TW201512174A - Cell proliferation inhibitors and conjugates thereof - Google Patents

Abstract

Disclosed herein are immunoconjugates comprising an inhibitor of Eg5 linked to an antigen binding moiety such as an antibody, useful for treating cell proliferative disorders. Also disclosed are novel inhibitors of Eg5 that can be used either alone or as part of an immunoconjugate to treat cell proliferation disorders. The Eg5 inhibitors include compounds of this formula as described herein:. The invention further provides pharmaceutical compositions comprising these compounds and immunoconjugates, optionally including a therapeutic co-agent, and methods to use these compounds, conjugates and compositions for treating cell proliferation disorders.

Description

Cell proliferation inhibitors and their combinations

The present invention provides compounds which inhibit cell proliferation by inhibiting Eg5 activity and are therefore suitable for the treatment of cell proliferative disorders associated with excessive Eg5 activity. The invention also provides a combination comprising an Eg5 inhibitor linked to an antigen binding moiety, and a pharmaceutical composition comprising such a combination. Also included are methods of using such compounds and conjugates to treat cell proliferative disorders, including cancer.

In recent years, a large number of studies have been directed to the use of antibodies to form cell proliferation inhibitors and cytotoxic agents by forming antibody-drug conjugates (ADCs) to eliminate specific cells targeted. An ADC typically contains an antibody selected for its ability to bind to a cell targeted for therapeutic intervention, which is linked to a drug selected for cytostatic or cytotoxic activity. Binding of an antibody to a targeted cell delivers the drug to the site required for its therapeutic effect, and thus reduces off-target activity, while improving the efficiency of utilizing the payload compound.

A variety of antibodies that recognize and selectively bind to targeted cells, such as cancer cells, have been disclosed for use in ADCs, and a variety of methods for attaching a payload (drug) compound, such as a cytotoxin, to an antibody have also been described. Although much work has been done on ADCs, only a few classes of cell proliferation inhibitors or cytotoxins have been widely used as ADC payloads. Even after the release of the first ADC approved for human use in the United States in 2000 (Mylotarg®, which was later withdrawn in the market), only a few chemical classes of pharmaceutical compounds (maystatin, auristatin (a few years later) Auristatin), calicheamycin and duocarmycin have entered clinical trials as ADC payloads. Antibody-Drug Conjugates: the Next Generation of Moving Parts , A. Lash, Start-Up , December 2011, 1-6. This implies the difficulty of identifying suitable classes of pharmaceutical compounds to produce an effective ADC payload. It is known to widely recognize the value of ADC as a therapeutic agent, particularly for the treatment of cancer, and therefore there remains a need for novel cell proliferation inhibitors that are suitable for use as a payload in ADCs.

The present invention encompasses novel Eg5 inhibitors, and methods of using Eg5 inhibitors as small molecule drugs or as pharmaceutical components (payloads) of antibody-drug conjugates (ADCs).

Eg5, also known as spindle kinesin or KSP, is a kinesin motor protein involved in the cross-linking of microtubules during mitosis and is therefore required for cell division. Eg5 inhibitors are known to be useful in the treatment of cell proliferative disorders, such as cancer (Rath and Kozielski , Nature Rev. Cancer , Vol. 12, 527-39 (2012); see also WO06/002236, WO2007/021794, WO2008/063912, WO2009 /077448, WO2011/128381, WO2011/128388 and WO2006/049835). Although many different chemical families of Eg5 inhibitors are known, they have not been used to date for ADCs. The present invention encompasses the use of an Eg5 inhibitor as a pharmaceutical payload for an ADC, and a novel Eg5 inhibitor suitable for use as an ADC payload and suitable for use as a small molecule drug. The invention further encompasses methods and intermediates suitable for incorporating certain Eg5 inhibitors into ADCs, and methods of using such novel compounds and conjugates to treat cell proliferative disorders.

The invention provides immunoconjugates (e.g., ADCs) comprising an Eg5 inhibitor linked to an antigen binding portion, such as an antibody or antibody fragment. Such conjugates comprising Eg5 inhibitors are useful for treating cell proliferative disorders, particularly when an Eg5 inhibitor is linked to an antibody that recognizes cancer cells and thus facilitates delivery of an Eg5 inhibitor to the cancer cells targeted by the challenge. Such immunoconjugates are particularly useful for treating certain cancers, as described in further detail herein. The information provided herein confirms that these immunoconjugates are potent cell proliferation inhibitors and are effective for treating some Types of cancer; not bound by theory, and its activity is attributed to the inhibition of Eg5 in cells.

In one aspect, the immunoconjugate of the invention comprises a compound of the formula:

Wherein Ab represents an antigen binding moiety; L represents a linkage group linking X to Ab; m is an integer from 1 to 4; n is an integer from 1 to 16; and X independently represents an Eg5 inhibitor at each occurrence.

In such immunoconjugates, X can be a compound of formula II as described herein, or any inhibitor of Eg5 having an IC50 of less than about 100 nM. A variety of such Eg5 inhibitors are known, including ispinesib, SB-743921, AZD4877, ARQ621, ARRY-520, LY2523355, MK-0731, EMD534085, and GSK-923295, and WO06/002236, WO2007. Eg5 inhibitors as described in WO2008/063912, WO2009/077448, WO2011/128381, WO2011/128388, and WO2006/049835.

Typically, in the immunoconjugates of the formula, m is 1 or 2, preferably 1; and n is 2-8, preferably from about 2 to about 6, more preferably between 3 and 5. Ab can be any suitable antigen binding moiety and is typically an antibody. Suitable antibodies are well known in the art and can be native antibody sequences, or they can be modified by, for example, protein engineering techniques to improve their suitability or activity. L may be any linker suitable for linking one or more X groups to an Ab; typically, L is attached to an lysine δ-amine group, or to a cysteine thiol group attached to an antibody. It can be a naturally occurring residue, or it can be introduced at a selected position in the antibody sequence.

Suitable options for X include the compounds of formula (II) disclosed herein, as well as monastrol (4-(3-hydroxyphenyl)-6-methyl-2-sulfinyl-3,4-di Hydrogen-1H-pyrimidine-5-carboxylic acid ethyl ester); (2S)-4-(2,5-difluorophenyl)-N-[(3 R ,4 S )-3-fluoro-1-methyl- 4-piperidinyl]-2,5-dihydro-2-(hydroxymethyl) -N -methyl-2-phenyl-1H-pyrrole-1-carboxamide (MK-0731, CAS 845256-65 -7); Litronesib (LY2523355, CAS 910634-41-2); and (2S)-2-(3-aminopropyl)-5-(2,5-difluorophenyl) - N -methoxy- N -methyl-2-phenyl-1,3,4-thiadiazole-3( 2H )-carbenamide (ARRY520); and AZ3146 (9-cyclopentyl-2) -[[2-methoxy-4-[(1-methylpiperidin-4-yl)oxy]-phenyl]amino]-7-methyl-7,9-dihydro-8 H -嘌呤-8-ketone).

In certain embodiments, the immunoconjugate has the formula (I)

Wherein Ab represents an antigen binding moiety, such as an antibody or antibody fragment; L represents a linkage group linking X to Ab by covalent or non-covalent linkage, which may optionally be attached to more than one X to Ab, and may or may not be Designed to promote in vivo cleavage; X represents the compound of formula (II) independently at each occurrence:

As further described below; m is an integer from 1 to 4, typically 1-2; and n is an integer from 1 to 16, preferably from 2 to 8.

The present invention provides a method for preparing an ADC using an Eg5 inhibitor, particularly a compound of the formula (II) or (III) as a payload (drug) to be delivered, and using the ADC to treat cell growth The method of illness.

The invention also provides modified compounds of formula (II), which are described herein as formula (IIA) and (IIB) and (IIC): such compounds comprise a reactive functional group and optionally one or more linkages The subcomponent is a structure of a compound of formula (II) to facilitate direct or indirect attachment of the compound to an antibody or antigen-binding fragment. These compounds are suitable for the preparation of immunoconjugates. Thus, in another aspect, the invention provides compounds of formula (IIA) and (IIB) and (IIC):

Wherein W comprises a reactive functional group which can be used to link (IIA) or (IIB) or (IIC) to a linker component or directly to Ab to provide an immunoconjugate of formula (I) and to provide for the use of such compounds A method of preparing an ADC.

In another aspect, the invention provides a novel Eg5 inhibitor of formula (III) as described herein, and a pharmaceutically acceptable salt thereof.

These compounds are novel Eg5 inhibitors and have anti-cancer activity as indicated herein. As demonstrated herein, it can be used as an ADC payload, or as other Eg5 inhibitors, which can be used as a small molecule therapeutic to treat a cell proliferative disorder.

In another aspect, the invention provides a mixture comprising at least one pharmaceutically acceptable carrier or excipient and optionally mixed with two or more pharmaceutically acceptable carriers or excipients. A pharmaceutical composition of formula (I) immunoconjugate or compound of formula (III), and methods of using such compositions to treat a cell proliferative disorder.

In another aspect, the invention provides a method of treating a condition characterized by excessive or undesired cell proliferation comprising administering to an individual in need of such treatment an effective amount of an immunoconjugate or formula of Formula (I) ( III) a compound or any subgenus thereof as described herein, or a pharmaceutical composition comprising the compound or immunoconjugate. The individual for treatment can be a mammal, and preferably a human. Conditions which can be treated by the compounds and methods described herein include various forms of cancer, such as stomach, bone marrow, colon, nasopharynx, esophageal and prostate tumors, gliomas, neuroblastoma, melanoma, breast cancer, lung cancer. , ovarian cancer, colorectal cancer, thyroid cancer, leukemia (such as chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non-Hodge Ginseng), bladder, kidney, stomach (eg gastrointestinal matrix tumor (GIST)), liver and pancreatic cancer and sarcoma. Other cell proliferative disorders that may be treated by such methods and compositions include diabetic retinopathy, liver and pulmonary fibrosis, Sjogren's syndrome, and lupus erythematosus.

The present invention includes compositions of formula (I)-(III) and subgenus thereof as described herein, and all stereoisomers (including diastereomers and enantiomers), tautomers and Its isotopically enriched forms (including deuterium substitutions), as well as the pharmaceutically acceptable salts of such compounds. The compositions of the present invention also comprise polymorphs of formula (I)-(III) (or a subformulae thereof) and salts thereof, especially pharmaceutically acceptable salts.

In another aspect, the invention provides an immunoconjugate comprising an improved linkage group that binds the antibody to a payload, such as a cytotoxin, including an Eg5 inhibitor as described herein. Such immunoconjugates comprise a linking group comprising a group of the formula -C(O)NR ²¹ - or -NR ²¹ -C(O)-, which may be a guanamine or an aminocarboxylic acid An ester wherein R ²¹ has the formula -(CH ₂ ) _1-4 -R ²² and R ²² is selected from the group consisting of -OH, -NH ₂ , N(R ²³ ) ₂ , COOR ²³ , CON(R ²³ ) ₂ , -( OCH ₂ CH ₂ O) _k -OCH ₂ CH ₂ OR ²³ and a polar group of -SO ₂ R ²³ wherein k is from 0 to 4 and each R ^{23 is} independently H or C _1-4 alkyl. These linking groups reduce aggregation of the immunoconjugate.

Figures 1A-1B. The average drug load (DAR, drug to antibody ratio) of the ADC was determined based on the heavy and light chain loadings.

Figures 2A-2E. Anti-proliferative activity of various compounds of formula (II) and (III) in cell culture.

Figures 3A-3L. Anti-proliferative activity of certain Eg5 inhibitors in a variety of cancer cell lines derived from different lineages.

Figures 4A-4V. The ADC is directed against the in vitro anti-proliferative activity of engineered cell lines against matched parental (Her2 low expression) cell lines against high Her2.

Figures 5A-5E. In vitro anti-proliferative activity of ADCs against cell lines with endogenous Her2 expression.

Figure 6 (A). The efficacy of the TBS-Cmpd 220 conjugate in HCC 1954 breast cancer xenografts.

Figure 6 (B). The efficacy of the TBS-Cmpd 220 conjugate in HCC 1954 breast cancer xenografts.

Figures 7 (A) and (B) show the efficacy of the TBS-Cmpd 220 conjugate in SK-OV-3ip xenografts.

Figure 8 shows the efficacy of the TBS-Cmpd 215 conjugate in SK-OV-3ip xenografts.

Figure 9 shows the efficacy of the TBS-Cmpd 223 conjugate in SK-OV-3ip xenografts.

Figure 10 (A) shows the degree of aggregation of a structure called ADC-110 as measured by size exclusion chromatography. The amount of aggregate detected was about 12% of the total detected conjugate.

Figure 10 (B) shows the amount of aggregation of ADC-111, which is about 2.4%.

Figure 10 (C) shows the amount of aggregation of ADC-112, which is about 2.7%.

Figure 11 shows the in vitro activities of ADC-110 and ADC-111 for various cell types.

Figure 12 shows the activity of a series of immunoconjugates with different payloads (from 5A, 5B, 5C, 5D, 5E and 5F of Table 5) linked to antibody cKitA. They all exhibit good to excellent activity against SK-OV-3ip in cell culture.

Figure 13 shows the activity of selected payloads bound to trastuzumab (TBS) against various tumor cell lines, demonstrating that various payloads of Formula II are active against various cancer cell lines. These payload compounds are found in Tables 5 and 6.

Figure 14 shows a comparison of representative inhibitors of the invention with Eg5 inhibitors of other compound classes; the compounds of Figure 14 are each linked to trastuzumab via a Val-Cit linker.

Figure 15 shows the activity of the trastuzumab immunoconjugate of the present invention against Her2 high performance and Her2 low expression cell lines compared to immunoconjugates with maytansin payload. Immunoconjugates with non-Her2 antigen binding agents are included for comparison.

Figure 16 shows treatment with trastuzumab and control conjugate (where the antigen binding group does not recognize tumor antigen) in combination with compound 5B (5 mg/kg dose and 10 mg/kg dose) with trastuzumab alone In vivo tumor growth inhibition results of mouse xenograft tumors (SK-OV-3ip).

Figure 17 shows an Eg5 inhibitor on trastuzumab (anti-Her2 antibody) compared to a control with a maytansin payload and a control lacking a payload and a control without a tumor-binding antibody. In vivo tumor growth inhibitory activity on a payload immunoconjugate-treated mouse xenograft tumor (SK_OV-3ip).

Figure 18 shows in vivo tumor growth inhibitory activity on mouse xenograft tumors (H526) treated with an immunoconjugate conjugated with an Eg5 inhibitor on cKitA (anti-cKit antibody).

Figure 19 shows a mouse xenograft tumor (H526) treated with an immunoconjugate conjugated with an Eg5 inhibitor on cKitA (anti-cKit antibody) compared to a cKitA conjugate containing a maytansin payload (DM1) In vivo tumor growth inhibitory activity.

Figure 20 shows mice bearing two xenograft tumors (H526 and SK-OV-3ip) treated with an immunoconjugate of Eg5 inhibitor payload on cKitA (anti-cKit) antibody and trastuzumab antibody On-site tumor growth inhibitory activity.

Figure 21 shows the activity of various Eg5 inhibitor immunoconjugates with trastuzumab antibodies tested on mouse xenograft SK-OV-3ip tumors. For comparison, the immunoconjugates have the same antibodies that bind to auristatin payload (MMAE) and maytansin payload (DM1).

Figure 22 shows the in vivo activity of several immunoconjugates in Table 5 in mouse xenograft tumors using Kadcyla® and anti-Her2 antibodies as comparators and vehicle alone as a control.

Figure 23 shows the in vivo activity of several immunoconjugates in Table 5 in mouse xenografts using Kadcyla® and Compound 6U (anti-Her2 antibody conjugate) and igG1 κ chain specific for viral glycoprotein gH As a comparator and a vehicle alone, a control was used.

Figure 24 shows the in vivo activity of several immunoconjugates in Table 5 in mouse xenografts using vehicle alone as a control. The immunoconjugates have an anti-cKit antibody that binds to the compound of Table 5, and the cell strain is sensitive to the ckit antibody.

Figure 25 shows the in vivo activity of several immunoconjugates in Table 5 in mouse xenografts using vehicle alone as a control. These immunoconjugates have anti-cKit antibodies that bind to the compounds in Table 5.

Figure 26 shows the in vivo activity of immunoconjugates with payload-linker compound 5B in mouse xenografts, and three different anti-cKit antibodies. cKitA is a parent antibody; cKitB and cKitC are cysteine engineered cKitA muteins, as described herein. Results were presented in two different dosing levels using vehicle alone as a control. At both doses, the cysteine engineered mutant antibodies each provide an immunoconjugate that is more active than the native antibody.

Figure 27 shows the in vivo activity of an immunoconjugate comprising the antibody cKitA binding to compound 5B using cKitA antibody (unbound) and vehicle as a control.

Figure 28 is a graph of in vitro (cell culture) activity of various immunoconjugates of the invention tested on a variety of different tumor cell types. The last plot (Figures 28-29) shows that in vitro activity is not significantly affected by small changes in DAR.

The following definitions apply unless otherwise expressly provided.

The term "amino acid" refers to typical, synthetic and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to typical amino acids. A typical amino acid is a protein amino acid encoded by the genetic code and includes alanine, arginine, aspartame, aspartic acid, cysteine, glutamic acid, glutamic acid, glycine Acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, valine, serine, threonine, tryptophan, tyrosine, valine And selenocysteine, pyrrole lysine and pyrroline-carboxy-isoamine. An amino acid analog refers to a compound having the same basic chemical structure as a typical amino acid (that is, an α-carbon bonded to a hydrogen, a carboxyl group, an amine group, and an R group), such as homoserine, orthraic acid, Adenine methionine, methyl methionine. Such analogs have a modified R group (eg, n-hamidoic acid) or a modified peptide backbone, but retain the same basic chemical structure as a typical amino acid.

The term "antigen-binding portion" as used herein refers to a moiety that is capable of specifically binding to an antigen, and includes, but is not limited to, antibodies and antibody fragments.

The term "antibody" as used herein refers to an immunoglobulin family polypeptide that is capable of binding a corresponding antigen non-covalently, reversibly, and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by a disulfide bond. Each heavy chain comprises a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as V _L) and a light chain constant region. The light chain constant region contains a domain, C _L . V _H and V _L regions can be further subdivided into regions of the conservative (called the framework regions (FR)) turn of the hypervariable regions (termed complementarity determining regions (CDR)). Each of V _H and V _{L is} composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody mediates binding of the immunoglobulin to a host tissue or factor comprising a plurality of cells of the immune system (eg, effector cells) and a first component (Clq) of the classical complement system.

The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies of the antibodies of the invention). Such antibodies can be of any isotype/category (eg, IgG, IgE, IgM, IgD, IgA, and IgY) or a subclass (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2).

Both light and heavy chains are divided into structural and functional homology regions. The terms "constant" and "variable" are used functionally. In this regard, it is understood the light chain (V _L) and heavy chain (V _H) variable domain portions determine antigen recognition and specificity of. Conversely, the constant domains of the light chain (C _L ) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental activity, Fc receptor binding, complement binding and the like. By convention, the numbering of the domains in the constant region increases as it goes further away from the antigen binding site or the amine terminus of the antibody. The N-terminus is a variable region and the C-terminus is a constant region; the CH3 and C _L domains actually comprise a carboxy-terminal domain of a heavy chain and a light chain, respectively.

The term "antigen-binding fragment" as used herein refers to retention specificity that interacts with an antigenic epitope (eg, by binding, steric hindrance, stabilization/destabilization, empty) One or more parts of the ability to distribute the antibody. Examples of binding fragments include, but are not limited to, single-chain Fv (scFv); disulfide-bonded Fv (sdFv); Fab fragment, F(ab') fragment, unit price consisting of VL, VH, CL, and CH1 domains Fragment; F(ab)2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond in the hinge region; an Fd fragment consisting of VH and CH1 domains; consisting of the VL and VH domains of the one-arm of the antibody Fv fragment; dAb fragment (Ward et al, Nature 341: 544-546, 1989), which consists of a VH domain; and an isolated complementarity determining region (CDR), or other epitope binding fragment of an antibody.

Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by independent genes, they can be joined by a synthetic linker using a recombinant method that enables them to be fabricated in which the VL and VH regions are paired to form a monovalent molecule. a single protein chain (referred to as single-chain Fv ("scFv"); see, eg, Bird et al, Science 242: 423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. 85: 5879-5883, 1988). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment". Such antibody binding fragments are obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as intact antibodies.

Antigen-binding fragments can also be incorporated into single domain antibodies, maximal antibodies, minimal antibodies, nanobodies, intrabodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, v-NARs, and double scFvs (see, for example, Hollinger and Hudson). , Nature Biotechnology 23: 1126-1136, 2005). The antigen-binding fragment can be grafted into a polypeptide-based backbone, such as a type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes a fibronectin polypeptide single antibody)

The antigen-binding fragment can be incorporated into a single-stranded molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with the complementary light-chain polypeptide form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8: 1057-1062, 1995; and U.S. Patent No. 5,641,870).

The term "single antibody" or "single antibody composition" as used herein refers to There are substantially identical amino acid sequences or polypeptides derived from the same genetic source, including antibodies and antigen-binding fragments. The term also encompasses antibody molecule preparations of a single molecule composition. The monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term "human antibody" as used herein includes antibodies having variable regions in which the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutant forms of human germline sequences or antibodies containing consensus framework sequences derived from human framework sequence analysis, for example As described by Knappik et al., J. Mol. Biol. 296: 57-86, 2000.

Human antibodies of the invention may include amino acid residues that are not encoded by human sequences (eg, mutations induced by in vitro random or site-specific mutagenesis or introduced by in vivo somatic mutation or conservative substitution to promote stability) Or manufacturing).

The term "humanized" anti-system as used herein refers to an antibody that retains the reactivity of a non-human antibody but is less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterpart. See, for example, Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44: 65-92 (1988); Verhoeyen et al, Science, 239 : 1534-1536 (1988); Padlan, Molec. Immun., 28: 489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994).

The term "specific binding" or "selective binding" when used in the context of describing the interaction between an antigen (eg, a protein) and an antibody, antibody fragment, or antibody-derived binding agent refers to a binding reaction to a protein The presence of antigens in heterologous populations of other biological agents, such as biological samples, such as blood, serum, plasma or tissue samples, is decisive. Thus, under certain specified immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least twice as background and does not substantially bind to other antigens present in the sample in significant amounts. In one embodiment, the binding of an antibody or binding agent having a particular binding specificity to a particular antigen is under the specified immunoassay conditions. At least ten (10) times and substantially not bound to other antigens present in the sample in significant amounts. Specific binding to an antibody or binding agent under such conditions may require that the antibody or binding agent has been selected for its specificity for a particular protein. This selection can be achieved by subtracting antibodies that cross-react with molecules or other subtypes from other species (eg, mice or rats), as appropriate or as appropriate. Alternatively, in some embodiments, antibodies or antibody fragments that cross-react with certain desired molecules are selected.

A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive for a particular protein. For example, solid phase ELISA immunoassays are routinely used to select antibodies that are immunoreactive with respect to proteins (see, eg, Harlow and Lane, Using Antibodies, A Laboratory Manual (1998), for description can be used to determine specific immune responses Forms and conditions of sexual immunoassay). A specific or selective binding reaction will typically produce a signal that is at least two times greater than the background signal and more typically at least 10 to 100 times greater than the background.

The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at multiple sites via weak non-covalent forces; the greater the interaction, the stronger the affinity.

The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. However, an isolated antibody that specifically binds to one antigen can be cross-reactive with other antigens. Furthermore, the isolated antibodies may be substantially free of other cellular material and/or chemicals.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of an amino acid residue. These terms apply to both classical amino acid polymers as well as non-classical amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

The term "immunoconjugate" or "antibody conjugate" as used herein refers to an antigen A binding moiety, such as an antibody or antigen-binding fragment thereof, is linked to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, and the like. The linkage can be a covalent bond, or a non-covalent interaction, and can include chelation. Various linkers known in the art can be employed to form immunoconjugates. Alternatively, the immunoconjugate can be provided as a fusion protein expressed as a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to a protein produced by joining two or more genes or gene fragments that originally encoded independent proteins, including peptides and polypeptides. Translation of the fusion gene produces a single protein with functional properties derived from each of the original proteins.

The term "cytotoxin" or "cytotoxic agent" as used herein refers to any agent that is detrimental to the growth and proliferation of cells and that can be used to reduce, inhibit or destroy cells or malignancies.

The term "anticancer agent" as used herein refers to any agent that can be used to treat a cell proliferative disorder, such as cancer, including but not limited to cytotoxic agents, chemotherapeutic agents, radiation therapy and radiotherapeutic agents, targeted antibiotics. Cancer agents and immunotherapeutics.

The term "drug moiety" or "payload" as used herein includes, but is not limited to, an Eg5 inhibitor, which refers to a chemical moiety that binds to or can bind to an antibody or antigen-binding fragment to form an immunoconjugate, and can include Attached to any part of an antibody or antigen-binding fragment. The immunoconjugate of the invention comprises, for example, an Eg5 inhibitor as a payload, but may also include one or more other payloads. For example, the drug moiety or payload can be an anticancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an antiparasitic agent, an antiviral agent, or an anesthetic. In certain embodiments, the drug moiety is selected from the group consisting of a V-ATPase inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, auristatin, and dolastatin (dolastatin), maytansinoid, MetAP (methionine aminopeptidase), nuclear export inhibitor of protein CRM1, DPPIV inhibitor, phosphonium transfer reaction inhibitor in mitochondria, protein synthesis inhibitor, Kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, proteasome inhibitors, kinesin inhibitors, HDAC Inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, and DHFR inhibitors. Suitable examples include auristatin, such as MMAE and MMAF; calicheamicin, such as gamma-calicheamicin; and maytansinoids, such as DM1, DM3, and DM4. Methods for linking each of these drug moieties to a linker compatible with the antibody and methods of the invention are known in the art. See, for example, Singh et al, (2009) Therapeutic Antibodies: Methods and Protocols, Vol. 525, 445-457. In addition, the payload can be a biophysical probe, a fluorophore, a spin label, an infrared probe, an affinity probe, a chelating agent, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, and a self. Spin label, DNA, RNA, protein, peptide, surface, antibody, antibody fragment, nanoparticle, quantum dot, liposome, PLGA particle, sugar or polysaccharide, reactive functional group (such as described herein) or ligably bindable a binding agent to another part or surface, and the like.

"Tumor" refers to neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues.

The term "anti-tumor activity" means a decrease in the rate, activity or metabolic activity of tumor cells. A possible way to demonstrate anti-tumor activity is to demonstrate a decrease in abnormal cell growth rate or tumor size stability or decrease during therapy. The activity can be analyzed using an approved in vitro or in vivo tumor model including, but not limited to, xenograft models, allograft models, MMTV models, and anti-tumor activities known in the art. Other known models.

The term "malignant tumor" refers to a non-benign tumor or cancer. The term "cancer" as used herein includes malignant tumors characterized by dysregulated or uncontrolled cell growth. Exemplary cancers include: carcinoma, sarcoma, leukemia, and lymphoma.

The term "cancer" includes primary malignant tumors (eg, tumors that do not migrate to sites at the site of non-primary tumor sites in the body) and secondary malignancies (eg, tumors derived from metastasis, ie, tumor cell migration) Secondary site different from the original tumor site At).

The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial agents) known to those skilled in the art. , antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrating agents, lubricants, sweeteners, flavoring agents, dyes and the like Combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pages 1289 to 1329). The use thereof in a therapeutic or pharmaceutical composition is encompassed except where any conventional carrier is incompatible with the active ingredient.

The term "therapeutically effective amount" of a compound of the invention refers to the invention that will cause an organism or medical response, such as an enzyme or protein activity, to reduce or inhibit, or ameliorate symptoms, alleviate the condition, slow or delay disease progression, or prevent disease, and the like. The amount of the compound. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the invention effective to at least partially alleviate, inhibit, prevent, and/or ameliorate a condition or disorder or disease when administered to an individual.

In another non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the invention effective to at least partially reduce or inhibit Eg5 activity when administered to a cell or tissue or non-cellular biomaterial or culture medium.

The term "individual" as used herein refers to an animal. Animals are typically mammals. An individual also refers to, for example, a primate (eg, human, male or female), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, and the like. In certain embodiments, the individual is a primate. In a particular embodiment, the individual is a human.

The term "inhibiting", "inhibiting" or "inhibiting" as used herein refers to alleviating or arresting a given condition, symptom or condition or disease, or significantly reducing the baseline activity of a biological activity or process. .

As used herein, in one embodiment, the term "treat", "treating" or "treatment" refers to amelioration of the disease or condition (ie, slowing or preventing or reducing) The development of a slow disease or at least one of its clinical symptoms). In another embodiment, "treat", "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter, including parameters that are not identifiable by the patient. In yet another embodiment, "treat", "treating" or "treatment" refers to physically (eg, stabilizing identifiable symptoms), physiologically (eg, stabilizing physical parameters) ) or both aspects of the disease or condition. In yet another embodiment, "treat", "treating" or "treatment" refers to preventing or delaying the progression of a disease or condition.

As used herein, an individual "needs" the treatment if it would benefit from treatment in biology, medicine, or quality of life.

The terms "a (a)", "an", "the", as used herein, are used in the context of the present invention (especially in the context of the claims), unless otherwise indicated herein. (the) and similar terms are to be construed as covering the singular and plural.

In certain embodiments, the modified immunoconjugates of the invention are described in terms of a "X group/antibody" ratio (eg, 1, 2, 3, 4, 5, 6, 7, or 8 or 12 or 16); The ratio corresponds to "n" in the formula (I). While this ratio has integer values for a particular binder molecule, it should be understood that the average is typically used to describe a sample containing multiple molecules because of the degree of heterogeneity within the immunoconjugate sample. The average load of the immunoconjugate sample is referred to herein as the "drug to antibody ratio" or DAR. In some embodiments, the DAR is between about 1 and about 16, and is typically about 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, at least 50% by weight of the sample is a compound having an average DAR of 2, and preferably, at least 50% of the sample is a product containing an average DAR of 1.5. Preferred embodiments include immunoconjugates wherein DAR is from about 2 to about 8, such as from about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In these embodiments, the DAR is "about q" means measured for DAR. The value is within ±20% of q, or preferably within ±10% of q.

The term "optical isomer" or "stereoisomer" as used herein, refers to any of the various stereoisomeric configurations that may be present for a given compound of the invention, and includes geometric isomers. It will be understood that the substituent may be attached to the palm center of the carbon atom. The term "puppet" refers to a molecule that has a property that does not overlap its mirror image, and the term "non-palphape" refers to a molecule that overlaps its mirror image. Thus, the invention includes enantiomers, diastereomers or racemates of the compounds. An "enantiomer" is a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. This term is used to refer to a racemic mixture where appropriate. A "diastereomer" is a stereoisomer that has at least two asymmetric atoms but which are not mirror images of each other. Absolute stereochemistry was specified according to the Cahn-Ingold-Prelog R-S system. When the compound is a pure enantiomer, the stereochemistry at each pair of palmitic carbons can be specified by R or S. The absolute configuration of an unknown analytical compound can be specified as (+) or (-) by the direction in which the plane-polarized light is rotated (right-handed or left-handed) at the wavelength of the sodium D-line. Certain compounds described herein contain one or more asymmetric centers or axes and may thereby result in enantiomers, diastereoisomers that may be defined as (R)- or (S)- in terms of absolute stereochemistry. Constructs and other stereoisomeric forms.

Depending on the choice of starting materials and procedures, the compounds may exist as one possible isomer or a mixture thereof, such as a pure optical isomer, or a mixture of isomers, such as a racemate and a mixture of diastereomers. , depending on the number of asymmetric carbon atoms. Unless otherwise stated, such as in the context of identifying a particular isomer, the invention is intended to include all such possible isomers, including racemic mixtures, mixtures of diastereomers, and optically pure forms. The optically active (R)- and (S)-isomers can be resolved using palmitic synthetic components or by preparation of palmitic reagents or using conventional techniques. If the compound contains a double bond, the substituent can be in an E or Z configuration. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent can have a cis or trans configuration. It is also intended to include all tautomeric forms.

The term "salt" or "salts" as used herein refers to a compound of the invention. An acid addition salt or a base addition salt. "Salt" includes, inter alia, "pharmaceutically acceptable salts." The term "pharmaceutically acceptable salts" refers to salts which retain the biological effectiveness and properties of the compounds of the invention and which are typically biologically or otherwise desirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts due to the presence of amine groups and/or carboxyl groups or the like.

The inorganic acid and the organic acid may be used to form a pharmaceutically acceptable acid addition salt such as acetate, aspartate, benzoate, besylate, bromide/hydrobromide, hydrogencarbonate/ Carbonate, hydrogen sulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlorophylline, citrate, ethane disulfonate, fumarate, glucoheptonate , gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, cisplatin Oleate, malonate, mandelate, methanesulfonate, methyl sulfate, naphthoate, naphthalene sulfonate, nicotinic acid, nitrate, octadecanoate, oleic acid Salt, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulphonate Salicylate, tartrate, tosylate and trifluoroacetate.

Inorganic acids which can form salts include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

Organic acids which can form salts include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methane Sulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

Inorganic bases which can form salts include, for example, ammonium salts and metals from columns I to X of the Periodic Table. In certain embodiments, the salt is produced from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; in particular, suitable salts include ammonium, potassium, sodium, calcium, and magnesium. salt.

Organic bases which can form salts include, for example, primary, secondary and tertiary amines; substituted amines, including naturally occurring substituted amines; cyclic amines; alkali ion exchange resins and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

The pharmaceutically acceptable salts of the present invention can be synthesized from basic or acidic moieties by conventional chemical methods. In general, the salts can be obtained by subjecting the free acid form of such compounds to a stoichiometric amount of a suitable base such as a hydroxide, carbonate, bicarbonate or the like of Na, Ca, Mg or K. The reaction is prepared or by reacting the free base form of such compounds with a stoichiometric amount of the appropriate acid. These reactions are typically carried out in water or in an organic solvent or a mixture of the two. In general, when practicable, it is desirable to use a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. A list of additional suitable salts can be found, for example, in "Remington's Pharmaceutical Sciences", 20th Edition, Mack Publishing Company, Easton, Pa., (1985); and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" , Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

Any formula given herein is also intended to indicate unlabeled forms of the compounds as well as isotopically labeled forms. An isotopically labeled compound has a structure depicted by the formula given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes which may be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, respectively. , ³¹ P, ³² P, ³⁵ S, ³⁶ Cl, ¹²⁵ I. The invention includes various isotopically labeled compounds as defined herein, such as compounds in which a radioisotope is present, such as ³ H and ¹⁴ C, or a compound in which a non-radioactive isotope such as ² H and ¹³ C is present. Such isotopically labeled compounds are suitable for metabolic studies (using ¹⁴ C); reaction kinetic studies (using, for example, ² H or ³ H); detection or imaging techniques such as positron emission tomography (PET) or single photons Computerized tomography (SPECT), including drug or tissue distribution analysis; or patient radiotherapy. In particular, ¹⁸ F or labeled compounds may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds of formula (I) can generally be replaced by conventional isotopically labeled reagents by conventional techniques known to those skilled in the art, or by methods analogous to those described in the accompanying examples and preparations. Label the reagent to prepare.

Furthermore, substitutions with heavy isotopes, especially with hydrazine (i.e., ² H or D), may afford certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements or improved therapeutic index. The concentration of the heavy isotope, in detail, can be determined by the isotope enrichment factor. The term "isotopic concentration factor" as used herein means the ratio between the isotope abundance of a given isotope and the natural abundance. If the substituent in the compound of the invention is represented by hydrazine, the isotope enrichment factor for each of the specified ruthenium atoms in the compound is at least 3500 (52.5% 各 on each designated ruthenium atom), at least 4000 (60% 氘 incorporation) At least 4500 (67.5% 氘 incorporation), at least 5000 (75% 氘 incorporation), at least 5500 (82.5% 氘 incorporation), at least 6000 (90% 氘 incorporation), at least 6333.3 (95% 氘 incorporation) At least 6466.7 (97% 氘 incorporation), at least 6600 (99% 氘 incorporation) or at least 6633.3 (99.5% 氘 incorporation).

The term "thiol-methyleneimine" as used herein refers to a group formed by the reaction of a thiol with maleimide, having the formula Wherein Y and Z are groups to be linked via a thiol-methylenediamine bond, and may comprise a linker component, an antibody or a payload.

As used herein, "cleavable" refers to the joining of two parts by covalent attachment. However, it decomposes under physiologically relevant conditions to cleave covalently linked linking groups or linker components, typically cleavable linking groups are cleaved in vivo in the intracellular environment faster than outside the cell. , causing the release of the payload to occur preferentially within the targeted cell. Cleavage can be enzymatic or non-enzymatic, but generally releases the payload from the antibody without degrading the antibody. Cleavage may leave a portion of the linking group or linker component attached to the payload, or it may release the payload without any linkage residue.

As used herein, "Pcl" refers to pyrroline carboxylate amides, for example

Wherein R ²⁰ is H which, when incorporated into the peptide, has the formula:

The corresponding compound wherein R ²⁰ is a methyl group is pyrrole lysine.

As used herein, "non-cleavable" refers to a linking group or linker component that is not particularly susceptible to decomposition under physiological conditions, for example, it is at least as stable as an antibody or antigen-binding fragment portion of an immunoconjugate. These linking groups are sometimes referred to as "stable", meaning that they are sufficiently resistant to degradation to maintain the payload attached to the antigen binding portion Ab until the Ab itself is at least partially degraded, ie, the degradation of Ab precedes the bonding group. The in vivo lysis of the group. Degradation of an antibody portion of an ADC having a stable or non-cleavable linking group may leave some or all of the linking groups, such as one or more amino acid groups attached to the antibody of the drug moiety of the payload or in vivo delivery .

The term "halogen" (or halo) as used herein refers to fluoro, bromo, chloro or iodo, especially fluoro or chloro. a halogen-substituted group and a moiety (such as a halogen-substituted alkyl (haloalkyl) group) It can be monohalogenated, polyhalogenated or fully halogenated.

The term "heteroatom" as used herein, unless otherwise provided, refers to a nitrogen (N), oxygen (O) or sulfur (S) atom, especially nitrogen or oxygen.

The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon moiety. Unless otherwise provided, alkyl refers to a hydrocarbon moiety having from 1 to 10 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, tert-butyl, n-pentyl, isuf Base, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-decyl, n-decyl And similar groups.

A substituted alkyl group is an alkyl group containing one or more substituents in place of hydrogen, such as 1, 2 or 3 substituents, up to the number of hydrogens present on the unsubstituted alkyl group. If not otherwise specified, suitable substituents for the alkyl group may be selected from the group consisting of halogen, CN, pendant oxy, hydroxy, C _1-4 alkoxy, substituted or unsubstituted C _3-6 cycloalkyl, substituted Or unsubstituted phenyl, amine, (C _1-4 alkyl)amino, di(C _1-4 alkyl)amino, C _1-4 alkylthio, C _1-4 alkyl sulfonate Mercapto, -C(=O)-C _1-4 alkyl, COOH, -COO(C _1-4 alkyl), -O(C=O)-C _1-4 alkyl, -NHC(=O C _1-4 alkyl and -NHC(=O)OC _1-4 alkyl. Preferred substituents for alkyl include halogen, CN, pendant oxy, hydroxy, C _1-4 alkoxy, C _3-6 cycloalkyl, phenyl, amine, (C _1-4 alkyl) amine , di(C _1-4 alkyl)amino, C _1-4 alkylthio, C _1-4 alkylsulfonyl, -C(=O)-C _1-4 alkyl, COOH, -COO (C _1-4 alkyl), -O(C=O)-C _1-4 alkyl, -NHC(=O)C _1-4 alkyl, and -NHC(=O)OC _1-4 alkyl. Unless otherwise specified, in some embodiments, a C _1-4 substituted alkyl group has 1-3 substituents.

The term "alkylene" as used herein refers to a divalent alkyl group having from 1 to 10 carbon atoms and two open valence states for attachment to other features. Unless otherwise provided, alkylene refers to a moiety having from 1 to 10 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. Representative examples of alkylene include, but are not limited to, methylene, ethyl, propyl, and isopropyl a base, an n-butyl group, a second butyl group, an isobutyl group, a thirylene group, a pentyl group, an isoamyl group, a neopentyl group, a hexyl group, a 3-methyl hexyl group, 2, 2-Dimethylamyl, 2,3-dimethylexopentyl, n-heptyl, octyl, fluorenyl, fluorenyl and the like. Substituted alkylene is an alkylene group containing one or more, such as 1, 2 or 3 substituents; unless otherwise specified, suitable substituents are selected from the substituents listed above for the alkyl group.

The term "haloalkyl" as used herein, refers to an alkyl group, as defined herein, substituted with one or more halo groups, as defined herein. The haloalkyl group can be a monohaloalkyl group, a dihaloalkyl group, a trihaloalkyl group, or a polyhaloalkyl group, including a perhaloalkyl group. The monohaloalkyl group may have one iodine, bromine, chlorine or fluorine in the alkyl group. Chlorine and fluorine are preferably on the alkyl or cycloalkyl group; fluorine, chlorine and bromine are usually preferably on the aryl or heteroaryl group. The dihaloalkyl and polyhaloalkyl groups may have two or more of the same halo atoms or a combination of different halo groups in the alkyl group. Polyhaloalkyl groups typically contain up to 12, or 10, or 8, or 6, or 4, or 3 or 2 halo groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloro Base, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. The perhalo-alkyl group means an alkyl group in which all hydrogen atoms are replaced by a halogen atom, such as a trifluoromethyl group.

The term "alkoxy" as used herein refers to alkyl-O-, wherein alkyl is as defined above. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and Similar group. Typically, the alkoxy group has from 1 to 10 or from 1 to 6 carbons, more typically from 1 to 4 carbon atoms.

"Substituted alkoxy" is an alkoxy group containing one or more, such as 1, 2 or 3 substituents on the alkyl portion of the alkoxy group. Unless otherwise specified, suitable substituents are selected from the substituents listed above for the alkyl group, except that the hydroxyl and amine groups are typically not present on the carbon of the oxygen directly attached to the substituted "alkyl-O" group. .

Similarly, such as "alkylaminocarbonyl", "alkoxyalkyl", "alkoxycarbonyl", Each alkyl moiety of the other groups of "alkoxy-carbonylalkyl", "alkylsulfonyl", "alkylsulfinyl", or "alkylamino" shall have the above-mentioned " The same meaning as stated in the definition of alkyl. When used in this manner, unless otherwise indicated, the alkyl group will typically be a 1-4 carbon alkyl group and will not be further substituted with groups other than the specified components. Unless otherwise specified, when the alkyl groups are substituted, suitable substituents are those specified above for the alkyl group.

The term "haloalkoxy" as used herein refers to haloalkyl-O-, wherein haloalkyl is as defined above. Representative examples of haloalkoxy include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, trichloromethoxy, 2-chloroethoxy, 2,2,2- Trifluoroethoxy, 1,1,1,3,3,3-hexafluoro-2-propoxy and the like. Typically, haloalkyl groups have from 1 to 4 carbon atoms.

The term "cycloalkyl" as used herein refers to a saturated or unsaturated, non-aromatic, monocyclic, bicyclic, tricyclic or spirocyclic hydrocarbon group of 3 to 12 carbon atoms: the cycloalkyl group may be unsaturated and thick. The ring may be a saturated, unsaturated or aromatic ring, with the proviso that the ring atom attached to the cycloalkyl group of the formula of interest is not an aromatic ring carbon. Unless otherwise provided, a cycloalkyl group means a cyclic hydrocarbon group having 3 to 9 ring carbon atoms or 3 to 7 ring carbon atoms. Preferably, unless otherwise specified, a cycloalkyl group is a saturated monocyclic ring having from 3 to 7 ring atoms.

The substituted cycloalkyl group is a cycloalkyl group substituted by 1 or 2 or 3 or more than 3 substituents (up to the number of hydrogens on the unsubstituted group). Typically, a substituted cycloalkyl group will have from 1 to 4 or from 1 to 2 substituents. Unless otherwise specified, suitable substituents are independently selected from the group consisting of halogen, hydroxy, thiol, cyano, nitro, pendant oxy, C _1-4 alkylimino, C _1-4 alkoxy Imino, hydroxyimino, C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, C _1-4 alkoxy, C _1-4 sulfanyl, C _2-4 Alkenyloxy, C _2-4 alkynyloxy, C _1-4 alkylcarbonyl, carboxy, C _1-4 alkoxycarbonyl, amine, C _1-4 alkylamino, di C _1-4 alkyl Amino, C _1-4 alkylaminocarbonyl, di C _1-4 alkylaminocarbonyl, C _1-4 alkylcarbonylamino, C _1-4 alkylcarbonyl (C _1-4 alkyl)amine group, a C _1-4 alkylsulfonyl group, sulfo C _1-4 alkyl amines and acyl C _1-4 alkyl sulfonic acyl group, wherein each of the previously mentioned hydrocarbon group (e.g. an alkyl group, an alkenyl group The alkynyl, alkoxy residue) may be further substituted with one or more groups which, at each occurrence, are independently selected from the list of preferred substituents for "alkyl" herein. Preferred substituents for cycloalkyl include C _1-4 alkyl, halogen, CN, pendant oxy, hydroxy, C _1-4 alkoxy, amine, (C _1-4 alkyl)amino, di C _1-4 alkyl)amino, C _1-4 alkylthio, C _1-4 alkylsulfonyl, -C(=O)-C _1-4 alkyl, COOH, -COO (C _{1 -4} alkyl), -O(C=O)-C _1-4 alkyl, -NHC(=O)C _1-4 alkyl and -NHC(=O)OC _1-4 alkyl.

Exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and the like. Exemplary bicyclic hydrocarbon groups include mercapto, fluorenyl, hexahydroindenyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1 Heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl and the like Group. Exemplary tricyclic hydrocarbon groups include adamantyl groups and the like.

Similarly, such as "cycloalkoxy", "cycloalkoxyalkyl", "cycloalkoxycarbonyl", "cycloalkoxy-carbonylalkyl", "cycloalkylsulfonyl", "halogen" Each cycloalkyl portion of the other group of the cycloalkyl group should have the same meaning as described in the definition of "cycloalkyl" as mentioned above. When used in such terms, a cycloalkyl group is typically a monocyclic 3-7 carbocyclic ring that is unsubstituted or substituted with 1-2 groups. When substituted as appropriate, the substituents are typically selected from the group consisting of C1-C4 alkyl groups and the groups recited above as suitable for cycloalkyl groups.

The term "aryl" as used herein refers to an aromatic hydrocarbon group having from 6 to 14 carbon atoms in the ring portion. Typically, the aryl group is a monocyclic, bicyclic or tricyclic aryl group having 6 to 14 carbon atoms, usually 6 to 10 carbon atoms, such as phenyl or naphthyl. Further, as used herein, the term "aryl" refers to an aromatic substituent which may be a single aromatic ring or a plurality of aromatic rings fused together. Non-limiting examples include phenyl, naphthyl, and 1,2,3,4-tetrahydronaphthyl, with the proviso that the tetrahydronaphthyl group is attached to the formula described via the carbon of the aromatic ring of the tetrahydronaphthyl group.

The aryl is substituted by 1 to 5 (such as 1 or 2 or 3) independently selected from the group consisting of substituents of the aryl group: hydroxyl, thiol, cyano, nitro, C _{1- 4-} alkyl, C _2-4 alkenyl, C _2-4 alkynyl, C _1-4 alkoxy, C _1-4 thioalkyl, C _2-4 alkenyloxy, C _2-4 alkynyloxy, Halogen, C _1-4 alkylcarbonyl, carboxyl, C _1-4 alkoxycarbonyl, amine, C _1-4 alkylamino, di C _1-4 alkylamino, C _1-4 alkylamine Carbocarbonyl, di-C _1-4 alkylaminocarbonyl, C _1-4 alkylcarbonylamino, C _1-4 alkylcarbonyl (C _1-4 alkyl) amine, C _1-4 alkylsulfonate a base, an amine sulfonyl group, a C _1-4 alkylamine sulfonyl group, and a C _1-4 alkylaminosulfonyl group, wherein each of the previously mentioned hydrocarbon groups (for example, an alkyl group, an alkenyl group, an alkynyl group, an alkane group) The oxy residue) may be further substituted with one or more groups, each of which is independently selected from the group listed above as a preferred substituent for the alkyl group. Preferred substituents for aryl are C _1-4 alkyl, halogen, CN, hydroxy, C _1-4 alkoxy, amine, (C _1-4 alkyl)amino, di(C _1-4 alkane Amino group, C _1-4 alkylthio group, C _1-4 alkylsulfonyl group, -C(=O)-C _1-4 alkyl group, COOH, -COO (C _1-4 alkyl group) , -O(C=O)-C _1-4 alkyl, -NHC(=O)C _1-4 alkyl, and -NHC(=O)OC _1-4 alkyl.

Similarly, each aryl moiety of other groups such as "aryloxy", "aryloxyalkyl", "aryloxycarbonyl", "aryloxy-carbonylalkyl" shall have the same as mentioned above The same meaning as stated in the definition of "aryl".

The term "heterocyclyl" as used herein, refers to a heterocyclic group which is saturated or partially unsaturated, but is not aromatic, and may be monocyclic or polycyclic (in polycyclic, especially bicyclic, tricyclic) Or in the case of a spiro ring; and has from 3 to 14, more usually from 4 to 10, and most preferably 5 or 6 ring atoms; one or more, preferably 1 to 4, especially 1 or 2 The ring atoms are heteroatoms independently selected from O, S and N (the remaining ring atoms are therefore carbon). Even though described as, for example, a C5-6 atomic ring, the heterocycle contains at least one heteroatom as a ring atom and has the stated number of ring atoms, such as 5-6 in this example. Preferably, the heterocyclic group has 1 or 2 of these heteroatoms as ring atoms, and preferably the heteroatoms are not directly bonded to each other. The bond ring (i.e., the ring attached to the formula of interest) preferably has from 4 to 12, especially from 5 to 7, ring atoms. A heterocyclic group can be fused to an aromatic ring, with the proviso that it is attached to the heterocyclic group of the formula of interest The child is not aromatic. The heterocyclic group can be attached to the formula of interest via a hetero atom (typically nitrogen) or a carbon atom of the heterocyclic group. The heterocyclic group may include a fused or bridged ring as well as a spiro ring, and only one ring of the polycyclic heterocyclic group needs to contain a hetero atom as a ring atom. Examples of the heterocyclic ring include tetrahydrofuran (THF), dihydrofuran, 1,4-dioxane, morpholine, 1,4-dithiane, piperazine, piperidine, 1,3-dioxolane, imidazolidinium. , imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropentan, oxathiolane, dithiolane, 1,3-dioxane, 1,3-dithiane, Oxetane, thiomorpholine and the like.

The substituted heterocyclic group is a heterocyclic group independently substituted with 1 to 5 (such as 1 or 2 or 3) substituents selected from the substituents described above for the cycloalkyl group.

Similarly, each heterocyclic moiety of the other groups such as "heterocyclyloxy", "heterocyclyloxyalkyl", or "heterocyclyloxycarbonyl" shall have the "heterocycle" as mentioned above. The same meaning as described in the definition of "base".

"Cyclic ether" as used herein, unless otherwise specified, refers to a heterocyclic ring containing from 4 to 7 ring atoms, which contains an oxygen atom as a ring member and, as appropriate, for a ring of 5 or more atoms. Two non-adjacent oxygen atoms. Typical examples include oxetane, tetrahydrofuran, tetrahydropyran, oxepane and 1,4-dioxane.

The term "heteroaryl" as used herein, refers to a 5-14 membered monocyclic or bicyclic or tricyclic aromatic ring system having from 1 to 8 heteroatoms as ring members; the heteroatoms being selected from the group consisting of N, O and S. . Heteroaryl and heterocycle may be referred to herein as, for example, _C5-6 heteroaryl or heterocycle: it is understood that when used in this description, the 5-6 refers to the total number of ring atoms, including carbon and heteroatoms; It may alternatively be referred to as a 5-6 membered heteroaryl or heterocyclic group. Typically, a heteroaryl is a 5-10 membered ring system containing at least one heteroatom as a ring member, such as a 5-6 membered monocyclic or 8-10 membered bicyclic group. Typical heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4- or 5-imidazolyl, 1-, 3-, 4- or 5- -pyrazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxan Azyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, 1- or 2-tetrazolyl, 2-, 3- or 4- Pyridyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-pyrazinyl and 2-, 4- or 5-pyrimidinyl.

The term "heteroaryl" also refers to a group in which a heteroaryl ring is fused to one or more aryl, cycloalkyl or heterocyclyl rings, wherein the group or point attached to the formula of interest is in the heteroaryl group. On the ring. Non-limiting examples include 1-pyridazinyl, 2-pyridazinyl, 3-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 7-pyridazinyl or 8-indole Pyridazinyl, 1-isoindolyl, 3-isoindolyl, 4-isoindolyl, 5-isodecyl, 6-isoindenyl or 7-isoindolyl, 2-indole , 3-indenyl, 4-indenyl, 5-indenyl, 6-fluorenyl or 7-fluorenyl, 2-oxazolyl, 3-oxazolyl, 4-oxazolyl, 5-oxazolyl, 6-carbazolyl or 7-oxazolyl, 2-indenyl, 4-indenyl, 5-indenyl, 6-fluorenyl, 7-fluorenyl or 8-indenyl, 1- Quinazinyl, 2-quinazinyl, 3-quinazozinyl, 4-quinazinyl, 6-quinazinyl, 7-quinazinyl, 8-quinolinazinyl or 9-quinazinyl, 2-quinoline , 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolinyl or 8-quinolinyl, 1-isoquinolinyl, 3-isoquinoline , 4-isoquinolyl, 5-isoquinolinyl, 6-isoquinolyl, 7-isoquinolinyl or 8-isoquinolinyl, 1-pyridazinyl, 4-pyridazinyl, 5 - pyridazinyl, 6-pyridazinyl, 7-pyridazinyl or 8-pyridazinyl, 2- Pyridyl, 3- Pyridyl, 4- Pyridyl, 5- Pyridyl or 6- Pyridyl, 2-quinazolinyl, 3-quinazolinyl, 5-quinazolinyl, 6-quinazolinyl, 7-quinazolinyl or 8-quinazolinyl, 3- Olinyl group, 4- Lolinyl, 5- Lolinyl, 6- Olinyl group, 7- Orolinyl or 8- Orolinyl, 2-acridinyl, 4-acridinyl, 6-acridinyl or 7-acridinyl, 1-4aH carbazolyl, 2-4aH carbazolyl, 3-4aH carbazolyl, 4- 4aH carbazolyl, 5-4aH carbazolyl, 6-4aH carbazolyl, 7-4aH oxazolyl or 8-4aH oxazolyl, 1-oxazolyl, 2-oxazolyl, 3-oxazolyl , 4-oxazolyl, 5-carbazolyl, 6-oxazolyl, 7-oxazolyl or 8-oxazolyl, 1-indolyl, 3-indolyl, 4-indolyl, 5 - porphyrin, 6-carbolinyl, 7-carbolinyl, 8-carbolinyl or 9-carbolinyl, 1-cyridinyl, 2-cyridinyl, 3-cyridinyl, 4-morphine Pyridyl, 6-cyridinyl, 7-cyridinyl, 8-cyridinyl, 9-cyridinyl or 10-cyridinyl, 1-acridinyl, 2-acridinyl, 3-acridinyl , 4-acridinyl, 5-acridinyl, 6-acridinyl, 7-acridinyl, 8-acridinyl or 9-acridinyl, 1- Pyridyl, 2- Pyridyl, 4- Pyridyl, 5- Pyridyl, 6- Pyridyl, 7- Pyridyl, 8- Pyridyl or 9- Pyridyl, 2-phinyl, 3-phinolinyl, 4-phinyl, 5- morpholinyl, 6-morpholinyl, 8- morpholinyl, 9-morpholinyl or 10- morpholinyl , 1-cyanozinyl, 2-cyanozinyl, 3-cyanozinyl, 4-cyanozinyl, 6-cyanoazinyl, 7-cyanoazinyl, 8-cyanoazinyl or 9-cyanoazinyl, 1 - phenothiazine, 2-morphothazinyl, 3-morphothazinyl, 4-morphothazinyl, 6-phutthiazinyl, 7-phutthiazinyl, 8-cythiazinyl, 9- Phenylthiazinyl or 10-cythiazinyl, 1-phinoxazinyl, 2-morphoxazinyl, 3-phinoxazinyl, 4-morphoxazinyl, 6-morphoxazinyl, 7-morphine Oxazinyl, 8-morphoxazinyl, 9-morphoxazinyl or 10-phinoxazinyl, 2-benzoisoquinolinyl, 3-benzoisoquinolinyl, 4-benzoisoquinoline , 5-benzoisoquinolyl, 6-benzoisoquinolyl, or 1-benzoisoquinolyl, 3-benzoisoquinolinyl, 4-benzoisoquinolinyl, 5- Benzoisoquinolyl, 6-benzoisoquinolyl, 7-benzoisoquinolyl, 8-benzoisoquinolinyl, 9-benzisoquinolinyl or 10-benzoisoquinoline , 2-thieno[2,3-b]furanyl, 3-thieno[2,3-b]furanyl, 4-thieno[2,3-b]furanyl or thieno[2,3 -b]furanyl, 2-7H-pyrazine [2,3-c]oxazolyl, 3-7H-pyrazino[2,3-c]oxazolyl, 5-7H-pyrazino[2,3-c]oxazolyl, 6-7H -pyrazino[2,3-c]oxazolyl, 7-7H-pyrazino[2,3-c]oxazolyl, 8-7H-pyrazino[2,3-c]carbazolyl , 9-7H-pyrazino[2,3-c]oxazolyl, 10-7H-pyrazino[2,3-c]oxazolyl or 11-7H-pyrazino[2,3-c ] carbazolyl, 2-2H-furo[3,2-b]-piperidyl, 3-2H-furo[3,2-b]-pyranyl, 5-2H-furo[3, 2-b]-piperidyl, 6-2H-furo[3,2-b]-piperanyl or 7-2H-furo[3,2-b]-pyranyl, 2-5H-pyridine And [2,3-d]-o-oxazinyl, 3-5H-pyrido[2,3-d]-o-oxazinyl, 4-5H-pyrido[2,3-d]-o -oxazinyl, 5-5H-pyrido[2,3-d]-o-oxazinyl, 7-5H-pyrido[2,3-d]-o-oxazinyl or 8-5H-pyridine And [2,3-d]-o-oxazinyl, 1-1H-pyrazolo[4,3-d]-oxazolyl, 3-1H-pyrazolo[4,3-d]- Azyl or 5-1H-pyrazolo[4,3-d]-oxazolyl, 2-4H-imidazo[4,5-d]thiazolyl, 4-4H-imidazo[4,5-d Thiazolyl or 5-4H-imidazo[4,5-d]thiazolyl, 3-pyrazino[2,3-d]pyridazinyl, 5-pyrazino[2,3-d]pyridazine Or 8-pyrazino[2,3-d]pyridazinyl, 2-imidazo[2,1-b]thiazolyl, 3-imidazole And [2,1-b]thiazolyl, 5-imidazo[2,1-b]thiazolyl or 6-imidazo[2,1-b]thiazolyl, 1-furo[3,4-c] Olinyl, 3-furo[3,4-c] Lolinyl, 6-furo[3,4-c] Lolinyl, 7-furo[3,4-c] Lolinyl, 8-furo[3,4-c] Orolinyl or 9-furo[3,4-c] Phytyl, 1-4H-pyrido[2,3-c]oxazolyl, 2-4H-pyrido[2,3-c]oxazolyl, 3-4H-pyrido[2,3-c] Carbazolyl, 4-4H-pyrido[2,3-c]carbazolyl, 5-4H-pyrido[2,3-c]oxazolyl, 6-4H-pyrido[2,3-c ]oxazolyl, 8-4H-pyrido[2,3-c]oxazolyl, 9-4H-pyrido[2,3-c]oxazolyl, 10-4H-pyrido[2,3- c] carbazolyl or 11-4H-pyrido[2,3-c]oxazolyl, 2-imidazo[1,2-b][1,2,4]triazinyl, 3-imidazo[ 1,2-b][1,2,4]triazinyl, 6-imidazo[1,2-b][1,2,4]triazinyl or 7-imidazo[1,2-b] [1,2,4]triazinyl, 7-benzo[b]thienyl, 2-benzoxazolyl, 4-benzoxazolyl, 5-benzoxazolyl, 6-benzoxan Azolyl or 7-benzoxazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl or 7-benzimidazolyl, 2-benzo Thiazolyl, 4-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl or 7-benzothiazolyl, 1-benzoxanthyl, 2-benzo Oxalyl, 4-benzoxanthyl, 5-benzoxanyl, 6-benzoxanyl, 7-benzoxanyl, 8-benzoxanyl or 9-benzoxanthene Base, 2-benzo Oxazinyl, 4-benzoxazinyl, 5-benzoxazinyl, 6-benzoxazinyl, 7-benzoxazinyl or 8-benzoxazinyl, 1-1H-pyrrole [1,2-b][2]benzodiazepine, 2-1H-pyrrolo[1,2-b][2]benzazepine, 3-1H-pyrrolo[1,2-b ][2]benzodiazepine, 5-1H-pyrrolo[1,2-b][2]benzazepine, 6-1H-pyrrolo[1,2-b][2]benzo N-decyl, 7-1H-pyrrolo[1,2-b][2]benzazepine, 8-1H-pyrrolo[1,2-b][2]benzazepine, 9- 1H-pyrrolo[1,2-b][2]benzazepine,10-1H-pyrrolo[1,2-b][2]benzazepine or 11-1H-pyrrolo[1 , 2-b] [2] benzazepine. Typical fused heteroaryl groups include, but are not limited to, 2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 5-quinolinyl, 6-quinolinyl, 7-quinolinyl or 8- Quinolinyl, 1-isoquinolyl, 3-isoquinolinyl, 4-isoquinolinyl, 5-isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl or 8-isoquine Orolinyl, 2-indenyl, 3-indenyl, 4-indenyl, 5-indenyl, 6-fluorenyl or 7-fluorenyl, 2-benzo[b]thienyl, 3 -Benzo[b]thienyl, 4-benzo[b]thienyl, 5-benzo[b]thienyl, 6-benzo[b]thienyl or 7-benzo[b]thienyl, 2 - benzoxazolyl, 4-benzoxazolyl, 5-benzoxazolyl, 6-benzoxazolyl or 7-benzoxazolyl, 2-benzimidazolyl, 4-benzo Imidazolyl, 5-benzimidazolyl, 6-benzimidazolyl or 7-benzimidazolyl and 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazole Or 7-benzothiazolyl.

The substituted heteroaryl group is a heteroaryl group containing one or more substituents selected from the substituents described above as being suitable for the aryl group.

Similarly, each heteroaryl portion of other groups such as "heteroaryloxy", "heteroaryloxyalkyl", or "heteroaryloxycarbonyl" shall have the definition of "heteroaryl" as mentioned above. The same meaning as described in the above.

In one aspect, the invention provides an immunoconjugate (eg, ADC) comprising an Eg5 inhibitor as a drug or payload, and compositions and methods for treating a cell proliferative disorder using such immunoconjugates or ADCs. Certain imidazole and triazole compounds are known in the art as Eg5 inhibitors and as therapeutic agents for the treatment of cell proliferative disorders, and can be used as ADC payloads; see, for example, WO2007/021794, WO2006/002236, WO2008/063912, WO2009/077448, WO2011/128381 and WO2011/128388. Other Eg5 inhibitors that are known in the art to be suitable for use as ADC payloads include, for example, WO2006/049835, U.S. Patent No. 7,504,405, U.S. Patent No. 7,939,539, and Rath and Kozielski, Nature Reviews: Cancer , Vol. 12, The compound disclosed in Figure 3 of 527-39 (August 2012).

An immunoconjugate comprising an Eg5 inhibitor as a payload (drug) comprises a combination of formula (I):

Wherein Ab represents an antigen binding moiety, such as an antibody or antibody fragment; L represents a linkage group linking X to Ab by covalent or non-covalent linkage, which may optionally be attached to more than one X to Ab, and may or may not contain A cleavable linker component; X represents an Eg5 inhibitor, such as a compound of formula (II) or formula (III) as described herein, or other Eg5 inhibitors, including Rath (Rath and Kozielski , Nature Rev. Cancer , s. Compounds disclosed in Volume 12, 527-39 (2012), including Ispins, SB-743921, AZD4877, ARQ621, ARRY-520, LY2523355, MK-0731, EMD534085, and GSK-923295, and WO06/002236 , E.g. An integer from 1 to 16, preferably from 2 to 8.

Certain aspects and examples of the invention are provided in the list of the following examples:

1. An immunoconjugate of formula (I):

Wherein Ab represents an antigen binding moiety; L represents a linkage group linking X to Ab; m is an integer from 1 to 4; n is an integer from 1 to 16; and X represents a group of formula (II)

It is linked to Ab by L, wherein: Z is N or CH; Ar ¹ is optionally substituted with up to 3 groups selected from halo, C _1-3 alkyl and C _1-3 haloalkyl Phenyl; Ar ² is phenyl or pyridyl or 4-6 atomic cyclic ether, and Ar ² optionally has up to two selected from halo, CN, C _1-3 alkyl, hydroxy, amine and C ₁ Substituted with a _-3 haloalkyl group; R ¹ is C _1-6 alkyl, -(CH ₂ ) _0-2 -C _3-6 cycloalkyl or contains up to two selected from N, O and S a -(CH ₂ ) _0-2 -C _4-7 heterocyclic group (4-7 membered heterocyclic ring) having a hetero atom as a ring member, wherein each C _1-6 alkyl group, C _3-6 cycloalkyl group or C _{4 -7} heterocyclic group optionally has up to three selected from halo, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, hydroxy, amine, pendant oxy, hydroxy Substituted by a C _1-4 alkyl group, an amino substituted C _1-4 alkyl group, a C _1-4 alkyl-amino group, and a COO (C _1-4 alkyl) group; R ² is H or C _1-4 alkyl; T is (CH ₂ ) _1-3 ; Y is selected from C _1-3 aminoalkyl, C _4-6 heterocyclyl and C _3-6 cycloalkyl, wherein C _{1- 3} aminoalkyl, C _4-6 heterocyclyl and C _3-6 cycloalkyl each optionally up to two selected from the group consisting of an amine group, a pendant oxy group, a halogen group, a hydroxyl group, a C _1-4 Alkyl, C _1-4 alkoxy, the hydroxy substituted C _1-4 alkyl, the substituted amino by C _1-4 alkyl, COOH, COO- (C _1-4 alkyl), - C ( =O) NH(C _1-4 alkyl), -C(=O)N(C _1-4 alkyl) ₂ and C _1-4 haloalkyl group substituted; A is NH, N (C _{1 -4} alkyl) or a bond between carbonyl and Q in formula (II); Q is selected from C _1-4 alkyl, -OC _1-4 alkyl, -(CH ₂ ) _0-2 -C _4-6 heterocyclyl, -(CH ₂ ) _0-2 -C _3-6 cycloalkyl, -(CH ₂ ) _0-2 -C _5-6 heteroaryl and -(CH ₂ ) _0-2 - Phenyl, and optionally up to three selected from halo, hydroxy, amine, -SH, -R, -OR, -SR, -SO ₂ R, -NHR, -O-glucuronate and - Substituted by a group of NR ₂ wherein each R is a C _1-6 alkyl group optionally substituted by halo, -SH, -NH ₂ , OMe or -OH; in some embodiments, R may also be C3-6 A cycloalkyl group or a 4-6 membered heterocyclic ring containing N, O or S as a ring member, and each R is independently substituted with a halo group, -SH, -NH ₂ , OMe or -OH.

Typically, m is 1 or 2, preferably 1; and n is 2-8, preferably from about 2 to about 4, or between 3 and 5.

In some such embodiments, R ¹ is C _1-6 alkyl, —(CH ₂ ) _0-2 -C _3-6 cycloalkyl, or contains up to two heteroatoms selected from N, O, and S. a -(CH ₂ ) _0-2 -C _4-7 heterocyclyl group as a ring member, wherein each C _1-6 alkyl group, C _3-6 cycloalkyl group or C _4-7 heterocyclic group is optionally up to three Selected from halo, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, hydroxy, amine, pendant oxy, C _1-4 alkyl substituted by hydroxy, C Substituted by a group of _1-4 alkyl-amino group and COO (C _1-4 alkyl group). In certain embodiments, each C _1-6 alkyl, C _3-6 cycloalkyl or C _4-7 heterocyclyl is selected from up to two selected from halo, C _1-4 alkyl, C _1- Substituted by a _4- haloalkyl group, a C _1-4 alkoxy group, a hydroxyl group, an amine group, and a group substituted with a hydroxy-substituted C _1-4 alkyl group, wherein a preferred substituent is selected from the group consisting of F, a hydroxyl group, a methoxy group, and an amine group. .

Certain examples of R ¹ include 4-tetrahydropyranyl and And as the case may be The dotted line indicates that each R ^{1 is} attached to the point of Formula II.

1A. In some of the foregoing embodiments, R ² is H.

1B. In some of the foregoing embodiments, A is a bond between a carbonyl group and Q. In other embodiments, A is NH. In some of the foregoing embodiments, when Y is a heterocyclic group or a cycloalkyl group, T is CH ₂ and when Y is a C _1-3 aminoalkyl group, T is CH ₂ or CH ₂ CH ₂ .

1C. In some of the foregoing embodiments, Q is 1 or 2 selected from the group consisting of hydroxyl, amine, thiol, amino-C _1-4 alkoxy or amino-C _1-4 alkylthio Group substituted C _1-4 alkyl. In other embodiments, Q is a ring selected from the group consisting of morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, piperazine, phenyl, and pyridine, wherein the ring is optionally up to two selected from the group consisting of C _1-4 alkyl groups, Substituents of halo, CN, hydroxy, amine, C _1-4 alkyl-amino, C _1-4 alkylsulfonyl and C _1-4 alkoxy.

1D. In some of the foregoing embodiments, Y is optionally up to two selected from the group consisting of halo, C _1-4 alkyl, hydroxy, amine, hydroxy-C _1-4 alkyl, and amine-C _1- A pyrrolidine ring substituted with a group of a ₄ alkyl group, a C _1-4 alkyl-amino group, and a C _1-4 alkoxy group. Preferred substituents for pyrrolidine include F, methyl, hydroxy and hydroxymethyl.

2. The immunoconjugate according to embodiment 1, wherein R ² is H.

3. The immunoconjugate according to embodiment 1 or embodiment 2, wherein Z is CH.

4. The immunoconjugate according to embodiment 1 or 2, wherein Z is N.

5. A conjugate according to any one of the immunized in Example 1 to 4, wherein R ¹ is tetrahydropyranyl and R ^1-yl ring optionally substituted by up to two substituents selected from oxo groups and methyl groups.

6. The immunoconjugate according to any of the preceding embodiments, wherein Ar ¹ is a dihalophenyl group. In certain embodiments, Ar ¹ is a 2,5-dihalophenyl group, for example, Ar ¹ can be a 2,5-difluorophenyl group.

In such embodiments, Ar ² may be phenyl, halophenyl, hydroxyphenyl or aminopyridine, such as phenyl, 3-fluorophenyl, 3-hydroxyphenyl, 3-amino-2-pyridine base.

7. The immunoconjugate according to any of the preceding embodiments, wherein the compound of formula (II) has the formula: Wherein in the formula (I), L is bonded to Y, or is attached to Q, or is attached to R ¹ in the formula (II). In a preferred embodiment, L is attached to an oxygen atom or an amine nitrogen which is part of the group Y or a part of the group Q.

The immunoconjugate according to any one of the preceding embodiments, wherein the compound of formula (II) has the formula:

Wherein L is attached to R ¹ and R ¹ is an optionally substituted alkyl group. In some such embodiments, R ¹ is C _3-6 alkyl of the formula -CMe ₂ (CH ₂ ) _0-2 -G-[L], wherein [L] indicates the point at which R ^{1 is} attached to L And G may be a bond, -O-, -NH-, -S-, -CONH- or -COO-. In some such embodiments, R ¹ is -C(Me) ₂ -(CH ₂ ) _0-2 -R ³⁰ , wherein R ³⁰ is hydroxy, carboxy or amine. In these embodiments, L is typically attached to R ¹ via a group R ³⁰ . Examples of R ¹ include -C(Me) ₂ -(CH ₂ ) _0-2 -O-[L], -C(Me) ₂ -(CH ₂ ) _0-2 -NH-[L], -C (Me) ₂ -(CH ₂ ) _0-2 -C(=O)-[L] and -C(Me) ₂ -(CH ₂ ) _0-2 -C(=O)-NH-[L], Wherein [L] indicates the point at which the compound of formula (II) is attached to linker L in formula I.

8. A conjugate according to any one of embodiments 1-7 immunized one of which R ¹ is 4-tetrahydropyranyl group. For example, R ¹ is . The tetrahydropyran ring may be optionally substituted with 1 or 2 substituents selected from the group consisting of a hydroxyl group, a methyl group, a methoxy group and a halogen group.

9. The immunoconjugate according to any of the preceding embodiments, wherein Q in formula (II) is a C _1-4 alkyl group substituted with 1 or 2 groups selected from the group consisting of hydroxyl and amine groups. Wherein A is NH or N (alkyl) Example of embodiment, Q is typically -CH ₂ OH, -CH ₂ NH ₂ or substituted with 1 or 2 substituents selected from -OH and -NH ₂ groups of the substituted C _{2 -4} alkyl. In the case where A is a bond, Q may be a C _1-3 alkyl group, optionally substituted with -OH and/or NH ₂ . The hydroxyl group or amine of the group Q can be used to link the compound of the formula (II) to L in the formula (I).

10. The immunoconjugate according to any of the preceding embodiments, wherein Y is pyrrolidine optionally substituted with 1 or 2 groups selected from the group consisting of fluorine, amine, hydroxy, methoxy and hydroxymethyl. In such embodiments, the pyrrolidine ring NH or the amine or hydroxyl group on the pyrrolidine ring can be the point at which the compound of formula (II) is attached to L in formula (I).

11. The immunoconjugate according to any of the preceding embodiments, wherein A is -NH-.

1B. The immunoconjugate of any of the preceding embodiments, wherein A is a bond.

12. The immunoconjugate of any of the preceding embodiments, wherein the linking group is cleavable. A cleavable linkage group includes a linker component that provides an enzymatic cleavage site in a cell, such as a dipeptide (eg, val-cit); a linker component that is pH sensitive and susceptible to cleavage within the cell, such as ruthenium or An imide; a disulfide linker component that tends to cleave inside the cell; or a glucuronidase-sensitive linker component, such as a phenyl ring on the aminobenzyloxy group A p-aminobenzyloxycarbonyl moiety having a -O-glucuronic acid group.

13. The immunoconjugate of any of embodiments 1-11, wherein the linking group is non-cleavable.

13A. The immunoconjugate of embodiment 13, wherein the linker is substituted with a polar group selected from the group consisting of -(CH ₂ ) _1-2 -COOH, -(CH ₂ ) _1-2 -OH, -COOH or -SO ₃ H , or a pharmaceutically acceptable salt thereof.

14. A compound of formula (III):

Or a pharmaceutically acceptable salt thereof, wherein: Z is N or CH; and Ar ¹ is optionally a group selected from the group consisting of halo, C _1-3 alkyl and C _1-3 haloalkyl Substituted phenyl; Ar ² is phenyl or pyridyl, and optionally up to two groups selected from halo, CN, C _1-3 alkyl, hydroxy, amine and C _1-3 haloalkyl Substituted; R ¹ is -(CH ₂ ) _0-2 -C _4-7 heterocyclyl or -(CH ₂ ) _0-2 -C _3-7 cycloalkyl, wherein C _4-7 heterocyclyl is contained Up to two 4-7 membered rings selected from hetero atoms of N, O and S as ring members, and C _4-7 heterocyclic groups and C _3-7 cycloalkyl groups each optionally up to three selected from halogen , C _1-4 alkyl (eg methyl), C _1-4 haloalkyl (eg trifluoromethyl), C _1-4 alkoxy, hydroxy, amine, pendant oxy, substituted by hydroxy C _1-4 alkyl, the substituted amino substituted with C _1-4 alkyl or COO (C _1-4 alkyl) of the group; optionally substituted with up to three substituents selected from halo, C _1-4 alkyl Substituting a group of C _1-4 alkoxy, pendant oxy or COO (C _1-4 alkyl); R ² is H or C _1-4 alkyl; T is (CH ₂ ) _1-3 ; Y is selected from C _1-2 aminoalkyl, C _4-6 heterocyclyl and C _3-6 cycloalkyl, wherein C _1-2 aminoalkyl C _4-6 heterocyclyl, and C _3-6 cycloalkyl each optionally substituted with up to two substituents selected from amino, oxo, halo, hydroxy, C _1-4 alkoxy, hydroxy substituted C of Substituted by a group of _1-4 alkyl, amino substituted C _1-4 alkyl, COOH, COO-(C _1-4 alkyl) and C _1-3 haloalkyl; A is NH, N (C _1-4 alkyl) or a bond between carbonyl and Q in formula (III); Q is selected from C _1-4 alkyl, -(CH ₂ ) _0-2 -C _4-6 heterocyclyl, -(CH ₂ ) _0-2 -C _{5-6heteroaryl} and -(CH ₂ ) _0-2 -phenyl, and Q optionally has up to three selected from the group consisting of halo, hydroxy, amine, -SH, a group substituted with -R, -OR, -SR, -SO ₂ R, -NHR, -O-glucuronate group and -NR ₂ wherein each R is optionally a halo group, -SH, -NH ₂ , OMe or -OH substituted C _1-6 alkyl.

In some such embodiments of formula (III), R ¹ is -(CH ₂ ) _0-2 -C _3-6 cycloalkyl or contains up to two heteroatoms selected from N, O and S as a ring a member-(CH ₂ ) _0-2 -C _4-7 heterocyclyl wherein each C _3-6 cycloalkyl or C _4-7 heterocyclyl optionally has up to three selected from halo, C _{1- 4-} alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, hydroxy, amine, pendant oxy, C _1-4 alkyl substituted by hydroxy, C _1-4 alkyl-amino and The group of COO (C _1-4 alkyl) is substituted. In certain embodiments, up to two C _3-6 cycloalkyl or C _4-7 heterocyclyl groups are selected from halo, C _1-4 alkyl, C _1-4 haloalkyl, C _1- the _{4-substituted} alkoxy, hydroxy, amino and hydroxy C _1-4 alkyl group substituted by the group, where preferred substituents are selected from F, hydroxy, methoxy and amino.

In certain embodiments, R ¹ is selected from the group consisting of 4-tetrahydropyranyl and

And depending on the situation The dotted line indicates the connection point of each R ¹ .

In some of the foregoing embodiments of formula (III), R ² is H.

In some of the foregoing examples of formula (III), A is a bond between a carbonyl group and Q. In other embodiments, A is NH. In some of the foregoing embodiments, when Y is a heterocyclic group or a cycloalkyl group, T is CH ₂ and when Y is a C _1-3 aminoalkyl group, T is CH ₂ or CH ₂ CH ₂ .

In some of the foregoing examples of formula (III), Q is 1 or 2 selected from the group consisting of hydroxyl, amine, thiol, amine-C _1-4 alkoxy or amine-C _1-4 alkyl sulphide a C _1-4 alkyl group substituted with a group. In other embodiments, Q is a ring selected from the group consisting of morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, piperazine, phenyl, and pyridine, wherein the ring is optionally up to two selected from the group consisting of C _1-4 alkyl groups, Substituents of halo, CN, hydroxy, amine, C _1-4 alkyl-amino, C _1-4 alkylsulfonyl and C _1-4 alkoxy.

In some of the foregoing examples of formula (III), Y is optionally up to two selected from the group consisting of halo, C _1-4 alkyl, hydroxy, amine, hydroxy-C _1-4 alkyl, amine- a pyrrolidine ring substituted with a C _1-4 alkyl group, a C _1-4 alkyl-amino group, and a C _1-4 alkoxy group. Preferred substituents for pyrrolidine include F, methyl, hydroxy and hydroxymethyl.

These novel Eg5 inhibitors can be used as low molecular weight pharmaceutical compounds to treat cancer, or they can be incorporated into ADCs for targeted in vivo delivery.

Example 15. Compound of embodiment 14, wherein R ¹ is tetrahydropyran-yl; In some embodiments, R ¹ is tetrahydropyran-4-yl.

16. A compound of formula (IIA) or (IIB),

Wherein Ar ¹ , Ar ² , Z, R ¹ , R ² , T, Q, Y and A are as defined in the above Example 1 with respect to formula (II), and Q* is selected from -CH ₂ O-, -CH ₂ S-, -CH ₂ -NH-, -CH ₂ -NMe-, -CH(Me)O-, -CH(OH)-CH ₂ O-, -CH(O-)-CH ₂ OH, -CH (OH)-CH ₂ NH-, -CH(NH-)-CH ₂ OH, -CH(O-)-CH ₂ NH ₂ , -CH(NH-)-CH ₂ OH, -CH(Me)S- , -CH(Me)NH-, -CH ₂ CH ₂ O-, -CH ₂ CH ₂ NH-, -CH ₂ CH ₂ S-, -CH(Me)CH ₂ O-, -CH(Me)CH ₂ S-, -CH(Me)CH ₂ NH-, Y* is selected from the group consisting of -CH(CH ₂ F)NH-, -CH ₂ NH-, Wherein R ¹⁰ and R ^{11 are} independently H, Me, OMe, F, CH ₂ F, CH ₂ OH, COOH, COO (C _1-4 alkyl), CONH (C _1-4 alkyl), CON (C) _1-4 alkyl) ₂ or OH; and W is a linking moiety comprising one or more linker components and a reactive functional group. Suitable linkage moieties having a reactive functional group such as maleimide are disclosed herein, including

Wherein each R ^{N is} independently H or CH ₂ CH ₂ -R ³⁰ , wherein R ³⁰ is a hydroxyl group, an amine group or a carboxyl group, and R ^{N is} preferably H; Wherein X represents a compound of formula (IIA) or IIB, and LG is a decomposing group suitable for providing a deuterating agent, such as Cl, -O-benzotriazole (-OBt), -O-azabenzotriazole (-OAt) ), -O-butanediamine, substituted phenoxy, -OC(O) (phenyl or substituted phenyl), -OC(O)(C _1-6 alkyl) or -OC (O)O(C _1-6 alkyl).

16B. Alternatively, the compound of formula (III) may have formula (IIC):

Wherein Ar ¹ , Ar ² , Z, R ² , T, Q, Y, W and A are as defined above for formula (III), and R ^1* is optionally substituted by a pendant oxy group, a hydroxyl group, an amine group or a carboxyl group. C _3-6 alkyl, for example R ^1* is -C(Me) ₂ -(CH ₂ ) _0-2 -R ³⁰ , wherein R ³⁰ is hydroxy, carboxy or amine; and W is one or more linkers a linking moiety of a component and a reactive functional group.

For example, W can be -L ¹ -L ² -L ³ -L ⁴ -L ⁵ -G, wherein G is a reactive functional group, and L ¹ , L ² , L ³ , L ^{4 ,} and L ⁵ are selected Linker components of the components from the above. A suitable reactive functional group (G) is a functional group having suitable reactivity to form a covalent linkage to an amino acid side chain of an amino acid in an antibody or antigen binding moiety, such as cysteine or -SH or -NH _{2 of} lysine. Examples of suitable reactive functional groups include maleimide, α-haloacetamide (halogen = Cl, Br or I), aldehyde (CHO), thiol (to form a disulfide), 2-Aminobenzaldehyde (ABA), 2-amino-benzophenone (ABP), 2-aminoacetophenone (AAP), carboxylate groups, and easy formation of indoleamine with free amine groups Esters, such as esters of N-hydroxybutylimine and its analogs. Suitable reactive functional groups ABA, AAP and ABP include the following groups:

These are placed in opposition to Pcl or Pyl at the end of the linking group selected as appropriate, as in Ou et al., Proc. Nat'l Acad. Sci. 2011, 108(26), 10437-42 Said to form a linking group, wherein L ¹ is

Wherein R ²⁰ is H or Me, and R ³⁰ is H, Me or phenyl.

Such embodiments of the invention are activated intermediates suitable for use in the preparation of a conjugate comprising an Eg5 inhibitor payload similar to the compounds of formula (II) and (III) above. In such embodiments, the compounds comprise a reactive functional group positioned at a position that is well tolerated even with a non-cleavable linker, such as a linkage group attached to the corresponding formula (II) The atom of Y or Q.

17. The compound of embodiment 16, wherein W comprises a group selected from the group consisting of -SH, -NH ₂ , -C(=O)H, -C(=O)Me, N-maleimide, and -NHC (= a reactive functional group of O)-CH ₂ -halo, -COOH and -C(=O)-OR', wherein the halogen is selected from the group consisting of Cl, Br and I, and -OR' is the cleavage moiety of the activated ester .

The compound of any one of embodiments 14-17, wherein Ar ¹ is a dihalophenyl group. Particularly suitable groups include 2,5-difluorophenyl, 2-fluoro-5-chlorophenyl and 2-chloro-5-fluorophenyl.

The compound of any one of embodiments 14-18, wherein Ar ² is phenyl or halophenyl. Particularly suitable groups include phenyl and 3-fluorophenyl.

The compound of any one of embodiments 14-19, wherein Z is CH.

The compound of any one of embodiments 14-19, wherein Z is N.

The compound of any one of embodiments 16-21, wherein R ¹ is 4-tetrahydropyranyl.

The compound of any one of embodiments 16-21, wherein R ¹ * is -C(Me) ₂ CH ₂ C(O)NH-[W], wherein [W] indicates the point of attachment of R ¹ * to W .

The compound of any one of embodiments 14-22, wherein R ² is H. In an alternate embodiment, R ² can be a methyl group.

The compound of any one of embodiments 14-23, wherein A is -NH-.

25. The compound of any one of embodiments 14-23 wherein A is a bond.

The compound of any one of embodiments 14-25, wherein T is CH ₂ or CH ₂ CH ₂ . Preferably, when Y or Y* is an aminoalkyl group such as -CH(CH ₂ F)NH ₂ or -CH ₂ NH ₂ , T is CH ₂ CH ₂ ; and when Y or Y* is as appropriate Substituted pyrrolidine, such as the following, T is -CH ₂ -:

27. The compound of any one of embodiments 14-26, wherein Y is selected from the group consisting of -CH(CH ₂ F)NH ₂ , Wherein R ¹⁰ and R ^{11 are} independently H, Me, OMe, F, CH ₂ F, CH ₂ OH, COOH, COO (C _1-4 alkyl) or OH.

In certain embodiments of the compounds, Y is selected from the group consisting of -CH(CH ₂ F)NH ₂ ,

A preferred embodiment of Y includes

Where [T] indicates the point of attachment of Y to T in the formula.

Preferred embodiments of Y* include

Where [T] indicates the point of attachment of Y* to T in the formula; and [W] indicates where Y* is connected to W.

28. The compound of any one of embodiments 14-27, wherein Q is selected from the group consisting of -CH ₂ OH, -CH ₂ -NH ₂ , -CH(Me)OH, -CH(OH)-CH ₂ OH, -CH (OH)-CH ₂ NH ₂ , -CH(NH ₂ )-CH ₂ OH, -CH(NH ₂ )-CH ₂ OH, -CH(Me)SH, -CH(Me)NH ₂ , -CH ₂ CH ₂ OH, -CH ₂ CH ₂ NH ₂ , -CH ₂ CH ₂ SH, -CH(Me)CH ₂ OH, -CH(Me)CH ₂ SH, -CH(Me)CH ₂ NH ₂ ,

Preferred examples of the combination-AQ include -CH ₂ OH, -CH(Me)OH, -NH-CH ₂ -CHOH-CH ₂ OH, -NH-CH ₂ -CH ₂ OH, and -NH-CHMe-CH ₂ OH. In particular, -AQ can be selected from

Where [CO] indicates the point of attachment of -A-Q to the carbonyl group in the formula.

29. The compound of embodiment 14, which is selected from the group consisting of the compounds of Table 1 and pharmaceutically acceptable salts thereof.

30. A pharmaceutical composition comprising a compound of any of embodiments 15-29, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

31. A combination comprising a therapeutically effective amount of a compound according to any one of embodiments 14-15, or a pharmaceutically acceptable salt thereof, and one or more therapeutically active adjuvants.

32. A method of treating a cell proliferative disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an immunoconjugate of any of embodiments 1-13, or a compound of any of embodiments 14-15, Or a pharmaceutically acceptable salt thereof.

33. The compound according to any one of embodiments 14-15, or the immunoconjugate of any of embodiments 1-13, or a pharmaceutically acceptable salt thereof, for use as a medicament.

34. The compound according to embodiment 33, or a pharmaceutically acceptable salt thereof, wherein the agent is for the treatment of cancer.

In certain embodiments, the cancer is selected from the group consisting of stomach, bone marrow, colon, nasopharynx, esophageal and prostate tumors, glioma, neuroblastoma, melanoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer. , leukemia (such as chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non-Hodgkin's), bladder, kidney, stomach ( For example, gastrointestinal matrix tumors (GIST), liver and pancreatic cancer and sarcoma.

The immunoconjugate of any of embodiments 1-13, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

36. The immunoconjugate according to embodiment 1, which has the formula selected from the group consisting of:

Wherein each R ^{N is} independently H or CH ₂ CH ₂ -R ³⁰ , wherein R ³⁰ is a hydroxyl group, an amine group or a carboxyl group, and R ^{N is} preferably H;

Wherein R ²⁰ is H or Me, and R ³⁰ is H, Me or phenyl.

And as appropriate

Specific examples include this:

And as appropriate

37. The immunoconjugate of any of embodiments 1-14, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

38. An immunoconjugate, Ab-L*-X, comprising a payload (X) linked to an antibody (Ab), wherein the linkage group L* comprises the formula -C(O)NR ²¹ - or -NR ^{a group of 21} -C(O)-, wherein R ²¹ has the formula -(CH ₂ ) _1-4 -R ²² , wherein R ²² is selected from the group consisting of -OH, -NH ₂ , N(R ²³ ) ₂ , COOR ²³ a polar group of CON(R ²³ ) ₂ , —(OCH ₂ CH ₂ O) _k —OCH ₂ CH ₂ OR ²³ and —SO ₂ R ²³ wherein k is from 0 to 4 and each R ^{23 is} independently H or C _1-4 alkyl. Preferably, X is an Eg5 inhibitor, including the compound of any of embodiments 14-29.

39. An immunoconjugate of formula (I):

Wherein Ab represents an antigen binding moiety; L represents a linkage group linking X to Ab; m is an integer from 1 to 4; n is an integer from 1 to 16; and X independently represents an Eg5 inhibitor at each occurrence.

In various embodiments, X is a compound according to any one of embodiments 14-29.

In certain such embodiments, X is a compound of this formula:

Wherein R ^4a is H, F or OH; R ^4b is H or F; and R ^{1 is} selected from Y ^{4 is} selected from And Q ^{4 is} selected from Wherein linker L is linked to X at Y ⁴ , Q ⁴ or R ¹ .

Preferred linkers L for these examples include the following, wherein [Ab] refers to the point of attachment to the antibody:

and Or when Q ⁴ is When L is

In the last option,

Combine to form this group -Q ⁴ -L:

40. The immunoconjugate of embodiment 39, wherein X is a compound selected from Table 1.

41. The immunoconjugate of embodiment 39 or 40, wherein m is 1 and the immunoconjugate is formed by reacting an Ab with a compound selected from Table 2.

42. An immunoconjugate prepared by reacting an antibody comprising at least one free thiol group with a maleimide compound selected from the group consisting of: and

In some embodiments of such immunoconjugates, the anti-system is selected from the group consisting of an anti-estrogen receptor antibody, an anti-progesterone receptor antibody, an anti-p53 antibody, an anti-HER-2 antibody, an anti-cKit antibody, an anti-EGFR antibody, an anti-tissue Protease D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti- CEA antibody, anti-retinoma protein antibody, anti-ras tumor protein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 Antibody, anti-CD9/p24 antibody, anti-CD1-antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, antibody CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA/CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD71 antibody, anti-CD95/Fas antibody , anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody , anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-τ antigen antibody, anti-fibrin antibody, anti-keratin antibody And anti-Tn-antigen antibodies.

Such immunoconjugates may have between 1 and 8, typically between 2 and 6, and preferably Drug to antibody ratio (DAR) between 3 and 5.

In the above enumerated examples, the Ab can be any antigen binding portion, and preferably an antigen or antigen fragment that recognizes a cell surface marker, such as a marker described herein as a target cell, such as a characteristic of a cancer cell.

In the exemplified examples, X can be any compound of formula (II) or (III), especially any of the compounds disclosed in Examples 1-11 above or Examples 14-15, and includes any of Table 1 substance. In a preferred embodiment of embodiment 36, the X is selected from the group consisting of

Where [L] indicates which atom of X is attached to the linking group described in Example 36.

In an additional embodiment, X has the formula:

Unless otherwise described, an Ab in any of the above embodiments can be any antigen binding portion, typically a portion of a characteristic antigen that recognizes a cell (such as a cancer cell) to which a pharmaceutical intervention is to be targeted. A wide variety of suitable antigens are well known in the art; specific antigens of particular interest are described herein. Typically, an Ab is an antibody, which may be isolated or constructed, and may be native or modified (engineered), or an antibody fragment that retains antigen-binding activity similar to an antibody.

L in the above examples may be any linking group linking the Ab to one or more X groups, including a single bond directly linking the Ab to the atom of the compound of formula (II). Suitable linkers for the ADC are well known in the art and can be used in the combinations of the present invention. L can be attached to the Ab at any suitable available location on the Ab. Typically, L is attached to an available amine nitrogen atom (i.e., a primary or secondary amine, rather than a decylamine), or a hydroxyl oxygen atom, or a sulfhydryl group, such as a cysteine.

In some such embodiments of formula (I), m is 1 or 2 and m is preferably 1.

In some such embodiments of formula (I), n is 1-10, typically 1-8 or 1-6, and preferably n is 1, 2, 3, 4 or 5.

In some embodiments of the compounds of Formula (II), IIA, IIB, and III, R ¹ is or includes a 3-6 membered cycloalkyl ring or a 4-6 membered heterocyclic group, and can be as in various enumerated embodiments The substitution. In some embodiments, R ¹ is an unsubstituted 5-6 membered heterocyclic group. In other embodiments, R ¹ is the substituted amine or hydroxyl 5-6 membered heterocyclic group, the amine or hydroxy optionally any one point of attachment of the linking group.

In any of the foregoing embodiments, L may comprise up to six further described herein as components of the linker ^{L 1, L 2, L 3} , L 4, L 5 and L ^6. Thus, for example, the immunoconjugate of Formula (I) can have Formula (IA):

Wherein Ab represents an antigen binding moiety; L ¹ , L ² , L ³ , L ⁴ , L ⁵ and L ⁶ each independently represent a linker component; n is an integer from 1 to 16; and X represents an Eg5 inhibitor, for example A compound of formula (II) or formula (III) as described herein.

These immunoconjugates can be depicted as equally or less, to indicate the linker component L ⁶ is connected to a compound of formula (II):

Wherein Ab represents an antigen-binding moiety; L ¹ , L ² , L ³ , L ⁴ , L ⁵ and L ⁶ each independently represent a linker component; n is an integer from 1 to 16; and Ar ¹ , Ar ² , R ¹ And R ² , T, Y, A, Q and Z are as defined herein with respect to formula (II) or formula (III). L ^{6 in} this formula is attached to the chemical structure shown: -L ⁶ - can be considered as a substituent of the group of formula (II) or (III). In some embodiments, L ^{6 is} attached to an atom of Q, Y or R ¹ , typically at the oxygen or nitrogen atom of Q, Y or R ¹ or a substituent thereof.

In such embodiments, each linker component may optionally be a bond to a group on either side of the linker component, such that in some embodiments, the compound of formula (IA) includes a linker that connects Ab to X. ⁰ , ¹ , ² , ³ , ⁴ , ⁵ or 6 of the components L ¹ , L ² , L ³ , L ⁴ , L ⁵ and L ⁶ .

Suitable linker components for forming the linking group L are known in the art and are used. Methods for constructing the linking group L are also known. These components include those commonly used to attach a group to an amino acid, such as a spacer of an alkyl and ethylene oxide oligomer, an amino acid of up to about 4 amino acids, and a short Peptides; and carbonyl, urethane, carbonate, urea, ester and guanamine linkages, and analogs thereof.

In some embodiments of such combinations, the L ¹ is selected from the group consisting of a reactive functional group formed upon reaction with a side chain of one of the amine groups, typically a cysteine thiol, or Free-NH _{2 of the} amine acid, or a Pcl or Pyl group engineered into the antibody. See, for example, Ou et al., PNAS 108 (26), 10437-42 (2011). Suitable -L ¹ - groups include, but are not limited to, a single bond, Particularly for use in attaching a cysteine residue to Ab; Especially for -NH ₂ attached to the amino acid residue of the Ab, wherein each p is 1-10, and each R is independently H or C _1-4 alkyl (preferably methyl);

Wherein R ²⁰ is H or Me, and R ³⁰ is H, Me or phenyl for bonding to a Pcl or Pyl group, wherein the thiol group shown is attached to the Pcl or Pyl of the engineered antibody. Amino acid moiety.

Suitable options for linking the subcomponents L ² , L ³ , L ⁴ and L ⁵ include, for example, alkyl-(CH ₂ ) _n - (where n is typically 1-10 or 1-6), ethylene glycol a unit (-CH ₂ CH ₂ O-) _n (wherein n is 1-20, typically 1-10 or 1-6), -O-, -S-, carbonyl (-C(=O)-), Indoleamine - C(=O)-NH- or -NH-C(=O)-; a guanamine or amine group containing -C(=O)-NR ²¹ - or -NR ²¹ -C(=O)- Formate, wherein R ²¹ is alkyl substituted with a polar substituent as described herein; ester-C(=O)-O- or -OC(=O)-; a ring system having two available attachment points , such as selected from phenyl (including 1,2-, 1,3-, and 1,4-disubstituted phenyl), C _5-6 heteroaryl, C _3-8 cycloalkyl (including 1,1- Disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl and 1,4-disubstituted cyclohexyl) and C _4-8 heterocyclyl rings and divalent rings of the specific examples depicted below; amine groups Acid-NH-CHR*-C=O- or -C(=O)-CHR*-NH-, or an amine derived from N attached to an adjacent structure (eg, attached to a maleimide nitrogen) a group having the formula [N]-CHR*-C(=O)-, wherein R* is a known amino acid (generally one of the classical amino acids, such as trp, ala, asp, lys, Gly and its class Analogous, also including side chains such as n-proline, orthraenic acid, homoserine, homocysteine, phenylglycine, citrulline, and other commonly designated alpha-amino acids, Amino acid polypeptides (eg, dipeptides, tripeptides, tetrapeptides, etc.), thiol-maleimide linkages (by adding -SH to maleimide), -S -CR ₂ - and other thiol ethers, such as -S-CR ₂ -C(=O)- or -C(=O)-CR ₂ -S-, wherein R is independently H or C at each occurrence _1-4 alkyl, -CH ₂ -C(=O)-, and disulfide (-SS-); and combinations of any of these components with other linker components described below, such as a bond, Non-enzymatic cleavage of a linker, a non-cleavable linker, an enzymatic cleavage linker, a light stable linker, a photocleavable linker or a linker comprising a self-decomposing spacer.

In some embodiments, each linker component L ¹ , L ² , L ³ , L ⁴ , L ^{5 ,} and L ⁶ is selected from the group consisting of: a bond, —(CH ₂ ) _q —, —(CR ₂ ) _q -, -(CH ₂ CH ₂ O) _q -, -(CH ₂ ) _q -NR-(CH ₂ ) _q -, -(CH ₂ ) _q -O-(CH ₂ ) _q -, -NH -CHR*-C(=O)-, -C(=O)-CHR*-NR-, -CHR*-C(=O)-, -C(=O)NR-, -NRC(=O) -, -C(=O)O-, -OC(=O)-, -NRC(=O)O-, -OC(=O)NR-, -(CH ₂ ) _q S(CH ₂ ) _q - ,

Wherein R ²⁰ is H or Me, and R ³⁰ is H, Me or phenyl, wherein each q is 0-10, preferably 0-6 or 1-6; each R, R ⁵ and R ^{6 are} independently H Or C _1-4 alkyl, R* is a side chain of a common amino acid such as gly, ala, trp, tyr, phe, leu, ile, val, asp, glu gln, asn, his, arg, lys, cys , met, ser, thr, phenylglycine, tert-butylglycine; R ^{7 is} independently selected from the group consisting of H, C _1-4 alkyl, phenyl, pyrimidine and pyridine; R ^{8 is} independently selected from R ^{9 is} independently selected from H, C _1-4 alkyl and C _1-6 haloalkyl; and any one or all of L ¹ to L ⁶ may be absent, that is, any or all of them may represent A bond between the two groups attached.

In certain embodiments, linker L comprises at least one of the formula -C(O)-NR ²¹ - or -NR ²¹ -C(O)- or -OC(=O)-NR ²¹ -, -NR ²¹ -C a linker component of (=O)-O-, wherein R ²¹ has the formula -(CH ₂ ) _1-4 -R ²² , wherein R ²² is such as -OH, -NH ₂ , N(R ²³ ) ₂ , COOR ^a polar group of ²³ , CON(R ²³ ) ₂ , -(OCH ₂ CH ₂ O) _k -OCH ₂ CH ₂ OR ²³ or -SO ₂ R ²³ wherein k is 0 to 4 and each R ^{23 is} independently H Or C _1-4 alkyl. It has been discovered that the use of this moiety in the linking group to which the antibody is attached to the payload reduces aggregation of the conjugate and thereby improves the solubility and efficacy of the immunoconjugate. In a preferred embodiment, R ²² is -(CH ₂ ) ₂ -OH or -(CH ₂ ) ₂ -COOH.

L ⁶ are particularly suitable option for the connection of the sub-component comprises, as explained herein the covalent bond, a carbonyl group [-C (= O) -] , Wherein G is an enzymatically cleavable group, such as a glucuronyl group, each q is 1-10, Z is a polar group such as -COOH or -SO ₃ H, and each R is independently H or C _{1- 4-} alkyl (preferably H or methyl).

In another aspect, the invention provides an immunoconjugate Ab-L*-X comprising a payload (X) linked to an antibody (Ab), wherein the linkage group L* comprises the formula -C ( O) a group of NR ²¹ - or -NR ²¹ -C(O)-, wherein R ²¹ has the formula -(CH ₂ ) _1-4 -R ²² , wherein R ²² is selected from -OH, -NH ₂ , N a polar group of (R ²³ ) ₂ , COOR ²³ , CON(R ²³ ) ₂ , —(OCH ₂ CH ₂ O) _k —OCH ₂ CH ₂ OR ²³ and —SO ₂ R ²³ wherein k is 0 to 4 and Each R ^{23 is} independently H or C _1-4 alkyl. The payload can be any suitable payload, such as a cytotoxin such as maytansinoid, auristatin, apocynin or amanita, or other known payloads having therapeutic utility in an ADC. In some embodiments of such immunoconjugates, X is an Eg5 inhibitor, such as described herein.

X in the formula I may be any Eg5 inhibitor, but is preferably a compound of formula II as described above, or any subclass of this formula as described in the enumerated examples, such as described above. a compound of formula (III). In some embodiments, X is a compound selected from Table 1. While formulas (II) and (III) describe "neutral" compounds, it is understood that in the context of the conjugate, X comprises an atom covalently attached to L or directly attached to the Ab.

Unless otherwise provided, X is attached to the linking groups of the above formulas via any available position. In some embodiments, X is attached to the linking group via a group represented by Q in formula (II) or formula (III) or a group represented by Y or an atom represented by R ¹ .

Similarly, an Ab can be any antigen binding moiety, including those described herein. Preferably, the Ab is an antibody which may be modified; for example, in addition to at least one of the Eg5 inhibitors of the invention, the Ab may also have other linked payloads. In embodiments in which the Ab is attached to the maleimide ring or to the -CH ₂ - or -S- of the linking group L, which is typically attached via the sulfur atom of the cysteine of Ab; In embodiments wherein the Ab is attached to the linking group at the carbonyl group of the linking group, it is typically attached via a nitrogen atom in the Ab, such as an amine to an amine.

The invention encompasses the use of any small molecule Eg5 inhibitor as a cytotoxic payload of an immunoconjugate. It is illustrated by, but not limited to, the Eg5 inhibitors of formula (II) and has been shown to cooperate with other classes of Eg5 inhibitors. In a preferred embodiment, the Eg5 inhibitor is a compound of formula (II) or (III), especially including any of the compounds of Table 1.

It should be understood that when a compound of formula (II) or (III) is part of an immunoconjugate, The valence is attached to the linking group L (or to the linker component that is part of L) or to itself to the Ab. Thus, in the immunoconjugate of the invention, the compound of formula (II) or (III) has an open valence state whereby it is covalently bonded to L (or directly linked to the Ab), preferably sufficiently tight for use in Delivery in vivo to inhibit or eliminate the targeted cells. Typically, linkage between an Eg5 inhibitor and an Ab involves the covalent attachment of an antigen binding moiety Ab to an Eg5 inhibitor, typically via a linking group comprising one or more linker components, such as those described herein.

In use, the Eg5 inhibitor will be released from the Ab before or more typically after the ADC reaches and binds to the antigen on the targeted cell: preferably, the Eg5 inhibitor is predominantly in the targeted cell, binding to the surface at the ADC The antigen is then released after internalization into the targeted cells. In some embodiments, the linking group L is designed to be cleavable and the Eg5 inhibitor is detached from the ADC after internalization.

In some embodiments, the linking group is not designed to be cleavable, and the release of the Eg5 inhibitor occurs when the antigen binding group (eg, an antibody) is degraded in vivo. Typically, degradation of Ab occurs within the target cell, as occurs by protease digestion. In such embodiments, at least a portion of the linking group L may remain attached to the Eg5 inhibitor X, with the proviso that the portion of the linking group L remaining on X does not interfere with inhibition of inhibition of Eg5. Submicron molar concentration affinity of agent X.

A variety of linking groups for use in ADCs are known (see, for example, Lash, Antibody-Drug Conjugates: the Next Generation of Moving Parts , Start-Up , December 2011, 1-6), and can be used in the present invention. Within the scope of the combination. The linking group can be a single covalent bond between the atom of the Eg5 inhibitor and the antibody atom; for example, Q can be an alkyl group, such as a methyl group, and A can be absent from formula (II), thereby providing Eg5 inhibitor:

To link this inhibitor to the antigen binding portion, it can be converted to a modified Eg5 inhibitor of the formula having iodide (I) as a reactive functional group:

The iodide compound α-haloacetamide can be directly reacted with a free thiol group on the antibody to provide an immunoconjugate of the formula: Wherein S is the sulfur atom of the cysteine residue of the antibody, and the linking group L in the formula (I) represents a covalent bond between CH ₂ and S.

In other embodiments of the combination of Formula (I), L may comprise ² , ³ , ⁴ , ⁵ , 6, or more than 6 linker components, such as L ¹ , L ² , L ³ , L ⁴ , L ⁵ And L ⁶ . A variety of linkers comprising a plurality of linker components are known in the art, and various linker components can be selected and combined to provide a practicable immunoconjugate of the invention. In certain embodiments, the immunoconjugate has the formula (IA):

Wherein Ab represents an antigen binding moiety; L ¹ , L ² , L ³ , L ⁴ , L ⁵ and L ⁶ represent a linker component; n is an integer from 1 to 16; and X represents an Eg5 inhibitor, eg as described herein A compound of formula (II) or formula (III).

In such compounds, L ^{1 is} typically selected from the group formed when the reactive functional group is reacted with a side chain typically used to bind one of the amino acid groups, such as a thiol of cysteine, or free of amine acid. -NH ₂ , or a Pcl or Pyl group engineered into an antibody. See, for example, Ou et al., PNAS 108 (26), 10437-42 (2011). Suitable -L ¹ - groups include, but are not limited to, a single bond as described above, Particularly for use in attaching a cysteine residue to Ab; Particularly for use in the attachment of an aminic acid residue to an Ab wherein each n is 1-10 and each R is independently H or C _1-4 alkyl (preferably methyl).

Suitable options for linking the subcomponents L ² , L ³ , L ⁴ and L ⁵ include, in addition to a bond, for example, alkyl-(CH ₂ ) _n - (where n is typically 1-10 or 1-6 ), ethylene glycol unit (-CH ₂ CH ₂ O-) _n (where n is typically 1-10 or 1-6), -O-, -S-, carbonyl (-C(=O)-), Indoleamine-C(=O)-NH- or -NH-C(=O)-, ester-C(=O)-O- or -OC(=O)-; a ring having two available attachment points, Such as divalent phenyl, C _5-6 heteroaryl, C _3-8 cycloalkyl or C _4-8 heterocyclic; amino acid -NH-CHR*-C=O- or -C(=O) -CHR*-NH-, or a group having the formula [N]-CHR*-C(=O)- derived from an amino acid attached to N (for example, to a maleimide nitrogen), Wherein R* is a known amino acid (usually one of the classical amino acids, and also includes, for example, n-decylamine, orthraenic acid, homoserine, homocysteine, phenylglycine, cucuramine a side chain of an acid and other specified α-amino acids, a polypeptide of a known amino acid (eg, a dipeptide, a tripeptide, a tetrapeptide, etc.), a thiol-methylenediamine bond (by Add -SH to maleimide), -S-CR ₂ - and other thiol ethers such as -S-CR ₂ -C(=O)- or -C(=O)-CR ₂ -S - where R is every time Present is independently H or C _{1-4 alkyl, -CH 2 -C (= O)} - and disulfide (-SS-); and any one of these components in connection with the following sub-components other Combinations of, for example, a linkage, a non-enzymatic cleavage linker, a non-cleavable linker, an enzymatic cleavable linker, a light stable linker, a photocleavable linker or a self-decomposing spacer.

In some embodiments, each linker component is selected from the group consisting of: a bond between the groups on either side (meaning that the linker component is effectively absent, thus the group to which it is flanked Linked together), -(CH ₂ ) _q -, -(CH ₂ CH ₂ O) _q -, -(CH ₂ ) _q -NR-(CH ₂ ) _q -, -NH-CHR*-C(=O )-, -CHR*-C(=O)-, -C(=O)-CHR*-NH-, -C(=O)NH-, -NHC(=O)-, -C(=O) O-, -OC(=O)-, -NHC(=O)O-, -OC(=O)NH-, -(CH ₂ ) _q S(CH ₂ ) _q -,

Wherein each q is 0-10, preferably 0-6 or 1-6; each R, R ⁵ and R ^{6 are} independently H or C _1-4 alkyl, and R ^{7 is} independently selected from H, C _{1- 4} alkyl, phenyl, pyrimidine and pyridine; R ^{8 is} independently selected from R ^{9 is} independently selected from the group consisting of H, C _1-4 alkyl and C _1-6 haloalkyl; and each R* represents a side chain of an amino acid, which may be an amino acid encoded by the genetic code Or an alpha-amino acid analog such as citrulline, tert-butylglycine, phenylglycine, homoseramine and the like; and any or all of these components may or may not It exists, that is, it can represent a bond between the two groups to which it is to be attached.

Preferred options for linking subcomponent L ⁶ include covalent bonds, carbonyl [-C(=O)-], Wherein G is an enzymatically cleavable group, such as a glucuronyl group, each n is 1-10, and each R is independently H or C _1-4 alkyl (preferably methyl).

Another aspect of the invention provides a linker that reduces ADC aggregation and thus improves ADC function and characteristics. It is well known that aggregation of ADCs can be detrimental to their activity, and aggregation depends on the payload and the characteristics of the linkers. Certain hydrophilic linkers have been used to reduce aggregation. Example 4 illustrates novel linkers that reduce aggregation (eg, linkers in ADC-111 and ADC-112). These novel linkers are included as a general formula -C(O)-NR ²¹ - or -NR ²¹ -C(O)- or -OC(=O)-NR ²¹ - or -NR ²¹ -C(=O)- a linker component of an N-substituted guanamine or urethane of O-, wherein R ²¹ is via, for example, a hydroxyl group, an amine group, a monoalkylamine or a dialkylamine, a carboxylate group, a carboxamide or An alkyl group substituted with a polar group of an alkylsulfonyl group. As demonstrated in Example 4 and Figures 10(A)-10(C), the addition of polar groups to the simple linker of the guanamine reduces the aggregation of the ADC, which in other cases exhibits significant aggregation, such as by size. Measurement by exclusion chromatography. Accordingly, the invention includes an ADC of the general formula Ab-L*-X, wherein Ab is an antibody, such as described herein, X is a payload, such as a cytotoxin or an Eg5 inhibitor, such as any of the Eg5 inhibitors described herein, And L* is a linker comprising a guanamine of the formula -C(O)-NR ²¹ - or NR ²¹ -C(O)- or a formula -OC(=O)-NR ²¹ - or -NR ²¹ -C ( =O)-O-carbamate, wherein R ²¹ has the formula -(CH ₂ ) _1-4 -R ²² , wherein R ²² is such as -OH, -NH ₂ , N(R ²³ ) ₂ , COOR ²³ , CON(R ²³ ) ₂ , -(OCH ₂ CH ₂ O) _k -OCH ₂ CH ₂ OR ²³ and -SO ₂ R ²³ polar groups, wherein k is 0 to 4 and each R ^{23 is} independently H Or C _1-4 alkyl. Preferred embodiments of the linker include those wherein R ²¹ has the formula -(CH ₂ ) _1-2 R ²³ ; preferred embodiments of R ²³ include a hydroxyl group and a carboxyl group.

Likewise, a variety of antigens associated with cancer cells are known, and antibodies that bind to such antigens can be used in immunoconjugates within the scope of the invention. For example, although the clinical candidate ADC reported in Lash utilizes only four payload categories, it includes at least 15 antigens associated with various targeted cells. Representative examples of immunoconjugates of the invention are described herein, but such examples do not limit the scope of the invention or the scope of the claims.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples of the invention, such as "such as"

Various enumerated embodiments of the invention are described herein. It will be appreciated that features specified in the various embodiments can be combined with other specified features to provide other embodiments of the invention. Example.

The pharmaceutically acceptable solvate of the present invention includes a solvate wherein the crystallization solvent is isotopically substituted (for example, D ₂ O, d ⁶ -acetone, d ⁶ -DMSO), and a solvent having a non-concentrated solvent. Things.

The compounds of the invention, i.e., compounds of formula (I) containing a group capable of acting as a donor and/or acceptor for hydrogen bonding, may be capable of forming a eutectic with a suitable eutectic former. Such eutectic crystals can be prepared from compounds of formula (I) by known eutectic formation procedures. The procedures include grinding, heating, co-subliming, co-melting or contacting the compound of formula (I) with a eutectic former in solution, and isolating the eutectic thereby formed. Suitable eutectic formers include the eutectic formers described in WO 2004/078163. Accordingly, the present invention further provides a eutectic comprising a compound of formula (I).

Any asymmetric atom of the compounds of the invention (e.g., carbon or an analog thereof) may exist in racemic or enantiomeric enriched form, such as (R)-, (S)- or (R, S)-configuration. . In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess of the (R)- or (S)-configuration, at least 60% enantiomeric excess, at least 70% enantiomeric Excess in bulk, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess; that is, for optical activity Compounds, it is generally preferred to use one enantiomer to substantially exclude the other enantiomer. If possible, the substituent at the atom having an unsaturated double bond may exist in the cis (Z)- or trans (E)- form.

Thus, as used herein, a compound of the invention may be in the form of one of the possible isomers, rotamers, singly isomers, tautomers or mixtures thereof, for example in a substantially pure geometry (shun Or isomers, diastereomers, optical isomers (enantiomers), racemates or mixtures thereof. As used herein, "substantially pure" or "substantially free of other isomers" means that the product contains less than 5% by weight and preferably less than 2% by weight of other isomers relative to the preferred isomer. body.

Based on physicochemical differences of the components, such as by chromatography and/or step-by-step Crystals, any mixture of the resulting isomers can be separated into pure or substantially pure geometric isomers or optical isomers, diastereomers, racemates.

Any resulting final product or intermediate racemate can be resolved to the optical enantiomer by known methods, for example by separation of its diastereomeric salts and release opticals with optically active acids or bases. Active acidic or basic compound. In particular, the basic moiety can be used to resolve a compound of the invention into its optical enantiomer, for example a salt formed by optically active acid by fractional crystallization, such as tartaric acid or benzoic acid. Tartaric acid, diethinyl tartaric acid, di-O, O'-p-toluamyl tartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. The racemic product can also be resolved by palm chromatography, for example by high pressure liquid chromatography (HPLC) using a palmitic adsorbent.

Furthermore, the compounds of the invention (including salts thereof) may also be obtained in the form of their hydrates or include other solvents used for their crystallization. The compounds of the invention may inherently or intentionally form solvates with pharmaceutically acceptable solvents, including water; therefore, the invention is intended to cover both fused and non-complexed forms. The term "solvate" refers to a molecular complex of a compound of the invention, including a pharmaceutically acceptable salt thereof, with one or more solvent molecules. These solvent molecules are solvent molecules commonly used in medical technology and are known to be harmless to the recipient, such as water, ethanol and the like. The term "hydrate" refers to a complex of solvent molecules that are water.

The compounds of the invention, including their salts, hydrates and solvates, can form polymorphs either inherently or intentionally.

Eg5 inhibitor

The ADC of the present invention may comprise any suitable Eg5 inhibitor, especially an inhibitor having a molecular weight of less than 1000 Da, preferably less than 700 Da. In some embodiments, the Eg5 inhibitor has an IC-50 concentration of less than 1 micromolar; in a preferred embodiment, the Eg5 inhibitor used as a payload has an IC of less than 100 nanomolar (nM) -50. The IC 50 used for this purpose can be measured as described in WO2006/002236. Suitable Eg5 inhibitors include Rath (Rath and Kozielski , Nature Rev. Cancer , Vol. 12, 527-39 (2012), including Ispins, SB-743921, AZD4877, ARQ621, ARRY-520 Eg5 inhibitors as described in LY2523355, MK-0731, EMD534085, and GSK-923295, and WO06/002236, WO2007/021794, WO2008/063912, WO2009/077448, WO2011/128381, WO2011/128388, and WO2006/049835: Preferred payloads are the compounds of formula (II) and (III) as described herein.

An Eg5 inhibitor of formula (II) or (III) used as an ADC payload can be attached to a linking group L at various positions on the inhibitor (or directly to the Ab); in some embodiments, II) The compound is attached to L via an atom of the group Q or Y or R ¹ . Any available valence state on a compound of formula (II) can be attached to L, but to facilitate the preparation of a conjugate or a modified Eg5 inhibitor of formula (IIA) or (IIB), attachment to L typically occurs at Q or Y. At the hetero atom (N, O or S). In some embodiments of the combination of formula (I), the compound of formula (II) comprises free-NH- or free-OH or free-SH for linking a compound of formula (II) to a linking group L. In some embodiments, the free -NH -, - OH or -SH in the formula (II) or a group Q Y or a part of R ^1. It should be noted that the free -NH- may be an amine group (-NH ₂ ), a cyclic amine (for example, -NH- in a cyclic group such as pyrrolidone, piperidine or morpholine) or a secondary acyclic amine. In each case, the -NH- group is preferably not part of the guanamine or is not bonded to the carbonyl group or to the aryl or heteroaryl ring, which combination will reduce its reactivity.

Methods of attaching such payloads to a linking group to construct a conjugate are known in the art. Typically, the free primary or secondary amine or hydroxyl group is linked by a oximation reaction using a linker comprising an activated ester such as N-hydroxybutylimine or a sulfonate substituted N-hydroxybutylimine. The components are combined to form an ester or guanamine linkage. Alternatively, the primary amine can be bound by forming a Schiff base with a carbonyl group of the linker component, typically -CH(=O) or -C(=O)Me. When the Eg5 inhibitor comprises a thiol group, the conjugate may comprise maleimide or α-haloacetamide (-NH-C(=O)-CH ₂ LG, wherein LG is Br, Cl Or the linker component of I) is formed, or it can be bound to a thiol-containing linker component or antigen-binding moiety by forming a disulfide bond.

In one aspect of the invention, an Eg5 inhibitor of formula (III) is provided. The compound of formula III can be used as a small molecule therapeutic, or it can be incorporated into the ADC as a payload.

Or a pharmaceutically acceptable salt thereof, wherein: Z is N or CH; and Ar ¹ is optionally a group selected from the group consisting of halo, C _1-3 alkyl and C _1-3 haloalkyl Substituted phenyl; Ar ² is phenyl or pyridyl, optionally up to two groups selected from halo, CN, C _1-3 alkyl, hydroxy, amine and C _1-3 haloalkyl Substituent; R ¹ is -(CH ₂ ) _0-2 -C _4-7 heterocyclyl, wherein the C _4-7 heterocyclyl contains up to two heteroatoms selected from N, O and S as ring members, and optionally substituted with up to three substituents selected from halo, C _1-4 alkoxy, hydroxy, amino, oxo, the hydroxy substituted C _1-4 alkyl, substituted by the C _1-4 alkyl group Substituted by a group of methyl, trifluoromethyl or COO(C _1-4 alkyl); optionally up to three selected from halo, C _1-4 alkyl, C _1-4 alkoxy, side Substituted by an oxy group or a group of -COO(C _1-4 alkyl); R ² is H or C _1-4 alkyl; T is (CH ₂ ) _1-3 ; Y is selected from C _1-2 amine group An alkyl group, a C _4-6 heterocyclic group and a C _3-6 cycloalkyl group, wherein the C _1-2 aminoalkyl group, the C _4-6 heterocyclic group and the C _3-6 cycloalkyl group are each as large as possible two substituents selected from amino, oxo, halo, hydroxy, C _1-4 alkoxy, The hydroxy-substituted C _1-4 alkyl, the substituted amino by C _1-4 alkyl, COOH, COO- (C _1-4 alkyl) and the C _1-3 haloalkyl group; A is NH , N(C _1-4 alkyl) or a bond between carbonyl and Q in formula (III); Q is selected from C _1-4 alkyl, -(CH ₂ ) _0-2 -C _4-6 Heterocyclyl, -(CH ₂ ) _0-2 -C _5-6 heteroaryl and -(CH ₂ ) _0-2 -phenyl, and Q optionally up to three selected from halo, hydroxy, amine Substituting groups of -SH, -R, -OR, -SR, -SO ₂ R, -NHR and -NR ₂ wherein each R is halo, -SH, -NH ₂ , OMe or -OH as appropriate Substituted C _1-6 alkyl.

Additional examples include compounds of formula (III) wherein R ¹ is C ₃ substituted with -OH, -NH ₂ , -COOH, -COO(C _1-4 alkyl), -CONMe ₂ , CONHMe or -CONH _{2 -5} alkyl; and all other features are as described above for formula (III).

In such compounds, Z can be CH or N; in various embodiments, Z is CH.

In such compounds, Ar ¹ can be a substituted phenyl group as described above, typically a disubstituted phenyl group such as a dihalophenyl group. In a preferred embodiment, Ar ¹ is a 2,5-dihalophenyl group such as 2,5-difluorophenyl, 2-chloro-5-fluorophenyl or 2-fluoro-5-chlorophenyl.

In such compounds, Ar ² can be a substituted phenyl or pyridine as described above, or an optionally substituted cyclic ether. In various embodiments, Ar ² is unsubstituted or monosubstituted phenyl or pyridine. Suitable substituents for the substituted Ar ² include a halo group, a hydroxyl group and an amine group; the substituent may be at any position, for example, it may be positioned between the Ar ² position attached to the imidazole/triazole ring in the formula.

In any of these embodiments of the compound of formula (III), R ² may be H or C _1-4 alkyl, typically H or Me, preferably H.

In any of these embodiments of the compound of Formula (III), R ¹ may be a substituted or unsubstituted heterocyclic group as described above; in some embodiments, R ¹ is a cyclic ether, For example, tetrahydropyran-4-yl, tetrahydropyran-3-yl, tetrahydrofuran-3-yl or oxetane-3-yl. Tetrahydro-pyran-4-yl may be preferred: When incorporated into the ADC, in this example, when the partial reduction of binding R ¹ is tert-butyl can occur when the composition of the aggregate, and therefore this section for the purpose of ADC Especially advantageous. Information confirming this benefit is included in Table 7 herein.

In any of these embodiments of the compound of formula (III), T can be methylene, ethyl or propyl. In a preferred embodiment, when Y is one of said heterocyclic groups or cycloalkyl groups, T is a methylene group, and when Y is an aminoalkyl group within the scope of formula (III), T is a sub Methyl or -CH ₂ CH ₂ -.

In any of these embodiments of the compound of formula (III), A can be a bond; in other embodiments, A is preferably -NH-.

In any of these embodiments of the compound of formula (III), Y can be an aminoalkyl or heterocyclic group as described above. In some embodiments, Y is an aminoalkyl group, such as 1-fluoro-2-amino-2-ethyl or 1-amino-2-ethyl or 1-methoxy-2-amino-2 - Ethyl. In some embodiments, Y is a pyrrolidine ring, such as pyrrolidin-3-yl, and may be substituted with F, CH ₂ F, CF ₃ , Me, or OH. In a preferred embodiment, Y is substituted at the 4 position of one of these group _{(F, CH 2 F, CF} 3, Me , or OH) of 3- pyrrolidinyl.

In any of these embodiments of the compound of formula (III), R ² can be H or C _1-4 alkyl; in some embodiments, R ² is H or methyl, preferably H.

Some examples of Eg5 inhibitors for use in the immunoconjugates of the invention include any of the compounds in Table 1, such as:

Similarly, any of the following Eg5 inhibitors can be used in the immunoconjugates of the invention:

In certain embodiments of a compound of Formula (II) or (III), R ¹ is a heterocyclic group, such as a cyclic ether, such as tetrahydropyranyl (eg, 4-tetrahydropyran): as with as compared butyl conjugate of R ^1, when used as ADC payload, a heterocyclic group at R ¹ in the compound of formula (II) reduce aggregation, so such compounds exhibit superior to known inhibitors of Eg5 The advantage.

Bonding group

The linking group L in the formula (I) may be a bond directly linking one of the payload compounds X to Ab (ie, L or each linker component may represent a bond to which the group to which it is attached) Or it may be a linking moiety comprising one or more linker components L ₁ , L ₂ , L ₃ , L ₄ , L ₅ , L _{6 ,} and the like. Some preferred linking groups are depicted herein. The linking group for the ADC typically contains two or more linker components that can be selected to facilitate assembly of the conjugate, or it can be selected to affect the conjugate properties. Linker components include chemical groups that are readily formed when attached to Ab to X, such as thiol-maleimide, thioether, guanamine, and ester; in targeted cells, on targeted cells, or A group that is susceptible to cleavage in vivo under conditions found around the cell, such as disulfides, guanidines, dipeptides (such as Val-Cit), substituted benzyloxycarbonyl groups, and the like; Oriented spacers in a suitable position of Ab, such as phenyl, heteroaryl, cycloalkyl or heterocyclyl and extended alkyl chains; and/or pharmacodynamic enhancing groups, such as via one or more polarities a group (carboxy, sulfonate, hydroxy, amine, amino acid, sugar) substituted alkyl, and one or more -NH- or -O- instead of a methylene alkyl chain, For example, glycol ether (-CH ₂ CH ₂ O-) _p , where p is 1-10, for example, it can enhance solubility or reduce intermolecular aggregation.

The linking group may be divalent, meaning that it may only bond one X group to the Ab, or it may be trivalent (capable of linking two X groups to the Ab), or it may be multivalent of. Trivalent, tetravalent, and multivalent linking groups can be used to increase the drug loading on the antibody, thereby increasing the drug to antibody ratio (DAR) without the need for additional sites on the antibody to link the linking groups. Such linking groups are known in the art, see, for example, Bioconjugate Chem., March-April 1999; 10(2): 279-88; US6638499; Clin Cancer Res October 15, 2004 10 ; 7063; WO2012 / 113847A1.

The linking group used in the immunoconjugate of formula (I) can be cleavable or non-cleavable. a cleavable linking group, such as a linking group containing a hydrazine, a disulfide, a dipeptide Val-Cit, and a linking group containing a glucuronidase cleavable pair of an aminobenzyloxycarbonyl moiety It is well known in the art and can be used. See, for example, Ducry et al, Bioconjugate Chem., Vol. 21, 5-13 (2010). For such immunoconjugates, the linking group is substantially stable in vivo until the immunoconjugate binds to or enters the cell, at which point the intracellular enzyme or intracellular chemical conditions (pH, reducing ability) cleave the linking group To release the Eg5 inhibitor.

Alternatively, a non-cleavable linking group can be used in the immunoconjugate of formula (I). Non-cleavable linkers lack structural components designed to degrade in cells, and thus their structure can vary substantially. See, for example, Ducry et al, Bioconjugate Chem., Vol. 21, 5-13 (2010). It is believed that such immunoconjugates enter the targeted cells and undergo proteolytic degradation of the antibody rather than breakdown of the linkage; therefore, at least a portion of the linking groups and even some antibodies or antibody fragments can remain attached to the Eg5 inhibitor. Formulas (IIA) and (IIB) and (IIC) represent activated Eg5 inhibitors having a linking group attached at a specific position in which linkages and/or residues of the antibody have been shown to not interfere The inhibition of Eg5; therefore, when a non-cleavable linking group is used, it is preferred to bond a linking group at a position represented by W in the formulae (IIA) and (IIB) and (IIC).

Particularly suitable linking groups are linking groups which reduce aggregation. The linking groups in compounds 367 and 368 herein have been shown to have the effect of reducing aggregation when used with the Eg5 inhibitors described herein. Therefore, the linking group of the formula (V) is also included in the present invention:

Wherein R ²¹ has the formula -(CH ₂ ) _1-4 -R ²² , wherein R ²² is selected from the group consisting of -OH, -NH ₂ , N(R ²³ ) ₂ , COOR ²³ , CON(R ²³ ) ₂ , -(OCH ₂ CH ₂ O) _k -OCH ₂ CH ₂ OR ²³ and -SO ₂ R ²³ polar groups, wherein k is 0 to 4 and each R ^{23 is} independently H or C _1-4 alkyl; and j is selected An integer from 1, 2, 3 and 4; and an immunoconjugate Ab-L*-X comprising a payload (X) linked to an antibody (Ab), wherein the linking group L* comprises the formula -C (O) a group of NR ²¹ - or -NR ²¹ -C(O)-. Preferably, the linking group has the formula (V). In formula (V), [PL] indicates the point of attachment of the payload, and [Ab] indicates the point of attachment to the antibody. The antibody is typically linked to L* via a sulfur atom of a cysteine residue, which may be a cysteine from a native antibody sequence or a cysteine introduced by protein engineering. Preferred polar groups for such linkers include a hydroxyl group and a carboxyl group, j is typically 2, 3 or 4, and R ²¹ is typically -(CH ₂ ) _2-3 -R ²³ .

Formula (IIA) and (IIB) and (IIC) compounds

The compounds of formula (IIA) and (IIB) and (IIC) comprise an Eg5 inhibitor linked to a reactive group and, optionally, one or more linker components which link the Eg5 inhibitor to the reactive group. Table 2 depicts examples of such compounds comprising an Eg5 inhibitor (such as the inhibitor shown in Table 1) plus a reactive functional group and, optionally, one or more linker components.

Items 508 and 509 are provided as comparative examples with Eg5 inhibitors known in the art but not in the scope of Formula II.

Antigen binding part

The antigen binding portion of formula (I) or (IA) can be any moiety that selectively binds to a cell surface marker found on a targeted cell type. In some aspects, an Ab is an antibody or antibody fragment (eg, an antigen-binding fragment of an antibody) that specifically binds to an antigen (eg, a tumor-associated antigen) that is predominantly or preferentially found on the surface of a cancer cell. In some aspects, Ab is specific Binding to cell surface receptor proteins or other cell surface molecules, cell survival regulators, cell proliferative regulators, molecules associated with tissue development or differentiation (eg, known or suspected to contribute functionally to tissue development or differentiation), lymph An antibody or antibody fragment of a molecule, a cytokine, a molecule involved in cell cycle regulation, a molecule involved in angiogenesis, or a molecule associated with angiogenesis (eg, known or suspected to contribute functionally to angiogenesis) (eg, Antigen-binding fragment). The tumor associated antigen can be a cluster differentiation factor (i.e., a CD protein). In some aspects of the invention, the antigen binding portion of the invention specifically binds to an antigen. In some aspects of the invention, the antigen binding portion of the invention specifically binds to two or more antigens as described herein, eg, the antigen binding portion of the invention is a bispecific or multispecific antibody or antigen thereof Combine the fragments.

Exemplary antibodies or antigen-binding fragments include, but are not limited to, anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2 antibodies, anti-cKit antibodies, anti-EGFR antibodies, anti-Cathepsin D antibodies , anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, Anti-retinoblastoma protein antibody, anti-ras tumor protein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti- CD9/p24 antibody, anti-CD1-antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, Anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA/CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD71 antibody, anti-CD95/Fas antibody, anti-CD99 Antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, antibody Ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambine light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-τ antigen antibody, anti-fibrin antibody, anti-keratin antibody and anti- Tn-antigen antibody.

In one embodiment, the antigen binding portion of the antibody-drug conjugate (ADC) of Formula (I) or (IA) specifically binds to a receptor encoded by the ErbB gene. The antigen binding portion can specifically bind to an ErbB receptor selected from the group consisting of EGFR, HER2, HER3 and HER4. The antigen binding portion may be an antibody that specifically binds to the extracellular domain (ECD) of the HER2 receptor and inhibits the growth of tumor cells that overexpress the HER2 receptor. The antibody may be a monoclonal antibody, such as a murine monoclonal antibody, a chimeric antibody or a humanized antibody. The humanized antibody can be huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (trastuzumab). The antibody can be an antibody fragment, such as a Fab fragment.

The antibodies used in the examples herein have the heavy and light chain sequences listed in Table 3. These sequences are identical to the sequence of trastuzumab and the antibody is referred to herein as "trastuzumab" or "TBS". Trastuzumab is therefore a suitable antibody for use in an immunoconjugate of formula (I) or (IA).

Antigen binding moieties of Formula I or IA include, but are not limited to, antibodies or antibody fragments (e.g., antigen binding fragments) directed against cell surface receptors and tumor associated antigens. Such tumor associated antigens are known in the art and can be prepared for the production of antibodies using methods and information well known in the art. In an attempt to find effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify, as compared to, one or more normal non-cancerous cells. Or a transmembrane or otherwise tumor-associated polypeptide that is specifically expressed on the surface of a plurality of specific types of cancer cells. Generally, such tumor-associated polypeptides are expressed in larger amounts on the surface of cancer cells as compared to those on non-cancerous cells. Identification of such tumor-associated cell surface antigen polypeptides provides the ability to specifically target cancer cells for destruction via antibody-based therapies.

Antibodies and antibody fragments (eg, antigen-binding fragments) suitable for use in the immunoconjugates of the invention include modified or engineered antibodies, such as modified to introduce a cysteine residue or an lysine residue in place of a native sequence. An antibody to at least one amino acid thereby providing a reactive site on the antibody or fragment to bind to the Eg5 inhibitor. Similarly, antibodies or antibody fragments can be modified to incorporate Pcl or pyrrole lysine (Noren et al, (1989) Science 14; 244 (4901): 182-188; Mendel et al, (1995) Annu Rev Biophys Biomol Struct. 24: 435-462) serves as a site for binding to an Eg5 inhibitor. Methods for combining such antibodies with payload or linker-payload combinations are known in the art.

Antigen binding moieties (e.g., antibodies and antigen binding fragments) suitable for use in the present invention may also have other modifications or binding to other moieties such as, but not limited to, polyethylene glycol tags, albumin, and other fusion polypeptides.

Gene production

The antibodies and antibody fragments (e.g., antigen-binding fragments) of the invention can be made by any means known in the art including, but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full length single plants Antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, and the like.

The invention further provides polynucleotides encoding the antibodies described herein, eg, polynucleotides encoding heavy or light chain variable regions or segments comprising a complementarity determining region as described herein.

Polynucleotide sequences can be produced by de novo solid phase DNA synthesis or by PCR mutations encoding existing sequences of antibodies or binding fragments thereof (e.g., sequences as described in the Examples below). Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al, Meth. Enzymol. 68:90, 1979; Brown et al, Meth. Enzymol. 68:109, The 1979 phosphodiester method; Beaucage et al., Tetra. Lett., 22: 1859, 1981, Aminophosphoric acid diethyl ester method; and U.S. Patent No. 4,458,066. Introduction of mutations into a polynucleotide sequence by PCR can be performed as follows: for example, PCR Technology: Principles and Applications for DNA Amplification, HAErlich (ed.), Freeman Press, NY, NY, 1992; PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Innis et al. (eds.), Academic Press, San Diego, CA, 1990; Mattila et al, Nucleic Acids Res. 19: 967, 1991; and Eckert et al., PCR Methods and Applications 1: 17, 1991.

Expression vectors and host cells for producing the above antibodies or antibody fragments are also provided in the present invention. A variety of expression vectors can be employed to represent polynucleotides encoding the antibody chains or binding fragments of the invention. Both viral-based expression vectors and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plastids, typically free vectors for expression of proteins or RNA, and human artificial chromosomes (see, eg, Harrington et al, Nat Genet 15:345, 1997). For example, non-viral vectors suitable for expressing polynucleotides and polypeptides in mammalian (eg, human) cells include pThioHis A, pThioHis B and pThioHis C, pcDNA3.1/His, pEBVHis A, pEBVHis B, and pEBVHis C (Invitrogen, San Diego, CA), MPSV vectors and many other vectors known in the art for expressing other proteins. Suitable viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses; vectors based on SV40, papilloma virus, HBP EB virus; vaccinia virus vectors and Semliki Forest virus (SFV) ). See Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.

The choice of expression vector depends on the host cell in which the vector is to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an antibody chain or fragment of the invention. In some embodiments, an inducible promoter is employed to prevent the inserted sequence from behaving outside of the induction conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. The transformed organism culture can be amplified under non-inducing conditions that are not biased toward the coding sequence population, and the host cell is more tolerant of the expression products of the coding sequences. In addition to the promoter, other regulatory elements may also be required or required for the effective performance of the antibody chains or fragments of the invention. Such elements typically include an ATG initiation codon and an adjacent ribosome binding site or other sequence. Furthermore, performance efficiencies can be enhanced by enhancers including cell systems suitable for use (see, for example, Scharf et al, Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al, Meth. Enzymol., 153: 516, 1987). For example, an SV40 enhancer or CMV enhancer can be used to increase expression in a mammalian host cell.

The expression vector can also provide a secretion signal sequence position to form a fusion protein with the polypeptide encoded by the inserted antibody sequence. More typically, the inserted antibody sequence is linked to the signal sequence prior to inclusion in the vector. Vectors intended to be used to receive sequences encoding the light and heavy chain variable domains of an antibody sometimes also encode a constant region or portion thereof. Such vectors render the variable region as a fusion protein with a constant region, thereby resulting in the production of intact antibodies or fragments thereof. Typically, the constant regions are human.

Host cells for docking and expressing the antibody chains of the invention may be prokaryotic or eukaryotic. E. coli is a prokaryotic host suitable for use in the selection and expression of the polynucleotides of the invention. Other suitable microbial hosts include bacilli, such as Bacillus subtilis; and other enterobacteriaceae, such as Salmonella, Serratia; and various Pseudomonas species. In such prokaryotic hosts, we may also make expression vectors, which typically contain expression control sequences that are compatible with the host cell (eg, replication) starting point). In addition, a variety of well-known promoters may be present, such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-endosinase promoter system, or a bacteriophage lambda promoter system. Such promoters typically control performance along with the operator sequence, and have ribosome binding site sequences and similar sequences for initiation and completion of transcription and translation. Other microorganisms, such as yeast, can also be used to express the antibodies or antibody fragments of the invention. Insect cells combined with a baculovirus vector can also be used.

In one aspect, mammalian host cells are used to express and produce the antibodies and antibody fragments of the invention. For example, it may be a fusion tumor cell line expressing an endogenous immunoglobulin gene (for example, a myeloma fusion tumor line as described in the Examples) or a mammalian cell line harboring an exogenous expression vector. Such cells include any normally lethal or normal or abnormal immortal animal or human cell. For example, a variety of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and fusion tumors. . The use of mammalian tissue cell cultures to express polypeptides is generally discussed, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, NY, NY, 1987. Expression vectors for use in mammalian host cells can include expression control sequences such as origins of replication, promoters and enhancers (see, eg, Queen et al, Immunol. Rev. 89:49-68, 1986), and necessary processing information sites. , such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Such expression vectors typically contain a promoter derived from a mammalian gene or a mammalian virus. Suitable promoters can be constitutive, cell type specific, stage specific and/or regulatable or regulatable. Suitable promoters include, but are not limited to, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone-inducible MMTV promoter, SV40 promoter, MRP polIII promoter, constitutive MPSV promoter A tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), a constitutive CMV promoter, and a promoter-enhancer combination known in the art.

The method for introducing a expression vector containing a polynucleotide sequence of interest varies depending on the type of the cellular host. For example, calcium chloride transfection is typically used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts (see generally Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, impact methods, viral particles, immunoliposomes, multivalent cations: nucleic acid conjugates, naked DNA, artificial virions, fusion Herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88: 223, 1997), enhanced DNA uptake by agents and ex vivo transduction. For long-term high yield production of recombinant proteins, stable performance will usually be required. For example, a cell line stably expressing an antibody chain or a binding fragment can be produced using the expression vector of the present invention, which contains a viral origin of replication or an endogenous expression element and a selectable marker gene. After introduction of the vector, the cells can be grown in the concentrated medium for 1-2 days and subsequently transferred to a selective medium. The use of a selectable marker is to confer resistance to selection and its presence allows for successful expression of the cell growth of the introduced sequence in a selective medium. Resistant, stably transfected cells can be propagated using tissue culture techniques appropriate to the cell type.

Immunoconjugate

The invention provides immunoconjugates comprising an Eg5 inhibitor linked to an antigen binding portion, such as an antibody or antibody fragment. Preferred immunoconjugates of the invention are immunoconjugates of formula (I) or (IA) as described herein. Methods for preparing such immunoconjugates are well known in the art. Preferred immunoconjugates include the immunoconjugates disclosed in Tables 5 and 6, and variants thereof having another antigen binding moiety in place of trastuzumab, in particular trastuzumab from an antibody selected from the list below Such conjugates of substitution: exemplary antibodies or antigen-binding fragments include, but are not limited to, anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2 antibodies, anti-cKit antibodies, anti-EGFR Antibody, anti-Cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-sugar Protein antibody, anti-CEA antibody , anti-retinoblastoma protein antibody, anti-ras tumor protein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody , anti-CD9/p24 antibody, anti-CD1-antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 An antibody, an anti-CD33 antibody, an anti-CD34 antibody, an anti-CD35 antibody, an anti-CD38 antibody, an anti-CD39 antibody, an anti-CD41 antibody, an anti-LCA/CD45 antibody, an anti-CD45RO antibody, an anti-CD45RA antibody, an anti-CD71 antibody, an anti-CD95/Fas antibody, Anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specificity Antigen antibody, anti-τ antigen antibody, anti-fibrin antibody, anti-keratin antibody and anti-Tn-antigen antibody.

In some embodiments, an immunoconjugate of the invention comprises an antibody or antibody fragment Ab having antigen binding activity, wherein the linking group L is attached to the Ab at the cysteine sulfur atom of Ab:

Wherein L and X are as defined for formula (I), and R' and R" are the amino acid side chains adjacent to the cysteine in the Ab. In these embodiments, -SL- typically comprises a thiol - maleimide linkages, and L optionally comprises additional linker components. In other embodiments, the conjugates comprise -S-CH ₂ -C(=O)-NH-L ² -L ^The linking group of ³ -L ⁴ -L ⁵ -L ⁶ - is bonded to X, wherein the linker components L ² , L ³ , L ⁴ , L ⁵ and L ^{6 are} as defined for formula (IA). It is well known in the art to form such combinations by reacting a compound having an alpha-haloacetamide or a maleimide with a sulfur atom of a cysteine residue in an antigen binding portion (antibody). Methods.

Preferred immunoconjugates include immunoconjugates comprising any of the following compounds (Tables 5 and 6) that bind to an antibody (AntiB), wherein the conjugate has the structure shown in the table, wherein AntiB-S- An antibody that is bonded to the maleimide ring via a sulfur atom of the cysteine residue of the antibody (S in the structure). In a preferred embodiment, the antibody (AntiB) is an antibody that recognizes an antigen expressed on a cancer cell. Suitable antigens are disclosed herein, including anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2 antibodies, anti-cKit antibodies, anti-EGFR antibodies, anti-Cathepsin D antibodies, anti-Bcl-2 Antibody, anti-E-Cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoma Protein antibody, anti-ras tumor protein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, Anti-CD1-antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti- CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA/CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD71 antibody, anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD100 antibody , anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, c-myc antibody, anti-cytokeratin antibodies, anti-λ light chain antibodies, anti-melanosomes antibody, anti-prostate specific antigen antibody, anti-τ-antigen antibody, an anti-fibrin antibody, anti-keratin antibody and the anti-antigen antibody Tn-.

The cysteine residue linking the antibody to the maleimide compound can be naturally present in the native antibody, or it can be engineered by proteins known in the art. The method is introduced into the antibody. Antibodies engineered to contain cysteine residues introduced by protein engineering are sometimes preferred. In particular, antibodies engineered to introduce cysteine in place of at least one of the following sites are particularly suitable for use in the immunoconjugates of the invention: heavy chain sites K360, E152 and S375; and light chains Residue K107. In particular, the combination of HC-K360C and LC-K107C and HC-E152C and HC-S375C is well suited. (EU number)

It will be appreciated that the antibody may contain more than one payload: in a typical embodiment, the conjugate contains 2-6, preferably 3-5, payload compounds (Eg5 inhibitor) in two heavy chains and two light chains The peptide consists of an antibody.

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. The pharmaceutical compositions can be formulated for specific administration routes, such as oral administration, parenteral administration, and rectal administration, and the like. In addition, the pharmaceutical compositions of the present invention may be in solid form (including, without limitation, capsules, troches, pills, granules, powders or suppositories) or in liquid form, including, without limitation, solutions, suspensions or emulsions. Such pharmaceutical compositions can be subjected to conventional medical procedures (such as sterilization) and / or may contain conventional inert diluents, lubricants or buffers and adjuvants (such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.).

The immunoconjugates of the invention are typically formulated as solutions or suspensions in aqueous buffers and/or isotonic aqueous solutions. It is typically formulated and administered at near neutral pH to protect the stability of the protein component, for example at a pH between 6 and 8, and may include a pharmaceutically acceptable salt. And / or buffer. Because protein components are typically produced by cells, they may contain relative ions found in the cells, such as phosphate, acetate, sodium, potassium, and the like, and such relative ions, if present, are typically not specific Identification or characterization. In addition, it is typically isolated and disposed of in a buffer solution (such as phosphate buffered saline) and is not expected to have any relative ion-influencing activity. Such immunoconjugates are typically administered parenterally by injection or by infusion. The method of formulation and administration is similar to the formulation and administration of other biobased drugs, such as antibody therapeutics, and is known to those skilled in the art.

The compound of formula (III) for use as a small molecule drug can be formulated for conventional routes and administered by conventional routes, such as orally, transdermally, parenterally, buccally, by inhalation or as a suppository.

Suitable compositions for oral administration include an effective amount of a compound of the invention in the form of a troche, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule or syrup or Tincture. The compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and the compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents. And preservatives to provide a pharmaceutically elegant and palatable preparation. Tablets may contain a mixture of the active ingredient in admixture with pharmaceutically acceptable non-toxic excipients suitable for the manufacture of tablets. Such excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulation and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin or gum arabic And lubricants such as magnesium stearate, stearic acid or talc. Lozenges are uncoated The coating may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. The formulation for oral use may be in the form of a hard gelatin capsule in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin or in which the active ingredient is mixed with a water or oil medium (for example, peanut oil, liquid paraffin or Olive oil) presented in the form of a mixed soft gelatin capsule.

Certain injectable compositions are isotonic aqueous solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, dissolution promoters, salts for regulating osmotic pressure and/or buffers. In addition, it may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods and contain from about 0.1% to about 75% or from about 1% to about 50% by weight of active ingredient.

Suitable compositions for transdermal administration comprise an effective amount of a compound of the invention and a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmacologically acceptable solvents to aid delivery through the skin of the host. For example, a transdermal device is in the form of a bandage comprising a bottom member, a reservoir containing a compound and, optionally, a carrier, and optionally delivering a compound to the host skin at a controlled and predetermined rate over an extended period of time. Control the barrier and the components that hold the device to the skin.

Suitable compositions for topical application (e.g., to the skin and eyes) include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations such as those delivered by aerosol or the like. Such surface delivery systems will be particularly suitable for dermal administration, for example for the treatment of skin cancer, for example in the form of day creams, lotions, sprays and the like for prophylactic use. Therefore, it is particularly suitable for use in surface (including make-up) formulations well known in the art. These materials may contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

As used herein, topical administration can also be by inhalation or intranasal administration. It can be in the form of a dry powder (alone, in the form of a mixture (for example, an anhydrous blend with lactose) or a mixed component granule (e.g., with phospholipids) is conveniently delivered from a pressurized container, pump, nebulizer, or aerosol device, either in a dry powder inhaler or in the form of an aerosol spray with or without the use of a suitable propellant.

The invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compound of the invention as an active ingredient, as water may promote degradation of certain compounds.

The anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low moisture containing ingredients and under low moisture or low moisture conditions. Anhydrous pharmaceutical compositions can be prepared and stored such that their anhydrous nature is maintained. Thus, anhydrous compositions are encapsulated using materials known to prevent exposure to water such that they can be included in a kit of suitable formulations. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (eg, vials), blister packs, and strip packs.

The invention further provides pharmaceutical compositions and dosage forms comprising one or more agents which reduce the rate at which the compound of the invention will decompose as an active ingredient. Such agents referred to herein as "stabilizers" include, but are not limited to, antioxidants such as ascorbic acid, pH buffers or salt buffers, and the like.

The compounds of formula I in free form or in salt form exhibit valuable pharmacological activity: as demonstrated herein, the compounds of formula (II) and (III) inhibit tumor cell growth and are therefore suitable for the treatment of cancer. As the data further confirms, such compounds can advantageously be delivered in the form of an payload of the ADC. Such conjugates as demonstrated herein demonstrate the in vivo activity against targeted cells and in vivo on tumors, as evidenced by effective growth inhibition of xenograft tumors representing different human cancers. Thus, an immunoconjugate of the invention comprising a payload of formula (II) or (III) linked to an antigen binding moiety (such as an antibody) is also suitable for the treatment of cancer, such as glioma, neuroblastoma, melanoma, breast cancer. , lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (such as chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non- Hodgkin's), bladder, kidney, stomach (eg gastrointestinal matrix tumors) (GIST)), liver and pancreatic cancer and sarcoma.

The compounds and immunoconjugates of the invention are particularly useful in the treatment of cancers which are known in the art to be inhibited by compounds which are active against Eg5, and which are described herein as being susceptible to inhibition by the compounds and conjugates of the invention. Suitable indications for treatment include, but are not limited to, stomach, bone marrow, colon, nasopharynx, esophageal and prostate tumors, gliomas, neuroblastoma, melanoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid Cancer, leukemia (eg chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non-Hodgkin's), bladder, kidney, stomach (eg gastrointestinal matrix tumors (GIST)), liver and pancreatic cancer and sarcoma.

Thus, as a further embodiment, the invention provides the use of a compound of formula (I) or (III) or any of the embodiments within the scope of formulas (I) and (III) as described herein for therapy. In another embodiment, the therapy is directed to a disease that can be treated by inhibiting Eg5. In another embodiment, the compounds of the invention are useful in the treatment of cancer, including but not limited to, breast cancer, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, myeloid leukemia, Lymphocytic leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), hairy cell leukemia And multiple myeloma.

Such methods typically comprise administering to a subject in need of such treatment an effective amount of a compound as described herein or a pharmaceutical composition comprising the compound. The compound can be administered by any suitable method, such as the methods described herein, and the administration can be repeated at intervals selected by the treating physician.

Thus, as a further embodiment, the invention provides the use of a compound of formula (I) or (III) or any of the compounds described herein for the manufacture of a medicament. In another embodiment, the agent is for treating a condition that can be treated by inhibiting Eg5. In another embodiment, the disease is selected from the group consisting of stomach, bone marrow, colon, nasopharynx, esophageal and prostate tumors, and nerves. Glioma, neuroblastoma, melanoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (eg chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic cells) Leukemia or T-ALL), lymphoma (especially non-Hodgkin's), bladder, kidney, stomach (eg gastrointestinal matrix tumor (GIST)), liver and pancreatic cancer and sarcoma.

For an individual of from about 50 to 70 kg, the pharmaceutical composition or combination of the invention may be from about 1 to 1000 mg of active ingredient, or from about 1 to 500 mg or from about 1 to 250 mg or from about 1 to 150 mg or from about 0.5 to 100 mg, or from about 1 to about 50 mg. The unit dose of the active ingredient. The therapeutically effective dose of the compound, pharmaceutical composition or combination thereof will depend on the species, weight, age and individual condition of the individual, the condition or disease being treated, or the severity thereof. A generally skilled physician, clinician or veterinarian can readily determine the effective amount of each active ingredient necessary to prevent, treat or inhibit the progression of a condition or disease.

A pharmaceutical combination is a fixed combination in the form of a dosage unit, or wherein the compound of the invention and a combination thereof (eg, another drug as described below, also referred to as "therapeutic agent" or "adjuvant") may be the same The time is administered separately or independently during the time interval, in particular wherein such time intervals allow a combination of collocations to exhibit cooperation, such as a synergistic effect. The individual components can be packaged in a kit or separately. One or both of such components (e.g., powder or liquid) may be reconstituted or diluted to the desired dosage prior to administration. The term "co-administered" or "combined administration" or a similar term thereof as used herein is intended to encompass the selection of a selected combination with a single individual (eg, a patient) in need thereof, and is intended to include that the reagent is not necessarily The same route of administration or treatment regimen administered at the same time. The term "pharmaceutical combination" as used herein means a product produced by mixing or combining more than one active ingredient and includes a fixed or non-fixed combination of the active ingredients. The term "fixed combination" means an active ingredient, such as a compound of the invention and a combination conjugate, administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means an active ingredient, such as a compound of the invention and a combination conjugate, administered to a patient simultaneously, in parallel or sequentially, in the form of separate entities. There is no specific time limit, wherein the administration provides a therapeutically effective amount of the two compounds in the patient. The latter is also applied to cocktail therapy, for example by administering three or more active ingredients.

The above dosage characteristics can be advantageously demonstrated in in vitro and in vivo tests using mammals (e.g., mice, rats, dogs, monkeys) or isolated organs, tissues, and formulations thereof. The compounds of the invention may be administered in vitro in the form of a solution (for example an aqueous solution) and, for example, in the form of a suspension or in an aqueous solution, enterally, parenterally, advantageously intravenously. The extracorporeal dose can be between about 10 ^-3 moles and ^10-9 moles. Depending on the route of administration, the therapeutically effective amount in vivo may range from about 0.1 to 500 mg/kg, or preferably from about 1 to 100 mg/kg.

The compounds of the invention may be administered simultaneously with or prior to or after one or more therapeutic adjuvants. The compounds of the invention may be administered by the same or different routes of administration, or together with the adjuvant in the same pharmaceutical composition.

In one embodiment, the invention provides a composition comprising a compound of formula (I) and at least one other therapeutic adjuvant as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy treats a disease or condition mediated by Eg5, such as cancer. The product provided as a combined preparation comprises a compound comprising formula (I) or (III) together with other therapeutic adjuvants in the same pharmaceutical composition, or a compound of formula (I) or (III) and other therapeutic adjuvants in separate forms, For example, a composition in the form of a kit.

In one embodiment, the invention provides a pharmaceutical composition comprising a compound of formula (I) or (III) and an additional therapeutic adjuvant. The pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier as described above.

Suitable adjuvants for use with the compounds and conjugates of the invention include anticancer agents, anti-allergic agents, anti-nausea agents (or anti-inflammatory agents), pain relieving agents, anti-inflammatory agents, cytoprotective agents, and combinations thereof.

Specific adjuvants contemplated for use in combination with the compounds and combinations disclosed herein include Arimidex®, Bicalut®, Blenoxane®, Butacaine Sulfate (Myleran®), Butacaine Sulfate Injection (Busulfex®), Capecita Xeloda®, N4-pentyloxycarbonyl-5-deoxy-5-fluorocytosine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®) ), Platinol®, Leustatin®, Cytoxan® or Neosar®, Cytarabine, Cytosar-U®, Cytarabine Injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegan), doxorubicin hydrochloride (Cerubidine®), trichostomycin liposomal injection (DaunoXome) ®), Dexamethasone, Taxotere®, Cranberry Hydrochloride (Adriamycin®, Rubex®), Etoposide (Vepesid®), Fludarabine Phosphate (Fludara®), 5-Fluorouracil (Adrucil) ®, Efudex®), Eulexin®, tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), Idamycin®, Isocyclophosphamide IFEX®), icyzine (Camptosar®), L-aspartate glutaminase (ELSPAR®), formazan tetrahydrofolate, Alkeran®, 6-mercaptopurine (Purinethol®), methylamine Folex®, Novantrone®, mylotarg, Pacificol (Taxol®), phoenix (钇90/MX-DTPA), pentose glycosides, with Carmustine implants Glydel®, Nolvadex®, Vumon®, 6-thioguanine, thiotepa, tiza Tirapazamine (Tirazone®), Hydrocamptin® for injection, Velban®, Oncovin® and Navelbine®.

Some patients may experience an allergic reaction to a compound of the invention and/or other anti-cancer agents during or after administration; therefore, anti-allergic agents are typically administered to minimize the risk of allergic reactions. Suitable anti-allergic agents include corticosteroids such as dexamethasone (eg Decadron®), beclomethasone (eg Beclovent®), hydrocorticosterone (also known as corticosterone, Hydrocorticosterone sodium succinate, sodium hydrocortisone phosphate, sold under the trade names Ala-Cort®, Hydrocorticosterone, Solu-Cortef®, Hydrocort Acetate® and Lanacort®, and nylon (trade name Delta-) Cortel®, Orapred®, Pediapred® and Prelone® are sold), prednisone (sold under the trade names Deltasoone®, Liquid Red®, Meticorten® and Orasone®), methylprednisolone (also known as 6-methylprednisolone, Methylprednisolone acetate, methylprednisolone sodium succinate, sold under the trade names Duralone®, Medralone®, Medrol®, M-Prednisol® and Solu-Medrol®; antihistamines such as diphenhydramine (eg Benadryl®) ), hydroxyzine and cyproheptadine; and bronchodilators such as beta-adrenergic receptor agonists, albuterol (eg Proventil®) and terbutaline (Brethine®).

Some patients may experience nausea during and after administration of a compound of the invention and/or an anticancer agent; therefore, anti-inflammatory drugs are used to prevent nausea (upper stomach) and vomiting. Suitable antiemetics include aprepitant (Emend®), ondansetron (Zofran®), granisetron HCl (Kytril®), lorazepam (Ativan®, ground) Decadron®, Compazine®, casopitant (Rezonic® and Zunrisa®), and combinations thereof.

Drug treatment to relieve pain experienced during the treatment period is typically prescribed to make the patient more comfortable. Common over-the-counter analgesics such as Tylenol® are commonly used. However, opioid analgesics such as hydrocodone/paracetamol or hydrocodone/acetaminophen (such as Vicodin®), morphine (such as Astramorph® or Avinza®), oxycodone (such as OxyContin® or Percocet®), Oxymorphone hydrochloride (Opana®) and fentanyl (such as Duragesic®) are also indicated for moderate or severe pain.

In the process of protecting normal cells from therapeutic toxicity and limiting organ toxicity, cytoprotective agents (such as neuroprotective agents, free radical scavengers, cardioprotective agents, anthracycline sprinkling neutralizers, nutrients and the like) () can be used as an adjuvant therapy. Suitable cytoprotective agents include Amifostine (Ethyol®), glutamic acid, and dexamethasone. (Tavocept®), Mesnex®, dexrazoxane (Zinecard® or Totect®), xaliproden (Xaprila®) and formazan tetrahydrofolate (also known as hyperthyroidism) Calcium tetrahydrofolate, erythromycin factor and aldehyde folate).

In one embodiment, the invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which comprises a compound of formula (I) or (III). In one embodiment, the kit includes components for retaining the compositions, such as a container, a separate vial, or a separate aluminum foil package. Examples of such kits are blister packs as are typically used for encapsulating tablets, capsules and the like.

The kit of the invention can be used, for example, for oral and parenteral administration of different dosage forms, administration of the individual compositions at different dosage intervals, or titration of the individual compositions relative to one another. To aid compliance, the kit of the invention typically includes a dosing guide.

In the combination therapies of the invention, the compounds of the invention and other therapeutic adjuvants may be made and/or formulated by the same or different manufacturers. Furthermore, the compounds of the invention and other therapeutic agents may be used in combination therapy: (i) prior to the release of the combination product to the physician (eg, where the kit comprises a compound of the invention and other therapeutic agents); (ii) in the administration Not previously performed by the physician (or under the direction of a physician); (iii) by the patient himself, for example, during sequential administration of the compounds of the invention and other therapeutic agents.

Accordingly, the invention provides the use of a compound of formula (I) or (III) for the treatment of a disease or condition mediated by Eg5, wherein the agent is prepared for administration with another therapeutic agent. The invention also provides the use of another therapeutic adjuvant for the treatment of a disease or condition, wherein the agent is administered with a compound of formula (I) or (III).

The invention also provides a compound of formula (I) or (III) for use in a method of treating a disease or condition mediated by Eg5, wherein the compound of formula (I) or (III) is prepared for use with another therapeutic agent Give it together. The invention also provides another therapeutic adjuvant for use in a method of treating a disease or condition mediated by Eg5, wherein the additional therapeutic adjuvant is prepared for administration with a compound of formula (I) or (III) . The invention also provides a method for treating a disease or condition mediated by Eg5 A compound of formula (I) or (III) wherein the compound of formula (I) or (III) is administered with another therapeutic adjuvant. The invention also provides another therapeutic adjuvant for use in a method of treating a disease or condition mediated by Eg5, wherein the additional therapeutic adjuvant is administered with a compound of formula (I) or (III).

The invention also provides the use of a compound of formula (I) or (III) for the treatment of a disease or condition mediated by Eg5, wherein the patient has been previously treated (e.g., within 24 hours) with another therapeutic agent. The invention also provides the use of another therapeutic agent for the treatment of a disease or condition mediated by Eg5, wherein the patient has been previously treated (e.g., within 24 hours) with a compound of formula (I) or (III).

resolve resolution

All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents and catalysts used to synthesize the compounds of the invention are either commercially available or can be synthesized by methods known to those skilled in the art (see, for example, Houben- Weyl 4th Edition 1952, Methods of Organic Synthesis , Thieme, Vol. 21). Furthermore, the compounds of the present invention can be produced by the following examples by way of organic synthesis methods known to those skilled in the art.

A variety of Eg5 inhibitors The compounds of formula (II) can be prepared according to methods known in the art, including those disclosed in WO2007/021794, WO2006/002236, WO2008/063912, WO2009/077448, WO2011/128381, and WO2011/128388.

Compounds of formula (III) can be prepared analogously using known methods in combination with the methods described herein. Illustrative examples of the synthesis of such compounds are provided in the following general schemes.

This process begins with a known protected palmitic amino acid having a THP group at the a-carbon and an ester formed with the appropriate a-haloacetophenone to provide the desired Ar < ¹ > group. Treatment with ammonium acetate provides a substituted imidazole in which the palmity is retained at the group on C-2 of the imidazole ring. The imidazole nitrogen can be alkylated with a weak base to introduce Ar ² CH ₂ -. Deprotecting the carbamate provides a free amine which can be formed by oxidation with Dess-Martin periodinane (DMP) or similar oxidation and Schiff base, followed by the use of cyanoborohydride or A reducing agent similar to the reduction of the imine is alkylated from a suitable primary alcohol. In this scheme, this step introduces a pyrrolidine ring, but other groups within the scope of formula (II) or (III) can similarly be introduced to provide different -TY groups for the compound of formula (II) or (III). Other compounds. The protecting group can be used as needed to, for example, allow various R ¹⁰ groups to be present on the pyrrolidine ring, or to provide a substitution on Ar ¹ or Ar ² as needed.

The free amine can then be deuterated with any suitable deuteration agent to introduce the appropriate -A-Q moiety using conventional methods known in the art. In an illustrative example in the scheme, a palmitic lactate derivative is used to introduce a thiol group in a protected form having a bond for the group A in formulae (II) and (III) And a protected hydroxyalkyl group for the group Q. Removal of the pyrrolidine ring nitrogen and the hydroxyl group on Q provides a compound of formula (III).

A variety of suitable pyrrolidine rings for the preparation of the compounds of the invention are known in the art, and Scheme 2 depicts the synthesis of some of them. The 3-pyrroline ring is known to be protected by benzyloxycarbonyl (CBZ) or other suitable protecting groups and is oxidized by m-chloroperoxybenzoic acid. The epoxide is then opened in the presence of copper (I) bromide with a Grignard reagent, such as vinyl Grenner, to provide a trans-disubstituted pyrrolidine. It can be used to prepare various pyrrolidine intermediates, and the enantiomers thereof can be easily separated. It can be used, for example, to prepare a fluorinated pyrrolidine ring as described in Scheme 2. The hydroxyl group can be replaced by F under stereochemical inversion using a fluorinating reagent such as perfluoro-1-butanesulfonium fluoride (PBSF) or DAST. The vinyl group can then be oxidized with osmium tetroxide and the resulting aldehyde can be reduced by conventional methods such as sodium borohydride to provide the palmitic pyrrolidine used in Scheme 1.

Scheme 3 illustrates the synthesis of a compound wherein A is -NH-, starting with the intermediate from Scheme 1. The phosgene in the dichloromethane, followed by the introduction of the appropriate amine, provides a protected intermediate, and removal of the protecting group can be accomplished under conventional conditions, such as palladium on carbon in methanol, using ammonium formate or hydrogen.

Scheme 3A illustrates a method of synthesizing a compound of formula (IIA) having a reactive functional group attached via a thiol group of a compound of formula (II) (in this case, maleimide), ie, L Connect to Q. The intermediate from Scheme 2 is converted to an activated oximation agent using bis(p-nitrophenol) carbonate to form a mixed carbonate having a p-nitrophenoxy leaving group. The mixed carbonate is then reacted with a suitable amine, followed by removal of the protecting group of the pyrrolidine ring nitrogen to provide a compound of formula (IIA) wherein W is maleimide, which is suitable for attachment to or to Ab The thiol reaction on the linker component. The product in this example will be considered a non-cleavable linker since the linker components present are not designed to be used at a rate that is faster than the rate at which the antibody will be ligated in the ADC of formula (I). Lysis in vivo.

Scheme 3B illustrates the use of the product of Scheme 1 to prepare a compound of formula (IIB). In this method, bis(p-nitrophenol) carbonate is used to form a mixed carbonate containing a coupling group of maleimide. The mixed carbonate is then used to deuterize the pyrrolidine nitrogen of the Eg5 inhibitor to provide a compound of formula (IIB) as indicated above. In this example, the reactive functional group of W in formula (IIB) is maleimide, and the linker component in group W includes a cleavable linker (val-cit), thus the compound A conjugate having a cleavable linking group that undergoes cleavage by cathepsin B is exemplified. The aminobenzyloxy carbazate linker component acts as a self-decomposing linker: once cathepsin B cleaves the val-cit dipeptide from the p-amino group, the benzyl carbamate spontaneously decomposes to release the Eg5 compound .

The invention further comprises any variant of the process according to the invention, wherein the intermediate product obtainable at any stage thereof is used as starting material and the remaining steps are carried out, or wherein the starting materials are formed in situ under the reaction conditions, or The reaction components are used in the form of their salts or optically pure materials.

The following examples are intended to illustrate the invention and should not be construed as limiting. The temperature is given in degrees Celsius. If not mentioned otherwise, all evaporation is carried out under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (= 20-133 mbar). The structure of the final product, intermediate and starting material is confirmed by standard analytical methods such as microanalysis and spectral characteristics (eg MS, IR, NMR). Abbreviations used are abbreviations commonly known in the art.

DCM: dichloromethane

DIAD: diisopropyl azodicarboxylate

DIPEA: diisopropylethylamine

DMF: N,N-dimethylformamide

ETOAC: ethyl acetate

TBSCl: third butyl dimethyl decyl chloride

TFA: trifluoroacetic acid

THF: tetrahydrofuran

All reactions were carried out using a commercial grade solvent under Ar without any further distillation. The reagents were used as commercial grades without further purification. Thin layer chromatography was carried out using a TLC aluminum plate with 0.2 mm silicone (Merck F ₂₅₄ ). Column chromatography was performed using a flash pre-filled Redisep® column using the ISCO Combiflash Companion system.

The following NMR spectra were recorded spectrometer at 23 ℃ or 29 ℃: Bruker 400MHz Bruker AVANCE 600MHz proton frequency and equipped with a ^{1.7mm 1 H {13 C, 15} N} CryoProbe TM. Preparative HPLC was performed on a Waters Autopurification system under the following conditions: Column Sunfire C18 30 x 100 mm, 5μ, eluted at 30 ml/min in a gradient of CH ₃ CN-CH ₃ CN in water + 0.1% TFA.

LC/MS data was generated using a Waters Acquity UPLC/SQD system using a photodiode array detector and a single quadrupole mass detector. Use the following conditions: Column: Waters Acquity HSS T3 1.8μm 2.1 x 50mm

Dissolving agent A: water + 0.05% formic acid + 3.75 mM ammonium acetate

Dissolving agent B: acetonitrile + 0.04% formic acid

Column temperature: 60 ° C

Injection volume 1μl, partial loop

PDA full scan 210-450nm and a user-selectable wavelength

Method A: LCMS_2_min

Flow 1.0ml/min

Stop time 2.00min

Gradient: time % A (solubilizer A) % B (solvent B)

Mass range ESI +/-: 100-1200m/z

Method B: LCMS_10_min

Flow 1.0ml/min

Stop time 10.00min

Mass range ESI +/-: 100-1600m/z

Method C: LC-MS column Acquity UPLC BEH C18 1.7 μm, 2.1*50 mm

5 minutes (flow 0.7 ml/min, solvent A: water + 0.1% formic acid, solvent B: ACN + 0.1% formic acid, gradient: 20 to 100% B in 4.3 min B)

When the LC method is not specified, if the residence time is less than 1.5 minutes, Method A is used and for the residence time between 1.5 and 10 minutes, Method B is used.

UPLCMS- Method D (Polarity Method, 2 minutes operation): System: Waters Acquity UPLC with Waters SQ detector.

Column: Acquity HSS T3 1.8μm 2.1 x 50mm.

Flow: 1 ml/min. Column temperature: 60 ° C.

Gradient: 1 to 98% B in 1.4 min, A = water + 0.05% formic acid + 3.75 mM B Ammonium acid, B = acetonitrile + 0.0.4% formic acid.

UPLCMS Method E (4 minutes operation)

System: Waters Acquity UPLC with Waters SQ detector.

Column: Sunfire C18 3.5μm 2.1x20mm.

Flow: 0.62 ml/min. Column temperature: 40 ° C.

Gradient: 5 to 100% B in 4 min, A = water + 0.1% trifluoroacetic acid, B = acetonitrile + 0.1% trifluoroacetic acid.

Preparative LC method

Normal phase chromatography - PrepLC method A

System: CombiFlash Rf200.

Column: RediSep Column Silica.

Gradient: 0 to 100% B, A = heptane, B = ethyl acetate.

Normal phase chromatography - PrepLC method B

System: CombiFlash Rf200.

Column: RediSep Column Silica.

Gradient: 0 to 100% B, A = dichloromethane, B = MeOH.

Preparative Reverse Phase Chromatography - PrepLC Method C

System: Waters Acquity Prep LC/MS with Waters SQ detector.

Column: Sunfire Preparative C18, 5μm, 30 x 100mm.

Flow: 30 ml/min.

Gradient: 5 to 100% B, A = acetonitrile + 0.1% trifluoroacetic acid, B + water + 0.1% trifluoroacetic acid.

Preparative Reverse Phase Chromatography - PrepLC Method D

System: Waters Acquity Prep LC/MS with Waters SQ detector.

Column: Sunfire Preparative C18, 5μm, 30 x 150mm.

Flow: 60 ml/min.

Gradient: 5 to 100% B, A = acetonitrile + 0.1% trifluoroacetic acid, B + water + 0.1% trifluoroacetic acid.

Synthesis of selected intermediates 2,5-dihydro-1H-pyrrole-1-carboxylic acid benzyl ester

To a solution of 2,5-dihydro-1H-pyrrole (30 g, 434 mmol, 96% from Alfa Aesar) in dioxane (1000 mL, 0.43 M solution) was added Cbz. After stirring at room temperature for 18 hours, the reaction mixture was concentrated to ca. 300 mL and diluted with 1000 mL EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na ₂ SO _4, filtered and concentrated in vacuo. The desired 2,5-dihydro-1H-pyrrole-1-carboxylic acid benzyl ester was obtained as a colorless oil (yield: 90.0 g). Rf = 0.6 (30% EtOAc in hexanes). ¹ H NMR (CDCl ₃ , 400 MHz): δ 7.32 (5H, m), 5.80 (2H, m), 5.77 (2H, s), 4.22 (4H, m). LC / MS (uplc): MH + 204.2,160.1 (-44), 0.86min.

6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylic acid benzyl ester

Add m-CPBA (44 g, to a solution of 2,5-dihydro-1H-pyrrole-1-carboxylic acid benzyl ester (33 g, 163 mmol; 90% from Aldrich) in dichloromethane (540 mL, 0.3 M solution). 340 mmol, 77% from Aldrich). After the reaction mixture was stirred at room temperature for 18 hours, 500 mL of a saturated aqueous Na ₂ CO ₃ solution was added, and the mixture was stirred at room temperature for 1 hour. The organic layer was separated, washed with water and brine, dried over anhydrous Na ₂ SO _4, filtered and concentrated in vacuo. The desired product was obtained as a yellow oil (yield: 29.5 g). Rf = 0.5 (30% EtOAc in hexanes). ¹ H NMR (CDCl ₃ , 400 MHz): δ 3.38 (2H, dd, J = 12.8, 6.0 Hz), 3.68 (2H, d, J = 3.6 Hz), 3.87 (2H, dd, J = 13.2, 19.6) , 5.11 (2H, s), 7.33 (5H, m). LC/MS (uplc): MH ⁺ 220.0, 0.69 min.

3-hydroxy-4-vinylpyrrolidine-1-carboxylic acid benzyl ester

To 6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylic acid benzyl ester (28.5 g, 130 mmol) and CuBr at -40 °C. A solution of SMe ₂ (26.7 g, 130 mmol) in anhydrous THF (260 mL, 0.5 M solution). The reaction mixture was then warmed to -20 ° C for 2 hours. After washed with saturated aqueous NH ₄ Cl (200mL) was quenched, the reaction mixture was extracted with EtOAc (500mL). The organic layer was washed with water and brine, dried over anhydrous Na ₂ SO _4, filtered and concentrated in vacuo. The desired trans-(+)-3-hydroxy-4-vinylpyrrolidine-1-carboxylic acid benzyl ester racemic mixture as a yellow oil was obtained by flash column chromatography, 48% yield (15.5 g ). Rf = 0.2 (30% EtOAc in hexanes). _{^{1 H NMR (CDCl 3, 400MHz}} ): δ 2.71 (1H, m), 3.28 (2H, m), 3.72 (2H, m), 4.11 (1H, m), 5.14 (2H, s), 5.16-5.23 ( 2H, m), 5.69 (1H, m), 7.33 (5H, m). LC / MS (uplc): MH + 248.0,0.78min.

Analysis of trans(±)-3-hydroxy-4-vinylpyrrolidine-1-carboxylic acid benzyl ester

A racemic mixture of trans (±)-3-hydroxy-4-vinylpyrrolidine-1-carboxylate (14g) was submitted to the Basel Separation Laboratory (Contact: Dr. Eric Francotte, Tel.+41 6169 62971 ). To obtain the desired enantiomer enriched of (3 S, 4 R) -3- hydroxy-4-vinyl-pyrrolidine-1-carboxylate (6.3g,> 99.5% ee) and non-desired (3 R , 4 S )-3-Hydroxy-4-vinylpyrrolidine-1-carboxylic acid benzyl ester (6.7 g, 99.5% ee ), 92% recovery.

(3R,4R)-3-fluoro-4-vinylpyrrolidine-1-carboxylic acid benzyl ester

Hydroxy-4-vinyl-pyrrolidine-1-carboxylate (5.0g, 20.2mmol) was added to the N in PhCF ₃ (81mL, 0.25M solution) to a solution of (3 S, 4 R), N-Diisopropylethylamine (53 mL, 303 mmol), triethylamine trihydrofluoride (19.8 mL, 121 mmol) and perfluoro-1-butanesulfonium fluoride (PBSF, 3.6 mL, 20.2 mmol). The resulting mixture was stirred at room temperature. After 60 and 120 minutes, additional perfluoro-1-butanesulfonium fluoride (3.6 mL, 20.2 mmol) was added. After 18 hours, the reaction mixture was transferred to a separatory funnel and extracted with 50mL 1.0N HCl (Warning! Large amount of heat is generated) twice, once washed twice with saturated aqueous NaHCO ₃ and washed with H ₂ O and brine. The organic phase was dried over anhydrous Na ₂ SO _4, filtered and concentrated to provide a crude brown oil. By flash column chromatography _{(SiO 2, 10% -30%} EtOAc in hexanes) was obtained as a yellow oil of pure (3 R, 4 R) -3- pyrrolidin-l-fluoro-4-vinyl Benzoyl formate, 81% yield (4.1 g). Rf = 0.55 (30% EtOAc in hexanes). _{^{1 H NMR (CDCl 3, 400MHz}} ): δ 7.37-7.25 (5H, m), 5.9 (1H, m), 5.24 (2H, m), 5.14 (2H, m), 5.03 (1H, dt, J = 52.8 , 3.2 Hz), 3.9-3.5 (3H, m), 3.53 (1H, q, J = 10.4 Hz), 2.83 (1H, m). ¹³ C NMR (CDCl _3, 100 MHz): δ 154.7, 154.6, 136.6, 131.89, 131.83, 128.48, 128.02, 127.94, 119.00, 118.94, 95.23, 94.47, 93.42, 92.67, 66.99, 66.94, 53.16, 52.94, 52.83, 52.60 , 48.17, 48.02, 47.91, 47.83, 47.2, 47.1. LC/MS (uplc): MH ⁺ 250.0, 0.93 min.

(3R,4S)-3-fluoro-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid benzyl ester

To (3 R , 4 R )-3-fluoro-4-vinylpyrrolidine-1-carboxylic acid benzyl ester (1.78 g, 7.15 mmol) in CH ₃ OH and H ₂ O (2:1, 18 mL) A solution of OsO ₄ in H ₂ O (3 mL 4% w/v solution, 0.5 mmol) was added to the solution. Then NaIO ₄ (4.6 g, 21.5 mmol) was added in one portion and the resulting mixture was stirred at room temperature. After 2 hours, the mixture was filtered to remove a white solid that precipitated and washed with EtOAc. The filtrate was concentrated in vacuo to remove most of the organic solvent. The residue was extracted with EtOAc 3 and a portion of the organic layers combined and dried over anhydrous Na ₂ SO _4, filtered and concentrated. The crude product (3 R, 4 S) -3- fluoro-4-acyl pyrrolidine-1-carboxylate used in the next step without further purification. LC/MS (uplc): MH ⁺ 208.2 (- 44)

To the above (3 R, 4 S) -3- fluoro-4-acyl pyrrolidine-1-carboxylate The crude product of ice was added NaBH ₄ (330mg in CH ₂ Cl ₂ (20mL), 14.30 mmol). The reaction was stirred at room temperature. Upon completion of the reaction, the crude mixture was acidified with 0.5 M HCl and stirred for 30 min. The reaction mixture was partitioned between CH ₂ Cl ₂ and water. The organic layer was washed with saturated NaHCO ₃ (twice) and water (twice), followed by ₂ SO ₄ Na sulfate and the solvent was evaporated under reduced pressure to produce the desired product as an oil of (3R, 4S) -3- fluoro Benzyl 4-(hydroxymethyl)pyrrolidine-1-carboxylate (1.7 g, 6.9 mmol, 97%). The crude product was used in the next step without any further purification. LC/MS (uplc): MH+ 254.2, 210.2 (- 44), 0.78 min.

2-((Tertibutoxycarbonyl)amino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid (R)-2-(2,5-difluorophenyl)-2- Ethyloxyethyl ester.

To (R)-2-((t-butoxycarbonyl)amino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid (4.82 g. 18.6 mmol) and K ₂ CO ₃ (2.3 g, 16.7 mmol) 2-Chloro-1-(2,5-difluorophenyl)ethanone (4.25 g, 22.3 mmol), KI (0.77 g, 4.6) were added successively to ice-cooled solution in acetone (372 mL). Mm). The reaction was allowed to reach room temperature with stirring. Upon completion of the reaction by LC/MS (uplc), mixture was cooled to 0 ° C and quenched with cold water (600 mL). After stirring at 0 ℃ 15min, the reaction mixture was filtered and washed with acetone / H ₂ O (1/3) washing the precipitate, there is provided as a solid of 2 - ((tert-butoxy carbonyl) amino) -2- ( (R)-2-(2,5-Difluorophenyl)-2-oxoethoxyethyltetrahydro-2H-piperazin-4-yl)acetate (4.1 g, 9.71 mmol, 52%). The solid was dried under high vacuum overnight and used in the next step without any further purification.

^{1 H-NMR (CDCl3,600MHz):} δ 7.66 (1H, m), 7.61 (1H, m), 7.49 (1H, m), 7.32 (1H, m), 5.33 (2H, m), 4.06 (1H, m), 3.86 (2H, m), 3.24 (2H, m), 2.01 (1H, m), 1.75-1.4 (13H, m). LC/MS (uplc): MH+ 414.1, 1.11 min (Method A).

(R)-((4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)carbamic acid tert-butyl ester .

To (2-(2,5-difluorophenyl)-2-((tert-butoxycarbonyl)amino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid To a solution of the oxoethyl ester (4.1 g, 9.71 mmol) in toluene (50 mL) was added EtOAc (15 g, 194 mmol). The resulting solution was heated under reflux (110 ° C). When by LC / MS (uplc, Method A) found that the reaction was complete, the mixture was cooled to room temperature and partitioned between EtOAC and H ₂ O. The organic layer was separated and washed (twice) with H ₂ O (twice) and saturated NaHCO ₃ solution, then dried over Na ₂ SO _4, filtered and evaporated under reduced pressure to produce a brown solid of the crude product (R )-((4-(2,5-Difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)carbamic acid tert-butyl ester (4 g , 9.7 mmol, 99%). The solid was dried under high vacuum overnight and used in the next step without any further purification. LC/MS (uplc): MH+ 394.2, 0.99 min (Method A).

(R)-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl) Methyl) tert-butyl methacrylate.

To (R)-((4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)carbamic acid tert-butyl ester (4g, 9.7mmol) and _{K 2 CO 3 (2.8g, 20.4mmol} ) in DMF (68mL) in a ice-cooled solution of benzyl bromide was added (1.4mL, 11.2mmol), and the reaction was stirred at 0 ℃ After 1 hour, it was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), 80 mL of water was added and upon addition, solids precipitated. The solid was filtered and washed with DMF / H ₂ O (1/1) and washed to provide (R) - ((1- benzyl-4- (2,5-difluorophenyl) lH-imidazol-2-yl (tetrahydro-2H-piperidin-4-yl)methyl)carbamic acid tert-butyl ester (3.7 g, 7.6 mmol, 73%). The solid was dried under high vacuum overnight and used in the next step without any further purification. ^{1 H-NMR (CDCl3,400MHz):} δ 7.85 (1H, m), 7.36 (4H, m), 7.21 (2H, m), 7.05 (1H, m), 6.87 (1H, m), 5.30 (1H, m), 5.22 (1H, m), 5.13 (1 H, d, m), 4.69 (1H, m), 4.00 (1 H, m), 3.81 (1H, m), 3.30 (2H, m), 2.13 (1H, m), 1.80 (1H, m), 1.45 (9H, m), 1.30 (1H, m), 1.01 (1H, m). LC/MS (uplc): MH+ 484.3, 1.34 min (Method A).

(R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methylamine.

To (R)-((1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl) carbamic acid tert-butyl ester (3.7g, 7.6mmol) in CH of the ₂ Cl ₂ (80mL) was added ice-cooled trifluoroacetic acid (20mL). The reaction mixture was stirred at room temperature. The crude mixture was diluted and product was partitioned between CH ₂ Cl ₂ and saturated NaHCO ₃ solution in CH ₂ Cl ₂ at the time by LC / MS (uplc, Method A) reaction was complete. The organic layer was separated and washed (twice) with saturated NaHCO ₃ solution (twice) and water, then dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure to yield the crude product (R) - (1- benzyl Methyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methylamine (2.8 g, 7.3 mmol, 95%) TFA salt. The crude product was dried under high vacuum overnight and used in the next step without any further purification. LC/MS (uplc): MH+ 384.2, 0.83 min (Method A).

(3R,4R)-3-((((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidyl) Methyl 4-methyl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate.

Was added to a solution of the Dess (3R, 4S) -3- fluoro-4- (hydroxymethyl) pyrrolidine-1-carboxylate (2.9g, 11.4mL) in CH ₂ Cl ₂ (80mL) - Martin high iodine (6.5 g, 15.20 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Upon completion of the reaction, the crude product (3R,4S)-3-fluoro-4-carbamimidyrrolidine-1-carboxylic acid benzyl ester was used in the next step as a solution without further work.

To (R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methylamine (2.9 g, 7.6 mmol) and sodium triethoxysulfonium borohydride (8.1 g, 38 mmol) in CH ₂ Cl ₂ (60 mL) was added (3R,4S)-3-fluoro-4- from the previous step A solution of benzyl pyrrolidine-1-carboxylate in CH ₂ Cl ₂ . The reaction mixture was stirred at room temperature for 2 hours. When the reaction mixture was diluted in CH ₂ Cl ₂ , it was partitioned between CH ₂ Cl ₂ and H ₂ O. The organic layer was separated and washed (twice) with saturated NaHCO ₃ solution (twice) and H ₂ O, then dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. After purification by normal phase column chromatography, the desired (3R,4R)-3-((((R)-(1-phenylmethyl-4-(2,5-difluorophenyl))-) 1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester ( PrepLC Method A , 2.7 g , 4.5 mmol, 59%). LC/MS (uplc): MH+ 619.3, 1.23 min (Method A).

General procedure for intermediate N-deuteration

i Pr ₂ EtN (7 mmol) and decyl chloride (6 mmol) were successively added to an ice-cooled solution of the amine (1 mmol) in CH ₂ Cl ₂ (0.1 M). The reaction mixture was stirred at room temperature. When by LC / MS (uplc, Method A) reaction was complete, the mixture was diluted CH ₂ Cl ₂ and was partitioned between CH ₂ Cl ₂ and H ₂ O. The organic layer was separated and washed (twice) with saturated NaHCO ₃ solution (twice) and H ₂ O, then dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The desired Cbz-guanamine payload was obtained after purification by normal phase column chromatography (PrepLC method A or C).

Synthesize selected linkers Linker 1.

a: TBSC1, imidazole, DMF, room temperature, 53% yield; b : maleimide, DIAD, PPh ₃ , -78...RT, THF, toluene, 62% yield; c: CF ₃ COOH, CH ₂ Cl ₂ , 100% yield.

(2-((tert-Butyldimethylmethyl)alkyl)oxy)ethyl)(2-hydroxyethyl)carbamic acid tert-butyl ester

Prepared as described in Liang, Qiren; De Brabander, Jef K., Tetrahedron, 2011 , Vol. 67, pp. 5046-5053.

(2-((t-butyldimethylmethylalkyl)oxy)ethyl)(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)B Tert-butyl carbamic acid

Under N ₂ : freshly prepared maleimide (yield) was added to a stirred solution of DIAD (0.237 mL, 1.221 mmol) and triphenylphosphine (320 mg, 1.221 mmol) in 10 mL of toluene at -78 °C. 118 mg, 1.221 mmol) and (2-((t-butyldimethylmethyl)alkyl)oxy)ethyl)(2-hydroxyethyl)carbamic acid tert-butyl ester (300 mg, 0.939 mmol) in 10 mL THF Solution in the middle. The mixture was allowed to warm to RT and stirred overnight, diluted with DCM and washed with water. Organics were dried over Na ₂ SO _4, filtered and adsorbed on Isolute. The desired product was obtained as a yellow solid (yield: 24 g, gradient 0 to 100% EtOAC in heptane, 232 mg, 0.582 mmol, 62%). A mixture of rotamers was observed by ¹ H-NMR at room temperature. ¹ H-NMR (DMSO, 600 MHz): δ 7.09 and 6.97 (2H, s), 3.68-3.63 (2H, m), 3.57-3.52 (2H, m), 3.20-3.14 (2H, m), 3.40-3.36 (2H, m), 1.31 (9H, s), 0.85 (9H, s), 0.02 (6H, s). LC/MS (Method A): MH+ 399.4.

1-(2-((2-hydroxyethyl)amino)ethyl)-1H-pyrrole-2,5-dione (linker 1 for ADC-1)

Under N ₂ : (2-((t-butyldimethylmethylalkyl)oxy)ethyl)(2-(2,5-di- oxo-2,5-dihydro-1H-pyrrole) tert-Butyl 1-ethyl)ethyl)carbamate (220 mg, 0.552 mmol) was dissolved in 20 mL DCM and TFA (2.126 mL, 27.6 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo, EtOAc (EtOAc m. Linker 1 was used without further purification. ¹ H-NMR (CD ₃ OD, 400 MHz): ¹ H-NMR (CD ₃ OD, 400 MHz): δ 6.94 (2H, s), 3.92-3.87 (2H, m), 3.83-3.79 (2H, m), 3.32-3.28 (2H, m), 3.23-3.18 (2H, m).

Synthetic linker 2

a: ethanolamine, 90% yield; b: di- t -butyl dicarbonate, TEA, THF, 50% yield; c: maleimide, D1AD, PPh ₃ , 43% yield; d: CF ₃ COOH, 60% yield.

3-((2-hydroxyethyl)amino)propionic acid tert-butyl ester

For example, Aebi, Johannes; Binggeli, Alfred; Green, Luke; Hartmann, Guido; Maerki, Hans P.; Mattei, Patrizio; Ricklin, Fabienne; Roche, Olivier, Patent: US2010/16282 A1, 2010; obtain

3-((t-butoxycarbonyl)(2-hydroxyethyl)amino)propionic acid tert-butyl ester

Under N _2, the 3 - ((2-hydroxyethyl) amino) propanoic acid tert-butyl ester (1304mg, 6.89mmol) was dissolved in 20mL THF, and the sequentially added triethylamine (0.960mL, 6.89mmol) Boc-anhydride (1.600 mL, 6.89 mmol). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and partitioned betweenEtOAc and brine. Organics were dried over Na ₂ SO _4, filtered and adsorbed on Isolute. The desired product was obtained as a colorless oil (yield: 80 g, gradient 0 to 100% EtOAC in heptane, 988 mg, 3.41 mmol, 50%).

3-((t-butoxycarbonyl)(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)propanoic acid tert-butyl ester

Under N _2: the DIAD (0.863mL, 4.44mmol) and triphenylphosphine (1164mg, 4.44mmol) at -78 deg.] C was added maleic acid (PEI) was prepared freshly in 10mL of toluene was stirred solution of ( 431 mg, 4.44 mmol) and a solution of 3-((t-butoxycarbonyl)(2-hydroxyethyl)amino)propionic acid tert-butyl ester (988 mg, 3.41 mmol) in 10 mL THF. The reaction mixture was allowed to warm to rt and stirred overnight. The reaction mixture was diluted with DCM and washed with water. Organics were dried over Na ₂ SO _4, filtered and adsorbed on Isolute. The desired product was obtained as a colorless oil (yield: 80 g, gradient 0 to 100% EtOAC in heptane, 903 mg, 1.48 mmol, 43%, purity by LC-MS, UV). 60%). LC/MS (Method A): MH.

3-((2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)propionic acid tert-butyl ester

Under N ₂ : 3-((t-butoxycarbonyl)(2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amine The third butyl propionate (870 mg, 2.361 mmol) was dissolved in 5 mL of DCM and the reaction mixture was cooled to -10 °C. TFA (5 mL, 64.9 mmol) was added. The reaction mixture was stirred at -10 °C for 3 hours, then concentrated in vacuo, EtOAc EtOAc m. It is about 50% pure as determined by purification in small batches.

100 mg of the crude compound was purified by reverse phase column chromatography to yield 50 mg of pure linker 2 (as trifluoroacetic acid salt).

¹ H-NMR (CDCl ₃ , 400 MHz): δ 6.76 (2H, s), 3.96-3.91 (2H, m), 3.39-3.29 (4H, m), 2.79-2.73 (2H, m), 1.48 (9H, s). LC/MS (Method A): MH.

Synthetic linker 4

3-((2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)methyl)azetidin-1-carboxylic acid tert-butyl ester

Diisopropyl azodicarboxylate (1.04 mL, 5.34 mmol) was added dropwise to a solution of triphenylphosphine (1.40 g, 5.34 mmol) in dry EtOAc The mixture was stirred for 5 minutes. 3-(hydroxymethyl)azetidin-1-carboxylic acid tert-butyl ester (1.00 g, 5.34 mmol) was then added to the reaction over 5 minutes. When maleimide (0.518 g, 5.34 mmol) was added, the resulting solution was stirred for another 5 minutes. The reaction mixture was allowed to warm to RT and stirred for additional 18 h. The reaction was concentrated to dryness. The crude product was purified by chromatography eluting elut elut elut elut elut elut elut elut elut elut /z[M+H] ⁺ 267.0; Method A.

2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)azetidin-1-yl 2,2,2-trifluoroacetate

To 3-((2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)methyl)azetidin-1-carboxylic acid tert-butyl ester (624 mg, 2.34 mmol) Trifluoroacetic acid (9.03 mL, 117 mmol) was slowly added to a solution in DCM (23.5 mL), and the mixture was stirred at RT for 30 min. The reaction was concentrated to dryness to yield a pale yellow solid of the desired product, 99% yield; UPLC-MS: Rt = 0.22min ; MS m / z [M + H] + 167.0; Method A.

Synthetic linker 5

(3-((2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)aminocarbamic acid Third butyl ester

To a solution of 3-tert-butoxycarbonyl)amino)propanoic acid (500 mg, 1.97 mmol) and HATU (1.50 g, 3.93 mmol) in DMF (20 ml), triethylamine (1.37 mL, 9.84 mmol), 1-(2-Aminoethyl)-1H-pyrrole-2,5-dione (500 mg, 1.97 mmol). The reaction was diluted with EtOAc and washed with EtOAc EtOAc. The organic layer was extracted, washed with saturated aqueous NaHCO _3. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by flash chromatography eluting eluting elut elut elut elut elut elut elut elut ^{/ z [M + H] +} 312.1; method A.

2-(2,2-difluoroacetic acid-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3- side of 2,2,2-trifluoroacetic acid Oxypropan-1-ammonium

To 3-((2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)methyl)azetidin-1-carboxylic acid tert-butyl ester (624 mg, 2.34 mmol) Trifluoroacetic acid (0.80 mL, 10.4 mmol) was slowly added to a solution in DCM (23.5 mL), and the mixture was stirred at RT for 30 min. The reaction was concentrated to dryness to yield a pale yellow solid of the desired product in quantitative yield; UPLC-MS: Rt = 0.24min ; MS m / z [M + H] + 212.1; Method A.

2-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl (4-nitrophenyl) carbonate

To a solution of 1-(2-hydroxyethyl)-1H-pyrrole-2,5-dione (250 mg, 1.77 mmol) in EtOAc (EtOAc) And bis(4-nitrophenyl) carbonate (701 mg, 2.30 mmol), and the reaction mixture was stirred at RT for 3 h. The reaction was extracted with water and DCM. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by 0-100% EtOAc in heptane eluting the chert chromatography to give a pale yellow solid of the title compound, 88% ^{yield; 1 H-NMR (DMSO,} 400MHz): δ 8.34- 8.32 (2H, m), 7.54-7.51 (2H, m), 7.09 (2H, s), 4.36-4.34 (2H, m), 3.81-3.79 (2H, m).

1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrrole-2,5-dione

Was added N- (methoxycarbonyl) maleic acyl imine of a solution of (2-amino ethoxy) ethanol saturated aqueous NaHCO ₃ (2.9mL, 29.0mmol) (150ml) at 0 ℃ (4.5 g, 29.0 mmol), and the reaction mixture was stirred at RT for 30 min and then stirred at RT for 3 hr. The reaction was extracted with DCM. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to yield the title compound as a pale yellow oil of 53% yield; UPLC-MS: Rt = 0.35min ; MS m / z [M + H] ⁺ 186.0; Method E.

2-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl (4-nitrophenyl) carbonate

In a similar manner to 2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl (4-nitrophenyl) carbonate, use 1- (2(2-Hydroxyethoxy)ethyl-1H)-pyrrole-2,5-dione to synthesize product; 63% yield; UPLC-MS: Rt = 1.80 min; MS m/z [M+H ] ⁺ 697.0; Method E.

Synthesis of sulfonate-substituted linkers

The general method for preparing this linker was adapted from the published method ( J. Med. Chem . 2011, Vol. 54, 3606-23); pentafluorophenyl ester was chosen instead of the N-hydroxybutanedipine used in the reference. Imine.

Synthesis Example 1. (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluoro) Phenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester.

LC/MS (uplc): MH+ 733.3, 1.36 min (Method A).

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionamide

To (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H) -imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester (115 mg, 0.14 mmol) Pd/C (30.4 mg, 0.03 mmol) and ammonium formate (108 mg, 1.7 mmol) were added in MeOH (2 mL). The reaction mixture was heated at 55 ° C for 1 hour. Upon completion, the reactants were filtered to remove Pd/C and the solvent was evaporated under reduced pressure to yield crude product (S)-1-(((R)-(1-phenylmethyl-4-(2,5) -difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)- Amino)-1-yloxypropan-2-yl ester. LC/MS (uplc): MH+ 599.2, 0.92 min. The crude product was used in the next step without any further purification.

Acetic acid (S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) from the Cbz protecting group removal step (tetrahydro-2H-piperidin-4-yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-yloxypropan-2- K ₂ CO ₃ (197 mg, 1.4 mmol) was added to a solution of EtOAc ( ₃ mL). The reaction was stirred at room temperature for 1 hour. Upon completion of the reaction by LC/MS (uplc, Method A), the crude mixture was filtered to remove solids, and when purified by reverse phase column chromatography, the desired product (S)-N-(( R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-N- (((3S,4R)-4-Fluoropyridin-3-yl)methyl)-2-hydroxypropionamide (PrepLC Method C), 60 mg, 0.09 mmol, 63%). The product was isolated as a TFA salt. ^{1 H-NMR (DMSO, 600MHz} ): δ 7.80 (1H, m), 7.75 (1H, m), 7.45-.7.25 (6H, m), 7.09 (1H, m), 5.71 (1H, m), 5.25 (2H, m), 5.11 (1H, m), 4.95 (1H, m), 4.05 (1H, m), 3.80 (1H, m), 3.35 (2H, m), 3.20 (1H, m), 2.90 ( 1H, m), 2.83 (1H, m), 2.73 (1H, m), 2.68 (1H, m), 2.22 (1H, m), 1.87 (1H, m), 1.45 (1H, m), 1.35 (1H) m), 1.25 (3H, m), 1.09 (1H, m), 0.67 (1H, m). Missing signals hidden under the solvent peak. LC/MS (upl): MH+ 557.2, 0.84 min (Method A).

General method of BOC protection

K ₂ CO ₃ ( ₂ mmol) and Boc anhydride (3 mmol) were added to a solution of urea (1 mmol) in MeOH (0.1M). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was filtered to remove solids, and the desired product was isolated after purification by normal phase column chromatography (PrepLC method A or B). Boc-urea.

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropionamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester.

General procedure for BOC protection: LC/MS (uplc): MH+ 657.3, 1.30 min (Method A).

Synthesis Example 2. (S)-2-Amino-N-((R)-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (Four Hydrogen-2H-piperazin-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-hydroxypropionamide

General procedure for removal of Cbz protecting groups. 18 mg, 0.023 mmol, 49%. ^{1 H-NMR (DMSO, 600MHz} ): δ 7.80 (1H, m), 7.75 (1H, m), 7.40 (2H, m), 7.32 (4H, m), 7.11 (1H, m), 5.27 (1H, m), 5.29 (1H, m), 5.14 (1H, m), 4.98 (1H, m), 3.82 (1H, m), 3.79 (1H, m), 3.65 (1H, m), 3.51 (1H, m) ), 3.40 (2H, m), 2.92 (1H, m), 2.84 (1H, m), 2.78 (1H, m), 2.59 (1H, m), 2.20 (1H, m), 2.01 (1H, m) , 1.88 (1H, m), 1.65 (1H, m), 1.45 (1H, m), 1.30 (1H, m), 1.18 (1H, m), 0.89 (1H, m), 0.65 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 572.2, 0.71 min (Method A).

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butoxycarbonyl)amino)-3-hydroxypropionamido)methyl)-4-fluoropyrrolidin-1 - tert-butyl formate.

General procedure for Boc protection: LC/MS (uplc): MH+ 772.2, 1.36 min (Method A).

Synthesis Example 3. (3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperidin-4-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzoate ester.

792 mg, 1.0 mmol, 76%. ^{1 H-NMR (DMSO, 600MHz} ): δ 7.70 (1H, m), 7.52 (1H, m), 7.30-7.10 (10H, m), 6.90 (1H, m), 5.78 (s, 1H), 5.37 ( 2H, bs), 5.34 (2H, m), 5.30 (1H, bs), 5.05 (1H, m), 4.92 (2H, bs), 4.25 (2H, bs), 3.87 (2H, m), 3.81 (1H) , m), 3.71 (1H, m), 3.64 (2H, m), 3, 43 (2H, m), 3.26 (1H, m), 2.61 (1H, m), 2.45 (1H, m), 2.01 ( 1H, m), 1.61-1.25 (4H, m), 1.12 (3H, m). LC/MS (uplc): MH+ 720.3, 1.25 min. (Method A).

Synthesis Example 4. (3R,4R)-3-(((3S,4R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazole) -2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3,4-dihydroxypyrrolidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1- Benzyl formate

2870 mg, 2.3 mmol, 60%. LC/MS (uplc): MH+ 748.2, 1.19 min (Method A).

Synthesis Example 5. (3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-3-(2,3-dihydroxypropyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester

670 mg, 0.9 mmol, 39%. LC/MS (uplc): MH+ 736.2, 1.16 min. (Method A).

(3R,4R)-3-((N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -Methylpyran-4-yl)methyl)-3-hydroxyazetidin-1-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate.

572 mg, 0.64 mmol, 66%. LC/MS (uplc): MH+ 718.2, 1.23 min (Method A).

(3R,4R)-3-((N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -Methylpyran-4-yl)methyl)-3-hydroxypiperidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate.

Isomer A: LC/MS (uplc): MH+ 746.2, 1.27 min (Method A).

Isomer B: LC/MS (uplc): MH+ 746.2, 1.28 min (Method A).

Cbz protecting group removal:

To a solution of Cbz-urea payload (1.0 mmol) in MeOH (0.1 M) was added Pd / C (10%, 0.2 mmol) and ammonium formate (12 mmol). The reaction was heated at 55 ° C for 30 minutes. Upon completion, the reaction was filtered to remove Pd/C and the desired urea was isolated on reverse phase column chromatography. (PrepLC method C or D).

Synthesis Example 6. 1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-((S)-1-hydroxyl) Prop-2-yl)urea

^{1 H-NMR (DMSO, 600MHz} ): δ 7.75 (2H, m), 7.40-7.25 (6H, m), 7.09 (1H, m), 5.90 (1H, bs), 5.36-5.30 (3H, m), 4.96 (1H, m), 4.71 (1H, m), 3.85 (2H, m), 3.79 (1H, m), 3.58 (2H, m), 3.29 (4H, m), 2.79 (2H, m), 2.57 (1H, bs), 2.17 (1H, bs), 1.72 (1H, m), 1.60 (1H, m), 1.38 (2H, m), 0.95 (2H, m), 1.08 (3H, bs). One signal is hidden under the solvent peak. LC/MS (uplc): MH+ 586.3, 0.86 min. (Method A).

1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl )-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-(2-hydroxyethyl)urea.

15 mg, 0.025 mmol, 19%. ¹ H-NMR (DMSO, 600 MHz): δ 7.78-7.68 (2H, m), 7.44 - 7.36 (2H, m), 7.36-7.25 (4H, m), 7.15-7.04 (1H, m), 6.45- 6.28 (1H, m), 5.45-521 (3H, m), 5.10-4.91 (1H, m), 4.71-4.57 (1H, m), 3.91-3.79 (1H, m), 3.67-3.52 (2H, m ), 3.28-3.14(4H,m), 3.09-2.91(1H,m),2.84-2.68(1H,m),2.24-2.12(1H,m),1.97-1.78(1H,m),1.59-1.49 (1H, m), 1.47-1.37 (1H, m), 1.36-1.27 (1H, m), 1.22-1.10 (1H, m), 0.79-0.60 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 572.2, 0.83 min (Method A).

Synthesis Example 7. (3S,4R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3,4-dihydroxypyrrolidine-1-carboxamide

^{1 H-NMR (DMSO, 600MHz} ): δ 7.73 (2H, bs), 7.38-7.25 (6H, m), 7.06 (1H, bs), 5.5 (2H, m), 4.95 (1H, m), 4.84 ( 2H, bs), 4.82 (1H, bs), 4.02 (2H, m), 3.81 (1H, m), 3.60 (1H, bs), 3.49 (2H, bs), 3.27 (1H, m), 3.13 (1H) , bs), 2.93 (1H, m), 2.72 (1H, m), 2.39 (1H, bs), 2.28 (1H, m), 1.85 (1H, bs), 1.73 (1H, m), 1.58 (1H, m), 1.03 (1H, m), 0.97 (1H, m), 0.28 (1H, m), 5H concealed at the solvent peak. LC/MS (uplc): MH+ 614.3, 0.82 min. (Method A).

Synthesis Example 8. 1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-3-(2,3-dihydroxypropyl)-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)urea

^{1 H-NMR (DMSO, 600MHz} ): δ 7.76 (2H, bs), 7.40-7.25 (6H, m), 7.09 (1H, bs), 6.36 (1H, bs), 5.40 (1H, bs), 5.30 ( 2H, m), 4.95 (1H, m), 4.79 (1H, bs), 4.60 (1H, bs), 3.84 (1H, m), 3.61 (1H, m), 3.56 (1H, m), 3.54 (1H) , bs), 3.29 (1H, m), 3.25 (4H, m), 3.10 (1H, m), 2.94 (1H, m), 2.68 (1H, m), 2.56 (1H, m), 2.13 (1H, m), 1.80 (1H, m), 1.49 (1H, m), 1.42 (1H, m), 1.35 (1H, m), 1.18 (1H, m), 0.71 (1H, m). Two signals are hidden under the solvent peak. LC/MS (uplc): MH+ 602.3, 0.78 min. (Method A).

N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl )-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-hydroxyazetidin-1-carboxamide.

45 mg, 0.073 mmol, 92%. LC/MS (uplc): MH+ 584.2, 0.84 min (Method A).

N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl )-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-hydroxypiperidine-1-carboxamide.

Isomer A: LC/MS (uplc): MH+ 612.30.88 min (Method A).

Isomer B: LC/MS (uplc): MH+ 612.30.89 min (Method A).

Synthesis Example 9. (3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid Butyl ester General procedure for the synthesis of Boc-urea payloads:

Urea coupling: To an ice-cooled solution of phosgene (20% in toluene, ₂ mmol) in CH ₂ Cl ₂ (0.1 M), EtOAc (1 mmol) and triethylamine (3 mmol) in CH ₂ Cl ₂ (1M) Solution in the middle. The reaction mixture was stirred at room temperature for 30 minutes. Upon completion of the reaction by LC/MS (uplc, Method A), the desired amine for urea (20 mmol) was added, and the reaction was stirred at 60 ° C for 2 hours, and then stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude solvent was evaporated under reduced pressure, and purified by normal phase column chromatography (Prep LC method A or B) to give the desired product.

0.49 mg, 0.69 mmol, 68%. LC/MS (uplc): MH+ 686.3, 1.25 min. (Method A).

(3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-3-(2-hydroxyethyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester.

1.05 g, 1.6 mmol, 85%. LC/MS (uplc): MH+ 782.2, 0.90 min (Method A).

Synthesis Example 10. (3R,4R)-3-(((3S,4R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazole) -2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3,4-dihydroxypyrrolidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1- Tert-butyl formate

817 mg, 1.09 mmol, 47%. LC/MS (uplc): MH+ 714.2, 1.18 min. (Method A).

Synthesis Example 11. (3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-3-(2,3-dihydroxypropyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

317 mg, 0.43 mmol, 49%. LC/MS (uplc): MH+ 702.2, 1.16 min. (Method A).

(3R,4R)-3-((N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-3-hydroxyazetidin-1-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester.

240 mg, 0.33 mmol, 73%. LC/MS (uplc): MH+ 684.2, 1.23 min (Method A).

(3R,4R)-3-((N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-3-hydroxypiperidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester.

Isomer A: LC/MS (uplc): MH+ 712.3, 129 min (Method A).

Isomer B: LC/MS (upcl): MH+ 712.3, 1.30 min (Method A).

Synthesis Example 12. (3R,4R)-3-((3-((S)-1-Azidopropan-2-yl)-1-((R)-(1-phenylmethyl-4-() 2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)ureido)methyl)-4- Fluoropyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro- 2H-piperidin-4-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester ( 100 mg, 0.15 mmol) p-toluenesulfonium chloride (40.3 mg, 0.21 mmol) was slowly added to an ice-cooled solution of CH ₂ Cl ₂ (0.8 mL) and pyridine (24 μl, 0.29 mmol). The reaction mixture was stirred at room temperature. Upon completion of the reaction, the reaction mixture was diluted in CH ₂ Cl ₂ and partitioned between H ₂ O and CH ₂ Cl ₂ . The organic layer was separated and washed twice with H ₂ O, dried over Na ₂ SO _4, filtered and evaporated under reduced pressure to produce the crude product (3R, 4R) -3 - ( (1 - ((R) - (1 -Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-((S)- 1-(Toluenesulfonyloxy)propan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (109 mg, 0.1 mmol, 71%). The crude mixture was used without further purification. LC/MS (uplc): 704.3 (-135). (Method A).

To (3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro- 2H-piperidin-4-yl)methyl)-3-((S)-1-(toluenesulfonyloxy)propan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1- To a solution of tert-butyl formate (109 mg, 0.1 mmol) in DMF (0.6 mL), sodium azide was added and the mixture was stirred at 70 °C. Upon completion of the reaction, the mixture was cooled to room temperature and diluted in EtOAc (5 mL). The reaction mixture was partitioned between H ₂ O and EtOAc. The organic layer was separated and washed twice with H ₂ O, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The desired product (3R,4R)-3-((3-((S)) was obtained after purification by column chromatography (gradient 30 to 100% EtOAC in heptane, 40 mg, 0.053 mmol, 52%). -1-azidopropan-2-yl)-1-((R)-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (four Hydrogen-2H-piperazin-4-yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester. ¹ H-NMR (DMSO, 600 MHz): δ 7.73 (1H, bs), 7.68 (1H, bs), 7.36-7.29 (6H, m), 7.09 (1H, bs), 6.29 (1H, bs), 5.34 ( 2H, m), 5.07 (1H, m), 3.97 (1H, m), 3.85 (1H, m), 3.70 (2H, m), 3.50 (1H, m), 3.37-3.20 (6H, m), 2.69 (1H, m), 2.58 (1H, m), 2.17 (1H, m), 1.96 (1H, m), 1.45 (1H, m), 1.30 (1H, m), 1.18 (9H, s), 1.12 ( 1H, m), 0.86 (2H, m). Three signals are hidden under the solvent peak. LC/MS (uplc): MH+ 711.4, 1.41 min. (Method A).

(3R,4R)-3-((3-((S)-1-Alanylpropan-2-yl)-1-((R)-(1-phenylmethyl-4-(2,5-di) Fluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4R)-3-((3-((S)-1-azidopropan-2-yl)-1-((R)-(1-phenylmethyl-4-(2,5) -difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tertidine was added triphenylphosphine (2mL) in a solution of the ester (100mg, 0.14mmol) in THF (111mg, 0.42mmol) and _{H 2 O (51μl, 2.81mmol)} . The reaction mixture was stirred at 50 °C. Upon completion of the reaction, the mixture was cooled to room temperature and diluted with EtOAc (5 mL). The reaction mixture was partitioned between EtOAc and H ₂ O. The organic layer was washed with H ₂ O, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. Isolated after column chromatography (gradient 0 to 10% MeOH in _{CH 2 Cl 2, 51mg, 0.071mmol} , 50%) the desired product (3R, 4R) -3 - ( (3 - ((S) -1-aminopropan-2-yl)-1-((R)-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (tetrahydro) -2H-piperidin-4-yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester. LC/MS (uplc): MH+ 685.4, 1.08 min. (Method A).

3-((S)-1-aminopropan-2-yl)-1-((R)-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazole-2 -yl)(tetrahydro-2H-piperidin-4-yl)methyl)-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)urea

To (3R,4R)-3-((3-((S)-1-aminopropan-2-yl)-1-((R)-(1-benzyl-4-(2,5-) Difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (40 mg, 0.058 mmol) in acetonitrile (1. Upon completion of the reaction, the crude product was filtered to remove the solid, and in reverse phase column chromatography (gradient 5% to 35% MeCN (+0.1% TFA) in H ₂ O (+0.1% TFA), 19.1 mg , 0.031 mmol, 53%), then the desired product 3-((S)-1-aminopropan-2-yl)-1-((R)-(1-phenylmethyl-4-(2, 5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-1-(((3S,4R)-4-fluoropyrrolidine-3 -yl)methyl)urea. The TFA salt was neutralized with a PL-HCO3 MP SPE column to give the free base. ^{1 H-NMR (DMSO, 600MHz} ): δ 7.75 (2H, bs), 7.40-7.28 (6H, m), 7.09 (1H, bs), 6.02 (1H, bs), 5.40-5.30 (3H, m), 4.95 (1H, m), 3.84 (1H, m), 3.74 (1H, m), 3.61 (2H, m), 3.34 (2H, m), 3.24 (2H, m), 2.95 (1H, m), 2.72 (1H, m), 2.66 (1H, m), 2.59 (1H, m), 2.55 (1H, m), 2.19 (1H, m), 1.82 (1H, m), 1.56 (1H, m), 1.42 ( 1H, m), 1.34 (1H, m), 1.119 (1H, m), 1.08 (3H, bs), 0.71 (1H, m). Two signals are hidden under the solvent peak. LC/MS (uplc): MH+ 585.3, 0.73 min. (Method A).

Synthesis Example 13.

(R)-2-(( Tertibutoxycarbonyl )amino)-3,3-dimethylpent-4-enoic acid Prepared as described in WO2005/54186 A2, 2005 ; P.48-49 (R)-2-((t-butoxycarbonyl)amino)-3,3-dimethylpent-4-enoate 2-(2,5-difluorophenyl)-2-oxooxy Ethyl ester

Under N ₂ : ice cooling to 2-chloro-1-(2,5-difluorophenyl)ethanone (1.974 g, 10.36 mmol) and K ₂ CO ₃ (1.074 g, 7.77 mmol) in 150 mL of acetone (R)-2-((Tertibutoxycarbonyl)amino)-3,3-dimethylpent-4-enoic acid (2.1 g, 8.63 mmol), KI (0.358 g, 2.158) were added successively to the solution. (mmol), the cooling bath was removed and the reaction mixture was stirred at RT for 3.5 h.

The reaction mixture was poured into crushed ice and extracted with DCM. Organics were dried over Na ₂ SO _4, filtered and adsorbed on Isolute. The desired product was obtained as a yellow solid after purified by column chromatography (jjjjjjjjjj ¹ H-NMR (DMSO, 400 MHz): δ 7.72-7.57 (2H, m), 7.55-7.45 (1H, m), 6.94 (1H, d, 8.9 Hz), 5.97 (1H, dd, 17.4, 10.7 Hz) , 5.40-5.25 (2H, m), 5.10-4.95 (2H, m), 4.11 (1H, d, 9.0 Hz), 1.39 (9H, s), 1.13 (6H, s). LC/MS (Method C): MH.

(R)-(1-(4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)carbamic acid Third butyl ester

(R)-2-((t-butoxycarbonyl)amino)-3,3-dimethylpent-4-enoate 2-(2,5-difluorophenyl)-2-oxooxy Ethyl ester (2.95 g, 7.42 mmol) was dissolved in 45 mL of toluene and ammonium acetate (11.44 g, 148 mmol) was added. The resulting mixture was heated to reflux for 40 hours.

The reaction mixture was cooled to RT, washed with water, saturated aqueous NaHCO ₃ and brine, dried over Na ₂ SO _4, filtered and concentrated in vacuo. The residue was dried at 40 ° C for 42 hours under reduced pressure. (2.69 g, 93%, 97% pure by LC-MS, UV) was taken as a yellow foam and used in the next step without further purification. LC/MS (Method A): MH.

(R)-(1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-ene-1 -based) tert-butyl carbamic acid

To (R)-(1-(4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1- at 0 °C K ₂ CO ₃ (1.911 g, 13.83 mmol), benzyl bromide (0.904 mL, 7.6 mmol) was added sequentially to a solution of butyl carbamic acid (2.69 g, 6.91 mmol) in 20 mL DMF. The resulting mixture was stirred at RT for 3 hours.

Ice water was added to produce a precipitate. The off-white solid was collected by filtration, washed with EtOAc EtOAc EtOAc (EtOAc) LC/MS (Method A): MH+ 4621.

(R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-ene-1- amine

Under N ₂ :(R)-(1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbutene- The 3-butyl-1-enyl-1-ylcarbamate (10.768 g, 23.03 mmol) was dissolved in 180 mL DCM and the reaction mixture was cooled to 0 ° C then TFA (44.4 mL, 576 mmol). The reaction mixture was stirred at 0 ° C for 5 minutes and then at RT for 1 hour. The residue was concentrated under reduced pressure, diluted with EtOAc EtOAc EtOAc. Extracted with DCM (3x), organics were dried over Na ₂ SO _4, filtered and concentrated. The desired product (8.7 g, 23.68 mmol, 100% yield, 97% pure) LC/MS (Method A): MH.

(3R,4R)-3-((((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2- Dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Under N ₂ : to (3R,4S)-3-fluoro-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (4.07 g, 18.55 mmol) in CH ₂ Cl ₂ (120 mL) Dess-Martin periodinane (13.11 g, 30.9 mmol) was added to the solution. The reaction mixture was stirred at room temperature for 30 minutes. Upon completion of the reaction, the crude product (3R,4S)-3-fluoro-4-carbamimidyrrolidine-1-carboxylic acid tert-butyl ester was used in the next step as a solution without further work.

(R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbutene-3 at 0 °C Add a solution of 1-en-1-amine (5.68 g, 15.46 mmol), sodium triethoxysulfonylborohydride (16.38 g, 77 mmol) and molecular sieves (20 g) in CH ₂ Cl ₂ (120 mL) from previous step A solution of (3R,4S)-3-fluoro-4-carbamimidyrrolidine-1-carboxylic acid tert-butyl ester in CH ₂ Cl ₂ . The reaction mixture was stirred at RT for 1 hour. The reaction mixture was diluted and washed with saturated NaHCO ₃ and brine, filtered with DCM. Organics were dried over Na ₂ SO _4, filtered and adsorbed on Isolute. The desired product was obtained after purification by column chromatography (330 g of EtOAc, EtOAc (EtOAc) LC/MS (Method B): MH+ 569.3, 6.60 min.

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H- Imidazolyl-2-yl)-2,2-dimethylbut-3-en-1-yl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Under N ₂ : containing (3R,4R)-3-((((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-) at 0 °C Imidazolyl-2-yl)-2,2-dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (3.03 g, 5.33 mmol) ) is the CH ₂ Cl ₂ (50mL) was added successively DIPEA (4.65mL, 26.6mmol), ( S) -2- prop-acetyl group acyl chloride (1.349mL, 10.66mmol). The resulting solution was stirred at 0 °C for 5 minutes, then warmed to RT and stirred for 2 hours. The reaction mixture was diluted with DCM and washed with saturated NaHCO _3, then washed with saturated NaCl. The organic layer was dried, concentrated and adsorbed onto an isolute. After purification by column chromatography (120 g of EtOAc, EtOAc (EtOAc) LC/MS (Method A): MH.

Synthesis Example 14. (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Under N ₂ :(3R,4R)-3-(((S)-2-ethoxycarbonyl-N-((R)-1-(1-benzyl-4-(2,5-di) Fluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid A solution of tributyl ester (1.5 g, 2.197 mmol) in THF (20 mL) was cooled to 0 <0>C and a solution of borane in THF (4.39 mL, 4.39 mmol, 1 M). The reaction mixture was allowed to warm to RT and stirred for 4 h. It was cooled to 0 ° C and quenched successively with 25 mL THF / EtOH 1:1, 40 mL of phosphate buffer (pH 7), and finally H ₂ O ₂ (2.244 mL, 21.97 mmol, 30% aqueous solution) was added and the reaction mixture was Stir overnight at RT. Brine was added to the reaction mixture and extracted 3 times with ETOAc. The organic layer was washed three times with cold saturated Na ₂ S ₂ O ₅ , water and brine, dried over Na ₂ SO ₄ , filtered and then applied to the isolute. After purification by column chromatography (80 g of EtOAc, EtOAc (EtOAc:EtOAc) LC/MS (Method A): MH. A mixture of rotamers was observed by ¹ H-NMR at room temperature. ¹ H-NMR (DMSO, 400 MHz): δ 7.85-7.65 (2H, m), 7.41-7.28 (6H, m), 7.15-7.05 (1H, m), 5.86 and 5.81 (1H, two single peaks, rotation Isomers, 5.40-5.17 (3H, m), 4.96 (1H, d, 15 Hz), 4.22-4.14 (1H, m), 3.95-3.70 (2H, m), 3.27-3.20 (1H, m), 3.15-2.97(1H,m),2.63-2.55(1H,m),2.22-2.14(1H,m),2.11(3H,s),1.70-1.49(5H,m),1.35-1.20(3H,m ), 1.09 (9H, s), 0.95 (6H, s).

(S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-hydroxy-2,2- Dimethylbutyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

After purification by reverse phase column chromatography, similar to the linker-payload combination The title compound (17 mg, 0.024 mmol, 85% yield, as TFA salt).

LC / MS (Method B): [M + H] + 559.3; Rt 3.25min. ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers, ratio about 3:1): δ 9.01 (1H, br s), 8.69 (1H, br s), 8.00 and 7.92 (1H, two isomers Two double peaks of 3.6 and 4.2 Hz), 7.45-7.30 (7H, m), 7.17-7.07 (1H, m), 5.87 and 5.85 (1H, two single peaks of two rotamers), 5.50 -5.00(3H,m), 4.65-4.58(1H,m), 4.30-3.90(2H,m),3.35-3.15(6H,m),2.42-2.32(1H,m),1.97-1.85(2H, m), 1.55-1.40 (1H, m), 1.40-1.20 (4H, m), 1.02 and 0.90 (3H, two single peaks of two rotamers), 0.82 and 0.68 (3H, two rotations) Two single peaks of the structure).

Synthesis Example 15.

Step 1: (R)-4-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3- Methyl)propanylamino)-4-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-3,3-dimethylbutyl acid

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-phenylmethyl-4-(2,5-di) at 0 °C Fluorophenyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (200 mg, 0.285 mmol) in a solution of acetone (volume: 7 ml), Jones reagent (2M, 0.856 mL, 1.712 mmol) was added dropwise. The yellow solution obtained was stirred at room temperature for 2.5 hours.

The excess Jones reagent was quenched with isopropanol (2 ml) at 0 ° C, then the mixture was concentrated, diluted with water and ethyl acetate (*3). The combined organic layer was dried with EtOAc EtOAcjjjjjjj

LC / MS (Method A): [M + H] + 715.3, Rt 1.27min.

Step 2: (R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-((( 3R,4R)-1-(Tertibutoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutyric acid

(R)-4-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl) Methyl) propylamino)-4-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-3,3-dimethylbutyric acid ( Step 1) (928 mg, 1.078 mmol) MeOH (1M, 3.23 mL, 3. The reaction mixture was diluted with water, evaporated, evaporated with EtOAc EtOAc The combined organic layers were dried <RTI ID=0.0></RTI> to <RTI ID=0.0></RTI> to <RTI ID=0.0>

LC / MS (Method A): [M + H] + 673.4, Rt 1.23min.

Step 3: (3R,4R)-3-(((S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazole- 2-yl)-2,2-dimethyl-4-(methylamino)-4-oxobutyl)-2-hydroxypropionylamino)methyl)-4-fluoropyrrolidine-1 -T-butyl formate

(R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-(((3R,4R) )-1-(Tertibutoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionamido)-3,3-dimethylbutyric acid (Step 2) ( 38 mg, 0.056 mmol) and 2M solution of methylamine in THF (0.113 mL, 0.226 mmol) were dissolved in DMF (volume: 2 ml), and DIPEA (0.049mL, 0.282mmol), HATU (32.2mg, 0.085mmol) ). The reaction mixture was stirred at room temperature for 1 hour. Dilute with EA and wash with brine (*3). The combined organic layers were dried <RTI ID=0.0></RTI> to <RTI ID=0.0></RTI> to <RTI ID=0.0></RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt;

LC / MS (Method A): [M + H] + 686.3, Rt 1.22min.

Step 4: (R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-((( 3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanylamino)-N,3,3-trimethylbutyramine

(3R,4R)-3-(((S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) -2,2-Dimethyl-4-(methylamino)-4-oxobutyl)-2-hydroxypropionamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid Tributyl ester (Step 3) (37 mg, 0.026 mmol, 49% pure) was dissolved in EtOAc (EtOAc:EtOAc) Concentration and purification by reverse phase chromatography gave the desired product (6.5 mg, 9.29.

LC / MS (Method B): [M + H] + 586.7, Rt 3.03min. ¹ H-NMR (DMSO, 600 MHz, a mixture of about 5:1 rotamers, some peaks of minor rotamers could not be removed): δ 8.99 (1.2H, br s), 8.71 (1.2 H, br s), 7.91-7.86 (0.2H, m), 7.84-7.81 (1H, m), 7.80-7.75 (1H, m), 7.69-7.65 (1H, m), 7.57 (0.2H, d, 3.6 Hz), 7.45-7.29 (6.5H, m), 7.15-7.10 (1.4H, m), 6.22 (1H, s), 5.78 (0.2H, s), 5.59 (0.2H, d, 16.2Hz), 5.48 (0.2H, d, 16.2 Hz), 5.34 (1H, d, 15.1 Hz), 5.17 (1H, d, 15.1 Hz), 5.10 (1H, d, 52.2 Hz), 4.83-4.77 (0.2H, m) , 4.57-4.51 (1H, m), 4.49-4.42 (0.2H, m), 4.06-4.01 (0.4H, m), 3.95-3.92 (2H, m), 3.35-3.24 (1.2H, m), 3.14 -3.00 (1.2H, m), 2.56 (0.6H, d, 4.2 Hz), 2.31-2.23 (1H, m), 2.19 (1H, d, 13.5 Hz), 2.10-2.00 (0.4H, m), 2.00 -1.92 (1H, m), 1.90 (1H, d, 13.5 Hz), 1.72-1.58 (1H, m), 1.35 (3H, d, 6.0 Hz), 1.15 (0.6H, s), 1.08 (0.6H, s), 1.05 (3H, s), 0.95 (3H, s), 0.77 (0.6H, d, 6.0 Hz). One CH ₃ group was hidden under the DMSO peak and OH was not seen.

Synthesis Example 16.

Step 1: (3R,4R)-3-(((2S)-2-Ethyloxy-N-((1R)-1-(1-benzyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-3,4-dihydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tertidine ester

Add (3R,4R)- to a stirred solution of 4-methylmorpholine-N-oxide (73.6 mg, 0.628 mmol) and osmium tetroxide 4% (0.082 mL, 10.47 μmol) in water (6 mL) 3-((())(2-ethyloxy)-N-((R)-1-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2- Benzyl-2,2-dimethylbut-3-en-1-yl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (143 mg, 0.209 mmol) in THF (4ml). The mixture was stirred at room temperature for 18 hours. The reaction mixture was washed with saturated Na ₂ S ₂ O ₅ solution was quenched and allowed to stir at room temperature for 1 hour and then extracted with DCM (3 times). The organic layer was dried over Na ₂ SO _4, filtered and concentrated to the desired product (157mg, 0.169mmol, 81% yield, 77% pure) 157 mg of generation, which was used without any further purification in the next step.

</RTI>^<RTIID=0.0></RTI>^</RTI>^< RTI ID=0.0>^</RTI> (2 diastereomers).

Step 2: (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-2,2-dimethyl-3-oxopropyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

(3R,4R)-3-(((2S)-2-Ethyloxy-N-((1R)-1-(1-phenylmethyl-4-(2,5-difluorophenyl))- 1H-imidazol-2-yl)-3,4-dihydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (step 1) (120 mg, 0.129 mmol) and K ₂ CO ₃ (35.6 mg, 0.258 mmol) were dissolved in THF (6 mL). Water (4 ml) containing NaIO4 (83 mg, 0.387 mmol) was added. The mixture was stirred at room temperature for 3 hours. The white precipitate was removed by filtration, then the filtrate was extracted three times with EA. Dried organic layer was successively washed with saturated Na ₂ S ₂ O _5, followed with brine Na ₂ SO _4, filtered and concentrated. Without further purification, 105 mg of the desired product (105 mg, 0.090 mmol, 70% yield, 59% pure) was obtained and used for the next step.

LC / MS (Method A): [M + H] + 685.4; Rt 1.42min.

Step 3: (R)-3-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3- Methyl)propanylamino)-3-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropane acid

Under N ₂ : (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-phenylmethyl-4-)) at 0 °C 2,5-Difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethyl-3-oxopropyl)propanylamino)methyl)-4-fluoropyrrolidine- Jones reagent 2M (0.372 mL, 0.745 mmol) was added dropwise to a solution of 1-carboxylic acid tert-butyl ester (Step 2) (102 mg, 0.149 mmol) in EtOAc (10 mL). The yellow solution obtained was stirred at room temperature for 3 hours. The excess Jones reagent was quenched with isopropanol (6 ml) at 0 ° C, then the reaction mixture was concentrated, diluted with water and ethyl acetate (*3). The combined organic layers were dried over Na ₂ SO _4, filtered and concentrated.

The desired product (81.5 mg, 0.078 mmol, 52% yield, 67% pure) was obtained and used in the next step without further purification.

LC / MS (Method A): [M + H] + 701.3; Rt 1.29min.

Step 4: (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-2,2-dimethyl-3-(methylamino)-3-oxopropyl)protonylamino)-4-fluoropyrrole Pyridin-1-carboxylic acid tert-butyl ester

(R)-3-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl) Methyl) propylamino)-3-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropanoic acid ( Step 3) (46 mg, 0.066 mmol) and MeOH (EtOAc m. DIPEA (0.057 mL, 0.328 mmol), HATU (37.4 mg, 0.098 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 24 hours. The mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried over Na ₂ SO _4, filtered and concentrated to yield 46.9 mg of the desired product (46.9mg, 0.066mmol, 100% yield), which was used without any further purification in the next step.

LC / MS (Method A): [M + H] + 714.3; Rt 1.29min.

Step 5: (S)-acetic acid 1-(((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2 -Dimethyl-3-(methylamino)-3-oxopropyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)- Amino)-1-oxopropan-2-yl ester

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-2,2-dimethyl-3-(methylamino)-3-oxopropyl)propanylamino)methyl)-4-fluoropyrrolidine-1 -T-butyl formate (step 4) (46.9 mg, 0.066 mmol) was dissolved in acetonitrile (2 ml) and water (1 ml). TFA (0.506 mL, 6.57 mmol) was added and the mixture was stirred at <RTIgt; The solution was diluted in EA, washed with saturated aqueous NaHCO _3, followed by brine. The organic layer was dried over Na ₂ SO _4, filtered and concentrated. Without further purification, 40.3 mg of the desired product (40.3 mg, 0.066 mmol, 100% yield) was obtained and used in the next step.

LC / MS (Method A): [M + H] + 614.3; Rt 0.89min.

Step 6: (R)-3-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-3-((S)-N-((( 3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-N,2,2-trimethylbutanamine

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-2,2-dimethyl-3-(methylamino)-3-oxopropyl)propanylamino)methyl)-4-fluoropyrrolidine-1 -T-butyl formate (step 5) (40.3 mg, 0.066 mmol) was dissolved in MeOH (3 mL). K ₂ CO ₃ (45.4 mg, 0.328 mmol) was added. The reaction mixture was stirred at room temperature for 40 minutes.

The mixture was filtered and submitted for purification by reverse phase chromatography. 8.9 mg of the expected product as a TFA salt (8.9 mg, 0.012 mmol, 19% yield, 94% pure).

</RTI>^< RTI ID=0.0></RTI>^</RTI> ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks of major rotamers): δ 9.04 (1H, br s), 8.85 (1H, br s), 7.76-7.70 (3H, m ), 7.45-7.31(6H,m), 7.13-7.07(1H,m),6.42(1H,s),5.25-5.20(2H,m),5.14-5.09(1H,m),4.57-4.51(1H , m), 3.92-3.85 (2H, m), 3.35-3.25 (1H, m), 3.20-3.05 (1H, m), 2.48 (3H, d, 4.5 Hz), 2.33-2.25 (1H, m), 1.98-1.88 (1H, m), 1.82-1.68 (1H, m), 1.36-1.32 (6H, m), 1.06 (3H, s).

Synthesis Example 17.

Step 1: (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-3-hydroxy-2,2-dimethylpropyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

At 0 ℃ to (3R, 4R) -3 under _{N 2 - (((S)} -2- acetyl group -N - ((R) -1- ( 1- benzyl-4- (2 ,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethyl-3-oxopropyl)propanylamino)methyl)-4-fluoropyrrolidine-1 - third carboxylate (232.6mg, 0.340mmol) (12ml) was added in the NaBH ₄ (19.28mg, 0.510mmol) in 2mL MeOH and THF, to give a clear solution. The reaction mixture was stirred at room temperature for 1 hour. The mixture was saturated NH at 0 ℃ ₄ Cl aqueous quenched, extracted with EA, dried over Na ₂ SO _4, filtered and concentrated.

Without further purification, 233 mg of the desired product (233 mg, 0.340 mmol, 100% yield, 100% pure) was obtained and used for the next step.

LC/MS (Method A): [M+H] ⁺ 687.3;

Step 2: (S)-Acetic acid 1-(((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-3-hydroxyl -2,2-dimethylpropyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-l-oxypropan-2-yl ester

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-3-hydroxy-2,2-dimethylpropyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (step 1) ( 26 mg, 0.038 mmol) was dissolved in acetonitrile (1 mL) and water (0.5 mL). Add TFA (0.292 mL, 3.79 mmol) and stir at 60 ° C for 1 small Time. The reaction mixture was diluted with EtOAc and washed with sat. NaHCOs. The organic layer was dried over Na2SO4, filtered and concentrated.

Without further purification, 22.2 mg of the desired product (22.2 mg, 0.038 mmol, 100% yield) was obtained and used in the next step.

LC/MS (Method A): [M+H] ⁺ 587.3; Rt 0.93 min

Step 3: (S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-3-hydroxy-2 ,2-dimethylpropyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

To (S)-acetic acid 1-(((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-3-hydroxy-2 ,2-dimethylpropyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-yloxypropan-2-yl ester (Step 2) ( 22.2mg, 0.038mmol) was stirred in methanol (2mL) was added in the _{K 2 CO 3 (26.2mg, 0.189mmol} ). The reaction mixture was stirred at room temperature for 1.5 hours. The mixture was filtered and submitted for purification by reverse phase chromatography. 8.8 mg of the expected product as a TFA salt (8.8 mg, 0.013 mmol, 35% yield, 100% pure).

LC / MS (Method B): [M + H] + 545.3; Rt 3.24min. ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks of major rotamers): δ 9.09 (1H, br s), 8.87 (1H, br s), 7.80-7.87 (1H, m ), 7.70-7.76 (1H, m), 7.30-7.44 (6H, m), 7.1-7.15 (1H, m), 6.03 (1H, s), 5.35-5.08 (3H, m), 4.60-4.55 (1H , m), 4.05-3.85 (2H, m), 3.35-3.20 (2H, m), 3.12-3.04 (2H, m), 2.41-2.31 (1H, m), 2.01-1.79 (2H, m), 1.35 (3H, d, 6.2 Hz), 0.90 (3H, s), 0.80 (3H, s).

Synthesis Example 18.

Step 1: (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-2,2-dimethyl-4-oxobutyl propyl propylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Under N ₂ : to (3R,4R)-3-(((S)-2-ethoxycarbonyl-N-((R)-1-(1-benzyl-4-(2,5-) Difluorophenyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl Dess-Martin periodinane (161 mg, 0.380 mmol) was added to a solution of EtOAc (EtOAc). The reaction mixture was stirred at room temperature for 35 minutes. The solution was diluted with EtOAc (EtOAc)EtOAc.

Without further purification, 199 mg of the desired product (199 mg, 0.285 mmol, 100% yield) was obtained and used in the next step.

LC/MS (Method A): [M+H] ⁺ 699.5; Rt 1.39min

Step 2: (3R,4R)-3-(((2S)-2-Ethyloxy-N-((1R,E)-1-(1-benzylmethyl-4-(2,5-di) Fluorophenyl)-1H-imidazol-2-yl)-4-((t-butylsulfinyl)imino)-2,2-dimethylbutyl)propanylamino)methyl) -4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-phenylmethyl-4-(2,5-II) at 50 °C Fluorophenyl)-1H-imidazol-2-yl)-2,2-dimethyl-4-oxobutyl propyl propylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid Butyl ester (250 mg, 0.358 mmol), 2-methylpropane-2-sulfinamide (Step 1) (217 mg, 1.789 mmol) and CuSO4.5H2O (447 mg, 1.789 mmol) were stirred in DCM (10 mL) . The reaction mixture was filtered and the precipitate was washed with additional DCM. The filtrate (= product) was concentrated in vacuo.

Without further purification, 496 mg (yield: </RTI> </RTI> <RTIgt;

LC / MS (Method A): [M + H] + 802.5; Rt 1.48min.

Step 3: (3R,4R)-3-(((2S)-2-Ethyloxy-N-((1R)-1-(1-benzyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-4-(1,1-dimethylethylsulfinamido)-2,2-dimethylbutyl)propanylamino)methyl)- 4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

(3R,4R)-3-(((2S)-2-Ethyloxy-N-((1R,E)-1-(1-benzylmethyl-4-(2,5-difluorophenyl) -1H-imidazol-2-yl)-4-((t-butylsulfinyl)imino)-2,2-dimethylbutyl)propanylamino)methyl)-4- Fluoropyrrolidine-1-carboxylic acid tert-butyl ester (Step 2) (496 mg, 0.353 mmol) was dissolved in MeOH (15 mL). NaBH ₄ (66.7 mg, 1.763 mmol) was added slowly. The reaction mixture was discolored (brown) and foamed. It was stirred at room temperature for 4 hours. The residue was diluted in EA, washed with water and then brine. The organic layer was dried over Na2SO4, filtered and concentrated.

Without further purification, 379 mg of the desired product (379 mg, 0.420 <RTIgt;

LC / MS (Method A): [M + H] + 804.7; Rt 1.42min.

Step 4: (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-4-amino-1-(1-phenylmethyl-4-(2, 3-Difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

(3R,4R)-3-(((2S)-2-Ethyloxy-N-((1R)-1-(1-phenylmethyl-4-)) at 0 ° C under N ₂ 2,5-Difluorophenyl)-1H-imidazol-2-yl)-4-(1,1-dimethylethylsulfinamido)- 2,2-dimethylbutyl)propanoid Amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (Step 3) (106 mg, 0.117 mmol) was dissolved in MeOH (3 mL). Dioxane (0.059 mL, 0.235 mmol) containing 4M HCl was added. The reaction mixture was stirred at 0&lt;0&gt;C for 1 h 20 min then quenched with sat. NaHCO3. The solution was diluted in EA, washed with water and then with brine. The organic layer was dried over Na2SO4, filtered and concentrated.

Without further purification, 82 mg of the desired product (82 mg, 0.076 mmol, 65% yield, 65% pure) was obtained and used for the next step.

LC / MS (Method A): [M + H] + 700.5; Rt 1.04min.

Step 5: (3R,4R)-3-(((S)-N-((R)-4-Ethylamino-1-(1-benzyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-2,2-dimethylbutyl)-2-ethenyloxypropylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-4-amino-1-(1-phenylmethyl-4-(2,5-di) Fluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (step 4) (44 mg, 0.041 mmol) and DIPEA (0.021 mL, 0.123 mmol) Ethyl chloride (4.35 μl, 0.061 mmol) was added. The mixture was stirred at room temperature for 1 hour and 20 minutes. The reaction mixture was diluted with EtOAc EtOAc m.

Without further purification, 30.3 mg of the desired product (30.3 mg, 0.041 mmol, 100% yield) was obtained and used in the next step.

LC / MS (Method A): [M + H] + 742.5; Rt 1.25min.

Step 6: (S)-acetic acid 1-(((R)-4-acetamido-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazole-2) -yl)-2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-yloxypropan-2-yl ester

(3R,4R)-3-(((S)-N-((R)-4-Ethylamino-1-(1-phenylmethyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-2,2-dimethylbutyl)-2-ethenyloxypropylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (step 5) (30.3 mg, 0.041 mmol) was dissolved in acetonitrile (2 ml) and water (1 ml). TFA (0.157 mL, 2.042 mmol) was added and the mixture was stirred at <RTIgt;

The solution was diluted in EA and washed with saturated NaHCO3 then brine. The organic layer was dried over Na2SO4, filtered and concentrated. Without further purification, 30.3 mg of the desired product (26.2 mg, 0.041 mmol, 100% yield) was obtained and used in the next step.

LC / MS (Method A): [M + H] + 642.4; Rt 0.84min.

Step 7: (S)-N-((R)-4-Ethylamino-1-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl -2,2-dimethylbutyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionamide

To (S)-acetic acid 1-(((R)-4-acetamido-1-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) -2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-yloxypropan-2-yl ester (step 6) (26.2mg, 0.041 mmol middle of) in methanol (2mL), was added _{K 2 CO 3 (28.2mg, 0.204mmol} ), and the reaction mixture was stirred at room temperature for 1 hour.

The solution was filtered and submitted for purification by reverse phase chromatography. 7.7 mg of the expected product as a TFA salt (7.7 mg, 0.011 mmol, 26% yield, 100% pure).

LC / MS (Method B): [M + H] + 600.4; Rt 3.09min. ¹ H-NMR (DMSO, 600 MHz): δ 9.06 (1H, br s), 8.78 (1H, br s), 7.93 (1H, d, 3.7 Hz), 7.80-7.74 (1H, m), 7.60-7.55 ( 1H, m), 7.44-7.30 (6H, m), 7.17-7.09 (1H, m), 5.84 (1H, s), 5.40-5.06 (3H, m), 4.62-4.56 (1H, m), 4.05- 3.90(2H,m), 3.35-3.21(2H,m), 2.90-2.80(2H,m), 2.45-2.35(1H,m),2.00-1.85(2H,m),1.73(3H,s), 1.38-1.41(1H,m), 1.35(3H,d,6.2Hz), 1.18-1.11(2H,m),0.93(3H,s),0.80(3H,s)

Synthesis Example 19.

Step 1: (R)-2-(((S)-2-Hydroxy-1-phenylethyl)amino)-2-(4-methyltetrahydro-2H-pyran-4-yl)acetonitrile

A solution of 4-methyltetrahydro-2H-piperidin-4-carbaldehyde (4.8 g, 37.5 mmol) in DCM (m. (S)-(+)-Phenylglycol (5.65 g, 41.2 mmol) was added and the reaction mixture was stirred at 0 ° C for 1 hour. Trimethyldecane cyanide (7.49 mL, 56.2 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was quenched with EtOAc (EtOAc)EtOAc. Organics were washed with NaOH (2M) and washed again, dried over Na ₂ SO _4, filtered and concentrated. Redissolved in THF (20 mL) and acidified with 10 mL EtOAc (EtOAc). It was then basified with NaOH (2M) and extracted with EA. Organics were dried over Na ₂ SO _4, filtered, concentrated and adsorbed on Isolute. The residue was purified by flash chromatography eluting EtOAc EtOAc (EtOAc) % yield) and 1.69 g (15% yield) of the desired diastereomer.

Analytical data of the desired diastereomer: LC/MS (Method A): [M+H] ⁺ 275.4, Rt 0.88 min. ^{1 H-NMR (DMSO, 400MHz} ): δ 7.40-7.25 (5H, m), 5.18 (1H, t, J = 5.7Hz), 3.90-3.82 (1H, m), 3.60-3.33 (6H, m), 3.09 (1H, d, J = 12.9 Hz), 2.63 (1H, d, J = 12.9), 1.68-1.36 (3H, m), 1.31-1.22 (1H, m), 1.10 (3H, s).

Analytical data of the second diastereomer: LC/MS (Method A): [M+H] ⁺ 275.4, Rt 0.82 min. ¹ H-NMR (DMSO, 400 MHz): δ 7.42-7.21 (5H, m), 4.79 (1H, t, J = 5.4 Hz), 3.81-3.62 (4H, m), 3.55-3.42 (4H, m), 2.69 (1H, dd, J = 9.5,4.7Hz), 1.71-1.55 (2H, m), 1.48-1.36 (1H, m), 1.34-1.22 (1H, m), 1.09 (3H, s).

(R)-2-((t-butoxycarbonyl)amino)-2-(4-methyltetrahydro-2H-pyran-4-yl)acetic acid

Step 2: (R)-2-(((S)-2-Hydroxy-1-phenylethyl)amino)-2-(4-methyltetrahydro-2H-pyran-4-yl)acetonitrile (Step 1) (4.17 g, 15.20 mmol) EtOAc (EtOAc m. Concentrate and co-evaporate with toluene (twice). It was used in the next step without any purification.

Step 3: The residue was dissolved in MeOH (vol.: 70 mL) and the mixture was purified with &lt _; RTI ID=0.0&gt;&gt;&gt; The reaction mixture was purified with hydrogen and then stirred at room temperature under hydrogen for 3 days until the starting material was consumed. Filtration through celite with MeOH and concentrated.

Step 4: The residue was suspended in DCM: (volume 40mL), are successively added diisopropylamine _{(6.50mL, 30.2mmol), Boc 2} O (3.31g, 15.2mmol) and the reaction mixture was stirred at RT 16 h. Concentrated, diluted with NaOH (0.2 M) and washed with DCM. The aqueous layer was acidified to pH 1 with HCl (4M) and product was extracted with EA. The organics were dried over Na ₂ SO _4, filtered and concentrated to give the desired product (1.89g, 6.57mmol, 44% yield). ¹ H-NMR (DMSO, 400 MHz): δ 12.59 (1H, s), 6.92 (1H, d, J = 9.2 Hz), 3.98 (1H, d, J = 9.2 Hz), 3.69-3.55 (2H, m) , 3.54-3.42 (2H, m), 1.70-1.45 (2H, m), 1.39 (9H, s), 1.36-1.21 (2H, m), 0.99 (3H, s).

(R)-2-((t-butoxycarbonyl)amino)-2-(4-methyltetrahydro-2H-pyran-4-yl)acetic acid 2-(2,5-difluorophenyl 2-oxoethyl ester

By using a method similar to that described in Scheme 1, by using (R)-2-((t-butoxycarbonyl)amino)-2-(4-methyltetrahydro-2H-pyran- 4-(4-)acetic acid was replaced by (R)-2-((t-butoxycarbonyl)amino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid to afford the title compound g, 16.14 mmol, 88% yield).

LC / MS (Method A): [M + H] + 428.2, Rt 1.16min.

(R)-((4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-methyltetrahydro-2H-piperidin-4-yl)methyl)aminocarboxylic acid Third butyl ester

By using a method similar to that described in Scheme 1, by using (R)-2-((t-butoxycarbonyl)amino)-2-(4-methyltetrahydro-2H-pyran- 4-(2,5-difluorophenyl)-2-oxoethyl acetate to replace (R)-2-((t-butoxycarbonyl)amino)-2-(tetrahydro) 2-(2,5-Difluorophenyl)-2-oxoethylacetate to give the title compound: mpjjjjjjjjj .

LC / MS (Method A): [M + H] + 408.2, Rt 1.06min.

(R)-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-methyltetrahydro-2H-pyran-4-yl) Methyl) butyl methacrylate

By using a method similar to that described in Scheme 1, by using (R)-((4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-methyltetrahydro) Replacement of (R)-((4-(2,5-difluorophenyl)-1H-imidazol-2-yl) with tetrabutyl 2H-piperazol-4-yl)methyl)carbamate The title compound was obtained as a pale yellow solid (6.58 g, 10.58 mmol, 78% yield). LC / MS (Method A): [M + H] + 498.3, Rt 1.44min.

(R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-methyltetrahydro-2H-pyran-4-yl)- amine

By using a method similar to that described in Scheme 1, by using (R)-((1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) ( Replacement of (R)-((1-benzyl-4-(2,5-difluorophenyl)-tert-butyl 4-methyltetrahydro-2H-piperidin-4-yl)methyl)carbamate -1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)carbamic acid tert-butyl ester to give the title compound as a pale yellow solid (4.23 g, 10.58 mmol, 99% Yield, as TFA salt).

LC / MS (Method A): [M + H] + 398.2, Rt 0.90min.

(3R,4R)-3-((((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-methyltetrahydro) -2H-piperidin-4-yl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester

By using a method similar to that described in Scheme 1, by using (R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (4) -methyltetrahydro-2H-piperidin-4-yl)methylamine replacement (R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methylamine to prepare the title Compound; colorless oil (360 mg, 0.55 mmol, 44% yield).

LC / MS (Method B): [M + H] + 633.3, Rt 6.05min.

(3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-methyl) Tetrahydro-2H-piperidin-4-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzoate ester

The title compound is prepared by a method analogous to that described in the general procedure for the synthesis of the urea compound.

Colorless oil (207 mg, 0.24 mmol, 42% yield, 85% pure).

LC / MS (Method A): [M + H] + 734.3, Rt 1.32min.

1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-methyltetrahydro-2H-pyran-4- Methyl)-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)urea

The title compound was prepared by a method analogous to the method described in the general procedure for Cbz.

The TFA salt was converted to the free base by passing through a PL-HCO ₃ cartridge. Colorless solid (8 mg, 0.012 mmol, 14% yield).

LC / MS (Method B): [M + H] + 600.2, Rt 3.71min. ¹ H-NMR (DMSO, 600 MHz) (mixture of rotamers): δ 7.87-7.80 (1H, m), 7.77-7.68 (1H, m), 7.43-7.26 (6H, m), 7.13-7.06 ( 1H,m),6.13-6.05(1H,m),5.67-5.62(1H,m),5.37-5.22(2H,m),5.94-4.91(1H,m),4.66(1H,br s),3.95 -3.75 (2H, m), 3.70-3.55 (2H, m), 3.46-3.38 (2H, m), 3.27-3.17 (2H, m), 2.97-2.81 (1H, m), 2.68-2.54 (1H, m), 2.08-2.02 (1H, m), 1.89-1.74 (1H, m), 1.56-1.44 (2H, m), 1.35-1.28 (2H, m), 1.17-0.98 (7H, m). Some signals are hidden under the solvent peak.

Synthesis Example 20.

Step 1: (S)-4-(((9H-茀-9-yl)methoxy)carbonyl)morpholine-2-carboxylic acid

(S)-morpholine-2-carboxylic acid hydrochloride [CAS 154731-81-4] (100 mg, 0.597 mmol) and sodium hydrogencarbonate (251 mg, 2.98 mmol) were dissolved in H ₂ O (3 ml) and added A solution of Fmoc-OSu [Aldrich, 82911-69-1] (302 mg, 0.895 mmol) in dioxane (4 mL). The RM was stirred at RT for 3 hours. Partitioned between EtOAc and NaHCO ₃ (saturated). The aqueous layer was washed with EA and then acidified to pH 3 (1M with HCl) and extracted. The organics were dried over Na ₂ SO _4, filtered and concentrated. Obtained 210 mg of the desired product as a colourless oil.

Colorless oil (210 mg, 0.582 mmol, 98% yield, 98% pure).

LC / MS (Method A): [M + H] + 354.1, [M + NH 4] + 371.1, Rt 0.93min.

Step 2: 2-(((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)aminecarboxamido)morpholine-4-carboxylic acid (S) )-(9H-茀-9-yl)methyl ester

(S)-4-(((9H-茀-9-yl)methoxy)carbonyl)morpholine-2-carboxylic acid (Step 1) (210 mg, 0.582 mmol) was dissolved in DCM (10 mL) DMF (7.95 μl, 0.103 mmol), hexanedichloride (0.081 mL, 0.924 mmol). The reaction mixture was stirred at room temperature for 1 hour, concentrated and evaporated twice with toluene. The residue was dissolved in 10 mL DCM and cooled to 0 &lt;RTI ID=0.0&gt;&gt;&gt;&gt; Finally, (3R,4R)-3-((((R)-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) (tetrahydrogen) was added dropwise. A solution of -2H-piperidin-4-yl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (120 mg, 0.205 mmol) in 5 mL DCM. The RM was stirred at 0 ° C for 15 minutes and then at room temperature for 2 hours. Diluted with NaHCO ₃ (sat.) And extracted with DCM, dried over Na ₂ SO _4, filtered and adsorbed on Isolute. The residue was purified by flash chromatography eluting elut elut elut

Colorless oil (135 mg, 0.142 mmol, 69% yield, 97% pure).

LC / MS (Method A): [M + H] + 920.4 Rt 1.53min.

Step 3: (S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro- 2H-piperidin-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The Fmoc group is removed by reaction with piperidine in DMF.

The title compound (23 mg, 0.028 mmol, mpqqqqqqqqqqq 65% yield, in the form of a double TFA salt).

LC / MS (Method B): [M + H] + 598.2, Rt 2.23min. ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers, 120 ° C): δ 7.83-7.77 (1H, m), 7.68 (1H, br s), 7.45-7.35 (3H, m), 7.30-7.22 (3H, m), 7.12-7.05 (1H, m), 5.42-5.13 (4H, m), 4.71 (1H, br s), 4.05-3.92 (2H, m), 3.90-3.80 (2H, m), 3.73-3.64(2H,m), 3.50-3.15(9H,m), 2.72-2.61(2H,m), 2.30-2.10(1H,m),1.50-1.20(3H,m),0.97-0.80(1H , m).

Step 1: 2-(((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H- Piperazin-4-yl)methyl)(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)amine-carboxyl)morpholine- 4-carboxylic acid (R)-(9H-fluoren-9-yl)methyl ester

The title compound was prepared by a method analogous to that described for the (S)- diastereomer. Purification by flash chromatography (40 g, EtOAc) elute

Colorless oil (135 mg, 0.113 mmol, 34% yield).

LC / MS (Method A): [M + H] + 920.3 Rt 1.54min.

(S)-Diastereomer was also obtained as a colorless oil (190 mg, 0.207 mmol, 62% yield).

LC / MS (Method A): [M + H] + 920.3 Rt 1.56min.

Step 2: (R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran 4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The Fmoc group is removed by reaction with piperidine in DMF.

The title compound (15 mg, 0.018 mmol, was obtained by the method of the method of the </ RTI></RTI></RTI><RTIgt; 98% yield, double TFA salt). LC / MS (Method B): [M + H] + 598.2, Rt 2.55min.

Synthesis Example 21. 4-Hydroxy-THP-core skeleton synthesis

4-((2R,5S)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)tetrahydro-2H-pentan-4-ol.

Pre-cooled (-78 ° C) to (S)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (5.4 mL, 30 mmol, 1 eq) in THF (60 mL) n BuLi (1.6 M in hexanes, 31.5 mmol, 1.1 eq) was added to the solution. The reaction mixture was stirred at -78 °C for 1 hour. After 1 h, a solution of dihydro-2H-piperidin-4(3H)-one (3 g, 30 mmol, 1 eq) in THF (40 mL). The reaction mixture was stirred at -20 °C. The reaction mixture was quenched with EtOAc (EtOAc EtOAc (EtOAc) The reaction mixture was partitioned between Et ₂ O and H ₂ O. The organic layer was separated, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by reverse phase column chromatography (PrepLC method B) to give the desired product (7.3 g, 25.7 mmol, 86%). LC/MS (uplc): MH+ 285.2, 0.96 min (Method A).

Methyl (R)-2-amino-(4-hydroxytetrahydro-2H-piperazin-4-yl)acetate.

To 4-((2R,5S)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)tetrahydro-2H-pentan-4-ol ( 4.3 g, 15.1 mmol, 1 eq) HCl (0.2 Nm 151 mL, 30.2 mmol, 2 eq). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was quenched with NaOH (1.0M) to pH = 8 and used in the next step as a solution. LC/MS (uplc): MH+ 190.1, 0.17 min (Method D, polar method).

Methyl (R)-2-(((benzyloxy)carbonyl)amino)-2-(4-hydroxytetrahydro-2H-pyran-4-yl)acetate.

To the solution of the previous step (6.62g, 14mmol, 1eq) was added _{NaHCO 3 (4.1g, 49mmol, 3.5eq} ) and from Cbz-Cl (5mL, 35mmol, 2.5eq). The reaction mixture was stirred at room temperature. When by LC / MS (uplc, Method A) reaction was complete, the reaction between (100 mL) and H ₂ O (100mL) the mixture was partitioned between EtOAc. The organic layer was separated, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by normal phase column chromatography (PrepLC method A) to give the desired product (5.4 g, 16.7 mmol, 60%). LC/MS (uplc): MH+ 324.2, 0.78 min.

(R)-2-(((benzyloxy)carbonyl)amino)-2-(4-((t-butyldimethylmethyl)alkyl)oxy)tetrahydro-2H-pyran-4- Methyl acetate.

Methyl (R)-2-(((benzyloxy)carbonyl)amino)-2-(4-hydroxytetrahydro-2H-pyran-4-yl)acetate (2.3 g, 6.4 mmol, 1 eq 2,6-Dimethylpyridine (5 mL, 42.9 mmol, 6.7 eq) and TBSOTf (5 mL, 21.8, 3.4 eq) were added to the ice-cooled solution in CH ₂ Cl ₂ (5 mL). The reaction mixture was stirred at room temperature. When by LC / MS (uplc, Method A) reaction was complete, the reaction mixture was partitioned between H ₂ O (50mL) and CH ₂ Cl ₂ (50mL). The organic layer was separated, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by EtOAc (EtOAc) (EtOAc). LC/MS (uplc): MH+ 438.2, 1.42 min (Method A).

(R)-2-(((benzyloxy)carbonyl)amino)-2-(4-((t-butyldimethylmethyl)alkyl)oxy)tetrahydro-2H-pyran-4- Base) acetic acid.

To (R)-2-(((benzyloxy)carbonyl)amino)-2-(4-((t-butyldimethylmethylalkyl)oxy)tetrahydro-2H-pyran-4 To a solution of methyl acetate (2.1 g, 4.9 mmol, 1 eq) in THF EtOAc (1. The reaction mixture was stirred at room temperature. Upon completion of the reaction (uplc, Method A), the reaction mixture was quenched with EtOAc (1.0M). The reaction was partitioned between _{2 O (60mL) EtOAc (60mL} ) and H. The organic layer was separated, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure, to form the desired product (2.2g, 4.8mmol, 99%) . The crude product was used without any further purification. LC/MS (uplc): MH+ 424.3, 1.25 min (Method A).

2-(((Benzyloxy)carbonyl)amino)-2-(4-((t-butyldimethylmethyl)alkyl)tetrahydro-2H-pyran-4-yl)acetic acid ( R)-2-(2,5-Difluorophenyl)-2-oxoethyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 578.3, 1.49 min (Method A).

(S)-((4-((t-butyldimethyl)alkyl)oxy)tetrahydro-2H-pyran-4-yl)(4-(2,5-difluorophenyl)-1H -imidazol-2-yl)methyl)carbamic acid benzyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 558.3, 1.48 min (Method A).

(S)-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-((t-butyldimethyl)alkyl)oxy Benzyl tetrahydro-2H-piperazin-4-yl)methyl)carbamate.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 648.6.3, 1.64 min (Method A).

(S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-((t-butyldimethylmethylalkyl)oxy) Tetrahydro-2H-piperidin-4-yl)methylamine.

To prepare this compound, the procedure described for the general Cbz protecting group removal is followed. The crude product was used without any further purification. LC/MS (uplc): MH+ 514.6, 1.40 min (Method A).

(3R,4R)-3-((((S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-((third Butyldimethylmercaptoalkyloxy)tetrahydro-2H-piperidin-4-yl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 715.6, 1.65 min (Method A).

(3R,4R)-3-((1-((S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) 4-(( Tert-butyldimethylmethylalkyl)oxy)tetrahydro-2H-piperidin-4-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl ) 1,4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (up lc): MH.s.

Synthesis Example 22. Cyclopropanol-core skeleton synthesis

Prepared according to the protocol starting from (R)-2-(((benzyloxy)carbonyl)amino)-2-(1-hydroxycyclopropyl)acetic acid: Esposito, A.; Paolo - Piras, P.; Ramazzotti, D.; Taddei, M. Org. Lett. 2001 , 3 , 3273-3275.

(R)-2-(((Benzyloxy)carbonyl)amino)-2-(1-((t-butyldimethylmethyl)alkyl)oxy)cyclopropyl)acetic acid.

(R)-2-(((Benzyloxy)carbonyl)amino)-2-(1-hydroxycyclopropyl)acetic acid (3 g, 11.3 mmol, 1 eq) and imidazole (2.3 g, 34 mmol, 3 eq) TBSCl (4.3 g, 28.3 mmol, 2.5 eq) was added to a solution in DMF (23 mL). The reaction mixture was stirred at room temperature. The reaction mixture was partitioned between EtOAc (EtOAc) (EtOAc) The organic layer was separated, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure to yield a yellow oil. The crude product was used without any further purification. LC/MS (uplc): MH+ 380.3, 1.25 min (Method A).

2-(((Benzyloxy)carbonyl)amino)-2-(1-((t-butyldimethylmethyl)alkyl)oxy)cyclopropyl)acetic acid (R)-2-(2, 5-Difluorophenyl)-2-oxoethyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 534.4, 1.51 min (Method A).

(S)-((1-((t-butyldimethylmethyl)alkyl)oxy)cyclopropyl)(4-(2,5-difluorophenyl)-1H-imidazol-2-yl) Phenyl phenyl carbamate.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 514.8, 1.47 min (Method A).

(S)-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-((t-butyldimethylmethyl)alkyloxy) Benzyl propyl)methyl)aminocarbamate.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 604.1, 1.64 min (Method A).

(S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-((t-butyldimethylmethylalkyl)oxy) Cyclopropyl)methylamine.

To prepare this compound, the procedure described for the general Cbz protecting group removal is followed. The crude product was used without any further purification. LC/MS (uplc): MH+ 470.3, 1.26 min (Method A).

(3R,4R)-3-((((S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-((third Butyl dimethyl decyl) oxy) cyclopropyl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH + 706.4, 1.62 min (Method A).

(3R,4R)-3-((1-((S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-(( Tert-butyldimethylmethylalkyl)oxy)cyclopropyl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine- Benzoic acid benzyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 807.6, 1.60 min (Method A).

(3R,4R)-3-((1-((S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-(( Tert-butyldimethylmethylalkyl)oxy)cyclopropyl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine- 1-butylic acid tert-butyl ester.

To prepare this compound, the protecting group exchange procedure described for similar compounds of the THP series (Cbz protecting group removal, Boc protection) was followed. The crude product was purified by normal phase chromatography (PrepLC method B).

Cbz protecting group removal: LC/MS (uplc): M+ 672.4, 1.25 min (Method A).

Boc protection: LC/MS (uplc): M+ 772.7.1.61 min (Method A).

Synthesis Example 23. 2-methoxypropyl-core skeleton synthesis

(R)-2-((Tert-butoxycarbonyl)amino)-3-methoxy-3-methylbutyric acid.

To NaH (60% mineral oil, 2.5 g, 64. mmol, 3 eq) and (R)-2-((t-butoxycarbonyl)amino)-3-hydroxy-3-methylbutyric acid (5 g, 21.4 mmol, 1 eq) EtOAc (1.6 mL, 25.7 mmol, EtOAc) Upon completion of the reaction by LC/MS (uplc, Method A), the reaction was quenched with HCl (1.0M) until pH = 3. The crude reaction was partitioned between EtOAc (100mL) and HCl (1.0M, 100mL) between the two layers were separated, and the organic layer was washed with HCl (1.0M, 3 x 100mL) , dried over Na ₂ SO _4, filtered and reduced The solvent was evaporated under pressure to give (R)-2-((t-butoxycarbonyl)amino)-3-methoxy-3-methylbutyric acid. The crude product was used in the next step without any further purification. LC/MS (uplc): MH+ 248.2, 0.79 mi (Method D, polar method).

2-((Tertibutoxycarbonyl)amino)-3-methoxy-3-methylbutyric acid (R)-2-(2,5-difluorophenyl)-2-oxoethoxy B ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 402.2, 1.20 min (Method A).

(S)-(1-(4-(2,5-Difluorophenyl)-1H-imidazol-2-yl)-2-methoxy-2-methylpropyl)carbamic acid tert-butyl ester .

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 382.3, 1.08 min (Method A).

(S)-(1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy-2-methylpropyl)amine Tert-butyl carboxylic acid.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 472.4, 1.44 min (Method A).

(S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy-2-methylpropan-1-amine .

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 372.4, 0.88 min (Method A).

(3R,4R)-3-((((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy) Tert-butyl 2-methylpropyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 573.7, 1.38 min (Method A).

(3R,4R)-3-((1-((S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy) Tert-butyl 2-methylpropyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH + 674.4, 1.34 min (Method A).

(3R,4S)-3-((((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy) Tert-butyl 2-methylpropyl)amino)methyl)-4-((t-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate.

To (S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy-2-methylprop-1- amine (0.7g, 0.72mmol) and tri acetyl group with sodium borohydride _{(8.1g, 38mmol) 2 Cl 2} (3mL) in the solution was added containing CH (3S, 4R) -3 - ( ( t-butoxide CH ₂ Cl ₂ (1 mL) of dimethyl dimethyl decyl) oxy)-4-methylpyridylpyrrolidine-1-carboxylate. The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was partitioned between CH ₂ Cl ₂ and H ₂ O. The organic layer was separated and washed (twice) with saturated NaHCO ₃ solution (twice) and H ₂ O, then dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by normal phase column chromatography (PrepLC method A) to give (3R,4S)-3-((((S)-1-(1-(phenyl)-4-(2,5-) Fluorophenyl)-1H-imidazol-2-yl)-2-methoxy-2-methylpropyl)amino)methyl)-4-((t-butyldimethylmethylalkyl)oxy Pyrrolidine-1-carboxylic acid tert-butyl ester (250 mg, 0.33 mmo, 46%). LC/MS (up lc): MH+ 685.9, 1.76 min (Method A).

(3R,4S)-3-(((S)-2-Ethyloxy-N-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-2-methoxy-2-methylpropyl)propanylamino)methyl)-4-((t-butyldimethylmethylalkyl)oxy)pyrrolidine 1-butylic acid tert-butyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): M + 799.5, 1.69 min (Method A).

(3R,4S)-3-(((S)-2-Ethyloxy-N-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-2-methoxy-2-methylpropyl)propanylamino)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester.

To (3R,4S)-3-(((S)-2-Ethyloxy-N-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)) -1H-imidazol-2-yl)-2-methoxy-2-methylpropyl)propanylamino)methyl)-4-((t-butyldimethylmethylalkyl)oxy)pyrrole To a solution of pyridine-1-carboxylic acid tert-butyl ester (108 mg, 0.13 mmol, 1 eq) in THF (2 mL) EtOAc (EtOAc (EtOAc) Upon completion of the reaction by LC/MS (uplc, method), the reactant was partitioned between NH ₄ Cl (saturated solution) and CH ₂ Cl ₂ . The organic layer was separated and washed with NaCl (saturated solution), dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (up lc): MH.

(3R,4S)-3-(((S)-N-((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) -2-methoxy-2-methylpropyl)-2-hydroxypropionylamino)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC method B). LC/MS (uplc): MH+ 643.3, 1.30 min (Method A).

(S)-N-((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy-2- Methylpropyl)-2-hydroxy-N-(((3S,4S)-4-hydroxypyrrolidin-3-yl)methyl)propanamine.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC method B). 17.6 mg, 0.031 mmol, 71%.

¹ H-NMR (DMSO, 600 MHz): δ 7.93-7.87 (1H, m), 7.80-7.71 (1H, m), 7.45-7.28 (6H, m), 7.15-7.06 (1H, m), 6.00-5.87 (1H, m), 5.46-5.31 (1H, m), 5.18-5.05 (1H, m), 4.93-4.77 (2H, m), 4.77-4.64 (1H, m), 3.69-3.53 (3H, m) , 2.95-2.83 (3H, m), 2.75-2.63 (1H, m), 2.39-2.28 (1H, m), 2.26-2.07 (1H, m), 1.59-1.45 (1H, m), 1.44-1.34 ( 3H, m), 1.33-1.22 (3H, m), 1.21-1.12 (1H, m), 0.79-0.69 (3H, m). LC/MS (uplc): MH+ 543.3, 0.91 min (Method A).

Synthesis Example 24. 2-Hydroxypropyl-core skeleton synthesis

(R)-2-(2,5-Difluorophenyl)-2-oxoethoxyethyl 2-((t-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoate.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 388.3, 1.04 min (Method A).

(S)-(1-(4-(2,5-Difluorophenyl)-1H-imidazol-2-yl)-2-hydroxy-2-methylpropyl)carbamic acid tert-butyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH+ 368.5, 0.96 min (Method A).

(S)-(1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-hydroxy-2-methylpropyl)carbamic acid Third butyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH ₂ + 459.5, 1.36 min (Method A).

(S)-1-Amino-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methylpropan-1-ol.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was used without any further purification. LC/MS (uplc): MH ₂ + 359.3, 0.80 min (Method A).

(3R,4R)-3-((((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-hydroxy- 2-methylpropyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 593.3, 1.24 min (Method A).

(3R,4R)-3-((((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-(( Tert-Butyldimethylmethylalkyl)oxy)-2-methylpropyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester.

(3R,4R)-3-((((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-hydroxyl) Benzyl-2-methylpropyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate (1.5 g, 0.94 mmol, 1 eq) in CH ₂ Cl ₂ (5 mL) 2,6-Dimethylpyridine (0.7 mL, 5.7 mmol, 6 eq) and TBSOTf (0.7 mL, 3.8 mL, 4 eq). The reaction was stirred at 0 °C. When by LC / MS (uplc, method) found that the reaction was complete, the reaction was partitioned between H ₂ O (20mL) and CH ₂ Cl ₂ (20mL). The organic layer was separated, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by normal phase chromatography (PrepLC method A) to give the desired product (0.13 mmol, 130 mg, 14%). LC/MS (uplc): MH+ 707.3, 1.70 min (Method A).

(3R,4R)-3-((1-((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2- ((Tertiary butyldimethylmethylalkyl)oxy)-2-methylpropyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoro Pyrrolidine-1-carboxylic acid benzyl ester.

To prepare this compound, follow the procedure described for similar compounds in the THP series. The crude product was purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): M+ 808.4, 1.64 min (Method A).

(3R,4R)-3-((1-((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2- ((Tertiary butyldimethylmethylalkyl)oxy)-2-methylpropyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoro Pyrrolidine-1-carboxylic acid tert-butyl ester.

To prepare this compound, follow the protecting groups described for similar compounds in the THP series. Exchange program (Cbz protection group removal, Boc protection). The crude product was purified by normal phase column chromatography (PrepLC Method A).

Cbz protecting group removal: LC/MS (uplc): M+ 674.4, 1.33 min (Method A).

Boc protection: LC/MS (uplc): M+ 774.4. 1.66 min (Method A).

Synthesis Example 25. (3S,4R)-3-((t-Butyldimethylmethylalkyl)oxy)-4-vinylpyrrolidine-1-carboxylic acid benzyl ester

Benzyl (3S,4R)-3-hydroxy-4-vinylpyrrolidine-1-carboxylate (4.00 g, 16.18 mmol) in DCM (54 ml). Treated with dimethyl decane chloride (2.93 g, 19.4 mmol), and the mixture was stirred at room temperature for 16 h. The mixture was quenched with aqueous saturated NH ₄ Cl and the organic layer was extracted with DCM. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to yield a pale yellow oil of the title compound, 102% yield; UPLC-MS: Rt = 1.56min ; MS m / z [M + H] ⁺ 362.2; Method A.

(3S,4S)-3-((t-butyldimethylmethylalkyl)oxy)-4-((R)-1,2-dihydroxyethyl)pyrrolidine-1-carboxylic acid benzyl ester

To (3S,4R)-3-((t-butyldimethylmethylalkyl)oxy)-4-vinylpyrrolidine-1-carboxylic acid benzyl ester (4.90 g, 13.6 mmol) at 0 °C A solution of potassium permanganate (1.63 g, 10.3 mmol) and sodium hydroxide (0.352 g, 8.81 mmol) in water (10 ml) was slowly added to a solution of a mixture of the third butanol (40 ml) and water (28 ml). . The reaction mixture was stirred at 0 ° C for 1 hour. The mixture was extracted with DCM. The organic layers were combined, dried over Na ₂ SO _4, filtered through diatomaceous earth and concentrated to dryness. The crude product was purified by flash chromatography eluting elut elut elut elut elut elut elut [M+H] ⁺ 396.1; Method A.

(3S,4S)-3-((t-butyldimethylmethylalkyl)oxy)-4-((R)-1,2-dihydroxyethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

To (3S,4S)-3-((t-butyldimethylmethylalkyl)oxy)-4-((R)-1,2-dihydroxyethyl)pyrrolidine-1- under argon A solution of benzyl formate (3.41 g, 8.62 mmol) in methanol (55 ml) was added 10% palladium/carbon (0.917 g, 0.862 mmol), ammonium formate (6.52 g, 103 mmol), and the reaction mixture at 50 ° C Stir for 30 minutes. The mixture was then cooled to RT, filtered over celite and washed with methanol. Di-tert-butyl dicarbonate (2.63 g, 12.1 mmol) was added to the filtrate and the reaction mixture was stirred at RT for 1 hour. The methanol was removed by evaporation and the reaction mixture was then extracted with ethyl acetate and saturated _aqueous NaHCO. Dried the organic layer was washed with brine, and the combined organic phases over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by EtOAc EtOAc (EtOAc) 1.25 min; MS m/z [M+H] ⁺ 3621.

(3S,4R)-3-((t-butyldimethylmethylalkyl)oxy)-4-methylpyridylpyrrolidine-1-carboxylic acid tert-butyl ester

To (3S,4S)-3-((t-butyldimethylmethylalkyl)oxy)-4-((R)-1,2-dihydroxyethyl)pyrrolidine-1- at 0 °C Sodium periodate (2.09 g, 9.79 mmol) was added to a solution of tributyl succinate (2.95 g, 8.16 mmol) in a mixture of methanol (44 ml) and water (11 ml), and the mixture was stirred at RT for 45 min. . The reaction mixture was filtered and the methanol was removed by evaporation. Water (15 ml) was added and the reaction was extracted with DCM. The organic layer was combined, dried over Na ₂ SO _4, filtered through diatomaceous earth and concentrated to dryness to give a pale yellow oil of the title compound, 100% ^{yield: 1 H NMR (400MHz, CDCl3} ) δ 9.69 (1H, Br s), 4.57-4.53 (1H, m), 3.71-3.49 (3H, m), 3.24 - 3.19 (1H, m), 3.01-2.95 (1H, m), 1.45 (9H, s), 0.88 (9H , s), 0.08 (6H, s).

(3R,4S)-3-((((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidyl)喃-4-yl)methyl)amino)methyl)-4-((t-butyldimethylmethylalkyl)oxy)pyrrolidine- 1-butyl formate

Add (R)-(1-benzyl-4-(2,5-difluorophenyl)- to a solution of sodium ethoxide borohydride (5.77 g, 27.2 mmol) in DCM (14 mL) 1H-Imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methylamine (2.09 g, 5.44 mmol). To the reaction mixture was slowly added (3S,4R)-3-((t-butyldimethylmethylalkyl)oxy)-4-carbamimidyrrolidine-1-carboxylic acid tert-butyl ester (2.69 g, 8.16). Methyl acetate (14 ml) was stirred and the mixture was stirred at RT for 16 h. The mixture was then quenched with water and the mixture was extracted with 1M aqueous Na ₂ S ₂ O ₃ and DCM. The organic layer was washed with saturated aqueous NaHCO _3, followed by brine. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut elut /z[M+H] ⁺ 697.8; Method A.

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanylamino)methyl)-4-((t-butyldimethylmethylalkyl)oxy)pyrrolidine 1-butylic acid tert-butyl ester

To (3R,4S)-3-((((R)-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) at 0 ° C Tetrahydro-2H-piperazin-4-yl)methyl)amino)methyl)-4-((t-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester ( 1.79 g, 2.57 mmol) of N-ethyl-N-isopropylpropan-2-amine (0.63 mL, 3.60 mmol), (S)-acetic acid 1- Chloro-1-oxopropan-2-yl ester (0.36 mL, 2.83 mmol). The reaction mixture was stirred at 0 ° C for 5 min, then allowed to warm to RT and stirred for 1.5 h. The reaction was quenched with saturated aqueous NaHCO ₃ and extracted with DCM. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by flash chromatography eluting elut elut elut elut elut elut elut /z[M+H] ⁺ 811.2; Method A.

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanamido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H) -imidazol-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanylamino)methyl)-4-((t-butyldimethylmethylalkyl)oxy)pyrrole Tetrabutylammonium fluoride (0.619 g, 2.37 mmol) was slowly added to a solution of pyridine-1-carboxylic acid tert-butyl ester (1.92 g, 2.37 mmol) in THF (12 mL). The reaction mixture was stirred at RT for 30 minutes. The reaction was quenched with aqueous saturated NH ₄ Cl and extracted with DCM. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by flash chromatography eluting eluting elut elut elut elut elut elut elut elut /z[M+H] ⁺ 696.9; Method A.

(3S,4R)-3-((((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidyl) Butan-4-yl)methyl)amino)methyl)-4-((t-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

In a similar manner to Example 25, the product was synthesized using (3R,4S)-3-hydroxy-4-vinylpyrrolidine-1-carboxylic acid benzyl ester instead; 105% yield; UPLC-MS: Rt=1.63 Min; MS m/z [M+H] ⁺ 697.0;

(3S,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanylamino)methyl)-4-((t-butyldimethylmethylalkyl)oxy)pyrrolidine 1-butylic acid tert-butyl ester

Quantitative yield; UPLC-MS: Rt = 1.63 min; MS m/z [M+H] ⁺ 811.0;

(3S,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanamido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

32% yield; UPLC-MS: Rt = 1.26 min; MS m/z [M+H] ⁺ 696.9;

Synthesis Example 26. (R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropan-1-amine

It was prepared as described in WO2008086122 A2, 2008 ; P.50-52.

(3R,4S)-3-((((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2- Dimethylpropyl)amino)methyl)-4-((t-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

In a similar manner to Example 1, (R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethyl was used. The product was synthesized by propyl-1-amine; 93% yield; UPLC-MS: Rt = 2.66 min; MS m/z [M+H] ⁺ 669.5;

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-2,2-dimethylpropyl)propanylamino)methyl)-4-((t-butyldimethylmethylalkyl)oxy)pyrrolidine-1- Tert-butyl formate

39% yield; UPLC-MS: Rt = 3.86 min; MS m/z [M+H] ⁺ 783.6;

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-2,2-dimethylpropyl)propanylamino)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

43% yield; UPLC-MS: Rt = 2.96 min; MS m/z [M+H] ⁺ 669.4;

Synthesis Example 27.

(3R,4S)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperazin-4-yl)methyl)-3-((R)-1-hydroxypropan-2-yl)ureido)methyl)-4-((t-butyldimethylmethyl)alkyloxy) Pyrrolidine-1-carboxylic acid tert-butyl ester

To a solution of phosgene (20% in toluene, 0.76 mL, 1.4 mmol) in DCM (7.2 mL), (3,,,,,,,,,,, -(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)-4-((Third A solution of tert-butyl dimethyl decyl) oxy) pyrrolidine-1-carboxylate (0.50 g, 0.717 mmol) and triethylamine (0.30 mL, 2.15 mmol) in DCM (7.2 mL). The reaction mixture was stirred at room temperature for 45 minutes. L-Propanamine (1.26 mL, 16.1 mmol) was added and the reaction was stirred at 40 ° C for 16 h. The reaction was concentrated to dryness. The crude product was purified by chromatography eluting elut elut elut elut elut elut elut elut elut elut /z[M+H] ⁺ 797.9; Method A.

(3R,4S)-3-((3-((R)-1-Ethyloxypropan-2-yl)-1-((R)-(1-phenylmethyl-4-(2,5) -difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4-((t-butyldimethyl decane) Tris-butyl oxy)pyrrolidine-1-carboxylate

(3R,4S)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-3-((R)-1-hydroxypropan-2-yl)ureido)methyl)-4-((t-butyldimethyl) Pyridyl (0.87 mL, 10.8 mmol), acetic anhydride (sodium butoxide) (0.43 g, 0.539 mmol) in anhydrous DCM (5.4 mL) 1.01 mL, 10.8 mmol). The reaction mixture was stirred at RT for 16 hours. The reaction was concentrated to dryness. The crude product was purified by EtOAc EtOAc (EtOAc) /z[M+H] ⁺ 840.0; Method A.

(3R,4S)-3-((3-((R)-1-Ethyloxypropan-2-yl)-1-((R)-(1-phenylmethyl-4-(2,5) -difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)ureido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tertidine ester

The product was synthesized in a similar manner to Example 1; 76% yield; UPLC-MS: Rt = 1.23 min; MS m/z [M+H] ⁺ 725.9;

Synthesis Example 28.

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazol-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanylamino)methyl)-4-((methylsulfonyl)oxy)pyrrolidine-1-carboxylic acid Third butyl ester

To (3R,4S)-3-(((S)-2-ethoxycarbonyl-N-((R)-(1-benzyl-4-(2,5-difluoro)) at 0 °C Phenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanylamino)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester Methane sulfonium chloride (0.74 mL, 9.47 mmol), triethylamine (1.3 mL, 9.47 mmol), and the reaction mixture was stirred at 0 ° C, 1.5 g (1. hour. The reaction was concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut elut /z[M+H] ⁺ 775.5; Method E.

(3R,4S)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropionylamino)methyl)-4-((methylsulfonyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H) -imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-((methylsulfonyl)oxy)pyrrolidine-1- Potassium carbonate (148 mg, 1.07 mmol) was added to a solution of EtOAc (EtOAc) (EtOAc) The reaction was diluted with brine and extracted with DCM. The combined organic extracts were dried over Na ₂ SO _4, filtered and concentrated to dryness to give the crude title pale yellow solid in quantitative yield of the compound was used without further purification in the next step; UPLC-MS: Rt = 2.54 min; MS m/z [M+H] ⁺ 733.4;

(3R,4S)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)-4-((methylsulfonate) Tris-butyl oxy)pyrrolidine-1-carboxylate

To (3R,4S)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (tetrahydro-2H-piperidin-4-yl)methyl)-2-hydroxypropionylamino)methyl)-4-((methylsulfonyl)oxy)pyrrolidine-1-carboxylic acid third Add 1H-imidazole (0.91 mg, 1.34 mmol), butyl dimethyl dimethyl dimethyl decane (202 mg, 1.34 mmol) and DMAP (1 mg) in a solution of butyl ester (655 mg, 0.894 mmol) in DCM (8.9 ml). , catalyst), and the reaction mixture was stirred at RT for 4 hours. Further, a third butyl chlorodimethyl decane (229 mg, 1.52 mmol), 1H-imidazole (0.55 mg, 0.804 mmol) was added, and the mixture was stirred for further 2 days. The reaction was diluted with brine and extracted with DCM. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting elut elut elut elut elut elut elut elut elut elut z[M+H] ⁺ 847.6; Method E.

(3R,4R)-3-Ethyloxy-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)pyrrolidine 1-butylic acid tert-butyl ester

To (3R,4S)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)-4-((methylsulfonate) Potassium acetate (12 mg, 0.123 mmol) was added to a solution of decyl)oxy)pyrrolidine-l-carboxylic acid in the butyl ester (52 mg, 0.061 mmol) in DMF (0.6 ml), and the reaction mixture was stirred at 110 ° C. 2 hours. The reaction was cooled to RT and diluted with water and extracted with EtOAc. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness to yield 88% yield of the product as a yellow solid of crude, i.e. without further purification in the next step; UPLC-MS: Rt = 3.41min; MS m / z [m + H] + 811.6; method E.

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)-4-hydroxypyrrolidine-1- Tert-butyl formate

To (3R,4R)-3-ethenyloxy-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H) -imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)pyrrole To a solution of pyridine-1-carboxylic acid tert-butyl ester (44 mg, 0.054 mmol) in MeOH (0.55 mL). The reaction was concentrated to dryness to give a pale yellow solid in quantitative yield of the title compound and used without further purification in the next step; UPLC-MS: Rt = 3.17min ; MS m / z [M + H] + 769.6; Method E.

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)-4-((methylsulfonate) Tris-butyl oxy)pyrrolidine-1-carboxylate

To (3R,4R)-3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazole) at 0 °C -2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)-4- Methyl sulfonium chloride (0.08 mL, 1.04 mmol) and triethylamine (0.15 mL, 1.04 mmol) were added to a solution of hydroxypyrrolidine-l-carboxylic acid tert-butyl ester (160 mg, 0.208 mmol) in EtOAc. And the reaction mixture was stirred at 0 ° C for 1.5 hours. The reaction mixture was concentrated to dryness EtOAcqqqqqqqqqqQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ Rt = 3.27 min; MS m/z [M+H] ⁺ 847.6;

(3S,4R)-3-azido-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazole) -2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)pyrrolidine- 1-butyl formate

To (3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)-4-((methylsulfonate) Add a sodium azide (0.014 g, 0.211 mmol) to a solution of decyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester (149 mg, 0.176 mmol) in DMF (1.8 mL). Stir at ° C for 18 hours. The reaction was quenched with water and extracted with TBME, and the organic extracts were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut elut /z[M+H] ⁺ 794.6; Method E.

Synthesis Example 29.

3-(1-((Tertidinoxycarbonyl)amino)-2-methoxy-2-oxooxyethylidene)azetidine-1-carboxylic acid benzyl ester

To 3-oxooxyazetidine-1-carboxylic acid benzyl ester (5.0 g, 24.4 mmol) and 2-((t-butoxycarbonyl)amino)-2-(dimethoxyphosphonium) 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a was added dropwise to a solution of methyl acetate (7.24 g, 24.4 mmol) in DCM (122 mL) Nitrogen hydrazine (4.4 mL, 29.2 mmol), and the reaction mixture was stirred at RT for 1 hour. The reaction was concentrated to dryness. Water was added and the reaction became acidic by the addition of 2N aqueous HCl. The reaction was then extracted with EtOAc. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by flash chromatography eluting eluting elut elut elut elut elut elut elut /z[M+Na] ⁺ 399.1; Method E;

3-(1-((Tertidinoxycarbonyl)amino)-2-methoxy-2-oxoethylethyl)azetidin-1-carboxylate

To 3-(1-((t-butoxycarbonyl)amino)-2-methoxy-2-oxooxyethylidene)azetidine-1-carboxylic acid benzyl ester under argon (8.0 g, 21.3 mmol) <RTI ID=0.0></RTI> The atmosphere was replaced by hydrogen and the resulting reaction mixture was stirred at RT for 2 h. The reaction was filtered through celite and then concentrated to dryness. The residue was dissolved in EtOAc (EtOAc) (EtOAc)EtOAc. The reaction was stirred at RT for 72 hours. The reaction was acidified with aq. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by flash chromatography eluting elut elut elut elut elut elut elut elut elut [M+H] ⁺ 379.4; Method A.

2-(1-((Benzyloxy)carbonyl)azetidin-3-yl)-2-((t-butoxycarbonyl)amino)acetic acid

To 3-(1-((t-butoxycarbonyl)amino)-2-methoxy-2-oxooxyethyl)azetidin-1-carboxylate at 0 ° C ( A solution of potassium carbonate (1.98 g, 14.3 mmol) in water (36 ml) was added dropwise. Methanol was removed under reduced pressure and the solution was brought to a pH of about 6 by the addition of 10% aqueous HCl. The reaction was then concentrated to dryness. The resulting solid was stirred at 200 mL DCM at RT for 2 h and then solid was removed by filtration. The filtrate was dried over Na ₂ SO _4, filtered and concentrated to dryness to give the title compound as a white solid of 89% of the yield, which was used without further purification in the next step; UPLC-MS: Rt = 0.92min ; MS m/z [MH] ^- 363.2; Method A.

3-(1-((Tertidinoxycarbonyl)amino)-2-(2-(2,5-difluorophenyl)-2-oxoethoxy)-2-oxoethoxy B Benzoazetidine-1-carboxylic acid benzyl ester

In a similar manner to Example 13, the use of 2-(1-((benzyloxy)carbonyl)azetidin-3-yl)-2-((t-butoxycarbonyl)amino) was used instead. Acetic acid to synthesize the product; 97% yield; UPLC-MS: Rt = 2.53 min; MS m/z [M+Na] ⁺ 541.3;

3-(((Tertidinoxycarbonyl)amino)(4-(2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)azetidin-1-carboxylate Methyl ester

81% yield; UPLC-MS: Rt = 1.82 min; MS m/z [M+H] ⁺ 499.3;

3-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((t-butoxycarbonyl)amino)methyl)azetidine Alkyl-1-carboxylate

42% yield; UPLC-MS: Rt = 2.71 min; MS m/z [M+H] ⁺ 589.4;

(R)-3-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((t-butoxycarbonyl)amino)methyl) Azetidine-1-carboxylate

3-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((t-butoxycarbonyl)amino)methyl)azetidine A racemic mixture of alkane-1-carboxylate (4.12 g) was submitted to the Basel Separation Laboratory (contact: Dr. Eric Francotte, Tel. +41 6169 62971). Obtaining the desired enantiomerically enriched (R)-3-(amino(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)- Benzyl azetidine-1-carboxylate (1.51 g, 99.3% ee ) and undesired (S)-3-(amino (1-benzyl-4-(2,5-di) Fluorophenyl)-1H-imidazol-2-yl)methyl)azetidin-1-carboxylate (1.53 g, 98.0% ee ), 74% recovery. The absolute configuration is established by x-ray crystallography of the title compound.

(R)-3-(Amino(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)azetidin-1-carboxylate Methyl ester

Quantitative yield; UPLC-MS: Rt = 1.93 min; MS m/z [M+H] ⁺ 489.3;

(3R,4R)-3-((((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-((phenyl) Oxy)carbonyl)azetidin-3-yl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid Butyl ester

34% yield; UPLC-MS: Rt = 2.27 min; MS m/z [M+H] ⁺ 690.5;

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(1-((benzyloxy)carbonyl)azetidin-3-yl)methyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid Tributyl ester

87% yield; UPLC-MS: Rt = 3.05 min; MS m/z [M+H] ⁺ 804.5;

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-azetidin-3-yl (1-phenylmethyl-4-(2,5) -difluorophenyl)-1H-imidazol-2-yl)methyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4R)-3-(((S)-2-ethoxycarbonyl-N-((R)-(1-benzyl-4-(2,5-difluoro) under argon Phenyl)-1H-imidazol-2-yl)(1-((benzyloxy)carbonyl)azetidin-3-yl)methyl)propanylamino)methyl)-4-fluoropyrrole Add a 10% palladium on carbon (54 mg, 0.051 mmol), ammonium formate (772 mg, 12.2 mmol) in a solution of pyridine-1-carboxylic acid tert-butyl ester (820 mg, 1.02 mmol) in methanol (10.2 ml). The mixture was stirred at RT for 2 hours. The reaction was filtered through celite and concentrated to dryness. Saturated aqueous NaHCO ₃ and the reaction extracted with DCM. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut elut M+H] ⁺ 670.4; Method E.

(3R,4R)-3-(((S)-N-((R)-azetidin-3-yl(1-benzylmethyl-4-(2,5-difluorophenyl)-)- 1H-imidazol-2-yl)methyl)-2-hydroxypropanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4R)-3-(((S)-2-ethoxycarbonyl-N-((R)-azetidin-3-yl (1-phenylmethyl-) at 0 °C 4-(2,5-Difluorophenyl)-1H-imidazol-2-yl)methyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (571 mg, 0.853) NaBH ₄ (213 mg, 5.63 mmol) was slowly added to aq. The reaction was quenched by the dropwise addition of saturated aqueous NH.sub.4Cl. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by EtOAc (EtOAc: EtOAc) Rt = 1.79 min; MS m/z [M+H] ⁺ 628.0;

(3R,4R)-3-(((S)-N-((R)-(1-Ethylazetidin-3-yl)(1-phenylmethyl-4-(2,5) -difluorophenyl)-1H-imidazol-2-yl)methyl)-2-hydroxypropanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The product is synthesized as (3R,4R)-3-(((S)-N-((R)-azetidin-3-yl(1-benzyl-4-(2,5-difluorobenzene) Obtained as a by-product of -1H-imidazol-2-yl)methyl)-2-hydroxypropionamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester; 19% yield; UPLC-MS: Rt = 2.27 min; MS m/z [M+H] ⁺ 670.3;

Synthesis Example 30.

2-(1-((Benzyloxy)carbonyl)piperidin-4-yl)-2-((t-butoxycarbonyl)amino)acetic acid

Prepared as described in WO02076450 A1, 2002 ; P.66.

4-(1-((Tertidinoxycarbonyl)amino)-2-(2-(2,5-difluorophenyl)-2-oxoethoxy)-2-oxoethoxy B Phenylpyridin-1-carboxylate

In a similar manner to Example 13, instead using 2-(1-((benzyloxy)carbonyl)piperidin-4-yl)-2-((t-butoxycarbonyl)amino)acetic acid to synthesize Product; 96% yield; UPLC-MS: Rt = 1.27 min; MS m/z [MH] ^- 545.0;

4-(((Tertidinoxycarbonyl)amino)(4-(2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)piperidine-1-carboxylic acid benzyl ester

103% yield; UPLC-MS: Rt = 1.22 min; MS m/z [MH] ^- 525.1;

4-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((t-butoxycarbonyl)amino)methyl)piperidine-1 - benzoic acid methyl ester

45% yield; UPLC-MS: Rt = 1.45 min; MS m/z [M+H] ⁺ 617.5;

(R)-4-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((t-butoxycarbonyl)amino)methyl) Piperidine-1-carboxylic acid benzyl ester

4-((1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((t-butoxycarbonyl)amino)methyl)piperidine-1 - A racemic mixture of benzyl formate (6.00 g) was submitted to the Basel Separation Laboratory (contact: Dr. Eric Francotte, Tel. +41 6169 62971). Obtaining the desired enantiomerically enriched (R)-4-((1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (( Trimethyloxycarbonyl)amino)methyl)piperidine-1-carboxylic acid benzyl ester (1.91 g, >99.5% ee ) and undesired (S)-4-((1-phenylmethyl-4-) (2,5-Difluorophenyl)-1H-imidazol-2-yl)((t-butoxycarbonyl)amino)methyl)piperidine-1-carboxylic acid benzyl ester (2.20 g, >99.5% Ee ), 69% recovery. The absolute configuration is established by x-ray crystallography of the title compound.

(R)-4-(amino(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)piperidine-1-methyl Acid benzyl ester

Quantitative yield; UPLC-MS: Rt = 2.01 min; MS m/z [M+H] ⁺ 517.3;

4-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(((3R,4R)-1-(Third Ethoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)amino)methyl)piperidine-1-carboxylic acid benzyl ester

79% yield; UPLC-MS: Rt = 2.32 min; MS m/z [M+H] ⁺ 718.4;

4-((R)-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)) Methyl)propanolamine)(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)piperidine-1-carboxylic acid benzyl ester

76% yield; UPLC-MS: Rt = 3.15 min; MS m/z [M+H] ⁺ 832.5;

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(piperidin-4-yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

56% yield; UPLC-MS: Rt = 1.94 min; MS m/z [M+H] ⁺ 698.4;

Synthesis Example 31.

(3S,4S)-3-(((tert-butyldimethylmethylalkyl)oxy)methyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

Prepared as described in WO06066896, 2006 ; P.385-388

(3R,4S)-3-((((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-) Piperidin-4-yl)methyl)amino)methyl)-4-((t-butyldimethylmethylalkyl)oxy)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester

To (3S,4S)-3-(((tert-butyldimethylmethylalkyl)oxy)methyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (5.23 g, To a solution of 15.1 mmol) in dry EtOAc (EtOAc) (EtOAc) This solution is then added to (R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl) A suspension of methylamine (5.80 g, 15.1 mmol) and sodium triethoxysulfonium borohydride (16.0 g, 76.0 mmol) in DCM (38 mL). The resulting mixture was stirred at RT for 18 hours. The mixture was then quenched with water and the mixture was extracted with 1M aqueous Na ₂ S ₂ O ₃ and DCM. The organic layer was washed with saturated aqueous NaHCO _3, followed by brine. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut elut z[M+H] ⁺ 711.4; Method A.

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-(((t-butyldimethylmethyl)alkyl)oxy) Pyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4S)-3-((((R)-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) at 0 ° C Tetrahydro-2H-piperazin-4-yl)methyl)amino)methyl)-4-(((t-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carboxylic acid Diisopropylethylamine (1.34 mL, 7.68 mmol), (S)-acetic acid 1-chloro-1-acetoxypropyl group were added sequentially to a solution of tributyl ester (3.9 g, 5.49 mmol) in DCM (55 mL) 2-Benzyl ester (0.83 mL, 6.58 mmol), and the mixture was warmed to RT and stirred 16 hr. The reaction was extracted with DCM and washed with saturated aqueous NaHCO _3. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by flash chromatography eluting eluting elut elut elut elut elut elut /z[M+H] ⁺ 825.6; Method E.

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanylamino)methyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H) -imidazol-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanylamino)methyl)-4-(((t-butyldimethylmethylalkyl)oxy) TBAF (1.69 g, 6.45 mmol) was added to a solution of &lt;RTI ID=0.0&gt;&gt; The reaction was diluted with saturated aqueous NH ₄ Cl and the reaction was extracted with DCM, and washed with brine and the organic layer. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by flash chromatography eluting eluting elut elut elut elut elut elut elut /z[M+H] ⁺ 711.4; Method E.

(3S,4R)-4-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanylamino)methyl)-1-(t-butoxycarbonyl)pyrrolidine-1-carboxylic acid

To (3R,4S)-3-(((S)-2-ethoxycarbonyl-N-((R)-(1-benzyl-4-(2,5-difluoro)) at 0 °C Phenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanylamino)methyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (300mg, 0.422mmol) in acetone (2.1 ml of) in the solution containing added dropwise 2M trioxide aqueous solution of H ₂ SO ₄ (1.3mL, 3.93mmol), and the reaction mixture was stirred at RT 1 hour. The excess Jones reagent was quenched by the dropwise addition of isopropanol (4 ml) at 0 °C. The reaction was concentrated to dryness. Water was added and the reaction was extracted with EtOAc. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to give 81% of the title compound as a solid the yield, which was used without further purification in the next step; UPLC-MS: Rt = 0.65min MS m/z [M+H] ⁺ 312.1; Method A.

Synthesis Example 32.

3-(2-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbutyl) 3-buten-1-yl)amine carbhydryl)phenyl)pyrrolidine-1-carboxylic acid tert-butyl ester

Add HATU (248 mg, 0.653 mmol), diiso) to a solution of 2-(1-(t-butoxycarbonyl)pyrrolidin-3-yl)benzoic acid (152 mg, 0.523 mmol) in DMF (1.5 mL) Propylethylamine (0.22 mL, 1.31 mmol) and (R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2 - dimethylbut-3-en-1-amine (160 mg, 0.435 mmol), and the obtained mixture was stirred at RT for 1 hour. The reaction was concentrated to dryness. The crude product was purified by EtOAc (EtOAc) elute elute elute elute Min; MS m/z [M+H] ⁺ 641.3;

3-(2-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-hydroxy-2,2- Dimethyl butyl) carbamoyl)phenyl)pyrrolidine-1-carboxylic acid tert-butyl ester

The product was synthesized in a similar manner to Example 14; 67% yield (yield of diastereomers); UPLC-MS: Rt = 2.54 and 2.58 min; MS m/z [M+H] ⁺ 659.3;

(4R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-(2-(1-(t-butoxycarbonyl) Pyrrolidin-3-yl)benzhydrylamino)-3,3-dimethylbutyric acid

28% yield (mixture of diastereomers); UPLC-MS: Rt = 2.64 and 2.65 min; MS m/z [M+H] ⁺ 673.1;

Synthesis Example 33. 3-(2-(methoxycarbonyl)phenoxy)azetidin-1-carboxylic acid tert-butyl ester

Add 3-hydroxyazetidine-1-carboxylic acid tert-butyl ester (1.124 g, 6.49 mmol) to a solution of methyl 2-fluorobenzoate (0.83 mL, 6.49 mmol) in DMF (16.2 mL) Cesium carbonate (10.6 g, 32.4 mmol), and the reaction mixture was stirred at 75 ° C for 18 hours. The reaction was extracted with water and DCM. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by flash chromatography eluting eluting elut elut elut elut elut elut elut elut M+Na] ⁺ ; Method E.

2-((1-(Tertidinoxycarbonyl)azetidin-3-yl)oxy)benzoic acid

A solution of 3-(2-(methoxycarbonyl)phenoxy)azetidin-1-carboxylic acid tert-butyl ester (325 mg, 1.06 mmol) in methanol (4.4 ml) and water (0.9 ml) Lithium hydroxide (127 mg, 5.29 mmol) was added and the mixture was stirred at RT for 18 h. The reaction was extracted with 1 M aqueous HCl and DCM. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to give a colorless solid of the title compound, 94% yield; UPLC-MS: Rt = 171min ; MS m / z [M + Na] + 330.0 Method E.

(R)-3-(2-((1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbutyl) 3--3-en-1-yl)aminomethane)phenoxy)azetidin-1-carboxylic acid tert-butyl ester

The product was synthesized in a similar manner to Example 9; 103% yield; UPLC-MS: Rt = 2.95 min; MS m/z [M+H] ⁺ 643.2;

(R)-3-(2-((1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-hydroxy-2,2- Dimethyl butyl)amine carbaryl)phenoxy)azetidine-1-carboxylic acid tert-butyl ester

30% yield; UPLC-MS: Rt = 2.53 min; MS m/z [M+H] ⁺ 661.2;

(R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-(2-((1-(t-butoxy)) Carbonyl)azetidin-3-yl)oxy)benzhydrylamino)-3,3-dimethylbutyric acid

32% yield; UPLC-MS: Rt = 2.67 min; MS m/z [M+H] ⁺ 675.1;

Synthesis Example 34.

(R)-3-((((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran)- 3-butyl)methyl)amino)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester

The product was synthesized in a similar manner to Example 4; 79% yield; UPLC-MS: Rt = 1.15 min; MS m/z [M+H] ⁺ 566.9;

(R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidyl) Butan-4-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester

91% yield; UPLC-MS: Rt = 1.25 min; MS m/z [M+H] ⁺ 667.9;

Additional payload instance: (3R,4S)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropionamido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H) -imidazol-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanamido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (150 mg, 0.215 mmol) Potassium carbonate (36 mg, 0.258 mmol) was added to a solution in MeOH (1 mL). Reaction ₄ Cl and extracted with DCM and saturated aqueous NH. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to give a clear oil of the title compound in quantitative yield; UPLC-MS: Rt = 1.20min ; MS m / z [M + H] + 655.5; Method A.

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-2-hydroxy-N-(((3S,4S)-4-hydroxypyrrolidin-3-yl)methyl)propanamide

The product was deprotected using a general scheme of removal of the protecting group; 61% yield; UPLC-MS: Rt = 0.78 min; MS m/z [M+H] ⁺ 555.5;

(3S,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropionamido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

Using (3R,4S)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (tetrahydro-2H-piperidin-4-yl)methyl)-2-hydroxypropionamido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester similarly produced by the product; Mass yield; UPLC-MS: Rt = 1.19 min; MS m/z [M+H] ⁺ 655.0;

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-2-hydroxy-N-(((3R,4R)-4-hydroxypyrrolidin-3-yl)methyl)propanamide

The product was deprotected using the general scheme 2 for removal of the protecting group; 54% yield; UPLC-MS: Rt = 0.79 min; MS m/z [M+H] ⁺ 555.5;

(3R,4S)-3-(((S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) -2,2-dimethylpropyl)-2-hydroxypropionamido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

Using (3R,4S)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) (tetrahydro-2H-piperidin-4-yl)methyl)-2-hydroxypropionamido)methyl)-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester similarly produced by the product; Mass yield; UPLC-MS: Rt = 2.88 min; MS m/z [M+H] ⁺ 627.3;

(S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropane 2-hydroxy-N-(((3S,4S)-4-hydroxypyrrolidin-3-yl)methyl)propanamide

The product was deprotected using the general scheme 2 for removal of the protecting group; 67% yield, as TFA salt; UPLC-MS: Rt = 1.94 min; MS m/z [M+H] ⁺ 527.2; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 7.88-7.87 (1H, m), 7.81-7.76 (1H, m), 7.41-7.28 (7H, m), 7.12-7.06 (1H , m), 5.80 (1H, s), 5.59-5.58 (1H, m), 5.34-5.30 (1H, m), 5.05-4.95 (2H, m), 4.72-4.65 (1H, m), 3.93-3.87 (1H, m), 3.75-3.66 (2H, m), 3.08-3.03 (1H, m), 2.73-2.64 (1H, m), 2.18-2.14 (1H, m), 1.75-1.58 (1H, m) , 1.30 - 1.28 (3H, m), 0.80 (9H, s).

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanylamino)methyl)-4-((pentylaminecarboxylidene)oxy)pyrrolidine-1- Tert-butyl formate

To (S)-acetic acid 1-(((R)-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran) 4-yl)methyl)(((3S,4S)-4-hydroxypyrrolidin-3-yl)methyl)amino)-1-oxopropan-2-yl ester (4.6 mg, 0.0066 mmol) And a solution of diisopropylethylamine (0.0011 mL, 0.066 mmol) in anhydrous DMF (0.07 ml), bis(4-nitrophenyl) carbonate (4.5 mg, 0.015 mmol), Stir for 1 hour at RT. Pentylamine (0.003 mL, 0.027 mmol) was added and the reaction was stirred at RT for 1.5 h. The reaction was extracted with water and EtOAc. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to give a pale yellow solid of the title compound, 99% yield; UPLC-MS: Rt = 1.16min ; MS m / z [M + H] ⁺ 810.5; Method E.

Amylaminocarbamic acid (3S,4S)-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazole-) 2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropionylamino)methyl)pyrrolidin-3-yl ester

Removal of the protecting group using a general scheme of removal of the protecting group; 43% yield; UPLC-MS: Rt = 1.96 min; MS m/z [M+H] ⁺ 668.5; Method E; ¹ H-NMR ( CDCl ₃ , 400 MHz, mixture of rotamers): δ 9.71 (2H, br), 7.72-7.67 (1H, m), 7.40-7.13 (7H, m), 7.04-6.92 (1H, m), 6.85-6.78 (1H,m), 5.67-5.64(1H,m),5.32-4.86(3H,m),4.65-4.38(1H,m),3.89-1.72(15H,m),1.51-1.07(13H,m) , 0.84-0.58 (3H, m).

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazol-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)propanylamino)methyl)-4-(pentylaminecarboxamido)pyrrolidine-1-carboxylic acid tertidine ester

Add HATU (88 mg, 0.232 mmol), (3S, 4R)-4-(((S)-2-Ethyloxy) in a solution of pentylamine (0.023 mL, 0.119 mmol) in DCM (0.18 mL) -N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)- a solution of propylamino)methyl)-1-(t-butoxycarbonyl)pyrrolidine-3-carboxylic acid (80 mg, 0.110 mmol) in DMF (0.18 ml), and the mixture was stirred at RT 1 hour. The reaction was extracted with water and EtOAc. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to give a pale yellow solid of the title compound in quantitative yield; UPLC-MS: Rt = 1.35min ; MS m / z [M + H] ⁺ 794.2; Method E.

(3S,4S)-4-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropanylamino)methyl)-N-pentylpyrrolidine-3-carboxamide

Using the general scheme of a protective group is removed by deprotection of the product; 43% yield, TFA salt; UPLC-MS: Rt = 0.94min ; MS m / z [M + H] + 652.5; Method A; ¹ H-NMR (CDCl ₃ , 400 MHz, mixture of rotamers): δ 10.31 (1H, br), 8.78 (1H, br), 7.94-7.66 (1H, m), 7.51-7.42 (1H, m), 7.32 -7.24 (2H, m), 7.17-7.15 (2H, m), 7.01-6.91 (2H, m), 6.84-6.80 (1H, m), 5.61-5.58 (1H, m), 5.20-5.16 (1H, m), 5.05-5.01 (1H, m), 4.67-4.49 (1H, m), 3.86-3.83 (1H, m), 3.63-2.54 (14H, m), 2.05-1.96 (1H, m), 1.44- 1.36 (2H, m), 1.32-1.13 (10H, m), 0.83-0.80 (3H, m).

(R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazole- 2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanamido)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester

In a similar manner to the example, (R)-3-((((R)-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Synthesis product of tetrahydro-2H-piperidin-4-yl)methyl)amino)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester; 59% yield; UPLC-MS: Rt = 1.36 min; MS m /z[M+H] ⁺ 680.9; Method A.

(R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) (tetrahydro) -2H-piperidin-4-yl)methyl)-2-hydroxypropionamido)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester

88% yield; UPLC-MS: Rt = 2.55 min; MS m/z [M+H] ⁺ 639.4;

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-2-hydroxy-N-((S)-pyrrolidin-3-ylmethyl)propanamide

Removing the protective group using General Scheme 2 Reaction of the product of deprotection; 50eq; 70% yield; UPLC-MS: Rt = 0.81min ; MS m / z [M + H] + 538.8; Method A; ¹ H- NMR (DMSO, 400 MHz, mixture of rotamers): δ 7:87-7.82 (1H, m), 7.51-7.52 (1H, m), 7.40-7.23 (4H, m), 7.09-7.03 (2H, m ), 6.92-6.86 (1H, m), 5.75-5.72 (1H, m), 5.36-5.32 (1H, m), 5.10-5.06 (1H, m), 4.57-4.52 (1H, m), 3.99-3.96 (1H, m), 3.80-3.76 (1H, m), 3.52-3.21 (4H, m), 2.97-2.91 (1H, m), 2.78-2.69 (2H, m), 2.55-2.50 (1H, m) , 2.08-2.04 (1H, m), 1.67-1.60 (2H, m), 1.50-1.47 (1H, m), 1.42-1.28 (6H, m), 0.90-0.80 (1H, m).

1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl )-3-((S)-1-hydroxypropan-2-yl)-1-((S)-pyrrolidin-3-ylmethyl)urea

Removing the protective group using General Scheme 2 Reaction of the product of deprotection; 50eq TFA; 60% yield; UPLC-MS: Rt = 0.82min ; MS m / z [M + H] + 584.3; Method A; ¹ H - NMR (DMSO, 400 MHz, mixture of rotamers): δ 7.84-7.82 (1H, m), 7.47-7.46 (1H, m), 7.35-7.24 (5H, m), 7.05-6.99 (1H, m) , 6.85-6.84 (1H, m), 5.92 (1H, s, br), 5.50-5.43 (2H, m), 5.10-5.03 (1H, m), 3.99-2.07 (17H, m), 1.64-0.75 ( 9H, m).

(S)-N-((R)-(1-Ethylazetidin-3-yl)(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazole -2-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

Removing the protective group using General Scheme 2 Reaction of the product of deprotection; 50eq; 45% yield; UPLC-MS: Rt = 1.45min ; MS m / z [M + H] + 570.1; Method E; ¹ H- NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.07 (1H, s, br), 8.72 (1H, s, br), 7.83-7.66 (2H, m), 7.4-7.28 (5H, m), 7.15-7.10(1H,m), 7.03-6.98(1H,m),6.23-5.65(1H,m),5.52-5.10(3H,m),4.54-4.44(1H,m),4.34-2.69(11H , m), 2.44 - 2.11 (2H, m), 1.70-1.55 (3H, m), 1.30-0.98 (3H, m).

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) 1-(Methoxycarbonyl)azetidin-3-yl)methyl)-2-hydroxypropanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4R)-3-(((S)-N-((R)-azetidin-3-yl(1-benzyl-4-(2,5-difluorophenyl)) -1H-imidazol-2-yl)methyl)-2-hydroxypropanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (20 mg, 0.027 mmol) in DCM (0.27 ml) Methyl chloroformate (0.0042 mL, 0.054 mmol), triethylamine (0.015 mL, 0.108 mmol) and DMAP (0.3 mg, 0.003 mmol) were added to the mixture and the mixture was stirred at RT for 1.5 hours. The reaction was extracted with saturated aqueous NaHCO ₃ and DCM. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to give a colorless solid of the title compound, 124% yield; UPLC-MS: Rt = 2.51min ; MS m / z [M + H] + 686.2; Method E.

3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((S)-N-(((3S,4R)- 4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanylamino)methyl)azetidin-1-carboxylate

Removing the protective group using General Scheme 2 Reaction of the product of deprotection; 50eq; 48% yield; UPLC-MS: Rt = 1.63min ; MS m / z [M + H] + 586.1; Method E; ¹ H- NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.05 (1H, s, br), 8.65 (1H, s, br), 7.83-7.66 (2H, m), 7.43-7.29 (5H, m), 7.15-7.10(1H,m), 7.00-6.98(1H,m),6.24-5.71(1H,m),5.51-5.09(3H,m),4.52-4.43(1H,m),4.22-2.76(14H , m), 2.47-2.10 (2H, m), 1.29-0.97 (3H, m).

4-((R)-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)) Methyl) propylamino)(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)piperidine-1-carboxylic acid 4-nitrobenzene ester

To (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H) -imidazol-2-yl)(piperidin-4-yl)methyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (80 mg, 0.115 mmol) in anhydrous DMF ( Diisopropylethylamine (0.20 mL, 1.15 mmol) and bis(4-nitrophenyl)carbonate (70 mg, 0.229 mmol) were added to the solution, and the mixture was stirred at RT for 1 hour. The reaction was extracted with water and EtOAc. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut elut elut elut elut ⁺ 863.0; Method A.

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) 1-(pentylamine-mercapto)piperidin-4-yl)methyl)-2-hydroxypropionamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

4-((R)-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)) Methyl) propylamino)(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)piperidine-1-carboxylic acid 4-nitrobenzene A solution of the ester (120 mg, 0.139 mmol) and pentylamine (0.024 mL, 0.209 <RTI ID=0.0> Additional pentylamine (0.048 mL, 0.417 mmol) was added and the reaction mixture was heated in a microwave to 100 &lt;0&gt;C for one hour. This cycle was repeated three more times until the reaction was completed. Potassium carbonate (23 mg, 0.167 mmol) was then added and the reaction was stirred at RT for 18 h. Subsequently the reaction, followed by washing with saturated aqueous NaHCO ₃ and DCM ₄ Cl solution and extracted with saturated NH. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness to give a yellow solid of the title compound in 60% yield; UPLC-MS: Rt = 2.75min ; MS m / z [M + H] + 769.6; Method E.

4-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((S)-N-(((3S,4R)- 4- Flurpyrrolidin-3-yl)methyl)-2-hydroxypropionylamino)methyl)-N-pentylpiperidine-1-carboxamide

The product was deprotected using a general scheme of removal of the protecting group; 50 eq; 50% yield, as TFA salt; UPLC-MS: Rt = 1.86 min; MS m/z [M+H] ⁺ 669.4; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.11-9.02 (1H, m), 8.84 (1H, br), 7.86-7.68 (2H, m), 7.41-7.38 (2H, m ), 7.35-7.31 (1H, m), 7.28-7.26 (1H, m), 7.15-7.07 (2H, m), 6.35-6.32 (1H, br), 5.62-5.02 (4H, m), 4.54-4.36 (1H, m), 4.01-3.18 (9H, m), 2.95-2.94 (2H, m), 2.69-2.02 (4H, m), 1.45-1.17 (11H, m), 0.94-0.84 (5H, m) , 0.49-0.40 (1H, m).

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-Ethylpiperidin-4-yl)(1-phenylmethyl-4-) (2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

By (S)-N-((R)-(1-ethenylazetidin-3-yl)(1-benzyl-4-(2,5-difluorophenyl)- 1H-imidazol-2-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionamide is formed in a similar reaction to give a product; Quantitative yield; UPLC-MS: Rt = 2.53 min; MS m/z [M+H] ⁺ 740.5;

(S)-N-((R)-(1-Ethylpiperidin-4-yl)(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2- Methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionamide

Removal of the protecting group using the general scheme 1 for the removal of the protecting group; 37% yield; UPLC-MS: Rt = 1.46 min; MS m/z [M+H] ⁺ 598.4; Method E; ¹ H-NMR ( DMSO, 400 MHz, mixture of rotamers): δ 8.97-8.80 (1H, m), 8.63-8.53 (1H, m), 7.79-7.62 (2H, m), 7.34-6.99 (7H, m), 5.59- 5.44 (1H, m), 5.33-4.95 (3H, m), 4.45-1.98 (14H, m), 1.88-1.79 (3H, m), 1.47-0.26 (7H, m).

4-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((S)-N-(((3R,4R)- Ethyl 1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionamido)methyl)piperidine-1-carboxylate

By (3R,4R)-3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-) (1-(Methoxycarbonyl)azetidin-3-yl)methyl)-2-hydroxypropanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester A similar reaction was formed, using ethyl chloroformate instead to give the product; quantitative yield; UPLC-MS: Rt = 2.94 min; MS m/z [M+H] ⁺ 770.5;

4-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((S)-N-(((3S,4R)- Ethyl 4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionylamino)methyl)piperidine-1-carboxylate

The product was deprotected using a general scheme of removal of the protecting group; 24% yield; UPLC-MS: Rt = 1.78 min; MS m/z [M+H] ⁺ 628.5; Method E; ¹ H-NMR ( DMSO, 400 MHz, mixture of rotamers): δ 9.04-8.96 (1H, m), 8.68-8.59 (1H, m), 7.87-7.70 (2H, m), 7.42-7.07 (7H, m), 5.64 5.52 (1H, m), 5.41-5.02 (3H, m), 4.53-4.35 (1H, m), 4.03-3.18 (11H, m), 2.86-2.57 (3H, m), 2.27-2.06 (1H, m ), 1.51-1.37 (1H, m), 1.31-0.40 (9H, m).

4-(((tert-butyldimethylmethylalkyl)oxy)methyl)pyrrolidin-2-one

To a solution of 4-(hydroxymethyl)pyrrolidin-2-one (1.00 g, 8.69 mmol) in EtOAc (EtOAc) (EtOAc) g, 10.4 mmol), and the reaction mixture was stirred at RT for 3 h. The reaction was concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut elut elut elut ] ⁺ 230.0; Method E.

1-((Benzyloxy)methyl)-4-(((t-butyldimethylmethyl)alkyl)oxy)methyl)pyrrolidin-2-one

To a solution of 4-(((tert-butyldimethylmethyl)alkyl)methyl)pyrrolidin-2-one (2.00 g, 8.72 mmol) in THF (29 mL) 9.59 mmol) and the reaction was stirred at RT for 30 min. ((Chloromethoxy)methyl)benzene (2.05 g, 13.1 mmol) was then added and the mixture was stirred at RT for 18 h. The reaction was quenched with aqueous saturated NH ₄ Cl and extracted with DCM. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut elut elut elut elut ] ⁺ 372.2; Method E.

1-((benzyloxy)methyl)-4-(hydroxymethyl)pyrrolidin-2-one

To 1-((benzyloxy)methyl)-4-(((t-butyldimethyl)alkyl)oxy)methyl)pyrrolidin-2-one (340 mg, 973 mmol) in THF (3.2 TBAF (254 mg, 0.973 mmol) was added to the solution in ml), and the mixture was stirred at RT for 1.5 hr. The reaction was quenched with aqueous saturated NH ₄ Cl and extracted with DCM. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by 0-100% EtOAc in heptane eluting the chert chromatography to give a clear oil, 90% _{^{yield; 1 H-NMR (CDCl 3}} , 400MHz): δ 7.55-7.7.27 (5H,m),4.89-4.85(2H,s),4.55-4.52(2H,s),3.65-3.53(3H,m),3.35-3.31(1H,m),2.56-2.48(2H,m) , 2.27-2.20 (1H, m).

4-((((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl) Methyl)amino)methyl)-1-((benzyloxy)methyl)pyrrolidin-2-one

The product was synthesized in a similar manner to Example 1 using 1-((benzyloxy)methyl)-4-(hydroxymethyl)pyrrolidin-2-one to give the product as a mixture of diastereomers. ; 36% yield; UPLC-MS: Rt = 1.88 min; MS m/z [M+H] ⁺ 601.4;

Acetic acid (2S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran) 4-yl)methyl)((1-((benzyloxy)methyl)-5-oxooxypyrrolidin-3-yl)methyl)amino)-1-yloxypropan-2- Base ester

The product was synthesized in a similar manner to Example 1; 35% yield; UPLC-MS: Rt = 2.59 min; MS m/z [M+H] ⁺ 715.5;

(2S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-N-((1-((benzyloxy)methyl)-5-oxoxypyrrolidin-3-yl)methyl)-2-hydroxypropionamide

To acetic acid (2S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran) 4-yl)methyl)((1-((benzyloxy)methyl)-5-oxoxypyrrolidin-3-yl)methyl)amino)-1-yloxypropane-2 Potassium carbonate (21 mg, 20.9 mmol) was added to a solution of <RTI ID=0.0></RTI><RTIgt; The reaction was quenched with aqueous saturated NH ₄ Cl and extracted with DCM. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness to give the product as a pale yellow solid, in 95% yield; UPLC-MS: Rt = 1.21min ; MS m / z [M + H] ⁺ 673.3; Method A.

(2S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-2-hydroxy-N-((5-oxooxypyrrolidin-3-yl)methyl)propanamide

(2S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4 -yl)methyl)-N-((1-((benzyloxy)methyl)-5-oxoxypyrrolidin-3-yl)methyl)-2-hydroxypropanamide (65 mg, 0.096 A solution of 1.25 M HCl in MeOH (1. <RTI ID=0.0></RTI></RTI><RTIgt; The reaction was quenched with saturated aqueous NaHCO ₃ and extracted with DCM. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by 0-100% EtOAc in heptane eluting the chert chromatography to give the title compound, 13% yield; UPLC-MS: Rt = 0.97min ; MS m / z [M + H] + 553.4; Method A; ¹ H-NMR (CDCl ₃ , 400 MHz, mixture of rotamers): δ 7.76-7.70 (1H, m), 7.45-7.42 (1H, m), 7.29-7.24 (3H, m), 7.14-7.13(2H,m), 7.00-6.92(1H,m),6.83-6.79(1H,m),5.66-5.63(1H,m),5.25-5.19(1H,m),5.01-4.95(1H , m), 4.33-4.26 (1H, m), 3.90-3.87 (1H, m), 3.71-3.68 (1H, m), 3.54-3.08 (5H, m), 2.91-2.74 (1H, m), 2.68 -2.59 (1H, m), 2.35-0.71 (8H, m).

(R)-3-(2-((1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethyl-) 4-tert-oxy-4-(pentylamino)butyl)amine-methylhydrazinyl)phenoxy)azetidin-1-carboxylic acid tert-butyl ester

To (R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-(2-((1-(t-butoxy)) Addition of HATU to a solution of carbonyl)azetidin-3-yl)oxy)benzhydrylamino)-3,3-dimethylbutyric acid (30 mg, 0.044 mmol) in DMF (0.45 ml) (20.3 mg, 0.053 mmol), diisopropylethylamine (0.04 mL, 0.222 mmol) and pentylamine (0.006 mL, 0.049 mmol), and the mixture was stirred at RT for 1 hour. Additional HATU (8.5 mg, 0.022 mmol), diisopropylethylamine (0.023 mL, 0.133 mmol) and pentamine (0.005 mL, 0.044 mmol). Reaction and 2M Na ₂ CO ₃ and extracted aqueous with EtOAc. The organic layers were combined, dried over Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut M+H] ⁺ 744.3; Method E.

(R)-2-(azetidin-3-yloxy)-N-(1-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazole-2 -yl)-2,2-dimethyl-4-oxo-4-(pentylamino)butyl)benzamide

The product was stripped of the protecting group using the general scheme of removal of the protecting group; 20 eq of TFA; 83% yield (TFA salt); UPLC-MS: Rt = 1.96 min; MS m/z [M+H] ⁺ 644.2; E; ¹ H-NMR (DMSO, 400MHz): δ 9.01-9.00 (1H, m), 8.94-8.85 (2H, m), 7.97-7.94 (1H, m), 7.78-7.74 (1H, m), 7.65 -7.63 (1H, m), 7.59-7.58 (1H, m), 7.46-7.42 (1H, m), 7.38-7.24 (6H, m), 7.11-7.06 (2H, m), 6.82-6.80 (1H, m), 5.56-5.39 (3H, m), 5.18-5.12 (1H, m), 4.50-4.46 (1H, m), 4.40-4.36 (1H, m), 4.30-4.24 (1H, m), 4.18- 4.12 (1H, m), 3.10-2.93 (2H, m), 2.77-2.74 (1H, m), 2.23-2.20 (1H, m), 1.38-1.31 (2H, m), 1.24-1.19 (4H, m ), 1.09 (3H, s), 0.94 (3H, s), 0.83-0.79 (3H, m).

Synthesis Example 35. Benzyl modification.

(R)-1-(4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-amine, TFA salt

11g(R)-(1-(4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)amino A solution of tert-butyl formate and 9 mL of TFA in 150 mL DCM was stirred at room temperature for 16 h. The reaction mixture was concentrated in vacuo to afford 18.8 g of brown/green oil. This material was used directly in the next step (assuming 100% conversion and 48.5% purity). UPLC-MS: Rt = 0.73 min; MS m/z [M+H] ⁺ 278.2;

(3R,4R)-3-((((R)-1-(4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylbutane-3 -en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester

Step 1: Place a stirred solution of (3R,4S)-3-fluoro-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid benzyl ester (8.28 g) in anhydrous DCM (100 mL) under argon The temperature of the solution is kept constant by using a water bath containing ambient temperature water. (In a similar reaction, we found that the temperature control is important: if the reaction mixture temperature a few degrees hotter, places certain extent occurs in addition to a fluorine-yl] Similarly, if the reaction at colder temperatures (0 ℃ -10 ℃) performed, the oxidation The reaction was not fast enough.) Next, Dess-Martin periodinane (15.85 g) was added in one portion and the reaction mixture was stirred at (about) 18 ° C for 20 minutes and then directly used in step 2.

Step 2: The reaction mixture of step 1 was filtered through a 0.45 μm PTFE syringe filter to (R)-1-(4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2 a stirred solution of dimethylbut-3-en-1-amine, TFA salt (18.84 g), and sodium triethyloxyborohydride (19.8 g) in anhydrous DCM (150 mL). Note: This solution was previously ultrasonicated to disperse and dissolve any bulk triethylphosphonium borohydride. Stirring was continued under an argon atmosphere while ensuring that the temperature did not exceed 20 ° C (by using a tap water bath). After 2 hours, LC-MS showed the reaction to actually complete 31% conversion to the desired product. The reaction was quenched by the addition of deionized water (150 mL) and the mixture was stirred vigorously at RT for one hour. The obtained inorganic solid was filtered through a No. 1 filter paper and the filtrate was slowly made alkaline by the addition of sodium hydrogencarbonate in portions while being vigorously stirred. Once the sodium bicarbonate addition was complete (no more foaming was observed), the two phase solution was transferred to a separatory funnel and the layers were separated. The aqueous layer was re-extracted with dichloromethane (2×100 mL). The combined organics were washed with EtOAc EtOAc m. This material was purified directly as follows:

System : Biotage SP4 Normal Phase Purification System

Stationary phase : 40+M gangue

Binary solvent system: polar solvent: 20% v/v methanol/dichloromethane, non-polar solvent: dichloromethane

Gradient : 0% non-polar/polar solvent was maintained for 25 minutes and then ramped up to 40% polar solvent over a 35 minute period and then maintained at this level for an additional 15 minutes at which time the product had dissolved from the column. The product-containing fractions were combined and concentrated in vacuo to yield 12.65 g of dark brown glassy oil which was found to be about 47% pure by LCMS. UPLC-MS: Rt = 1.15 min; MS m/z [M+H] ⁺ 514.3;

This material was used directly in the next step.

(3R,4R)-3-((((R)-1-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy)benzyl)-1H) -imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester

To (3R,4R)-3-((((R)-1-(4-(2,5-difluorophenyl)-1H-imidazol-2-yl) at room temperature under argon -2,2-Dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester (12.65 g) and methanesulfonic acid 3-(A) Potassium carbonate (8.02 g) was added to a solution of oxymethoxy) phenylmethyl ester (8.57 g, prepared according to Bioorg. Med. Chem. 2006 , 14 , 1771-1784) in anhydrous DMF (100 ml). A water condenser was assembled and the mixture was stirred at 75 ° C for 3 hours at which time LC-MS showed the reaction was completed. The reaction mixture was cooled to rt and partitioned between EtOAc (EtOAc) After extraction, the aqueous phase was re-extracted with ethyl acetate (150 mL). The combined organics were washed with EtOAc EtOAc m. Crude product yield: 19.88 g. This material was purified directly as follows: System : Biotage SP4 Normal Phase Purification System

Stationary phase : 40+M gangue

Binary solvent system: polar solvent: ethyl acetate, non-polar solvent: n-heptane

Gradual gradient :

1. 0% polar solvent --> maintain for 5 minutes

2. 0% polar solvent --> ramp up to 5% polar solvent after 15 minutes

3. 5% polar solvent --> maintain for 10 minutes

4. 5% polar solvent --> ramp up to 7% polar solvent after 10 minutes

5. 7% polar solvent --> 20 minutes

6. 7% polar solvent --> ramp up to 12% polar solvent after 10 minutes

7. 12% polar solvent --> maintain for 3 minutes

8. 12% polar solvent --> ramp up to 20% polar solvent after 10 minutes

9. 20% polar solvent --> maintain for 10 minutes

10. 20% polar solvent --> ramp up to 25% polar solvent after 10 minutes

11. 25% polar solvent --> maintain for 30 minutes

The desired product begins to elute from the column between 7% polar solvent and 12% polar solvent. The product-containing fractions were combined and concentrated in vacuo to give a brown oil. Yield: 7.7 g, found to be 65% pure by LC-MS. UPLC-MS: Rt = 1.25 min; MS m/z [M+H] ⁺ 664.5;

This material is used directly in the next step

(3R,4R)-3-((((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-(methoxymethoxy)) phenyl) -1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester Similar to (3R,4R)-3-((((R)-1-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy))phenyl)) Preparation of -1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester

UPLC-MS: Rt = 5.73 min; Method B.

This material is used directly in the next step

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-() Methoxymethoxy)benzyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)propanylamino)methyl)-4-fluoro Pyrrolidine-1-carboxylic acid benzyl ester

(3R,4R)-3-((((R)-1-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy)benzyl)-1H) -imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester (3.85 g) in anhydrous The solution in DCM (50 mL) was cooled to 0 °C using an ice bath. To this solution, DIPEA (1.978 mL) and (S)-acetic acid 1-chloro-1-oxopropan-2-yl ester (0.956 mL, Fluka) were added, and turbid smoke was observed. The dark orange reaction mixture was allowed to warm to room temp. and stirring was continued for 1 hour, at which time LC-MS showed the reaction was completed and converted to the desired product. The volatiles were removed under high vacuum and the crude material was purified directly as follows: System : Biotage SP4 normal phase purification system

Stationary phase : 40+M gangue

Binary solvent system: polar solvent: ethyl acetate, non-polar solvent: n-heptane

Gradual gradient :

1. 0% polar solvent --> maintain 10 minutes

2. 0% polar solvent --> ramp up to 46% polar solvent after 20 minutes

3. 46% polar solvent --> maintain for 30 minutes. As of this stage, all the required products are Has been dissolved from the column. The operation stops at this moment.

The product-containing fractions were combined and concentrated in vacuo to give a brown oil.

Yield: 2.545 g, found to be 95% pure by LC-MS. UPLC-MS: Rt = 1.49 min; MS m/z [M+H] ⁺ 777.5;

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro) -5-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)propanylamino)methyl) -4-fluoropyrrolidine-1-carboxylic acid benzyl ester

(3R,4R)-3-((((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-(methoxymethoxy)) phenyl) Benzyl-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylic acid benzyl ester (1.2 g) The solution in anhydrous DCM (20 mL) was cooled to 0. To this solution, DIPEA (0.822 mL) and (S)-acetic acid 1-chloro-1-oxopropan-2-yl ester (0.397 mL, Fluka) were added, and turbid smoke was observed. The dark orange reaction mixture was allowed to warm to room temp. and stirring was continued for 1 hour, at which time LC-MS showed the reaction was completed and converted to the desired product. The reaction mixture was then partitioned between deionized water (100 mL) and dichloromethane (EtOAc). After extraction, the aqueous phase was re-extracted with dichloromethane (70 mL). The combined organics were washed with EtOAc EtOAc m. Yield: 1.4g UPLC-MS: Rt = 1.46min; MS m / z [M + H] + 796.5; Method A.

This material has the appropriate purity for direct use in the next step without further purification Chemical.

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-() Methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine- Benzoic acid benzoate

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-() Methoxymethoxy)benzyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)propanylamino)methyl)-4-fluoro A solution of pyrrolidine-1-carboxylic acid benzyl ester (2.545 g) in dry THF (25 mL) was cooled to 0 &lt;0&gt;C using an ice bath and then treated by dropwise addition of 1.0 M borane THF complex (12.45 mL). . The reaction mixture was allowed to warm to room temperature and stirring was continued for 4 h. The reaction mixture was then cooled to 0.degree. C. and excess borane was quenched by successively adding 1:1 mixture THF: EtOH (70mL), pH 7.0 phosphate buffer (90mL) and 30% hydrogen peroxide solution (4.45mL). The reaction mixture was stirred at room temperature for 16 h and then brine (15 mL) was then evaporated. The mixture was transferred to a sep. funnel and extracted with ethyl acetate (2×200 mL). The combined organics were washed with EtOAc EtOAc EtOAc EtOAc.

Yield: 2.5917 g, found to be 61% pure by LC-MS. This material was used in the next step without purification. UPLC-MS: Rt = 1.29 min; MS m/z [M+H] ⁺ 795.6;

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro) -5-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4- Fluoropyrrolidine-1-carboxylate

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-) Fluoro-5-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)propanylamino)methyl A solution of -4-fluoropyrrolidine-1-carboxylic acid benzyl ester (1.4 g) in dry THF (25 mL) was placed under argon and cooled to 0 °C using an ice bath. It was then treated by dropwise addition of 1.0 M borane THF complex (6.27 mL). The reaction mixture was allowed to warm to room temperature and stirring was continued for 4 h. The mixture was then cooled to 0 ° C and excess borane was quenched by successive addition of a 1:1 mixture of THF: EtOH (70mL), pH 7.0 phosphate buffer (90mL) and 30% hydrogen peroxide solution (2.24mL). The mixture was stirred at room temperature for 16 hours and then saturated brine (15 mL) was then added to mixture. The mixture was transferred to a sep. funnel and extracted with ethyl acetate (2×200 mL). The combined organics were washed with EtOAc EtOAc EtOAc (EtOAc) Yield: 1.445 g, found to be 74% pure by LC-MS. This material was used in the next step without purification. UPLC-MS: Rt = 1.28 min; MS m/z [M+H] ⁺ 814.7;

(S)-acetic acid 1-(((R)-1-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy)benzyl)-1H-imidazole) -2-yl)-4-hydroxy-2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-yloxy Prop-2-yl ester

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl))-) 1-(3-(Methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)- Ammonium formate (2.508 g) and 5 wt% palladium on carbon (423 mg) were added to a solution of 4-fluoropyrrolidine-1-carboxylic acid benzyl ester (2.59 g) in methanol (50 ml). The resulting black suspension was stirred at 50 ° C for 2 hours under an argon atmosphere, at which time LC-MS showed the reaction was completed. The reaction mixture was filtered through a No. 1 filter paper, and the palladium catalyst cake was washed with methanol (2×30 ml) and dichloromethane (30 ml). The filtrate was then used directly in the next step. (Which assumes 100% conversion, 60% pure.) UPLC-MS: Rt = 0.85min; MS m / z [M + H] + 661.8; Method A.

(S)-acetic acid 1-(((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-(methoxymethoxy)) phenyl) -1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino) -1-sided oxypropan-2-yl ester

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl))-) 1-(3-Fluoro-5-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino) Ammonium formate (2.489 g) and 5 wt% palladium on carbon (280 mg) were added to a solution of benzylmethyl -4-fluoropyrrolidine-1-carboxylate (1.445 g) in methanol (20 ml). The resulting black suspension was stirred at 50 ° C for 1.5 hours under an argon atmosphere, at which time LC-MS showed the reaction was completed. The reaction mixture was filtered through a No. 1 filter paper, and the palladium catalyst cake was washed with methanol (2×30 ml) and dichloromethane (30 ml). The filtrate was then used directly in the next step. (It assumes 100% conversion, 76% purity.)

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-() Methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine- 1-butyl formate

(S)-Acetic acid 1-(((R)-1-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy))benzyl) -1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)- Additional methanol (50 mL) and BOC anhydride (0.864 g) were added to the filtrate of 1-side oxypropan-2-yl ester . The reaction mixture was stirred at RT for 30 minutes at which time LC-MS showed &lt;EMI ID&gt; The volatiles were concentrated in vacuo and EtOAcqqqqqqq The mixture was transferred to a sep. funnel and after extraction, the aqueous was extracted with ethyl acetate (100 mL). Note: An emulsion is formed during the extraction process. This emulsion was resolved by adding a small amount of saturated brine to a separatory funnel . The combined organics were washed with aq. EtOAc (EtOAc)EtOAc. Crude product yield: 2.695 g, 56% pure by LC-MS at 254 nm. Purification was carried out as follows: System : Biotage SP4 Normal Phase Purification System

Stationary phase : 25+M gangue

Binary solvent system: polar solvent: ethyl acetate, non-polar solvent: n-heptane

Gradual gradient :

1. 0% polar solvent --> maintain for 5 minutes

2. 0% polar solvent --> ramp up to 80% polar solvent after 30 minutes

3. 80% polar solvent --> 25 minutes

4. 80% polar solvent --> ramp up to 100% polar solvent after 10 minutes

5. 100% polar solvent --> maintain for 15 minutes

The product-containing fractions were combined and concentrated in vacuo to give a white foam. Yield: 1.25 g, 87% pure by LC-MS at 254 nm. UPLC-MS: Rt = 1.30 min; MS m/z [M+H] ⁺ 761.6;

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro) -5-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanamide Tert-butyl)methyl)-4-fluoropyrrolidine-1-carboxylate

To a solution of freshly prepared (S) - acetic acid 1 - (((R) -1- (4- (2,5- difluorophenyl) -1- (3-fluoro-5- (methoxymethoxy Benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl) Additional methanol (20 mL) and BOC anhydride (0.767 g) were added to the filtrate of the amino)-1-oxopropan-2-yl ester . The reaction mixture was stirred at room temperature for 1 hour at which time LC-MS showed &lt;EMI ID&gt; The volatiles were concentrated in vacuo and purified directly as follows: System : Biotage SP4 normal phase purification system

Stationary phase : 25+M gangue

Binary solvent system: polar solvent: ethyl acetate, non-polar solvent: n-heptane

Gradual gradient :

1. 0% polar solvent --> maintain for 5 minutes

2. 0% polar solvent --> ramp up to 50% polar solvent after 30 minutes

3. 50% polar solvent --> maintain for 10 minutes

4. 50% polar solvent --> ramp up to 80% polar solvent after 20 minutes

5. 80% polar solvent --> maintain for 10 minutes

The product-containing fractions were combined and concentrated in vacuo to yield an almost clear and colorless waxy oil. Yield: 0.676 g, 96% pure by LC-MS at 254 nm. UPLC-MS: Rt = 1.28 min; MS m/z [M+H] ⁺ 780.5;

(S)-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-hydroxybenzyl)-1H-imidazol-2-yl)-4-hydroxyl -2,2-dimethylbutyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

To (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-) (methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine 6M HCl (0.998 mL) was added to a solution of 1- butyl formate (50 mg) in acetonitrile (1 ml) and MeOH was stirred at 60 ° C for 3 hours, at which time LC-MS showed the reaction was completed and completely converted to Needed. The mixture was filtered through a 0.2 [mu]m PTFE cartridge filter and the resulting clear solution was purified by reverse phase preparative HPLC method C. The product-containing fractions were combined and concentrated in vacuo to give an off-white powder. Yield: 12.9 mg UPLC-MS: Rt = 0.77 min; MS m/z [M+H] ⁺ 575.2; UPLC-MS: Rt = 2.87 min; MS m/z [M+H] ⁺ 575.2;

(S)-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-hydroxybenzyl)-1H-imidazol-2-yl) 4-hydroxy-2,2-dimethylbutyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionamide

To (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-) Fluoro-5-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4 To a solution of fluoropyrrolidine-1-carboxylic acid tert-butyl ester (50 mg) in acetonitrile (1 ml) was added 6M HCl (1.03 mL) and the mixture was stirred at 60 ° C for 2 hours, at which time LC-MS showed that the reaction was completed. Complete conversion to the desired product. The mixture was filtered through a 0.2 [mu]m PTFE cartridge filter and the resulting clear solution was purified by reverse phase preparative HPLC method C. The product-containing fractions were combined and lyophilized overnight to yield an off-white loose powder. Yield: 19.4mg UPLC-MS: Rt = 0.70min; MS m / z [M + H] + 593.1; Method A. UPLC-MS: Rt = 2.59 min; MS m/z [M+H] ⁺ 593.2; ^{1 H NMR (600MHz, DMSO-} d 6) δ 10.10 (s, 1H), 9.04 (s, 1H), 8.71 (s, 1H), 7.91 (s, 1H), 7.80-7.73 (m, 1H), 7.41 -7.30 (m, 1H), 7.18-7.09 (m, 1H), 6.69-6.57 (m, 2H), 6.52 (dd, J = 16.2, 10.3 Hz, 1H), 5.80 (s, 1H), 5.39-5.21 (m, 2H), 4.97 (d, J = 15.5 Hz, 1H), 4.64 - 4.57 (m, 1H), 4.08 - 3.98 (m, 1H), 3.95 - 3.85 (m, 1H), 3.37 - 3.27 (m , 2H), 3.23 (dt, J = 9.9, 5.0 Hz, 2H), 2.48-2.36 (m, 1H), 2.08-1.95 (m, 1H), 1.94-1.86 (m, 1H), 1.51 (ddd, J = 23.3, 13.9, 6.7 Hz, 1H), 1.35 (t, J = 7.0 Hz, 3H), 1.30-1.22 (m, 1H), 0.93 (s, 3H), 0.84 (s, 3H).

(R)-4-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)) Propionylamino)-4-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy)benzyl)-1H-imidazol-2-yl) -3,3-dimethylbutyric acid

To (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-) (methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine To the cooled (0 ° C) solution of 1-butylic acid tert-butyl ester (520 mg) in anhydrous DMF (10 mL) was added EtOAc. After the addition was completed, the dark brick red/yellow orange RM was stirred overnight at RT, at which time the color had turned dark brown. RM was partitioned between ethyl acetate (150 mL) and deionized water (100 mL). The mixture was transferred to a sep. funnel and after extraction the aqueous was extracted with ethyl acetate (2×100 mL). The combined organics were washed with EtOAc (EtOAc)EtOAc. The crude product yield: 563.8mg UPLC-MS: Rt = 1.27min; MS m / z [M + H] + 775.4; [MH] - 773.4 Method A.

(R)-4-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidine- 3-yl)methyl)propanylamino)-4-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-(methoxymethoxy)phenylmethyl) -1H-imidazol-2-yl)-3,3-dimethylbutyric acid

To (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3-) Fluoro-5-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4 To a cooled (0 ° C) solution of fluoropyrrolidine-1-carboxylic acid tert-butyl ester (300 mg) in anhydrous DMF (5 mL) was added EtOAc (EtOAc). After the addition was completed, the dark brick red/yellow orange RM was stirred overnight at RT, at which time the color had turned dark brown. RM was partitioned between ethyl acetate (100 mL) and deionized water (100 mL). The mixture was transferred to a sep. funnel and after extraction the aqueous was extracted with ethyl acetate (2×100 mL). The combined organics were washed with EtOAc (EtOAc)EtOAc. The crude product yield: 322mg UPLC-MS: Rt = 1.26min; MS m / z [MH] - 791.2 Method A.

(R)-4-(4-(2,5-difluorophenyl)-1-(3-hydroxybenzyl)-1H-imidazol-2-yl)-4-((S)-N-( ((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanylamino)-N,3,3-trimethylbutyramine

Step 1: To (R)-4-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidine-3) -yl)methyl)propanylamino)-4-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy)benzyl)-1H-imidazole- HATU (124 mg) was added to a solution of 2-yl)-3,3-dimethylbutyric acid (60 mg) and DIPEA (189 uL) in anhydrous DMF (2 mL). The RM was stirred at RT for 10 minutes and then methylamine hydrochloride (73 mg) was added. The RM was stirred overnight at RT, at which time LC-MS showed the reaction was complete. The volatiles were removed under high vacuum and the crude residue was dissolved in 1:1 mixture acetonitrile: water (4ml). The RM was filtered through a 0.2 [mu]m PTFE cartridge filter and the resulting clear solution was divided into 2x2 mL batches and purified by reverse phase preparative HPLC method C. The product-containing fractions from the two runs were combined and concentrated in vacuo to give a light blue solid: yield: 15 mg. This material was used directly in the next step: Step 2: Removal of BOC and MOM protecting groups : acetonitrile (1 ml) and 6M HCl (1.08 mL) were added to the crude product from step 1. The RM was stirred at 50 °C for 2 hours at which time LC-MS showed the reaction was completed and the protecting group was removed to yield the desired final product. The RM was filtered through a 0.2 [mu]m PTFE cartridge filter and then purified by reverse phase preparative scale HPLC Method C. The product containing fractions were lyophilized overnight to yield a white loose powder. Yield: 8.9 mg.

UPLC-MS: Rt = 2.41 min; MS m/z [M+H] ⁺ 603.3;

1H-NMR (HSQC) consistent with the target structure.

(R)-4-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-hydroxybenzyl)-1H-imidazol-2-yl)-4-((S) -N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanyl)-N,3,3-trimethylbutyramine

Step 1 : To (R)-4-((S)-2-Ethyloxy-N-(((3R,4R)-1-(T-butoxycarbonyl)-4-fluoropyrrolidine-3) -yl)methyl)propanylamino)-4-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-(methoxymethoxy)phenylmethyl)- HATU (173 mg) was added to a solution of 1H-imidazol-2-yl)-3,3-dimethylbutyric acid (60 mg) and DIPEA (264 mL). The RM was stirred at RT for 10 minutes and then methylamine hydrochloride (102 mg) was added. The RM was stirred at RT for 2 hours at which time LC-MS showed the reaction was completed. The RM was concentrated under high vacuum to give a brown waxy oil. This material was used directly in the next step.

Step 2: Removal of BOC, MOM and thiol protecting groups : The crude product from Step 1 was dissolved in acetonitrile (2 mL) and 6M HCl (1.5 mL). The RM was stirred at 60 ° C for 2 hours at which time LC-MS showed the reaction was completed and the protecting group was removed to give a final product. RM was then partitioned between deionized water (50 mL) and ethyl acetate (50 mL). After extraction, the aqueous phase was re-extracted with ethyl acetate (70 mL). The combined organics were washed with EtOAc EtOAc (EtOAc)EtOAc. RM was dissolved in 2:1 water: acetonitrile solution (4 mL) and this solution was filtered through a 0.2 [mu]m PTFE cartridge filter. The resulting solution was divided into 2 x 2 mL batches and purified by reverse phase preparative scale HPLC Method C. The product-containing fractions were combined and then lyophilized overnight to yield a white loose powder. Yield: 18.9 mg.

UPLC-MS: Rt = 2.41 min; MS m/z [M+H] ⁺ 618.3;

1H-NMR (HSQC) consistent with the target structure.

Synthetic payload-linker combination

Cleavable linker: MC-ValCit-PABC ADC: Add i Pr ₂ EtN (6 eq) and 4-((S)-2-carbonate to a solution of the payload (0.05 mmol, 1 eq) in DMSO (0.1 M) ((S)-2-(6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutanamine) 5-5-ureidopentylamino)benzyl (4-nitrophenyl) ester (0.09 mmol, 1.6 eq). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude product was filtered to remove solids and purified directly by reverse phase column chromatography (using PrepLC method C or D) to afford TFA salt. Required MC-ValCit-PABC-payload.

3-((())-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin喃-4-yl)methyl)-2-hydroxypropylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid (3R,4R)-4-((S)-2-((S) -2-(6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutyrylamino)-5-urea Benzymidino) phenylmethyl ester.

^{1 H-NMR (DMSO, 600MHz} ): δ 9.98 (1H, m), 8.09 (1H, m), 7.80 (1H, m), 7.74 (1H, m), 7.71 (2H, m), 7.56 (2H, m), 7.37 (3H, m), 7.31 (4H, m), 7.09 (1H, m), 7.00 (2H, m), 6.01 (1H, m), 5.68 (1H, m), 5.28 (1H, m), 5.18 (1H, m), 5.15 (2H, m), 5.01 (1H, m), 4.88 (1H, m), 4.73 (1H, m) 4.50 (1H, m), 4.39 (1H, m) , 4.21 (1H, m), 3.80 (1H, m), 3.50-3.25 (8H, m), 3.37 (2H, m), 3.03 (1H, m), 2.98 (1H, m), 2.82 (1H, m ), 2.60 (1H, m), 2.30 (1H, m), 2.20 (1H, m), 2.18 (1H, m), 2.14 (1H, m), 2.13 (1H, m), 2.12 (1H, m) , 1.98 (1H, m), 1.71 (1H, m), 1.50 (3H, m), 1.49-1.35 (3H, m), 1.27 (3H, m), 1.23 (1H, m), 1.10 (1H, m ), 0.98 (1H, m), 0.86 (6H, m), 0.70 (1H, m). Missing peaks hidden under the solvent peak. LC/MS (uplc): M+: 1155.5, 1.07 min (Method A).

3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid (3R,4R)-4-((S) -2-((S)-2-(6-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutanthene Amino)-5-ureidoisoamylamino) phenylmethyl ester.

^{1 H-NMR (DMSO, 600MHz} ): δ 9.99 (1H, m), 8.09 (1H, m), 7.80 (1H, m), 7.71 (2H, m), 7.57 (3H, m), 7.35 (2H, m), 7.30 (4H, m), 7.23 (1H, m), 7.09 (1H, m), 7.00 (2H, s), 5.97 (2H, m), 5.35 (2H, m), 5.07 (2H, m ), 4.89 (1H, m), 4.77 (1H, m), 4.42 (1H, m), 4.20 (1H, m), 3.84 (1H, m), 3.79 (1H, m), 3.64 (2H, m) , 3.40-3.23 (8H, m), 3.03 (1H, m), 2.96 (2H, m), 2.81 (1H, m), 2.57 (1H, m), 2.25 (1H, m), 2.15 (2H, m ), 2.00 (1H, m), 1.66 (2H, m), 1.52-1.25 (8H, m), 1.21 (2H, m), 1.07 (3H, m), 0.84 (6H, m), 0.77 (1H, m). Missing peaks hidden under the solvent peak. LC/MS (uplc): M+ 1184.5, 1185.5 (+H), 1.04 min (Method A).

3-(((3S,4R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-3,4-dihydroxypyrrolidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid (3R,4R)-4-( (S)-2-((S)-2-(6-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-yl Butylamino)-5-ureidopentylamino) phenylmethyl ester.

^{1 H-NMR (DMSO, 600MHz} ): δ 9.99 (1H, m), 8.08 (1H, m), 7.80 (1H, m), 7.71 (2H, m), 7.58 (2H, m), 7.36 (2H, m), 7.21 (2H, m), 7.20 (2H, m), 7.17 (2H, m), 7.09 (1H, m), 7.00 (2H, s), 6.01 (1H, m), 5.75 (1H, m ), 5.37 (1H, m), 5.13 (1H, m), 4.89 (3H, m), 4.41 (1H, m), 4.21 (1H, m), 4.06 (1H, m), 3.98 (1H, m) , 3.82 (1H, m), 3.41 (1H, m), 3.37 (2H, m), 3.32 (2H, m), 3.13 (2H, m), 3.05 (1H, m), 2.97 (2H, m), 2.38 (1H, m), 2.35-2.05 (3H, m), 1.97 (2H, m), 1.70 (2H, m), 1.65 (2H, m), 1.60-1.40 (6H, m), 1.36 (2H, m), 1.19 (2H, m), 1.04 (1H, m), 1.01 (1H, m), 0.87 (6H, m), 0.46 (2H, m). Missing peaks hidden under the solvent peak. LC/MS (uplc): M+ 1212.4, 2.48 min.

((S)-2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidyl) 4-((S)-2)methyl-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)aminocarbamate -((S)-2-(6-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutanamine )-5-ureido-amylamino) phenylmethyl ester.

¹ H-NMR (DMSO, 600MHz): δ 10.03-9.92 (1H, m), 9.09-8.90 (1H, m), 8.66-8.47 (1H, m), 8.14-7.96 (1H, m), 7.84-7.70 (3H, m), 7.68-7.53 (2H, m), 7.47-7.36 (2H, m), 7.36-7.21 (7H, m), 7.17-7.06 (1H, m), 7.06-6.94 (2H, m) , 6.34-6.11 (1H, m), 6.10-5.90 (1H, m), 5.41-5.09 (4H, m), 5.05-4.89 (2H, m), 4.46-4.31 (1H, m), 4.31-4.14 ( 1H, m), 3.91-3.79 (3H, m), 3.79-3.69 (1H, m), 3.65-3.53 (1H, m), 3.52-3.27 (6H, m), 3.26-3.16 (1H, m), 3.16-3.07(2H,m), 3.07-2.99(1H,m),2.99-2.89(1H,m),2.63-2.47(2H,m),2.45-2.29(1H,m),2.27-2.04(2H , m), 2.03-1.86 (2H, m), 1.77-1.65 (1H, m), 1.65-1.55 (1H, m), 1.55-1.31 (7H, m), 1.30-1.14 (4H, m), 1.11 -0.96 (3H, m), 0.91 - 0.78 (6H, m), 0.75 - 0.61 (1H, m). Missing peaks hidden under the solvent peak. LC/MS (uplc): M+ 1183.6.

3-((())-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin喃-4-yl)methyl)-2-hydroxypropylamino)methyl)-4-hydroxypyrrolidine-1-carboxylic acid (3R,4S)-4-((S)-2-((S) -2-(6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutyrylamino)-5-urea Pentylmethylamino) phenylmethyl ester

10% yield. UPLC-MS: Rt = 1.01 min; MS m/z [M+H] ⁺ 1153.4;

3-((())-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin喃-4-yl)methyl)-2-hydroxypropionylamino)methyl)pyrrolidine-1-carboxylic acid (R)-4-((S)-2-((S)-2-(6- (2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutyrylamino)-5-ureidopentadelamyl) Benzyl methyl ester

14% yield; UPLC-MS: Rt = 1.09 min; MS m/z [M+H] ⁺ 1137.4;

Suitable linker: a) urethane linkage

i Pr ₂ EtN (0.9 mmol, 10 eq), bis(4-nitrophenyl) carbonate (0.14 mmol, 1.6) was added sequentially to a solution of Boc- </ RTI> (0.09 mmol, 1 eq) in DMF (0.1 M). Eq) and stirred at room temperature for 1 hour. Upon completion of the reaction by LC/MS (uplc, method), a linker (3 eq) was added and the reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the solvent was evaporated under reduced pressure, and purified by normal phase column chromatography (PrepLC method B) to give the desired product (Boc-LP). .

b) guanamine linkage

To a solution of the carboxylic acid linker (0.23 mmol, 1.8 eq), HATU (0.27 mmol, 2.1 eq) in DMF (0.3M), i Pr ₂ EtN (1.4 mmol, 11 eq), followed by Boc-load ( 0.13 mmol, 1 eq). The reaction mixture was stirred at room temperature. When by LC / MS (uplc, Method A) reaction was complete, the reaction mixture was diluted in EtOAc and partitioned between EtOAc and H ₂ O. The organic layer was separated and washed with H ₂ O (twice), dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The desired product was obtained after purification by normal phase column chromatography (PrepLC method B).

c) Click Chemistry

To the hydride-Boc-support (0.05 mmol, 1 eq) and 1-(prop-2-yn-1-yl)-1H-pyrrole-2,5-dione (0.078 mmol, 1.5 eq) in THF ( A solution of CuSO ₄ .5H ₂ O and sodium ascorbate in H ₂ O (1 M) was added to the solution in 0.1 M). The reaction mixture was stirred at room temperature. When by LC / MS (uplc, Method A) reaction was complete, the reaction mixture was diluted in EtOAc and partitioned between EtOAc and H ₂ O. The organic layer was separated, washed with H ₂ O, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The desired product was isolated by normal phase column chromatography (PrepLC Method B).

d) sulfo-GMBS coupling

Was added to Boc- payload _{i Pr 2 EtNH (0.30mmol, 6eq} ) and 1- (2,5-oxo-2 was the ₂ Cl ₂ (0.1M) solution (0.05mmol, 1eq) in CH, 5-Dihydro-1H-pyrrol-1-yl)-4-oxo-4-(perfluorophenoxy)butane-2-sulfonic acid (0.15 mmol, 3 eq). The reaction was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the solvent was evaporated under reduced pressure. The desired product as a mixture of diastereomers was separated by normal phase column chromatography (PrepLC Method B).

General scheme 1 for connecting sub-connections.

Add i Pr ₂ EtN (10 eq), bis(4-nitrophenyl) carbonate (2.2 eq), and react at room temperature in a solution of Boc-payload (1 eq) in dry DMF (0.1 M). Stir for 30 minutes. Upon completion by LC/MS (uplc), a linker (4 eq) was added and the reaction was stirred at room temperature for 1 hour. The reaction was quenched with water and extracted with EtOAc. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting eluting elut elut

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-(((2-(2,5-di- oxo-2), 5-Dihydro-1H-pyrrol-1-yl)ethyl)amine-methylmethyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

80% yield; UPLC-MS: Rt = 1.24 min; MS m/z [M+H] ⁺ 863.1;

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-(((2-(2-(2,5-di)oxy) -2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)amine carbhydryl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

Using General Scheme 1, but using 1.5 eq of bis(4-nitrophenyl)carbonate, 1.5 eq of 2-(2-(2,5-di- oxo-2,5-dihydro-1H-pyrrole) The product was synthesized as the amine and 15 eq of DIPEA to give the desired product, 32% yield; UPLC-MS: Rt = 2.62 min; MS m/z [M+H] ⁺ 907.5 Method E.

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-(((6-(2,5-di- oxo-2, 3-Dihydro-1H-pyrrol-1-yl)hexyl)amine,carinyl)oxy)pyrrolidine-1-carboxylic acid, tert-butyl ester

Using General Scheme 1, but using 2,2,2-trifluoroacetic acid 6-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)hexan-1-ammonium as the amine The product was synthesized, to form the desired product, 86% yield; UPLC-MS: Rt = 1.35min ; MS m / z [m + H] + 919.2; method A.

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-((3-((2,5-di- oxo-2), 3-Dihydro-1H-pyrrol-1-yl)methyl)azetidin-1-ylcarbonyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

Using General Scheme 1, but using 1.5 eq of bis(4-nitrophenyl)carbonate, 1.5 eq of 2,2,2-trifluoroacetic acid 3-((2,5-di- oxo-2,5-di) Hydrogen-1H-pyrrol-1-yl)methyl)azetidin-1-indole as an amine and 15 eq of DIPEA to give the product to give the desired product, 39% yield; UPLC-MS: Rt = 2.66 min; MS m/z [M+H] ⁺ 889.4;

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-(((3-((2-(2,5-di-oxyloxy)) Benzyl-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)aminocarbazyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

Using General Scheme 1, but using 1.5 eq of bis(4-nitrophenyl)carbonate, 1.5 eq of 2,2,2-trifluoroacetic acid 3-((2-(2,5-di- oxo-2), 5-Dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropan-1-ammonium as the amine and 15 eq of DIPEA to give the desired product to give the desired product, 80% yield; - MS: Rt = 2.66 min; MS m/z [M+H] ⁺ 934.3;

(3S,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-(((2-(2,5-di- oxo-2), 5-Dihydro-1H-pyrrol-1-yl)ethyl)amine-methylmethyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

81% yield; UPLC-MS: Rt = 1.27 min; MS m/z [M+H] ⁺ 863.3;

(3S,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-(((2-(2-(2,5-di)oxy) -2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)amine carbhydryl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

50% yield; UPLC-MS: Rt = 1.28 min; MS m/z [M+H] ⁺ 907.3;

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-2,2-dimethylpropyl)propanylamino)methyl)-4-(((2-(2,5-di- oxo-2,5-di) Hydrogen-1H-pyrrol-1-yl)ethyl)amine-mercapto)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

67% yield; UPLC-MS: Rt = 3.05 min; MS m/z [M+H] ⁺ 835.5;

(3R,4S)-3-((3-((R)-1-Ethyloxypropan-2-yl)-1-((R)-(1-phenylmethyl-4-(2,5) -difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4-(((2-(2,5-) Bis-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amine-methylhydrazine)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

70% yield; UPLC-MS: Rt = 1.26min , MS m / z [M + H] + 892.5; Method E.

(3R,4S)-3-((3-((R)-1-Ethyloxypropan-2-yl)-1-((R)-(1-phenylmethyl-4-(2,5) -difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperazin-4-yl)methyl)ureido)methyl)-4-(((2-(2-(2) ,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)amine-mercapto)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester

45% yield; UPLC-MS: Rt = 1.28 min; MS m/z [M+H] ⁺ 936.3;

(R)-3-((S)-2-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro- 2H-piperidin-4-yl)methyl)-14-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)-5-methyl-3,8- Di-tert-butyl-7,12-dioxa-2,4,9-triazatetradecyl)pyrrolidine-1-carboxylic acid tert-butyl ester

31% yield; UPLC-MS: Rt = 1.29 min; MS m/z [M+H] ⁺ 878.2;

(3R,4R)-3-((1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-3-((S)-1-((3-((2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl) Methyl)azetidin-1-carbonyl)oxy)propan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

34% yield; UPLC-MS: Rt = 1.28 min; MS m/z [M+H] ⁺ 878.2;

General scheme for connecting sub-connections 2

Add i Pr ₂ EtN (3 eq), 2-(2,5-di- oxo-2,5-dihydro-1H carbonate) to a solution of Boc-payload (1 eq) in dry DMF (0.2M). Pyrrrol-1-yl)ethyl (4-nitrophenyl) ester (1.1 eq), and the reaction was stirred at room temperature for 1 hour. Upon completion by LC/MS (uplc), the reaction was quenched with water and extracted with ethyl acetate. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting eluting elut elut

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) 1-((2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)carbonyl)azetidin-3-yl)methyl) 2-hydroxypropionylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

42% yield; UPLC-MS: Rt = 2.49 min; MS m/z [M+H] ⁺ 795.3;

4-((R)-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)) Methyl)propanylamino)(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)methyl)piperidine-1-carboxylic acid 2-(2- (2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl ester

32% yield; UPLC-MS: Rt = 2.74 min; MS m/z [M+H] ⁺ 909.6;

General scheme for connecting sub-connections 3

i Pr ₂ EtN (11 eq), Boc-payload (1 eq) containing DMF (1:9 ratio) was added sequentially to a solution of the carboxylic acid linker (1.8 eq), HATU (2.1 eq) in DCM (0.2M) With DCM). The reaction mixture was stirred at RT for 1 hour. When by LC / MS (uplc) found that the reaction was complete, the reaction mixture was diluted in EtOAc and partitioned between EtOAc and H ₂ O. The organic layer was combined, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by chromatography eluting eluting eluting elut elut

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(1-(3-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)propanyl)piperidin-4-yl)methyl Propylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

99% yield; UPLC-MS: Rt = 2.58 min; MS m/z [M+H] ⁺ 849.5;

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(1-(3-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanyl)piperidine -4-yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

99% yield; UPLC-MS: Rt = 2.59 min; MS m/z [M+H] ⁺ 893.5;

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) 1-(3-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)propanyl)azetidin-3-yl)methyl)-2- Hydroxypropylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

43% yield; UPLC-MS: Rt = 2.32 min; MS m/z [M+H] ⁺ 779.2;

General scheme for connecting sub-connections 4

To a solution of Boc-payload (1.0 eq) and i Pr ₂ EtN (7.0 eq) in DMF (0.2M), HATU (2.0 eq) and amine linker (2.0 eq) were added sequentially. The reaction mixture was stirred at RT for 1 hour. When by LC / MS (uplc) found that the reaction was complete, the reaction mixture was diluted in EtOAc and partitioned between EtOAc and H ₂ O. The organic layer was combined, dried over Na ₂ SO _4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by chromatography eluting eluting eluting elut elut

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-((2-(2,5-di- oxo-2,5) -Dihydro-1H-pyrrol-1-yl)ethyl)amine-methylpyridyl)pyrrolidine-1-carboxylic acid tert-butyl ester

53% yield; UPLC-MS: Rt = 2.46 min; MS m/z [M+H] ⁺ 847.0;

(3R,4S)-3-(((S)-2-Ethyloxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)propanylamino)methyl)-4-((2-(2-(2,5-di- oxy)- 2,5-Dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminecarboxyl)pyrrolidine-1-carboxylic acid tert-butyl ester

27% yield; UPLC-MS: Rt = 2.48 min; MS m/z [M+H] ⁺ 890.9;

3-(2-(((R)-1-(1-(()))))))) , 5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4-oxobutyl butyl carboxamide Phenyl)pyrrolidine-1-carboxylic acid tert-butyl ester

General scheme 4 using a linker ligation, but using 1.2 eq HATU, 1.1 eq amine linker and 5 eq DIPEA to synthesize the product to give the desired product, 89% yield; UPLC-MS: Rt = 2.47 and 2.50 min; MS m /z[M+H] ⁺ 795.4; Method E.

(R)-3-(2-((1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((2-(2) , 5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4-oxobutyl butyl carboxamide Phenoxy)azetidin-1-carboxylic acid tert-butyl ester

General scheme 4 using a linker ligation, but using 1.2 eq HATU, 1.1 eq amine linker and 5 eq DIPEA to synthesize the product to give the desired product, 96% yield; UPLC-MS: Rt = 2.44 min; MS m/z [M+H] ⁺ 797.3; Method E.

(3R,4S)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)-4-(4-((2) , 5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-ylpyrrolidine-1-carboxylic acid third Butyl ester

To (3S,4R)-3-azido-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-2-((t-butyldimethylmethylalkyl)oxy)propanylamino)methyl)pyrrolidine 1-butylic acid tert-butyl ester (40 mg, 0.050 mmol) and 1-(prop-2-yn-1-yl)-1H-pyrrole-2,5-dione (10 mg, 0.076 mmol) in MeCN (0.3 ml) A solution of iodinated ketone (I) (10 mg, 0.050 mmol) in water (0.3 ml), triethylamine (0.07 mL, 0.050 mmol), and the mixture was stirred for 1 hour at RT. The reaction mixture was filtered with EtOAc EtOAc (EtOAc)EtOAc. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The crude product was purified by chromatography eluting eluting elut elut elut elut elut elut elut elut /z[M+H] ⁺ 929.6; Method E.

General Scheme 1 for simultaneous removal of protecting groups from N-BOC plus -O-acetate or -O-TBS

The matrix (1.0 eq) was dissolved in a 2:1 mixture of acetonitrile and water (0.1M). TFA (50 eq) was added and the reaction mixture was stirred at 60 ° C until complete (7-72 s) as found by UPLC-MS. The reaction was filtered and purified by reverse phase column chromatography. The product as a white powder in the form of a TFA salt was isolated upon lyophilization.

(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (3S,4S)-4-(((S)-N- ((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)- 2-hydroxypropionylamino)methyl)pyrrolidin-3-yl ester

41% yield; UPLC-MS: Rt = 0.86 min; MS m/z [M+H] ⁺ 721.1; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 9.11-8.63 (2H, m), 7.91-7.51 (2H, m), 7.45-7.05 (8H, m), 7.04-6.92 (2H, m), 5.65-4.98 (3H, m), 4.94-4.82 (1H, m), 4.55-4.44 (1H, m), 3.89-2.84 (13H, m), 2.69-1.70 (4H, m), 1.55- 0.48 (7H, m).

(2-(2-(2,5-Di-Ethoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid (3S,4S)-4-( ((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4 -yl)methyl)-2-hydroxypropionylamino)methyl)pyrrolidin-3-yl ester

24% yield; UPLC-MS: Rt = 0.89 min; MS m/z [M+H] ⁺ 765.1; Method A; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 9.07-8.71 ( 2H, m), 7.89-7.73 (2H, m), 7.42-7.07 (8H, m), 7.02-6.99 (2H, m), 5.65-5.49 (1H, m), 5.40-5.03 (2H, m), 4.49-4.47 (1H, m), 3.92-2.91 (18H, m), 2.69-2.10 (3H, m), 1.53-0.52 (7H, m).

(6-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)hexyl)carbamic acid (3S,4S)-4-(((S)-N-(((())) (R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-2 -hydroxypropionylamino)methyl)pyrrolidin-3-yl ester

37% yield; UPLC-MS: Rt = 0.95 min; MS m/z [M+H] ⁺ 777.2; Method A; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 8.96-8. 2H,m),7.88-7.54(2H,m),7.43-7.26(6H,m),7.17-7.07(2H,m),7.01-7.00(2H,m),5.65-5.04(3H,m), 4.94-4.85 (1H, m), 4.50-4.42 (1H, m), 3.94-2.83 (14H, m), 2.68-1.75 (3H, m), 1.52-0.51 (15H, m).

3-((2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)methyl)azetidin-1-carboxylic acid (3S,4S)-4-(( (S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-2-hydroxypropionylamino)methyl)pyrrolidin-3-yl ester

40% yield; UPLC-MS: Rt=0.90 min; MS m/z [M+H] ⁺ 747.2; Method A; ¹ H-NMR (CDCl ₃ , 400 MHz, mixture of rotamers): δ 10.25-9.50 (2H,m), 7.93-7.56(2H,m), 7.46-7.27(5H,m),7.22-6.91(2H,m),6.76-6.74(2H,m),5.98-4.44(3H,m) , 4.05-3.58 (10H, m), 3.45-3.18 (3H, m), 3.09-2.19 (9H, m), 1.40-0.64 (7H, m).

(3-((2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)aminocarbamic acid (3S,4S)-4-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropionamido)methyl)pyrrolidin-3-yl ester

26% yield; UPLC-MS: Rt = 0.84 min; MS m/z [M+H] ⁺ 792.1; Method A; ¹ H-NMR (CDCl ₃ , 400 MHz, mixture of rotamers): δ 9.97-9.51 (2H,m), 7.88-7.61(2H,m),7.44-7.29(5H,m),7.14-6.81(2H,m),6.73-6.69(2H,m),5.98-4.44(5H,m) , 3.97-3.94 (1H, m), 3.75-2.32 (22H, m), 1.43-0.66 (7H, m).

(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (3R,4R)-4-(((S)-N- ((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)- 2-hydroxypropionylamino)methyl)pyrrolidin-3-yl ester

30% yield; UPLC-MS: Rt = 0.87 min; MS m/z [M+H] ⁺ 720.3; Method A; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 8.96-8.85 ( 2H,m), 7.92-7.66 (2H, m), 7.42-6.89 (10H, m), 5.66-5.03 (3H, m), 4.54-4.43 (1H, m), 4.08-2.93 (18H, m), 1.52-0.51 (7H, m).

(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (3S,4S)-4-(((S)-N- ((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyl Propylamino)methyl)pyrrolidin-3-yl ester (2-(2-(2,5-Di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid (3R,4R)-4-( ((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4 -yl)methyl)-2-hydroxypropionylamino)methyl)pyrrolidin-3-yl ester

42% yield; UPLC-MS: Rt = 0.89 min; MS m/z [M+H] ⁺ 765.2; Method A; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 8.97-8.85 ( 2H,m),7.93-7.66(2H,m),7.42-6.84(10H,m),5.67-5.04(3H,m), 4.55-4.43(1H,m),4.09-2.93(21H,m), 1.52-0.51 (7H, m).

(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (3S,4S)-4-(((S)-N- ((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropyl)-2-hydroxyl Propylamino)methyl)pyrrolidin-3-yl ester

60% yield; UPLC-MS: Rt = 2.05 min; MS m/z [M+H] ⁺ 693.2;

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-(3-(2,5-) Bilateral oxy-2,5-dihydro-1H-pyrrol-1-yl)propanyl)piperidin-4-yl)methyl)-N-((3S,4R)-4-fluoropyrrolidine -3-yl)methyl)-2-hydroxypropanamide

30% yield; UPLC-MS: Rt = 1.57 min; MS m/z [M+H] ⁺ 707.4; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 8.98-8.90 (1H, m), 8.64 - 8.56 (1H, m), 7.78-7.61 (2H, m), 7.34-7.18 (6H, m), 7.06-7.02 (1H, m), 6.93 (2H, s), 5.60-5.44 (1H, m), 5.34-4.96 (3H, m), 4.46-4.29 (1H, m), 4.19-1.97 ( 17H, m), 1.42 - 0.29 (7H, m).

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-(3-(2-(2) , 5-two Oleoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanyl)piperidin-4-yl)methyl)-N-((3S,4R)-4- Flupiryrridin-3-yl)methyl)-2-hydroxypropanamide

29% yield; UPLC-MS: Rt = 1.59 min; MS m/z [M+H] ⁺ 751.5; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.02-8.93 (1H, m), 8.66-8.57 (1H, m), 7.85-7.69 (2H, m), 7.42-7.24 (6H, m), 7.15-7.07 (1H, m), 7.01 (2H, s), 5.67-5.51 (1H, m), 5.42-5.03 (3H, m), 4.53-4.35 (1H, m), 4.27-2.05 ( 22H, m), 1.51-0.34 (7H, m).

4-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((S)-N-(((3S,4R)- 4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionylamino)methyl)piperidine-1-carboxylic acid 2-(2-(2,5-di- oxo-2,5- Dihydro-1H-pyrrol-1-yl)ethoxy)ethyl ester

21% yield; UPLC-MS: Rt = 1.70 min; MS m/z [M+H] ⁺ 767.5;

(3S,4S)-4-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropionylamino)methyl)-N-(2-(2,5-di-oxo-2,5-dihydro- 1H-pyrrol-1-yl)ethyl)pyrrolidine-3-carboxamide

28% yield; UPLC-MS: Rt = 1.77 min; MS m/z [M+H] ⁺ 705.3; Method E; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 8.64-8.51 ( 2H,m),8.34-8.32(1H,m),7.82-7.67(2H,m),7.43-7.27(6H,m),7.15-7.09(1H,m),7.02-7.00(2H,m), 5.62-4.47 (1H, m), 5.36-5.01 (2H, m), 4.51-4.39 (1H, m), 3.89-2.99 (18H, m), 1.83-0.50 (7H, m).

(3S,4S)-4-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-2-hydroxypropanylamino)methyl)-N-(2-(2-(2,5-di- oxo-2,5-) Dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)pyrrolidine-3-carboxamide

75% yield; UPLC-MS: Rt = 1.80 min; MS m/z [M+H] ⁺ 749.4; Method E; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 8.62-8.58 ( 2H,m), 8.32-8.27(1H,m),7.83-7.64(2H,m),7.43-7.27(6H,m),7.15-7.09(1H,m),7.04-7.02(2H,m), 5.63-4.46(1H,m), 5.36-5.03(2H,m),4.44-4.40(1H,m),3.88-3.08(21H,m),2.84-2.62(1H,m),1.91-0.51(7H , m).

(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (3S,4S)-4-((1-((R)) -(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(( S)-1-hydroxypropan-2-yl)ureido)methyl)pyrrolidin-3-yl ester

29% yield; UPLC-MS: Rt = 0.87 min; MS m/z [M+H] ⁺ 750.2;

(2-(2-(2,5-Di-Ethoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid (3S,4S)-4-( (1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)- 3-((S)-1-hydroxypropan-2-yl)ureido)methyl)pyrrolidin-3-yl ester

48% yield; UPLC-MS: Rt = 0.88 min; MS m/z [M+H] ⁺ 794.2;

General scheme for the removal of the overall protection base 2

The matrix (1.0 eq) was dissolved in DCM (0.1M) and TFA (20-50 eq) was added and the reaction mixture was stirred at RT for 1-2 hours. The reaction was filtered and purified by reverse phase column chromatography. The product as a white powder in the form of a TFA salt was isolated upon lyophilization.

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-N-(((3S,4S)-4-(4-((2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)) -1H-1,2,3-triazol-1-yl)pyrrolidin-3-yl)methyl)-2-hydroxypropanamide

50 eq TFA, 24% yield; UPLC-MS: Rt = 1.65 min; MS m/z [M+H] ⁺ 715.4; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.04-8.82 (2H, m), 8.09-8.04 (1H, m), 7.88-7.76 (1H, m), 7.71-7.60 (1H, m), 7.42-7.05 (9H, m), 5.58-5.55 (1H, m), 5.39-4.98 (5H, m), 4.68-4.66 (2H, m), 4.01-2.46 (10H, m), 1.47- 0.45 (9H, m).

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-(3-(2,5-) Bilateral oxy-2,5-dihydro-1H-pyrrol-1-yl)propanyl)azetidin-3-yl)methyl)-N-((3S,4R)-4- Flupiryrridin-3-yl)methyl)-2-hydroxypropanamide

50 eq TFA; 26% yield; UPLC-MS: Rt = 1.46 min; MS m/z [M+H] ⁺ 679.2; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.04 (1H, s, br), 8.69 (1H, s, br), 7.83-7.65 (2H, m), 7.43-7.29 (6H, m), 7.15-7.10 (1H, m), 7.02-6.97 (2H, m), 6.25-5.65 (1H, m), 5.52-5.07 (3H, m), 4.56-4.26 (1H, m), 4.16- 2.76 (17H, m), 2.48-2.08 (3H, m), 1.30-0.97 (3H, m).

3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)((S)-N-(((3S,4R)- 4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionamido)methyl)azetidin-1-carboxylic acid 2-(2,5-di- oxo-2,5- Dihydro-1H-pyrrol-1-yl)ethyl ester

50 eq TFA; 60% yield; UPLC-MS: Rt = 1.65 min; MS m/z [M+H] ⁺ 695.3; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.05 (1H, s, br), 8.69 (1H, s, br), 7.84-7.66 (2H, m), 7.43-7.29 (6H, m), 7.15-7.10 (1H, m), 7.02-6.98 (2H, m), 6.25-5.70 (1H, m), 5.51-5.08 (3H, m), 4.51-4.26 (1H, m), 4.15- 2.76 (17H, m), 2.43-2.07 (3H, m), 1.29-0.97 (3H, m).

3-((2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)methyl)azetidin-1-carboxylic acid (S)-2-(3-( (R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-3 -(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester

50eq TFA; 75% yield; UPLC-MS: Rt = 1.78 min; MS m/z [M+H] ⁺ 778.3;

N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((2-(2,5-di) Oleoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4-oxobutyl)-2-(pyrrolidine-3 -yl)benzamide

20 eq. TFA; mixture of diastereomers; 88% yield; UPLC-MS: Rt = 1.72 min; MS m/z [M+H] ⁺ 695.2; Method E; ¹ H-NMR (DMSO, 400 MHz): δ 9.50-9.46(1H,m), 8.87-8.82(2H,m), 8.12-8.10(1H,m), 7.80-7.73(1H,m),7.53-7.26(11H,m),7.10-7.06( 1H, m), 6.97 (2H, s), 5.54-5.38 (3H, m), 3.76-3.57 (1H, m), 3.54-2.89 (8H, m), 2.87-2.80 (1H, m), 2.30- 1.82 (3H, m), 1.10-1.08 (3H, m), 0.86-0.85 (3H, m).

(R)-2-(azetidin-3-yloxy)-N-(1-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazole-2 -yl)-4-((2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl- 4-sided oxybutyl)benzamide

20 eq TFA; 78% yield; UPLC-MS: Rt = 1.70 min; MS m/z [M+H] ⁺ 697.2; Method E; ¹ H-NMR (DMSO, 400 MHz): δ 9.02-9.00 (1H, m ), 8.87 (2H, s, br), 8.09-8.06 (1H, m), 7.79-7.75 (1H, m), 7.64-7.62 (1H, m), 7.59-7.58 (1H, m), 7.47-7.42 (1H, m), 7.36-7.26 (6H, m), 7.12-7.06 (2H, m), 6.85 (2H, s), 6.82-6.80 (1H, m), 5.53-5.38 (3H, m), 5.18 -5.12 (1H, m), 4.52-4.45 (1H, m), 4.40-4.36 (1H, m), 4.28-4.22 (1H, m), 4.18-4.12 (1H, m), 3.46-3.42 (2H, m), 3.35-3.27 (1H, m), 3.19-3.11 (1H, m), 2.74-2.70 (1H, m), 2.20-2.17 (1H, m), 1.03 (3H, s), 0.89 (3H, s).

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (S)-2-(3- ((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)- 3-((S)-pyrrolidin-3-ylmethyl)ureido)propyl ester

40% yield; UPLC-MS: Rt = 1.72 min; MS m/z [M+H] ⁺ 778.4;

General procedure for the removal of the BOC protecting group for the linker-payload:

Trifluoroacetic acid (0.5 mL, 6.5 mmol) was added to a mixture of &lt;RTI ID=0.0&gt;0&gt; Upon completion of the reaction by LC/MS (uplc, Method A), the crude product was purified by reverse phase column chromatography (using PrepLC method C or D). The product was isolated as a TFA salt.

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-4-(((2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(2-hydroxyethyl) Aminomethyl)oxy)-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1- Tert-butyl formate

The use of (S)-2-amino-3-(2,5-di- oxo-2,5- at the effective hydroxyl group by a method similar to that described in the attachment of the payload to the linker component The title compound was prepared as the title compound (3H-H-H-H-pyrrol-l-yl)-propionic acid (3M).

The desired product was used in the next step without further purification.

LC / MS (Method A): [M + H] + 967.5, Rt 1.42min.

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (S)-2-(3- ((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)- 3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester

Boc-linker-payload: LC/MS (uplc): MH+ 896.6, 1.31 min (Method A).

5 mg, 0.005 mmol, 67%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.05 (1H, m), 8.67 (1H, m), 7.77 (3H, m), 7.39 (2H, m), 7.32 (2H, m), 7.27 (2H, m), 7.10 (1H, m), 7.01 (2H, m), 6.33 (1H, m), 5.34 (2H, m), 5.25 (1H, m), 5.20 (1H, m), 3.96 (2H, m ), 3.89 (1H, m), 3.83 (1H, m), 3.73 (1H, m), 3.60 (1H, m), 3.56 (4H, m), 3.39 (2H, m), 3.35 (1H, m) , 3.30 (1H, m), 3.09 (2H, m), 2.59 (1H, m), 2.54 (1H, m), 2.26 (1H, m), 2.00 (1H, m), 1.45 (1H, m), 1.25-1.05 (2H, m), 1.10 (3H, m), 0.75 (1H, m). Missing signals hidden under the solvent peak. LC/MS (up lc): MH.

(6-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)hexyl)carbamic acid (S)-2-(3-((R)-(1- Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S,4R) )-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester.

Boc-linker-payload: LC/MS (uplc): M+ 908.6, 1.37 min (Method A).

4.5 mg, 0.005 mmol, 53%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.02 (1H, m), 8.65 (1H, m), 7.77 (2H, m), 7.39 (2H, m), 7.32 (2H, m), 7.27 (2H, m), 7.10 (1H, m), 7.01 (1H, m), 6.99 (2H, m), 6.32 (1H, m), 5.33 (2H, m), 5.24 (1H, m) 5.20 (1H, m) , 3.94 (1H, m), 3.83 (1H, m), 3.72 (1H, m), 3.60 (1H, m), 3.38 (3H, m), 3.32 (3H, m), 3.21 (1H, m), 2.95 (2H, m), 2.60 (1H, m), 2.53 (1H, m), 2.27 (1H, m), 2.00 (1H, m), 1.47 (2H, m), 1.36 (2H, m), 1.21. (5H, m), 1.09 (4H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH + 808.5, 1.0 min (Method A).

N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl )-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-iodoacetamide.

Boc-linker-payload: LC/MS (uplc): MH+ 753.3, 1.40 min (Method A).

Linker-payload: LC/MS (uplc): MH+ 653.2, 0.94 min (Method A).

(2-(2-(2-Iodoethylamino)ethoxy)ethyl)aminocarbamic acid (S)-2-(3-((R)-(1-phenylmethyl-4-(2) ,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidine- 3-yl)methyl)ureido)propyl ester

Boc-linker-payload: LC/MS (uplc): M+ 984.3, 1.27 min (Method A).

Linker-payload: LC/MS (uplc): MH+ 884.4, 0.91 min (Method A). (6-(2-iodoethylamino)hexyl)carbamic acid (S)-2-(3-((R)-(1-benzylmethyl-4-(2,5-difluoro) Phenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)- Ureido)propyl ester

Boc-linker-payload: LC/MS (uplc): MH+ 996.6, 1.33 min (Method A).

4 mg, 0.004 mmol, 40%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.00 (1H, m), 8.63 (1H, m), 8.19 (1H, m), 7.77 (3H, m), 7.39 (2H, m), 7.30 (1H, m), 7.27 (2H, m), 7.10 (1H, m), 7.05 (1H, m), 6.31 (1H, m), 5.33 (2H, m) 5.23 (1H, m), 5.19 (1H, m) , 3.94 (4H, m), 3.83 (1H, m), 3.71 (1H, m), 3.60 (2H, m), 3.47 (1H, m), 3.34 (3H, m), 3.20 (1H, m), 3.02 (2H, m), 2.95 (2H, m), 2.58 (1H, m), 2.54 (1H, m), 2.25 (1H, m), 2.00 (1H, m), 1.37 (5H, m), 1.24 (5H, m), 1.08 (3H, m), 0.69 (1H, m). Missing signals hidden under the solvent peak. LC/MS (up lc): MH.

1-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl )-3-((S)-1-(4-((2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2, 3-Triazol-1-yl)propan-2-yl)-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)urea.

Boc-linker-payload: LC/MS (uplc): MH+ 846.5, 1.31 min (Method A).

5.5 mg, 0.006 mmol, 86%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.07 (1H, m), 8.52 (1H, m), 8.03 (1H, m), 7.79 (1H, m), 7.75 (1H, m), 7.38 (2H, m), 7.32 (2H, m), 7.24 (2H, m), 7.11 (1H, m), 7.09 (2H, m), 6.42 (1H, m), 5.30-5.15 (4H, m), 4.67 (2H) , m), 4.42 (2H, m), 4.21 (1H, m), 3.81 (1H, m), 3.58 (1H, m), 3.39 (3H, m), 3.30 (1H, m), 3.20 (1H, m), 2.53 (1H, m), 2.47 (1H, m), 2.30 (1H, m), 1.90 (1H, m), 1.35 (1H, m), 1.25 (1H, m), 1.11 (4H, m ), 0.65 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH.s.

(2-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (S)-1-((( R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)((( 3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-oxopropan-2-yl ester

Boc-linker-payload: LC/MS(uplc): MH+ 867.4, 1.33min (square Law A).

2 mg, 0.002 mmol, 36%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.11 (1H, m), 8.80 (1H, m), 7.78 (2H, m), 7.51 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.22 (2H, m), 7.10 (1H, m), 7.02 (2H, m), 5.61 (1H, m), 5.52 (1H, m), 5.23 (1H, m), 5.09 (1H, m ), 5.02 (1H, m), 3.83 (1H, m), 3.75 (1H, m), 3.62 (1H, m), 3.54 (4H, m), 3.30 (1H, m), 3.23 (2H, m) , 3.09 (2H, m), 2.66 (1H, m), 2.61 (1H, m), 2.31 (1H, m), 2.18 (1H, m), 1.42 (3H, m), 1.38 (2H, m), 1.10 (1H, m), 0.67 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 767.3, 0.92 min (Method A).

(6-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)hexyl)carbamic acid (S)-1-(((R)-(1-phenyl) 4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)((3S,4R)-4-fluoro Pyrrolidin-3-yl)methyl)amino)-1-oxopropan-2-yl ester

Boc-linker-payload: LC/MS (uplc): MH+ 879.6, 1.40 min (Method A).

3.2 mg, 0.0034 mmol, 27%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.20-9.06 (1H, m), 8.97-8.33 (1H, m), 7.83-7.73 (2H, m), 7.56-7.46 (1H, m), 7.44-7.36 (2H, m), 7.35-7.28 (2H, m), 7.27-7.19 (2H, m), 7.17-7.08 (1H, m), 7.03-6.96 (2H, m), 5.66-5.59 (1H, m) , 5.59-5.43 (1H, m), 5.26-4.98 (3H, m), 3.88-3.77 (1H, m), 3.77-368 (1H, m), 3.36-3.27 (4H, m), 3.27-3.17 ( 2H,m),3.07-2.96(1H,m),2.95-2.82(1H,m),2.72-2.56(2H,m), 2.37-2.24(1H,m),2.23-2.05(1H,m), 1.52-1.28 (9H, m), 1.28-1.16 (2H, m), 1.16-0.97 (3H, m), 0.73-0.56 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 779.4, 1.01 min (Method A).

(2-(2-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethyl)aminocarboxylic acid (S -2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4) -yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester

Boc-linker-payload: LC/MS (uplc): M+ 940.4, 1.29 min (Method A).

4 mg, 0.004 mmol, 47%. ^{1 H-NMR (DMSO, 600MHz} ): δ 8.95 (1H, m), 8.45 (1H, m), 7.79 (1H, m), 7.76 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.27 (2H, m), 7.11 (1H, m), 7.06 (1H, m), 7.03 (2H, m), 6.32 (1H, m), 5.34 (2H, m), 5.15-5.25 (2H , m), 3.96 (1H, m), 3.91 (1H, m), 3.85 (1H, m), 3.75 (1H, m), 3.60 (1H, m), 3.55-3.25 (12H, m), 3.18 ( 1H, m), 3.11 (2H, m), 2.65 (2H, m), 2.51 (1H, m), 2.28 (1H, m), 2.00 (1H, m), 1.40 (2H, m), 1.23 (1H) , m), 1.10 (3H, m), 0.65 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+: 840.3, 0.94 min (Method A).

(2-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)aminocarboxylic acid (S)-2-(3-((R)-(1) -Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S, 4R)-4-Fluoropyridin-3-yl)methyl)ureido)propyl ester.

Boc-linker-payload: LC/MS (uplc): MH+ 852.4, 1.28 min (Method A).

10 mg, 0.011 mmol, 69%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.03 (1H, m), 8.64 (1H, m), 7.78 (1H, m), 7.77 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.27 (2H, m), 7.20 (1H, m), 7.08 (1H, m), 7.01 (2H, m), 6.92 (1H, m), 6.30 (1H, m), 5.34 (2H, m ), 5.22 (2H, m), 3.90 (3H, m), 3.80 (1H, m), 3.74 (1H, m), 3.60 (1H, m), 3.33 (2H, m), 3.20 (1H, m) , 3.13 (2H, m), 2.61 (1H, m), 2.54 (1H, bs), 2.28 (1H, m), 1.99 (1H, m), 1.45 (1H, m), 1.30-1.15 (2H, m ), 1.11 (3H, m), 0.68 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH.s.

(2-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid 2-(3-((R)) -(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(( (3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)ethyl ester.

Boc-linker-payload: LC/MS (uplc): MH+ 882.2, 1.26 min (Method A).

14 mg, 0.015 mmol, 71%. ¹ H-NMR (DMSO, 600 MHz): δ 9.11-9.00 (1H, m), 8.75-8.60 (1H, m), 7.81-7.71 (2H, m), 7.43-7.36 (2H, m), 7.36-7.24 (4H, m), 7.14-7.06 (1H, m), 7.05-7.00 (2H, m), 7.00-6.95 (1H, m), 6.79-6.65 (1H, m), 5.38-5.11 (4H, m) , 4.02-3.95 (2H, m), 3.71-3.64 (1H, m), 3.64-3.59 (1H.m), 3.59-3.54 (2H, m), 3.54-.344 (3H, m), 3.44-3.26 (7H,m), 3.26-3.16(1H,m),3.13-3.04(2H.m), 2.65-2.44(2H,m),2.43-2.26(1H,m),2.02-1.83(1H,m) , 1.45-1.33 (1H, m), 1.30-1.12 (2H, m), 0.78-0.60 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 782.2, 0.90 min (Method A).

N-((S)-2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)-3-(2-(2, 5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamine.

Boc-linker-payload: LC/MS (uplc): MH+ 880.4, 1.25 min (Method A).

10.5 mg, 0.011 mmol, 89%. ¹ H-NMR (DMSO, 600 MHz): δ 9.06 (1H, m), 8.70 (1H, m), 7.79 (1H, m), 7.78 (1H, m), 7.76 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.28 (2H, m), 7.10 (1H, m), 7.02 (2H, m), 6.35 (1H, m), 5.40-5.10 (4H, m), 3.83 (3H) , m), 3.72 (1H, m), 3.45 (2H, m), 3.37-3.25 (4H, m), 3.25-3.05 (4H, m), 2.57 (1H, m), 2.50 (2H, m), 2.42 (1H, m), 2.30 (2H, m), 1.92 (1H, m), 1.40 (1H, m), 1.25 (1H, m), 1.22 (1H, m), 1.04 (3H, m), 0.70 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 780.3, 0.93 min (Method A).

((S)-2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidyl)喃-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)carbamic acid 2-(2-(2, 5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl ester.

Boc-linker-payload: LC/MS (uplc): MH+ 896.2, 1.27 min (Method A).

9 mg, 0.009 mmol, 40%. ¹ H-NMR (DMSO, 600 MHz): δ 9.11-8.96 (1H, m), 8.74-8.59 (1H, m), 7.82-7.69 (2H, m), 7.45-7.36 (2H, m), 7.35-7.20 (5H,m), 7.15-7.06(1H,m),7.05-6.98(2H,m),6.34-6.15(1H,m),5.38-5.12(4H,m),4.08-3.96(2H,m) , 3.90-3.79 (2H, m), 3.79-3.67 (1H, m), 3.40-3.27 (4H, m), 3.26-3.18 (1H, m), 3.12-2.98 (2H, m), 2.65-2.46 ( 3H, m), 2.46-2.30 (1H, m), 2.04-1.87 (1H, m), 1.46-1.34 (1H, m), 1.31-1.12 (2H, m), 1.09-0.97 (3H, m), 0.78-0.60 (1H, m). Missing signals hidden under the solvent peak. LC/MS (up lc): MH.

N-((S)-2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)-6-(2,5-di Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)hexylamine.

Boc-linker-payload: LC/MS (uplc): MH+ 878.3, 1.28 min (Method A).

19 mg, 0.02 mmol, 31%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.05 (1H, m), 8.72 (1H, m), 7.97 (1H, m), 7.77 (2H, m), 7.40 (2H, m), 7.35 (2H, m), 7.29 (2H, m), 7.11 (1H, m), 7.01 (2H, m), 6.40 (1H, m), 5.33 (2H, m), 5.28-5.20 (2H, m), 3.84 (2H) , m), 3.70 (1H, m), 3.60 (1H, m), 3.45 (1H, m), 3.37 (2H, m), 3.33 (2H, m), 3.22 (2H, m), 3.10 (1H, m), 2.57 (2H, m), 2.51 (2H, m), 2.44 (1H, m), .2.07 (2H, m), 1.92 (1H, m), 1.49 (3H, m), 1.38 (1H, m), 1.24 (1H, m), 1.19 (2H, m), 1.04 (3H, m), 0.70 (1H, m). LC/MS (uplc): MH + 778.2, 0.96 min (Method A).

(2-(2-(2,5-Di-Ethoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid (3S,4R)-1-( ((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)( ((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)aminecarboxylidene-4-hydroxypyrrolidin-3-yl ester and (2-(2-(2,5-two-side) Oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (3R,4S)-1-(((R)-(1-phenylmethyl-4) -(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)((3S,4R)-4-fluoropyrrolidine- 3-yl)methyl)amine carbaryl)-4-hydroxypyrrolidin-3-yl ester.

Two different isomers were prepared by sequential protection-removal of the 2 OH to give the corresponding linker-positional isomers as pure isomers. The structural determination of the specified isomers was not performed.

Isomer A: Boc-linker-payload: LC/MS (uplc): M+ 1038.7, 1.50 min (Method A).

Isomer B: Boc-linker-payload: LC/MS (uplc): MH+ 1039.2, 1.49 min (Method A).

Isomer A: 2.3 mg, 0.002 mmol, 30%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.04 (1H, m), 8.74 (1H, m), 7.76 (2H, m), 7.39 (2H, m), 7.33 (2H, m), 7.25 (2H, m), 7.09 (2H, m), 7.02 (2H, m), 567 (1H, m), 5.38 (1H, m), 5.25 (1H, m), 4.93 (1H, m), 4.71 (1H, m ), 4.27 (1H, m), 3.80 (1H, m), 3.71 (2H, m), 3.57 (4H, m), 3.40 (2H, m), 3.30 (4H, m), 3.27 (4H, m) , 3.11 (2H, m), 2.81 (1H, m), 2.37 (1H, m), 2.22 (1H, m), 2.17 (1H, m), 1.70 (1H, m), 1.20 (1H, m), 1.05 (1H, m), 0.68 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH.s.

Isomer B: 6.2 mg, 0.006 mmol, 43%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.03 (1H, m), 8.76 (1H, m), 7.75 (2H, m), 7.39 (2H, m), 7.33 (2H, m), 7.25 (2H, m), 7.10 (2H, m), 7.03 (2H, m), 5.71 (1H, m), 5.37 (1H, m), 5.25 (1H, m), 4.89 (1H, m), 4.77 (1H, m ), 4.24 (1H, m), 3.83 (1H, m), 3.78 (1H, m), 3.58 (4H, m), 3.51 (3H, m), 3.40 (2H, m), 3.29 (1H, m) , 3.14 (2H, m), 3.10 (2H, m), 2.80 (1H, m), 2.36 (2H, m), 2.30 (1H, m), 2.13 (1H, m), 1.72 (1H, m), 1.08 (1H, m), 0.88 (1H, m), 0.33 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH.s.

(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (3R,4S)-1-(((R)-(1) -Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)((3S,4R)- 4-fluoropyrrolidin-3-yl)methyl)aminecarboxylidene-4-hydroxypyrrolidin-3-yl ester and (2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (3S,4R)-1-(((R)-(1) -Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)((3S,4R)- 4-Fluoropyrrolidin-3-yl)methyl)amine carbhydryl)-4-hydroxypyrrolidin-3-yl ester.

Two different isomers were prepared by sequential protection-removal of the 2 OH to give the corresponding linker-positional isomers as pure isomers. The structural determination of the specified isomers was not performed.

Isomer A: Boc-linker-payload: LC/MS (uplc): MH+ 995.2, 1.52 min (Method A).

Isomer B: Boc-linker-payload: LC/MS (uplc): M+ 994.2, 1.51 min (Method A).

Isomer A: 11 mg, 0.012 mmol, 58%. ¹ H-NMR (DMSO, 600MHz): δ 9.09-8.94 (1H, m), 8.71-8.53 (1H, m), 7.82-7.69 (2H, m), 7.42-7.36 (2H, m), 7.36-7.21 (5H,m), 7.16-7.06(1H,m),7.03-6.97(2H,m),5.72-5.62(1H,m),5.44-5.34(1H,m),5.34-5.16(1H,m) , 4.94-4.86 (1H, m), 4.74-4.66 (1H, m), 4.33-4.21 (1H, m), 3.88-3.76 (1H, m), 3.76-3.65 (2H, m), 3.65-3.53 ( 1H, m), 3.22-3.16 (4H, m), 3.16-3.03 (2H, m), 2.90- 2.77 (1H, m), 2.45-2.10 (3H, m), 1.77-1.67 (1H, m), 1.12-0.99(1H,m), 0.91-0.81(1H,m),0.34-0.15(1H,m).1H,m),4.74-4.66(1H,m),4.33-4.21(1H,m), 3.88-3.76(1H,m), 3.76-3.65(2H,m), 3.65-3.53(1H,m),3.22-3.16

Isomer B: 10 mg, 0.0011 mmol, 94%, ¹ H-NMR (DMSO, 600 MHz): δ 9.11-8.94 (1H, m), 8.80-8.57 (1H, m), 7.84-7.72 (2H, m) , 7.44-7.36(2H,m), 7.36-7.28(3H,m), 7.28-7.20(2H,m),7.14-7.07(1H,m),7.05-6.99(2H,m),5.79-5.69( 1H, m), 5.43-5.33 (1H, m), 5.31-5.16 (1H, m), 4.91-4.82 (1H, m), 4.82-4.72 (1H, m), 4.27-4.17 (1H, m), 3.92-3.74(2H,m),3.24-3.02(4H,m),2.83-2.71(1H,m),2.44-2.25(3H,m),2.14-2.03(1H,m),1.76-1.64(1H , m), 1.12-0.98 (1H, m), 0.92-0.79 (1H, m), 0.37-0.19 (1H, m). 9.11-8.94 (1H, m), 8.80-8.57 (1H, m), 7.84 -7.72 (2H, m), 7.44-7.36 (2H, m), 7.36-7.28 (3H, m), 7.28-7.20 (2H, m), (6-(2,5-di- oxy-2, 5-Dihydro-1H-pyrrol-1-yl)hexyl)carbamic acid (3R,4S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl) -1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)aminecarboxamide 4-hydroxypyrrolidin-3-yl ester and (6-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl) carbamic acid (3S, 4R)-1-(((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)aminecarboxamido)-4-hydroxy Pyrrolidin-3-yl ester.

Two different isomers were prepared by sequential protection-removal of the 2 OH to give the corresponding linker-positional isomers as pure isomers. The structural determination of the specified isomers was not performed.

Isomer A: Boc-linker-payload: LC/MS (uplc): M+ 1050.3, 1.57 min (Method A).

Isomer B: Boc-linker-payload: LC/MS (uplc): M+ 1050.3, 1.57 min (Method A). Isomer A: 12 mg, 0.012 mmol, 83%. ¹ H-NMR (DMSO, 600MHz): δ 9.06-8.93 (1H, m), 8.74-8.61 (1H, m), 7.84-7.69 (2H, m), 7.45-7.28 (4H, m), 7.28-7.21 (2H,m), 7.18-7.06(2H,m),7.05-6.96(2H,m), 5.70-5.59(1H,m),5.43-5.33(1H,m),5.33-5.17(1H,m) , 4.99-4.88 (1H, m), 4.75-4.63 (1H, m), 4.33-4.20 (1H, m), 3.85-3.75 (1H, m), 3.75-3.63 (2H, m), 3.63-3.52 ( 1H, m), 3.30-3.19 (3H, m), 3.17-3.05 (1H, m), 3.01-2.89 (2H, m), 2.87-2.74 (1H, m), 2.44-2.08 (3H, m), 1.77-1.62 (1H, m), 1.54-1.44 (2H, m), 1.43-1.32 (2H, m), 1.31-1.13 (4H, m), 1.09-0.95 (1H, m), 0.93-0.79 (1H , m), 0.31-0.15 (1H, m). Missing signals hidden under the solvent peak. LC/MS (up lc): MH.s.

Isomer B: 9.5 mg, 0.0095 mmol, 72%. ¹ H-NMR (DMSO, 600 MHz): δ 9.07-8.93 (1H, m), 8.76-8.63 (1H, m), 7.80-7.70 (2H, m), 7.43-7.36 (2H, m), 7.36-7.28 (2H,m), 7.27-7.21(2H,m),7.18-7.13(1H,m),7.13-7.06(1H,m),7.04-6.98(2H,m),5.80-5.63(1H,m) , 5.42-5.32 (1H, m), 5.32-5.12 (1H, m), 4.97-4.83 (1H, m), 4.82-4.72 (1H, m), 4.23-4.17 (1H, m), 3.89-3.72 ( 2H,m), 3.72-3.56(2H,m),3.31-3.24(2H,m),3.21-3.05(2H,m),3.03-2.88(2H,m),2.86-2.71(1H,m), 2.44-2.23(2H,m),2.21-1.99(1H,m),1.80-1.65(1H,m),1.55-1.43(2H,m),1.43-1.32(2H,m),1.31-1.14(4H m), 1.13 - 0.98 (1H, m), 0.94 - 0.80 (1H, m), 0.40 - 0.33 (1H, m). Missing signals hidden under the solvent peak.

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (R)-1-(3- ((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)- 3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)-3-hydroxypropan-2-yl ester.

Boc-linker-payload: LC/MS (uplc): M+ 1026.3, 1.51 min (Method A), 7.10 min (Method B).

6.1 mg, 0.006 mmol, 51%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.15- 8.96 (1H, m), 8.69-8.48 (1H, m), 7.83-7.67 (2H, m), 7.47-7.37 (2H, m), 7.37-7.21 (4H,m), 7.18-7.07(1H,m),7.07-6.99(2H,m),6.98-6.86(1H,m), 6.80-6.59(1H,m),5.38-5.09(4H,m) , 4.78-4.63 (1H, m), 3.88-3.77 (1H, m), 3.54-3.43 (4H, m), 3.42-3.26 (6H, m), 3.25-3.14 (2H, m), 3.15-2.98 ( 2H,m),2.61-2.44(3H,m),2.43-2.25(2H,m),2.00-1.78(1H,m),1.43-1.30(1H,m),1.30-1.08(2H,m), 0.76-0.57 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH.s.

(2-(2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid (R)-3-(3- ((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)- 3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)-2-hydroxypropyl ester.

Boc-linker-payload: LC/MS (uplc): M+ 1026.3, 1.52 min (Method A), 7.17 min (Method B).

4.0 mg, 0.004 mmol, 70%. ¹ H-NMR (DMSO, 600 MHz): δ 9.09-8.93 (1H, m), 8.59-8.37 (1H, m), 7.85-7.70 (2H, m), 7.45-7.36 (2H, m), 7.36-7.23 (4H, m), 7.15-7.06 (2H, m), 7.06-7.01 (2H, m), 6.68-6.56 (1H, m), 5.40-5.12 (4H, m), 4.06-3.95 (1H, m) , 3.90-3.77 (2H, m), 3.77-3.65 (2H, m), 3.26-2.97 (5H, m), 2.64-2.46 (3H, m), 2.46-2.27 (1H, m), 1.98-1.81 ( 1H, m), 1.45-1.36 (1H, m), 1.30-1.10 (2H, m), 0.77-0.60 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 812.2, 0.86 min (Method A), 3.41 min (Method B).

(2-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (R)-1-(3-((R)-(1) -Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S, 4R)-4-Fluoropyridin-3-yl)methyl)ureido)-3-hydroxypropan-2-yl ester.

Boc-linker-payload: LC/MS (uplc): M+ 982.3, 1.51 min (Method A).

3.8 mg, 0.0041 mmol, 41%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.09-8.89 (1H, m), 8.55-8.35 (1H, m), 7.84-7.69 (2H, m), 7.45-7.36 (2H, m), 7.36-7.23 (4H,m), 7.16-7.07(2H,m),7.05-6.98(2H,m),6.73-6.58(1H,m),5.40-5.10(4H,m),4.87-4.64(2H,m) , 3.88-3.79 (1H, m), 3.72-3.64 (1H, m), 3.63-3.56 (1H, m), 3.25-3.03 (6H, m), 2.43-2.26 (1H, m), 1.96-1.80 ( 1H, m), 1.44-1.32 (1H, m), 1.29-1.09 (2H, m), 0.75-0.57 (1H, m). 9.09-8.89 (1H, m), 8.55-8.35 (1H, m), 7.84-7.69(2H,m), 7.45-7.36(2H,m),7.36-7.23(4H,N-((S)-2-(3-((R)-(1-phenylmethyl-4-) (2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrole Pyridin-3-yl)methyl)ureido)propyl)-4-((2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane Formamide.

Boc-linker-payload: LC/MS (uplc): M+ 904.3, 1.29 min (Method A).

31 mg, 0.032 mmol, 85%. ¹ H-NMR (DMSO, 600 MHz): δ 9.14-8.91 (1H, m), 8.76-8.59 (1H, m), 7.94-7.83 (1H, m), 7.83-7.68 (2H, m), 7.48-7.37 (2H,m), 7.37-7.23(4H,m),7.18-7.07(1H,m),7.06-6.98(2H,m),6.54-6.24(1H,m),5.43-5.14(4H,m) , 3.91-3.76 (2H, m), 3.76-3.65 (1H, m), 3.65-3.54 (1H, m), 3.52-3.28 (4H, m), 3.28-3.13 (3H, m), 3.13 - 3.01 ( 1H,m), 2.67-2.47(2H,m), 2.47-2.31(1H,m),2.14-2.00(1H,m),2.00-1.86(1H,m),1.77-1.66(2H,m), 1.66-1.57(2H,m),1.57-1.47(1H,m),1.44-1.35(1H,m),1.34-1.10(4H,m),1.08-0.99(3H,m),0.97-0.83(2H , m), 0.78-0.60 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH + 804.2, 0.93 min (Method A).

(2-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid 1-(((R)-(((())) 1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)(((3S,4R)) 4-fluoropyrrolidin-3-yl)methyl)aminecarboxyl)azetidin-3-yl ester.

Boc-linker-payload: LC/MS (uplc): MH+ 894.2, 1.26 min (Method A).

23 mg, 0.024 mmol, 61%. ¹ H-NMR (DMSO, 600 MHz): δ 9.15-8.97 (1H, m), 8.77-8.60 (1H, m), 7.88-7.78 (1H, m), 7.78-7.69 (1H, m), 7.43-7.37 (2H,m), 7.37-7.29(3H,m), 7.28-7.21(2H,m),7.14-7.08(1H,m),7.05-6.99(2H,m),5.56-5.44(1H,m) , 5.43-5.21(2H,), 5.16-4.96(2H,m), 4.46-4.35(1H,m),4.12-4.03(1H,m),4.03-3.95(1H,m),3.88-3.80(1H , m), 3.46-3.24 (6H, m), 3.26-3.13 (1H, m), 3.13-.303 (2H, m), 2.76-2.62 (1H, m), 2.58-2.34 (3H, m), 1.95-1.80 (1H, m), 1.68-1.53 (1H, m), 1.15-0.93 (1H, m), 0.49-0.30 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH + 794.2, 0.91 min (Method A).

(2-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid 1-(((R)-(((())) 1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)(((3S,4R)) 4-Fluoropyrrolidin-3-yl)methyl)amine-mercapto)piperidin-3-yl ester.

A mixture of diastereomers is obtained which is isolated prior to coupling of the linkers.

Boc-linker-payload, separated into pure diastereomers.

Isomer A, LC/MS (uplc): M+ 922.3, 1.33 min (Method A).

Isomer B, LC/MS (uplc): M+ 922.3, 1.34 min (Method A).

Isomer A: 10 mg, 0.010 mmol, 47%. ¹ H-NMR (DMSO, 600 MHz): δ 9.11-8.93 (1H, m), 8.87-8.69 (1H, m), 7.85-7.74 (1H, m), 7.74-7.64 (1H, m), 7.45-7.36 (2H,m), 7.36-7.28(2H,m), 7.27-7.18(2H,m),7.15-7.06(1H,m),7.06-6.97(3H,m),5.69-5.59(1H,m) , 5.46-5.36, (1H, m), 5.31-5.13 (1H, m), 4.72-4.62 (1H, m), 4.62-4.54 (1H, m), 3.71-3.60 (2H, m), 3.59-3.46 (6H,m), 3.46-3.31(4H,m),3.31-3.16(2H,m),3.15-3.00(4H,m),2.99-2.90(1H,m),2.89-2.75(1H,m) , 2.48-2.30 (2H, m), 2.29-2.10 (1H, m), 1.94-1.80 (1H, m), 1.79-1.59 (2H, m), 1.58-1.48 (1H, m), 1.47-1.36 ( 1H, m), 1.19-1.04 (1H, m), 0.89-0.76 (1H, m), 0.55-0.38 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 822.3, 0.96 min (Method A).

Isomer B: 22.5 mg, 0.022 mmol, 52%. ¹ H-NMR (DMSO, 600 MHz): δ 9.19-8.99 (1H m), 8.92-8.76 (1H, m), 7.81-7.73 (1H, m), 7.71-7.63 (1H, m), 7.45-7.37 ( 2H,m), 7.37-7.27(2H,m), 7.26-7.20(2H,m),7.14-7.06(2H,m),7.05-6.99(2H,m),5.58-5.47(1H,m), 5.42-5.31 (1H, m), 5.25-5.07 (1H, m), 4.69-4.61 (1H, m), 4.56-4.47 (1H, m), 3.90-3.70 (2H, m), 3.65-3.46 (6H , m), 3.45-3.33 (5H, m), 3.33-3.20 (1H, m), 3.17-2.98 (4H, m), 2.98-2.86 (2H, m), 2.50-2.21 (3H, m), 1.99 -1.87 (1H, m), 1.79-1.71 (1H, m), 1.71-1.63 (1H, m), 1.63-1.43 (2H, m), 1.22-1.06 (1H, m), 0.90-0.76 (1H, m), 0.55-0.39 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 822.3, 0.97 min (Method A).

(3-((2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)aminocarbamic acid (S)-2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran) 4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester.

Boc-linker-payload: LC/MS (uplc): MH+ 923.2, 1.22 min (Method A).

6.1 mg, 0.006 mmol, 37%. ¹ H-NMR (DMSO, 600 MHz): δ 9.05 (1H, m), 8.64 (1H, m), 8.1 (1H, m), 7.78 (2H, m), 7.40 (2H, m), 7.33 (2H, m), 7.27 (2H, m), 7.12 (1H, m), 7.01 (2H, m), 6.97 (1H, m), 6.31 (1H, m), 5.34 (2H, m), 5.25 (1H, m ), 5.20 (1H, m), 3.94 (3H, m), 3.80 (1H, m), 3.75 (1H, m), 3.50 (1H, m), 3.45 (2H, m), 3.25 (2H, m) , 3.15 (4H, m), 2.59 (1H, m), 2.50 (1H, m), 2.26 (1H, m), 2.17 (2H, m), 2.01 (1H, m), 1.45 (1H, m), 1.30-1.20 (2H, m), 1.10 (3H, m), 0.68 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): 823.2, 0.90 min (Method A).

(2-(2-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid 2-amino-3-(( (R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)(( (3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-3-oxopropyl propyl ester.

Boc-linker-payload, mixture of diastereomers, LC/MS (uplc): M+ 982.3, 1.39 min and 1.40 min (Method A).

The product was obtained as an inseparable mixture of diastereomers (4:1, LC/MS, Method A). 17.8 mg, 0.017 mmol, 70%. ¹ H-NMR (DMSO, 600 MHz) major diastereomer: δ 9.34-9.21 (1H, m), 9.19-9.05 (1H, m), 8.71-8.55 (3H, m), 7.94-7.86 (1H , m), 7.82-7.72 (1H, m), 7.45-7.37 (2H, m), 7.37-7.31 (2H, m), 7.31-7.26 (1H, m), 7.26-7.21 (2H, m), 7.16 -7.08 (1H, m), 7.06-7.01 (2H, m), 5.56-5.49 (1H, m), 5.41-5.35 (1H, m), 5.35-5.22 (1H, m), 5.19-5.07 (1H, m), 4.56-4.47 (1H, m), 4.38-4.29 (1H, m), 4.19-4.10 (1H, m), 4.00-3.90 (1H, m), 3.78-3.68 (1H, m), 3.63 3.54(3H,m), 3.54-3.48(3H,m),3.31-3.22(1H,m),3.22-3.02(3H,m),2.83-2.71(1H,m),2.68-2.56(1H,m ), 2.46-2.35 (1H, m), 2.02-1.83 (1H, m), 1.45-1.33 (1H, m), 1.28-1.18 (1H, m), 1.01-0.86 (1H, m), 0.52-0.36 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 782.3, 0.78 min (primary) and 0.79 min (minor) (Method A).

N-((S)-2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H) -piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)-3-(2,5-di Oleoxy-2,5-dihydro-1H-pyrrol-1-yl)propanamide

Boc-linker-payload: LC/MS (uplc): MH+ 836.2, 1.26 min (Method A).

4 mg, 0.004 mmol, 24%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.05 (1H, m), 8.67 (1H, m), 8.14 (1H, m), 7.79 (1H, m), 7.75 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.27 (2H, m), 7.10 (1H, m), 7.02 (2H, m), 6.35 (1H, m), 5.35-5.25 (4H, m), 3.81 (2H) , m), 3.75 (1H, m), 3.62 (3H, m), 3.45 (2H, m), 3.37 (2H, m), 3.16 (2H, m), 3.07 (1H, m), 2.57 (1H, m), 2.41 (1H, m), 2.39 (2H, m), 1.90 (1H, m), 1.45 (1H, m), 1.25 (1H, m), 1.15 (1H, m), 1.02 (3H, m ), 0.70 (1H, m). Missing signals hidden under the solvent peak. LC/MS (up lc): MH.s.

4-(((S)-2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) (tetrahydro- 2H-piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)amino)-1-(2) , 5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)-4-oxobutane-2-sulfonic acid.

Boc-linker-payload, mixture of diastereomers: LC/MS (uplc): MH+ 930.5, 1.01 min (isomer A) and 1.02 min (isomer B), (Method A).

The mixture of diastereomers was separated using reverse phase chromatography (PrepLC Method A) after removal of the Boc protecting group.

Isomer A, 31 mg, 0.031 mmol, 10%. ¹ H-NMR (DMSO, 600 MHz): δ 9.08-8.48 (1H, m), 8.90-8.75 (1H, m), 7.94-7.84 (1H, m), 7.81-7.68 (2H, m), 7.47-7.36 (2H, m), 7.35-7.23 (4H, m), 7.15-7.04 (1H, m), 7.03-6.94 (2H, m), 6.20-6.08 (1H, m), 5.46-5.20 (4H, m) , 3.95-3.67(6H,m), 3.29-3.11(2H,m), 2.90-2.64(4H,m), 2.59-2.44(3H,m),2.13-1.91(1H,m),1.78-1.59( 1H, m), 1.52-1.36 (1H, m), 1.35-1.23 (1H, m), 1.19-1.07 (1H, m), 1.06-0.94 (3H, m), 0.70-0.53 (1H, m). Missing signals hidden under the solvent peak. 9.08-8.48 (1H, m), 8.90-8.75 (1H, m), 7.94-7.

Isomer B, 35 mg, 0.017 mmol, 11%. ¹ H-NMR (DMSO, 600 MHz): δ-NMR (DMSO, 600 MHz): mmol, 11%.m), 7.94-7.84 (1H, m), 7.81-7.68 (2H, m), 7.47-7.36 (2H , m), 7.35-7.23 (4H, m), 7.15-7.04 (1H, m), 7.03-6.94 (2H, m), 6.20-6.08 (1H, m), 5.46-5.20 (4H, m), 3.95 -3.67(6H,m), 3.29-3.66(5H,m), 3.29-3.16(2H,m),2.84-2.74(1H,m),2.73-2.58(1H,m),2.57-2.42(5H, m), 2.08-1.97 (1H, m), 1.71-1.55 (1H, m), 1.42-1.24 (2H, m), 1.21-1.08 (1H, m), 1.07-0.95 (3H, m), 0.69- 0.51 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 830.5, 0.89 min. (Method A).

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (S)-2-(3- ((S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-hydroxycyclopropyl)methyl)-3-((( 3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester.

Boc-linker-payload, LC/MS (uplc): M+ 982.5, 1.64 (Method A).

17.7 mg, 0.018 mmol, 66%. ¹ H-NMR (DMSO, 600 MHz): δ 9.20-9.08 (1H, m), 8.91-8.71 (1H, m), 7.89-7.81 (1H, m), 7.81-7.74 (1H, m), 7.40-7.36 (2H, m), 7.35-7.29 (2H, m), 7.27-7.23 (2H, m), 7.15-7.09 (1H, m), 7.05-7.00 (3H, m), 6.38-6.28 (1H, m) , 5.37-5.14(4H,m), 4.03-3.78(4H,m),3.77-3.68(1H,m),3.61-3.52(2H,m),3.52-3.45(2H,m),3.44-3.29( 4H,m),3.13-3.03(2H,m),3.03-2.93(1H,m),2.57-2.42(3H,m),1.16-1.06(3H,m),0.69-0.50(3H,m), 0.50-0.36 (1H, m). LC/MS (uplc): MH+ 768.3, 0.94 min (Method A).

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (S)-2-(3- ((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-hydroxy-2-methylpropyl)-3- (((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester.

Boc-linker-payload, LC/MS (uplc): M+ 984.5, 1.65 min (Method A).

4.1 mg, 0.004 mmol, 54%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.26-9.05 (1H, m), 9.00-8.76 (1H, m), 7.99-7.84 (1H, m), 7.83-7.71 (1H, m), 7.51-7.24 (6H,m), 7.19-7.09(1H,m),7.08-6.95(3H,m),6.55-6.37(1H,m),5.50-5.15(4H,m),4.09-3.88(2H,m) , 3.88-3.74 (2H, m), 3.60-3.53 (3H, m), 3.53-.344 (3H, m), 3.44-3.23 (4H,), 3.15-3.01 (2H, m), 2.70-2.59 ( 1H, m), 2.39-2.12 (1H, m), 2.08-1.93 (1H, m), 1.17-1.09 (3H, m), 1.09-0.98 (3H, m), 0.97-0.78 (3H, m). LC/MS (uplc): MH+ 770.6, 0.95 min (Method A).

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (S)-2-(3- ((S)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-hydroxytetrahydro-2H-pyran-4-yl)- 3-((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester.

Boc-linker-payload, LC/MS(uplc): M+ 1026.6, 1.58min (method A).

11.3 mg, 0.012 mmol, 32%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.17-8.95 (1H, m), 8.77-8.59 (1H, m), 8.02-7.88 (1H, m), 7.87-7.74 (1H, m), 7.45-7.38 (2H,m), 7.38-7.32(2H,m),7.32-7.27(2H,m),7.18-7.11(1H,m),7.06-6.99(3H,m),6.51-6.34(1H,m) , 5.55-5.10 (4H, m), 4.02-3.88 (4H, m), 3.88-3.71 (2H, m), 3.69-3.60 (1H, m), 3.60-3.54 (3H, m), 3.54-3.45 ( 3H,m),3.44-3.37(2H,m), 3.36-3.24(3H,m),3.16-3.02(2H,m),2.72-2.58(1H,m), 2.40-2.20(1H,m), 2.14-1.93 (1H, m), 1.52-1.33 (2H, m), 1.21-1.02 (5H, m). LC/MS (uplc): MH.s.

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (S)-2-(3- ((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy-2-methylpropyl)- 3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester.

Boc-linker-payload, LC/MS (uplc): MH+ 884.6, 1.36 min (Method A). 25 mg, 0.026 mmol, 82%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.10-8.93 (1H, m), 8.78-8.61 (1H, m), 7.96-7.83 (1H, m), 7.82-7.70 (1H, m), 7.48-7.26 (6H, m), 7.20-7.08 (1H, m), 7.08-6.98 (3H, m), 6.55-6.38 (1H, m), 5.65-5.45 (1H, m), 5.45-5.25 (2H, m) , 5.25-5.08 (1H, m), 3.62-3.53 (2H, m), 3.53-3.45 (2H, m), 3.45-3.35 (2H, m), 3.34-3.17 (2H, m), 3.16-3.05 ( 2H, m), 3.05-2.92 (3H, m), 2.58-2.39 (2H, m), 2.26-2.02 (1H, m), 2.02-1.82 (1H, m), 1.42-1.23 (3H, m), 1.23-1.06 (3H, m), 0.92-0.73 (3H, m). LC/MS (uplc): MH+ 784.4, 1.01 min (Method A).

(2-(2-(2,5-Di-Ethoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid (3S,4S)-4-( ((S)-N-((S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-methoxy-2 -methylpropyl)-2-hydroxypropionylamino)methyl)pyrrolidin-3-yl ester

Boc-linker-payload, LC/MS (uplc): MH+ 895.3, 1.37 min (Method A).

26.8 mg, 0.031 mmol, 79%. ¹ H-NMR (DMSO, 600 MHz): δ 9.06-8.90 (1H, m), 8.88-8.73 (1H, m), 8.72-8.63 (1H, m), 7.96-7.91 (1H, m), 7.90-7.69 (2H,m), 7.45-7.30(8H,m), 7.29-7.24(1H,m),7.19-7.07(3H,m),7.06-6.98(3H,m),6.00-5.84(1H,m) , 5.62-5.32 (2H, m), 5.17-5.03 (2h, m), 4.92-4.82 (2H, m), 4.81-4.70 (3H, m), 4.62-4.49 (2H, m), 4.11-3.99 ( 1H,m), 3.97-3.86(2H,m),3.83-3.73(1H,m),3.62-3.45(7H,m),3.44-3.28(4H,m),3.22-3.05(5H,m), 3.04-2.96(1H,m),2.92-2.71(4H,m),2.43-2.33(1H,m),2.04-1.85(2H,m),1.66-1.51(m,1H),1.50-1.41(1H m), 1.36-1.30 (3H, m), 1.30-1.24 (3H, m), 1.08-0.98 (1H, m), 0.89-0.79 (2H, m), 0.77-0.65 (3H, m). A mixture of rotamers can be observed by NMR. LC/MS (uplc): MH+ 753.3, 0.98 min (Method A).

(S)-2-((((R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)) -N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutoxy)carbonyl)amino) -3-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid

The title compound (8 mg, 17) was prepared by the method described in the general method (60 ° C, 3 days) of the removal of the Boc protecting group from the linker-payload combination after purification by reverse phase column chromatography. %, in TFA salt).

LC / MS (Method B): [M + H] + 769.4, Rt 3.21min. ^{1 H-NMR (DMSO, 400MHz} ): δ 12.89 (1H, br s), 8.97 (1H, br s), 8.54 (1H, br s), 7.97 (1H, d, 3.9Hz), 7.80-7.70 (1H , m), 7.45-7.30 (7H, m), 7.17-7.08 (1H, m), 6.99 (2H, s), 5.84 (1H, s), 5.40 (1H, d, 15, 3Hz), 5.25 (1H , d, 56 Hz), 5.06 (1H, d, 15.3 Hz), 4.63-4.54 (1H, m), 4.21-4.11 (1H, m), 4.06-3.88 (2H, m), 3.80-3.63 (4H, m ), 3.34 - 3.23 (2H, m), 2.40 - 2.35 (1H, m), 2.00-1.85 (1H, m), 1.58-1.46 (1H, m), 1.35 (3H, d, 6.2 Hz), 1.32 1.21 (1H, m), 0.95 (3H, s), 0.71 (3H, s). 1H was hidden under DMSO and OH was not seen.

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-4-(((2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(2-hydroxyethyl) Aminomethylmercapto)oxy)-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Under N ₂ : to (3R,4R)-3-(((S)-2-ethoxycarbonyl-N-((R)-1-(1-benzyl-4-(2,5-) Difluorophenyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl DIPEA (0.299 mL, 1.712 mmol) and bis(4-nitrophenyl) carbonate (39.9 mg, 0.131 mmol) were added sequentially to a solution of EtOAc (40 mg, 0.057 mmol). The reaction mixture was stirred at room temperature for 16 hours. Addition of 1-(2-((2-hydroxyethyl)amino)ethyl)-1H-pyrrole-2,5-dione (linker 1, TFA) upon completion by LC/MS (uplc) (66.1 mg, 0.114 mmol), and the mixture was stirred at room temperature for 16 h. Addition of additional 1-(2-((2-hydroxyethyl)amino)ethyl)-1H-pyrrole-2,5-dione (linker 1, TFA salt) (66.1 mg, 0.114 mmol), The mixture was stirred at room temperature for 16 hours. Add cold water and ethyl acetate, the organic layer was washed twice with brine, dried over Na ₂ SO _4, filtered and the solvent removed in vacuo. Adsorbed on Isolute. After purification by column chromatography (4 g of EtOAc, EtOAc (EtOAc:EtOAc)

LC / MS (Method A): [M + H] + 911.5, Rt 1.27min.

(2-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(2-hydroxyethyl)carbamic acid (R)-4-(1- Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidine-3 -yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutyl ester

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-4-(((2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(2-hydroxyethyl) Aminomethyl hydrazinyloxy)-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (18 mg, 0.017 mmol) was dissolved in A mixture of acetonitrile (1 mL) and water (0.500 mL). TFA (0.065 mL, 0.840 mmol) was added and the reaction mixture was stirred at 60 ° C for 7 days. The desired product (9 mg, 59% in TFA salt) was obtained after purification by reverse phase column chromatography.

LC / MS (Method B): [M + H] + 769.5, Rt 3.35min. ¹ H-NMR (DMSO, 600 MHz, ratio of the mixture of the isomers: 1:1): δ 9.02 (1H, br s), 8.68 (1H, br s), 7.98 (1H, s), 7.82-7.73 (1H, m), 7.44 - 7.31 (6H, m), 7.16-7.10 (1H, m), 6.97 and 6.96 (2H, two unimodal, rotamers), 5.84 and 5.84 (1H, two single peaks, rotation Isomer), 5.41 (1H, d, 15.4 Hz), 5.27 (1H, d, 54 Hz), 5.06 (1H, d, 15.4 Hz), 4.62-4.57 (1H, m), 4.05-3.90 (2H, m ), 3.82-3.67 (2H, m), 3.57-3.49 (2H, m), 3.46-3.07 (8H, m), 2.42-2.36 (1H, m), 1.98-1.85 (2H, m), 1.58-1.49 (1H, m), 1.38-1.23 (4H, m), 1.01 and 0.97 (3H, two monomodal, rotamers), 0.69 and 0.67 (3H, two monomodal, rotamer).

(3R,4R)-3-((R)-3-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-14-(2,5 - Bis-oxy-2,5-dihydro-1H-pyrrol-1-yl)-2-((S)-2-hydroxypropionyl)-4,4-dimethyl-8-sideoxy T-butyl -7,12-dioxa-2,9-diazatetradecyl)-4-fluoropyrrolidine-1-carboxylate

The title compound is prepared at the effective hydroxyl group by a method analogous to that described in the attachment of the payload to the linker component. Not purified. Colorless oil (40 mg, 0.023 mmol, 55% yield, 52% pure).

LC / MS (Method A): [M + H] + 912.2, Rt 1.34min.

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid (R)-4-(1- Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidine-3 -yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutyl ester

The title compound was prepared by the method described in the general method (60 ° C, 29 hr) of the Boc protecting group removed from the </ RTI><RTIgt; Purified by reverse phase column chromatography, colourless solid (19 mg, <RTIgt; LC / MS (Method B): [M + H] + 769.3, Rt 3.79min. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.04 (1H, br s), 8.73 (1H, br s), 7.97-7.93 (1H, m), 7.78-7.72 (1H, m), 7.44-7.30 (6H , m), 7.16-7.09 (1H, m), 7.00 (2H, s), 6.93-6.88 (1H, m), 5.84 (1H, s), 5.40 (1H, d, 15 Hz), 5.25 (1H, d , 54 Hz), 5.06 (1H, d, 15 Hz), 4.62-4.56 (1H, m), 4.05-3.65 (4H, m), 3.45-3.40 (2H, m), 3.37-3.15 (2H, m), 3.12 -3.05(2H,m),2.42-2.33(1H,m),2.00-1.85(2H,m),1.60-1.50(1H,m),1.35(3H,d,5.9Hz),1.32-1.24(1H , m), 0.93 (3H, s), 0.73 (3H, s).

(3R,4R)-3-((R)-2-((S)-2-Ethyloxypropanyl)-3-(1-benzylmethyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-32-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)-4,4-dimethyl-8- Trioxybutyl-7,12,15,18,21,24,27,30-octaoxa-2,9-diazatridecyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the effective hydroxyl group by a method analogous to that described in the attachment of the payload to the linker component. Not purified. Colorless oil (28 mg, 0.024 mmol, crude).

LC / MS (Method A): [M + H] + 1175.4, [M + NH 4] + 1192.5, Rt 1.34min.

(23-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18,21-heptaoxadisane (R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)- 4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutyl ester

The title compound was prepared by a method analogous to the method described in the general method of the Boc protecting group removal (RT, 1 hr). Purified by reverse phase column chromatography. Colorless solid (2.3 mg, 2.005 [mu]mol, 9% yield, 100% pure).

LC / MS (Method B): [M + H] + 1033.3; Rt 4.02min.

(3R,4R)-3-(((S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) )-4-((2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4- Trioxybutyl butyl)-2-hydroxypropionylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Purified by column chromatography (40 g EtOAc, EtOAc (EtOAc) Colorless oil (500 mg, 0.604 mmol, 60% yield, 96% pure).

LC / MS (Method A): [M + H] + 795.6, Rt 1.20min.

(R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-(((3R,4R) )-1-(Tertibutoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanylamino)-3,3-dimethylbutyric acid (980 mg, 1.005 mmol) And N-(2-aminoethyl) maleimide (TFA salt, 511 mg, 2.010 mmol) was dissolved in DMF (30 ml), and DIPEA (0.878 mL, 5.03 mmol), HATU ( 573 mg, 1.508 mmol). The reaction mixture was stirred at RT for 1 hour. The reaction mixture was diluted with EA and washed with brine (3×). The organic layers were combined and dried them with Na ₂ SO _4, filtered and concentrated. The residue was purified by EtOAc EtOAc EtOAc EtOAc (EtOAc

LC / MS (Method A): [M + H] + 795.6, Rt 1.20min.

(R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-N-(2-(2,5-di-oxy)- 2,5-Dihydro-1H-pyrrol-1-yl)ethyl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)- 2-hydroxypropionylamino)-3,3-dimethylbutyramine

The title compound was prepared by a method analogous to the method described in the general method of the Boc protecting group removal (RT, 1 hr). Purified by reverse phase chromatography, colorless solid (112 mg, 0.137 mmol, 55% yield, as TFA salt).

(3R,4R)-3-(((S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) )-4-((2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4- The side oxybutyl)-2-hydroxypropionamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (200 mg, 0.252 mmol) was dissolved in DCM (4 mL). TFA (1.94 mL, 25.2 mmol) was added and the mixture was stirred at RT for 1 hour. Concentration and purification by reverse phase chromatography gave a colourless solid (112 mg, 0.137 <RTIgt;

LC / MS (Method B): [M + H] + 695.4, Rt 3.02min. ¹ H-NMR (DMSO, 600 MHz, ratio of rotamer mixture 5:1, some peaks of minor rotamers are not explicitly specified): δ 8.96 (1.2H, br s), 8.66 (1.2H, br s), 7.85-7.80 (2H, m), 7.80-7.75 (1H, m), 7.44-7.31 (6.8H, m), 7.16-7.09 (1.6H, m), 7.01 (0.4H, s), 6.99 (2H, s), 6.17 (1H, s), 5.72 (0.2H, s), 5.55 (0.2H, d, 15.9 Hz), 5.46 (0.2H, d, 15.9 Hz), 5.33 (1H, d, 15.0) Hz), 5.17-5.05 (2.2H, m), 4.80-4.74 (0.2H, m), 4.57-4.50 (1H, m), 4.47-4.41 (0.2H, m), 4.04-3.99 (0.4H, m ), 3.95-3.90 (2H, m), 3.41 (2H, t, 6.0 Hz), 3.34 - 2.98 (5.2H, m), 2.31-2.22 (1.2H, m), 2.12 (1H, d, 13.8Hz) , 2.08-2.02 (0.2H, m), 1.99-1.89 (1.2H, m), 1.82 (1H, d, 13.8Hz), 1.73-1.58 (1H, m), 1.35 (3H, d, 6.0Hz), 1.11 (0.6H, s), 1.04 (0.6H, s), 1.00 (3H, s), 0.92 (3H, s), 0.77 (0.6H, d, 6.0 Hz).

(3R,4R)-3-(((S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) )-4-((2-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)amino)-2,2 -Dimethyl-4-oxobutyl)-2-hydroxypropionylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Purified by column chromatography (24 g EtOAc, EtOAc (EtOAc) Colorless oil (108 mg, 0.109 mmol, 44% yield, 85% pure).

LC / MS (Method A): [M + H] + 839.5, Rt 1.21min.

(R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-N-(2-(2-(2,5-two-side) Oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidine-3 -yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutyramine

General method for removal of Boc protecting groups by a similar linker-payload combination The title compound was prepared by the method of the method described in (RT, 1 hr). Purified by reverse phase chromatography to give EtOAc (EtOAc:EtOAc:

LC / MS (Method B): [M + H] + 739.4, Rt 3.20min. ¹ H-NMR (DMSO, 600 MHz, ratio of rotamer mixture 5:1, some peaks of minor rotamers were unremovably identified): δ 8.93 (1.2H, br s), 8.61 (1.2H, br s), 7.86-7.76 (2.6H, m), 7.72-7.67 (1H, m), 7.60-7.56 (0.2H, m), 7.46-7.30 (7.2H, m), 7.16-7.09 (1.8H, m ), 6.99 (2.4H, br s), 6.19 (1H, s), 5.69 (0.2H, s), 5.58 (0.2H, d, 15.0 Hz), 5.48 (0.2H, d, 15.0 Hz), 5.33 ( 1H, d, 15.0 Hz), 5.20-5.05 (2.2H, m), 4.81-4.75 (0.2H, m), 4.58-4.51 (1.2H, m), 4.05-3.90 (2.4H, m), 3.37- 3.25 (7.2H, m), 3.15-3.00 (5.2H, m), 2.60-2.55 (1.2H, m), 2.32-2.25 (2H, m), 2.21 (1H, d, 13.8Hz), 2.12-2.02 (0.4H, m), 2.00-1.90 (2.2H, m), 1.74-1.60 (1H, m), 1.35 (3H, d, 6.6 Hz), 1.13 (0.6H, s), 1.07 (0.6H, s ), 1.04 (3H, s), 0.93 (3H, s), 0.77 (0.6H, d, 5.4 Hz).

(3R,4R)-3-(((S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) -3((2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-3- Ortho-hydroxypropyl)-2-hydroxypropionylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Not purified. Crude compound

LC / MS (Method A): [M + H] + 781.4, Rt 1.19min.

(R)-3-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-N-(2-(2,5-di-oxy)- 2,5-Dihydro-1H-pyrrol-1-yl)ethyl)-3-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)- 2-hydroxypropionylamino)-2.2-dimethylpropanamide

The title compound was prepared by a method analogous to the method described in the general method of the Boc protecting group removal (RT, 1 hr). Purified by reverse phase chromatography to give a colorless solid (yield: </RTI><RTIgt; LC / MS (Method B): [M + H] + 681.2; Rt 3.13min. ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks of major rotamers): δ 8.95 (1H, br s), 8.67 (1H, br s), 7.84-7.73 (2H, m) , 7.65 (1H, d, 3.7 Hz), 7.45-7.28 (6H, m), 7.15-7.05 (1H, m), 6.94 (2H, s), 6.42 (1H, s), 5.22-5.18 (2H, m ), 5.16-5.07 (1H, m), 4.57-4.51 (1H, m), 3.95-3.85 (2H, m), 3.40-3.25 (3H, m), 3.15-3.00 (3H, m), 2.30-2.22 (1H, m), 1.97-1.87 (1H, m), 1.80-1.65 (1H, m), 1.34-1.30 (6H, m), 1.02 (3H, s).

(3R,4R)-3-(((S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) -3((2-(2-(2,5-di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)amino)-2,2 -Dimethyl-3-oxopropyl)-2-hydroxypropionylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Not purified. Crude compound

LC / MS (Method A): [M + H] + 825.4, Rt 1.20min.

(R)-3-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-N-(2-(2-(2,5-two-side) Oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)-3-((S)-N-(((3S,4R)-4-fluoropyrrolidine-3 -yl)methyl)-2-hydroxypropylamino)-2,2-dimethylpropanamide

The title compound was prepared by a method analogous to the method described in the general method of the Boc protecting group removal (RT, 1 hr). Purified by reverse phase chromatography to give a colourless solid (yield: </RTI> <RTIgt;

LC / MS (Method B): [M + H] + 725.3; Rt 3.28min. ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks of major rotamers): δ 8.93 (1H, br s), 8.63 (1H, br s), 7.83-7.72 (1H, m) , 7.71-7.66(2H,m), 7.46-7.26(6H,m),7.13-7.06(1H,m),6.99(2H,s),6.43(1H,s),5.25-5.17(2H,m) , 5.16-5.03 (1H, m), 4.57-4.51 (1H, m), 3.95-3.83 (2H, m), 3.46-3.39 (2H, m), 3.34-3.21 (3H, m), 3.19-3.01 ( 4H, m), 2.98-2.86 (1H, m), 2.30-2.22 (1H, m), 1.97-1.87 (1H, m), 1.75-1.60 (1H, m), 1.40 (3H, s), 1.34 ( 3H, d, 6.2 Hz), 1.02 (3H, s).

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-3-(((2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amine formazan Tert-butyl ester of oxy)-2,2-dimethylpropyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylate

The title compound is prepared at the effective hydroxyl group by a method analogous to that described in the attachment of the payload to the linker component. Not purified. Colorless oil (27 mg, 0.032 mmol, crude).

LC / MS (Method A): [M + H] + 853.4, Rt 1.33min.

(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (R)-3-(1-benzylmethyl-4-( 2,5-Difluorophenyl)-1H-imidazol-2-yl)-3-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)- 2-hydroxypropionyl)-2,2-dimethylpropyl ester

The title compound was prepared by the method described in the general method (60 ° C, 17 hr) of the Boc protecting group removed from the linker-payload combination. Colorless solid (4 mg, 4.85 [mu]mol, 15% yield, 100% pure).

</RTI>^<RTIID=0.0></RTI>^</RTI> ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks of major rotamers): δ 9.01 (1H, br s), 8.73 (1H, br s), 7.90 (1H, d, 3.8 Hz ), 7.80-7.74 (1H, m), 7.44-7.30 (6H, m), 7.16-7.08 (2H, m), 6.99 (2H, s), 6.04 (1H, s), 5.36 (1H, d, 15.2) Hz), 5.22-5.04 (2H, m), 4.59-4.54 (1H, m), 4.05-3.85 (2H, m, overlap with water peak), 3.80-3.72 (2H, m), 3.45 (2H, t, 6.0Hz), 3.40-3.25(2H,m), 3.14-3.08(2H,m), 2.36-2.28(1H,m),2.00-1.90(1H,m),1.80-1.68(1H,m),1.34 (3H, d, 6.2 Hz), 0.87 (3H, s), 0.80 (3H, s).

(3R,4R)-3-((R)-2-((S)-2-Ethyloxypropanyl)-3-(1-benzylmethyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-13-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)-4,4-dimethyl-7- Tert-butyl-6,11-dioxa-2,8-diazatridecyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the effective hydroxyl group by a method analogous to that described in the attachment of the payload to the linker component. Not purified. Colorless oil (37 mg, 0.042 mmol, crude).

LC / MS (Method A): [M + H] + 897.5, Rt 1.34min.

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamic acid (R)-3-(1- Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-3-((S)-N-(((3S,4R)- 4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionylamino)-2,2-dimethylpropyl ester

The title compound was prepared by the method described in the general method (60 ° C, 16 hr) of the Boc protecting group removed from the linker-payload combination. Purification by reverse phase chromatography gave the desired product. A colorless solid of TFA salt (7.7 mg, 7.98 smol, 19% yield, 90% pure).

LC / MS (Method B): [M + H] + 755.3; Rt 3.60min. ¹ H-NMR (DMSO, 600 MHz rotamer mixture, peak of major rotamer is reported): δ 9.00 (1H, br s), 8.70 (1H, br s), 7.90 (1H, d, 3.9 Hz) , 7.84-7.74(1H,m), 7.45-7.32(6H,m),7.16-7.09(1H,m),7.01(2H,s),6.94(1H,t,5.9Hz),6.07(1H,s ), 5.37 (1H, d, 15.3 Hz), 5.21-5.06 (2H, m), 4.60-4.53 (1H, m), 4.07-3.85 (2H, m), 3.80-3.70 (2H, m, with water peak Overlap), 3.60-3.22 (7H, m, overlap with water peak), 3.20-3.00 (3H, m), 2.35-2.27 (1H, m), 2.00-1.90 (1H, m), 1.85-1.70 (1H, m), 1.34 (3H, d, 6.1 Hz), 0.91 (3H, s), 0.83 (3H, s).

Step 1: (3R,4R)-3-((R)-18-azido-3-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazole-2- 2-((S)-2-hydroxypropionyl)-4,4-dimethyl-6-oxirane-10,13,16-trioxa-2,7-diaza-10- Octadecyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

(R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-(((3R,4R) )-1-(t-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanylamino)-3,3-dimethylbutyric acid (60 mg, 0.043 mmol) And 11-azido-3,6,9 trioxa-11-amine (0.017 mL, 0.086 mmol) was dissolved in DMF (2 mL), and DIPEA (0.037 mL, 0.214 mmol), (24.42 mg, 0.064 mmol). The reaction mixture was stirred at room temperature for 1 hour. Dilute with EA and wash with brine (*3). The combined organic layers were dried and concentrated to give EtOAc (EtOAc).

LC / MS (Method A): [M + H] + 873.5, Rt 1.27min.

Step 2: (3R,4R)-3-((R)-3-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-18-( 4-((2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-2- ((S)-2-hydroxypropionyl)-4,4-dimethyl-6-oxirane-10,13,16-trioxa-2,7-diazaoctadecyl)- 4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

To (3R,4R)-3-((R)-18-azido-3-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl )-2-((S)-2-hydroxypropionyl)-4,4-dimethyl-6-o-oxy-10,13,16-trioxa-2,7-diaza Alkyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (step 1) (37 mg, 0.043 mmol) and 1-(prop-2-yn-1-yl)-1H-pyrrole-2,5- A solution of CuI (9.34 mg, 0.049 mmol) in water (1 mL), TEA (6.83 μl, 0.049 mmol) was added in hexanes (1. The reaction mixture was stirred at room temperature for 60 hours. The reaction mixture was diluted with ethyl acetate, filtered, washed with brine and dried over Na ₂ SO _4. Purification by column chromatography (12 g oxime, 0 to 20% DCM in methanol). Colorless oil (24 mg, 0.013 mmol, 27% yield, 55% pure).

LC / MS (Method A): [M + H] + 1008.5, Rt 1.15min.

Step 3: (R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-N-(2-(2-(2-() 2-(4-((2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1-yl) Ethoxy)ethoxy)ethoxy)ethyl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxyl Propionamide-3,3-dimethylbutyramine

The title compound was prepared by a method analogous to the method described in the general method of the Boc protecting group removal (RT, 1 hr). Purification by reverse phase chromatography gave the desired product (yield: 2.8 g, 2.60.

LC / MS (Method B): [M + H] + 908.5, Rt 3.08min. ¹ H-NMR (DMSO, 600 MHz, ratio of rotamer mixture 5:1, some peaks of minor rotamers are not explicitly specified): δ 8.97 (1.2H, br s), 8.69 (1.2H, br s), 7.96 (1.2H, s), 8.03-7.93 (0.2H, m), 7.85-7.75 (3.4H, m), 7.59-7.55 (0.2H, m), 7.45-7.29 (7.4H, m) , 7.16-7.09 (1.6H, m), 7.07 (2H, s), 6.21 (1H, s), 5.72 (0.2H, s), 5.59 (0.2H, d, 16.2Hz), 5.48 (0.2H, d , 16.2 Hz), 5.34 (1H, d, 15.0 Hz), 5.20-5.06 (2.2H, m), 4.82-4.76 (0.2H, m), 4.65 (2.4H, s), 4.57-4.52 (1H, m ), 4.50-4.44 (2.8H, m), 4.07-4.02 (0.2H, m), 3.97-3.93 (2H, m), 3.38 (2.4H, t, 5.1 Hz), 3.52-3.40 (10H, m) , 3.32-3.25 (2.4H, m), 3.17-3.12 (2.4H, m), 3.07-3.02 (0.4H, m), 2.32-2.25 (1.2H, m), 2.22 (1H, d, 15.0Hz) , 2.12-2.01 (0.6H, m), 2.00-1.90 (2.2H, m), 1.75-1.60 (1H, m), 1.36 (3H, d, 6.0 Hz), 1.15 (0.6H, s), 1.09 ( 0.6H, s), 1.06 (3H, s), 0.94 (3H, s), 0.78 (0.6H, d, 6.0 Hz).

(3R,4R)-3-((S)-2-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4 -A Tetrahydro-2H-piperidin-4-yl)methyl)-14-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)-5-methyl- 3,8-di-oxy-7,12-dioxa-2,4,9-triazatetradecyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the effective hydroxyl group by a method analogous to that described in the attachment of the payload to the linker component. Purification by flash chromatography (24 g, silica gel) eluting with DCM / MeOH. Colorless oil (70 mg, 0.077 mmol, 51% yield).

LC / MS (Method A): [M + H] + 910.4, Rt 1.35min.

(2-(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)aminocarboxylic acid (S)-2-(3- ((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4-methyltetrahydro-2H-pyran-4-yl) Methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester

General method for removal of Boc protecting groups by a similar linker-payload combination The title compound was prepared by the method of the method described in (RT, 1 hr). Purification by reverse phase chromatography gave the desired product (35 mg, <RTIgt;

LC / MS (Method B): [M + H] + 810.2, Rt 4.10min. ¹ H-NMR (DMSO, 400 MHz): δ 8.94 (1H, bs), 8.48 (1H, bs), 7.92 (1H, d, 3.9 Hz), 7.78-7.70 (1H, m), 7.45-7.25 (7H, m), 7.15-7.07 (1H, m), 7.03 (2H, s), 6.45 (1H, d, 7.4 Hz), 5.56 (1H, s), 5.39 (1H, d, 15.3 Hz), 5.30-5.07 ( 2H,m),4.05-3.85(4H,m),3.84-3.70(1H,m), 3.60-3.45(6H,m),3.43-3.20(6H,m),3.12-3.02(2H,m), 2.47-2.37(1H,m), 2.30-2.10(1H,m),1.82-1.67(1H,m),1.54-1.35(2H,m),1.30-1.20(1H,m),1.17-1.05(6H , m), 0.97-0.89 (1H, m).

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-4-(3-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)propanylamino)-2,2-di Methylbutyl) propylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the payload amine by a method analogous to that described in the attachment of the payload to the linker component. Not purified (36 mg, purity 38%, crude product, yield 69%, 0.016 mmol).

LC / MS (Method A): [M + H] + 851.5; Rt 1.25min.

(S)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-(3-(2, 5-two Oleoxy-2,5-dihydro-1H-pyrrol-1-yl)propanylamino)-2,2-dimethylbutyl)-N-((3S,4R)-4-fluoropyrrole Pyridin-3-yl)methyl)-2-hydroxypropanamide

The title compound was prepared by the method described in the general method (60 ° C, 20 hr) of the Boc protecting group removed from the linker-payload combination. Purification by reverse phase chromatography gave a colourless solid (yield: 6 <RTIgt;

LC / MS (Method B): [M + H] + 709.4; Rt 3.27min. ¹ H-NMR (DMSO, 600 MHz): δ 9.05 (1H, br s), 8.75 (1H, br s), 7.93 (1H, d, 3.8 Hz), 7.80-7.74 (1H, m), 7.74-7.66 ( 1H, m), 7.44-7.30 (6H, m), 7.16-7.09 (1H, m), 6.99 (2H, s), 5.84 (1H, s), 5.38 (1H, d, 15.4 Hz), 5.25-5.19 (1H, m), 5.09 (1H, d, 15.4 Hz), 4.63-4.55 (1H, m), 4.05-3.90 (2H, m), 3.58-3.53 (2H, m, overlap with water peak), 3.35- 3.15(2H,m), 2.86-2.78(2H,m), 2.41-2.35(1H,m),2.31-2.23(2H,m),1.97-1.85(2H,m),1.41-1.32(4H,m ), 1.17-1.06 (1H, m), 0.92 (3H, s), 0.79 (3H, s).

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-4-(3-(2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanylamino -2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the payload amine by a method analogous to that described in the attachment of the payload to the linker component. Not purified (33 mg, yield 100%, crude product, 0.037 mmol).

</RTI>^< RTI ID=0.0></RTI>^</RTI>

(S)-N-((R)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-(3-(2- (2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanylamino)-2,2-dimethylbutyl)-N-(( (3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

The title compound was prepared by the method described in the general method (60 ° C, 20 hr) of the Boc protecting group removed from the linker-payload combination. Purification by reverse phase chromatography gave a colourless solid (10 mg, 11.02.

LC / MS (Method B): [M + H] + 753.4; Rt 3.33min. ¹ H-NMR (DMSO, 600 MHz): δ 9.05 (1H, br s), 8.75 (1H, br s), 7.93 (1H, d, 3.6 Hz), 7.80-7.74 (1H, m), 7.57-7.52 ( 1H, m), 7.45-7.30 (6H, m), 7.16-7.07 (1H, m), 7.01 (2H, s), 5.84 (1H, s), 5.38 (1H, d, 15.3 Hz), 5.32-5.17 (1H, m), 5.09 (1H, d, 15.3 Hz), 4.63-4.55 (1H, m), 4.05-3.85 (2H, m, overlap with water peak), 3.55-3.51 (4H, m), 3.47- 3.43(2H,m), 3.40-3.15(2H,m), 2.90-2.80(2H,m),2.42-2.32(1H,m), 2.23-2.16(2H,m),1.92-1.88(1H,m ), 1.45 - 1.30 (4H, m), 1.20 - 10.10 (1H, m), 0.93 (3H, s), 0.80 (3H, s).

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-4-(3-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)propanyl) Morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the payload amine by a method analogous to that described in the attachment of the payload to the linker component. Purification by flash chromatography (24 g, silica gel) eluting with DCM / MeOH. Colorless oil (50 mg, 0.059 mmol, 72% yield).

</RTI>^<RTIID=0.0></RTI>

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-4-(3-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)propanyl)-N-(((3S,4R)) -4-fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by the method described in the general method (RT, 6 hr) of the Boc protecting group removal of the linker-payload combination. Purification by reverse phase chromatography gave a colorless solid (12 mg, EtOAc (EtOAc)

LC / MS (Method B): [M + H] + 749.2; Rt 3.27min. ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers, 120 ° C): δ 7.83-7.77 (1H, m), 7.67 (1H, d, 3.7 Hz), 7.45-7.32 (3H, m), 7.30- 7.15 (3H, m), 7.10-7.07 (1H, m), 6.93 (2H, s), 5.40-5.10 (4H, m), 4.40-4.15 (1H, m), 4.05-3.85 (4H, m), 3.76-3.62 (4H, m), 3.55-3.25 (7H, m), 2.95-2.85 (1H, m), 2.75-2.55 (5H, m), 2.37-2.20 (1H, m), 1.55-1.25 (3H , m), 1.05-0.85 (1H, m).

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-4-(3-(2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)B Oxy)propanyl)morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the payload amine by a method analogous to that described in the attachment of the payload to the linker component. Purification by flash chromatography (12 g, silica gel) eluting with DCM / MeOH. Yellow oil (30 mg, 0.034 mmol, 57% yield).

LC / MS (Method A): [M + H] + 893.3; Rt 1.27min.

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-4-(3-(2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanyl)-N- (((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by a method analogous to the method described in the general method (RT, 4 hr) for the removal of the Boc protecting group of the linker-payload combination. Purification by reverse phase chromatography gave a colourless solid (yield: <RTIgt;

</RTI>^<RTIID=0.0></RTI>^</RTI>

(3R,4R)-3-(((S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-4-(6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexanyl) Morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the payload amine by a method analogous to that described in the attachment of the payload to the linker component. Purification by flash chromatography (4 g, silica gel) eluting with DCM / MeOH. Yellow oil (13 mg, 0.014 mmol, 35% yield).

</RTI>^<RTIID=0.0></RTI>

(S)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-4-(6-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)-N-(((3S,4R)) -4-fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by the method described in the general method (RT, 6 hr) of the Boc protecting group removal of the linker-payload combination. Purification by reverse phase chromatography gave a colorless solid (10 mg, 10.72.

LC / MS (Method B): [M + H] + 791.3; Rt 3.72min.

(3R,4R)-3-(((R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-4-(3-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)propanyl) Morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the payload amine by a method analogous to that described in the attachment of the payload to the linker component. Purification by flash chromatography (12 g, silica gel) eluting with DCM / MeOH. Colorless oil (15 mg, 0.018 mmol, 60% yield).

LC / MS (Method A): [M + H] + 849.2; Rt 1.30min.

(R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-4-(3-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)propanyl)-N-(((3S,4R)) -4-fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by the method described in the general method (RT, 3 hr) of Boc. Purification by reverse phase chromatography gave a colourless solid (15 mg, <RTIgt;

LC / MS (Method B): [M + H] + 749.1; Rt 3.56min.

(3R,4R)-3-(((R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Tetrahydro-2H-piperazin-4-yl)methyl)-4-(6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexanyl) Morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the payload amine by a method analogous to that described in the attachment of the payload to the linker component. Flash chromatography by dissolving with DCM/MeOH (4 g, silicone resin) purification. Yellow oil (22 mg, 0.025 mmol, 84% yield).

</RTI>^<RTIID=0.0></RTI>^</RTI>

(R)-N-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- Methyl)-4-(6-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)-N-(((3S,4R)) -4-fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by the method described in the general method (RT, 6 hr) of the Boc protecting group removal of the linker-payload combination. Purification by reverse phase chromatography gave a colourless solid (15 mg, <RTIgt;

LC / MS (Method B): [M + H] + 791.2; Rt 3.88min.

(3R,4R)-3-((S)-2-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(4 -methyltetrahydro-2H-piperidin-4-yl)methyl)-32-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)-5-A -3,8-di-oxy-7,12,15,18,21,24,27,30-octaoxa-2,4,9-triazadodecyl)-4-fluoro Pyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is prepared at the effective hydroxyl group by a method analogous to that described in the attachment of the payload to the linker component. Not purified. Yellow oil (63 mg, 0.054 mmol, 83% yield, crude).

LC / MS (Method A): [M + H] + 1174.6,), [M + NH 4] + 1191.7, Rt 1.35min.

(23-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18,21-heptaoxadisane (S)-2-(3-((R)-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)) Methyltetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl ester

The title compound was prepared by a method analogous to the method described in the general method of the Boc protecting group removal (RT, 1 hr). Pure by reverse phase chromatography The desired product (10 mg, 8.33 μmol, 16% yield, TFA salt) was obtained.

LC / MS (Method Bv2): [M + H] + 1074.4, Rt 4.49min.

(3-(N-((R)-1-(3-Benzyl-7-chloro-4-yloxy-3,4-dihydroquinazolin-2-yl)-2-methylpropane) 4-(benzyl)amino)propyl)aminocarbamate 4-((S)-2-((S)-2-(6-(2,5-di- oxo-2), 5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutylideneamino)-5-ureidopentylamino)benzyl ester

The title compound is prepared by a method analogous to that described in the general procedure for the attachment of the linker to the amine in the payload compound of formula (II). Purification by reverse phase chromatography gave colorless solid (8 mg, 6.74.

LC / MS (Method B): [M + H] + 1115.6; Rt 5.71min. ¹ H-NMR (DMSO, 400 MHz): δ 9.96 (1H, s), 8.23 (1H, d, J = 8.6 Hz), 8.07 (1H, d, J = 7.5 Hz), 7.83-7.74 (2H, m) , 7.65 (1H, dd, J = 8.6, 2.1 Hz), 7.61 - 7.54 (2H, m), 7.41 - 7.10 (12Hm), 7.00 (2H, s), 6.77 (1H, t, J = 5.9 Hz), 5.97 (2H, br s), 5.88 (1H, d, J = 16.3 Hz), 5.53 (1H, d, J = 10.5 Hz), 5.07 (1H, d, J = 16.3 Hz), 4.89 - 4.75 (2H, m), 4.43-4.33 (1H, m), 4.19 (1H, dd, J = 8.6, 6.7 Hz), 3.40-3.20 (6H, m), 3.08-2.90 (2H, m), 2.79-2.68 (1H, m), 2.46-2.40 (2H, m), 2.31 (3H, s), 2.24-2.04 (2H, m), 2.02-1.90 (1H, m), 1.76-1.10 (10H, m), 0.92-0.79 ( 9H, m), 0.48 (3H, d, J = 6.4 Hz).

(3-(N-((R)-1-(3-benzyl-7-chloro-4-yloxy-4H-) Alken-2-yl)-2-methylpropyl) -4-methylbenzhydrylamino)propyl)aminocarbamic acid 4-((S)-2-((S)-2-(6- (2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutyrylamino)-5-ureidopentadelamyl) Benzyl methyl ester

The title compound is prepared by a method analogous to that described in the general procedure for the attachment of the linker to the amine in the payload compound of formula (II). Purification by reverse phase chromatography gave a colourless solid (26mg, <RTIgt;

LC / MS (Method B): [M + H] + 1115.5; Rt 5.58min. ¹ H-NMR (DMSO, 400 MHz): δ 9.96 (1H, s), 8.11 - 8.04 (2H, m), 7.90 (1H, s), 7.83 - 7.76 (1H, d, J = 8.6 Hz), 7.61 7.52(3H,m), 7.29-7.11(12H,m), 7.00(2H,s),5.99(2H,br s), 5.76(1H,d,J=10.0Hz),4.90-4.77(2H,m ), 4.42-4.32 (1H, m), 4.23-4.12 (2H, m), 3.95-3.85 (1H, m), 3.42-3.25 (3H, m), 3.06-2.88 (2H, m), 2.64-2.53 (6H, m), 2.31 (3H, s), 2.25-2.05 (1H, m), 2.02-1.87 (1H, m), 1.75-1.65 (1H, m), 1.65-1.29 (7H, m), 1.25 -1.10 (3H, m), 0.97 (3H, d, J = 6.6 Hz), 0.87-0.79 (6H, m), 0.57-0.50 (3H, m).

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-4-(((2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(2-hydroxyethyl) Aminomethylmercapto)oxy)-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Under N ₂ : to (3R,4R)-3-(((S)-2-ethoxycarbonyl-N-((R)-1-(1-benzyl-4-(2,5-) Difluorophenyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl DIPEA (0.374 mL, 2.140 mmol) and bis(4-nitrophenyl)carbonate (50 mg, 0.164 mmol) were added sequentially to a solution of EtOAc (50 mg, EtOAc). The reaction mixture was stirred at room temperature for 16 hours. Addition of 1-(2-((2-hydroxyethyl)amino)ethyl)-1H-pyrrole-2,5-dione (linker 1, TFA) upon completion by LC/MS (uplc) Salt (180 mg, 0.604 mmol), and the mixture was stirred at room temperature for 16 h. Add cold water and ethyl acetate, the organic layer was washed twice with brine, dried over Na ₂ SO _4, filtered and the solvent removed in vacuo. Adsorbed on Isolute. After purification by column chromatography (4 g of EtOAc, EtOAc (EtOAc) LC/MS (Method A): MH.

(3R,4R)-3-((R)-2-((S)-2-Ethyloxypropanyl)-3-(1-benzylmethyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-9-(2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-4,4, 14,14-tetramethyl-8,12-di-oxy-7,13-dioxa-2,9-diazapentadecyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester

Under N ₂ : to (3R,4R)-3-(((S)-2-ethoxycarbonyl-N-((R)-1-(1-benzyl-4-(2,5-) Difluorophenyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl DIPEA (0.374 mL, 2.140 mmol) and bis(4-nitrophenyl)carbonate (50 mg, 0.164 mmol) were added sequentially to a solution of EtOAc (50 mg, EtOAc). The reaction mixture was stirred at room temperature for 16 hours. Addition of 3-((2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino) when found by LC/MS (uplc) Tributyl butyl propionate (linker 2, TFA salt) (54.6 mg, 0.143 mmol), and the reaction mixture was stirred at room temperature for 16 h. Add cold water and ethyl acetate, the organic layer was washed twice with brine, dried over Na ₂ SO _4, filtered and the solvent removed in vacuo. Adsorbed on Isolute. After purification by column chromatography (4 g of EtOAc, EtOAc (EtOAc) LC/MS (Method A): MH.

3-(((R)-4-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-( ((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropionylamino)-3,3-dimethylbutoxy)carbonyl)(2-(2,5) -di- oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)propionic acid

(3R,4R)-3-((R)-2-((S)-2-Ethyloxypropanyl)-3-(1-benzylmethyl-4-(2,5-difluorobenzene) -1H-imidazol-2-yl)-9-(2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-4,4, 14,14-tetramethyl-8,12-di-oxy-7,13-dioxa-2,9-diazapentadecyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl The ester (40 mg, 0.040 mmol) was dissolved in a mixture of acetonitrile (1 mL) and water (1 mL). TFA (0.155 mL, 2.010 mmol) was added and the mixture was stirred at <RTI ID=0.0># </RTI> <RTIgt;

The desired product (13 mg, 35% in TFA salt) was obtained after purification by reverse phase column chromatography. LC/MS (Method B): MH. The mixture of rotamers was observed by ¹ H-NMR at room temperature and some of the peaks were hidden under water. ¹ H-NMR (DMSO, 600 MHz): δ 12.27 (1H, br s), 8.98 (1H, br s), 8.63 (1H, br s), 7.96 (1H, s), 7.76 (1H, s), 7.45 -7.30 (6H, m), 7.16-7.09 (1H, m), 6.97 and 6.96 (2H, two unimodal, rotamer), 5.83 (1H, s), 5.45-5.37 (1H, m), 5.26 (1H, d, 54 Hz), 5.09-5.03 (1H, m), 4.62-4.57 (1H, m), 4.04-3.90 (2H, m), 3.82-3.66 (2H, m), 2.46-2.35 (3H , m), 2.00-1.85 (2H, m), 1.55-1.20 (5H, m), 1.03 and 0.96 (3H, two unimodal, rotamers), 0.67 and 0.63 (3H, two single peaks, Rotamer).

(2-(2,5-Di-Sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (R)-4-(1-benzyl-4-( 2,5-Difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)- 2-hydroxypropionyl)-3,3-dimethylbutyl ester

(3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-) 1H-imidazol-2-yl)-4-(((2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)aminecarboxamido)oxy Tert-butyl-2,2-dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (50 mg, 0.073 mmol) dissolved in acetonitrile (1 mL) and water ( In a mixture of 0.5 mL). TFA (0.222 mL, 2.88 mmol) was added and the mixture was stirred at <RTIgt;

The desired product (17 mg, 34% in TFA salt) was obtained after purification by reverse phase column chromatography. LC/MS (Method B): MH. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.01 (1H, br s), 8.68 (1H, br s), 7.96 (1H, s), 7.76 (1H, s), 7.45-7.30 (6H, m), 7.15-7.03 (2H, m), 6.99 (2H, s), 5.84 (1H, s), 5.40 (1H, d, 15 Hz), 5.25 (1H, d, 54 Hz), 5.06 (1H, d, 15 Hz), 4.62-4.56(1H,m),4.05-3.65(4H,m), 3.45-3.40(2H,m),3.37-3.15(2H,m),3.12-3.05(2H,m),2.42-2.33(1H , m), 2.00-1.85 (2H, m), 1.60-1.50 (1H, m), 1.35 (3H, d, 5.9 Hz), 1.32-1.24 (1H, m), 0.93 (3H, s), 0.73 ( 3H, s).

(2-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (R)-4-(4-(2,5-difluoro) Phenyl)-1-(3-hydroxybenzyl)-1H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl) )methyl)-2-hydroxypropionyl)-3,3-dimethylbutyl ester

Step 1 (a)-Activation : to (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorobenzene) 1-(3-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino) To a stirred solution of 1,4- fluoropyrrolidine-1-carboxylic acid tert-butyl ester (128 mg) in DMF (4 mL), DIPEA (304 &lt;RTIgt; The mixture was stirred at RT for 2 h at which time LC-MS showed the reaction was completed and converted to the desired intermediate.

Step 1 (b) - Amine solution : To the above crude product mixture was added N-(2-aminoethyl) maleimide imine trifluoroacetate (98 mg). The RM was stirred for an additional 2 hours at 50 °C at which time LC-MS showed conversion to the desired intermediate. The RM was transferred to a sep. funnel and deionized water (40 mL) and ethyl acetate (40 mL) was then applied. After extraction, the aqueous layer was extracted with EtOAc EtOAc EtOAc (EtOAc m. . Crude product yield: 270 mg. This material was used directly in the next step.

Step 2: Removal of BOC and MOM and thiol protecting groups : The crude product from step 1 (b) was dissolved in acetonitrile (1 ml) and 6M HCl (1 mL) was added. The RM was stirred at 60 ° C for 3 hours, at which time LC-MS showed that the reaction was almost complete, and the protecting group was removed to give a final product. The RM was filtered through a 0.2 [mu]m PTFE cartridge filter and the resulting clear solution was purified by reverse phase preparative HPLC method C. The product containing fractions were lyophilized overnight to yield a white loose powder. Yield: 24.7 mg.

UPLC-MS: Rt = 0.85 min; MS m/z [M+H] ⁺ 741.3;

UPLC-MS: Rt = 3.30 min; MS m/z [M+H] ⁺ 741.3;

1H-NMR (HSQC) consistent with the target structure.

(2-(2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamic acid (R)-4-(4-(2,5-difluoro) Phenyl)-1-(3-fluoro-5-hydroxybenzyl)-1H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidine -3-yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutyl ester

Step 1 : A stirred solution of triphosgene (119 mg) in dry DCM (5 mL) was cooled to 0 ° C and placed under argon atmosphere. (3R,4R)-3-(((S)-2-Ethyloxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-) 3-fluoro-5-(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanylamino)methyl) A solution of -4-fluoropyrrolidine-l-carboxylic acid tert-butyl ester (80 mg) and DIPEA (0.258 ml) in DCM (2 mL). Stirring was continued for 2 hours at RT.

Step 2 : The above crude RM was cooled to 0 ° C and N-(2-aminoethyl) maleimide imine trifluoroacetate (150 mg) and DIPEA (0.258 ml) were added dropwise to DCM (3 mL) Solution in ). The RM was allowed to warm to RT and stirring was continued for 10 minutes. The temperature was then increased to 50 ° C and stirring was continued for 1 hour at which time LC-MS showed conversion to the desired intermediate. The RM was concentrated under high vacuum to give a dark orange oil. This material was used directly in the next step.

Step 3: Removal of BOC, MOM and thiol protecting groups : The crude product from Step 2 was dissolved in acetonitrile (2 mL) and 6M HCl (1.6 mL). The RM was stirred at 60 ° C for 2 hours at which time LC-MS showed the reaction was completed and the protecting group was removed to give a final product. The RM was filtered through a 0.2 [mu]m PTFE cartridge filter and the resulting solution was divided into 2x2 mL batches and purified by reverse phase preparative HPLC method C.

The product-containing fractions were combined and then lyophilized overnight to yield a white loose powder. Yield: 34.3 mg

UPLC-MS: Rt = 0.74 min; MS m/z [MH] ^- 757.3;

UPLC-MS: Rt = 2.94 min; MS m/z [M+H] ⁺ 759.2;

^{1 H NMR (600MHz, DMSO- d} 6) δ 10.10 (s, 1H), 9.03 (s, 1H), 8.70 (s, 1H), 7.93 (s, 1H), 7.81-7.73 (m, 1H), 7.41 -7.29(m,1H),7.19-7.10(m,1H),7.06(t, J =6.2Hz,1H),6.98(s,2H),6.70-6.58(m,2H),6.53(d, J =10.5 Hz, 1H), 5.79 (s, 1H), 5.37-5.21 (m, 2H), 4.95 (d, J = 15.6 Hz, 1H), 4.60 (q, J = 6.3 Hz, 1H), 4.03 (dd , J =16.1, 4.4 Hz, 1H), 3.89 (dd, J = 15.9, 7.4 Hz, 1H), 3.82 (q, J = 8.3 Hz, 2H), 3.65-3.37 (m, 2H), 3.37-3.17 ( m, 2H), 3.08 (q, J = 5.9 Hz, 2H), 2.47-2.33 (m, 1H), 2.07-1.84 (m, 2H), 1.69 (dt, J = 14.2, 7.0 Hz, 1H), 1.35 (d, J = 6.2 Hz, 4H), 0.95 (s, 3H), 0.77 (s, 3H).

(R)-4-(4-(2,5-difluorophenyl)-1-(3-hydroxybenzyl)-1H-imidazol-2-yl)-N-(2-(2,5- Bis-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl) )methyl)-2-hydroxypropionylamino)-3,3-dimethylbutyramine

Step 1 : To (R)-4-((S)-2-Ethyloxy-N-(((3R,4R)-1-(T-butoxycarbonyl)-4-fluoropyrrolidine-3) -yl)methyl)propanylamino)-4-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy)benzyl)-1H-imidazole- HATU (82 mg) was added to a solution of 2-yl)-3,3-dimethylbutyric acid (80 mg) and DIPEA (189 uL) in anhydrous DMF (2 mL). The RM was stirred at RT for 10 minutes and then N-(2-aminoethyl) maleimide diamine trifluoroacetate (92 mg) was added. The RM was stirred at RT over the weekend when LC-MS showed the reaction was complete. RM was partitioned between dichloromethane (40 mL) and deionized water (40 mL). The mixture was transferred to a sep. funnel and after extraction the aqueous was extracted with dichloromethane (40 mL). The combined organics were washed with EtOAc (EtOAc)EtOAc. Crude product yield: 251 mg. The product still contains some DMF, but is used directly in the next step:

Step 2: Removal of BOC, MOM and thiol protecting groups : To the crude product from step 1, acetonitrile (4 ml) and 6M HCl (1.2 mL) were added. The RM was stirred at 60 ° C for 2 hours at which time LC-MS showed the reaction was complete and the protecting group was removed to yield the desired final product. The RM was filtered through a 0.2 [mu]m PTFE cartridge filter and the resulting clear solution was divided into 2x 2.6 mL batches and purified by reverse phase preparative HPLC method C. The product-containing fractions were combined and lyophilized overnight to yield a beige/creamy loose powder. Yield: 7 mg.

UPLC-MS: Rt = 2.72 min; MS m/z [M+H] ⁺ 711.3;

1H-NMR (HSQC) consistent with the target structure.

(R)-4-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-hydroxybenzyl)-1H-imidazol-2-yl)-N-(2-( 2,5-di-oxy-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidine -3-yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutyramine

Step 1: To (R)-4-((S)-2-Ethyloxy-N-(((3R,4R)-1-(t-butoxycarbonyl)-4-fluoropyrrolidine-3) -yl)methyl)propanylamino)-4-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-(methoxymethoxy)phenylmethyl)- HATU (147 mg) was added to a solution of 1H-imidazol-2-yl)-3,3-dimethylbutyric acid (144 mg) and DIPEA (450 uL) The RM was stirred at RT for 10 minutes and then N-(2-aminoethyl) maleimide diamine trifluoroacetate (164 mg) was added. The RM was stirred at RT for 2 hours at which time LC-MS showed the reaction was completed to afford intermediate. The RM was concentrated in vacuo and the residue was taken in EtOAc (3 mL). The solution was filtered through a 0.2 [mu]m PTFE cartridge filter and divided into 2 x 1.5 mL batches. These batches were purified by reverse phase preparative scale HPLC Method C.

The product-containing fractions were combined and concentrated in vacuo to give a white powder. Yield: 43 mg.

Step 2: Removal of BOC, MOM and thiol protecting groups : The product from Step 1 was dissolved in acetonitrile (2 mL) and 6M HCl (1.0 mL). The RM was stirred at 60 ° C for 2 hours at which time LC-MS showed that the reaction was actually completed and the protecting group was removed to give a final product. The RM was filtered through a 0.2 [mu]m PTFE cartridge filter and the resulting solution was divided into 2x2 mL batches and purified by reverse phase preparative HPLC method C.

The product-containing fractions were combined and then lyophilized overnight to yield a white loose powder. Yield: 23.2 mg.

UPLC-MS: Rt = 2.56 min; MS m/z [MH] ^- 727.4;

1H-NMR (HSQC) consistent with the target structure.

Cysteine metabolites: General procedure for the synthesis of cysteine metabolites:

To a solution of Boc-linker-available (0.1 mmol, 1 eq) in acetonitrile (1 mL) EtOAc (1 mmol The reaction mixture was stirred at room temperature for 1 hour. The crude product (Boc-Cys-metabolite) was used in the next step when the reaction was found to be completed by LC/MS (uplc, Method A).

TFA (150 mmol) was added to the solution from the previous step and the mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude product was subjected to reverse phase column chromatography (PrepLC method C or D), gradient MeCN (+0.1% TFA) to H ₂ O (+0.1) Purified by % TFA). The product was isolated as a TFA salt.

(2R)-2-amino-3-((1-((1R,5S)-1-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-) 2-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-5-methyl-3,8-di- oxy-l-(tetrahydro-2H-per喃-4-yl)-7,12-dioxa-2,4,9-triazatetradecane-14-yl)-2,5-di-oxypyrrolidin-3-yl)thio ) Propionic acid.

Boc-Cys-metabolite: LC/MS (uplc): MH+ 1017.6, 1.07 min. (Method A).

21 mg, 0.018 mmol, 16%. ¹ H-NMR (DMSO, 600 MHz): δ 9.17-8.59 (1H, m), 8.81 - 8.59 (1H, m), 8.58-8.31 (3H, m), 7.83-7.69 (2H, m), 7.45-7.36 (2H,m), 7.35-7.21(4H,m),7.14-6.98(2H,m),6.43-6.23(1H,m),5.38-5.28(2H,m),5.27-5.10(2H,m) , 4.31-4.21 (1H, m), 4.15-4.03 (1H, m), 4.00-3.80 (4H, m), 3.78-3.68 (1H, m), 3.27-3.15 (5H, m), 3.14 - 3.02 ( 4H, m), 2.65-2.47 (4H, m), 2.33-2.15 (1H, m), 2.07-1.90 (1H, m), 1.46-1.32 (1H, m), 1.28-1.15 (2H, m) 1.14 -1.02 (3H, m), 0.79-0.58 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 917.6, 0.80 min. (Method A).

(3R)-6-((1R,5S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-(((3S) , 4R)-4-fluoropyrrolidin-3-yl)methyl)-5-methyl-3,8,16-trityloxy-1-(tetrahydro-2H-pyran-4-yl)- 7,12-Dioxa-2,4,9,15-tetraazaheptadecan-17-yl)-5-oxothiomorpholine-3-carboxylic acid.

4 mg, 0.0037 mmol, 99%. ¹ H-NMR (DMSO, 600 MHz): δ-NMR (DMSO, 600 MHz): 4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-(((3) , 4R)-4-fluoropyrrolidin-3-yl)methyl)-5-methyl-3,8,16-trityloxy-42-6.21 (1H, m), 5.40-5.09 (4H, m ), 4.38-4.26 (1H, m), 4.06-3.80 (5H, m), 3.80-3.68 (1H, m), 3.68-3.55 (2H, m), 3.26-3.07 (8H, m), 3.05-2.91 (1H, m), 2.80-2.69 (1H, m), 2.66-2.54 (1H, m), 2.36-2.15 (1H, m), 2.09-1.91 (1H, m), 1.47-1.35 (1H, m) , 1.32-1.15 (2H, m), 1.14-1.04 (3H, m), 0.78-0.59 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): M+ 917.4, 084 min.

(2R)-2-amino-3-((1-((1-((S))-2-(3-((R)-(1-phenylmethyl-4-(2,5-difluoro)) Phenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)- Urinyl)propyl)-1H-1,2,3-triazol-4-yl)methyl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid.

Boc-Cys-metabolite: LC/MS (uplc): MH+ 967.5, 1.06 min. (Method A).

10 mg, 0.009 mmol, 62%. ^{1 H-NMR (DMSO, 600MHz} ): δ 9.20-8.90 (1H, m), 8.69-8.23 (4H, m), 8.08-7.97 (1H, m), 7.83-7.70 (2H, m), 7.41-7.36 (2H,m), 7.35-7.29(2H,m), 7.26-7.22(2H,m),7.14-7.08(1H,m),6.58-6.31(1H,m),5.39-5.07(4H,m) , 4.72-4.58 (2H, m), 4.49-4.34, (2H, m), 4.33-4.06 (4H, m), 3.92-3.81 (2H, m), 3.79-3.67 (2H, m), 3.11-2.98 (1H, m), 2.70-2.43 (4H, m), 2.38-2.18 (1H, m), 2.06-1.80 (1H, m), 1.41-1.29 (1H, m), 1.28-1.19 (1H, m) , 1.18-1.02 (4H, m), 0.75-0.55 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 867.5, 0.78 min. (Method A).

(2R)-2-amino-3-((1-(4-((()())))) Phenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)- Urinyl)propyl)amino)-4-oxooxy-2-sulfobutyl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid.

Boc-cys-metabolite (mixture of diastereomers): LC/MS (uplc): MH+ 1051.5, 0.97 min. (Method A).

The mixture of diastereomers was separated using reverse phase chromatography (PrepLC Method A) after removal of the Boc protecting group.

Isomer A, 27 mg, 0.024 mmol, 8%. ¹ H-NMR (DMSO, 600 MHz): δ 9.12-8.96 (1H, m), 8.86-8.78 (1H, m), 8.48-8.24 (3H, m), 7.97-7.84 (1H, m), 7.81-7.65 (2H, m), 7.48-7.37 (2H, m), 7.36-7.24 (4H, m), 7.17-7.01 (1H, m), 6.18-6.01 (1H, m), 5.49-5.16 (4H, m) , 4.34-4.20 (1H, m), 4.15-4.05 (1H, m), 3.96-3.66 (6H, m), 2.87-2.64 (4H, m), 2.61-2.39 (3H, m),, 2.13-1.97 (1H, m), 1.93-1.58 (2H, m), 1.50-1.36 (1H, m), 1.35-1.20 (1H, m, 1.19-0.93 (4H, m), 0.71-0.52 (1H, m). The signal hidden under the solvent peak was absent. LC/MS (uplc): MH+ 951.5, 0.79 min. (Method A).

Isomer B, 28 mg, 0.025 mmol, 8%. ¹ H-NMR (DMSO, 600 MHz): δ 9.14-8.96 (1H, m), 8.86-8.61 (1H, m), 8.51-8.24 (3H, m), 8.11 - 7.89 (1H, m), 7.80-7.65 (2H, m), 7.49-7.38 (2H, m), 7.37-7.23 (4H, m), 7.16-7.01 (1H, m), 6.01-5.90 (1H, m), 5.49-5.17 (4H, m) , 4.35-4.19 (1H, m), 4.16-3.96 (2H, m), 3.92-3.66 (6H, m), 3.11-2.98 (1H, m), 2.89-2.77 (1H, m), 2.75-2.59 ( 1H,m), 2.58-2.44(4H,m),2.10-1.93(1H,m),1.72-1.56(1H,m),1.41-1.24(2H,m),1.21-1.09(1H,m), 1.08-0.95 (3H, m), 0.75-0.53 (1H). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 95. (Method A).

(2R)-2-amino-3-((1-((1R,5S)-1-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-) 2-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-5-methyl-3,8-di- oxy-l-(tetrahydro-2H-per喃-4-yl)-7-oxa-2,4,9-triazapentadecan-15-yl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid .

Boc-Cys-metabolite: LC/MS (uplc): M+ 1029.7, 1.12 min (Method A).

Cys-metabolite: LC/MS (uplc): M+ 929.6, 0.83 min (Method A).

(2R)-2-amino-3-((1-((1R,4S)-1-(1-benzylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-) 2-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-4-methyl-3,6-di- oxy-l-(tetrahydro-2H-per喃-4-yl)-5,10-dioxa-2,7-diazadodecane-12-yl)-2,5-di- oxypyrrolidin-3-yl)thio)propyl acid.

Boc-Cys-metabolite: LC/MS (uplc): M+ 988.5, 1.10 min (Method A).

Cys-metabolite: LC/MS (uplc): M+ 888.5, 0.79 min (Method A).

(3R)-6-((1R,4S)-1-(1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2-(((3S) , 4R)-4-fluoropyrrolidin-3-yl)methyl)-4-methyl-3,6,14-trilateral oxy-1-(tetrahydro-2H-pyran-4-yl)- 5,10-Dioxa-2,7,13-triazapentadecan-15-yl)-5-oxothiomorpholine-3-carboxylic acid.

Cys-metabolite: LC/MS (uplc): M+ 888.5, 0.83 min (Method A).

(2R)-2-amino-3-((1-(6-((((()))(((((((((())))) Phenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amine Andyl-1-yloxypropan-2-yl)oxy)carbonyl)amino)hexyl)-2,5-di- oxypyrrolidin-3-yl)thio)propionic acid.

Boc-Cys-metabolite: LC/MS (uplc): M+ 1000.5, 1.14 min (Method A).

Cys-metabolite: LC/MS (uplc): M+ 900.4, 0.83 min (Method A).

(2R)-2-amino-3-((1-(3-((S))-2-(((R)-(1-phenylmethyl-4-(2,5-difluorophenyl))) -1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)aminecarboxamide )morpholinyl)-3-oxopropyl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid

Step 1: To a solution containing (3R,4R)-3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazole) 2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-4-(3-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl) Benzyl)morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (25 mg, 0.029 mmol) in acetonitrile (volume: 1.5 ml) and water (volume L-cysteine (5.35 mg, 0.044 mmol) was added to 0.500 ml), and RM was stirred at RT for 2 hr.

Step 2: HCl (25% aq.) (EtOAc (EtOAc) (EtOAc) (EtOAc) concentrate.

Purification by reverse phase chromatography gave a colorless solid, &lt;RTI ID=0.0&gt;&gt;

</RTI>^< RTI ID=0.0></RTI>

(2R)-2-amino-3-((1-(2-((R)-4-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-imidazole-2) -yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-3,3-dimethyl Butylamino)ethyl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid

Step 1: To (3R,4R)-3-(((S)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)-4-((2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2,2-di Methyl-4-oxobutyl)-2-hydroxypropionylamino)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (30 mg, 0.038 mmol) in acetonitrile (volume: 1.5 ml) L-cysteine (13.7 mg, 0.113 mmol) was added to water (volume: 0.500 ml), and RM was stirred at RT for 16 hours.

Step 2: TFA (0.145 mL, 1.887 mmol) was added and EtOAc was stirred at RT for 60 hr. concentrate.

Purification by reverse phase chromatography gave a colorless solid double TFA salt (37 mg, 0.035 mmol, 93% yield, 99% pure).

LC / MS (Method B): [M + H] + 816.3; Rt 2.37min.

(2R)-2-amino-3-((1-(2-(2-((R)-4-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-3,3 -Dimethylbutyrylamido)ethoxy)ethyl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid

Step 1: To (3R,4R)-3-(((S)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-) Imidazolyl-2-yl)-4-((2-(2-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)amino -2,2-Dimethyl-4-oxobutyl)-2-hydroxypropionamido)methyl)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester (54 mg, 0.055 mmol, L-cysteine (19.9 mg, 0.164 mmol) was added to 85% pure acetonitrile (volume: 1.5 ml) and water (volume: 0.500 ml), and RM was stirred at RT for 16 hours.

Step 2: TFA (0.211 mL, 2.74 mmol) was added and EtOAc was stirred at 60 &lt;0&gt;C for 4 h. concentrate.

Purification by reverse phase chromatography gave a colorless solid, &lt;RTI ID=0.0&gt;&gt;

LC / MS (Method B): [M + H] + 860.5; Rt 2.50min.

(2S)-2-amino-3-((1-((S))-2-(((())))) -1H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-) 3,3-Dimethylbutoxy)carbonyl)amino)-2-carboxyethyl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid

11 mg of a colorless powder of TFA salt (11 mg, 9.84 μmol, 13.2% yield, 100% pure) was obtained.

LC / MS (Method B): [M + H] + 890.3; Rt 2.54 / 2.63min ( two diastereomers isomer).

The cysteine metabolites of compounds 5A, 5D and 5E were prepared and were highly potent Eg5 inhibitors with IC-50 <0.5 nM, 0.5 nM and 0.6 nM, respectively.

General scheme for the synthesis of dissimilated products 2

To a solution of (L)-cysteine (10 eq) in water (0.1 M) was added Boc-protected linker-avail (1.0 eq) of DMF (0.1 M) and the reaction was stirred at room temperature 1 hour. It was reacted with ethyl acetate. Dried composition organic extracts were Na ₂ SO _4, filtered and concentrated to dryness. The residue was dissolved in DCM (0.1M) and trifluoroacetic acid (50 eq). The reaction was stirred at RT for 2 hours and then concentrated to dryness. The crude product was purified by EtOAc (EtOAc) elute

(2R)-2-amino-3-((1-(2-(((((((((((((((((((((((((((( 4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-piperidin-4-yl)methyl)-2-hydroxypropylamino)methyl)pyrrole Pyridin-3-yl)oxy)carbonyl)amino)ethyl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid

49% yield; UPLC-MS: Rt = 1.66 and 1.67 min; MS m/z [M+H] ⁺ 842.3; ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers and diastereomers): δ 7.99-6.77 (12H, m), 5.64-4.99 (3H, m), 4.87-4.85 (1H, m ), 4.53-4.51 (1H, m), 4.03-2.05 (25H, m), 1.51-0.57 (7H, m).

Rearranged cysteine metabolites: (3R)-6-(2-((2-((R)-4-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4) -((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-3,3-dimethylbutylamamine Ethyl)amino)-2-oxoethyl)-5-oxothiomorpholine-3-carboxylic acid

(2R)-2-amino-3-((1-(2-((R)-4-(1-phenylmethyl-4-(2,5-difluorophenyl)-1H-imidazole-2) -yl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropylamino)-3,3-dimethyl Isobutyryl)ethyl)-2,5-di-oxypyrrolidin-3-yl)thio)propionic acid (17 mg, 0.016 mmol) was dissolved in acetonitrile (volume: 0.5 mL) and 1.5 mL was added. PBS buffer (pH 7.2). The RM was stirred at RT for 10 days. Purification by reverse phase chromatography gave a colorless solid, &lt;RTI ID=0.0&gt;&gt;

LC / MS (Method B): [M + H] + 816.5; Rt 2.54min.

Synthetic ADC

The ADC was obtained and characterized as described below in the general binding procedure.

General method for the binding of a linker-payload (L-P) to an antigen binding moiety

The payload binds to the antigen binding portion (eg, IgG1 κ or λ of the antibody) at the partially reduced hinge and interchain disulfide in a 2-step method. The antibody at a concentration of 5-10 mg/ml in PBS containing 2 mM EDTA was first partially reduced by 50 mM mercaptoethylamine (added as a solid) at 37 ° C for 1 to 1.5 hours. Desalting and adding 1% w/v PS-20 cleaner Thereafter, the partially reduced antibody (1-2 mg/ml) was reacted overnight at 4 ° C with a linker-paying compound in an amount of 0.5-1 mg per 10 mg of antibody, and the compound was dissolved in DMSO or other suitable solvent at 10 mg/ml. in. The antibody-drug conjugate was purified by protein A chromatography. After baseline washing with PBS, the conjugate was lysed with 50 mM citrate (pH 2.7), 140 mM NaCl, neutralized and sterile filtered. The ADCs described in Tables 5-6 were prepared according to this procedure. This procedure produces an average drug load of 4-6 payload molecules per antibody: specific examples of products of this general procedure are shown in Tables 5-6, which identify antibodies ligated to a sequence with trastuzumab The linkage group - the payload portion, and provides a measured DAR value and % aggregation data for each combination. Biological data from selected combinations of Tables 5-6 are provided in the following sections and related figures.

A different approach is used for antibodies in which a new cysteine residue is introduced by protein engineering as a binding site. In order to reduce all disulfide bonds between the primary disulfide bond and the cysteine or GSH adduct of the engineered cysteine residue, freshly prepared DTT is added to the Cys of the previously purified antibody. In the mutant, to a final concentration of 20 mM. After incubation with DTT for 1 hour at 37 °C, the mixture was dialyzed against PBS for 3 days at 4 °C with daily buffer exchange to remove DTT and reoxidize native disulfide bonds. An alternative method is to remove the reducing reagent via a desalting column Sephadex G-25. Once the protein was sufficiently reduced, 1 mM oxidized ascorbate (dehydroascorbic acid) was added to the desalted sample and reoxidized for 20 hours. Reoxidation restores intrachain disulfide, while dialysis allows for the removal of cysteine and glutathione linked to the newly introduced cysteine. (These methods are to produce similar results, but follow previously described in the literature of attempts to re-oxidation scheme using CuSO ₄ to cause protein precipitation. The examples herein are all using the dialysis solution.

The reoxidized antibody was bound to compound 223 (5B) by incubating 5 mg/ml antibody with 0.35 mM maleimide compound in 50 mM sodium phosphate buffer (pH 7.2) for 1 hour. The completeness of the reaction was monitored by RP-HPLC and typically obtained as DAR of 3-4. turn off The sample prepared by this method had a DAR of 3.2 for cKitA. The engineered antibody produced slightly higher DAR under the same conditions, with DAR = 3.9 for cKitB-5B conjugate and DAR = 4.0 for cKitC-5B conjugate. The DAR measurements are further verified by MS.

The following ADC was prepared by the above procedure using an anti-Her2 antibody (such as trastuzumab) as an antibody (referred to herein as "TBS"), demonstrating the versatility and efficacy of the Eg5 inhibitor of Formula II as a payload compound. Characterization data (DAR and % aggregation) of the numerous immunoconjugates of Ab = TBS are presented in Table 7; the structure of ADC No. 110 to No. 133 ADC is shown here.

ADC-112.

ADC-115.

ADC-118.

ADC-121.

ADC-124.

ADC-127.

ADC-131.

Characterization of antibody-drug conjugates

Immunoconjugates prepared as described above are characterized by LC/MS as illustrated in Figure 1 for an immunoconjugate. Binding typically provides a linker that binds to an antibody - effective negative A mixture of conjugates having a different number of copies of the loaded portion. Mass spectrometry confirmed that the linker-payload group was attached to the light and/or heavy chains in each of the conjugates in Tables 5-6. The conjugate was characterized in terms of average drug loading (DAR, drug to antibody ratio) and aggregation (expressed in %) of the sample.

The DAR values of the reduced and deglycosylated samples were extrapolated from the LC-MS data. As illustrated in Figure 1 for the combination of trastuzumab with Compound No. 223 (TBS-Compound 223), LC/MS allows quantification of the average number of payload (drug) molecules attached to the antibody in the ADC. HPLC separates antibodies into light and heavy chains, and the heavy chain (HC) and light chain (LC) are distinguished by the number of linker-payload groups per chain. Mass spectrometry data enables the identification of component species in the mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, and the like. The average drug to antibody ratio (DAR) of the ADC can be calculated from the average load on the LC and HC chains (see Figure 1B). The DAR of a given conjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains. In this example, the DAR is 5.8.

Some conjugates were found to form aggregates, and the conjugates described herein were also characterized by the degree of aggregation. Aggregation was measured by analytical size exclusion chromatography (Superdex 200 5/150 GL operating in PBS). It was observed that the degree of aggregation depends on the linker-payload and DAR. Combinations with a lower percentage of aggregation may be more suitable for some purposes, and thus immunoconjugates exhibiting less than 50% aggregation and preferably less than 20% aggregation may be preferred. The data also demonstrates that the degree of aggregation of a given payload can be manipulated by selecting a linkage group and DAR. Table 7 also indicates that the linking groups of the respective conjugates are stable or cleavable.

The structure of some of these ADCs (ADC-110 to ADC-133) was found in the previous section. Note that ADC-127 and ADC-128 are comparative examples, and the payload is outside the scope of equation (II). Some ADCs appear more than once in the table, indicating different formulations of the ADC, indicating % aggregation and some changes in DAR. Up to 15-20% change in DAR caused less change in observed biological activity, see Figures 28-29.

While the examples in Table 7 are conjugated to trastuzumab, several linker-payload combinations of the invention have also been conjugated to other antibodies and antigen binding moieties for different antigens. A description of other antibodies and an overview of their characterization and activity are provided in the following activity examples, and characterization data for a number of such combinations are provided below. A conjugate having an antibody against other tumor cell antigens exhibits a growth inhibitory effect on cells exhibiting a high level of antigen recognized by an antibody in the ADC, and a growth inhibitory effect on cells lacking the antigen. This demonstrates that an ADC having an Eg5 inhibitor of formula (II) as its payload can be used to target different cell lines or tumor types by selecting antibodies that recognize antigens on the targeted cell line, thereby providing an effective binding of the present invention. Antibodies that target other cells and antigens evidence of.

The cKitA antibody and the anti-gH antigen binding portion are described in the Examples, see Example 5 and Example 8.

Activity Example 1. In vitro antiproliferative activity of Eg5 inhibitor

The compound of formula (II) is structurally similar to the known Eg5 inhibitors. Its in vitro activity at the target site was verified by measuring the IC50 against Eg5. The table below provides IC-50 data for selected compounds: the lower limit for the analytical conditions used is 0.0005 μM.

Substance and method Cell line

In order to generate a cell line that overexpresses Her2, MDA-MB-231 breast cancer cells stably transduced with a lentiviral construct (pLenti 6.3 (Invitrogen); driven by a cellular giant virus enhancer-promoter) encoding Her2 antigen The mutant version (NM_004448; codon K753M) lacks kinase activity and is therefore a non-oncogene, but is still recognized by the anti-Her2 antibody. The cell line MDA-MB-231/Her2 mutant type 16 ("pure line 16") overexpressing Her2 was isolated by fluorescence-activated cell sorting and selection with blasticidin. Mock-transduced MDA-MB-231-M40 cell lines (transduced with lentivirus but not exogenous Her2) were isolated in the same manner. Pure line 16 and parental MDA-MB-231 cultures were maintained by relaying RPMI-1640 growth medium supplemented with 10% (v/v) fetal bovine serum. The surrogate model SK-OV-3ip was isolated by successive passage of SK-OV-3 cells in the peritoneal cavity of mice to select cells that thrived in the rodent host. SK-OV-3ip cells were maintained by relaying in McCoy's 5A growth medium supplemented with 10% (v/v) fetal bovine serum. T47D2 cells are variants of the T47D breast cancer cell line. Other cell lines exhibiting a high level of Her2 antigen include breast cancer cell line HCC1954 and ovarian cancer cell line OE-19. The gastric cancer cell line NCI-N87 is a moderate Her2 performer that provides additional efficacy testing. MDA-MB-231 parental and M40 cell lines and Her2-negative breast cancer cell line MDA-MB-468 were used to assess selectivity for Her2 expression. Anti-cKit ADCs (cKitA, cKitB and cKitB) were tested on high cKit NCI-H526 cells (small cell lung cancer cell lines) and cKit negative MDA-MB-468 cells. All other cell lines are readily available from standard suppliers. All cell lines were maintained by successive passages in a humidified 37 ° C incubator containing 5% CO ₂ atmosphere.

Cell proliferation using antibody-drug conjugate treatment

On day 0, cells were seeded in 96 wells of clear bottom black wall culture dish (Costar #3603) in 90 μL of growth medium at 3000 cells per well. On day 1, antibody-drug conjugates were diluted in cell growth medium at a 10-fold higher than final concentration starting at 90 [mu]g/mL and down to 1.5 ng/mL via 3-fold serial dilution. The conjugate dilution (10 [mu]L/well) was then added to a 96 well cell culture dish; the final concentration was reduced from 9000 ng/mL to 0.15 ng/mL. Prepare double or triple replicate samples. The cells were placed in a humidified 37 ° C incubator with a 5% CO ₂ atmosphere. On day 5 or 6, the 96-well plate was removed from the incubator and allowed to equilibrate to room temperature. Cells TiterGlo 2 (50 μL/well; Promega #G7571) were added to each well and agitated for 10 minutes. Bioluminescence (indicating relative ATP content) was measured using Wallac MicroBeta photometry.

Cell proliferation treated with Eg5 inhibitor

On day 0, cells were seeded in 96 wells of clear bottom black wall culture dish (Costar #3603) in 90 μL of growth medium at 3000 cells per well. On day 1, the Eg5 inhibitor was diluted in cell growth medium at a 10-fold higher than the final concentration, starting at 1000 nM and down to 51 pM via 3-fold serial dilution. Inhibitor dilutions (10 [mu]L/well) were then added to 96 well cell culture dishes; final concentrations ranged from 100 nM to 5.1 pM. Prepare double or triple replicate samples. The cells were placed in a humidified 37 ° C incubator with a 5% CO ₂ atmosphere. On day 5 or 6, the 96-well plate was removed from the incubator and allowed to equilibrate to room temperature. Cell TiterGlo 2 (50 μL/well; Promega #G7571) was added to each well and agitated for 10 minutes. Bioluminescence (indicating relative ATP content) was measured using Wallac MicroBeta photometry.

Definition and derivation of performance values

The Cell TiterGlo 2 data for the replicate samples were averaged and then corrected for untreated cells. Dose response curves were derived using a four parameter logic model (S-type dose response model #205) provided by the additional XL-Fit Suite Software (IDBS) used as MicroSoft Excel.

Fit=(A+((B-A)/(1+((C/x)^D))))))

Inv=(C/(((B-A)/(y-A))-1)^(1/D))))

Res=(y-fit)

EC50 = concentration of the test article when the fitted Cell TiterGlo 2 signal is 50% of the signal produced by the untreated cells.

IC50 = concentration of the test article when the fitted Cell TiterGlo 2 signal reduces the signal difference between the untreated cells and the maximum effect of the test article by 50%. For example, if the maximum effect is that the signal reduction is reduced to 40% of the untreated cells, the IC50 is the concentration at which the fitted dose response curve reaches 70% of the untreated cells. The IC50 is equivalent to the parameter "C" in the fitting algorithm provided above.

result

Cell proliferation in the presence of an Eg5 inhibitor is performed as described in the above methods. After 5 or 6 days, cell counts were determined using Cell TiterGlo 2 Reagent (Figure 2, except Figures 2 (A)-(B)), which were incubated for 3 days. The data for the double replicate samples were averaged and the mean was then corrected for the average of untreated cells or cells treated at the lowest tested concentration. Both calibration methods produce comparable results. Comparison of Compound #2 (Table 1) and Compound #14 (Table 1) in Figures 2 (E)-(F) was performed using a single sample of each concentration, which was corrected for the lowest test concentration. Information on the cellular activity of selected inhibitors of formula (II) and on comparative Eg5 inhibitors is provided in the table below.

These Eg5 inhibitors have antiproliferative activity against cell lines from a variety of different lineages, indicating that these molecules have broad potential as ADC payloads and as cancer therapeutics. The inhibitory concentrations in these assays are generally within the tight range of any given compound, and the observed changes in maximal inhibition appear to be consistent with cell cycle arrest in the G2/M transition phase, followed by apoptosis; Wide variations between cell lines can explain changes in maximal compound effects.

A variety of anti-proliferative activities were observed when compared in a panel of Eg5 inhibitors. Note that the order of compound potency is generally maintained between one cell line and another. Cellular potency is influenced by a number of factors, including the intrinsic inhibition of Eg5 enzyme activity and the permeability of cell membranes to compounds. For example, Compound #77 contains a carboxylic acid that will be deprotonated in large amounts at physiological pH, which may explain why this compound is slightly less potent than other compounds in this test (Figure IE).

A variety of chemical scaffold displays confer strong anti-proliferative activity. For example, Figures 2(A)-(E) show examples of Eg5 inhibitors in the third butyl and THP series (R ¹ = tributyl or 4-tetrahydropyranyl, respectively), and A THP series inhibitor with core urea (A = NH) or core guanamine (A = bond). Examples from each series inhibited proliferation at a concentration of Yanamimus. Compounds with relatively low potency have also been observed: in some cases, compounds with lower potency are disproportionately effective as cell proliferation inhibitors when delivered to cells in the form of antibody-drug conjugates.

Figures 3(A)-(L) illustrate examples of anti-proliferative activity of specific Eg5 inhibitors against various cancer cell lines derived from different lineages (see Table 8 below). Although the potency does vary, all cell lines in these lineages are sensitive to compounds of formula (II) and (III).

Activity Example 2. In vitro antiproliferative activity of Eg5 inhibitor ADC

Cell proliferation in the presence of an antibody-drug conjugate ("ADC") having an anti-Her2 trastuzumab antibody ("TBS") and an Eg5 inhibitor was performed as described in the above method. 5 Or after 6 days, cell counts were determined using Cell TiterGlo 2 reagent. The data for the double replicate samples were averaged and the mean was then corrected for the mean of untreated cells. Figures 4A-V show paired dose response plots illustrating the antiproliferative effect of ADC on high Her2 pure lineage 16 cells versus low Her2 parental MDA-MB-231 cells. Regardless of the specific linker chemistry used, the activity of the ADC is highly sensitive to elevated Her2 performance.

4A-H illustrate the in vitro efficacy of an ADC using a linker designed to cleave in lysosomes to release an unmodified Eg5 inhibitor inside a target cell. Combinations of different payloads (from different family members) with two different cleavable linkers (containing the dipeptide glutamate-citrulline or glycan-glucuronide) are generally anti-proliferative for pure lineage 16 but for The anti-proliferative properties of the parental MDA-MB-231 cells were much worse.

4G-H illustrate the selective activity of TBS-Compound 312, which incorporates the Eg5 inhibitor compound #77 containing a carboxylate group. The free compound has moderate anti-proliferative activity (Fig. 2E), which may be due to poor membrane permeability at extracellular pH (about 7). When released in an acidic lysosome (pH about 5), the carboxylate group will be partially geologically removed, thereby removing charge and improving membrane permeability, which in turn explains the activity of the ADC.

It is contemplated that different properties can be obtained via the use of a non-cleavable linker whereby the intracellular metabolism of the ADC will result in an payload and an adduct coupled to a linker derived from one or more amino acids of the antibody. The in vitro anti-proliferative activity of an exemplary ADC having a non-cleavable linker is illustrated in Figures 4I-R. This series contains linkers that are joined at the Eg5 region of the inhibitor known or predicted to be not involved in Eg5 binding. The linker shown in Figures 4I-R varies with respect to the mode of attachment to the payload and to the antibody cysteine. In addition, linkers vary in length and predicted physical properties such as lipophilicity and conformational flexibility.

All of these ADCs are linked to the antibody cysteine via a maleimide group in the linker, except for the TBS-compound 300 (Fig. 4K, L, O, P), which uses an iodoethylamine group. The group is subjected to thiol coupling. The Her2 selective in vitro potency of this ADC confirmed that maleimide was not required for antiproliferative activity.

A non-cleavable linker ligated to a payload at a different site was used to generate an ADC having the in vitro activity as illustrated in Figures 4S-V. Furthermore, attachment of the lin-bonding group ker at different positions on the payload does not prevent Her2-dependent cellular potency from being achieved, and it is confirmed that the linkage point of the linking group on the compound of the formula (II) can be varied.

To confirm that ADC can inhibit proliferation of endogenous Her2-expressing cells, selected ADCs with anti-Her2 antibodies were incubated with HCC1954 (Fig. 5A, B) or SK-OV-3ip (Fig. 5C-E) cells. ADCs with cleavable or non-cleavable linkers derived from different linker-payload structure families inhibit proliferation of these high Her2 cell lines.

Activity Example 3. In vivo efficacy evaluation of Eg5 inhibitor ADC

Compounds of the invention that bind to trastuzumab (TBS) also demonstrated significant activity in xenograft tumor models based on implantation of human tumor cell lines into immunodeficient nude mice. As previously described (Sausville and Burger, 2006), studies using this tumor xenograft mouse have provided valuable insight into the in vivo efficacy of anti-cancer agents. Specifically, in vivo efficacy studies were performed using nu/nu mice subcutaneously injected with 5.0 x 10 ⁶ SK-OV-3ip cells (Yoneda et al., 1998) or HCC1954 cells. These cell lines were selected based on previous in vitro potency assays that revealed their high sensitivity to the aforementioned Eg5 inhibitor ADCs in an antigen-dependent manner. After the tumor reached a size of about 200-250 mm ³ , the Eg5 inhibitor ADC was intravenously injected in a single dose at a dose of 0.3 mg/kg to 10 mg/kg, depending on the experiment, wherein each treatment group contained 9 mice. Tumor volume was monitored twice weekly after administration of the antibody-drug conjugate. All animal studies were performed according to Guide for the Care and Use of Laboratory Animals (NIH publication; National Academy Press, 8th edition, 2001).

Figures 6 (A) and 6 (B) show the efficacy of a linker-payload compound No. 220 ADC (TBS-compound 220) with trastuzumab for HCC1954 breast cancer xenograft tumors in mice. Figure 6 (A) shows tumor size changes over a period of about 55 days using a single dose of TBS-Compound 20 conjugate of 1, 2 or 3 mg/kg, which contains Compound 12 (Table 1) was used as a payload. The 1 mg/kg dose showed a modest reduction in tumor growth compared to the control without the Eg5 inhibitor, while the 2 mg/kg and 3 mg/kg doses prevented tumor growth during the test. Figure 3b shows the results of the 3 mg/kg and 6 mg/kg doses which prevent tumor enlargement.

Animals treated with TBS alone (anti-Her2 antibody without Eg5 inhibitor) (Figure 6 (B)) at 6 mg/kg (corresponding to the amount of antibody in the highest conjugate dose tested) allowed tumor volume during the test period It is about four times larger and appears to be similar to a vehicle-only control treatment. Other animals received the isotype control-Compound 220 conjugate, which is a conjugate with the same payload linked to antibodies that do not target HCC1954 cells. At similar doses of the Eg5 inhibitor, the isotype control exhibited minimal tumor growth inhibition relative to the TBS control, but was significantly less effective in inhibiting tumor growth than the TBS-Compound 220 conjugate targeting HCC1954 cells, except at 6 mg/kg. At the dose, the isotype control exhibited almost the same activity as TBS-Compound 220.

Figures 7 (A) and 7 (B) show similar results in SKOV3ip xenografts. A single dose of the combination of Compound 220 and TBS was administered to mice at doses of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, and 10 mg/kg, which delivered a dose of 5 to 96 μg/kg. Compound 12. In this xenograft, a 3 mg/kg dose of TBS-Compound 220 conjugate demonstrated potent tumor growth inhibition. In this tumor model, the isotype control also showed some tumor growth inhibition, but was lower than the TBS conjugate, and the trastuzumab antibody alone had a smaller growth inhibitory effect relative to the vehicle control.

Figure 8 summarizes tumor growth of SKOV3ip xenografts after administration of a single dose of a combination of Compound 215 and trastuzumab (TBS-Compound 215) at 5 mg/kg and 10 mg/kg. A 5 mg/kg dose achieved significant growth inhibition, while a 10 mg/kg dose reduced tumors. In this experiment, a 5 mg/kg dose of trastuzumab alone (no Eg5 inhibitor) and a 5 mg/kg dose of an isotype control (compound 215 that binds to antibodies against SKOV3ip tumor cells) appeared to inhibit tumors. Growing. In addition, they are not as good as TBS-compound 215 knots The compound is effective at comparable doses.

Figure 9 summarizes the administration of a single dose of Compound 223 (comprising Compound 17 as a payload) to trastuzumab (TBS-Compound 223, or TBS-5B) at 5 mg/kg and 10 mg/kg. Tumor growth of SKOV3ip xenografts. A 5 mg/kg dose achieved significant growth inhibition, while a 10 mg/kg dose resulted in tumor arrest. In this experiment, a single trastuzumab (no Eg5 inhibitor) at a dose of 10 mg/kg moderately inhibited tumor growth. An isotype control at 5 mg/kg and 10 mg/kg dose (Compound 215 bound to antibodies that do not recognize SKOV3ip tumor cells) did not significantly inhibit tumor growth compared to vehicle treated tumors.

Activity Example 4. Novel linkers that reduce ADC aggregation.

In this example, the ADC is constructed from a simple linker and a payload compound that produces a significant and undesired amount of aggregation. The antibody used in each of the ADCs in this example was TBS. A construct called ADC-110 uses unbranched linkers and exhibits about 12% aggregation. See Figure 10 (A). Modification of the linker of this ADC (see ADC-111 and ADC-112) to introduce polar groups of deuterated nitrogen reduces aggregation to less than 3%. See Figure 10 (B) and Figure 10 (C).

Payload and payload + link connection points for each of these ADCs:

Thus, ADC-111 consists of the antibody trastuzumab linked to the maleimide group of compound 367, and ADC-112 is the same antibody linked to the maleimide group of compound 368. composition. ADC-110 consists of compound 366 and trastuzumab. The linkers in these ADCs are considered to be non-cleavable. These ADC confirmed, Formula II, IIC and III compounds may be used as the R ¹ and the target binding moiety (e.g. an antibody, even when a non-cleavable linker midnight) of the connection point.

Figure 11 shows the in vitro efficacy of ADC-110 and ADC-111 against several cancer cell lines, demonstrating that these conjugates are highly active against cell lines with high Her2 content (SKOV3ip and MB231-M16), with Her2 but Cells that are less sensitive to trastuzumab ADC have low activity, which may be due to Her2 turnover (MB231-W6) and have much lower activity on Her2-negative cells (MB468), which are immediately Eg5 The inhibitor is highly sensitive. The SKOV3ip and MDA-MB231-M16 cell lines have high Her2 expression and are therefore expected to be sensitive to these trastuzumab conjugates. MB231-W6 also has Her2 performance, but is less sensitive to trastuzumab ADC, which may be due to the turnover rate of Her2. MDA-MB468 is Her2 negative but highly sensitive to Eg5 inhibitors.

Figure 11 (A) shows that SKOV3ip cells are highly sensitive to ADC-110 and ADC-111 and are expected to be attributed to high Her2 levels on these cells. The sensitivity of the MB468 cell line is much lower, as expected based on its Her2 deficiency. Figure 11B shows that the MB321-M16 cell line is more sensitive than MB321-W6 and is believed to be due to the lower turnover of the Her2 antigen in MB321-W6 cells, resulting in less effective ADC internalization. Figure 11 (C) compares SKOV3ip to two low-sensitivity cell lines MB468 and MB231-W6. The data in Figure 11 also shows the branching link in ADC-111. The sub-linker does not significantly affect its activity relative to the unbranched linker in ADC-110. Thus, the branching linker reduces the aggregation of the ADC without interfering with efficacy.

Similarly, ADC was prepared using antibody TBS and payload/linker compound 6D (see Table 6). This ADC has a linker having a carboxylate group directly on the alkyl chain of the linker. Other ADCs lacking the same carboxylate group on the linker were prepared. The ADC with a carboxylic acid group on the linker has DAR = 4.9 and 3% aggregation, while the corresponding ADC lacking a carboxylic acid group on the linker has DAR = 4.2, but aggregates to 11.6%. Again, the polar groups on the linker significantly reduce aggregation. These ADCs all exhibit antibody-dependent inhibition of tumor cell growth in cell culture on Her2+ cell lines, but ADCs with carboxylic acids on the linker are significantly more active against MB468 cells (breast cancer).

Activity Example 5. Activity of immunoconjugates targeting other antigens.

Immunoconjugates were prepared by binding to the antibody designated cKitA by the payload shown in Table 5. This antibody recognizes a different antigen from an antibody called trastuzumab, and trastuzumab is selective for the antigen cKit (also known as CD117). cKit is found on hematopoietic stem and progenitor cells and is associated with mast cell tumors, gastrointestinal matrix tumors (GIST), germ cell tumors, and some leukemias. Thus, the immunoconjugates of the invention having an anti-cKit antibody are useful for treating such conditions.

A conjugate having a drug to antibody ratio (DAR) between 3.0 and 4.5 is thus prepared by the above method. The activity of the immunoconjugate was tested in a cell line expected to be recognized by the anti-cKit antibody cKitA. Figure 12 shows inhibition of cell growth by six of these immunoconjugates. Although activity varied with payload, it was extremely active at concentrations below 1 [mu]g/mL of immunoconjugate, with the most activity at about 1 ng/mL, demonstrating effective cell growth inhibition.

Activity Example 6. Comparison of immunoconjugates with linker changes.

Immunoconjugates were prepared from the compounds of Tables 5 and 6 by the methods described herein. These compounds contain similar Eg5 inhibitors as payloads but with different linkers. In each case, the payload is linked via a group corresponding to R ¹ in Formula II. An antibody referred to herein as TBS is used. Each of the immunoconjugates has a DAR between 3 and 5. These immunoconjugates are tested for cell growth inhibition against four different cell lines that are expected to have altered sensitivity to antibodies. All cell lines were found to be inhibited by each compound. The change in linker within the scope of Formula II as described herein has a measurable but generally modest effect on cell growth inhibition as illustrated by the inhibition curve in Figure 13. A connection point of the linker, i.e., a nitrogen atom in the azacyclic ring at R ¹ is cyclobutane ring, under the test conditions did significantly reduce activity. The data demonstrates that a variety of linkers are suitable for use in immunoconjugates of formula I containing a compound of formula II. It is noteworthy that placing the hydroxyl group on the phenyl ring corresponding to Ar2 does not significantly increase or decrease the activity in the cell culture.

Activity Example 7. Comparison of Eg5 inhibitors of Formula II with other Eg5 inhibitors as ADC payloads.

Immunoconjugates were prepared from the payloads 6N (payload-linker compound 509) and 6P (compound 508) in Table 2. These payloads are known to be effective Eg5 inhibitors in the literature and represent classes of compounds other than the compound of formula II. For comparison, immunoconjugates with a compound of formula II (compound 220) as a payload and containing the same "val-cit" cleavable linker as 6N and 6P were tested along with other classes of Eg5 inhibitors. While other classes of Eg5 inhibitors exhibit activity as a payload, their immunoconjugates are about 10 times less active than immunoconjugates with a compound of formula II payload, as illustrated in Figure 14.

In vivo activity

General procedure: Female nu / nu mice (Harlan Laboratories, Livermore, CA) was injected subcutaneously in the right flank at 5x10 ⁶ th suspended SK-OV-3ip1 tumors (BD Biosciences) in a total volume of 200μL HBSS / 50% Matrigel ^TM cell. Alternatively, female SCID / Beige mice (Harlan Laboratories, Livermore, CA) was injected subcutaneously in the right flank suspended in a total volume of 200μL ^{HBSS / 50% Matrigel TM (BD} Biosciences) 5x10 6 th H526 tumor cells. All animal studies were performed according to Guide for the Care and Use of Laboratory Animals (NIH publication; National Academy Press, 8th edition, 2001).

For efficacy studies, mice were randomly assigned between 7-10 days post implantation and animals with an average tumor volume of approximately 225 ^mm3 were included in the study. Typically, at least 5 mice are used for each treatment group, and the number of mice in each group is indicated in the graph outlining the test results. Mice were administered intravenously via the lateral tail vein with antibody-drug conjugate or vehicle (50 mM citrate, 140 mM NaCl, pH 7.3).

Dosing was started from day 0, and tumor xenografts were measured in two dimensions (L and W) twice a week using a digital caliper. Tumor volume was calculated as (L x W ² )/2. Body weight was measured twice a week and clinical observations were recorded daily. Tumor volume and body weight were captured and stored by StudyDirector software (StudyLog, South San Francisco, CA). Animals were humanely sacrificed after a 50-day study period.

Active example 8.

The immunoconjugate of Formula 5B (TBS-5B) in Table 5 in which the antibody (AntiB) is trastuzumab is prepared as described herein. In cell analysis, the immunoconjugate with the same antibody and known ADC payload (DM1) (TBS-DM1) and AntiB in it is an immunoconjugate of igG1 κ chain specific for viral glycoprotein gH (Immunoconjugate: gH-5B) compared. Figure 15 shows the results of cell proliferation of TBS-5B, TBS-DM1 and gH-5B in a cell line with high Her2 expression (SK-OV-3ip, human ovarian cancer cell line). The activity of the combination with 5B as a payload was comparable to that of a combination with DM1 as a payload, confirming the high efficacy of the Eg5 inhibitor as an ADC payload. As expected, the conjugate with gH as the antibody component exhibited minimal activity in the same cell line, confirming that the activity of the TBS-5B conjugate is dependent on the TBS antibody. For comparison, 5B-TBS was also tested on cell lines with low Her2 performance. As expected, the immunoconjugate is inactive against cell lines lacking the antigen (Her2) targeted by its antibody. This confirms that the activity of 5B is dependent on the matching of the antibody of the immunoconjugate to the performance of its targeted antigen on the cell.

Next, the immunoconjugates were tested in SK-OV-3ip xenograft tumors in mice in vivo. This xenograft responded poorly to TBS-DM1.

Figure 16 shows the effective tumor growth inhibition of the immunoconjugate TBS-5B of the present invention. Two control groups were used: one control group was treated with TBS antibody alone (TBS), and the other control group was treated with 5B and IgG κ-binding conjugate (gH-5B) specific for glycoprotein gH. By day 20, the TBS-5B immunoconjugate produced normal dose response and statistically significant tumor suppression relative to vehicle and gH controls using the indicated criteria. At the 10 mg/kg dose, there was almost no tumor growth at 30 days. Each group had 9 mice and none of them exhibited significant body weight loss during treatment. Controls have lower tumor growth inhibition using the indicated criteria, And not statistically significant under the test conditions. These results demonstrate that Eg5 inhibitors are effective ADC payloads for the treatment of tumors targeted by their antibodies in vivo, and the efficacy depends on both the Eg5 inhibitor and the antibody that matches the cell line.

Figure 17 illustrates Eg5 immunoconjugates in vivo in SK-OV-3ip xenografts in mice compared to conjugates containing the ADC used in clinical trials and linked to the same antibody (TBS) payload (DM1) Tumors are more active. Again, the TBS-5B immunoconjugate almost completely stopped tumor growth at a dose of 10 mg/kg, and by 30 days, using the indicated criteria, was statistically significant relative to the control. A comparable dose of TBS-SMCC-DM1 was less effective and did not reach statistical significance by 30 days. The activity of treatment with a single antibody (TBS) or with DM1 or 5B linked to gH viral glycoprotein IgG is much lower. This confirms that the Eg5 inhibitor payload is at least as effective as other payload categories that have been successful in clinical trials (DM1 is the payload of the FDA-approved immunoconjugate Kadcyla®) and its in vivo efficacy is dependent on the immunoconjugate Specific recognition of antibodies to targeted tumor cells.

Activity Example 9: In vivo efficacy of using other antibodies

An immunoconjugate of an Eg5 inhibitor of the invention comprising a specific antibody (herein referred to as "cKit antibody") linked to a cKit antigen is prepared. The cKit conjugate was tested on human small cell lung cancer xenograft tumors (H526) in mice. (n=5; no significant weight loss in any of the groups) Figure 18 shows H526 tumor growth inhibition at 6.5 mg/kg dose of immunoconjugate with payload-linker 5B on the first cKit antibody (cKitA). The activity at lower doses was much lower, and neither the cKitA antibody alone nor the Eg5 payload-linker combination (5B) bound to the antigen-binding group specific for viral glycoprotein gH showed measurable tumor growth. .

Figure 19 summarizes the results of comparison of cKitA immunoconjugates with Eg5 inhibitors (5B) and SMCC-DMl (n=5 for each curve; no significant weight loss for any group). The cKitA immunoconjugate conjugated to 5B or SMCC-DM1 displayed tumor growth inhibition of H526 xenograft tumors in mice. Using the indicated standard, the SMCC-DM1 conjugate was at a dose of 10 mg/kg. Non-statistically significant tumor growth inhibition was exhibited at 5 mg/kg. The 5B conjugate produced an inhibitory effect at 5 mg/kg, which was statistically significant and comparable to the 10 mg/kg dose of the SMCC-DM1 conjugate, and the 5B (Eg5 inhibitor) conjugate at the 10 mg/kg dose. The activity is much higher. The control showed no activity against the conjugate of 5B and gH, which is specific for the viral glycoprotein gH.

Activity Example 10. Crossover experiment.

For this experiment, two different xenograft tumors were implanted in each mouse, ie SK-OV-3ip was implanted on one side of the body and H526 was implanted on the other side. The SK-OV-3ip cell line was Her2 positive (Her2+) and lacked cKit (cKit-), while the H526 cell line was Her2 negative (Her2-) and cKit positive (cKit+). Each mouse was then treated with vehicle or one of three immunoconjugates: gH-5B, TBS-5B or cKitA-5B. As shown in Figure 20, [Her2+, cKit-] tumor growth was strongly inhibited by TBS-5B, as expected due to its expected binding to TBS antibodies. None of the other treatments significantly affected tumor growth. Similarly, [Her2-, cKit+] tumor growth was only inhibited by the cKit-5B conjugate and was not affected by any other treatment. This crossover experiment confirmed that the tumor suppressor is due to the immunoconjugate and does not occur due to the antibody or the payload of the release, and that only the intact immunoconjugate of the antibody to the antigen on the tumor is effective.

Activity Example 11. Effect of linker changes

This example illustrates the effect of linker changes on the in vivo efficacy of immunoconjugates with Eg5 inhibitors similar to the payload (5B, 5H, 5G and 5A, see Table 5). Four immunoconjugates were prepared using different linkers to attach similar Eg5 inhibitors to trastuzumab antibodies. Figure 21 summarizes the activity of immunoconjugates that are similarly effective at inhibiting SK-OV-3ip tumor xenografts in mice at a dose of 10 mg/kg. The activity of these immunoconjugates is also similar to the immunoconjugate (TBS-MC-MMAF) of the same antibody (TBS) and MMAF (the payload for ADC in clinical trials), and has a better than the same antibody. The immunoconjugate conjugate (TBS-SMCC-DM1) is more active. As expected, using the same effective negative binding to antibodies specific for viral glycoprotein (gH) Controls loaded with unconjugated antibody TBS showed little or no inhibition of tumor growth. (9 mice per group (n=9) for each immunoconjugate tested; no significant weight loss in any group).

Activity Example 12. In vivo activity of different Eg5 payload/linker combinations

This example compares different linker junctions and linkers, comparing the in vivo efficacy of an immunoconjugate with an Eg5 inhibitor linked by a linker at two different positions as a payload, using two different for each junction The linkers (5B, 5E, 5F and 5D, see Table 5) were all bound to trastuzumab antibodies. Figure 22 summarizes the activity of immunoconjugates that inhibit SK-OV-3ip tumor xenograft growth in mice at a dose of 10 mg/kg. The activity of these immunoconjugates is similar and is equivalent or better when compared to the TBS-SMCC-DM1 conjugate. As expected, controls using unbound antibody (TBS) exhibited little growth inhibition. (8 mice per group (n=8) for each immunoconjugate tested; no significant weight loss in any group).

Activity Example 13. Activity of different Eg5 immunoconjugates against NCI-N87 xenografts

This example compares the in vivo efficacy of a tumor cell line (NCI-N87, a gastric tumor cell line) with various Eg5 inhibitors as a payload and a linker immunoconjugate to a more difficult to inhibit. Four immunoconjugates with Eg5 inhibitor payloads (5B, 5E, 5D and 6U, see Tables 5-6) were prepared using different Eg5 inhibitor-payload combinations, all of which bind to trastuzumab antibodies . Figure 23 summarizes the activity of immunoconjugates that inhibit N87 tumor xenograft growth in mice at a dose of 10 mg/kg. The activity of these immunoconjugates varied in this model and compared to the trastuzumab-SMCC-DMl immunoconjugate. All immunoconjugates appeared to inhibit tumor growth; using the indicated criteria, the 5B and 6U and DM1 conjugates achieved statistical significance under the test conditions. As expected, controls using unbound antibody (TBS) exhibited little growth inhibition. (8 mice per group (n=8) for each immunoconjugate tested; no significant weight loss in any group).

Activity Example 14. Activity of Eg5 immunoconjugates against H526 xenografts

This example compares the in vivo efficacy of a combination of various Eg5 inhibitors that bind to cKit antibodies as a payload immunoconjugate to a tumor cell line (H526) expressing cKit. Six immunoconjugates with Eg5 inhibitor payloads (5B, 5E, 5F, 5C, 5A and 5D, see Table 5) were prepared using different Eg5 inhibitor-payload combinations, all of which were combined with cKit antibody (cKitA) Combine. Figure 24 summarizes the activity of immunoconjugates that inhibit H526 tumor xenograft growth in mice at a dose of 5 mg/kg. (For each immunoconjugate tested, 5 mice per group (n=5); no significant weight loss in any group). The payload/linker combinations 5E and 5D were more effective at the 5 mg/kg dose, achieving statistically significant tumor growth inhibition using the indicated criteria.

Figure 25 summarizes the activity of the same immunoconjugate on H526 xenografts at 10 mg/kg. (For each immunoconjugate tested, 5 mice per group (n=5); no significant weight loss in any group). The activity of these immunoconjugates at 10 mg/kg varied in this model, with 5D appearing to be the most effective and long lasting; using the indicated criteria, both 5D and 5E achieved statistically significant tumor growth inhibition at this dose.

Activity Example 15. Comparison of cKit antibodies in Eg5 inhibitor conjugates

This example compares the in vivo efficacy of immunoconjugates with different cKit antibodies and Eg5 inhibitor payloads against tumor cell lines (H526) expressing cKit. Two modified cKit antibodies were prepared by the following general procedure using cKit A as a starting material. By using as previously described (Meissner et al., Biotechnol Bioeng 75:. 197-203 ( 2001)) of the transient transfection method of co-transfection of heavy and light chain plasmid, expression cKitA antibodies in 293 Freestyle ^TM cells Cys mutant. DNA plastids for co-transfection were prepared using the Qiagen plastid preparation kit according to the manufacturer's protocol. 293 Freestyle ^TM cells at 37 [deg.] C in 5% CO ₂ under a Freestyle ^TM expression medium (Invitrogen) cultured in suspension. One day before transfection, the cells split to 0.7 x 10 ⁶ cells/ml in fresh medium. Day of transfection, cells reach a density typically 1.5 x 10 ⁶ cells / ml. The cells were transfected with a ratio of 1:1 heavy and light chain plastids using the PEI method (Meissner et al., 2001). The transfected cells were further cultured for 5 days. The culture medium was harvested by centrifuging the culture at 2000 xg for 20 minutes and filtering through a 0.2 micron filter. Using protein ^{A-Sepharose TM (GE Healthcare Life} Sciences) antibody was purified from the culture medium by filtration of the performance. By eluting buffer (pH 3.0) from Protein A-Sepharose ^TM column eluting antibody IgG, and neutralized immediately with 1M Tris-HCl (pH 8.0) , followed by buffer exchange to PBS.

Engineered Cys ADCs have been reported to be more tolerant in mouse and rat animal models than ADCs made by binding to partially reduced native disulfides or via native lysine residues. To assess the difference in in vivo efficacy between an ADC that binds to an engineered Cys antibody and an ADC that binds to a partially reduced native disulfide bond, the Eg5 linker-payload compound 223 is bound to the antibody cKitA HC-E152C-S375C Double mutants (cKitB: these immunoconjugates are called cKitB-compound 223 or cKitB-5B) and cKitA HC-K360C-LC-K107C double mutants (cKitC: these immunoconjugates are called cKitC-compound 223 Or cKitC-5B) and wild-type cKitA antibody (immunoconjugate cKitA-compound 223 or cKitA-5B). (The residue number is EU number)

Engineered antibodies contain cysteine residues that have recently been introduced at specific positions and are found to be particularly suitable for payload attachment. The antibody cKitB is a modified form of cKitA with two cysteine residues engineered into its heavy chain, and cKitC has a cysteine residue engineered into its heavy chain and a caspase The amino acid residue is engineered into a modified form of cKitA in its light chain. Since cKitB and cKitC each have two heavy chains and two light chains, the modified antibody has four newly added cysteine residues, which are suitable for binding without the need to reduce interchain disulfide. Thus an immunoconjugate (DAR = 4) to which four payload groups are attached can be prepared. Each cKit antibody was conjugated to the payload/linker combination 5B using the methods described herein to compare the effects of the modified antibody sequences.

Preparation of antibody cKitA and mutant cKit B according to the protocol described in Example 5 cKitC. The reduction and reoxidation of cKit B and cKitC were carried out according to the protocol described in Example 6. The reoxidized antibody was bound to Compound 223 by incubating 5 mg/ml antibody with 0.35 mM Compound 223 in 50 mM sodium phosphate buffer (pH 7.2) for 1 hour. The completeness of the reaction was monitored by RP-HPLC and DAR of 3.9 and 4.0 was obtained for the cKitB and cKitC conjugates, respectively. The DAR measurements are further verified by MS. ADCs were shown to be effective and in vitro cell kill assays and have pharmacokinetic properties similar to unbound antibodies in mice without tumors.

An ADC having a compound 223 bound to the native disulfide bond of cKitA was prepared as follows in a 2-step process. The antibody at a concentration of 5-10 mg/ml in PBS containing 2 mM EDTA was first partially reduced by 50 mM mercaptoethylamine (added as a solid) at 37 ° C for 1 hour. After desalting and addition of 1% w/v PS-20 detergent, the partially reduced antibody (1-2 mg/ml) was reacted overnight at 4 ° C with compound 223 in an amount of 0.5-1 mg per 10 mg of antibody, which was 10 mg/ml was dissolved in DMSO. The ADC was purified by protein A chromatography. After baseline washing with PBS, the conjugate was lysed with 50 mM citrate (pH 2.7), 140 mM NaCl, neutralized and sterile filtered. The average DAR is 3.2.

Features of the three cKit ADCs: cKitA-5B: DAR=3.2, aggregated 0.8%

cKitB-5B: DAR=3.9, aggregate 1.5%

cKitC-5B: DAR=4.0, aggregate 3.2%

Immunoconjugates having the following combinations of payload and cKit and trastuzumab antibodies and mutant antibodies were prepared and characterized by the same method. Note that engineered antibodies always provide near DAR of 4, and if four added cysteine residues in each antibody complex bind to the payload, then the DAR is the expected load:

Figure 26 summarizes the activity of two immunoconjugates made from cysteine engineered cKit antibodies that inhibit H526 tumor xenograft growth in mice at doses of 5 mg/kg and 10 mg/kg. . (For each immunoconjugate tested, 6 mice per group (n=6); no significant weight loss in any group). It is possible that the immunoconjugate is more active than the cKitA conjugate at both doses because the engineered antibody can form a conjugate without interfering with the native disulfide bridge structure. Thus, when an immunoconjugate of an Eg5 inhibitor is active against various cKit antibodies, including unmodified, this confirms that the protein is engineered to introduce a new cysteine residue into the constant region and to use neocysteine. The residue serves as a point of attachment for the payload/linker group to provide an improved immunoconjugate.

Additional activity data on tumor cell growth inhibition

Figure 28 provides additional information demonstrating that the immunoconjugates of the invention inhibit tumor cell growth. The data in Figure 28 demonstrate that these immunoconjugates inhibit the growth of a variety of tumor cell lines, including SK-OV-3ip, MDA-MB-231, HCC1954, MDA-MB-468, MDA-MB-231-M40, MDA. -MB-231-M16, H526 and NCI-N87. In each case, as expected, the activity of the ADC was determined in part by the performance level of the antigen recognized by the antibody of the ADC on the treated cells. Additional information confirms that structural changes in the range of formula (II) generally produce active ADCs, even if the level of activity varies between cell lines. The data further confirms that a variety of linkers can be used and that the linker can be attached to a position on the groups R1, Y and Q.

A summary of in vitro cell growth inhibition of various ADCs of the invention is provided in the table below. The first column lists Table 2, 5 or 6 from the payload/linker combination used in the ADC. The compound ID number; the second column indicates which antibody is used in the ADC. The third column identifies the cell line to which the ADC is directed: for most ADCs, activity is measured against high antigen cell lines and against low antigen cell lines, stable activity is expected in high antigen cell lines, and antibodies are expected in low antigen cell lines. Delivery is ineffective and therefore the activity is expected to be much lower. Activity was reported as absolute AC50 (ng/mL), Ainf (%), and relative EC50 (ng/mL). Information about the methods used for this information is provided after the table.

For the cell line in the previous table:

Definition and derivation of performance values

The cell TiterGlo 2 data of the replicate samples were averaged and then corrected for untreated cells. Analysis included wells of untreated cells, reflecting uninhibited cell growth. The average of the controls was used to correct the results from the treated samples to the % scale. The analysis of the corrected data was performed using a 4-parameter logistic model with standard analytical data analysis software (Helios software application).

The four parameters include Ainf (the largest active flat line area, generally found under high [ADC]), A0 (the smallest active flat line area, generally found under low [ADC]), and the two flat lines on the y-axis. The ADC concentration at the midpoint between the zones, and n, the slope of the fitted curve at the midpoint between the two flattened zones.

For each compound, the software derives three measures, namely absolute AC50, relative EC50 and Ainf. Absolute AC50 ("Abs AC50" in the table) is the ADC concentration where the fitted curve spans 50% of the Y-axis. The relative EC50 ("Rel EC50" in the table) is the ADC concentration at the midpoint between the A0 and Ainf of the fitted curve.

Comparative immunoconjugates:

TBS-SMCC-DM1: antibody = trastuzumab; linker = SMCC; payload = DM1: DAR is about 3.5

MC-MMAF conjugate using a maleimide hexamethylene linker:

Claims (47)

An immunoconjugate of formula (I): Wherein A represents an antigen-binding moiety; L represents a linkage group linking X to Ab; m is an integer from 1 to 4; n is an integer from 1 to 16; and X independently represents formula (II) at each occurrence Group It is linked to the Ab by L, wherein: Z is N or CH; Ar ¹ is optionally substituted with up to 3 groups selected from halo, C _1-3 alkyl and C _1-3 haloalkyl Phenyl; Ar ² is phenyl or pyridyl, and Ar ² optionally has up to two groups selected from the group consisting of halo, CN, C _1-3 alkyl, hydroxy, amine and C _1-3 haloalkyl Substituted; R ¹ is C _1-6 alkyl, -(CH ₂ ) _0-2 -C _3-6 cycloalkyl or contains up to two heteroatoms selected from N, O and S as ring members - (CH ₂ ) _0-2 -C _4-7 heterocyclic group, wherein each C _1-6 alkyl group, C _3-6 cycloalkyl group or C _4-7 heterocyclic group optionally has up to three selected from a halogen group , C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, hydroxy, amine, carboxyl, pendant oxo, hydroxy substituted C _1-4 alkyl, Substituted by an amine-substituted C _1-4 alkyl group and a COO (C _1-4 alkyl) group; R ² is H or C _1-4 alkyl; T is (CH ₂ ) _1-3 ; From C _1-3 aminoalkyl, C _4-6 heterocyclyl and C _3-6 cycloalkyl, wherein C _1-3 aminoalkyl, C _4-6 heterocyclyl and C _3-6 naphthenic group each optionally substituted with up to three substituents selected from amino, oxo, halo, hydroxy, C _1-4 alkyl, C _1-4 alkoxy, hydroxy-substituted C _1-4 alkyl, the substituted amino C _1-4 alkyl, COOH, COO- (C _1-4 alkyl), - C (= O) NH (C 1-4 alkyl), - C (=O) substituted by a group of N(C _1-4 alkyl) ₂ and C _1-4 haloalkyl; A is NH, N(C _1-4 alkyl) or a carbonyl group in formula (II) a bond between Q; Q is selected from C _1-4 alkyl, -OC _1-4 alkyl, -(CH ₂ ) _0-2 -C _4-6 heterocyclyl, -(CH ₂ ) _0-2 -C _3-6 cycloalkyl, -(CH ₂ ) _0-2 -C _5-6 heteroaryl and -(CH ₂ ) _0-2 -phenyl, and optionally up to three selected from halo, a group substituted with a hydroxyl group, an amine group, -SH, -R, -OR, -SR, -SO ₂ R, -NHR, -O-glucuronate, and -NR ₂ , wherein each R is C _{a 1-6} alkyl group, a C3-6 cycloalkyl group or a 4- to 6-membered heterocyclic ring containing N, O or S as a ring member, and each R is independently independently a halo group, -SH, -NH ₂ , OMe or -OH substitution. The immunoconjugate of claim 1, wherein R ² is H. An immunoconjugate according to claim 1 or 2, wherein Z is CH. An immunoconjugate according to claim 1 or 2, wherein Z is N. The immunoconjugate of claim 1 or 2, wherein R ¹ is tetrahydropyran, and R ^{1 is} optionally substituted with up to two groups selected from the group consisting of pendant oxy groups and methyl groups. The immunoconjugate of claim 1 or 2 wherein Ar ¹ is a dihalophenyl group. The immunoconjugate of claim 1 or 2, wherein the compound of formula (II) has the formula: Where L is connected to Y, or to Q, or to R ¹ . The immunoconjugate of claim 1 or 2 wherein R ¹ is 4-tetrahydropyranyl. The immunoconjugate of claim 1 or 2, wherein R ¹ is -C(Me) ₂ -(CH ₂ ) _0-2 R ³⁰ , wherein R ³⁰ is -OH, COOH or NH ₂ and L is attached to R ¹ . The immunoconjugate of claim 1 or 2, wherein Q is a C _1-4 alkyl group substituted with 1 or 2 groups selected from the group consisting of a hydroxyl group and an amine group. The immunoconjugate of claim 1 or 2, wherein Y is pyrrolidone substituted with a halo, an amine or a hydroxy group, as appropriate. An immunoconjugate according to claim 1 or 2 wherein A is -NH-. The immunoconjugate of claim 1 or 2, wherein the linking group is cleavable. The immunoconjugate of claim 1 or 2, wherein the linkage group is non-cleavable. A compound of formula (III): Or a pharmaceutically acceptable salt thereof, wherein: Z is N or CH; and Ar ¹ is optionally a group selected from the group consisting of halo, C _1-3 alkyl and C _1-3 haloalkyl Substituted phenyl; Ar ² is phenyl or pyridyl, optionally up to two groups selected from halo, CN, C _1-3 alkyl, hydroxy, amine and C _1-3 haloalkyl Substituted; R ¹ is -(CH ₂ ) _0-2 -C _4-7 heterocyclyl or -(CH ₂ ) _0-2 -C _3-7 cycloalkyl, wherein the C _4-7 heterocyclic group contains Two heteroatoms selected from N, O and S are used as ring members, and the C _4-7 heterocyclic group and the C _3-7 cycloalkyl group are optionally up to three selected from a halogen group and a C _1-4 alkane. group, C _1-4 alkoxy, hydroxy, amino, oxo, the hydroxy substituted C _1-4 alkyl, the substituted amino by C _1-4 alkyl, C _1-4 haloalkyl and a group substituted with COO (C _1-4 alkyl); or R ¹ is a C _3-6 alkyl group substituted with -OH, -COOH or -NH ₂ ; R ² is H or C _1-4 alkyl; Is (CH ₂ ) _1-3 ; Y is selected from C _1-2 aminoalkyl, C _4-6 heterocyclic and C _3-6 cycloalkyl, wherein C _1-2 aminoalkyl, C _{4 -6} heterocyclyl and C _3-6 cycloalkyl each optionally up to three selected from the group consisting of an amine group, a pendant oxy group, a halogen group, a hydroxyl group, a C _1-4 alkoxy group Group, the hydroxy substituted C _1-4 alkyl, the substituted amino by C _1-4 alkyl, COOH, COO- (C _1-4 alkyl), CONH (C _1-4 alkyl), CON ( Substituting a group of C _1-4 alkyl) ₂ and C _1-3 haloalkyl; A is NH, N(C _1-4 alkyl) or a bond between a carbonyl group in formula (III) and Q; Q is selected from the group consisting of C _1-4 alkyl, -(CH ₂ ) _0-2 -C _4-6 heterocyclyl, -(CH ₂ ) _0-2 -C _5-6 heteroaryl and -(CH ₂ ) _0-2 -phenyl, and Q optionally has up to three groups selected from the group consisting of halo, hydroxy, amine, -SH, -R, -OR, -SR, -SO ₂ R, -NHR and -NR ₂ Group substitution wherein each R is a C _1-6 alkyl group optionally substituted with up to three groups selected from halo, -SH, -NH ₂ , OMe or -OH. The compound of claim 15, wherein R ¹ is tetrahydropyranyl. The compound of claim 15, wherein R ¹ is a group of the formula -C(Me) ₂ -(CH ₂ ) _0-2 R ³⁰ wherein R ³⁰ is -OH, COOH or NH ₂ . A compound of formula (IIA) or (IIB) or (IIC): Wherein Ar ¹ , Ar ² , Z, R ¹ , R ² , T, Q, Y and A are as defined in claim 1 and Q* is selected from the group consisting of -CH ₂ O-, -CH ₂ S-, -CH ₂ -NH-, -CH ₂ -NMe-, -CH(Me)O-, -CH(OH)-CH ₂ O-, -CH(CH ₂ OH)-O-, -CH(OH)-CH ₂ NH -, -CH(CH ₂ OH)-NH-, -CH(CH ₂ NH ₂ )-O-, -CH(CH ₂ OH)-NH-, -CH(Me)S-, -CH(Me)NH -, -CH ₂ CH ₂ O-, -CH ₂ CH ₂ NH-, -CH ₂ CH ₂ S-, -CH(Me)CH ₂ O-, -CH(Me)CH ₂ S-, -CH(Me ) CH ₂ NH-, Y* is selected from the group consisting of -CH(CH ₂ F)NH-, -CH ₂ NH-, Wherein R ¹⁰ and R ^{11 are} independently H, Me, OMe, F, CH ₂ F, CH ₂ OH, COOH, CONH (C _1-4 alkyl), CON(C _1-4 alkyl) ₂ , COO ( C _1-4 alkyl) or OH; R ^{1 *} is selected from C _3-6 alkyl substituted by hydroxyl, amine or carboxyl; and W is one or more linker components and one reactive functional group The key part. The compound of claim 18, wherein W comprises a group selected from the group consisting of -SH, -NH ₂ , -C(=O)H, -C(=O)Me, N-maleimide, and -NHC (=O) a reactive functional group of -CH ₂ -halo, -COOH and -C(=O)-OR', wherein the halo group is selected from the group consisting of Cl, Br and I, and -OR' is the cleavage moiety of the activated ester. The compound of any one of claims 15 to 19, wherein Ar ¹ is a dihalophenyl group. The compound of any one of claims 15 to 19, wherein Ar ² is phenyl or halophenyl or hydroxyphenyl. The compound of any one of claims 15 to 19, wherein Z is CH. The compound of any one of claims 15 to 19, wherein Z is N. The compound of any one of claims 15, 18 and 19, wherein R ¹ is 4-tetrahydropyranyl. The compound of any one of claims 15 to 19, wherein R ² is H. The compound of any one of claims 15 to 19, wherein A is -NH-. The compound of any one of claims 15 to 19, wherein A is a bond. The compound of any one of claims 15 to 19, wherein T is CH ₂ or CH ₂ CH ₂ . The compound of any one of claims 15 to 19, wherein Y is selected from the group consisting of -CH(CH ₂ F)NH ₂ , Wherein R ¹⁰ and R ^{11 are} independently H, Me, OMe, F, CH ₂ F, CH ₂ OH, COOH, COO (C _1-4 alkyl) or OH. The compound of any one of claims 15 to 29, wherein Q is selected from the group consisting of -CH ₂ OH, -CH ₂ -NH ₂ , -CH(Me)OH, -CH(OH)-CH ₂ OH, -CH ( OH)-CH ₂ NH ₂ , -CH(NH ₂ )-CH ₂ OH, -CH(NH ₂ )-CH ₂ OH, -CH(Me)SH, -CH(Me)NH ₂ , -CH ₂ CH ₂ OH, -CH ₂ CH ₂ NH ₂ , -CH ₂ CH ₂ SH, -CH(Me)CH ₂ OH, -CH(Me)CH ₂ SH, -CH(Me)CH ₂ NH ₂ , The compound of claim 15 which is selected from the group consisting of the compounds of Table 1 and pharmaceutically acceptable salts thereof. A pharmaceutical composition comprising a compound according to any one of claims 15 to 31, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers. A combination comprising a therapeutically effective amount of a compound of any one of claims 15 to 31, or a pharmaceutically acceptable salt thereof, and one or more therapeutically active adjuvants. Use of an immunoconjugate according to any one of claims 1 to 14 or a compound of any one of claims 15 to 17 or a pharmaceutically acceptable salt thereof for the manufacture of a therapeutic cell proliferative disorder Pharmacy. The compound of any one of claims 15 to 17, or a pharmaceutically acceptable salt thereof, It is used as a medicament. The compound of claim 35, wherein the agent is for treating cancer. An immunoconjugate according to claim 1 or 2 for use as a medicament. The immunoconjugate of claim 37, wherein the agent is for treating cancer. An immunoconjugate according to claim 1 or 2 for use in the treatment of cancer. An immunoconjugate Ab-L*-X comprising a payload (X) linked to an antibody (Ab), wherein the linkage group L* comprises the formula -C(O)NR ²¹ - or -NR ²¹ -C a group of (O)-, wherein R ²¹ has the formula -(CH ₂ ) _1-4 -R ²² , wherein R ²² is selected from the group consisting of -OH, -NH ₂ , N(R ²³ ) ₂ , COOR ²³ , CON ( R ²³ ) ₂ , -(OCH ₂ CH ₂ O) _k -OCH ₂ CH ₂ OR ²³ and -SO ₂ R ²³ polar groups, wherein k is 0 to 4 and each R ^{23 is} independently H or C _{1- 4} alkyl. An immunoconjugate of formula (I): Wherein Ab represents an antigen binding moiety; L represents a linkage group linking X to Ab; m is an integer from 1 to 4; n is an integer from 1 to 16; and X independently represents an Eg5 inhibitor at each occurrence. The immunoconjugate of claim 41, wherein X is a compound selected from Table 1. The immunoconjugate of claim 41, wherein m is 1, and the immunoconjugate is formed by reacting an Ab with a compound selected from Table 2. A compound selected from Table 5 or an immunoconjugate thereof. An immunoconjugate selected from the group consisting of the immunoconjugates of Table 5, wherein AntiB represents an antibody. The immunoconjugate of any one of claims 1, 2, and 41 to 45, wherein the antigen-binding portion is an antibody having at least one non-native cysteine residue introduced into the constant region, wherein the linking group L Attached to the non-native cysteine residue. The immunoconjugate of claim 46, wherein m is 1 and n is between 1 and 5, preferably about 2 or about 4.