 mg, 0.114 mmol, 1 equiv.) in anhydrous DMF (2 mL) was added sodium azide (15 mg, 0.229 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 2 hours at 80 °C. The reaction was quenched by water and the aqueous layer was extracted 5 with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over NazSO4, and concentrated. LC-MS: calculated [M-H4]+ 808.43, found 809.00.
[0363] To a solution of compound 75 (92 mg, 0.114 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (8 mg, 0.342 mmol, 3.0 equiv.) at room température. The mixture 10 was stirred at room température for another 1 hr. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 694.36, found 694.94.
Structure 6.3b ((S)-3-(4-(4-((20-azido-3,6,9,12,15,18-hexaoxaicosvl)oxv)naphthalen-Iyl)Dhenvl)-3-(2-(4-((4-methvlDvridin-2-vl)aniino)b»tanamido)acetamido)propanoic acid)
[0364] To a solution of compound 69 (100 mg, 0.152 mmol, 1 equîv.) and azido-PEG?-OTs (154 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (100 mg, 0.305 mmol, 2 equîv.) at 0 °C. The reaction mixture was stirred at 40 °C overnîght. The reaction was quenched by saturated NaHCOs solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over NaaSCL, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase, and the product was eluted with
2-3% methanol in DCM. LC-MS: calculated [M+FI]+ 988.50, found 989.14.
[0365] To a solution of compound 21 (112 mg, 0.113 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (8 mg, 0.340 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over NaiSOi, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the resîdue and the mixture was stirred at room température for another 3 hours. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 874.43, found 875.08.
Structure 6.4b ((S)-3-(4-(4-((35-azido-3,6,9,1245,lS,2L24,27,30,33undecaoxapentatriacontvDoxv)naphthalen-l-vl)phenyD-3-(2-(4-((4-methylpyrÎdin-220 yl)amino)butanamido)acetamido)propanoic acid)
124
[0366] To a solution of compound 69 (80 mg, 0.122 mmol, 1 equiv.) and azido-PEG|2-OTs (184 mg, 0.244 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2COj (80 mg, 0.244 mmol, 2 equiv.) 5 at 0 °C. The reaction mixture was stirred at 40 °C for 5 hours. The reaction was quenched by saturated
NaHCOj solution (10 mL) and the aqueous layer was extractcd with ethyl acetate (3x10 mL). The organîc phase was combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 2-3% methanol in DCM. LCMS: calculated [M+H]+ 1208.63, found 1209.21.
79
[0367] To a solution of compound 82 (100 mg, 0.0972 mmol, L0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (7 mg, 0.292 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, 15 dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator, LC-MS: calculated [M+H]+ 1094.56, 1095.05.
125
Synthesis of Structure 7b ((Rf3-(4~(4-(2-(2-(2~azidoethoxy)ethoxy)ethoxy)naphthalen-lyl)phenyi)-3-(2-(4-((4-methylpyridin-2-yl)aniino)butanamido)acetamido)propanoic acid).
Br 84
[0368] To a solution of compound 84 (1.0 g, 2.90 mmol, 1 equiv.) and potassium carbonate (0.60 g, 4.36 mmol, 1.5 equiv.) in anhydrous DMF (10 mL) was added methyl iodide (362 uL, 5.81 mmol, 2.0 equiv.) at room température. The reaction mixture was stirred at room température 1 hr. LC-MS: calculated [M+H]+ 358.06, found 358.34.
[0369] Compound 85 (1.0 g, 2.791 mmol, 1.0 equiv.) was cooled by ice bath. HCl in dioxane (7.0 mL, 27.91 mmol, 10 equiv.) was added into the flask. The reaction was warmed to room température and stirred for another 1 hour. The solvent was removed by rotary evaporator and the product was directly used without further purification. LC-MS: calculated [M+H]+ 258.01, found 257.97.
[0370] To a solution of compound 64 (790 mg, 2.248 mmol, 1 equiv.), compound 86 (728 mg, 2.473 mmol, 1.10 equiv.), and TBTU (866 mg, 2.698 mmol, 1.20 equiv.) in anhydrous DMF (15 mL) was added diisopropylethylamine (1J 75 mL, 6.744 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched by saturated NalICOj aqueous solution (10 mL) and the product was extracted with ethyl acetate (3 x 20 mL). The organic phase was combined, dried overNaiSCU, and concentrated. The product was purîfied by
126
CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. LC-MS: calculated [M+H]+ 591.17, found 591.49.
[0371] Compound 87 (200 mg, 0.338 mmol, 1.0 equiv.), compound 65 (141 mg, 0.507 mmol, 1-5 5 equiv.), XPhos Pd G2 (5.3 mg, 0.068 mmol, 0.02 equiv.), and K3PO4 (143 mg, 0.676 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 times). Then, THF (8 mL) and water (2 mL) were added via syringe. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at room température for overnight. The reaction was quenched with water 10 (10 mL), and the aqueous phase was extracted with ethyl acetate (3*10 mL). The organic phase was dried overNa2SO4 and concentrated. LC-MS: calculated [M+H]-t- 745.35, found 746.08.
[0372] To a solution of compound 88 (0.247 g, 0.331 mmol, I equiv.) in ethyl acetate (10 mL) was added 10% Pd/C ( 100 mg) at room température. The reaction mixture was stirred at room température 15 for overnight. The catalyst was removed by filtration through Celite® and the product was used directly without further purification. LC-MS: calculated [M+H]+ 655.31, found 655.96.
[0373] To a solution of compound 89 (50 mg, 0.076 mmol, 1 equiv.) and azido-PEGa-OTs (50 mg, 0.152 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (50 mg, 0.152 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred for 72 hr at room température. The reaction was quenched by saturated NaHCOa solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3 x 10 mL), The organic phase was combined, dried over Na2SÛ4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 4% MeOH in DCM. LC-MS: calculated [M+H]+ 812.39, found 813.14.
Structure 7b
[0374] To a solution of compound 90 (36 mg, 0.0443 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (3 mg, 0.133 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3 x 10 mL). The organic phase was combined, dried over NaaSOj, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 698.32, found 698.90.
Synthesis of Structure 8b ((S)-3~(4-(7-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)naphthalen-lyl)phenyl)-3-(2-(4f(4-methylpyridin-2-yl)ammo)butanamido)acetamido)propanoic acid).
62 93
[0375] To a solution of compound 92 (1.0 g, 4.48 mmol, 1 equiv.), and compound 62 (1.06 mL, 8.96 mmol, 2 equiv.) in anhydrous DMF (10 mL) was added K2CO3 (1.24 g, 8.96 mmol, 2 equiv.) at room température. The reaction mixture was stirred at 80 °C overnight. The reaction was quenched by saturated NaHCCh solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SÜ4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 5% ethyl acetate in hexane.
[0376] To a solution of compound 94 (0.5 g, 1.596 mmol, LO equiv.) in anhydrous THF (10 mL) was added n-BuLi in hexane (0.96 mL, 2.394 mmol, 1.5 equiv.) drop-wise at -78 °C. The reaction was kept at -78 °C for another 1 hour. Triisopropylborate (0.553 mL, 2.394 mmol, 1.5 equiv.) was then added into the mixture at -78 °C. The reaction was then warmed up to room température and stirred for another 1 hour. The reaction was quenched by saturated NH4CI solution (20 mL) and the pH was adjusted to 3. The aqueous phase was extracted with EtOAc (3 x 20 mL) and the organic phase was combined, dried overNajSCL, and concentrated. The solid was triturated with hexane and filtered. The product was used directly without further purification. LC-MS: calculated [M-HJ277.11, found 277.35.
[0377] Compound 96 (100 mg, 0.169 mmol, 1.0 equiv.), compound 95 (70 mg, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (2.7 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfiîled with nitrogen (this process was repeated a total of 3 times). Then, THF (8 mL) and water (2 mL) were added via syrînge. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at room température for overnight. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over NasSO4, and concentrated. The compound was separated by CombiFlash® using silica gel as the statîonary phase and was eluted with 3% methanol in DCM.
[0378] To a solution of compound 97 (0.116 g, 0.157 mmol, I equiv.) in ethyl acetate (10 mL) was added 10% Pd/C ( 100 mg) at room température. The reaction mixture was stirred at room température for overnight. The catalyst was removed by filtration through Celite® and the product was used 15 directly without further purification. LC-MS: calculated [M+H]+ 655.3 I, found 655.87.
[0379] To a solution of compound 98 (87 mg, 0.133 mmol, 1 equiv.) and azido-PEGî-OTs (87 mg, 0.266 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2COj (87 mg, 0.266 mmol, 2 equiv.) at room température. The reaction mixture was stirred at 40 °C for 6 hours. The reaction was 5 quenched by saturated NaHCO? solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organîc phase was combined, dried over NazSOi, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% MeOH in DCM. LC-MS; calculated [M+H]+ 812.39, found 813.05.
[0380] To a solution of compound 99 (65 mg, 0.0801 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (6 mg, 0.240 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by FIC1 (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organîc phase was combined, dried over Na2SÛ4, and concentrated, TFA (0.5 mL) and DCM (0.5 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 698.32, found 698.99.
131
Synthesîs of Structure 9b ((14S,17R)-l-azido-14-(4-((4-methylpyridin~2-yl)atnino)butanamido)17-(4-(naphthalen-l -yl)phenyl)-l 5-oxo~3,6,9,12-tetraoxa-16-azanonadecan-19-oic acid).
[0381] Compound 102 (0.19 g, 0.468 mmol, 1.0 equiv.) was cooled by ice bath. HCl in dioxane (2.35 5 mL, 9.37 mmol, 20 equiv.) was added into the flask. The reaction was warmed to room température and stirred for another 1 hr. The solvent was removed by rotary evaporator and the product was directly used without further purification. LC-MS: calculated [M+H]+ 306.14, found 306.51.
103
[0382] To a solution of compound 23 (110 mg, 0.188 mmol, 1 equiv.), compound 103 (71 mg, 0.207 10 mmol, 1.10 equiv.), and TBTU (72.7 mg, 0.226 mmol, 1.20 equiv.) m anhydrous DMF (2 mL) was added diisopropylethylamine (0.1 mL, 0.566 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hour. The reaction was quenched by saturated NaHCOa aqueous solution (10 mL) and the product was extracted with ethyl acetate (3x10 mL). The organic phase w'as combined, dried over NazSCL, and concentrated. The product was purified by 15 CombiFlash® using silîca gel as the stationary phase and was eluted with 3-4% methanol in DCM.
LC-MS: calculated [M+HJ+ 870.43, found 871.12.
[0383] To a solution of compound 104 (110 mg, 0.126 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (9 mg, 0.379 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hour. The pH was adjusted to 3.0 by HCl (6N) 5 and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hours. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 756.36, found 756.88.
Synthesis of Structure 10b ((S)-3~(4-(5-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)riaphthalen-lyl)pheHyl)-3-(2~(4-((4-methylpyridin-2-yl)amîfto)butauamido)acetamido)propanoÎc acid).
Br
[0384] To a solution of compound 106 (1.0 g, 4.48 mmol, I equiv.), and compound 62 (1.06 mL, 8.96 mmol, 2 equiv.) in anhydrous DMF (10 mL) was added CS2CO3 (2.92 g, 8.96 mmol, 2 equiv.) 15 at room température. The reaction mixture was stirred at room température overnight. The reaction was quenched by water solution (20 mL) and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over NajSCh, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 5% ethyl acetate in hexane.
133
[0385] To a solution of compound 107 (1.188 g, 3.793 mmol, 1.0 equiv.) in anhydrous THF (10 mL) was added n-BuLi in hexane (2.27 mL, 5.689 mmol, 1.5 equiv.) drop-wise at -78 °C. The reaction was kept at -78 °C for another 1 hour. Triisopropylborate (1.31 mL, 5.689 mmol, 1-5 equiv.) was then added into the mixture at -78 °C. The reaction was then warmed up to room température and stirred for another 1 hour. The reaction was quenched by saturated NH4CI solution (20 mL) and the pH was adjusted to 3. The aqueous phase was extracted with EtOAc (3 x 20 mL) and the organic phase was combined, dried overNaîSO4, and concentrated. The solid was triturated with hexane and fdtered. The product was used directly without further purification. LC-MS; calculated [M-H]-,
[0386] Compound 96 (100 mg, 0.169 mmol, 1-0 equiv.), compound 108 (70 mg, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (2.7 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 times). Then, THF (8 mL) and water (2 mL) were added via syringe. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at room température for ovemight. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The compound was separated by CombiFlash® using si 1 ica gel as the stationary phase and was eluted with 3% methanol in DCM. LC-MS: calculated [M+H]+ 745.35, found 745.99.
[0387] To a solution of compound 109 (0.135 g, 0.1 SI mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd/C (100 mg) at room température. The reaction mixture was stirred at room température for overnight. The catalyst was removed by filtration through Celite® and the product was used directly without further purification. LC-MS: calculated [M+H]+ 655.31, found 655.87.
[0388] To a solution of compound 110 (50 mg, 0.0764 mmol, l equiv.) and azido-PEG5-OTs (64 mg, 0.152 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (50 mg, 0.152 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCOî solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over NasSOi, and concentrated. The product was purified by CombiFlash® using silica gel as the statîonary phase and was eluted with 4% methanol in DCM. The yield is 62%. LC-MS: calculated [M+H]+ 900.44, found 901.19.
Structure 10b
[0389] To a solution of compound 111 (43 mg, 0.0478 mmol, LO equiv.) in THF (2 mL) and water (2 ntL) was added lithium hydroxide (3.4 mg, 0.143 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) 5 and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 786.37, found 787.04.
Synthesîs of Structure 11b ((S)-3-(4-(4-((14-azîdo-3,6,9,12-tetraoxatetradecyi)oxy)naphthalen-lyl)ph euyl)~3-((S)-l~(4-((4-methyIpyridin-2-yl)amino)butanoyl)pyrrolidine~2carboxamido)propanoic acid).
113
[0390] To a solution of compound 22 (500 mg, 1.698 mmol, 1 equiv.), compound 113 (295 mg, 15 1.783 mmol, 1.05 equiv.), and TBTU (654 mg, 2.038 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (0.888 mL, 5.096 mmol, 3 equîv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched by saturated NaHCOj aqueous solution (10 mL) and the product was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was purified by 20 CombiFlash® using silica gel as the stationary phase and was eluted with 2-3% methanol in DCM.
The yield is 98.72%. LC-MS: calculated [M+H]+ 406.23, found 406.07.
[0391] To a solution of compound 114 (0.68 g, 1.676 mmol, 1 equiv.) in THF (5 mL) and H2O (5 mL) was added lithium hydroxide (0.12 g, 5.030 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for 1 hr, the reaction 5 mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic layer was combined, dried over NazSCL, and concentrated. The product was used without further purification. LC-MS: calculated [M+H]+ 392.21, found 392.39.
[0392] To a solution of compound 115 (300 mg, 0.766 mmol, I equiv.), compound 116 (237 mg, 10 0.804 mmol, 1.05 equiv.), and TBTU (295 mg, 0.919 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (0.400 mL, 2.299 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched by saturated NaHCOa aqueous solution (10 mL) and the product was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried overNazSCL, and concentrated. The product was purified by 15 CombiFlash® using silica gel as the stationary phase and was ekited with 3-4% methanol in DCM.
The yîeld is 83%. LC-MS: calculated [M+H]+ 631.21, found 63L46.
[0393] Compound 118 (100 mg, 0.158 mmol, 1.0 equiv.), compound 65 (66 mg, 0.237 mmol, 1.5 equiv.), XPhos Pd G2 (2.5 mg, 0.0032 mmol, 0.02 equiv.), and K3PO4 (67 mg, 0.316 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 tîmes). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at 40 °C for 1 hr. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The compound was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 3% methanol in DCM. The yield was 96%. LC-MS: calculated [M+H]+ 785.38, found 785.69.
[0394] To a solution of compound 119 (0.120 g, 0.153 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd/C (100 mg) at room température. The reaction mixture was stirred at room température
for overnîght. The catalyst was removed by filtration through Celite® and the product was used directly without further purification. LC-MS: calculated [M+H]+ 695.34, found 695.66.
[0395] To a solution of compound 120 (83 mg, 0.119 mmol, 1 equiv.) and azido-PEG^-OTs (100 5 mg, 0.239 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (78 mg, 0.239 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCCh solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 10 4% methanol in DCM. The yield was 79%. LC-MS: calculated 940.47, found 941.16.
[0396] To a solution of compound 121 (89 mg, 0.0947 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (6.8 mg, 0.284 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over NaîSO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 826.41, found 827.10.
Synthesis of Structure 12b ((S)-3-(4-(4-((14~azido-3,6,9,12tetraoxatetradecyl)oxy)benzo[d]oxazol- 7-yl)ph enyl)-3-(2-(4-((4-methylpyridin-2yl)amino)butanamido)acetamido)propanoic acid).
[0397] To a solution of compound 123 (1.0 g, 7.40 mmol, 1 equiv.), and compound 62 (1.32 mL, 11.10 mmol, 1.5 equiv.) in anhydrous DMF (10 mL) was added CS2CO3 (3.62 g, 11.10 mmol, 1.5 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred overnight. The reaction was quenched by w'ater (10 mL). The aqueous phase was extracted with ethyl acetate (3 x 10 mL) and the organic phase was combined, dried over anhydrous Na2SÛ4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 5-7% ethyl acetate in hexane. 85% yield.
Br
[0398] To a solution of compound 124 (1.425 g, 6.326 mmol, 1 equiv.) in anhydrous acetonitrile (20 mL) was added N-bromosuccinimide (1.216 g, 6.832 mmol, 1.08 equiv.) at 0 °C portion-wise. The reaction mixture was kept at 0 °C for another 30 min and then allow'ed to warm to room température and stirred overnight. The solvent was removed under reduced pressure and the residue was purified by CombiFlash® using silica gel as the stationary phase. The product was eluted with 4-5% ethyl 15 acetate in hexane. 65% yield. LC-MS: calculated [M+H]+ 303.99. found 304.08.
[0399] The mixture of compound 125 (1.339 g, 4.402 mmol, l equiv.), bis(pinacolato)diboron (2.236 g, 8.805 mmol, 2 equiv.), potassium acetate (0.864 g, 8.805 mmol, 2 equiv.) and Pd(dppf)Cl2 (161 mg, 0.220 mmol, 0.05 equiv.) in 15 mL of anhydrous 1,4-dioxane was stirred at 100 °C under
140 nitrogen for S hours. After concentration, the residue was partitioned between H2O and DCM, the aqueous phase was extracted with DCM, and the combined organic layer was washed with brine, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 15-20% ethyl acetate in hexane. LC-MS: calculated [M+H]+
[0400] Compound 96 (200 mg, 0.338 mmol, 1.0 equiv.), compound 126 (178 mg, 0.507 mmol, 1.5 equiv.), XPhos Pd G2 (5.3 mg, 0.0068 mmol, 0.02 equiv.), and K3PO4 (143 mg, 0.676 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then 10 evacuated and backfilled with nitrogen (this process was repeated a total of 3 tîmes). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at 40 °C for 1 hr. The reaction was quenched with saturated NaHCOj (10 mL), and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over Na2SÜ4, and concentrated. The compound w'as separated by CombiFlash® 15 using silica gel as the stationary phase and was eluted with 2-3% methanol in DCM. LC-MS:
calculated [M+H]+ 736.33, found 736.89.
141
[0401] To a solution of compound 127 (0.219 g, 0.297 mmol, 1 equiv.) in ethyl acetate ( 10 mL) was added 10% Pd/C (100 mg) at room température. The reaction mixture was stirred at room température overnight. The catalyst was removed by filtration through Celite® and the product was used directly without further purification. LC-MS: calculated [M+H]+ 646.28, found 646.78.
[0402] To a solution of compound 128 (73 mg, 0.113 mmol, I equiv.) and azido-PEGs-OTs (94 mg, 0.226 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (74 mg, 0.226 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCOs solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the statîonary phase and was eluted with 4% methanol in DCM. The yield is 80%. LC-MS: calculated [M+H]+ 891.42, found 892.00.
[0403] To a solution of compound 129 (43 mg, 0.0478 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (3.4 mg, 0.143 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for I hour. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over NaiSOj, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LCMS: calculated [M+H]+ 777.35, found 777.94.
142
Synthesis of Structure 13b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5,6,7,8tetrahydronaphthalen-l-yl)phenyl)-3-(2-(4-((4-methylpyridin-2yl)amino)butanamido)acetatnido)propanoic acid).
B2pin2
Br KOAc
Pd(dppf)Ci2
OH 5
[0404] The mixture of compound I (300 mg, 1.321 mmol, 1 equiv.), bis(pinacolato)diboron (671 mg, 2.642 mmol, 2 equiv.), potassium acetate (389 mg, 3.963 mmol, 2 equiv.) and Pd(dppf)Ch (48 mg, 0.066 mmol, 0.05 equiv.) in 10 mL of anhydrous 1,4-dioxane was stirred at 80 °C under nitrogen overnight. After concentration, the residue was partitioned between H2O and DCM, the aqueous 10 phase was extracted with DCM, and the combined organic layer was washed with brîne, dried over
Na2SO4, and concentrated. The product was purified by CombiFlash® usîng silica gel as the stationary phase and was eluted with 10% ethyl acetate in hexane. LC-MS: calculated [M-H]- 273.17, found 273.29.
[0405] Compound 1 (100 mg, 0.169 mmol, 1.0 equiv.), compound 2 (70 mg, 0.253 mmol, 1.5 equiv.),
XPhos Pd G2 (2.7 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfllled with nitrogen (this process was repeated a total of 3 times). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at 40 °C for 3 hr. The reaction was then cooled to room température and left overnight. The reaction was quenched with saturated NaHCCL (10 mL), and the aqueous phase was extracted with ethyl acetate (3*10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The compound was separated by CombiFlash® using silica gel as the stationary
[0406] To a solution of compound I (30 mg, 0.0455 mmol, I equiv.) and azido-PEGs-OTs (38 mg, 5 0.091 1 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (30 mg, 0.091 1 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCÛ3 solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over NasSOi, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 10 4% methanol in DCM. The yield is 70%. LC-MS: calculated [M+H]+ 904.47, found 904.88.
[0407] To a solution of compound 1 (29 mg, 0.0321 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (2.3 mg, 0.0962 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over NazSÛ4, and concentrated. TFA (4 mL) and DCM (2 mL) were added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 790.41, found 790.64.
144
Synthesis of Structure 14b ((S)-3-(4'f(14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-2r(trifluoromethoxy)-[lrl r-biphenyl]-4-yl)-3-(2-(4-((4-methylpyridin-2yl)amino)butanamido)acetamido)propanoic acid).
[0408] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (118 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nîtrogen (this process was repeated a total of 3 times). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 10 min 10 and the reaction was kept at 40 °C for overnight. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 * 10 mL). The organic phase was combined, dried over NaiSOi, and concentrated. The compound was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 2-4% methanol in DCM. LC-MS:
calculated [M+H]+ 779.32, found 779.65.
[0409| To a solution of compound 1 (0.19 g, 0.244 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd/C (100 mg) at room température. The reaction was evacuated and backfilled with
145 hydrogen (this process was repeated for 3 times.). The reaction mixture was stirred at room température for overnight. The catalyst was removed by filtration through Celite® and the product was used directly without further purification. LC-MS: calculated [M+H]+ 689.27, found 689.54.
[0410] To a solution of compound 1 (80 mg, 0.116 mmol, 1 equiv.) and azido-PEGs-OTs (97 mg, 0.232 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CssCOs (76 mg, 0.232 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCCh solution (10 mL) and the aqueous layer was extracted with ethyl 10 acetate (3x5 mL). The organîc phase was combined, dried over NazSOi, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 34% methanol in DCM. The yîeld was 82%. LC-MS: calculated [M+H]+ 934.41, found 935.04.
[0411] To a solution of compound 1 (90 mg, 0.0964 mmol, 1.0 equiv.) in THF (2 mL) and water (2 15 mL) was added lithium hydroxide (7 mg, 0.289 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organîc phase was combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC20 MS: calculated [M+H]+ 820.34, found 820.89.
146
Synthesis of Structure 15b ((S)-3-(3-(5-((14-azido-3f6,9,12-tetraoxatetradecyl)oxy)naphthalen-lyl)phenyl)-3-(2-(4-((4-methyIpyridin-2-yl)amino)butanamido)acetanùdo)propanoic acid).
Mel
K2CO3
[0412] To a solution of compound 1 (1.0 g, 2.90 mmol, I equiv.) and potassium carbonate (0.60 g, 4.36 mmol, 1.5 equiv.) in anhydrous DMF (10 mL) was added methyl iodide (362 uL, 5.81 mmol, 2.0 equiv.) at room température. The reaction mixture was stirred at room température for 1 hr. The reaction was then quenched with water (20 mL) and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over anhydrous Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 15% ethyl acetate in hexane. LC-MS: calculated [Μ+Ή]+ 358.06, found 358.18.
[0413] Compound 1 (858 mg, 1.677 mmol, 1.0 equiv.) was cooled by ice bath. HCl in dioxane (8.4 mL, 33.54 mmol, 20 equiv.) was added into the flask. The reaction was warmed to room température and stirred for another 1 hr. The solvent was removed by rotary evaporator and the product was directly used without further purification. LC-MS: calculated [M+H]+ 258.01, found 258.08.
[0414| To a solution of compound 1 (640 mg, 1.821 mmol, 1 equiv.), compound 2 (590 mg, 2.003 mmol, 1.10 equiv.), and TBTU (702 mg, 2.185 mmol, 1.20 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (0.952 mL, 5.464 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched by saturated NaHCOi aqueous solution (10 mL) and the product was extracted with ethyl acetate (3x10 mL).
147
The organic phase was combined, dried overNajSOi, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. LC-MS: calculated [M+H]+ 591.17, found 591.40.
[0415] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (106 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and KjPCL (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 tîmes). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 10 min 10 and the reaction was kept at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3*10 mL). The organic phase was combined, dried overNaiSCL, and concentrated. The compound was separated by CombiFlash® usîng silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. LC-MS: calculated [M+H]+
745.35, found 745.99.
[0416] To a solution of compound 1 (0.189 g, 0.253 mmol, I equiv.) in ethyl acetate (10 mL) was added 10% Pd/C (100 mg) at room température. The reaction was evacuated and backfilled with hydrogen (this process was repeated for 3 times.). The réaction mixture was stirred at room
148 température for overnight. The catalyst was removed by filtration through Celite® and the product was used directly without further purification. LC-MS: calculated [M+H]+ 655.31, found 655.42.
[0417] To a solution of compound 1 (80 mg, 0.122 mmol, 1 equiv.) and azido-PEGs-OTs (102 mg, 5 0.244 mmol, 2 equiv,) in anhydrous DMF (2 mL) was added CS2CO3 (80 mg, 0.244 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCOi solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SÛ4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 110
2% methanol in DCM. The yield is 90%. LC-MS: calculated 900.44, found 901.10.
[0418] To a solution of compound 1 (100 mg, 0.111 mmol, LO equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (8 mg, 0.333 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the 15 aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LCMS: calculated [M+H]+ 786.37, found 786.95.
149
Synthesis of Structure 16b ((S)-3-(4-(4-((14-azido-3,6>9>12-tetraoxatetradecyl)oxy)naphthalen-lyl)pb eny 1)-3-((R)-1-(4-((4-methylpyridin-2-yl)amino)butanoyl)pyrrolidine-2carboxamido)propanoic acid).
[0419] To a solution of compound 1 (500 mg, 1.698 mmol, l equiv.), compound 2 (295 mg, 1.783 mmol, 1.05 equiv.), and TBTU (654 mg, 2.038 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (0.888 mL, 5.096 mmol, 3 equîv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched by saturated NaHCOj aqueous solution (10 mL) and the product was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried overNa2SÛ4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 2-3% methanol in DCM. The yield is 98.43%. LC-MS: calculated [M+H]+ 406.23, found 406.34.
[0420] To a solution of compound 1 (0.678 g, 1.672 mmol, 1 equiv.) in THF (10 mL) and H2O (10 mL) was added lithium hydroxide (0.12 g, 5.016 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for 1 hr, the reaction mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic layer was combined, dried over NaiSO4, and concentrated. The product was used without further purification. LC-MS: calculated [M+H]+ 392.21, found 392.39.
[0421] To a solution of compound 1 (130 mg, 0.332 mmol, 1 equiv.), compound 2 (125 mg, 0.348 mmol, 1.05 equiv.), and TBTU (128 mg, 0.398 mmol, 1.2 equiv.) in anhydrous DMF (5 mL) was added diisopropylethylamine (0.174 mL, 0.996 mmol, 3 equiv.) at 0 °C. The reaction mixture was 5 warmed to room température and stîrred for another 1 hr. The reaction was quenched by saturated NaHCOj aqueous solution (10 mL) and the product was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried overNa2SO4, and concentrated. The product was purîfied by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. The yield is 86%. LC-MS: calculated [M+H]+ 695.34, found 695.93.
[0422] To a solution of compound 1 (80 mg, 0.115 mmol, 1 equiv.) and azido-PEG5-OTs (96 mg, 0.230 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (75 mg, 0.230 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCO3 solution (10 mL) and the aqueous layer was extracted with ethyl 15 acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 45% methanol in DCM. The yield is 60%.
151
[0423] To a solution of compound 1 (65 mg, 0.0691 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (5 mg, 0.207 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SÛ4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the resΐdue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LCMS: calculated [M+H]+ 826.41, found 827.01.
Synthesis of Structure 17b ((S)-3-(4-(7-((14-azido-3,6,9,12tetraoxatetradecyl)oxy)benzo[b]thiophen~4-yl)phenyl)-3-(2-(4-((4-methyipyridin-2yi)amino)hutanamido)acetamido)propanoic acid).
Br
[0424] A solution of bromine (1.877 g, 1 1.745 mmol, 1.05 equiv.) in dry tetrachloromethane (20 15 mL) was added dropwise during 1.5 hours to a stirred solution of compound I (1.837 g, 11.186 mmol, 1 equiv.) in tetrachloromethane (20 mL) at 0 0 C. After a further hour at 0 ° C, the organic layer was washed with water and brine, dried over Na2SO4, concentrated to give a residue, which was purified by CombiFlash® using silica gel as the stationary phase. The product was eluted with pure hexane with impurities.
152 ]0425] To a dichloromethane (20 ml) solution of compound 1 (2.70 g, 11.105 mmol, 1.0 equiv.), under nitrogen atmosphère, at 0 °C, boron trifluoride dimethyl sulfide complex (3.5 mL, 33.317 mmol, 3.0 equiv.) was added and stirred at room température for 20 hours. The reaction mixture was cooled to 0 °C and quenched with saturated NH4CI solution (20 mL). The aqueous phase was 5 extracted with ethyl acetate (3 x 20 mL) and the organic phase was combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using si 1 ica gel as the stationary phase and was eluted with 5% ethyl acetate in hexane. LC-MS: calculated [M-H]- 226.92, found 227.03.
[0426] To a solution of compound 1 (1.838 g, 8.023 mmol, 1 equiv.), and compound 2 (1.906 mL, 10 16.04 mmol, 2 equiv.) in anhydrous DMF (10 mL) was added CszCOj (5.228 g, 16.04 mmol, 2 equiv.) at room température. The reaction mixture was stirred at room température ovemight. The reaction was quenched by water (20 mL) and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over NazSOi, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 2-3% ethyl acetate in 15 hexane.
[0427] To a solution of compound 1 (2.22 g, 6.954 mmol, LO equiv.) in anhydrous THF (20 mL) was added n-BuLi in hexane (4.17 mL, 10.43 mmol, 1.5 equiv.) drop-wise at -78 °C. The reaction was kept at -78 °C for another I hr. Triisopropylborate (2.40 mL, 10.43 mmol, 1.5 equiv.) was then added into the mixture at -78 °C. The reaction was then warmed up to room température and stirred for another 1 hr. The reaction was quenched by saturated NH4CI solution (20 mL) and the pH was adjusted to 3. The aqueous phase was extracted with EtOAc (3 x 20 mL) and the organic phase was combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using
153 siIica gel as the stationary phase and was eluted with 4-6% methanol in DCM. LC-MS: calculated
[M-H]- 283.07, found 283.20.
[0428] Compound 1 (400 mg, 0.676 mmol, 1.0 equiv.), compound 2 (288 mg, 1.01 mmol, 1.5 equiv.), 5 XPhos Pd G2 (10 mg, 0.0135 mmol, 0.02 equiv,), and K3PO4 (287 mg, 1.352 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilied with nitrogen (this process was repeated a total of 3 times). Then, THF (8 mL) and water (2 mL) were added via syringe. The mixture was bubbled with nitrogen for 10 min and the reaction was kept at 40 °C for 2 hours. The reaction was quenched with saturated NaHCOj solution 10 (10 mL) , and the aqueous phase was extracted with ethyl acetate (3 * 10 mL). The organic phase was combined, dried overNasSCU, and concentrated. The compound was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. LC-MS: calculated [M+H]+ 751.3 1, found 751.84.
[0429] To a solution of compound 1 (0.50 g, 0.666 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd/C (100 mg) at room température. The reaction was evacuated and backfilled with hydrogen (this process was repeated for 3 times.), The reaction mixture was stirred at room
154
température for overnight. The catalyst was removed by filtration through Celite® and the product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 5% methanol in DCM. LC-MS: calculated [M+H]+ 661.26, found 661.73.
[0430] To a solution of compound 1 (130 mg, 0.196 mmol, 1 equiv.) and azido-PEG5-OTs (164 mg,
0.393 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2COj (128 mg, 0.393 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCCh solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 34% methanol in DCM. The yield is 82%. LC-MS: calculated [M+H]+ 906.40, found 906.95.
[0431] To a solution of compound I (147 mg, 0.162 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (12 mg, 0.486 mmol, 3.0 equiv.) at room température. The mixture 15 was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (2 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: 20 calculated [M+H]+ 792.33, found 792.89.
155
Synthesis of Structure 18b ((S)-3-(4-(6-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-2yl)phenyl)-3~(2-(4-((4-methylpyrîdin~2~yl)atnino)butanamido)acetamido)propanoic acid).
[0432] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (71.5 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfllled with nitrogen (thîs process was repeated a total of 3 times). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 10 min 10 and the reaction was kept at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried overNasSCL, and concentrated. The compound was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 2-3% méthanol in DCM. LC-MS: calculated [M+H]+ 655.31, found 655.87.
[0433] To a solution of compound 1 ( 160 mg, 0.244 mmol, 1 equiv.) and azido-PEG5-OTs (204 mg, 0.488 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CsKOî (160 mg, 0.488 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 60 °C. The reaction was quenched by saturated NaHCO3 solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over NaîSCU, and concentrated. The
156
product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 34% methanol in DCM. The yield was 30%. LC-MS: calculated [M+U]+ 900.44, found 901.01.
[0434] To a solution of compound 1 (67 mg, 0.0744 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (5 mg, 0.223 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH wras adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over NajSCL, and concentrated. TFA (2 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 10% methanol in DCM. LC-MS: calculated [M+H]+ 786.37, found 786.86.
Synthesis of Structure 19b ((S)-3-(3-(6-((14-azido-3,6>9J2-tetraoxatetradecyl)oxy)naphthalen-2yl)ph enyl)-3-(2-(4-((4-methylpyrtdin-2-yl)amino) butanamido)acetamido)propanoic acidf
[0435] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (71.5 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K.3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 times). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 10 min and the reaction was kept at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined,
157
dried overNasSO*, and concentrated. The compound was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 2-3% methanol in DCM. LC-MS: calculated [M+H]+
655.31, found 655.78.
[0436] To a solution of compound 1 (104 mg, 0.158 mmol, 1 equiv.) and azido-PEGs-OTs (132 mg,
0.317 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (103 mg, 0.317 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 60 °C. The reaction was quenched by saturated NaHCOi solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over NajSCU, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 34% methanol in DCM. LC-MS: calculated [M+H]+ 900.44, found 901.01.
[0437] To a solution of compound 1 (125 mg, 0.138 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (10 mg, 0.416 mmol, 3.0 equiv.) at room température. The mixture 15 was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 20 12% methanol in DCM. LC-MS: calculated [M+H]+ 786.37, found 786.86.
158
Synthesis of Structure 20b ((S)-3-(3-(4-((14-azido~3,6t9,12~tetraoxatetradecyl)oxy)naphthalen-Iyl)phenyl)-3-(2~(4-((4-methylpyridin~2~yl)amino)butanamido)acetamido)propanoic acid).
[0438] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (102 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 times). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 10 min 10 and the reaction was kept at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethy 1 acetate (3 χ 10 mL). The organîc phase was combined, dried overNasSOi, and concentrated. The compound was separated by CombiFlash® using si 1 ica gel as the stationary phase and was eluted with 2-3% methanol in DCM. LC-MS: calculated [M+H]+ 655.31, found 655.78.
[0439] To a solution of compound I (160 mg, 0.244 mmol, 1 equiv.) and azido-PEG5-OTs (204 mg, 0.488 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 ( 159 mg, 0.488 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 60 °C. The reaction was quenched by saturated NaHCCh solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organîc phase was combined, dried over NazSCh, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 34% methanol in DCM. LC-MS: calculated [M+H]+ 900.44, found 901.01.
[0440] To a solution of compound 1 (125 mg. 0.138 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (10 mg, 0.416 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the 5 aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over
NazSO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 8-12 % methanol in DCM. LC-MS: calculated [Μ+Η]+ 786.37, found 786.86.
Synthesis of Structure 22b ((S)-3-(4f4-((14-azido-3>6,9>12-tetraoxatetradecyl)oxy)naphthalen-lyl)phenyl)-3-((S)-2-(4-((4-methylpyriditi-2-yl)amino)butanamÎdo)propanamido)propanoic acid).
Compound 1 Compound 2
[0441] To a solution of compound 1 (250 mg, 0.85 mmol), L-alanine methyl ester hydrochloride sait (130 mg, 0.93 mmol), and TBTU (327 mg, 1.02 mmol) in DMF (2 mL) was added DIPEA (329 mg, 444 pL, 2.55 mmol) at 0 °C. The reaction mixture was warmed to room température and stirred for 1 hour. The réaction was quenched with sat. NH4CI (aq) solution (0.75 mL) and deionized water (1 mL) then extracted with ethyl acetate (3 mL), The aqueous layer was further extracted with ethyl acetate (2x3 mL). The combined organic phase was washed with sat. NaHCOa (aq) solution (2 mL). The organic layer was dried over NazSCM, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 2: 294 mg (91%). [M+H] calculated for C19H29NÎO5: 380.46, found: 380.33.
Compound 2
Compound 3
[0442] To a solution of compound 2 (294 mg, 0.77 mmol) in THF (4.5 mL) and deionized water (3 mL) at 0 °C was added a solution of lithium hydroxide (56 mg, 2.32 mmol) in deionized water (1 mL). The reaction was warmed to room température and stirred for 40 minutes. The reaction mixture was acidified to pH=3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3x10 mL). The combined organic phase was dried over NazSCU, filtered, and concentrated. Compound 3 was used without further purification. Yield of compound 3: 267 mg (94%). [M+H] calculated for Ci^NjOs: 366.43, found: 366.19.
Compound 3
DIEA
Compound 4
[0443] To a solution of compound 3 (267 mg, 0.73 mmol), compound 3a (288 mg, 0.80 mmol), and
TBTU (282 mg, 0.88 mmol) in DMF (3 mL) was added DIPEA (283 mg, 382 pL, 2.19 mmol) at 0 °C. The reaction mixture was warmed to room température and stirred for 1 hour. The réaction mixture was quenched with sat. NHæl (aq) solution (1.5 mL) and deionized water (1.5 mL) then extracted with ethyl acetate (12 mL). The aqueous layer was further extracted with ethyl acetate (2 x 12 mL). The combined organic phase was washed with half sat. NH4CI (aq) solution (10 mL), half sat. NaHCO3 (aq) solution (10 mL), and sat. NaCl (aq) solution (10 mL). The organic layer was dried over NazSCU, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 4: 342 mg (70%). [M+H] calculated for C38H44N4O7: 669.79, found: 669.74.
161
Compound 4
Compound 5
[0444] To a solution of compound 4 (150 mg, 0.22 mmol) and azido-PEGj-OTs (187 mg, 0.49 mmol) in anhydrous DMF ( 1.2 mL) was added Cs2COj (146 mg, 0.49 mmol). The reaction mixture was stirred at 60 °C for 3 hours. The reaction mixture was quenched with sat. NaHCCh (aq) solution (10 mL) and deionized water (5 mL) then extracted with ethyl acetate (7.5 mL). The aqueous layer was further extracted with ethyl acetate (2 x 7.5 mL). The combined organic phase was dried over NajSCL, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-4% methanol in DCM. Yield of compound 5: 142 mg (69%). [M+H] calculated for CasHôsNïOh: 915.06, found: 914.96.
1Q Compound 5 Structure 22b
[0445] To a solution of compound 5 ( 142 mg, 0.16 mmol) in THF (2 mL) and deionized water ( 1.5 mL) at 0 °C was added a solution of lithium hydroxide (11 mg, 0.47 mmol) in deionized water (6.5 mL). The reaction was warmed to room température and stirred for 1 hour. The reaction mixture was acidified to pH=3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3 x 15 8 mL). The combined organic phase was dried over NajSCX filtered, and concentrated. To the crude residue was added TFA (2.0 mL) and water (100 pL). The reaction mixture was stirred for
1.5 hours at room température. The solvent was removed under reduced pressure, and the residue was coevaporated with acetonitrile:toluene [1:1] (2 x 20 mL). The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-13% methanol in DCM. Yield of
Structure 22b: 100 mg (80%). [M+H] calculated for C42H53N7O9: 800.92, found: 800.81.
162
Synthesis of Structure 23b ((S)-3~(4-(4f(14-azido-3,6,9,12-tetraoxatetradeeyl)oxy)naphthalen-lyi)phenyl)-3-((S)-3-methyl-2-(4f(4-methylpyridin-2yl)amino)butanamido)butanamido)propanoic acid).
Compound 1 Compound 2
[0446] To a solution of compound 1 (250 mg, 0.85 mmol), L-vaiine methyl ester hydrochloride sait (157 mg, 0.93 mmol), and TBTU (327 mg, 1.02 mmol) in DMF (2 mL) was added DIPEA (329 mg, 444 gL, 2.55 mmol) at 0 °C. The réaction mixture was warmed to room température and stirred for 1 hour. The reaction was quenched with sat. NH4C1 (aq) solution (0.75 mL) and deionized water (1 mL) then extracted with ethyl acetate (3 mL). The aqueous layer was further extracted with ethyl acetate (2x3 mL). The combined organic phase was washed with sat. NaHCOs (aq) solution (2 mL). Ί he organic layer was dried over NasSOi, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silîca gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 2: 297 mg (86%). [M+H] calculated for C21H33N3O5: 408.5 I, found: 407.87.
Compound 2 _
Compound 3
[0447] To a solution of compound 2 (297 mg, 0.73 mmol) in THF (4.5 mL) and deionized water (3 mL) at 0 °C was added a solution of lithium hydroxide (52 mg, 2.19 mmol) in deionized water (1 mL). The reaction was warmed to room température and stirred for 40 minutes. The reaction mixture was acidified to pH=3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3x10 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. Compound 3 was used without further purification assuming 100% yield. [M+H] calculated for C2ÛH31N3O5: 394.49, found: 393.83.
Compound 4
[0448] To a solution of compound 3 (287 mg, 0.73 mmol), compound 3a (287 mg, 0.80 mmol), and TBTU (281 mg, 0.88 mmol) in DMF (3 mL) was added DIPEA (283 mg, 382 pL, 2.19 mmol) at 0 °C. The reaction mixture was warmed to room température and stirred for I hour. The reaction mixture was quenched with sat. NH4CI (aq) solution (2.5 mL) and deionized water (2.5 mL) then extracted with ethyl acetate (12 mL). The aqueous layer was further extracted with ethyl acetate (2 x 12 mL). The combined organic phase was washed with half sat. NH4CI (aq) solution (10 mL), half sat. NaHCOs (aq) solution (10 mL), and sat. NaCl (aq) solution (10 mL). The organic layer was dried over Na2SÛ4, filtered, and concentrated. The crude mixture was separated by
CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 4: 374 mg (74%). [M+H] calculated for C40H4SN4O7: 697.84, found: 697.46.
Compound 4
Compound 5 (0449] To a solution of compound 4 (150 mg, 0.215 mmol) and azido-PEGs-OTs (180 mg, 0.43 mmol) in anhydrous DMF (1.2 mL) was added Cs2CO3 (140 mg, 0.43 mmol). The reaction mixture was stirred at 60 °C for 3 hours. The reaction mixture was quenched with sat. NaHCO3 (aq) solution (10 mL) and deionized water (5 mL) then extracted with ethyl acetate (7.5 mL). The aqueous layer was further extracted with ethyl acetate (2 x 7.5 mL). The combined organic phase was dried overNa2SO4, filtered, and concentrated. The crude mixture was separated by
164
CombiFlash® using silica gel as the stationary phase with 0-4% methanol in DCM. Yield of compound 5: 134 mg (66%). [M+H] calculated for C50H67N7O11: 943.12, found: 942.96.
Structure 23b
Compound 6
[0450] To a solution of compound 5 (134 mg, 0.14 mmol) in THF (2 mL) and deionized water (1.5 5 mL) at 0 °C was added a solution of lithium hydroxide (10 mg, 0.43 mmol) in deionized water (0.5 mL). The reaction was warmed to room température and stirred for 1 hour. The reaction mixture was acidified to pH=3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3 x 8 mL). The combined organic phase was dried overNazSCX filtered, and concentrated. To the crude residue was added TFA (1.9 mL) and water (95 pL). The reaction mixture was stirred for 1.5 10 hours at room température. The solvent was removed under reduced pressure, and the residue was coevaporated with acetonitrile:toluene [1:1] (2 x 20 mL). The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-10% methanol in DCM. Yield of Structure 23b: 36 mg (30.5%). [M+H] calculated for C44H57N7O9: 828.97, found 828.90.
Synthesis oj Structure 24b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-lyl)phenyl)-3-((S)-2-(4-((4-methylpyridin-2-yl)amino)hutanamido)-3~ phenylpropanantido)propanoic acid).
[0451] To a solution of compound 1 (200 mg, 0.679 mmol, 1 equiv.), compound 2 (161 mg, 0.747 20 mmol, 1.2 equiv.), and TBTU (261 mg, 0.815 mmol, 1.2 equiv.) in anhydrous DMF (4 mL) was added diisopropylethylamine (0.355 mL, 2.038 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCCh solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was separated by
165
CombiFlash® using silica gel as the stationary phase and was eluted with 2-3 % methanol in DCM.
LC-MS: calculated [M+H]+ 456.24, found 456.12.
[0452] To a solution of compound 1 (300 mg, 0.658 mmol, 1 equiv.) tn THF (5 mL) and H2O (5 mL) 5 was added lithium hydroxide (47 mg, 1.975 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for 1 hr, the reaction mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic layer was combined, dried over Na2SO4, and concentrated. The product was used without further purification. LC-MS: calculated [M+H]+ 442.23, found 442.08.
OH
[0453] To a solution of compound 1 (290 mg, 0.656 mmol, I equiv.), compound 2 (258 mg, 0.722 mmol, 1.1 equiv.), and TBTU (253 mg, 0.788 mmol, 1.2 equiv.) in anhydrous DMF (5 mL) was added diisopropylethylamine (0.343 mL, 1.970 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated 15 NaHCOs solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over NasSCL, and concentrated. The product was separated by CombiFlash® using silica gel as the stationaty phase and was eluted with 3-4 % methanol in DCM. LC-MS: calculated [M+H]+ 745.35, found 745.63.
166
[0454] To a solution of compound 1(113 mg, 0.151 mmol, 1 equiv.) and azîdo-PEG5-OTs (126 mg, 0.303 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (99 mg, 0.303 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCOj solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the statîonary phase and was eiuted with 34% methanol in DCM. LC-MS: calculated [M+H]+ 990.49, found 990.87.
Structure 24b
[0455] To a solution of compound 1 (140 mg, 0.141 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (10 mg, 0.424 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another ] hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the statîonary phase and eiuted with 6-10 % methanol in DCM. LC-MS: calculated [M+H]+ 876.42, found 876.88.
Synthesis of Structure 25b ((S)-3-(4-(4-((14-azido-3,6,9,I2-tetraoxatetradecyl)oxy)naphthalen-l20 yl)phenyl)-3-((S)-3- (benzyloxy)-2-(4-((4-methylpyridiu-2yl)amino)butanamido)propanamido)propanoic acid).
167
[0456] To a solution of compound 1 (100 mg, 0.339 mmol, 1 equiv.), compound 2 (92 mg, 0.373 mmol, 1.1 equiv.), and TBTU (131 mg, 0.407 mmol, 1.2 equiv.) in anhydrous DMF (4 mL) was added diisopropylethylamine (0.178 mL, 1.019 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCOs solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 2-4 % methanol in DCM. LC-MS: calculated [M+H]+ 486.25, found 486.37.
[0457] To a solution of compound 1 (160 mg, 0.329 mmol, 1 equiv.) in THF (5 mL) and H2O (5 mL) was added lithium hydroxide (23 mg, 0.988 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for I hr, the reaction mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate IS (3x10 mL) and the organic layer was combined, dried over Na2SO4, and concentrated. The product was used without further purification. LC-MS: calculated [M+H]+ 472.24, found 472.32.
OH
[0458] To a solution of compound 1 (1600 mg, 0.339 mmol, 1 equiv.), compound 2 (133 mg, 0.373 mmol, 1.1 equiv.), and TBTU (130 mg, 0.815 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was 20 added diisopropylethylamine (0.177 mL, 1.018 mmol, 3 equiv.) at 0 °C. The reaction mixture was
168 warmed to room température and stirred for another I hr. The reaction was quenched with saturated NaHCOj solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 2-3 % methanol in DCM.
LC-MS: calculated [M+H]+ 775.36, found 775.87.
[0459] To a solution of compound 1 (140 mg, 0.180 mmol, I equiv.) and azido-PEG5-OTs (150 mg, 0.361 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (117 mg, 0.361 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hours at 40 °C. The reaction was quenched by saturated NaHCCL solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over NajSCL, and concentrated. The product was purîfied by CombiFlash® using silica gel as the stationary phase and was eluted with 34% methanol in DCM. LC-MS: calculated [M+H]+ 1020.50, found 1020.88.
[0460] To a solution of compound 1 (170 mg, 0.166 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (12 mg, 0.499 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hours. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) was added into the residue and the mixture
169 was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 6-10 % methanol in DCM. LC-MS: calculated [M+H]+ 906.43, found 906.95.
Synthesis of Structure 27b ((S)-3-(3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3f5-dimethyilH-pyrazoi-l-yl)phenyl)-3-(2-(4~((4-methylpyridin-2-yl)amino)butanamido) acetamido)propanoic acid).
Me!
K2CO3
[0461] To a solution of compound 1 (3.0 g, 8.71 mmol, 1 equiv.) and potassium carbonate (1.806 g, 13.073 mmol, 1.5 equiv.) in anhydrous DMF (10 mL) was added methyl iodide (1.085 mL, 17.431 mmol, 2.0 equiv.) at room température. The reaction mixture was stirred at room température for 1 hr. The reaction was then quenched with water (20 mL) and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combîned, dried over anhydrous NazSCh, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 15% ethyl acetate in hexane. LC-MS: calculated [M+H]+ 358.06, found 358.15.
Cul K2CO3 C8H1BN2
[0462] The mixture of compound I (200 mg, 0.558 mmol, I equiv.), compound 2 (169 mg, 0.837 20 mmol, 1,5 equiv.), copper (1) iodide (106 mg, 0.558 mmol, LO equiv.), potassium carbonate (154 mg, 1.116 mmol, 2.0 equiv.) and trans-N,N'-dimethylcyclohexane- L2-diamine (88 pL, 0.558 mmol, 1.0 equiv.) in anhydrous DMF (5 mL) was backfilled with nitrogen 3 times. The mixture was stirred at 120 °C for 24 hrs. The mixture was cooled to room température and was
170 concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 30-40% ethyl acetate in hexane. LC-MS: calculated [M+H]+ 480.24, found 480.43.
[0463] Compound I (30 mg, 0.0626 mmol, 1.0 equiv.) was cooled by ice bath. HCl in dioxane (0.313 mL, 1.25 mmol, 20 equiv.) was added into the flask. The reaction was warmed to room température and stirred for another 1 hr. The solvent was removed by rotary evaporator and the product was directly used without further purification. LC-MS: calculated [M+H]+ 380.19, found 380.33.
[0464] To a solution of compound I (10 mg, 0.0571 mmol, 1 equiv.), compound 2 (26 mg, 0.0628 mmol, 1.1 equiv.), and TBTLJ (22 mg, 0.0685 mmol, 1.2 equiv.) in anhydrous DMF (I mL) was added diisopropylethylamine (0.030 mL, 0.171 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another I hr. The reaction was quenched with saturated 15 NaHCOj solution (5 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over NasSOi, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4 % methanol in DCM. LC-MS: calculated [M+HJ+ 537.26, found 537.41.
171
[0465] Compound 1 (30 mg, 0.0626 mmol, 1.0 equiv.) was cooled by ice bath. HCl in dioxane (0.313 mL, 1.25 mmol, 20 equiv.) was added into the flask. The reaction was warmed to room température and stirred for another 1 hr. The solvent was removed by rotary évaporator and the product was directly used without further purification. LC-MS: calculated [M+H]+ 437.21, found 437.31.
[0466] To a solution of compound 1 (20 mg, 0.0569 mmol, 1 equiv.), compound 2 (26 mg, 0.0626 mmol, 1.1 equiv.), and TBTU (22 mg, 0.0683 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.03 mL, 0.170 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The réaction was quenched with saturated NaHCOs solution (5 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 4-5 % methanol in DCM.
LC-MS: calculated [M+H]+ 713.36, found 713.85.
172
[0467] To a solution of compound 1 (0.033 g, 0.0463 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd/C (20 mg) at room température. The reaction mixture was stirred with hydrogen gas at room température for overnight. The catalyst was removed by filtration through Celite® and the product was used directly without further purification. LC-MS: calculated [M+HJ+ 623.31, found 623.56.
[0468] To a solution of compound 1(16 mg, 0.0257 mmol, 1 equiv.) and azido-PEG5-OTs (22 mg, 0.0514 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (17 mg, 0.0514 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hrs at 40 °C. The reaction was quenched by saturated NaHCOj solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over NasSCL, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. LC-MS: calculated [M+H]+ 868.45, found 868.96.
173
Structure 27b
[0469] To a solution of compound 1 (5 mg, 0.0058 mmol, 1.0 equiv.) in THF (1 mL) and water (1 mL) was added lithium hydroxide (1 mg, 0.0346 mmol, 6.0 equiv.) at room température. The mixture was stirred at room température for another 1 hrs. The pH was adjusted to 3.0 by HCl (6N) 5 and the aqueous phase was extracted with EtOAc (3 x 10 mL). The organic phase was combined, dried over NasSCL, and concentrated. TFA (1 mL) and DCM (1 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 754.38, found 755.
Synthesis of Structure 29b ((S)-3-(4-(3-((l4-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthaien-1yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid).
[0470] Compound 1 (100 mg, 0.169 mmol, 1.0 equiv.), compound 2 (68 mg, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (3 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 times). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 10 min and the reaction was kept at 40 °C for 2 hrs. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The compound was separated by CombiFlash®
174 using silica gel as the stationary phase and was eluted with 4% methanol in DCM. LC-MS:
calculated [M+H]+ 655.31, found 656.
[0471] To a solution of compound 1 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEG5-OTs (127 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (100 mg, 0.305 mmol, 2 equiv.) at room température. The reaction mixture was stirred for 3 hrs at 40 °C. The reaction was quenched by saturated NaHCOj solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SÛ4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with
3-4% methanol in DCM. LC-MS: calculated [M+H]+ 900.44, found 901.
[0472] To a solution of compound 1 (125 mg, 0.138 mmol, 1.0 equiv.) in THF (1 mL) and water (1 mL) was added lithium hydroxide (10 mg, 0.416 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hrs. The pH was adjusted to 3.0 by HCl (6N) 15 and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (3 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator. The product was used directly without further purification. LC-MS: calculated [M+H]+ 786.37, found 787.
175
Synthesîs of Structure 30b ((S)-N-(l-azido-21-(4-(naphthalen-l-yl)phenyl)-19,23-dioxo3,6,9,12,15-pentaoxa-18,22-diazatetracosan-24-yl)-4-((4-methylpyridin-2-yl)amino)butanamide).
[0473] Compound 1 (100 mg, 0.169 mmol, 1.0 equiv.), compound 2 (43 mg, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (3 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mîxed in a round-bottom flask. The flask was sealed with a screw-cap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 times). Then, THF (5 mL) and water (1 mL) were added via syringe. The mixture was bubbled with nitrogen for 10 min and the réaction was kept at 40 °C for 2 hrs. The reaction was quenched with water ( ! 0 mL) , and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over NasSOi, and concentrated. The compound was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. LC-MS: calculated [M+H]+ 639.31, found 640.
[0474] To a solution of compound 1 (90 mg, 0.140 mmol, 1 equiv.) in THF (5 mL) and FhO (5 mL) was added lithium hydroxide (10 mg, 0.422 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for 1 hr, the reaction mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic layer was combined, dried over Na2SO4, and concentrated. The product was used without further purification. LC-MS: calculated [M+H]+ 625.29, found 625.36.
176
[0475] To a solution of compound 1 (88 mg, 0.140 mmol, 1 equiv.), compound 2 (48 mg, 0.154 mmol, 1.1 equiv.), and TBTU (54 mg, 0.169 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.074 mL, 0.422 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCOs solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over NazSCL, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 4-6 % methanol in DCM. LC-MS: calculated [M+H]+ 913.47, found 913.70.
[0476] To a solution of compound I (93 mg, 0.101 mmol, 1.0 equiv.) in DCM (2 mL) was added
TFA (3 mL) and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using siIica gel as S the stationary phase. The product was eluted with 10-12% methanol in dîchloromethane. LC-MS:
calculated [M+H]+ 813.42, found 813.68.
Synthesis of Structure 31b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-lyt)phenyl)-3-((S)-3-hydroxy-2-(4-((4-methylpyridin-210 yl)amino)butanamido)propanatnido)propanoic acid).
[0477] To a solution of compound 1 (150 mg, 0.509 mmol, 1 equiv.), compound 2 (87 mg, 0.560 mmol, 1.1 equiv.), and TBTU (196 mg, 0.196 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.074 mL, 0.422 mmol, 3 equiv.) at 0 °C. The reaction mixture was
178 * warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCOa solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, drîed overNa2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 4-6 % methanol in DCM.
LC-MS: calculated [M+H]+396.21, found 396.17.
[0478] To a solution of compound I (196 mg, 0.495 mmol, 1 equiv.) in THF (5 mL) and H2O (5 mL) was added lithium hydroxide (35 mg, 1.486 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stîrrîng at room température for 1 hr, the reaction mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3 x 10 mL) and the organic layer was combined, dried over Na2SO4, and concentrated. The product was used without further purification. LC-MS: calculated [M-+-H]+ 382.19, found 382.13.
[0479] To a solution of compound 1 (189 mg, 0.495 mmol, 1 equiv.), compound 2 (195 mg, 0.545 mmol, 1.1 equiv.), and TBTU (190 mg, 0.595 mmol, 1.2 equiv.) în anhydrous DMF (5 mL) was added diisopropylethy lamine (0.259 mL, 1.486 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another I hr. The reaction was quenched with saturated NaHCCh solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried overNa2SÜ4, and concentrated. The product was separated by
CombiFlash® using silica gel as the stationary phase and was eluted with 4-6 % methanol in DCM. LC-MS: calculated [M+H]+ 685.32, found 685.58.
[0480] To a solution of compound 1 (75 mg, 0.109 mmol, 1 equiv.) and azido-PEG5-OTs (91 mg, 0.219 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (71 mg, 0.219 mmol, 2 equiv.) at room température. The reaction mixture was stirred ovemîght at 40 °C. The réaction was quenched by saturated NaHCOs solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organîc phase was combined, dried overNazSCh, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 4% methanol in DCM. The yîeld is 29%. LC-MS: calculated [M+H]+ 930.45, found 930.90.
[0481] To a solution of compound 1 (30 mg, 0.0323 mmol, 1.0 equiv.) in THF (1 mL) and water (1 mL) was added lithium hydroxîde (2.3 mg, 0.0968 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hrs. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organîc phase was combined, dried over NaiSOi, and concentrated. TFA (2 mL) and DCM (1 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase. The product was eluted with 12-15% methanol in dichloromethane. LC-MS: calculated [M+H]+ 816.39, found 816.92.
180
Synthesis of Structure 32b ((S)-4-(((S)-l-(4-(4-((14-a7,ido-3,6,9,12tetraoxatetradecyl)oxy)naphthalen~l-yl)phenyl)~2-carhoxyethyl)amino)-3f4-((4-methylpyridin-2yl)amino)butanamido)-4-oxobutanoic acid).
O
OH
[0482] To a solution of compound 1 (100 mg, 0.404 mmol, 1 equiv.), compound 2 (160 mg, 0.444 mmol, 1.1 equiv.), and TBTU (155 mg, 0.485 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.21 1 mL, 1.213 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated 10 NaHCOs solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SÛ4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 2-3 % methanol in DCM. LC-MS: calculated [M+H]+ 551.23, found 551.45.
[0483] Compound 1 (0.164 g, 0.297 mmol, 1.0 equiv.) was cooled by ice bath. HCl in dioxane (0.745 mL, 2.978 mmol, 10 equiv.) was added into the flask, The reaction was warmed to room température and stirred for another 1 hr. The solvent was removed by rotary evaporator and the
181 product was dîrectly used without further purification. LC-MS: calculated [M+H]+ 451.18, found
451.35.
[0484] To a solution of compound I (100 mg, 0.404 mmol, 1 equiv.), compound 2 (160 mg, 0.444 mmol, 1.1 equiv.), and TBTU (155 mg, 0.485 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.211 mL, 1.213 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCOs solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over NajSCU, and concentrated. The product was separated by
CombiFlash® using silica gel as the stationary phase and was eluted with 3-5 % methanol in DCM. LC-MS: calculated [M+H]+ 727.33, found 727.53.
[0485] To a solution of compound 1 (150 mg, 0.206 mmol, 1 equiv.) and azido-PEG5-OTs (172 mg, 0.412 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (134 mg, 0.412 mmol, 2 equiv.) at room température. The reaction mixture was stirred overnight at room température. The reaction was quenched by saturated NaHCOj solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase
182 and was eluted with 4% methanol in DCM. The yîeld is 29%. LC-MS: calculated [M+H]+ 940.45, found 940.71.
[0486] To a solution of compound i (30 mg, 0.0344 mmol, 1.0 equiv.) in THF (I mL) and water (I mL) was added lithium hydroxide (2.5 mg, 0.103 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hrs. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SÛ4, and concentrated. TFA (2 mL) and DCM (1 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase. The product was eluted with 20% methanol in dichloromethane. LC-MS; calculated [M+H]+ 844.38, found 844.56.
Synthesis of Structure 33b ((S)-3-((S)-6-aminû-2-(4-((4-methylpyridin-215 yl) amino)hutanamido)hexanamido)-3-(4-(4-((14-azido-3,6,9,12tetraoxatetradecyl)oxy)naphthalen-l-yl)phenyl)propanoic acid).
[0487] To a solution of compound 1 (150 mg, 0.509 mmol, 1 equiv.), compound 2 (166 mg, 0.560 mmol, 1.1 equiv.), and TBTU (196 mg, 0.611 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was
183 added diisopropylethylamine (0.266 mL, 1.528 mmol, 3 equiv.) at 0 °C. The réaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCOj solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was separated by
CombiFlash® using silica gel as the stationary phase and was eluted with 3-5 % methanol in DCM. LC-MS: calculated [M+H]+ 537.32, found 537.23.
[0488] To a solution of compound 1 (230 mg, 0.428 mmol, 1 equiv.) in THF (5 mL) and H2O (5 mL) was added lithium hydroxide (31 mg, 1.285 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température . After stirring at room température for 1 hr, the reaction mixture was acidifled by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic layer was combined, dried over NasSCM, and concentrated. The product was used without further purification. LC-MS: calculated [M+H]+ 523.31, found 523.55.
[0489] To a solution of compound 1 (230 mg, 0.440 mmol, 1 equiv.), compound 2 (173 mg, 0.484 mmol, 1.1 equiv,), and TBTU (170 mg, 0.528 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.230 mL, 1.320 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCO? solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried overNasSCL. and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 4-6 % methanol in DCM. LC-MS: calculated [M+H]+ 826.43, found 826.65.
185
[0490] To a solution of compound 1 (150 mg, 0.181 mmol, 1 equiv.) and azido-PEG5-OTs (113 mg, 0.272 mmol, 1.5 equiv.) in anhydrous DMF (2 mL) was added Cs2COî (118 mg, 0.363 mmol, 2 equiv.) at room température. The reaction mixture was stirred at 40 °C for 3 hrs. The reaction was quenched by saturated NaHCCL solution (5 mL) and the aqueous layer was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SÛ4, and concentrated. The product was punfied by CombiFlash® using silica gel as the stationary phase and was eluted with 4% methanol in DCM. The yield is 66%. LC-MS: calculated [M+H]+ 1071.57, found 1071.89.
[0491] To a solution of compound 1 (130 mg, 0.121 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (8.7 mg, 0.364 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another I hrs. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (3 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase.
The product was eluted with 20% methanol in dichloromethane. LC-MS: calculated [M+H]+ 857.45, found 857.64.
Synthesis of Structure 34b ((5)-3-(4-(4-((14-azido-3,6t9,12-tetraoxatetradecyl)oxy)naphthalen-l2 0 yl)phenyl)-3~((S)-4-methyl-2-(4-((4-methylpyridin-2yl)atnino)butanamido)pentanamido)propanoic acid).
[0492] To a solution of compound 1 (150 mg, 0.509 mmol, 1 equiv.), compound 2 (101 mg, 0.560 mmol, 1.1 equiv.), and TBTU (196 mg, 0.611 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.266 mL, 1.528 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCOs solution (5 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and was eluted with 3-5 % methanol in DCM. LC-MS: calculated [M+H]+ 422.26, found 422.36.
[0493] To a solution of compound I (186 mg, 0.441 mmol, 1 equiv.) in THF (3 mL) and H2O (3 mL) was added lithium hydroxide (31 mg, 1.323 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture w'as warmed to room température. After stirring at room température for 1 hr, the reaction mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic layer was combined, dried over Na2SO4, and concentrated. The product was used without further purification. LC-MS: calculated [M+H]+ 408.24, found 408.23.
OH k 187
[0494] To a solution of compound 1 (168 mg, 0.412 mmol, 1 equiv.), compound 2 (162 mg, 0.453 mmol, 1.1 equiv.), and TBTU (159 mg, 0.494 mmol, 1.2 equiv.) in anhydrous DM F (2 mL) was added dîisopropylethylamine (0.215 mL, 1.237 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated 5 NaHCÛ3 solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried overNa2SÛ4, and concentrated. The product was separated by CombiFlash® using silica gel as the statîonary phase and was eluted with 2-4 % methanol in DCM. LC-MS: calculated [M+H]+ 711.37, found 711.69.
[0495] To a solution of compound 1 (150 mg, 0.206 mmol, 1 equiv.) and azîdo-PEG5-OTs (132 mg, 0.317 mmol, 1.5 equiv.) in anhydrous DMF (2 mL) was added CS2CO3 (137 mg, 0.422 mmol, 2 equiv.) at room température. The reaction mixture was stirred at 40 °C for 3 hrs. The reaction was quenched by saturated NaHCO? solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3x10 mL). The organic phase was combined, dried over Na2SÛ4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. The yield is 82%. LC-MS: calculated [M+H]+ 956.51, found 956.64.
188
[0496] To a solution of compound 1(160 mg, 0.167 mmol, 1,0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (12 mg, 0.502 mmol, 3.0 equiv.) at room température. The mixture was stirred at room température for another 1 hrs. The pH was adjusted to 3.0 by HCl (6N) and the aqueous phase was extracted with EtOAc (3x10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. TFA (3 mL) and DCM (2 mL) was added into the residue and the mixture was stirred at room température for another 3 hr. The solvent was removed by rotary evaporator and the product was separated by CombiFlash® using silica gel as the stationary phase. The product was eluted with 8-10% methanol in dichloromethane. LC-MS: calculated [M+H]+ 842.44, found 842.67.
Synthesis of Structure 35b ((S)-3-(4f4~((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-3~ yl)ph enyl)-3f(2S,3R)-3-hydroxy-2-(4-((4-methylpyridin-2yl)amino)butanamido)butanamido)propanoic acid).
[0497] To a vial containîng L-threonine-OMe HCl (1.000 g, 5.896 mmol, 1.3 eq) was added compound 1 (1.335 g, 4.535 mmol, I eq), dimethylaminopyridine (0.277 g, 2.268 mmol, 0.5 eq), and CH2CI2 (13.3 mL). To the mixture was added diisopropylamine (2.054 mL, 11.792 mmol, 2.6 eq) and the resultîng solution was cooled to 0 °C. EDC*HC1 (1.130 g, 5.896 mmol, 1.3 eq) was added and the reaction was allowed to stir at 0 °C for 30 minutes before warming to room température. The reaction w'as determined to be complété after 16 hours by HPLC and was transferred to a separatory funnel, washed with 66% saturated NH4CI (4 x 20 mL) and saturated NH4CI (20 mL). The organic layer was dried over Na2SO4 and concentrated to yield a viscous oH (1.7588 g, 94.7%) which was carried directly into the next step. LC-MS: calculated [M+H]+: 410.22, found 410.03
k 189
[0498] Compound 1 was dissolved in MeOH (4.5 mL) and to the mixture was added a 2.0 M solution of LiOH (9.1 mL). The reaction was stirred for 1.5 h and concentrated to remove MeOH. The mixture was then acidified to pH = 4 with 20% KHSO4 and extracted with EtOAc (3x15 mL). The combined organic was washed with brîne (20 mL), dried over Na2SO4, and concentrated to obtain 3 as a solid (1.5095 g, 88.9% yield). LC-MS: calculated [M-H]': 394.21, found 394.37. Ή NMR (400 MHz, Chloroform-rT) δ 8.26 (d, 1 H), 7.27 - 7.24 (m, 1 H), 7.23 (s, 1 H), 6.95 (ddd, 1 H), 4.60 (dd, lH),4.39(qd, 1 H), 3.97 - 3.77 (m, 2H), 2.36 (s, 3H), ), 2.41-2.23 (m, 2H), 1.98-1.84 (m,2H), 1.45 (s, 9H), 1.19 (d,3H).
OH
[0499] A via! was changed with compound 1 (0.200 g, 0.506 mmol, 1 eq), TBTLÎ (0.195 g, 0.607 mmol, 1.2 eq), DMF (2.0 mL) and DIEA (0.264 mL, 1.517 mmol, 3.0 eq). The reaction was stirred for 2 minutes before the addition of 2 (0.253 g, 0.708 mmol, 1.4 eq). After completion, the reaction was diluted with sat. aq. NaHCOj (10 mL), extracted with EtOAc (3x5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated. The crude material was purified via column chromatography, eluting with 0-20% MeOH in CH2C12 to obtain the product (150.8 mg, 42.7% yield). LC-MS: calculated [M+H]+: 699.33, found 699.53
[0500] To a vial containing compound 1 (0.151 g, 0.216 mmol, 1 eq) was added CS2CO3 (0.106 g, 0.324 mmol, 1.5 eq) and DMF (1.9 mL). Ns-PEG5-OTs (0.135 g, 0.324 mmol, 1.5 eq) was added
I 190 to the mixture, and the reaction stirred at 40 °C. After completion, the reaction was diluted with EtOAc (10 mL), sat. aq. NaHCO3 (5 mL) and water (5 mL). The layers were separated and aqueous extracted a total of 3 x 10 mL with EtOAc. The combined organic layers were dried over Na2SÜ4 and concentrated. The crude material was purifïed via column chromatography, eluting with 0-20%
MeOH in CH2CI2 to obtain the product (103 mg, 50.4% yield). LC-MS: calculated [M+H]+: 944.47, found 944.56
[0501] To a vial containing compound 1 (0.103 g, 0.109 mmol, 1 eq) was added MeOH (1.5 mL) and 2.0 M LiOH (2.0 mL). The reaction was stirred at room température, then concentrated to remove MeOH, acidified with 20% KHSO4 to pH = 2. To the mixture was added EtOAc (5 mL) and water (4 mL). The aqueous layer was extracted with EtOAc (3x5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated to yield the product (0.0879 g, 86.9%). LC-MS: calculated [M+H]+: 930.45, found 930.56.
[0502] To a vial containing compound 1 (0.0879 g, 0.0945 mmol, 1 eq) was added CH2CI2 (0.3 mL) and trifluoroacetic acid (0.64 mL). The solution was stirred at room température. After completion (>97% product), the reaction was concentrated, co-evaporating with toluene (3 mL) and then acetonitrîle (2x3 mL). The product was obtained with additional TFA présent (115.6 mg).
191
Synthesis of Structure 36b ((S)-3-(4~(4f(14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-lyl)phenyl)-3-((2S,3S)-3~methyl-2-(4f(4~methylpyridin-2yl)amino)butanamido)pentanamido)propanoic acid).
EDC*HCI, DIEA
DMAP CH2CI2, 0 °C to RT
[0503] To a vial containing L-isoleucine-OMe HCl (1.000 g, 5.505 mmol, 1.3 eq) was added compound 1 (1.246 g, 4.234 mmol, 1 eq), dimethylaminopyridine (0.259 g, 2.117 mmol, 0.5 eq), and CH2CI2 (12.5 mL). To the mixture was added dîisopropy lamine (2.054 mL, 11.792 mmol, 2.6 eq) and the resulting solution was cooled to 0 °C. EDC*HC1 (1.055 g, 5.505 mmol, 1.3 eq) was added and the reaction was allowed to stîr at 0 °C for 30 minutes before warming to room température. The réaction was determined to be complété after 16 hours by HPLC and was transferred to a separatory funnel, washed with 66% saturated NH4CI (4 x 20 mL) and saturated NH4CÎ (1x20 mL). The organîc layer was dried over Na2SÛ4 and concentrated to yield a viscous oil (1.8634 g, wet with CH2CI2) which was carried directly into the next step. LC-MS: calculated [M+H]+: 422.26, found 422.00.
[0504] Compound 1 was dissolved in MeOH (4.2 mL) and to the mixture was added a 2.0 mL solution of LiOH (8.5 mL). The reaction was stirred for 1.5 h and concentrated to remove MeOH. The mixture was then acidified to pH = 4 with 20% KHSO4 and extracted with EtOAc (3x15 mL). The combined organîc was washed with brine (20 mL), dried over NasSOi, and concentrated to obtain the product as a viscous oil (1.6123 g, 93.4% yield across two steps). LC-MS: calculated [Μ-H]’; 406.24, found 406.43. 'HNMR (400 MHz, Chlorofornwf) § 8.23 (d, 1H), 7.12 (d, 1H), 6.95 - 6.88 (m, 1 H), 4.58 (dd, 1H), 3.99 - 3.83 (m, 2H), 2.35 - 2.34 (s, 3H), 2.30 (hept, 2H), 2.00 1.84 (m, 4H), 1.45 (s, 9H), 0.91 (m, 6H).
[0505] A vial was changed with compound 1 (0.200 g, 0.491 mmol, 1 eq), TBTU (0.189 g, 0.589 mmol, 1.2 eq), DMF (2.0 mL) and DIEA (0.256 mL, 1,472 mmol, 3.0 eq). The réaction was stirred for 2 minutes before the addition of 2 (0.246 g, 0.687 mmol, 1.4 eq). After completion, the reaction was dîluted with sat. aq. NaHCO? (10 mL), extracted with EtOAc (3x5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated. The crude materia! was purified via column chromatography, eluting with 0-20% MeOH in CH2Ch to obtain the product ¢0.3024 mg, 86.7% yield). LC-MS: calculated [M+H]+: 711.37, found 711.51.
[0506] To a vial containing compound 1 (0.170 g, 0.238 mmol, 1 eq) was added CS2CO3 (0.116 g,
0.358 mmol, 1.5 eq) and DMF (2.1 mL). Nj-PEG5-OTs (0.149 g, 0.358 mmol, 1.5 eq) was added to the mixture, and the reaction stirred at 40 °C. After completion, the reaction was diluted with EtOAc (10 mL), sat. aq. NaHCOj (5 mL) and water (5 mL). The layers were separated and aqueous extracted a total of 3x10 mL with EtOAc. Combined organic layers were dried over Na2SO4 and concentrated. The crude material was purified via column chromatography, eluting with 0-20% MeOI-I in CH2CI2 to obtain the product (0.1645 g, 72.1% yield). LC-MS: calculated [M+H]+: 956.51, found 956.78.
[0507] To a vial containing compound 1 (0.164 g, 0.172 mmol, 1 eq) was added MeOH (2.0 mL) and 2.0 M LiOH (3.0 mL). The reaction was stirred at room température and monitored by HPLC. Additional LiOH (33 mg, 1.38 mmol, 8 eq), water (5 mL) and MeOH (4 mL) was required to dissolve the material and drive the reaction. HPLC revealed the formation of two new peaks, thought to be diastereomers. Upon reaching >94% conversion, the reaction was concentrated to remove MeOH, acidified with 20% KHSO4 to pH = 2. To the mixture was added EtOAc (5 mL) and water (4 mL). The aqueous layer was extracted with EtOAc (4x5 mL). The combined organic layers were washed with brine (10 mL), dried over Na^SOi, and concentrated to yield the product (0.1417 g, 87.4%). LC-MS: calculated [M+H]+: 942.49, found 942.56.
[0508] To a vial containing compound 1 (0.1417 g, 0.1504 mmol, 1 eq) was added CH2CI2 (0.5 mL) and trifluoroacetic acid (1.0 mL). The solution was stirred at room température. After completion (>97% product), the reaction was concentrated, co-evaporating with toluene (3 mL) and then acetonitrile (2x3 mL). The product was obtained with additional TFA présent (150.3 mg). Two peaks were présent through the reaction for both starting material and product. LC-MS: calculated [M+H]+: 842.44, found 842.56. Both product peaks were found to hâve the same mass, indicating the presence of diastereomers.
Synthesis of Structure 3 7b ((S)-3-(4-(4-((l4-azido-3,6,9,12-tetraoxatetradecy l)oxy)nuph thulen-1yl)phenyl)-3-((R)-3-methyl-2-(4-((4-methylpyridin-2yl)amino)butanamido)butanamido)propanoîc acid).
2
[0509] To a solution of compound 1 (150 mg, 0.509 mmol, I equiv.), compound 2 (94 mg, 0.560 mmol, 1.1 equiv.), and TBTU (196 mg, 0.611 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.266 mL, 1.528 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room température and stirred for another 1 hr. The reaction was quenched with saturated NaHCO2 solution (10 mL) and the aqueous phase was extracted with ethyl acetate (3x5 mL). The organic phase was combined, dried overNa2SO4, and concentrated. The product was separated by CombiFlash and was eluted with 2-3 % methanol in DCM. Yield: 205 mg (99%).
[0510] To a solution of compound 1 (207 mg, 0.508 mmol, 1 equiv.) in THF (5 mL) and H2O (5 mL) was added lithium hydroxide (36 mg, 1.523 mmol, 3 equiv.) portion-wise at 0 °C. The reaction mixture was warmed to room température. After stirring at room température for 1 hr, the reaction mixture was acidified by HCl (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3x10 mL) and the organic layer was combined, dried overNa2SO4, and concentrated. The product was used without further purification. Yield: 180 mg (91 %).
Compound 5
Structure 37b
[0511] To a solution of compound 3 (180 mg, 0.46 mmol), compound 3a (180 mg, 0.50 mmol), and TBTU (176 mg, 0.55 mmol) in DMF (2.5 mL) was added DIPEA (177 mg, 239 pL, 1.37 mmol) at 0 °C. The reaction mixture was warmed to room température and stirred for 1 hour. The reaction mixture was quenched with sat. NH4CI (aq) solution (1.75 mL) and deionized water (1.75 mL) then extracted with ethyl acetate (8 mL). The aqueous iayer was further extracted with ethyl acetate (2 x 8 mL). The combined organic phase was washed with half sat. NFLCl (aq) solution (6 mL) and half sat. NaHCOj (aq) solution (6 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 4: 295 mg (92%). [M+H]+ calculated for C40H48N4O7: 697.84, found: 697.82.
[0512] To a solution of compound 4 (200 mg, 0.29 mmol) and azido-PEGs-OTs (240 mg, 0.57 mmol) in anhydrous DMF (2.5 mL) was added Cs2CCb (187 mg, 0.57 mmol). The reaction mixture was stirred at 60 °C for 2 hours. The reaction mixture was quenched with sat. NaHCOs (aq) solution (15 mL) and deionized water (7.5 mL) then extracted with ethyl acetate (10 mL). The aqueous Iayer was further extracted with ethyl acetate (2 x 10 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 5: 97 mg (36%). [M+H]+ calculated for C50H67N7O11: 943.15, found: 942.96.
196
[0513] To a solution of compound 5 (94 mg, 0.10 mmol) in THF (1.5 mL) and deiomzed water (1 mL) was added a solution of lithium hydroxide (7.2 mg, 0.30 mmol) in deionized water (0.5 mL). The reaction mixture was stirred for I hour then acidified to pH=3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3x5 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. To the crude residue was added TFA (1.34 mL) and water (67 pL). The reaction mixture was stirred for 1.5 hours at room température. The solvent was removed under reduced pressure, and the residue was coevaporated with acetonitrile:toluene [1:1] (2 x 20 mL). The crude mixture was separated by CombiFlash using silica gel as the stationary phase with 0-10% methanol in DCM. Yield of Structure 37b: 44 mg (53%). [M+H]+ calculated for C44H57N7O9: 828.97, found: 828.63.
Example 2. Synthèses of Tridentate ανβό Integrin Ligands and Conjugation of ανβό Integrin Ligands to Cargo Molécules (RNAi Agents),
[0514] The ανβό integrin ligands can be conjugated to one or more RNAi agents useful for inhibiting the expression of one or more targeted genes. The ανβό integrin ligands facilitate the delîvery of the RNAi agents to the targeted cells and/or tissues. Example 1, above, described the synthesis of certain ανβό integrin ligands disclosed herein. The following describes the general procedures for the synthèses of certain ανβό integrin lîgand-RNAi agent conjugates that are illustrated in the non-limiting Examples set forth herein.
[0515] A. Synthesis of RNAi Agents RNAi agents can be synthesized using methods generally known in the art. For the synthesis of the RNAi agents illustrated in the Examples set forth herein, the sense and antisense strands of the RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Depending on the scale, a MerMade96E® (Bioautomation), a MerMadel2® (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Synthèses were performed on a solid support made of controlled pore glass (CPG, 500 Â or 600Â, obtaîned from Prime Synthesis, Aston, PA, USA). AH RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2f-O-methyl phosphoramidites were used: (5'-O-dimethoxytrîtyl-N6(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5'-Odimethoxy-trity!-N4-(acetyl)-2'-O-methyl-cytidine-3Γ-O-(2-cyanoethyl-N,N-diisopropyl·amino) phosphoramidite, (5r-O-dimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine3'-0-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluorophosphoramidîtes carried the same protectîng groups as the 2'-O-methyl RNA amidites. 5'dimethoxytrityl-2'-O-methyl-inosine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites
197 were purchased from Glen Research (Virginia). The inverted abasic (3-O-dimethoxytntyl-2deoxyrîbose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, MA, USA). The following UNA phosphoramidites were used: 5'-(4,4’Dimethoxytrityl)-N6-(benzoyl)-2',3'-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,Ndiisopropyl)]-phosphoramidite, 5-(4,4'-Dimethoxytrityl)-N-acetyl-2r,3'-seco-cytosine, 2'-benzoyl3 - [(2-cyanoethy I )-(N ,N -di i so -propy [ )] -phosphoram id ite, 5 -(4,4'-D i meth oxytrity 1 )-N-isobutyry I 2',3'-seco-guanosine, 2f-benzoyl-3f-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'(4,4'-Dimethoxy-trîtyl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N- diîso-propyl)]phosphoramidite. TFA aminolink phosphoramidites were also commercially purchased (ThermoFisher).
[0516] In some examples, the ανβ6 integrin ligands disclosed herein are conjugated to the RNAi agents by linking the components to a scaffold that includes a tri-alkyne group. In some examples, the tri-aikyne group is added by using a tri-alkyne-containîng phosphoramidite, which can be added at the 5’ terminal end of the sense strand of an RNAi agent. When used in connection with the RNAi agents presented in certain Examples herein, tri-alkynecontaining phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while ail other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3Â) were added. 5-Benzylthio-l H-tetrazole (BTT, 250 mM in acetonitrile) or 5-Ethylthio1 H-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 min (RNA), 90 sec (2’ Ο-Me), and 60 sec (2’ F). In order to introduce phosphorothîoate linkages, a 100 mM solution of 3-phenyl 1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was employed.
[0517] Alternatively, where the ανβό integrin ligands are conjugated to the RNAi agents via a trialkyne scaffold, instead of using a phosphoramidite approach, tri-alkyne-containing compounds can be introduced post-synthetically (see, for example, section E, below). When used in connection with the RNAi agents presented in certain Examples set forth herein, when attaching a tri-alkyne group post-synthetically to the 5’ end of the sense strand the 5’ terminal nucléotide of the sense strand was functionalized with a nucléotide that included a primary amine at the 5’ end to facilitate attachment to the tri-alkyne-containing scaffold. TFA aminolink phosphoramidite was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3Â) were added. 5-Benzylthio-l H-tetrazole (BTT, 250 mM in acetonitrile) or 5-Ethylthio-l H-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 min (RNA), 90 sec (2’ Ο-Me), and 60 sec (2’ F). In order to introduce phosphorothîoate linkages, a 100 mM solution of 3-phenyl 1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was employed.
198
[0518] 13. Cleavage and deprotection of support bound oligomer. After fînalization of the solid phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt. % methylamîne in water and 28% to 31% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30 °C. The solution was evaporated and the solid residue was reconstituted in water (see below).
[0519] C. Purification. Crude oligomers were purified by anionic exchange H P LC using a TSKgel SuperQ-5PW 13μηι column and Shimadzu LC-8 System. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 16/40 column packed with Sephadex G-25 fine with a running buffer of lOOmM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile or fiItered water.
[0520] D. Annealing. Complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1 x PBS (Phosphate-Buffered Saline, 1x, Corning, Cellgro) to form the RNAi agents. Some RNAi agents were lyophilized and stored at -15 to -25 °C. Duplex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 1 x PBS. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to détermine the duplex concentration. The conversion factor used was either 0.037 mg/(mL'cm), or, alternatively for some experiments, a conversion factor was calculated from an experimentally determined extinction coefficient.
[0521] E. Conjugation of Tri-alkyne scaffold. Either prior to or after annealing, the 5' or 3' amine functionalized sense strand of an RNAi agent can be conjugated to a tri-alkyne scaffold. Example tri-alkyne scaffold structures that can be used in forming the constructs disclosed herein include the following:
[0522] The following describes the conjugation of tri-alkyne scaffold to the annealed duplex: Amine 5 functionalized duplex was dissolved in 90% DMSO/10% H2O, at -50-70 mg/mL. 40 eq triethylamine was added, followed by 3 eq tri-alkyne-PNP. Once complété, the conjugale was precipitated twice in a solvent System of Ix phosphate buffered salîne/acetonitrile (1:14 ratio), and dried.
[0523] F. Conjugation of ανβό Integrin Ligands. Either prier to or after annealîng, the 5' or 3' 10 tridentate alkyne functionalized sense strand is conjugated to the ανβό Integrin Ligands. The following example describes the conjugation of ανβό integrin ligands to the annealed duplex: Stock solutions of 0.5M Tris(3-hydroxypropyltriazolylmethyl)amine (ΤΗΡΤΑ), 0.5M of Cu(ll) sulfate pentahydrate (Cu(II)SO4 5 H2O) and 2M solution of sodium ascorbate were prepared in deionized water. A 75 mg/mL solution in DMSO of ανβό integrin ligand was made. In a 1.5 mL centrifuge tube 15 containing tri-alkyne functionalized duplex (3mg, 75pL, 40mg/mL in deionized water, -15,000 g/mol), 25 pL of IM Hepes pH 8.5 buffer is added. After vortexing, 35 pL of DMSO was added and the solution is vortexed. ανβό integrin ligand was added to the reaction (6 eq/duplex, 2 eq/alkyne,
200 ~15pL) and the solution is vortexed. Using pH paper, pH was checked and confirmed to be pH -8. In a separate 1.5 mL centrifuge tube, 50 pL of 0.5M THPTA was mixed with lOuL of0.5M Cu(II)SO4 5 H2O, vortexed, and incubated at room temp for 5 min. After 5 min, THPTA/Cu solution (7.2 pL, 6 eq 5:1 THPTA:Cu) was added to the reaction vial, and vortexed. Immediately afterwards, 2M ascorbate (5 pL, 50 eq per duplex, 16.7 per aïkyne) was added to the réaction vial and vortexed. Once the reaction was complété (typically complété in 0.5-1 h), the reaction was immediately purified by non-denaturîng anion exchange chromatography.
[0524] G. Functionalization ofThiol group on Cysteine Linker. in some examples, a cysteine linker can be used to facilitate conjugation of the ανβό integrin ligands to the RNAi agent. Either prior to or after annealing, the 5’ or 3’ tridentate alkyne-Cys(Stbu)-PEG2 functionalized sense strand is functionalized with a maleimide-containing moiety, or can be reduced and left as the free thîol, as shown in the following structure:
[0525] The following exampie describes the modification of the tri-alkyne-Cys(Stbu)-PEG2-duplex with N-ethyl maleimîde: Trî-alkyne-Cys(Stbu)-PEG2-duplex (35 mg) was dissolved in 500 pL deionized H2O. HEPES buffer (I M, pH 8.5, 82 pL), was added to the reaction, and the solution was vortexed. A solution of 1 M Dithiothreîtol (DTT, 100 eq, 236 pL) was added and the solution was placed on a vortex shaker for 3 h. After confirmation of réduction of the disulfide by denaturing RPHPLC, the conjugate was precipitated three times in a solvent system of Ix phosphate buffered saline/acetonitrile (1:14 ratio). The precipitated pellet was reconstituted in 0.5 mL of 0.1 M HEPES, pH 6.5, and N-ethyl maleimîde (3 mg, 10 eq) was added to the solution, and placed on a vortex mixer for-15 min. After completion ofthe reaction, the conjugate was precipitated three times in a solvent system of Ix phosphate buffered saline/acetonitrile (1:14 ratio), desalted, and dried.
Example 3. ανβό Integrin Ligand Binding Activity.
201
[0526] As reported in the following Table 1, IC50 binding data was obtained for the ανβό integrin ligands of Structures 1 and 2:
Table 1. IC50 Binding Activity.
Group IC50 (nM)
ανβ3 ανβ5 ανβό
Structure 1 not active not active 13
Structure 2 not active not active 129
[0527] Azide-functionalized structures (i.e., Structures 1b and 2b) were examined for IC50 under conditions typically used and known in the art. As shown in Table 1, above. Structures 1 and 2 showed sélective binding to ανβό integrin.
Example 4. In Vivo Intratracheal Administration of RNAi Agents Targeting Alpha-ENaC Conjugated to ανβό Integrin Ligands in Rats.
[0528] RNAi agents that included a sense strand and an antisense strand were synthesized according to phosphoramidite technology on solid phase in accordance with general procedures known in the art and commonly used in oligonucleotide synthesis as set forth in Example 2 herein. The RNAi agents included an antisense strand having a nucleobase sequence at least partially complementary to the gene expressing the alpha subunit of the amiloride-sensitive épithélial sodium channel (commonly referred to as alpha-ENaC or SCNN1 A). The alpha-ENaC RNAi agents were designed to be capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of alphaENaC in a sequence spécifie manner, thereby inhibiting expression of the alpha-ENaC gene. The RNAi agent used in this Example (AD04835) was comprised of modified nucléotides and more than one non-phosphodiester linkage, and included the following nucléotide sequences:
Sense strand sequence (5’ -> 3’):
(NH2-Cû)sgscugugcaAfCfCfagaacaaauas(învAb) (SEQ ID NO:1)
Antisense strand sequence (5’ 3’) cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc (SEQ ID NO:2), wherein (invAb) represents an invereted (3’-3’ linked) abasic deoxyribonucleotide; s represents a phosphorothîoate linkage; a, c, g, and u represent 2’-O-methyl adenosine, cytidîne, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, cytidîne, guanosine, or uridine, respectively; cPrpu represents a 5’-cyclopropyl phosphonate-2'-O-methyl uridine (see, e.g.,
202
Table A); and (NFb-Ce) represents a Cê terminal amine to facditate targetmg ligand conjugatton as desired (see, e.g., Table A).
[0529] As the person of ordinary skill în the art would clearly understand, the nucléotide monomers are linked by standard phosphodiester linkages except where inclusion of a phosphorothîoate linkage, as shown in the modified nucléotide sequences disclosed herein, replaces the phosphodiester linkage typically présent in an oligonucleotide.
[0530] On study day 1 and day 2, male Sprague-Dawley rats were administered a dose of 200 microliters intratracheally via a microsprayer device (Penn Century, Philadelphia, PA), which included the following dosing groups:
(1 ) 5% dextrose in water vehicle (D5W);
(2) 3.0 mg/kg of an alpha-ENaC RNAi agent (AD04835) without a ligand (“naked RNAi agent”), formulated in 5% dextrose in water (d5w); or (3) 3.0 mg/kg of an alpha-ENaC RNAi agent (AD04835) conjugated to a tridentate ανβό integrin ligand of Structure 1, formulated in d5w.
[0531] The sanie alpha-ENaC RNAi agent was used in Groups 2 and 3. For Group 3, the terminal amine (NHi-Cô) présent on the 5' terminal end of the sense strand of the RNAi agent was then conjugated to a scaffold that included three terminal alkyne groups. The alkyne groups were then conjugated to the azide functional group présent on Structure 1b, thereby forming a tridentate ανβό integrin ligand of Structure I. General synthetic procedures are described in Example 2, above.
[0532] Four (4) rats were dosed per group. Rats were euthanized on study day 5, and total RNA was isolated from both lungs following collection and homogenization. mRNA abundance of alphaENaC was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 2. Relative alpha-ENaC Expression of mRNA Normalized to Control of Example 4.
Group Relative Expression (Géométrie Mean) Lower / Upper 95% Confidence Interval
(1) 5% dextrose vehicle 1.000 0.81 /1.23
(2) Naked RNAi agent (no ligand) 0.36 0.07/ 1.79
(3) tridentate ανβό integrin ligand Structure 1 [(ανβό integrin ligand Structure 1)3-RNAî agent] 0.19 0.05/0.59
[0533] As shown in Table 2 above, the ανβό integrin ligand of Structure 1 în tridentate form conjugated to the alpha-ENaC RNAi agent (i.e., Group 3) showed increased relative knockdown of
203 alpha-ENaC mRNA (approximately 8i% knockdown), compared to naked RNAi agent (64% knockdown) without any ligand (i.e., Group 2) and the vehicle control, in vivo.
Example 5. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting Alpha5 ENaC Conjugated to ανβό Integrin Ligands in Rats.
[0534] In the following examples, various RNAi agents are used as cargo molécules to test the delivery of a cargo molécule via an ανβό integrin to a cell of interest. Certain of the RNAi agents used herein are described in US 62/679,549, which is incorporated herein by reference in its entirety. [0535] On study day 1, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration 10 administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 3. Dosing Groups of Rats in Example 5.
Group RNAi Agent and Dose Dosing Reginien
1 Isotonie saline (no RNAi agent) Single OP dose on day 1
2 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD04835) conjugated to the trîdentate ανβό integrin ligand of Structure 2, formulated in isotonie saline. Single OP dose on day 1
3 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD04835) conjugated to the trîdentate ανβό integrin ligand of Structure 5.1, formulated in isotonie saline. Single OP dose on day 1
4 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD04835) conjugated to the trîdentate ανβό integrin ligand of Structure 5.2, formulated in isotonie saline. Single OP dose on day 1
5 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD04835) conjugated to the trîdentate ανβό integrin ligand of Structure 6, formulated in isotonie saline. Single OP dose on day 1
6 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD04835) conjugated to the trîdentate ανβό integrin ligand of Structure 6.1, formulated in isotonie saline. Single OP dose on day 1
7 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD04835) conjugated to the trîdentate ανβό integrin ligand of Structure 6.2, formulated in isotonie saline. Single OP dose on day 1
[0536] The RNAi agents were synthesized having nucléotide sequences directed to target the human 15 alpha-ENaC gene, and included a functionalized amine reactive group (NFh-Cé) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The respective ανβό integrin ligands were then conjugated to the RNAi agents via a trîdentate scaffold that included a cysteine-n-ethyl-maleimide linker. For the RNAi agent-ανβό integrin ligand conjugales of Example 5, the RNAi agent as well as the scaffold/linker structures, were consistent for each of the Groups 220532
204
7. Thus, the only variable for Groups 2 through 7 was the spécifie ανβ6 integrin ligand (each in tridentate form) that was used. The RNAÎ agent-ανβό integrin ligand conjugates of Exampie 5 had structures represented by the following:
wherein represents the RNAi agent, and “avb6 Ligand” represents the respective ligand Structure. The structure of the RNAi agent used in this Example (AD04835) is set forth in Example 4, above.
[0537] Five (5) rats were dosed in each Group (n=5). Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNNI A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 4. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 5.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.195 0.243
Group 2 (RNAi agent-Cys-(n-ethyl-Mal)-PEG2tridentate ανβ6 integrin ligand Structure 2) 0.543 0.114 0.145
Group 3 (RNAi agent-Cys-(n-ethyLMal)-PEG2tridentate ανβ6 integrin ligand Structure 5.1) 0.541 0.138 0.185
205
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 4 (RNAi agent-Cys-(n-ethyl-Mal)-PEG2tridentate ανβό integrin ligand Structure 5.2) 0.522 0.151 0.212
Group 5 (RNAi agent-Cys-(n-ethyl-Mal)-PEG2tridentate ανβό integrin ligand Structure 6) 0.399 0.108 0.148
Group 6 (RNAi agent-Cys-(n-ethyl-Mal)-PEG2tridentate ανβό integrin ligand Structure 6.1 ) 0.351 0.100 0.139
Group 7 (RNAi agent-Cys-(n-ethyl-Mal)-PEG2tridentate ανβό integrin ligand Structure 6.2) 0.568 0.061 0.068
[0538] As shown in Table 4 above, each of the alpha-ENaC RNAi agents showed a réduction in mRNA expression in rats compared to control. For example, Group 6 (AD04835-tridentate-Structure 6.1) showed approximately a 65% réduction (0.351) in average rENaC mRNA expression compared to control; Group 2 (AD04835-tridentate-Structure 2) showed approximately a 46% réduction (0.543) in average rENaC mRNA expression compared to control; and Group 4 (AD04835tridentate-Structure 5.2) showed approximately a 48% réduction (0.522) in average rENaC mRNA expression compared to control.
1Q Example 6. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting AlphaENaC Conjugated to ανβό Integrin Ligands in Rats.
[0539] On study day 1, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 5. Dosing Groups of Rats in Example 6.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) Single OP dose on day 1
2 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD05347), conjugated to a tridentate ανβό integrin ligand of Structure 2 that included a glutaric linker (Le., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
3 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD05453), conjugated to a tridentate ανβό integrin ligand of Structure 2 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
4 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD05453), conjugated to a tridentate ανβό integrin ligand of Single OP dose on day 1
206
Group RNAi Agent and Dose Dosing Rcgimen
Structure 6 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline.
5 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD05453), conjugated to a tridentate ανβό integrin ligand of Structure 6.1 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
6 0.5 mg/kg of alpha-ENaC double-stranded RNAi agent (AD05453), conjugated to a tridentate ανβό integrin ligand of Structure 7 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
[0540] The RNAi agents were synthesized havîng nucléotide sequences directed to target the human alpha-ENaC gene, and included a functionalized amine reactive group (NHz-Cû) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The RNAi agents used in this Example were comprised of modified nucléotides and more than one non-phosphodiester linkage, and included the following nucléotide sequences: AD05347:
Sense strand sequence (5’ 3’):
(NH2-C6)cscugugcaAfCfCfagaacaaauas(invAb) (SEQ ID NO;3)
Antisense strand sequence (5’ -> 3’) cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc (SEQ ID NO:2), and
AD05453:
Sense strand sequence (5’ 3’):
(NH2-C6)cscugugcaAæfCfagaacaaauas(invAb) (SEQ ID NO:3)
Antisense strand sequence (5’ -> 3’) usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg (SEQ ID NO:4), wherein (invAb) represents an invereted (3’-3* linked) abasic deoxyribonucleotide; s represents a phosphorothioate linkage; a, c, g, and u represent 2’-O-methyl adenosine, cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, cytidine, guanosine, or uridine, respectively; cPrpu represents a 5’-cyclopropyl phosphonate-2'-O-methyl uridine (see, e.g.. Table A); and (NHj-Cô) represents a Cô terminal amine to facîlitate targeting ligand conjugation as desired (see, e.g., Table A).
207
[0541] ForGroups 2, 3, 4, 5, and 6, the respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a glutaric linker (via addition of glutaric acid), as depicted in the following Structure 300a:
(Structure 300a), wherein
structure.
represents the RNAi agent, and “avbô Ligand” represents the respective ligand
208
[0542] For Groups 7 and 8, the respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure having the structure depicted in Structure 330a:
(Structure 330a), wherein zAWWvW represents the RNAi agent, and “avbô Ligand” represents the respective ligand structure.
[0543] Four (4) rats were dosed in Groups 1, 3, 4, 6, and 7 (n=4); five (5) rats were dosed in Groups 5 and 8 (n=5); and three (3) rats were dosed in Group 2 (n=3). Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to
GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 6. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 6.
Group TD Number of animais (n=) Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 4 1.000 0.137 0.159
Group 2 (0.5 mg/kg AD05347glutaric-tridentate ανβό integrin ligand Structure 2) 3 0.486 0.090 0.110
209
Group ID Number of animais (n=) Average Relative rENaC mRNA expression Low (error) High (error)
Group 3 (0.5 mg/kg AD05453glutaric-tridentate ανβό integrin ligand Structure 2) 4 0.615 0.066 0.074
Group 4 (0.5 mg/kg AD05453glutaric-tridentate ανβό integrin ligand Structure 6) 4 0.512 0.119 0.156
Group 5 (0.5 mg/kg AD05453glutarîc-tridentate ανβό integrin ligand Structure 6.1) 5 0.494 0.101 0.127
Group 6 (0.5 mg/kg AD05453glutaric-trîdentate ανβό integrin ligand Structure 7) 4 0.743 0.104 0.121
[0544] As shown in Table 6 above, each of the alpha-ENaC RNAi agents showed a réduction in mRNA expression in rats comparcd to control. For example, Group 5 (AD05453-tridentate ανβό integrin ligand Structure 6.1) showed approximately a 51% réduction (0.494) in average rENaC 5 mRNA expression compared to control, and Group 3 (AD05453-tridentate ανβό integrin ligand
Structure 2) showed approximately a 38% réduction (0.615) in average rENaC mRNA expression compared to control. Further, Group 5 (which included ανβό integrin ligand Structure 6.1) showed improvement over Group 6 (which included ανβό integrin ligand Structure 7), indicating a chirality dependence of (s), as found in Structure 6.1, over (r) as found in Structure 7, for the ανβό integrin 10 ligands.
Exaniple 7. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting AlphaENaC Conjugated to ανβό Integrin Ligands in Rats.
[0545] On study day l, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the foliowing dosing Groups:
Table 7. Dosing Groups of Rats in Example 7.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) Single OP dose on day 1
2 0.5 mg/kg ofalpha-ENaC RNAi agent AD05347 conjugated to a tridentate ανβό integrin ligand of Structure 2 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
210
Group RNAi Agent and Dose Dosing Regimen
3 0.5 mg/kg of alpha-ENaC RNAi agent AD05347 conjugated to a tridentate ανβό integrin ligand of Structure 6.1 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
4 0.5 mg/kg of alpha-ENaC RNAi agent AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 2 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
5 0.5 mg/kg of alpha-ENaC RNAi agent AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 9 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
6 0.5 mg/kg of alpha-ENaC RNAi agent AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 6 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
7 0.5 mg/kg of alpha-ENaC RNAi agent AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 8 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day I
8 0.5 mg/kg of alpha-ENaC RNAi agent AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 6.1 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
9 0.5 mg/kg of alpha-ENaC RNAi agent AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 10 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
10 0.5 mg/kg of alpha-ENaC RNAi agent AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 11 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. Single OP dose on day 1
11 0.5 mg/kg of alpha-ENaC RNAi agent AD05453 conjugated to a tridentate peptide-based épithélial cell targeting ligand via the amine (NH2-C&) linkage on the 5’ terminal end of the sense strand that further included a 20 kilodalton (kDa) PEG moiety, formulated in isotonie saline. Single OP dose on day 1
[0546] The RNAi agents were synthesîzed having nucléotide sequences directed to target the human alpha-ENaC gene, and included a functionalized amine réactivé group (NH2-Cô) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nucléotide sequences for the RNAi agents used in this Example are set forth in Example 6, above. For Groups 2, 3, 4, 5, 6, 7, S, 9, and 10, the respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/Iinker structure that included a glutaric linker as depicted in Structure 300a,
I 211 shown in Example 6, above. For Group 11, the épithélial cell targeting ligands were comprised of RGD-mimetic peptides that are known to bind to ανβό integrin and included a 20 kDa PEG moiety as a pharmacokinetic (PK) modulator.
[0547] Four (4) rats were dosed in each Group (n=4). Rats were sacriflced on study day 9, and total
RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 8, Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 7.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.162 0.193
Group 2 (0.5 mg/kg AD05347-glutaric-tridentate ανβό integrin ligand Structure 2) 0.469 0.101 0.129
Group 3 (0.5 mg/kg AD05347-glutarîc-tridentate ανβό integrin ligand Structure 6.1) 0.358 0.078 0.100
Group 4 (0.5 mg/kg AD05453-glutaric-tridentate ανβό integrin ligand Structure 2) 0.562 0.086 0.102
Group 5 (0.5 mg/kg AD05453-glutaric-tridentate ανβό integrin ligand Structure 9) 0.620 0.168 0.230
Group 6 (0.5 mg/kg AD05453-glutaric-tridentate ανβό integrin ligand Structure 6) 0.559 0.099 0.120
Group 7 (0.5 mg/kg AD05453-glutarîc-trîdentate ανβό integrin ligand Structure 8) 0.691 0.072 0.081
Group 8 (0.5 mg/kg AD05453-glutaric-tridentate ανβό integrin ligand Structure 6.1) 0.454 0.055 0.063
Group 9 (0.5 mg/kg AD05453-glutaric-tridentate ανβό integrin ligand Structure 10) 0.454 0.080 0.097
Group 10 (0.5 mg/kg AD05453-glutarictridentate ανβό integrin ligand Structure 11 ) 0.577 0.1 13 0.140
Group 11 (0.5 mg/kg AD05453-tridentate peptide-based épithélial cell targeting ligand20kDa PEG) 0.558 0.057 0.064
[0548] As shown in Table 8 above, each of the alpha-ENaC RNAi agents showed a réduction in mRNA expression in rats compared to control. For example, Group 3 (AD05347-glutaric-tridentate ανβό integrin ligand Structure 6.1 ) showed approximately a 64% réduction (0.358) in average rENaC mRNA expression compared to control, and Group 8 (AD05453-glutaric-tridentate ανβό integrin ligand Structure 6.1) showed approximately a 55% réduction (0.454) in average rENaC mRNA expression compared to control. Further, the ανβό integrin ligands in Example 7 (i.e., Structure 2,
A 212
Structure 6, Structure 6.1, Structure 8, Structure 9, Structure 10, and Structure 11) ail showed comparable knockdown levels to the tridentate peptide-based épithélial cell targeting ligand that further included a relatively bulky 20 kilodalton PEG moiety to enhance the pharmacokinetic effect of Group 11.
Example 8. In Vivo Intratracheal Administration of RNAi Agents Targeting Alpha-ENaC Conjugated to ανβό Integrin Ligands in Rats
[0549J On study day 1 and day 2, male Sprague-Dawley rats were administered a dose of 200 mîcroliters intratracheally via a microsprayer device (Penn Century, Philadelphia, PA), which 10 included the following dosing Groups:
Table 9. Dosing Groups of Rats in Example 8.
Group RNAi Agent and Dose Dosing Régi m en
1 Isotonie saline (no RNAi agent) IT dose on day 1 and day 2
2 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a monodentate peptide-based épithélial cell targeting ligand via the amine (NHj-Cô) linkage on the 5' terminal end of the sense strand that further included a 20 kilodalton (kDa) PEG moiety, a cysteine linker, and an FCFP peptide linker, formulated in isotonie saline. IT dose on day 1 and day 2
3 0.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a tridentate ανβ6 integrin ligand ofStructure 1 that included a cysteine linker (i.e., having the structure represented in Structure 331a), formulated in isotonie saline. IT dose on day 1 and day 2
4 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a tridentate ανβό integrin ligand ofStructure 1 that included a cysteine linker (i.e., having the structure represented in Structure 331a), formulated in isotonie saline. IT dose on day 1 and day 2
5 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to atridentate ανβό integrin ligand ofStructure 1 that included a cysteine-n-ethyl-maleimide linker (i.e., having the structure represented in Structure 330a), formulated in isotonie saline. IT dose on day 1 and day 2
6 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to atridentate peptide-based épithélial cell targeting ligand via the amine (NFh-Cô) linkage on the 5’ terminal end ofthe sense strand that further included a 20kDa PEG moiety and a cysteine linker, formulated in isotonie saline IT dose on day 1 and day 2
7 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a tridentate ανβό integrin ligand of Structure 1 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. IT dose on day 1 and day 2
213
[0550] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, and included a functionalized amine reactive group (NHï-Cs) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nucléotide sequences for the RNAi agents used in this Example are set forth in Example 4, above.
[0551] For Groups 3 and 4, the ανβό integrin ligand of Structure I was conjugated to the RNAi agents via a tridentate scaffold and lînker structure that included a cysteine linker as depicted in the following Structure 331a:
the respective ligand structure.
[0552] For Group 5, the ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold and linker structure that included a cysteine-n-ethyl-maleimide linker as depicted in Structure 330a, shown in Example 6, above. For Group 7, the ανβό integrin ligands were conjugated 15 to the RNAi agents via a tridentate scaffold and linker structure that included a glutaric acid linker as depicted in Structure 300a, shown in Example 6, above. For Groups 2 and 6, the peptide-based épithélial cell targeting ligands were comprised of RGD-mimetic peptides and included a 20kDa PEG moiety as a pharmacokinetic (PK) modulator.
[05531 The same alpha-ENaC RNAi agent was used in each of Groups 2 through 7.
B 214
[0554] Five (5) rats were dosed in each of Groups 1, 2, 3, 4, 5, and 6 (n=5), and four (4) rats were dosed in Group 7 (n=4). Rats were sacrificed on study day 8, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as 5 fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 10. Average Relative rENaC mRNA Expression at Sacrifice (Day 8) in Example 8.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.143 0.167
Group 2 (1.5 mg/kg AD04835-Cys-FCFPmonodentate peptide-based ligand-PEG20kDa) 0.354 0.078 0.100
Group 3 (0.5 mg/kg AD04835-Cys-tridentate ανβό integrin ligand of Structure 1) 0.695 0.215 0.312
Group 4 (1.5 mg/kg AD04835-Cys-tridentate ανβό integrin ligand of Structure 1) 0.438 0.077 0.093
Group 5 (1.5 mg/kg AD04835-Cys-(n-ethylMal)-tridentate ανβό integrin ligand of Structure 1) 0.349 0.083 0.108
Group 6 (1.5 mg/kg AD04835-Cys-PEG20ktridentate peptide-based ligand) 0.643 0.070 0.079
Group 7 (1.5 mg/kg AD04835-glutaric-tridentate ανβό integrin ligand of Structure I) 0.648 0.184 0.256
[0555] As shown in Table 10 above, each of the alpha-ENaC RNAi agents showed a réduction in 10 mRNA expression in rats compared to control. For example, Group 5 (comprising AD04835-Cys(n-ethyl-Mal)-trîdentate ανβό integrin ligand Structure 1) showed approximately a 65% réduction (0.358) in average rENaC mRNA expression compared to control, which was comparable to the level of knockdown achieved in Group 2, which had a peptide-based épithélial cell targeting ligand that also included a 20kDa PEG moiety as a pharmacokinetic modulator.
Example 9. In Vivo Intratracheal Administration of RNAi Agents Targeting Alpha-ENaC Conjugated to ανβ6 Integrin Ligands in Rats
[0556] On study day 1 and day 2, male Sprague-Dawley rats were administered a dose of 200 microliters intratracheally via a microsprayer device (Penn Century, Philadelphia, PA), which 20 included the following dosing Groups:
Table 11. Dosing Groups of Rats in Example 9.
215
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) IT dose on day 1 and day 2
2 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a tridentate ανβό integrin ligand of Structure 1 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. IT dose on day 1 and day 2
3 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a tridentate ανβό integrin ligand of Structure 2 that included a glutaric linker (i.e., having the structure represented in Structure 300a), formulated in isotonie saline. IT dose on day 1 and day 2
6 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a tridentate ανβό integrin ligand of Structure 2 that included a cysteine linker (i.e., having the structure represented in Structure 331a), formulated in isotonie saline. IT dose on day 1 and day 2
[0557] The RNAi agents were synthesized having nncleotide sequences directed to target the human alpha-ENaC gene, and included a functionalized amine reactive group (NEh-Ce) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nncleotide sequences for the RNAi agents used in this Example are set forth in Example 4, above. For Groups 2 and 3, the respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a glutaric linker as depicted in Structure 300a, shown in Example 6, above. For Group 6 the respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a cysteine linker as depicted in Structure
331a, shown in Example 8, above.
[0558] The same alpha-ENaC RNAi agent was used in each of Groups 2 through 8.
Five (5) rats were dosed in Group 1 (n=5), and four (4) rats were dosed in each of Groups 2 and 3 (n=4). Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenîzation. Alpha-ENaC (SCNNIA) mRNA expression was quantîtated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 12. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 9.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.165 0.197
Group 2 (1.5 mg/kg AD04835-glutaric-tridentate ανβό integrin ligand Structure 1) 0.545 0.121 0.156
216
Group 3 (1.5 mg/kg AD04835-glutarîc-tridentate ανβ6 integrin ligand Structure 2) 0.483 0.038 0.041
Group 6 (1.5 mg/kg AD04835-Cys-tridentate ανβ6 integrin ligand Structure 2) 0.237 0.125 0.267
[0559] As shown in Table 12 above, each of the alpha-ENaC RNAi agents showed a réduction in mRNA expression in rats compared to control. For example, Group 3 (RNAi agent-glutarictridentate ανβό integrin ligand Structure 2) showed approximately a 52% réduction (0.483) in average rENaC mRNA expression compared to control, and Group 6 (RNAi agent-Cys-tridentate ανβό integrin ligand Structure 2) showed approximately a 76% réduction (0.237) in average rENaC mRNA expression compared to control.
Example 10. In vivo Intratracheal Administration of RNAi Agents Targeting Alpha-ENaC 10 Conjugated to ανβό Integrin Ligands in Rats.
[0560] On study day 1 and day 2, male Sprague-Dawley rats were administered a dose of 200 microliters intratracheal ly via a microsprayer device (Penn Century, Philadelphia, PA), which included the following dosing Groups:
[0561] Table 13. Dosing Groups of Rats in Example 10.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) IT dose on day 1 and day 2
2 1.0 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a monodentate peptide-based épithélial cell targeting ligand via the amine (NHi-Cé) linkage on the 5’ terminal end of the sense strand that further included a 20kDa PEG moiety, a cysteine linker, and an FCFP peptide linker, formulated in isotonie saline. IT dose on day 1 and day 2
3 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to atridentate ανβό integrin ligand of Structure 1 that included a cysteine linker (i.e., having the structure represented in Structure 331a), formulated in isotonie saline. IT dose on day 1 and day 2
4 1.5 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to atridentate ανβό integrin ligand of Structure 2 that included a cysteine linker (i.e., having the structure represented in Structure 331a), formulated in isotonie saline. IT dose on day 1 and day 2
5 1.0 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a trîdentate ανβό integrin ligand of Structure 2 that included a cysteine linker (i.e., having the structure represented in Structure 331a), formulated in isotonie saline. IT dose on day 1 and day 2
217
6 0.50 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a tridentate ανβό integrin ligand of Structure 2 that included a cysteine linker (i.e., having the structure represented in Structure 331a), formulated in isotonie saline. IT dose on day 1 and day 2
7 0.10 mg/kg of alpha-ENaC RNAi agent AD04835 conjugated to a tridentate ανβό integrin ligand of Structure 2 that included a cysteine linker (i.e., having the structure represented in Structure 331a), formulated in isotonie saline. IT dose on day 1 and day 2
[0562] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, and included a functionalized amine reactive group (NH2-Cs) at the 5' tenninal end ofthe sense strand to facilitate conjugation tothe ανβό integrin ligands. The nucléotide sequences 5 for the RNAi agents used in this Example are set forth in Example 4, above. For Groups 3, 4, 5, and , the respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a cysteine linker as depicted în Structure 331a, shown in Example 8, above. For Group 2, the targeting ligands were comprised of RGD-mimetic peptides and included a 20kDa PEG moiety as a pharmacokinetic (PK) modulator and an FCFP peptide linker.
[0563] The saine alpha-ENaC RNAi agent was used in each of Groups 2 through 7.
[0564] Five (5) rats were dosed in each Group (n=5). Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAP DH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% 15 confidence interval).
Table 14. Average Relative rENaC mRNA Expression at Sacrifice (Day 8) in Example 10.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.164 0.196
Group 2 (1.0 mg/kg AD04835-Cys-PEG20kDaFCFP-PEGso-peptide-based épithélial cell targeting ligand) 0.531 0.132 0.176
Group 3 (1.5 mg/kg AD04835-Cys-trîdentate ανβό integrin ligand Structure 1) 0.451 0.156 0.238
Group 4 (1.5 mg/kg AD04835-Cys-tridentate ανβό integrin ligand Structure 2) 0.418 0.077 0.094
Group 5 (1.0 mg/kg AD04835-Cys-tridentate ανβό integrin ligand Structure 2) 0.436 0.043 0.048
Group 6 (0.5 mg/kg AD04835-Cys-tridentate ανβό integrin ligand Structure 2) 0.537 0.049 0.054
Group 7 (0.1 mg/kg AD04835-Cys-tridentate ανβό integrin ligand Structure 2) 0.616 0.069 0.078
B 218
As shown in Table 14 above, each of the alpha-ENaC RNAi agents showed a réduction in mRNA expression in rats compared to control. Notably, Group 5 (1.0 mg/kg AD04835-Cys-trîdentate ανβό integrin ligand Structure 2) showed a nuinerically superior level of inhibition of alpha-ENaC expression compared to Group 2 (1.0 mg/kg ADO4835-Cys-PEG2OkDa-FCFP-PEG2o-peptide-based épithélial cell targeting ligand), despite not including a large 20 kilodalton PEG moiety as pharmacokinetic modulator (Group 5 = approximately 56% knockdown (0.436); Group 2 = approximately 47% knockdown (0.531)).
Exam pie 11. In Vivo Oroph aryngeal Aspiration A dministration of RNAi Agents Targeting A IphaENaC Conjugated to ανβ6 Integrin Ligands in Rats.
[0565] On study day 1, day 2, and day 3, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 15. Dosing Groups of Rats in Example 11.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) OP dose administered on each of days 1, 2, and 3
2 0.01 mg/kg of AD05453 (“naked RNAi agent”), formulated in isotonie saline. OP dose administered on each of days 1, 2, and 3
3 0.05 mg/kg of AD05453 (“naked RNAi agent”), formulated in isotonie saline OP dose administered on each of days 1, 2, and 3
4 0.15 mg/kg of AD05453 (“naked RNAi agent”), formulated in isotonie saline OP dose administered on each of days 1,2, and 3
5 0.50 mg/kg of AD05453 (“naked RNAi agent”), formulated in isotonie saline OP dose administered on each of days 1, 2, and 3
6 0.01 mg/kg of AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 6.1, formulated in isotonie saline. OP dose administered on each of days 1, 2, and 3
7 0.05 mg/kg of AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 6.1, formulated in isotonie saline. OP dose administered on each of days 1, 2, and 3
8 0.15 mg/kg of AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 6.1, formulated in isotonie saline. OP dose administered on each of days 1, 2, and 3
9 0.50 mg/kg of AD05453 conjugated to a tridentate ανβό integrin ligand of Structure 6.1, formulated in isotonie saline. OP dose administered on each of days 1,2, and 3
219
[0566] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, and included a functionalized amine reactive group (NFh-Cé) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nucléotide sequences for the RNAi agents used in this Example are set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scafïbld/linker structure that included a glutaric linkeras depicted in Structure 300a, shown in Example 6, above.
[0567] Five (5) rats were dosed in each of Groups 1, 3, 4, 5, 8, and 9 (n=5), and six (6) rats were dosed in Groups 2, 6, and 7 (n=6). Rats were sacrificed on study day 9, and total RNA was îsolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 16. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 11.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.199 0.249
Group 2 (0.01 mg/kg AD05347 (naked)) 1.016 0.219 0.279
Group 3 (0.05 mg/kg AD05347 (naked)) 0.881 0.157 0.192
Group 4 (0.15 mg/kg AD05347 (naked)) 0.638 0.179 0.250
Group 5 (0.50 mg/kg AD05347 (naked)) 0.354 0.076 0.097
Group 6 (0.01 mg/kg AD05453-glutarictridentate ανβό integrin ligand Structure 6.1) 0.646 0.058 0.063
Group 7 (0.05 mg/kg AD05453-glutarictridentate ανβό integrin ligand Structure 6.1) 0.432 0.044 0.049
Group 8 (0.15 mg/kg AD05453-glutarictridentate ανβό integrin ligand Structure 6.1) 0.319 0.034 0.038
Group 9 (0.50 mg/kg AD05453-glutarictridentate ανβό integrin ligand Structure 6.1) 0.254 0.043 0.052
[0568] As shown in Table 16 above, each of the alpha-ENaC RNAi agents conjugated to the avb6 integrin ligand having Structure 6.1 (in tridentate form) showed a réduction in mRNA expression in rats compared to control. Further, at each dosage level, the alpha-ENaC RNAi agents conjugated to the avbô integrin ligand having Structure 6.1 outperformed the alpha-ENaC RNAi agents administered naked, showing a ligand effect on delivery of the RNAi agent, (e.g., compare Groups 2 and 6; Groups 3 and 7; Groups 4 and 8; and Groups 5 and 9).
Example 12. Additional ανβό Integrin Ligand Binding Activity.
220
[0569] As reported in the following Table 17, additional IC50 binding data was obtained for the ανβό integrin ligands of Structures 2, 6.1,7, and 23 used in certain Examples herein:
Table 17. IC50 Binding Activity.
Group IC50 (nM) ανβό
Structure 2 205.4
Structure 6.1 1.6
Structure 7 381.5
Structure 23 759.7
[0570] Azide-functionalîzed structures (i.e.. Structures 2b and 6.1b, 7b, and 23b) were examined for IC50 under conditions typically used and known in the art. As shown in Table 17, above, Structure 6.1 showed potent binding activity to ανβό integrin (1C50 = 1.6 nM).
Example 13. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting AlphaENaC Conjugated to ανβό Integrin Ligands in Rats.
[0571] On study day 1, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 18. Dosing Groups of Rats in Example 13.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) Single OP dose admînistered on day 1
5 (0.5 mg/kg AD05347 tridentate ανβό integrin ligand Structure 1 ) Single OP dose admînistered on day 1
6 (0.5 mg/kg AD05347 tridentate ανβό integrin ligand Structure 2) Single OP dose admînistered on day I
7 (0.5 mg/kg AD05347 tridentate ανβό integrin ligand Structure 5) Single OP dose admînistered on day 1
[0572] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, and those including the AD05347 duplex included a functionalized amine reactive group (NHz-Ce) at the 5' terminal end of the sense strand to facilitate conjugatîon to the ανβό integrin ligands. The nucléotide sequences for RNAi agent AD05347 is set forth in Example 6, above. The
B 221 respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a glutarîc linker as depicted in Structure 300a. shown in Example 6, above.
[0573] Five (5) rats were dosed in each group (n=5). Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR. normalized to GAPDH expression and expressed as fraction of vehîcle control group (géométrie mean, +/- 95% confidence interval).
Table 19. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) m Example 13.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.044 0.046
Group 5 (0.5 mg/kg AD05347 tridentate ανβό integrin ligand Structure 1) 0.449 0.088 0.109
Group 6 (0.5 mg/kg AD05347 tridentate ανβό integrin ligand Structure 2) 0.487 0.049 0.055
Group 7 (0.5 mg/kg AD05347 tridentate ανβό integrin ligand Structure 5) 0.715 0.078 0.087 '
Example 14. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting AlphaENaC Conjugated to ανβό Integrin Ligands in Rats.
[0574] On study day 1, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 20. Dosing Groups of Rats in Example 14.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) Single OP dose administered on day 1
2 (0.5 mg/kg AD05347- tridentate ανβό integrin ligand Structure 2) Single OP dose administered on day 1
3 (0.5 mg/kg AD05453- tridentate ανβό integrin ligand Structure 6.1) Single OP dose administered on day 1
4 (0.5 mg/kg AD05453- tridentate ανβό integrin ligand Structure 6.3) Single OP dose administered on day 1
5 (0.5 mg/kg AD05453- tridentate ανβό integrin ligand Structure 6.4) Single OP dose administered on day I
[0575] The RNAi agents were synthesized having nucléotide sequences dîrected to target the human alpha-ENaC gene, and those including the AD05347 and AD05453 duplex included a functionalized
Μ 222 amine reactive group (NH^-Cè) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nucléotide sequences for the RNAi agents used in this Example are set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a trîdentate scaffold/linker structure that included a glutarîc linker as depicted in Structure 5 300a, shown in Example 6, above.
[0576] Four (4) rats were dosed in each group (n=4). Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantîtated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% 10 confidence interval).
Table 21. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 14.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.164 0.197
Group 2 (0.5 mg/kg AD05347-tridentate ανβό integrin ligand Structure 2) 0.418 0.051 0.058
Group 3 (0.5 mg/kg AD05453-tridentate ανβό integrin ligand Structure 6.1) 0.472 0.071 0.084
Group 4 (0.5 mg/kg AD05453-tridentate ανβό integrin ligand Structure 6.3) 0.534 0.059 0.066
Group 5 (0.5 mg/kg AD05453-tridentate ανβό integrin ligand Structure 6.4) 0.620 0.105 0.127
Example 15. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting Alpha15 ENaC Conjugated to ανβό Integrin Ligands in Rats.
[0577] On study day 1, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 22. Dosing Groups of Rats in Example 15.
Group RNAi Agent and Dose Dosing Regîmen
1 Isotonie saline (no RNAi agent) Single OP dose admînistered on day 1
4 (0.5 mg/kg AD05453-tridentate ανβό integrin ligand Structure 6.1) Single OP dose admînistered on day 1
9 (0.5 mg/kg AD05453-trîdentate ανβό integrin ligand Structure 2) Single OP dose admînistered on day 1
11 (0.5 mg/kg AD0545 3-trîdentate ανβό integrin ligand Structure 12) Single OP dose admînistered on day 1
223
12 (0.5 mg/kg AD05453-tridentate ανβό integrin ligand Structure 13) Single OP dose administered on day 1
[0578] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, the RNAi agents including a functionalized amine reactive group (NHî-Cô) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nucleotîde sequences for RNAÎ agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a glutaric linker as depicted in Structure 300a, shown in Example 6, above.
[0579] Four (4) rats were dosed in each of groups 1-9 and 12 (n=4). Three (3) rats were dosed in groups 10 and 11 (n=3). Rats were sacrificed on study day 7, and total RNA was isolated from both lungs following collection and homogenizatîon. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, nonnalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 23. Average Relative rENaC mRNA Expression at Sacrifice (Day 7) in Example 15.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.058 0.062
Group 4 (0.5 mg/kg AD05453- tridentate ανβό integrin ligand Structure 6.1) 0.606 0.217 0.338
Group 9 (0.5 mg/kg AD05453- tridentate ανβό integrin ligand Structure 2) 0.705 0.136 0.169
Group 11 (0.5 mg/kg AD05453- tridentate ανβό integrin ligand Structure 12) 0.703 0.093 0.108
Group 12 (0.5 mg/kg AD05453- tridentate ανβό integrin ligand Structure 13) 0.711 0.086 0.098
Example 16./« Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting AlphaENaC Conjugated to ανβό Integrin Ligands in Rats.
[0580] On study day 1, male Sprague Dawley rats were dosed via oropharyngeal COP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 24. Dosing Groups of Rats in Example 16.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) Single OP dose administered on day 1
224
2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) Single OP dose administered on day 1
3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 14) Single OP dose administered on day 1
4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 15) Single OP dose administered on day 1
[0581] The RNAi agents were synthesized having nucléotide sequences directed to target the hutnan alpha-ENaC gene, the RNAi agents including a functionalized amine reactive group (NHs-Cô) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nucléotide sequences for RNAi agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a glutaric linker as depicted in Structure 300a, shown in Example 6, above.
[0582] Four (4) rats were dosed in each group. Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 25. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 16.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.084 0.092
Group 2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) 0.597 0.163 0.224
Group 3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 14) 0.674 0.115 0.139
Group 4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 15) 0.533 0.047 0.052
Example 17. In Vivo Oropharyngeal Aspiration Administration of RJSAi Agents Targeting AiphaENaC Conjugated to avft6 Integrin Ligands in Rats.
[0583] On study day 1, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups;
Table 26. Dosing Groups of Rats in Example 17.
Group RNAi Agent and Dose Dosing Regimen
225
1 Isotonie saline (no RNAi agent) Single OP dose administered on day 1
2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) Single OP dose administered on day 1
3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 16) Single OP dose administered on day 1
4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 11) Single OP dose administered on day 1
[0584] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, the RNAi agents including a functionalized amine réactivé group (NH2-C&) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The 5 nucléotide sequences for RNAi agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a glutarîc linker as depicted in Structure 300a, shown in Example 6, above.
[0585] Five (5) rats were dosed in each group, except for group 4, which had four (4) rats dosed. Rats were sacrificed on study day 9, and total RNA was îsolated from both lungs foliowing collection 10 and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantîtated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 27. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 17.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.195 0.242
Group 2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) 0.489 0.168 0.257
Group 3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 16) 0.872 0.104 0.118
Group 4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 1 1) 0.625 0.126 0.158
Example 18. In Vivo Oropharyngeal Aspiration A dministration of RNAi Agents Targeting AlphaENaC Conjugated to ανβό Integrin Ligands in Rats.
[0586] On study day 1, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the foliowing dosing Groups:
Table 28. Dosing Groups of Rats in Example 18.
Group RNAi Agent and Dose
Dosing Regimen
226
1 Isotonie saline (no RNAi agent) Single OP dose administered on day 1
2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) Single OP dose administered on day 1
3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 17) Single OP dose administered on day 1
4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 15) Single OP dose administered on day 1
[0587] The RNAi agents were synthesized having nucléotide sequences dîrected to target the human alpha-ENaC gene, the RNAi agents includîng a functionalized amine reactive group (NH^-Cô) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nucléotide sequences for RNAi agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a glutaric linker as depîcted in Structure 300a, shown in Example 6, above.
[0588] Four (4) rats were dosed in each group. Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 29. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 18.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.140 0.162
Group 2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) 0.622 0.035 0.037
Group 3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 17) 0.818 0.101 0.116
Group 4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 15) 0.628 0.101 0.120
Example 19. In Vivo Oropharyngeal Aspiration Administration of RNAiAgents Targeting AlphaENaC Conjugated to ανβό Integrin Ligands in Rats.
[0589] On study day 1. male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 30. Dosing Groups of Rats in Example 19.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) Single OP dose administered on day 1
2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) Single OP dose administered on day 1
3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 15) Single OP dose administered on day 1
4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 18) Single OP dose administered on day 1
5 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 19) Single OP dose administered on day 1
6 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 20) Single OP dose administered on day 1
[0590] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, the RNAi agents including a functionalized amine reactive group (Nlh-Cô) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The 5 nucléotide sequences for RNAi agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/Iinker structure that included a glutaric linker as depicted in Structure 300a, shown in Example 6, above.
[0591] Four (4) rats were dosed in each group. Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) 10 mRNA expression was quantîtated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 31. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 19.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.121 0.138
Group 2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) 0.503 0.074 0.086
Group 3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 15) 0.700 0.079 0.089
Group 4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 18) 0.742 0.137 0.169
Group 5 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 19) 0.837 0.186 0.239
Group 6 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 20) 0.589 0.078 0.090
228
Example20. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting AlphaENaC Conjugated to ανβ6 Integrin Ligands in Rats,
[0592] On study days I and 2, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 32. Dosîng Groups of Rats in Example 20.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) OP dose administered on days 1 and 2
2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) OP dose administered on days I and 2
3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 22) OP dose administered on days 1 and 2
4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 23) OP dose administered on days 1 and 2
5 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 24) OP dose administered on days 1 and 2
6 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 25) OP dose administered on days 1 and 2
7 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 15) OP dose administered on days 1 and 2
[0593] The RNAi agents were synthesîzed having nucléotide sequences directed to target the human alpha-ENaC gene, the RNAi agents including a functionalized amine reactive group (NHs-Cô) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The nucléotide sequences for RNAi agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaflbld/linker structure that included a glutaric linker as depicted in Structure 300a, shown in Example 6, above.
[0594] Four (4) rats were dosed in each group, except for group 1, which had three (3) rats dosed. Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 33. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 20.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
• 229
Group 1 (isotonie saline) 1.000 0.164 0.197
Group 2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) 0.400 0.057 0.066
Group 3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 22) 0.483 0.170 0.263
Group 4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 23) 0.339 0.042 0.048
Group 5 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 24) 0.493 0.125 0.168
Group 6 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 25) 0.416 0.089 0.1 13
Group 7 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 15) 0.473 0.052 0.058
Example 21. In P7w Oropharyngeal Aspiration Administration of RNAi Agents Targeting AlphaENaC Conjugated to ανβό Integrin Ligands in Rats.
[0595] On study days 1 and 2, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) 5 aspiration administration with 200 microliters using a pipette, according to the following dosîng
Groups:
Table 34. Dosing Groups of Rats in Example 21.
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAi agent) OP dose administered on days 1 and 2
2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) OP dose administered on days 1 and 2
4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 27) OP dose administered on days 1 and 2
[0596] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, the RNAi agents including a functionalized amine réactivé group (NEh-Cs) at the 5' terminal end of the sense strand to facilitate conjugatîon to the ανβό integrin ligands. The nucléotide sequences for RNAi agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAÎ agents via a tridentate scaffold/iinker structure that included a glutaric linker as depicted in Structure 300a, shown in Example 6, above.
[0597] Five (5) rats were dosed in each group, except group 2, which had six (6) rats dosed. Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control 20 group (géométrie mean, +/- 95% confidence inter val).
230
Table 35. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 21.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.150 0.176
Group 2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) 0.380 0.108 0.151
Group 4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 27) 0.41 1 0.051 0.058
Example 22. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting Alpha5 ENaC Conjugated to ανβό Integrin Ligands in Rats,
[0598] On study days 1 and 2, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 36. Dosing Groups of Rats in Example 22.
Group RNAi Agent and Dose Dosing Regimen
l Isotonie saline (no RNAi agent) OP dose administered on days 1 and 2
2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1 ) OP dose administered on days 1 and 2
3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 29) OP dose administered on days 1 and 2
4 (0.5 mg/kg ADO5453 tridentate ανβό integrin ligand Structure 30) OP dose administered on days 1 and 2
5 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 31) OP dose administered on days I and 2
6 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 32) OP dose administered on days 1 and 2
7 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 33) OP dose administered on days 1 and 2
[0599] The RNAi agents were synthesized having nucléotide sequences directed to target the human alpha-ENaC gene, the RNAi agents including a functionalized amine reactive group (NHz-Cs) at the 5' terminal end of the sense strand to facilitate conjugation to the ανβό integrin ligands. The 15 nucléotide sequences for RNAi agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a tridentate scaffold/linker structure that included a glutaric linker as depicted in Structure 300a, shown in Example 6, above.
231
[0600] Four (4) rats were dosed in each group. Rats were sacrîficed on study day 9, and total RNA was isoiated from both lungs following collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/-95% confidence 5 interval).
Table 37. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 22.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.179 0.218
Group 2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) 0.511 0.132 0.178
Group 3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 29) 0.455 0.024 0.025
Group 4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 30) 0.637 0.047 0.050
Group 5 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 31 ) 0.505 0.079 0.093
Group 6 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 32) 0.534 0.135 0.181
Group 7 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 33) 0.560 0.145 0.196
Example 23. In Vivo Oropharyngeal Aspiration Administration of RNAi Agents Targeting Alpha10 ENaC Conjugated to ανβό Integrin Ligands in Rats.
[0601] On study days 1 and 2, male Sprague Dawley rats were dosed via oropharyngeal (“OP”) aspiration administration with 200 microliters using a pipette, according to the following dosing Groups:
Table 38. Dosing Groups of Rats in Exampie 23,
Group RNAi Agent and Dose Dosing Regimen
1 Isotonie saline (no RNAÎ agent) OP dose adminîstered on days 1 and 2
2 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 6.1) OP dose adminîstered on days 1 and 2
3 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 29) OP dose adminîstered on days 1 and 2
4 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 34) OP dose adminîstered on days 1 and 2
5 (0.5 mg/kg AD05453 tridentate ανβό integrin ligand Structure 35)___ OP dose adminîstered on days 1 and 2
232
6 (0.5 mg/kg AD05453 trîdentate ανβό integrin ligand Structure 36) OP dose administered on days 1 and 2
7 (0.5 mg/kg AD05453 trîdentate ανβό integrin ligand Structure 37) OP dose administered on days 1 and 2
[0602] The RNAi agents were synthesized having nucieotide sequences directed to target the human alpha-ENaC gene, the RNAi agents including a functionalized amine reactive group (NFh-Cô) at the 5' terminal end of the sense strand to facilîtate conjugation to the ανβό integrin ligands. The nucieotide sequences for RNAi agent AD05453 is set forth in Example 6, above. The respective ανβό integrin ligands were conjugated to the RNAi agents via a trîdentate scaffold/lînker structure that included a glutaric linker as depicted in Structure 300a, shown in Example 6, above.
[0603] Four (4) rats were dosed in each group. Rats were sacrificed on study day 9, and total RNA was isolated from both lungs following collection and homogenizatîon. Alpha-ENaC (SCNN1A) mRNA expression was quantitated by probe-based quantitative PCR, normalized to GAPDH expression and expressed as fraction of vehicle control group (géométrie mean, +/- 95% confidence interval).
Table 39. Average Relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 23.
Group ID Average Relative rENaC mRNA expression Low (error) High (error)
Group 1 (isotonie saline) 1.000 0.1 17 0.132
Group 2 (0.5 mg/kg AD05453 trîdentate ανβό integrin ligand Structure 6.1 ) 0.368 0.079 0.100
Group 3 (0.5 mg/kg AD05453 trîdentate ανβό integrin ligand Structure 29) 0.429 0.033 0.036
Group 4 (0.5 mg/kg AD05453 trîdentate ανβό integrin ligand Structure 34) 0.465 0.103 0.132
Group 5 (0.5 mg/kg AD05453 trîdentate ανβό integrin ligand Structure 35) 0.449 0.053 0.060
Group 6 (0.5 mg/kg AD05453 trîdentate ανβό integrin ligand Structure 36) 0.501 0.043 0.047
Group 7 (0.5 mg/kg AD05453 trîdentate ανβό integrin ligand Structure 37) 0.443 0.049 0.055
Other Embodiments
[0604] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not 1 imit the scope of
the invention, which is defined by the scope of the appended daims. Other aspects, advantages, and modifications are within the scope of the following daims.

Claims (39)

1. An ανβό integrin ligand comprising the structure:
rP2 (Formula I), or a pharmaceutîcally acceptable sait thereof, wherein, n is an înteger from 0 to 7;
J is C-H or N;
Zis OR13,N(R,3)2 or SR13;
R1 is H, optionally substituted Ci-Cô alkyl, OH, COOH, CON(R5)2, OR6, or R1 comprises a cargo molécule, wherein each R6 is independently H or Ci-Cô alkyl, and R6 is H or Ci-Cô alkyl;
R2, RP1 and RP2 are each independently H, halo, optionally substituted cycio alkyl ene, 15 optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene, or R2, RP1 and RP2 may comprise a cargo molécule;
R10 îs H or optionally substituted alkyl;
R11 is H or optionally substituted alkyl, or R11 and R1 together with the atoms to which they are attached form an optionally substituted heterocycle;
20 R12 is H or optionally substituted alkyl;
each R13 is independently H, optionally substituted alkyl, or R13 comprises a cargo molécule;
R14 is optionally substituted alkyl; and wherein at least one of R1, R2, R13, RP1 and RP2 comprises a cargo molécule.
2. An ανβό integrin ligand comprising the structure:
(Formula II),
235 or a pharamaceutically acceptable sait thereof, wherein, n is an integer from 0 to 7 (i.e., n is 0, 1,2, 3, 4, 5, 6, or 7);
J is C-H or N;
R1 is H, Ci-C6 alkyl, CH(R3)(R4), OH, COOH, CH2CH2CH2NH2, CONHR5, OR6, or R1 comprises a cargo molécule, wherein R3 is H or Ci-Ce alkyl, R4 is H, Ci-Cô alkyl, R5 is H or Ci-Cé alkyl, and R6 is H or Ci-C6 alkyl;
R2 is optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted hetero cycloalkylene, optionally substituted heteroarylene, or R2 comprises a cargo molécule,
R10 is H or optionally substituted alkyl;
R11 is H or optionally substituted alkyl, or R11 and R1 together with the atoms to which they are attached form an optionally substituted heterocycle;
R12 is H or optionally substituted alkyl;
R13 is H or optionally substituted alkyl;
R14 is optionally substituted alkyl;
wherein at least one of R1 or R2 comprises a cargo molécule.
3. The ανβό integrin ligand of claim 1 comprising the structure:
(Formula III), or a pharmaceutically acceptable sait thereof, wherein, n is an integer from 1 to 7 (i.e., n is 1,2, 3, 4, 5, 6, or 7);
R7 includes one or more cargo molécules; and
R8 is one or more optionally substituted divalent cyclic moieties having 2, 3, 4, 5, 6, 7, S, 9, or 10 carbon atoms, such as cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycioheptyl), cycloalkenyl (e.g., cyclopentenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, or cycloheptcnyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, pyrimidinyl, pyridazinyl, pyrrole, pyrazole, imidazoJe, thiophcne, benzothiophene, thîazole, benzothiazole, furan, oxazole, isoxazole, benzofuran, indole, ïndazole, benzimidazole, oxadîazole, 1,2,320532
236 triazole, 1,2,4-triazole, tetrazole, quinolinyl, isoquinolinyl, or quinoxalinyl), or heterocyclyl (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, dioxane, or dioxolane).
4. The ανβό integrin ligand of claim 1 comprising the structure:
or a pharmaceutically acceptable sait thereof, wherein, n is an integer from 1 to 7 (i.e., n is 1, 2, 3, 4, 5, 6, or 7); and R9 comprises one or more cargo molécules.
10
5. The ανβό integrin ligand of any one of daims 1 through 3, wherein n is 3.
6. The ανβό integrin ligand of any one of daims 1 through 3, wherein n is 4.
7. An ανβό integrin ligand selected from the group consisting of:
(Structure la).
237
(Structure 7 a),
238
(Structure 12a),
239 H ? H X /-/ /N. // OH Ti N TT y TT 1 H il 1 11 Υχ ° /Λ/ 0 O ôo X (Structure 13a), H 9 H YfX -- ^ 'ΐ γ YYf H O /L 0 rj /L ,0. ,F U TF X (Structure 14a), H j? H /·\Χ /--/ /N^ /-/ /OH Τΐ Ί N X T Y H O /L 0 ULxY ex X (Structure 15a), Cl H X/X/ z\ ZX zN^./Nx/x ΌΗ H O O JL 0 X œ X (Structure 16a), 20532
240
X (Structure 18a),
241
X (Structure 29a),
242
(Structure 30a),
(Structure 31 a).
(Structure 33a),
243
(Structure 34a),
x (Structure 35a),
(Structure 36a), and
X (Structure 37a), or a pharmaceuticany acceptable sait thereof, wherein X comprises a cargo molécule.
244
8. An ανβό integrin ligand selected from the group consisting of:
246
(Structure 6.4);
247
(Structure 7);
(Structure 8);
(Structure 9);
248
(Structure 12);
249
(Structure 14);
(Structure 15);
250
251
(Structure 27);
252
(Structure 32);
253
(Structure 35);
(Structure 36); and
254
(Structure 37), or a pharmaceuticaliy acceptable sait thereof, wherein $ indicates the point of connection to a moiety comprising a cargo molécule.
S
9. The ανβό integrin ligand of claim 8, wherein the ligand has the structure:
(Structure 6.1), or a pharmaceuticaliy acceptable sait thereof, wherein * indicates the point of connection to a moiety comprising a cargo molécule.
10
10. The ανβό integrin ligand of any one of daims 1-9, wherein the cargo molécule is an active pharmaceutical ingrédient or a prodrug.
11. The ανβό integrin ligand of any one of daims 1-9, wherein the cargo molécule comprises a small molécule, an antibody, an antibody fragment, an immun oglobulin, a
15 monoclonal antibody, a label or marker, a lipid, a natural or modified nucieic acid, a natural or modified nucieic acid oligonucleotide, a natural or modified nucieic acid polynucleotide, a peptide, an aptamer, a polymer, a polyamine, a protein, a toxin, a vitamin, a polyethylene glycol, a hapten, a digoxigenin, a biotin, a radioactive atom or molécule, or a fluorophore.
255
12. The ανβό integrin ligand ot any one of daims 1-9, wherein the cargo molécule comprises an RNAi agent.
13. The ανβό integrin ligand of any one of claims 1-12, further comprising a polyethylene glycol linker having 2-20 ethylene oxide units.
14. A structure comprising the ανβό integrin ligand of any one of claims 1-13, a lînking group, and a scaffold, wherein the the structure is bound to the cargo molécule.
15. The structure of claim 14, wherein the structure comprises the ανβό integrin ligand in monodentate fonn.
16. The structure of claim 14, wherein the structure comprises the ανβό integrin ligand in bidentate form.
17. The structure of daim 14, wherein the structure comprises the ανβό integrin ligand in tridentate form.
18. The structure of daim 14, wherein the structure comprises the ανβό integrin ligand in tetradentate form.
19. The structure of daim 14, wherein the scaffold is of the formula:
256
(Structure 300a), wherein
represents an RNAi agent, and “avb6 Ligand” represents the respective ligand structure and linking agent.
20. An ανβό integrin ligand precursor comprising the structure:
or a phannaceutically acceptable sait thereof, wherein, n is an integer from 0 to 7;
J is C-H or N;
Z is OR13, N(R,3h or SR13;
257
R1 is H, optionally substituted Ci-C6 alkyl, OH, COOH, CON(R5)2, OR6, or R1 comprises a linking group conjugated to a reactive group, wherein each R5 is independently H or Ci-Cô alkyl, and R6 is H or Ci-Cô alkyl;
R2, RP1 and RP2 are each independently H, optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene, or R2, RP1 and RP2 may comprise a linking group conjugated to a reactive group;
R10 is H or optionally substituted alkyl;
R11 îs H or optionally substituted alkyl, or Ru and R1 together with the atoms to which they are attached form an optionally substituted heterocycle;
R12 îs H or optionally substituted alkyl;
each R13 is independently H, optionally substituted alkyl, or R13 comprises a linking group conjugated to a reactive group;
R14 is optionally substituted alkyl; and wherein at least one of R1, R2, R13, RP1 and RP2 comprises a linking group conjugated to a reactive group.
21. The ανβό integrin ligand precursor of daim 20, wherein the linking group is a PEG linker.
22. The ανβό integrin ligand precursor of daim 20, wherein the PEG linker comprises 220 PEG units.
23. The ανβό integrin ligand precursor of daim 20, wherein the réactivé group is an azide.
24. The ανβό integrin ligand precursor of daim 20, wherein the linking group conjugated to a reactive group is of the structure:
wherein n is an integer from 2 to 20 and * indicates the point of connection to the structure of Formula 1b.
25. An ανβό integrin ligand precursor sdected from the group consisting of:
d C)— # \—/ IZ / ZI Σ\ i 1 L c >O >° >o < iz iz IZ > Λ °=? Λ o=<b^ y° H , , , \ Z- ) IZ rj n // \__/ Ά / Z^A /TA / / \ - r <->1 $ y 'ί / \ θ--\ /---\ \ / \ JT ( O 00 \ / 7 / ( \=d \ >=/ \=/ \ \ oA o4 L ω ) 0 o=( \ / ___/ / \ / / ,__k _1 d-r ό o / - °a <\ /) °y ( /Tvfv7 \ 1 O 0 O ° \ / \ r\ / \ T T / )=/ \=z \ O / ) A # °=( 20532 OH (Structure 1b), (Structure 2b), N3 (Structure 5b), n3 (Structure 5.1b),
259
(Structure 5.2b),
260
261
(Structure 9b),
(Structure 10b),
(Structure 11b),
(Structure 12b),
262
(Structure 16b),
263
(Structure 17b),
264
265
266
(Structure 32b),
267
or a pharmaceutically acceptable sait thereof.
26. A composition comprising the ανβό integrin ligand of any of claims 1-13 or the structure of any of claims 14-19, and a pharmaceutically acceptable excipient.
27. The composition of claim 26, wherein the ανβό integrin ligand is conjugated to an oligonucleotide-based compound that is capable of inhibi ting the expression of a target gene in an épithélial cell.
28. The composition of claim 26, wherein the ανβό integrin ligand is conjugated to an RNAi agent that is capable of inhibiting the expression of a target gene in an épithélial cell.
29. The composition of daim 26, wherein the ανβό integrin ligand is conjugated to an RNAi agent that is capable of inhibiting the expression of a target gene in a bronchiolar épithélial cell.
268
30. An m vitro method of delivering one or more cargo molécules to a cell, the method comprising administering to the cell an ανβό integrin ligand of any of daims 1-13 or the structure of any of daims 14-19.
31. An ανβό integrin ligand of any of daims 1-13, the structure of any of daims 14-19, or a composition of any of daims 26-29 for use in a method of delivering one or more cargo molécules to a cell or tissue of a subject in vivo.
32. Use of an ανβό integrin ligand of any of daims 1-13 or the structure of any of daims 14-19 in the manufacture of a composition for delivering one or more cargo molécules to a cell or tissue of a subject in vivo.
33. The method of daim 30 or the ligand, the structure or the composition for use of daim 31 or the use of daim 32, wherein the cell is selected from the group consisting of: type 1 and IT alveolar épithélial cell, goblet cell, secretory épithélial cell, ciliated épithélial cell, corneal and conjunctîval épithélial cell, dermal épithélial cell, cholangiocyte, enterocyte, ductal épithélial cell, glandular épithélial cell, and épithélial tumors (carcinomas).
34. The method of daim 30 or the ligand, the structure or the composition for use of daim 31 or the use of daim 32, wherein the one or more cargo molécules comprises an oligonucleotide-based compound.
35. The method or the ligand, the structure or the composition for use, or the use of daim 34, wherein the oligonucleotide-based compound is an RNAi agent.
36. A composition that includes an oligonudeotide-based compound conjugated to an ανβό integrin ligand of any of daims 1-13 for use in a method of inhibiting the expression of a target gene in a cell in vivo.
37. Use of an oligonudeotide-based compound conjugated to an ανβό integrin ligand of any of daims 1-13 in the manufacture of a composition for inhibiting the expression of a target gene in a cell in vivo.
* 269
38. The composition for use of claim 36 or the use of claim 36, wherein the cell is selected from the group consisting of: type T and II alveolar épithélial cell, goblet cell, secretory épithélial cell, ciliated épithélial cell, comeal and conjunctival épithélial cell, dermal épithélial cell, cholangiocyte, enterocyte, ductal épithélial cell, glandular épithélial cell, 5 and épithélial tumors (carcinomas).
39. The composition for use or the use of any one of daims 36 to 38 wherein the oligonucleotide-based compound is an RNAi agent.
OA1202000151 2017-11-01 2018-10-31 Integrin ligands and uses thereof. OA20532A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US62/580,398 2017-11-01
US62/646,739 2018-03-22
US62/679,549 2018-06-01

Publications (1)

Publication Number Publication Date
OA20532A true OA20532A (en) 2022-10-27

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