Heteroaryl sulfone-based conjugation handles, methods for their preparation, and their use in synthesizing antibody-drug conjugates

Abstract

To provide a linker technology that possesses comparable high selectivity and reactivity to cysteine but avoids instability associated with maleimide linker components.SOLUTION: The invention provides novel heteroaryl sulfone-based conjugation handles of a compound expressed by a formula (I): where Het is pyrimidine or pyrazine ring, each E is -C(R1)2-, each R1 is H, C1-C6 alkyl etc., each X is independently an amino acid, each Y is independently an amino acid, each Z is independently a spacer element, D is an active agent or a moiety that binds to an active agent. Also provided are methods for their preparation, their use in synthesizing antibody-drug conjugates, and the resulting antibody-drug conjugates made with components having heteroaryl sulfone-based conjugation handles.

Description

[Technical field]

[0001] The present invention is directed to novel linker compounds and moieties useful for incorporation into and making polypeptide conjugates, including antibody drug conjugates (ADCs), as well as related linker payload compounds (LPs).The present invention further relates to compositions comprising the above-described linkers, linker payloads, and antibody drug conjugates, as well as methods of using these payloads, payload linkers, and antibody drug conjugates to treat pathological conditions, including cancer. [Background technology]

[0002] Antibody drug conjugates (ADCs) are therapeutic modalities consisting of monoclonal antibodies attached to small molecule cytotoxic payloads. The antibody portion of the ADC serves as a transport vehicle that recognizes and binds to protein antigens expressed in tumor tissues. Localized delivery and release of the payload within or near malignant cells allows targeted delivery of potent cytotoxic agents to diseased tissues while reducing damage to antigen-negative normal tissues. ADC design seeks to combine the advantages of large and small molecule drugs and eliminate the weaknesses associated with each approach. Thus, many ADCs are designed to minimize the systemic toxicity of the free drug and enhance the antitumor activity of the targeting vehicle, e.g., monoclonal antibodies (mAbs).

[0003] Several ADCs currently under investigation contain maleimide-based linkers. In these ADCs, antibody conjugation is typically accomplished as shown below.

[0004] [ka]

[0005] The use of such conjugation in many investigational ADC constructs can be attributed to the high reactivity and selectivity of maleimides for cysteine ​​residues. More specifically, ADCs can be constructed by incubating an antibody with a linker payload that contains a maleimide conjugation handle that reacts with exposed thiols of the antibody.

[0006] However, there are numerous examples of instability directly associated with the maleimide moiety. For example, researchers at Seattle Genetics have shown that maleimide-based ADCs known as 1F6-C4v2-mc-MMAF and 1F6-C4v2-mc-vc-MMAF undergo degradation (manifested in reduced drug-antibody ratios) upon exposure to plasma proteins with a half-life of approximately 7 days. (Alley et al., "Contribution of Linker Stability to the Activities of Anticancer immunoconjugates," Bioconjugate Chem., 2008, 19, 759.) Alley further provides mass spectrometry evidence for serum albumin mc-MMAF adduct formation, which is believed to occur via thioether fragmentation of the ADC and subsequent capture of the resulting maleimide by albumin at Cys34, as shown therein.

[0007] [ka]

[0008] Similar maleimide-related instability has also been shown to exist by plasma stability assays and glutathione (GSH) incubation studies, where the ADC has been shown to undergo a reduction in drug-to-antibody ratio (DAR) in the presence of GSH, as shown below.

[0009] [ka] [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Patent Application No. PCT / IB2015 / 050280 (WO2015 / 110935) [Patent Document 2] International Patent Application No. PCT / IB2016 / 051465 [Patent Document 3] EP0404097 [Patent Document 4] WO93 / 11161 [Patent Document 5] International Patent Application No. PCT / IB2012 / 056234 [Patent Document 6] WO2013 / 093809 [Patent Document 7] WO2014 / 068443 [Patent Document 8] WO2012 / 059882 [Patent Document 9] WO2015 / 015448 [Patent Document 10] WO2015 / 162563 [Patent Document 11] U.S. Patent No. 4,816,567 [Patent Document 12] U.S. Patent No. 4,816,397 [Patent Document 13] U.S. Patent No. 5,585,089 [Patent Document 14] International Publication No. WO87 / 02671 [Patent Document 15] European Patent No. 0184187 [Patent Document 16] European Patent Application Publication No. 0171496 [Patent Document 17] European Patent Application Publication No. 0173494 [Patent Document 18] International Publication No. WO86 / 01533 [Patent Document 19] European Patent Application Publication No. 012023 [Patent Document 20] U.S. Patent No. 5,225,539 [Patent Document 21] U.S. Patent No. 5,625,126 [Patent Document 22] U.S. Patent No. 5,633,425 [Patent Document 23] U.S. Patent No. 5,569,825 [Patent Document 24] U.S. Patent No. 5,661,016 [Patent Document 25] U.S. Patent No. 5,545,806 [Patent Document 26] International Publication No. WO97 / 34631 [Patent Document 27] US5,773,001 [Patent Document 28] US6,214,345B1 [Patent Document 29] US8,039,273_B2 [Patent Document 30] US8,568,728B2 [Patent Document 31] WO2013056070A2 [Patent Document 32] US2013 / 0129753A1 [Patent Document 33] US8399403B2 [Patent Document 34] US2010 / 210593A1 [Patent Document 35] WO2007 / 11968A2 [Non-patent literature]

[0011] [Non-Patent Document 1] Alley et al., “Contribution of Linker Stability to the Activities of Anticancer immunoconjugates,” Bioconjugate Chem., 2008, 19, 759 [Non-Patent Document 2] Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). [Non-Patent Document 3] Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917 [Non-Patent Document 4] Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994). [Non-Patent Document 5] Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448 [Non-Patent Document 6] Jones et al., 1986, Nature 321:522-525 [Non-Patent Document 7] Riechmann et al., 1988, Nature 332:323-329 [Non-Patent Document 8] Presta, 1992, Curr.Op.Struct.Biol.2:593~596 [Non-Patent Document 9] Edited by SP Parker, McGraw-Hill Dictionary of Chemical Terms, McGraw-Hill Book Company, New York (1984) [Non-Patent Document 10] Eliel and Wilen, Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York (1994). [Non-Patent Document 11] Laguzza et al., 1989, J. Med. Chem., 32(3):548-55 [Non-Patent Document 12] Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002) [Non-Patent Document 13] Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308~7312 [Non-Patent Document 14] Kozbor et al., 1983, Immunology Today 4:72-79 [Non-Patent Document 15] Olsson et al., 1982, Meth. Enzymol. 92:3-16 [Non-Patent Document 16] Kabat E et al., 1980, J. Immunology 125(3):961-969 [Non-Patent Document 17] Berter et al., 1988, Science 240:1041-1043 [Non-Patent Document 18] Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439~3443 [Non-Patent Document 19] Liu et al., 1987, J.Immunol.139:3521~3526 [Non-Patent Document 20] Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214~218 [Non-Patent Document 21] Nishimura et al., 1987, Cancer. Res. 47:999-1005 [Non-Patent Document 22] Wood et al., 1985, Nature 314:446-449 [Non-Patent Document 23] Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559 [Non-Patent Document 24] Morrison, 1985, Science 229:1202~1207 [Non-Patent Document 25] Oi et al., 1986, BioTechniques 4:214 [Non-Patent Document 26] Verhoeyan et al., 1988, Science 239:1534 [Non-Patent Document 27] Beidler et al., 1988, J. Immunol. 141:4053~4060 [Non-Patent Document 28] Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93 [Non-Patent Document 29] Jespers et al., 1994, Biotechnology, 12:899-903 [Non-Patent Document 30] Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381. [Non-Patent Document 31] Marks et al., 1991, J. Mol. Biol., 222:581 [Non-Patent Document 32] Quan and Carter, 2002, The rise of monoclonal antibodies as therapeutics, In Anti-IgE and Allergic Disease, Jardieu and Fick (eds.), Marcel Dekker, New York, NY, Chapter 20, pp. 427-469 [Non-Patent Document 33] Goodson, Medical Applications of Controlled Release, op. cit., vol. 2, pp. 115-138 (1984) [Non-Patent Document 34] Langer, Science 249:1527~1533 (1990) [Non-Patent Document 35] "Remington's Pharmaceutical Sciences", E.W. Martin [Non-Patent Document 36] Dubowchik, GM et al., Bioconjug Chem, 13, 855-69 (2002) [Non-Patent Document 37] Yu-Xiu, Liu; Ming-Bo, Cui;Qi-Qi, Zhao;Qing-Min, Wang;Ying, Liu;Run-Qiu, Huang, Reduction of pyrimidine derivatives by LiAlH4, Journal of Chemical Research, 2007, #8, p.490~493 Summary of the Invention [Problem to be solved by the invention]

[0012] In light of the above, it would be advantageous to develop and employ linker technology that has comparable high selectivity and reactivity towards cysteine, but avoids the instability associated with the maleimide linker component. [Means for solving the problem]

[0013] The present invention relates to non-maleimide-based ADC linkers and linker components, their preparation, and their use in the preparation of ADCs that are more stable than the corresponding maleimide-based ADCs. In many cases, this improved stability results in ADCs with improved therapeutic indices and / or other advantageous properties.

[0014] Thus, the invention provides specific cysteine-selective covalent reactive groups (CRGs) that serve as versatile conjugation handles that can undergo complete conjugation with any antibody of interest, typically within about one day, providing stable achiral ADC linkers with a substantially unchanged DAR for more than one week under physiological conditions.

[0015] The present invention also relates to non-maleimide-based linkers and linker components of use beyond the ADC field, including linkage and conjugation of other biological entities, such as peptides, proteins, RNA (including mRNA, siRNA), and single- and double-stranded DNA, which may or may not contain cysteine-containing, or simply sulfur-containing, moieties.

[0016] The present invention also relates to non-maleimide-based linkers and linker components of use beyond the ADC field, including the linkage and conjugation of multiple biological entities, such as peptides, proteins, RNA (including mRNA, siRNA), and single- and double-stranded DNA, which may or may not contain cysteine-containing, or simply sulfur-containing, moieties to one another.

[0017] The present invention also provides linker payloads that include non-maleimide-based linker components, particularly heterocyclic linkers bonded to sulfones and amides, for use in and beyond the ADC field, and have the formula (I):

[0018] [ka] and pharma-ceutically acceptable salts or solvates thereof, wherein Het is a monocyclic, bicyclic or polycyclic heteroaryl ring system having 1-4 heteroatoms, wherein the carbon atom on said ring system bonded to -SO2- is adjacent to at least one heteroatom on said ring system, said heteroatoms being selected from the group consisting of N, O and S; Each E is -C(R 1 )2-, -OC(R 1 )2-C(R 1 )2- (wherein r is at least 2), and -C(R 1 )2-C(R 1 )2-O-, where s is at least 1; Each R 1 are independently selected from the group consisting of H, C1-C6 straight or branched alkyl, C2-C6 straight or branched alkenyl, and C2-C6 straight or branched alkynyl; R 2 is a C1-C alkyl group optionally substituted with halogen or haloalkyl; 10 C5-C optionally substituted with alkyl, halogen or haloalkyl 12 is aryl, each X is independently an amino acid, and each amino acid X is the same or different; each Y is independently an amino acid, and each amino acid Y is the same or different; each Z is independently a spacer element, and each spacer element is the same or different; m is 0 to 5, n is 0 to 5, p is 0 to 2, q is 0 to 10, r is 0 to 2, s is 0 to 2, t is 0 to 1, and w is 1 to 2; D is a cytotoxic agent.

[0019] In the above embodiment, w is preferably 1.

[0020] It has been found that such linker payloads having heterocycles bonded to sulfones and amides can form ADCs that are surprisingly stable compared to ADCs formed using maleimide linkers or even other non-maleimide linkers. Without wishing to be bound by theory, such ADCs are typically based on the well-known S N It is believed to be formed (i.e., "conjugated") via an Ar reaction mechanism.

[0021] The present invention further relates to ADCs formed using the non-maleimide-based linker payloads described above, which have a heterocycle attached to an amide, such ADCs having the formula (II):

[0022] [ka] and pharma- ceutically acceptable salts or solvates thereof, wherein P is a peptide sequence containing at least one naturally occurring or engineered cysteine ​​residue, or P is a moiety containing at least one free sulfur atom; S is the sulfur atom in P, Het is a monocyclic, bicyclic or polycyclic heteroaryl ring system having 1-4 heteroatoms, wherein the carbon atom on said ring system bonded to -S(O2)- is adjacent to at least one heteroatom on said ring system, said heteroatoms being selected from the group consisting of N, O and S; Each E is -C(R 1 )2-, -OC(R 1 )2-C(R 1 )2- (wherein r is at least 2), and -C(R 1 )2-C(R 1 )2-O-, where s is at least 1; Each R 1 are independently selected from the group consisting of H, C1-C6 straight or branched alkyl, C2-C6 straight or branched alkenyl, and C2-C6 straight or branched alkynyl; each X is independently an amino acid, and each amino acid X is the same or different; each Y is independently an amino acid, and each amino acid Y is the same or different; each Z is independently a spacer element, and each spacer element is the same or different; m is 0 to 5, n is 0 to 5, p is 0 to 2, q is 0 to 10, r is 0 to 2, s is 0 to 2, t is 0 to 1, and w is 1 to 2; D is a cytotoxic agent.

[0023] In the above embodiment, w is preferably 1.

[0024] The present invention also relates to the above linker payloads and ADCs, wherein the Het variable moiety is

[0025] [ka] is selected from.

[0026] The present invention further relates to the above linker payloads and ADCs, wherein the Het variable moiety is

[0027] [ka] where any bond on the selected Het may be joined with an S (ie, the Het structure may "point" in either direction).

[0028] In another embodiment of the present invention there is provided a compound identified herein, or a pharma- ceutically acceptable salt or solvate thereof, D is a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, -(OCH2CH2)p-, -CO2H, -NH2, OH, -N3,

[0029] [ka] is selected from the group consisting of:

[0030] In one aspect of this embodiment, there is provided a compound as identified herein, or a pharma- ceutically acceptable salt or solvate thereof, wherein D is a cytotoxic moiety. In another aspect, there is provided a compound as described in claim 6, or a pharma- ceutically acceptable salt or solvate thereof, wherein D is an anthracycline, an auristatin, a splicostatin, a CBI dimer, a CPI dimer, a CTI dimer (including "mixed" dimers containing CBI, CPI and / or CTI dimer components, as described in International Patent Applications PCT / IB2015 / 050280 (WO2015 / 110935) and PCT / IB2016 / 051465), an enantiomeric dimer, a cytotoxic moiety ... Selected from the group consisting of diynes (e.g., calicheamicin, esperamicin or neocarzinostatin), duocarmycins, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, SN38, tubulysin, hemiasterin, camptothecins, combretastatins, dolastatins, indolinobenzodiazepine dimers, pyrrolobenzodiazepine dimers and pladienolides, and stereoisomers, isosteres, analogs or derivatives thereof. For example, embodiments in which the cytotoxic agent D is an auristatin selected from the group consisting of dolastatin, MMAD, MMAE, MMAF, PF-06380101, PF-06463377, PF-06456780, PF-06843982, PF-06874082, PF-06852321, PF-06757725, PF-06859944 and PF-06698970.

[0031] Further embodiments of the invention include those in which D is something other than a cytotoxic agent, for example, D is a moiety with therapeutic properties (i.e., a therapeutic agent) and includes peptide(s), protein(s), nucleic acid(s), growth factor(s), antiviral agent(s), or immunological agent(s), or D is fluorophore(s) or other imaging agent(s). As in the case where D is a cytotoxic agent, the invention includes modulation of the stability of compounds in which D is other than a cytotoxic agent.

[0032] In yet another embodiment of the present invention there is provided a compound as described herein, or a pharma- ceutically acceptable salt or solvate thereof, Z is Z A -(Z B ) 1~3 or (Z B ) 1~3 -Z A and Z A is Z A1 -Z A2 or Z A2 -Z A1 and Z A1 is absent or -C(O)-, -C(S)-, -C(O)NR-, -C(O)C1-C6 alkyl-, -C(O)NR-C1-C6 alkyl-, -C1-C6 alkyl(OCH2CH2) 1~6 -, -C(O)C1-C6 alkylNRC(O)-, -C(O)C1-C6 alkyl(OCH2CH2) 1~6 -, -C1~C6 alkyl (OCH2CH2) 1~6 -C(O)-, -C 1~ C6 alkyl-SSC 1~ C6 alkylNRC(O)CH2-, -C 1~ C6 alkyl (OCH2CH2) 1~6 NRC(O)CH2-, -C(O)C 1~ C6 alkyl-NRC(O)C 1~6 Alkyl-, -N=CR-phenyl-OC 1~ C6 alkyl-, -N=CR-phenyl-OC 1~ C6 alkyl-C(O)-, -C(O)-C 1~ C6 alkyl (OCH2CH2) 1~6 NRC(O)-, -C(O)C 1~ C6 alkyl-phenyl (NR-C(O)C 1~ C6 alkyl) 1~4 -, -C(O)C 1~ C6 alkyl (OCH2CH2) 1~6 -NRC(O)C 1~ C6 alkyl-, -C 1~C6 alkyl-, -S-, -C(O)-CH(NR-C(O)C1-C6 alkyl)-C1-C6 alkyl- and (-CH2-CH2-O-) 1~20 and one or more components selected from the group consisting of: Z A2 are -PABA-, -PABC-, -C(O)(CH2) n C(O)- or absent, Z B does not exist or is -C 1~ C6 alkylene-, -NRC3~C8-heterocyclylNR-, -NRC3~C8-carbocyclylNR-, -NRC1~C6 alkylNR-, -NRC1~C6 alkylene-, -S-, -NR-, -NRNR-, -O(CR2) 1~4 SS(CR2) 1~4 N(R)-, -NR C1-C6-alkylenephenyleneNR-, -NR C1-C6 alkylenephenyleneSO2NR-, -OC 1~ C6 alkyl S-SC 1~ C6 alkylC(COOR)NR-, -NRC(COOR)C 1~ C6 alkyl S-SC 1~ C6 alkylO-.

[0033] In yet another embodiment of the present invention there is provided a compound as described herein, or a pharma- ceutically acceptable salt or solvate thereof, Z is

[0034] [ka] It is.

[0035] In a further embodiment of the invention,

[0036] [ka]

[0037] [ka]

[0038]

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[0048] [ka]

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[0050] [ka]

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[0054] [ka]

[0055] [ka]

[0056] [ka] or a pharma- ceutically acceptable salt or solvate thereof.

[0057] In a further embodiment of the present invention, there is provided a pharmaceutical composition comprising a compound as described herein, or a pharma- ceutically acceptable salt or solvate thereof, and a pharma- ceutically acceptable excipient.

[0058] In a further embodiment of the present invention, a method of treating cancer is provided, comprising administering to a patient in need thereof a therapeutically effective amount of a compound or pharmaceutical composition as described herein. In one aspect of this embodiment, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric (stomach) cancer, testicular cancer, leukemia and lymphoma. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] The present invention is directed to heteroarylsulfone linkers containing amide functional groups, related linker payloads that react with thiol functional groups on cysteine ​​residues, and the resulting conjugates formed by these reactions. Such conjugates include antibody drug conjugates containing therapeutic and / or cytotoxic components, as well as methods of using them to treat cancer and other pathological conditions. The present invention also relates to in vitro, in situ, and in vivo methods of using such compounds and / or conjugates for the detection, diagnosis, or treatment of mammalian cells or associated pathological conditions.

[0060] Definitions and Abbreviations Unless otherwise noted, the following terms and phrases, as used herein, are intended to have the following meanings: When trade names are used herein, the trade name includes the product formulation, generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless the context dictates otherwise.

[0061] The term "antibody" (or "Ab" or "AB") is used herein in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. Intact antibodies have two main regions: a variable region and a constant region. The variable region binds to and interacts with a target antigen. The variable region contains complementarity determining regions (CDRs) that recognize and bind to specific binding sites on a particular antigen. The constant region can be recognized by and interact with the immune system (see, e.g., Janeway et al., 2001, Immuno. Biology, 5th ed., Garland Publishing, New York). Antibodies may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody may be from any suitable species. In some embodiments, the antibody is of human or murine origin. The antibody may be, for example, human, humanized or chimeric.

[0062] The terms "specifically bind" and "specific binding" refer to the binding of an antibody to a predetermined antigen. An antibody typically binds to at least about 1×10 7 M -1 and further binds to a given antigen with an affinity that is at least twice as great as the affinity with which it binds to a non-specific antigen other than the given antigen or an antigen closely related thereto (e.g., BSA, casein).

[0063] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0064] The term "monoclonal antibody" specifically includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, but which exhibit the desired biological activity.

[0065] An "intact antibody" refers to an antibody that contains an antigen-binding variable region, as well as a light chain constant domain (C L ), and the heavy chain constant domain C H1 , C H2 , C H3 and C H4 The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0066] An intact antibody may have one or more "effector functions," which refer to those biological activities attributable to the Fc region of an antibody (e.g., a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include complement dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis.

[0067] "Antibody fragments" preferably include portions of intact antibodies that contain the antigen-binding region or a variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment(s), fragment(s) produced by a Fab expression library, or epitope-binding fragments of any of the above that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).

[0068] The term "variable" in the context of antibodies refers to certain parts of the variable domains of antibodies that vary widely in sequence and are used in the binding and specificity of each particular antibody for its particular antigen. This variability is concentrated in three segments called "hypervariable regions" in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). Each of the variable domains of naturally occurring heavy and light chains contains four FRs connected by three hypervariable regions.

[0069] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally refer to the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and 31-35 (H1), 50-65 (H2), and 95-102 (L3) in the heavy chain variable domain; Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and 26-32 (H1), 53-55 (142), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). FR residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0070] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H and V L Fv polypeptides typically comprise V domains, which are present in a single polypeptide chain. H and V L It further comprises a polypeptide linker between the domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0071] The term "bispecific antibody" refers to small antibody fragments having two antigen-binding sites, which fragments comprise a variable light domain (VL domain) in the same polypeptide chain. L ) connected to a variable weight domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, generating two antigen-binding sites. Bispecific antibodies are described in more detail in, for example, EP0404097; WO93 / 11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0072] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a recipient hypervariable region have been replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin have been replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FRs being those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596.

[0073] As used herein, "isolated" means separated from (a) a natural source, such as a plant or animal cell, or cell culture, or (b) other components of a synthetic organic chemical reaction mixture. As used herein, "purified" means that when isolated, the isolate contains at least 95%, and in another embodiment at least 98%, of the compound (e.g., conjugate) by weight of the isolate.

[0074] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. The contaminants of that natural environment are materials that would prevent the antibody from being used diagnostically or therapeutically, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver staining. Since at least one component of the antibody's natural environment will no longer be present, an isolated antibody also includes an antibody in its original location in a recombinant cell. Ordinarily, however, an isolated antibody is prepared by at least one purification step.

[0075] An "apoptosis-inducing" antibody is one that induces programmed cell death as determined by Annexin V binding, DNA fragmentation, cell shrinkage, endoplasmic reticulum expansion, cell fragmentation, and / or formation of membrane vesicles (referred to as apoptotic bodies). The cell is a tumor cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. A variety of methods are available to assess cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by Annexin binding, DNA fragmentation can be assessed by DNA laddering, and nuclear / chromatin condensation along with DNA fragmentation can be assessed by an increase in hypodiploid cells.

[0076] The term "therapeutically effective amount" refers to an amount of drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of drug can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that the drug can inhibit the growth of existing cancer cells and / or kill existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or by determining the response rate (RR).

[0077] The term "substantial amount" refers to a majority of the population of a mixture or sample, i.e., greater than 50%.

[0078] The term "intracellular metabolites" refers to compounds resulting from intracellular metabolic processes or reactions on an antibody drug conjugate (ADC). The metabolic processes or reactions can be enzymatic processes, such as proteolytic cleavage of a peptide linker of an ADC. Intracellular metabolites include, but are not limited to, antibodies and free drugs that have undergone intracellular cleavage after entry, diffusion, uptake or transport into a cell.

[0079] The terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction or the like within a cell on an ADC, whereby a covalent attachment, e.g., a linker between a drug moiety and an antibody, is cleaved, resulting in the free drug or other metabolite of the conjugate dissociated from the antibody within the cell. The cleaved portion of the ADC is thus an intracellular metabolite.

[0080] The term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug administered to a patient. Bioavailability is a constant term that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the systemic circulation from an administered dosage form.

[0081] The term "cytotoxic activity" refers to the cell killing, cytostatic or antiproliferative effects of an ADC, or an intracellular metabolite of said ADC. Cytotoxic activity is measured using the IC50, which is the concentration (molar or mass) per unit volume at which half of the cells survive. 50 It may also be expressed as a value.

[0082] "Disorder" refers to any condition that can benefit from treatment with drug or antibody-drug conjugate.This includes chronic and acute disorders or diseases, including pathological conditions that predispose mammals to the disorder of interest.Non-limiting examples of disorders that can be treated herein include benign and malignant cancers; leukemia and lymphatic malignancies, neuronal, glial, astrocyte, hypothalamus and other glands, macrophage, epithelial, interstitial and blastocoel disorders; and inflammatory, angiogenic and immune disorders.

[0083] The terms "cancer" and "cancerous" refer to or describe a physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancer cells.

[0084] Examples of "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds and poultry. In an exemplary embodiment, the patient is a human.

[0085] The term "treat" or "treatment", unless otherwise indicated by context, refers to therapeutic treatment and prophylactic measures to prevent recurrence, the purpose of which is to inhibit or delay (alleviate) the development or spread of undesirable physiological changes or disorders, such as cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., non-worsening) state of disease, slowing or delaying the progression of disease, remission or palliation of the pathology, and remission (whether partial or total). "Treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to having the condition or disorder.

[0086] In the context of cancer, the term "treating" includes any or all of inhibiting the growth of tumor cells, cancer cells, or tumors, inhibiting the replication of tumor cells or cancer cells, alleviating the overall tumor burden or reducing the number of cancer cells, and ameliorating one or more symptoms associated with the disease.

[0087] In the context of autoimmune disease, the term "treating" includes any or all of inhibiting the replication of cells associated with the autoimmune pathology, including, but not limited to, cells that produce autoimmune antibodies, alleviating the autoimmune antibody load, and ameliorating one or more symptoms of the autoimmune disease.

[0088] In the context of an infectious disease, the term "treating" includes any or all of inhibiting the growth, proliferation or replication of the pathogen causing the infectious disease, and ameliorating one or more symptoms of the infectious disease.

[0089] The term "package insert" is used to refer to instructions typically included in commercially available packages of therapeutic products that contain information about the indication(s), usage, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic product.

[0090] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such names include progeny. The terms "transformant" and "transformed cell" include the primary subject cell and culture or progeny derived therefrom, regardless of the number of transfers. It is also understood that not all progeny are exactly the same in DNA content due to deliberate or accidental mutations. Mutant progeny that have the same function or biological activity as screened for in the original transformed cell are included. Different names are also intended, but such cases will be clear from the context.

[0091] Unless otherwise indicated, the term "alkyl" by itself or as part of another term refers to a straight or branched chain saturated hydrocarbon having the indicated number of carbon atoms (e.g., "C1-C8" alkyl refers to an alkyl group having 1 to 8 carbon atoms). When the number of carbon atoms is not indicated, the alkyl group has 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms. Representative linear C1-C8 alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl, while branched C1-C8 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl, and unsaturated C2-C8 alkyls include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

[0092] Unless otherwise indicated, "alkylene," by itself or as part of another term, refers to a saturated branched or straight chain or cyclic hydrocarbon radical having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane, of the stated number of carbon atoms, typically 1 to 18 carbon atoms. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,2-ethylene (-CH2CH2-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and the like. "C1-C 10 " A straight chain alkylene has the formula -(CH2) 1~10 - is a straight chain saturated hydrocarbon group. 10Examples of alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decalene. In certain embodiments of the invention, alkylene has 1 to 9, 1 to 8, 1 to 7, and 1 to 6 carbons.

[0093] Unless otherwise indicated, the term "heteroalkyl", by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, which is fully saturated or contains one to three degrees of unsaturation, and consists of the stated number of carbon atoms and one to three heteroatoms selected from the group consisting of O, N, Si, S and / or P, or a combination thereof, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N and S may be located at any interior position of the heteroalkyl group. The heteroatom Si may be located at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive.

[0094] "Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

[0095] "Haro (C 1~6 "-alkyl)" refers to a C substituted with 1 to 3 or 1 to 2 halo groups. 1~6 -Alkyl group, C 1~6 -Alkyl and halo are as defined herein. This term includes, for example, CF3.

[0096] Unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent group derived from a heteroalkyl (as discussed above). For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini.

[0097] Unless otherwise indicated, "aryl," by itself or as part of another term, means a substituted or unsubstituted monovalent aromatic hydrocarbon radical of 6 to 20 carbon atoms, preferably 6 to 14 carbon atoms, derived by removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. Substituted aromatic groups (e.g., aryl groups) can be substituted with one or more, preferably one to five, of the following groups: C1-C8 alkyl, -O-(C1-C8 alkyl), -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN. Each R' is independently selected from -H, C1-C8 alkyl, C1-C8 heteroalkyl, and aryl, preferably unsubstituted aryl. In some embodiments, the substituted aromatic group can further include one or more of -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', and -SR'.

[0098] The term "heteroaryl", also referred to herein as "Het" (sometimes shown in a circle), as used herein, refers to a 5-14 membered, e.g., 5-6 membered, aromatic heterocyclic ring having at least one heteroatom selected from nitrogen, oxygen, and sulfur and containing at least one carbon atom. Heteroaryls can be monocyclic, bicyclic, or tricyclic ring systems. Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl, pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, and quinoxalinyl. Heteroaryl may be optionally substituted. Exemplary substituents include, but are not limited to, -X, -R, -O-, -OR, -SR, -S - , -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R, -C(=O)NR2, -SO3 - , -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3 2- , PO3H2, -AsO2H2, -C(=O)R, -C(=O)X, -C(=S)R, -CO2R, -CO2 - , -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2, -C(=NR)NR2, C1~C 20 Heteroalkyl, C6-C 20 Aryl, C3-C8 heterocyclyl, C3-C 20 Carbocyclyl, protecting group or prodrug moieties are included, where each X is independently a halogen: -F, -Cl, -Br, or -I, and each R is independently -H or C1-C6 alkyl.

[0099] The terms "arylene" and "heteroarylene" refer to divalent versions of "aryl" and "heteroaryl", respectively, as well as other terms incorporating "aryl" and "heteroaryl".

[0100] "Hydroxy" refers to the group --OH.

[0101] "Substituted alkyl" means an alkyl in which one or more hydrogen atoms are each independently replaced with a substituent. Exemplary substituents include, but are not limited to, -X, -R, -O-, -OR, -SR, -S - , -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R, -C(=O)NR2, -SO3 - , -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3 2- , PO3H2, -AsO2H2, -C(=O)R, -C(=O)X, -C(=S)R, -CO2R, -CO2 - , -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2, -C(=NR)NR2, C1~C 20 Heteroalkyl, C6-C 20 Included are aryl, C3-C8 heterocyclyl, protecting group or prodrug moiety, where each X is independently a halogen -F, -Cl, -Br, or -I, and each R is independently -H or C1-C6 alkyl. Substituted alkyl substituted with halogen is sometimes referred to herein as haloalkyl. Aryl, alkylene, heteroalkylene, and other groups containing or not containing alkyl or alkylene moieties as described herein may also be similarly substituted.

[0102] Unless otherwise indicated, "aralkyl," by itself or as part of another term, means an alkyl group, as defined above, substituted with an aryl group, as defined above.

[0103] Unless otherwise indicated, "heteroatom" refers to oxygen, nitrogen, sulfur, or phosphorus. Each heteroatom in a structure may be the same or different.

[0104] Unless otherwise indicated, "C3-C8 heterocyclyl," by itself or as part of another term, refers to a mono- or divalent, substituted or unsubstituted, aromatic or non-aromatic, monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also referred to as ring members) and 1 to 4 heteroatom ring members independently selected from N, O, P, or S, and derived by the removal of one hydrogen atom from a ring atom of the parent ring system. Similarly, unless otherwise indicated, "C3-C 10 "Heterocyclyl" by itself or as part of another term refers to a monovalent or divalent, substituted or unsubstituted, aromatic or non-aromatic, monocyclic or bicyclic ring system having 3 to 10 carbon atoms (also referred to as ring members) and 1 to 4 heteroatom ring members independently selected from N, O, P, or S, and obtained by removing one hydrogen atom from a ring atom of the parent ring system. One or more N, C, or S atoms in a heterocyclyl can be oxidized. The ring containing the heteroatom can be aromatic or non-aromatic. When the term "heterocyclyl" is used without reference to a specific number of carbons, heterocyclyl groups having more than 10 carbons, for example rings or ring systems having 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons, also include C3-C6 rings. 10It is possible and included with heterocyclyl. Similarly, when the term "heterocyclyl" is used without reference to a specific number of carbons, heterocyclyl groups having less than three carbons, for example rings having one or two, are possible and included. The term "heterocycloalkyl" refers to a non-aromatic heterocyclyl ring or ring system, in which all carbon atoms are saturated (i.e., bonded to hydrogen or another substituent as described below, and there are no double or triple bonds). In certain embodiments, heterocycloalkyl groups typically have 3-5 members and 1-2 heteroatoms. In certain embodiments, heterocycloalkyl can be epoxy.

[0105] Unless otherwise specified, the heterocyclyl is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Representative examples of C3-C8 heterocyclyl include, but are not limited to, tetrahydrofuranyl, oxetanyl, pyranyl, pyrrolidinyl, piperidinyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiopene), furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, and tetrazolyl. C3-C8 heterocyclyl, or C3-C 10 Heterocyclyl can be substituted with up to seven groups, including but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, -OR', aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(=O)2R', -S(O)R', halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where each R' is independently selected from -H, C1-C8 alkyl, C1-C8 heteroalkyl, and aryl. In some embodiments, the substituted heterocyclyl can also include one or more of -NHC(=NH)NH2, -NHCONH2, -S(=O)2R', and -SR'.

[0106] Unless otherwise specified, "C3~C 20 "Carbocyclyl" by itself or as part of another term is a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19- or 20-membered monovalent substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocyclic ring derived from the removal of one hydrogen atom from a ring atom of a parent ring system. Preferably, the ring is a C3-C 20 Carbocyclyls contain 3 to 14, or 3 to 6 carbons. Representative C3-C8 carbocyclyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, cyclooctadienyl, bicyclo(1.1.1.)pentane, and bicyclo(2.2.2.)octane. A C3-C8 carbocyclyl group can be unsubstituted or substituted with up to seven groups, including but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, -OR', aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(=O)2R', -S(=O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where each R' is independently selected from -H, C1-C8 alkyl, C1-C8 heteroalkyl, and aryl. "C3-C8 carbocyclo" is the corresponding divalent moiety.

[0107] Unless otherwise indicated, "heteroaralkyl," by itself or as part of another term, means an alkyl group, as defined above, substituted with an aromatic heterocyclyl group, as defined above.

[0108] Unless otherwise indicated, "C3-C8 carbocyclyl," by itself or as part of another term, is a 3-, 4-, 5-, 6-, 7-, or 8-membered mono- or divalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocyclic ring obtained by removing one or two hydrogen atoms from a ring atom of a parent ring system. 10 "Carbocyclyl" by itself or as part of another term is a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered mono- or divalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocyclic ring obtained by removing one hydrogen atom from a ring atom of a parent ring system. Representative C3-C8 carbocyclyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, cyclooctadienyl, bicyclo(111)pentane, and bicyclo(222)octane. A C3-C8 carbocyclyl group, or a C3-C8 10 Carbocyclyl groups may be unsubstituted or substituted with up to seven groups including, but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, -OR', aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(=O)2R', -S(=O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, C1-C8 alkyl, C1-C8 heteroalkyl and aryl. When the term "carbocyclyl" is used without reference to a specific number of carbons, carbocyclyl groups having more than 10 carbons, for example ring systems having 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons, are considered to be C-C 10Also possible and encompassed in conjunction with carbocyclyl. The term "cycloalkyl" refers to a carbocyclyl ring or ring system in which all carbon atoms are saturated (i.e., bonded to a hydrogen or to another substituent as described below, and there are no double or triple bonds).

[0109] The term "chiral" refers to molecules that have the property of being non-superimposable on their mirror image partners, while the term "achiral" refers to molecules that are superimposable on their mirror image partners.

[0110] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0111] "Diastereomer" refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated by high resolution analytical procedures, such as electrophoresis and chromatography.

[0112] The stereochemical definitions and rules used herein generally follow those of S.P. Parker, ed., McGraw-Hill Dictionary of Chemical Terms, McGraw-Hill Book Company, New York (1984); and Eliel and Wilen, Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York (1994). Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the molecule around its chiral center(s). The prefixes d and l, or (+) and (-), are used to indicate the sign of rotation of plane-polarized light by the compound, with (-) or l meaning that the compound is levorotatory. Compounds with the prefixes (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0113] An amino acid "derivative" includes an amino acid having a substitution or modification by covalent attachment of the parent amino acid, e.g., by alkylation, glycosylation, acetylation, phosphorylation, etc. Also included within the definition of "derivative" are one or more analogs of an amino acid, e.g., having substituted linkages, and other modifications known in the art.

[0114] "Natural amino acid" refers to, unless the context indicates otherwise, arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine.

[0115] The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of a compound. The compound typically contains at least one amino group and is thus capable of forming an acid addition salt with this amino group. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, hydrogen sulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have multiple charged atoms in its structure. The example where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0116] The term "loading" or "drug loading" or "payload loading" refers to or refers to the average number of payloads per antibody in an ADC molecule ("payload" or "s) is used interchangeably herein with "drug" or "s). Drug loading can range from 1 to 20 drugs per antibody. This is sometimes referred to as the DAR, or drug-to-antibody ratio. The compositions of ADCs described herein typically have a DAR of 1 to 20, and in certain embodiments, 1 to 8, 2 to 8, 2 to 6, 2 to 5, and 2 to 4. Typical DAR values ​​are 2, 4, 6, and 8. The average number of drugs per antibody, or DAR value, can be characterized by conventional means, such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC. Quantitative DAR values ​​can also be determined. In some cases, isolation, purification, and characterization of homogenous ADCs with specific DAR values ​​can be achieved by means such as reverse-phase HPLC or electrophoresis. The DAR can be limited by the number of attachment sites on the antibody. For example, when the attachment is a cysteine ​​thiol, the antibody may have only one or several cysteine ​​thiol groups, or may have only one or several sufficiently reactive thiol groups through which the linker unit may be attached. In some embodiments, the cysteine ​​thiol is the thiol group of a cysteine ​​residue that forms an interchain disulfide bond. In some embodiments, the cysteine ​​thiol is the thiol group of a cysteine ​​residue that does not form an interchain disulfide bond. Typically, less than the theoretical maximum drug moiety is conjugated to the antibody during the conjugation reaction. The antibody may contain many lysine residues that do not react with, for example, the linker or linker intermediate. Only the most reactive lysine groups may react with the reactive linker reagent.

[0117] Generally, antibodies do not contain many, if any, free and reactive cysteine ​​thiol groups that can be linked to drugs via linkers. Most cysteine ​​thiol residues in antibodies exist as disulfide bridges and must be reduced with a reducing agent, such as dithiothreitol (DTT). Antibodies can be subjected to denaturing conditions to expose reactive nucleophilic groups, such as lysine or cysteine. The loading amount (drug / antibody ratio) of ADC can be controlled in several different ways, including (i) limiting the molar excess of drug-linker relative to antibody, (ii) limiting the reaction time or temperature of conjugation, and (iii) partial or limited reductive conditions for cysteine ​​thiol modification. When more than one nucleophilic group reacts with drug-linker, the resulting product is a mixture of ADCs with a distribution of one or more drug moieties per antibody. The average number of drugs per antibody can be calculated from the mixture, for example, by a double ELISA antibody assay specific for antibody and specific for drug. Individual ADCs can be identified in the mixture by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography.

[0118] Below is a list of abbreviations and definitions that may not be otherwise defined or set forth in this application. DMSO (referring to dimethyl sulfoxide), HRMS (referring to high resolution mass spectrometry), DAD (referring to diode array detection), TFA (referring to 2,2,2-trifluoroacetic acid or trifluoroacetic acid), TFF (referring to tangential flow filtration), EtOH (referring to ethanol), MW (referring to molecular weight), HPLC (referring to high performance liquid chromatography), preparative HPLC (referring to preparative high performance liquid chromatography), etc. (referring to other), trityl (referring to 1,1',1''-ethane-1,1,1-triyltribenzene), THF (referring to tetrahydrofuran), NHS (referring to 1-hydroxy-2,5-pyrrolidinedione), Cbz (referring to carboxybenzyl), eq. (referring to equivalents), n-BuLi (referring to n-butyl lithium), OAc (referring to acetate), MeOH (referring to methanol), i-Pr (referring to isopropyl or propan-2-yl), NMM (referring to 4-methylmorpholine), and "-" (referring to data not available at this time in the table).

[0119] As used herein, "H / C" or "H-(C)" refers to trastuzumab (sold under the trade name HERCEPTIN®), or a substantially similar monoclonal antibody that blocks the HER2 / neu receptor, attached (to a linker or to a compound of the invention) through one of its cysteine ​​residues.

[0120] As used herein, "CD33-11A1-v1417-(C)" refers to an anti-CD33 antibody that is a monoclonal antibody that binds to CD33, linked (to a linker or to a compound of the invention) via one of its cysteine ​​residues.

[0121] As used throughout this application, numbering of amino acid residues (eg, alanine at position 114) is based on the EU index of the Kabat method.

[0122] As used herein, "H-(A114C)" refers to an engineered trastuzumab, a monoclonal antibody that blocks the HER2 / neu receptor, attached (to a linker or to a compound of the invention) through one of its engineered cysteines substituted for an alanine at position 114 of the heavy chain.

[0123] As used herein, "H-(kK183C)" refers to an engineered trastuzumab, a monoclonal antibody that blocks the HER2 / neu receptor, attached (to a linker or to a compound of the invention) via its engineered cysteine ​​substituted at the lysine at position 183 of the light (kappa) chain.

[0124] As used herein, "H-(E380C)" refers to an engineered trastuzumab, a monoclonal antibody that blocks the HER2 / neu receptor, attached (to a linker or to a compound of the invention) via its engineered cysteine ​​substituted for glutamate at position 380 of the heavy chain.

[0125] As used herein, "CD33-11A1-v1417-(K290C)-(K334C)" refers to an engineered anti-CD33 antibody that is a monoclonal antibody that binds to CD33 and is linked (to a linker or a compound of the invention) via its engineered cysteines substituted at the lysine at position 290 and at the lysine at position 334 of the heavy chain.

[0126] As used herein, "CD33-11A1-v1417-(K334C)-(K392C)" refers to an engineered anti-CD33 antibody that is a monoclonal antibody that binds to CD33 and is linked (to a linker or a compound of the invention) via its engineered cysteines substituted at the lysine at position 334 and at the lysine at position 392 of the heavy chain.

[0127] Generally, as used herein, "H-((AA1)###(AA2))" (where (AA1) and (AA2) are the first and second amino acids) refers to engineered trastuzumab, a monoclonal antibody that blocks the HER2 / neu receptor, which is attached to a compound of the invention via its engineered (AA2) substituted in (AA1) at position ### of the heavy chain, where ### represents the position of the relevant amino acid(s). Similar notations referring to "k" or "kappa" indicate a substitution on the kappa form of the light chain.

[0128] Similarly, as used herein, "H-((AA1)###(AA2)-((AA3)####(AA4))" (where (AA1), (AA2), (AA3) and (AA4) are the first, second, third and fourth amino acids) refers to an engineered trastuzumab, a monoclonal antibody that blocks the HER2 / neu receptor, which is attached to a compound of the invention via its engineered (AA2) substituted at (AA1) at position ### (where ### represents the position of the relevant amino acid(s)) of the heavy chain and also attached to a compound of the invention via its engineered (AA4) substituted at (AA3) at position #### (where #### represents the position of the relevant amino acid(s)) of the heavy chain. Similar notation referring to "k" or "kappa" indicates a kappa-morphic substitution of the light chain.

[0129] Generally, as used herein, "CD33-11A1-v1417-((AA1)###(AA2)) (where (AA1) and (AA2) are the first and second amino acids) refers to an engineered anti-CD33 antibody that is a monoclonal antibody that binds to a compound of the invention via its engineered (AA2) substituted in (AA1) at position ### (where ### represents the position of the relevant amino acid(s)) of the heavy chain, and binds to CD33. Similar notations referring to "k" or "kappa" indicate a kappa-type substitution of the light chain.

[0130] Similarly, as used herein, "CD33-11A1-v1417-(AA1)###(AA2)+(AA3)####(AA4) / (AA2)###+(AA4)####" (where (AA1), (AA2), (AA3) and (AA4) are the first, second, third and fourth amino acids) refers to an engineered trastuzumab, a monoclonal antibody that blocks the HER2 / neu receptor, which is attached to a compound of the invention via its engineered (AA2) substituted at (AA1) at position ### (where ### represents the position of the relevant amino acid(s)) of the heavy chain and which is attached to a compound of the invention via its engineered (AA4) substituted at (AA3) at position #### (where #### represents the position of the relevant amino acid(s)) of the heavy chain. Similar notations referring to "k" or "kappa" indicate substitutions on the kappa form of the light chain.

[0131] As used herein, "-PABC-" or "PABC" refers to a compound of the structure:

[0132] [ka] or a variant thereof.

[0133] As used herein, "-PABA-" or "PABA" refers to a compound having the structure:

[0134] [ka] or a variant thereof.

[0135] Antibody Units (Ab or AB) As noted above, the term "antibody" (or "Ab" or "AB") is used herein in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. Additionally, while certain aspects of the invention described herein refer to antibody drug conjugates, it is further envisioned that the antibody portion of the conjugate may be replaced with one that specifically binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target cell population. For example, instead of containing an antibody, the conjugates of the invention may contain a targeting molecule that binds, complexes, or reacts with a receptor, antigen or other receptive moiety of the cell population sought to be therapeutically or otherwise biologically modified. Examples of such molecules include smaller molecular weight proteins, polypeptides or peptides, lectins, glycoproteins, non-peptides, vitamins, nutrient transport molecules (such as, but not limited to, transferrin), or any other cell-binding molecule or substance. In certain embodiments, the antibody or other such targeting molecule acts to deliver the drug to a specific target cell population with which the antibody or other targeting molecule interacts.

[0136] In another aspect, the present invention relates to an antibody drug conjugate compound of formula II, wherein P is selected from the group consisting of trastuzumab, trastuzumab variants (e.g., trastuzumab variants disclosed herein or in International Patent Application No. PCT / IB2012 / 056234), oregovomab, edrecolomab, cetuximab, vitronectin receptor (α vβ3), alemtuzumab, anti-HLA-DR antibodies, including humanized anti-HLA-DR antibodies for the treatment of non-Hodgkin's lymphoma, 131I Lym-1, anti-HLA-Dr10 antibodies, including mouse anti-HLA-Dr10 antibodies for the treatment of non-Hodgkin's lymphoma, anti-CD33 antibodies, anti-CD22 antibodies, including humanized anti-CD22 mAbs for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma, labetuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, and gemtuzumab.

[0137] Heteroatoms that may be present on an antibody unit include sulfur (in one embodiment, from a sulfhydryl group of the antibody), oxygen (in one embodiment, from a carbonyl, carboxyl, or hydroxyl group of the antibody), and nitrogen (in one embodiment, from a primary or secondary amino group of the antibody). These heteroatoms may be present on the antibody in its native state, e.g., a naturally-occurring antibody, or may be introduced into the antibody by chemical modification.

[0138] In one embodiment, the antibody units have sulfhydryl groups and the antibody units are linked through the sulfhydryl group's sulfur atom.

[0139] In yet another embodiment, the antibody has natural or engineered cysteine ​​residues that react with the compounds of the invention and thus interact with the sulfur atoms of the antibody units and with R 2 A thioether bond can be formed consisting of a carbon atom bearing an SO2 group. See, for example, WO2013 / 093809 ("Engineered Antibody Constant Regions for Site-Specific Conjugation and Methods and Uses Therefor"); and WO2014 / 068443 ("Spliceostatin Analogs").

[0140] Additionally, free sulfur or sulfhydryl groups can be introduced by other well-known methods, as described in the following paragraphs.

[0141] In another embodiment, the antibody has a lysine residue that can react with an activated ester (such esters include, but are not limited to, N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl esters), thus forming an amide bond consisting of the nitrogen atom of the antibody unit and a carbonyl. For example, in one aspect of this embodiment, the antibody unit has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's Reagent).

[0142] In another embodiment, the antibody unit has one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups. For example, in one aspect of this embodiment, the antibody unit has one or more carbohydrate groups that can be oxidized to provide aldehyde groups (see, for example, Laguzza et al., 1989, J. Med. Chem., 32(3):548-55). The corresponding aldehydes can form bonds with reactive sites, such as compounds that contain hydrazine or hydroxylamine moieties, and also those that contain one or more free or masked sulfhydryl groups. Other protocols for the modification of proteins for drug attachment or association are described in Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002), which is incorporated herein by reference.

[0143] In another embodiment, the antibody unit has one or more glutamine residues that can react with primary amines under conditions enzymatically catalyzed by transglutaminase, thereby forming an amide bond consisting of the side chain carbonyl group of the glutamine residue on the antibody unit and the amine. In one aspect of this embodiment, the glutamine residue can be an endogenous glutamine, an engineered glutamine-containing tag, and / or an endogenous glutamine made reactive by antibody engineering, or an engineered transglutaminase (e.g., with altered substrate specificity), as described in WO2012 / 059882 ("Engineered Polypeptide Conjugates and Methods for Making Thereof Using Transglutaminase"), WO2015 / 015448 ("Engineered Polypeptide Conjugates Using Transglutaminase"), and WO2015 / 162563 ("Antibody-Drug Conjugates with High Drug Loading"), the entire contents of each of which are incorporated herein by reference. In another aspect of this embodiment, the antibody unit has one or more glutamine residues that can be chemically modified to introduce one or more sulfhydryl groups. Such modifications can be carried out under conditions catalyzed by transglutaminase, in which an amide bond is formed between the side chain carbonyl of a glutamine residue and a primary amine that also contains one or more free or masked sulfhydryl groups.

[0144] When the conjugate comprises a non-immunoreactive protein, polypeptide, or peptide unit instead of an antibody, useful non-immunoreactive protein, polypeptide, or peptide units include, but are not limited to, transferrin, epidermal growth factor ("EGF"), bombesin, gastrin, gastrin releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors ("TOPs"), such as TGF-α and TGF-β, vaccinia growth factor ("VGF"), insulin and insulin-like growth factors I and II, somatostatin, lectins, and apoproteins from low density lipoproteins.

[0145] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies against a particular antigenic determinant (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against an antigen of interest can be prepared by using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture.

[0146] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric monoclonal antibodies.Human monoclonal antibodies can be produced by any of a number of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982, Meth. Enzymol. 92:3-16).

[0147] The antibody may also be a bispecific antibody. Methods for making bispecific antibodies are known in the art and are discussed below.

[0148] The antibody may be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to a target cell (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen) or other antibody that binds to a tumor cell or matrix. In this context, "functional activity" means that the fragment, derivative or analog is capable of eliciting anti-anti-idiotypic antibodies that recognize the same antigen as that recognized by the antibody from which the fragment, derivative or analog is derived. Specifically, in exemplary embodiments, the antigenicity of the idiotype of an immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequences that specifically recognize the antigen. To determine which CDR sequences bind to an antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., BIAcore assay) (for the location of CDR sequences, see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. Immunology 125(3):961-969).

[0149] Other useful antibodies include fragments of antibodies, such as, but not limited to, F(ab')2 fragments, Fab fragments, Fvs, single chain antibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, or any other molecule with the same specificity as an antibody.

[0150] In addition, recombinant antibodies, for example, chimeric and humanized monoclonal antibodies, which contain both human and non-human parts, can be produced using standard recombinant DNA technology, are useful antibodies. Chimeric antibodies are molecules in which different parts are derived from different animal species, for example, variable regions derived from mouse monoclonals, and human immunoglobulin constant regions. (See, for example, U.S. Patent No. 4,816,567; and U.S. Patent No. 4,816,397, the entire contents of which are incorporated herein by reference). Humanized antibodies are antibody molecules from non-human species that have one or more complementarity determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. (See, for example, U.S. Patent No. 5,585,089, the entire contents of which are incorporated herein by reference).Such chimeric and humanized monoclonal antibodies can be made by recombinant DNA techniques known in the art, for example, as described in International Publication No. WO 87 / 02671; European Patent No. 0184187; European Patent Application Publication No. 0171496; European Patent Application Publication No. 0173494; International Publication No. WO 86 / 01533; U.S. Patent No. 4,816,567; European Patent Application Publication No. 012023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA, each of which is incorporated herein by reference in its entirety. 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Patent No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060.

[0151] Fully human antibodies are particularly desirable and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but that can express human heavy and light chain genes. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For a review of this technology for producing human antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, e.g., U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806, each of which is incorporated by reference in its entirety. Other human antibodies can be obtained commercially, for example, from Abgenix, Inc. (now Amgen, Freemont, Calif.) and Medarex (Princeton, NJ).

[0152] Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection". In this approach, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope. (See, e.g., Jespers et al., 1994, Biotechnology, 12:899-903). Human antibodies can also be produced using a variety of techniques known in the art, including phage display libraries (see, e.g., Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581; Quan and Carter, 2002, The rise of monoclonal antibodies as therapeutics, In Anti-IgE and Allergic Disease, Jardieu and Fick, eds., Marcel Dekker, New York, NY, Chapter 20, pp. 427-469).

[0153] In other embodiments, the antibody is a fusion protein of an antibody, or a functionally active fragment thereof, e.g., the antibody is fused via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus of the amino acid sequence of another protein (or a portion thereof, preferably at least a 10, 20 or 50 amino acid portion) that is not an antibody. Preferably, the antibody or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain.

[0154] That is, antibodies include analogs and derivatives that are modified by the covalent attachment of any type of molecule, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not limited to, antibody derivatives and analogs include those that are further modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular antibody units or other proteins, etc. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, analogs or derivatives can contain one or more unnatural amino acids.

[0155] The antibody may have modifications (e.g., substitutions, deletions, or additions) in amino acid residues that interact with Fc receptors. In particular, the antibody may have modifications in amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97 / 34631, which is incorporated herein by reference in its entirety).

[0156] Antibodies immunospecific for cancer cell antigens can be obtained commercially or produced by any method known to those of skill in the art, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a similar database, the literature, or by routine cloning and sequencing.

[0157] In certain embodiments, known antibodies can be used for the treatment of cancer. Antibodies immunospecific for cancer cell antigens can be obtained commercially or produced by any method known to those skilled in the art, such as recombinant expression technology. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a similar database, literature, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, OVAREX, a mouse antibody for the treatment of ovarian cancer; mouse IgG, for the treatment of colorectal cancer. 2a PANOREX (Glaxo Wellcome, NC), an antibody; cetuximab ERBITUX (Imclone Systems Inc., NY), an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers, e.g., head and neck cancer; Vitaxin (MedImmune, Inc., MD), a humanized antibody for the treatment of sarcoma; CAMPATH I / H (Leukosite, MA), a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); SMART ID10 (Protein Design, Inc., NY), a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma. Labs, Inc., CA); ONCOLYM (Techniclone, Inc., CA), a radiolabeled murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; ALLOMUNE (BioTransplant, CA), a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; and CEACIDE (Immunomedics, NJ), a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0158] In an attempt to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or other tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cell(s).Often, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells.The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction by antibody-based therapy.

[0159] Linker Units Linker is a bifunctional compound that can be used to link drug and antibody to form antibody drug conjugate (ADC).Such conjugate is useful, for example, in forming immunoconjugates against tumor-associated antigens.Such conjugates allow selective delivery of cytotoxic drugs to tumor cells.In ADC, linker plays the role of attaching payload to antibody.

[0160] The conjugates of the present invention contain linkers that may incorporate a cleavage element that, when cleaved chemically or enzymatically, ultimately results in the release of a drug or active agent that may be a derivative of the drug. Examples of cleavage elements include, but are not limited to, chemically cleaved acylhydrazones, such as those described in US5,773,001, enzymatically cleaved dipeptide or dipeptide-p-aminobenzylcarbamate sequences, such as those described in US6,214,345B1, enzymatically cleaved glucuronide or glycoside moieties, such as those described in US8,039,273_B2 and US8,568,728B2, etc. In certain embodiments of the present invention, additional self-immolative spacer elements, such as p-aminobenzylcarbamate moieties, substituted or unsubstituted N,N-diaminoethyl or N,N-diaminopropyl moieties, may be incorporated into the linker unit.

[0161] In certain embodiments of the invention, the linker may be "non-cleavable," in which case there are no chemically or enzymatically sensitive bonds within the linker. In these cases, the drug or active agent is expected to be released via enzymatic catabolism of the antibody itself, thereby releasing the drug with the linker and one or more amino acids from the intact antibody.

[0162] In certain embodiments of the invention, a linker unit may be attached to the drug first, and then the drug-linker element is attached to the antibody or antibody fragment in a separate transformation.

[0163] In other embodiments of the invention, a linker unit may be attached to the antibody or antibody fragment first, and then the drug or drug derivative is attached to the antibody linker or antibody fragment linker in a separate transformation.

[0164] Synthesis of Compounds and Antibody-Drug Conjugates Thereof The compounds and conjugates of the invention include those that can be made using the synthetic procedures outlined below in the examples. As described in more detail below, the compounds and conjugates of the invention can be prepared using a section of the linker unit that has a reactive site. In one embodiment, a second section of the linker unit is introduced that has a second reactive site that is selectively reactive to the thiol group of a cysteine ​​residue (or the selenol group of a selenocysteine ​​residue). As described in more detail below, the conjugates can be prepared using a section of the linker that has a reactive site for binding to a drug, probe, or imaging agent, and introducing another section of the linker unit that has a reactive site for an antibody. In one embodiment, the linker unit has a reactive site that has an electrophilic group that is selectively reactive with a thiol nucleophilic group present on an antibody unit, for example, an antibody. The electrophilic group provides a convenient site for antibody attachment. The heteroatom of the nucleophilic group of the antibody is reactive to the electrophilic group on the linker unit to form a covalent bond to the linker unit.

[0165] The compounds and conjugates of the invention also include those that can be made using stepwise preparations.

[0166] Certain compounds of formula I of the present invention may be prepared by nucleophilic aromatic substitution reactions (S N The thiol moiety present on the polypeptide or antibody unit contains a thiol-reactive electrophilic group capable of receiving a thiol-reactive electrophilic group, R 2 The reaction proceeds with the electrophilic group with displacement of the SO group. This reaction proceeds with the sulfur atom on the polypeptide or antibody unit and the R 2 A novel covalent bond between the aromatic carbon atom of the electrophilic group that previously carried the SO2 group is realized. In one aspect of the invention, the thiol group of the polypeptide or antibody unit may be naturally occurring in free or capped form, or may be present in a naturally occurring sulfide bond and may require prior treatment with a reagent, such as dithiothreitol (DTT), cysteine, tris-(2-carboxyethyl)phosphine (TCEP), that results in the uncapping of the thiol group with the release of a reactive thiol. In another aspect of the invention, the thiol group of the polypeptide or antibody unit may be introduced by engineering and may be present in free or capped form and may require prior treatment with a reagent, such as dithiothreitol (DTT), cysteine, tris-(2-carboxyethyl)phosphine (TCEP), that results in the uncapping of the thiol group with the release of a reactive thiol. In another aspect of the invention, a thiol group of a polypeptide or antibody unit may be introduced by chemical or enzymatic reaction of the polypeptide or antibody unit with a compound that contains a reactive moiety for attachment to the polypeptide or antibody unit and also contains one or more free, capped or protected thiol groups, which may require prior treatment with a reagent such as dithiothreitol (DTT), cysteine, tris-(2-carboxyethyl)phosphine (TCEP), etc., that results in uncapping of the thiol group with release of a reactive thiol.

[0167] In embodiments of the invention, the reaction of thiol groups present on a polypeptide or antibody unit can undergo selective reaction with the thiol-reactive electrophilic groups in Formula I in the presence of other potential nucleophilic groups, including, for example, amine groups present on a lysine side chain, amidine groups present on an arginine side chain, imidazole groups present on a histidine side chain, alcohol groups present on serine or threonine side chains, or phenol groups present on a tyrosine side chain.

[0168] Compositions and Methods of Administration In other embodiments, another aspect of the invention relates to a pharmaceutical composition comprising an effective amount of a compound of the invention and / or its antibody-drug conjugate, and a pharma- ceutically acceptable carrier or vehicle. In certain embodiments, the composition is suitable for administration to animals or humans.

[0169] The pharmaceutical composition may be in any form that allows the composition to be administered to a patient. For example, the composition may be in solid or liquid form. Typical routes of administration include, but are not limited to, parenteral, ocular, and intratumoral. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, or intrasternal injection or infusion techniques. In one aspect, the composition is administered parenterally. In a particular embodiment, the composition is administered intravenously.

[0170] The pharmaceutical composition can be formulated to allow the compound of the invention and / or its antibody drug conjugate to be bioavailable upon administration of the composition to a patient. The composition can take the form of one or more dosage units, for example, a tablet can be a single dosage unit and a container of the compound of the invention and / or its antibody drug conjugate in liquid form can hold multiple dosage units.

[0171] Materials used in the preparation of pharmaceutical compositions may be non-toxic in the amounts used.It is clear to those skilled in the art that the optimal dosage of active ingredient(s) in pharmaceutical compositions depends on various factors.Relevant factors include, but are not limited to, the type of animal (e.g., human), the specific form of the compound of the present invention and / or its antibody drug conjugate, the mode of administration, and the composition used.

[0172] The pharma- ceutically acceptable carrier or vehicle can be solid or particulate, so that the composition is, for example, in tablet or powder form. The carrier(s) can be liquid. Additionally, the carrier(s) can be particulate.

[0173] The composition may be in the form of a liquid, such as a solution, emulsion or suspension. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may also be included.

[0174] Liquid compositions, whether they are solutions, suspensions or other similar forms, can also contain one or more of the following: sterile excipients, such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, cyclodextrins, propylene glycol or other solvents that can serve as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetic acid, citric acid, phosphates or amino acids, and agents for regulating osmotic pressure, such as sodium chloride or dextrose. Parenteral compositions can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass, plastic or other materials. Saline is an exemplary adjuvant. Injectable compositions are preferably sterile.

[0175] The amount of the compound of the present invention and / or its antibody drug conjugate that is effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.In addition, in vitro or in vivo assays can be used in some cases to help identify the optimal dosage range.The exact dosage to be used in the composition also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances.

[0176] The composition contains an effective amount of the compound of the present invention and / or its antibody drug conjugate such that a suitable dosage is obtained. Typically, this amount is at least about 0.01% of the compound of the present invention and / or its antibody drug conjugate by weight of the composition. In an exemplary embodiment, the pharmaceutical composition is prepared so that a parenteral dosage unit contains an amount of the compound of the present invention and / or its antibody drug conjugate in the range of about 0.01% to about 2% by weight.

[0177] For intravenous administration, the composition can contain about 0.01 to about 100 mg of the compound of the present invention and / or its antibody drug conjugate per kg of patient body weight. In one embodiment, the composition can contain about 1 to about 100 mg of the compound of the present invention and / or its antibody drug conjugate per kg of patient body weight. In another embodiment, the amount administered is in the range of about 0.1 to about 25 mg / kg of body weight of the compound of the present invention and / or its antibody drug conjugate.

[0178] In general, the dosage of the compound of the present invention and / or its antibody-drug conjugate administered to a patient is typically about 0.01 mg / kg to about 20 mg / kg of the patient's body weight. In one embodiment, the dosage administered to a patient is about 0.01 mg / kg to about 10 mg / kg of the patient's body weight. In another embodiment, the dosage administered to a patient is about 0.1 mg / kg to about 10 mg / kg of the patient's body weight. In yet another embodiment, the dosage administered to a patient is about 0.1 mg / kg to about 5 mg / kg of the patient's body weight. In yet another embodiment, the dosage administered is about 0.1 mg / kg to about 3 mg / kg of the patient's body weight. In yet another embodiment, the dosage administered is about 1 mg / kg to about 3 mg / kg of the patient's body weight.

[0179] The compound of the present invention and / or its antibody drug conjugate can be administered by any convenient route, for example, by infusion or bolus injection. Administration can be systemic or local. Various delivery systems, for example, encapsulation in liposomes, microparticles, microcapsules, capsules, etc., are known and can be used to administer the compound of the present invention and / or its antibody drug conjugate. In certain embodiments, multiple compounds of the present invention and / or its antibody drug conjugate are administered to a patient.

[0180] In certain embodiments, it may be desirable to administer one or more of the compounds of the present invention and / or their antibody drug conjugates locally in the area that requires treatment.This can be achieved, for example, but not limited to, by local infusion during surgery; local application, for example, in conjunction with wound dressing after surgery; by injection; by using a catheter; or by using an implant (the implant being of a porous, non-porous, or gelatinous material, including a membrane, for example, a silastic membrane, or a fiber).In one embodiment, administration may be by direct injection at the site (or former site) of cancer, tumor, or neoplastic or pre-neoplastic tissue.In another embodiment, administration may be by direct injection at the site (or former site) of autoimmune disease manifestation.

[0181] In yet another embodiment, the compound of the present invention and / or its antibody drug conjugate can be delivered in a controlled release system, such as, but not limited to, a pump, or various polymeric materials can be used.In yet another embodiment, the controlled release system can be placed near the target of the compound of the present invention and / or its antibody drug conjugate, for example, the liver, thereby requiring only a fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) can be used.

[0182] The term "carrier" refers to a vehicle, adjuvant, or additive with which the compound or its antibody drug conjugate is administered. Such pharmaceutical carriers may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. The carrier may be saline solution, etc. In addition, auxiliary agents, stabilizers, and other agents may be used. In one embodiment, the compound or conjugate and the pharma- ceutically acceptable carrier are sterile when administered to a patient. When the compound or conjugate is administered intravenously, water is an exemplary carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

[0183] The composition can take the form of a solution, pellet, powder, sustained release formulation, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences", EW Martin.

[0184] In one embodiment, the compound of the present invention and / or its antibody drug conjugate is formulated by conventional procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly humans. Typically, the carrier or vehicle for intravenous administration is a sterile, isotonic, aqueous buffer solution. If necessary, the composition can also include a solubilizing agent. The composition for intravenous administration can optionally include a local anesthetic, such as lignocaine, to relieve pain at the site of injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, such as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the compound of the present invention and / or its antibody drug conjugate is administered by injection, it can be dispensed, for example, in an infusion bottle containing sterile pharmaceutical grade water or saline. When the compound of the present invention and / or its antibody drug conjugate is administered by injection, an ampoule of sterile water for injection, or saline can be provided so that the ingredients can be mixed prior to administration.

[0185] The composition can contain various materials that modify the physical form of solid or liquid dosage unit.For example, the composition can contain materials that form a coating shell around active ingredient.The materials that form the coating shell are typically inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredient can be placed in gelatin capsules.

[0186] Whether in solid or liquid form, the compositions can include pharmacological agents used in the treatment of cancer.

[0187] Therapeutic uses of compounds and antibody drug conjugates thereof - Patents.com Another aspect of the present invention relates to methods of using the compounds of the present invention and antibody drug conjugates thereof for treating cancer.

[0188] The compounds of the present invention and / or their antibody drug conjugates are useful for inhibiting the proliferation of tumor or cancer cells, inducing apoptosis in tumor or cancer cells, or treating cancer in patients. The compounds of the present invention and / or their antibody drug conjugates can therefore be used in a variety of settings for the treatment of cancer in animals. The conjugates can be used to deliver the compounds of the present invention to tumor or cancer cells. Without being bound by theory, in one embodiment, the antibody of the conjugate binds to or associates with an antigen associated with a cancer or tumor cell, and the conjugate can be taken up (internalized) into the tumor or cancer cell by receptor-mediated endocytosis or other internalization mechanisms. The antigen can be attached to the tumor or cancer cell, or can be an extracellular matrix protein associated with the tumor or cancer cell. In certain embodiments, once inside the cell, one or more specific peptide sequences are enzymatically or hydrolytically cleaved by one or more tumor or cancer cell-associated proteases, resulting in the release of the compounds of the present invention from the conjugate. The released compounds of the present invention are then free to move within the cell and induce cytotoxic or cytostatic activity. The conjugate can also be cleaved by intracellular proteases to release the compound of the invention. In an alternative embodiment, the compound of the invention is cleaved from the conjugate on the outside of the tumor or cancer cell, and the compound of the invention subsequently enters the cell.

[0189] In certain embodiments, the conjugates provide conjugation-specific tumor or cancer drug targeting, thus reducing the general toxicity of the compounds of the invention.

[0190] In another embodiment, the antibody unit binds to a tumor cell or a cancer cell.

[0191] In another embodiment, the antibody unit binds to a tumor cell or cancer cell antigen that is on the surface of the tumor cell or cancer cell.

[0192] In another embodiment, the antibody unit binds to a tumor cell or cancer cell antigen that is an extracellular matrix protein associated with tumor cells or cancer cells.

[0193] The specificity of the antibody unit for a particular tumor or cancer cell can be important for determining which tumor or cancer is most effectively treated.

[0194] Particular types of cancers that can be treated with the compounds of the invention and / or their antibody drug conjugates include, but are not limited to, carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, skin, stomach, and testis; and blood-borne cancers, including, but not limited to, leukemia and lymphoma.

[0195] Multimodal Therapy for Cancer: Cancer, including but not limited to tumors, metastases, or other diseases or disorders characterized by uncontrolled cell proliferation, can be treated or inhibited by administration of the compounds of the invention and / or antibody drug conjugates thereof.

[0196] In another embodiment, a method of treating cancer is provided, comprising administering to a patient in need thereof an effective amount of a compound of the present invention and / or its antibody drug conjugate, and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is one with which the treatment of cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is one with which the treatment of cancer has been found to be refractory. The compound of the present invention and / or its antibody drug conjugate can be administered to a patient who has also undergone surgery as a treatment for cancer.

[0197] In some embodiments, the patient also undergoes additional treatment, such as radiation therapy.In certain embodiments, the compound of the present invention and / or its antibody drug conjugate is administered simultaneously with chemotherapy or radiation therapy.In another specific embodiment, the chemotherapy or radiation therapy is administered before or subsequent to the administration of the compound of the present invention and / or its antibody drug conjugate.

[0198] The chemotherapy agents may be administered over a series of sessions. Any one or a combination of chemotherapy agents, such as standard chemotherapy agent(s), may be administered.

[0199] Furthermore, the method of treating cancer with the compounds of the present invention and / or their antibody drug conjugates is provided as an alternative to chemotherapy or radiation therapy, which have proven or can prove too toxic, e.g., resulting in unacceptable or intolerable side effects for the treated subject. The treated patient can optionally be treated with another cancer treatment, e.g., surgery, radiation therapy, or chemotherapy, depending on which treatment is found to be acceptable or tolerable.

[0200] The compound of the present invention and / or its antibody drug conjugate can also be used in in vitro or ex vivo mode for the treatment of certain cancers, including, but not limited to, leukemia and lymphoma, and such treatment involves autologous stem cell transplantation.This can include a multi-step process of collecting the animal's autologous hematopoietic stem cells, eradicating all cancer cells from them, then eradicating the animal's remaining bone marrow cell population by administering high doses of the compound of the present invention and / or its antibody drug conjugate, with or without concomitant high doses of radiation therapy, and reinjecting the stem cell transplant into the animal.Supportive care is then provided while bone marrow function is restored, and the patient is allowed to recover.

[0201] The invention is further described in the following examples, which are not intended to limit the scope of the invention. EXAMPLES

[0202] Experimental procedure Experiments were generally performed under an inert atmosphere (nitrogen or argon), especially when oxygen- or moisture-sensitive reagents or intermediates were used. Commercially available solvents and reagents, including anhydrous solvents, were generally used without further purification where appropriate (generally Sure-Seal™ products from Aldrich Chemical Company, Milwaukee, Wisconsin). Products were generally dried under vacuum before undergoing further reactions or animal testing. Mass spectrometry data are reported from liquid chromatography mass spectrometry (LCMS), atmospheric pressure chemical ionization (APCI) or gas chromatography mass spectrometry (GCMS) instrumentation. Chemical shifts for nuclear magnetic resonance (NMR) data are expressed in parts per million (ppm, δ) referenced to residual peaks from the deuterated solvent used.

[0203] For syntheses that refer to procedures in other examples or methods, reaction protocols (reaction length and temperature) may vary. In general, reactions were followed by thin layer chromatography, LCMS or HPLC, and were subjected to workup, if appropriate. Purification may vary from experiment to experiment. In general, the solvents and solvent ratios used for eluents / gradients were selected to achieve appropriate retention times.

[0204] HRMS was performed on an Agilent 6220TOF LC / MS.

[0205] HPLC, LCMS, and SEC conditions used for analysis Protocol A: Column: Waters Acquity UPLC BEH, 2.1 mm x 50 mm, C18, 1.7 μm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 0.1 min, 5% to 95% B over 0.7 min, 95% B over 0.1 min; Flow rate: 1.25 mL / min, Temperature: 60 °C; Detection: 200-450 nm; MS(+) range 100-1200 Daltons; Injection volume: 5 μL; Instrument: Waters Acquity.

[0206] Protocol B: Column: Phenomenex Luna C18(2), 150 x 3.0 mm, 5 μm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (v / v); Gradient: 0% to 100% B over 8.5 min; Flow rate: 1.5 mL / min, Temperature: not controlled; Detection: DAD, 210 nm; Injection volume: 10 μL; Instrument: Agilent 1100 HPLC

[0207] Protocol C: Column: Phenomenex Luna C18(2), 150 x 3.0 mm, 5 μm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 1.5 min, 5% to 100% B over 8.5 min, then 100% B for 1 min; Flow rate: 0.75 mL / min, Temperature: 25 °C; Detection: DAD, 215 nm, 254 nm; MS(+) range 150-2000 Daltons; Injection volume: 10 μL; Instrument: Agilent 1200 LCMS.

[0208] Protocol D: Column: Waters Acquity UPLC BEH, 2.1 mm x 50 mm, C18, 1.7 μm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 0.1 min, 5% to 95% B over 0.7 min, 95% B over 0.1 min; Flow rate: 1.25 mL / min, Temperature: 60 °C; Detection: 200-450 nm; MS(+) range 100-1200 Daltons; Injection volume: 5 μL; Instrument: Waters Acquity.

[0209] Protocol E: Column: Waters Acquity HSS T3, C18, 2.1 × 50 mm, 1.7 μm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 0.1 min, 5% to 95% B over 2.5 min, 95% B, 0.35 min; Flow rate: 1.25 mL / min, Temperature: 60 °C; Detection: 200-450 nm; MS(+) range 100-2000 m / z; Injection volume: 5 μL; Instrument: Waters Acquity UPLC.

[0210] Protocol F: Column: YMC-pack ODS-A, 150 mm x 4.6 mm, 5 μm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 0% to 95% B over 10 min, 95% B over 5 min; Flow rate: 1.0 mL / min. Detection: 220 nm.

[0211] Protocol G: Column: YMC-pack ODS-A, 150 mm x 4.6 mm, 5 μm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 65% to 100% B over 10 min, 100% B over 5 min; Flow rate: 1.0 mL / min. Detection: 220 nm.

[0212] Protocol H: Column: Phenomenex Luna C18(2), 150 × 3.0 mm, 5 μm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (v / v); Gradient: 0% to 100% B over 20 min; Flow rate: 1.5 mL / min, Temperature: not controlled, Detection: DAD 210 nm; Injection volume: 10 μL; Instrument: Agilent 1100 HPLC.

[0213] Protocol I: Column: Waters Acquity HSS T3, C18, 2.1 × 50 mm, 1.7 μm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 0.1 min, 5% to 95% B over 5 min, 95% B, 0.35 min; Flow rate: 1.25 mL / min, Temperature: 60 °C; Detection: 200-450 nm; MS (+) range 100-2000 m / z; Injection volume: 5 μL; Instrument: Waters Acquity UPLC.

[0214] Protocol J: Column: Ultimate XB-C18, 50 mm x 3.0 mm, 3 μm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 1% to 5% B over 1 min, 5% to 100% B over 5 min, 100% B over 2 min, then 1% B over 2 min; Flow rate: 1.2 mL / min. Detection: 220 nm.

[0215] Protocol K: Column: Ultimate XB-C18, 30 mm x 5.0 mm, 3 μm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 1% to 5% B over 1 min, 5% to 100% B over 5 min, 100% B over 2 min, then 1% B over 2 min; Flow rate: 1.2 mL / min. Detection: 220 nm.

[0216] Protocol L: Column: Agilent Poroshell300SB-C8, 75×2.1 mm, 2.6 μm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: Initial conditions: 20% B to 45% B over 4 min; Flow rate: 1.0 mL / min, Temperature: 60 °C; Detection: 220 nm; MS(+) range 400-2000 Da; Injection volume: 10 μL; Instrument: Agilent1100LC, Waters MicromassZQ MS. Deconvolution was performed using MaxEnt1.

[0217] Protocol M: Column: TSK-gel G3000SWxl, 300 × 7.8 mm, 10 μm; Mobile phase: phosphate buffer solution with 2% acetonitrile (PBS, 1×, pH 7.4); Isocratic; Flow rate: 1 mL / min, Temperature: Room temperature; Injection volume: 5 μL; Instrument: Agilent 1100 HPLC.

[0218] Protocol N: Column: GE Superdex200 (5 / 150GL); Mobile phase: phosphate buffer solution with 2% acetonitrile (PBS, 1x, pH 7.4); Isocratic; Flow rate: 0.25 mL / min, Temperature: Room temperature; Injection volume: 10 μL; Instrument: Agilent 1100 HPLC.

[0219] Protocol O: Column: Waters Acquity HSS T3, C18, 2.1 × 50 mm, 1.7 μm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B for 0.1 min, 5% to 95% B for 0.1–1.0 min, 95% B for 1.0–1.1 min; Flow rate: 1.25 mL / min, Temperature: 60 °C; Detection: 200–450 nm; MS(+) range 100–2000 m / z; Injection volume: 5 μL; Instrument: Waters Acquity UPLC and Waters Acquity SQ systems.

[0220] Protocol P: Column: Waters Acquity HSS T3, C18, 2.1 × 50 mm, 1.7 μm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 0.1 min, 5% to 95% B over 0.1–2.6 min, 95% B for 2.6–2.95 min; Flow rate: 1.25 mL / min, Temperature: 60 °C; Detection: 200–450 nm; MS(+) range 100–2000 m / z; Injection volume: 5 μL; Instrument: Waters Acquity UPLC and Waters Acquity SQ systems.

[0221] Protocol Q: Column: Waters Xbridge, C18, 2 x 50 mm, 5 μm; Mobile phase A: 10 mM NH4HCO3 in water; Mobile phase B: acetonitrile; Gradient: 1-5% B over 0.6 min, 5-100% B over 0.6-3.4 min; Flow rate: 0.8 mL / min, Temperature: room temperature; Injection volume: 10 μL; Instrument: Agilent 1200 HPLC.

[0222] HPLC conditions used for purification: Method A: Column: Phenomenex Luna C18, 100 x 30 mm, 10 μm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in methanol (v / v); Gradient: 10% to 90% B over 20 min; Flow rate: 20 mL / min, Temperature: not controlled, Detection: DAD 210 nm, 254 nm; Injection volume: variable; Instrument: Gilson.

[0223] Method B: Column: Phenomenex Luna C18(2), 150×21.2 mm, 5 μm; Mobile phase A: 0.02% trifluoroacetic acid in water; Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile; Gradient: 1% B over 1.5 min, 1% to 50% B over 8.5 min; Flow rate: 27 mL / min; Detection: DAD 210-360 nm; MS(+) range 150-2000 Daltons; Instrument: Waters FractionLynx.

[0224] Method C: Column: Phenomenex Luna C18, 100 x 30 mm, 10 μm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in methanol (v / v); Gradient: 20% to 80% B over 20 min; Flow rate: 20 mL / min, Temperature: not controlled, Detection: DAD 210 nm, 254 nm; Injection volume: variable; Instrument: Gilson.

[0225] Method D: Column: Phenomenex Synergi Max-RP C12, 250 x 50 mm, 10 μm; Mobile phase A: 0.2% formic acid in water (v / v); Mobile phase B: acetonitrile (v / v); Gradient: 15% to 45% B over 30 min, 95% B over 5 min; Flow rate: 80 mL / min, Temperature: not controlled, Detection: UV 210 nm, 254 nm; MS(+) range: 100-1000 Daltons; Injection volume: variable; Instrument: Shimadzu LC-20AP.

[0226] In some instances, some minor modifications to the elution conditions for the LCMS and HPLC preparative purification conditions were made, such as, but not limited to, changes in gradients, flow rates, or modifier composition. When applicable, such modifications are indicated in the specific procedures listed herein.

[0227] General steps: General Procedure A: Fmoc Removal Using Diethylamine To a solution of the Fmoc-containing compound in dichloromethane, diethylamine was added to give a ratio of diethylamine:dichloromethane of 1:1 to 1:10. The reaction progress was monitored by LCMS (or HPLC or TLC). The reaction was usually complete within 3 hours. The solvent was removed in vacuo or, in some cases, concentrated under a stream of nitrogen, and the residue was purified by reverse phase chromatography.

[0228] General Procedure B: Amide coupling with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU). To a stirred solution of amine (1.0 equiv.) and acid (1.0-2.0 equiv.) in N,N-dimethylformamide (DMF) or N,N-dimethylacetamide (DMA) was added HATU (1.0-2.0 equiv.) followed by N,N-diisopropylethylamine (2.0-4.0 equiv.). Reaction progress was monitored by LCMS (or HPLC or TLC). Reactions were usually complete within 3 h. The solvent was removed in vacuo. The residue was purified by silica gel or reverse phase chromatography. Note: All LCMS data is based on Protocol A (1.5 min method) unless otherwise noted.

[0229] Preparation of 5-(methylsulfonyl)pyrazine-2-carboxylic acid (#1)

[0230] [ka] A mixture of 5-methylsulfanyl-pyrazine-2-carboxylic acid (0.73 g, 4.3 mmol, 1 equiv.) and oxone (15.4 g, 22 mmol, ca. 6 equiv.) in acetone (30 mL) was vigorously stirred at 45° C. for approximately 20 h. The cooled (room temperature) reaction mixture was filtered, the filter cake was washed with acetone (2×20 mL) and acetonitrile (10 mL), and the filtrate was concentrated under reduced pressure. The crude material was purified by reverse phase chromatography on a 100 g C18 column using a gradient elution of 0-30% acetonitrile in water containing 0.1% formic acid over 20 min to give 5-(methylsulfonyl)pyrazine-2-carboxylic acid, #1 (0.25 g, 29%) as a white solid. LCMS (Protocol E): m / z 202.9 [M+H] + , retention time = 0.23 min; 1 H NMR (400 MHz, DMSO): δ 9.35-9.32 (m, 2H), 3.40 (s, 3H).

[0231] Intermediate #2 Preparation of 2-(methylsulfonyl)benzo[d]thiazole-6-carboxylic acid (#2)

[0232] [ka] 2-(Methylsulfonyl)benzo[d]thiazole-6-carboxylic acid (30 mg, 8%) was synthesized from 2-(methylsulfanyl)-1,3-benzothiazole-6-carboxylic acid (compound 12 in WO2013056070A2) (0.33 g, 1.44 mmol), oxone (7.0 g, 11 mmol), and acetone (10 mL) by following a procedure similar to that of 5-(methylsulfonyl)pyrazine-2-carboxylic acid, #1, above. LCMS (Protocol E): m / z 257.9 [M+H] + , retention time = 0.85 min; 1 H NMR (400 MHz, DMSO): δ 13.45 (br s, 1H), 9.02-9.00 (m, 1H), 8.37-8.22 (m, 1H), 8.24-8.21 (m, 1H), 3.62 (s, 3H).

[0233] Preparation of N,2-dimethyl-N-[6-({[5-(methylsulfonyl)pyrazin-2-yl]carbonyl}amino)hexanoyl]alanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#6)

[0234] [ka] Step 1. Synthesis of N-(6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-N-methylhexanamido)-2-methylpropanoic acid (#3) To a solution of 6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}hexanoic acid (1 g, 3 mmol) in N,N-dimethylformamide (5 mL, 0.15 M) was added HATU (1.108 g, 2.8 mmol) followed by N,N-diisopropylethylamine (1 mL, 5 mmol). This reaction mixture was added dropwise to a solution of 2-methyl-2-(methylamino)propanoic acid (332 mg, 2.83 mmol) and N,N-diisopropylethylamine (1 mL, 5 mmol) in N,N-dimethylformamide (5 mL). The resulting brown solution was stirred at room temperature. After 90 min, LCMS analysis indicated the reaction was complete. The reaction mixture was diluted with dichloromethane and washed with a 1N aqueous solution of hydrochloric acid (x3), the organic layer was dried over anhydrous sodium sulfate, concentrated to dryness, and the resulting residue was purified by silica gel chromatography (50% to 100% ethyl acetate in heptane with 0.1% acetic acid). The pooled product-containing fractions were evaporated to give the title compound #3 (415 mg, 30%) as a pinkish gum. LCMS: m / z 453.20 [M+H] + , retention time = 0.89 minutes; 1 H NMR (500 MHz, chloroform-d) δ ppm 7.76 (d, J=7.6 Hz, 2 H) 7.60 (d, J=7.3 Hz, 2 H) 7.39 (t, J=7.5 Hz, 2 H) 7.28 - 7.34 (m, 2 H) 4.33 - 4.45 (m, 2 H) 4.17 - 4.28 (m, 1 H) 3.12 - 3.22 (m, 2 H) 2.97 (s, 3 H) 2.30 - 2.39 (m, 2 H) 1.58 - 1.68 (m, 2 H) 1.47 - 1.56 (m, 2 H) 1.43 (s, 6 H) 1.31 - 1.39 (m, 2 H).

[0235] Step 2. Synthesis of N-(6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}hexanoyl)-N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#4) To a solution of #3 (194 mg, 0.429 mmol) in tetrahydrofuran (2 mL) in an ice-cooled water bath, N-methylmorpholine (140 μL) was added, followed by dropwise addition of isopropenyl chloroformate (47.8 μL, 1.3 mmol). After 1 h, the reaction mixture was added dropwise to a solution of N-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalanine (Example #114 in US2013 / 0129753A1) (211 mg, 0.311 mmol) in isopropanol (1 mL) and tetrahydrofuran (1 mL). After 2 hours, LCMS analysis indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure and the residue was then purified by reverse phase chromatography using a modified version of purification method A, utilizing a gradient elution of 20-100% acetonitrile in water containing 0.02% acetic acid over 20 min. The pooled product-containing fractions were lyophilized to afford the title compound #4 (82 mg, 25%) as a white solid. LCMS: m / z 1053.6 [M+H] + , retention time = 2.10 min; HPLC (protocol B): retention time = 8.034 min.

[0236] Step 3. Synthesis of N-(6-aminohexanoyl)-N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, trifluoroacetate salt (#5) According to general procedure A above, #5 (59 mg) was synthesized as an amorphous glass from #4 (81 mg, 0.077 mmol), dichloromethane (1 mL, 0.08 M) and diethylamine (0.3 mL, 3 mmol) after purification by reverse phase chromatography using a modified version of purification method A (utilizing gradient elution of 20-70% acetonitrile in water containing 0.02% acetic acid over 20 min). LCMS: m / z 831.5 [M+H] + , retention time = 0.69 min; HPLC (protocol B): retention time = 5.240 min.

[0237] Step 4. Synthesis of N,2-dimethyl-N-[6-({[5-(methylsulfonyl)pyrazin-2-yl]carbonyl}amino)hexanoyl]alanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#6) To a solution of 5-(methylsulfonyl)pyrazine-2-carboxylic acid, #1 (5.5 mg, 0.027 mmol), and N,N-diisopropylethylamine (3 mL, 0.017 mmol) in N,N-dimethylformamide (0.1 mL) was added HATU (7.9 mg, 0.020 mmol). After approximately 15 min, a solution of #5 (13.5 mg, 0.016 mmol) and N,N-diisopropylethylamine (6 mL, 0.033 mmol) in N,N-dimethylformamide (0.2 mL) was added to the reaction mixture. After 45 min, LC-MS analysis indicated the reaction was complete, and the mixture was directly purified by reverse-phase chromatography by a modified version of purification method A (utilizing a gradient elution of 20-95% acetonitrile in water containing 0.02% acetic acid over 20 min) to give the title compound A (1.9 mg, 12%). LC-MS: m / z 1013.5 [MH + ], 1039.5[M+Na + ] retention time = 1.61 min; HPLC (protocol H): retention time = 10.090 min.

[0238] Preparation of N,2-dimethyl-N-[6-({[2-(methylsulfonyl)-1,3-benzothiazol-6-yl]carbonyl}amino)hexanoyl]alanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#7)

[0239] [ka] 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-NMM) (11.6 mg, 0.042 mmol) was added followed by N-methylmorpholine (10 μL) to a mixture of 2-(methylsulfonyl)-1,3-benzothiazole-6-carboxylic acid, #2 (11.0 mg, 0.043 mmol) in tetrahydrofuran (0.5 mL) at room temperature. After 1 h, this mixture was added to a solution of #5 (21 mg, 0.022 mmol) in N,N-dimethylacetamide (1 mL). After an additional 1 h, LCMS showed the reaction was complete. The reaction mixture was diluted with acetonitrile and purified by reverse phase chromatography using a modified version of purification method A (utilizing a gradient elution of 20-80% acetonitrile in water containing 0.02% acetic acid over 20 min) to give the title compound #7 (3.3 mg, 3%). LCMS: m / z 1070.4 [M+H] + , retention time = 0.89 min; HPLC (protocol B): retention time = 5.722 min;

[0240] Preparation of 2-methyl-1-{23-[2-(methylsulfonyl)pyrimidin-5-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#10)

[0241] [ka] Step 1. Synthesis of 1-[1-(9H-fluoren-9-yl)-3,25-dioxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-yl]-2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#8) According to general procedure B above, after silica gel chromatography (gradient: 10-100% acetone in heptane), 2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (US200301124) was obtained. #8 (238 mg, 59%) was synthesized as a white foam from Example #239 in 13 / 0129753A1 (237 mg, 0.302 mmol), 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosane-25-oic acid (201 mg, 0.332 mmol), N,N-diisopropylethylamine (210 μL, 1.21 mmol), N,N-dimethylformamide (3 mL) and HATU (133 mg, 0.332 mmol). LCMS: m / z 1344.1 [M+H] + , retention time = 2.33 min; HPLC (Protocol F): retention time = 10.301 min; 1 H NMR (400 MHz, CDCl3): δ 7.76 (d, 2H), 7.59 (d, 2H), 7.39 (m, 2H), 7.33 (m, 2H), 7.26-6.83 (m, 5H), 5.44 (br, 1H), 4.72-4.62 (m, 2H), 4.41 (d, 2H), 4.16 (m, 2H), 3.76-3.31 (m, 39H), 3.08-2.99 (m, 5H), 2.60-2.25 (m, 6H), 1.93-1.46 (m, 13H), 1.46 (m, 1H), 1.38 (s, 9H), 1.19 (m, 4H), 0.99-0.84 (m, 13H).

[0242] Step 2. Synthesis of 1-(21-amino-4,7,10,13,16,19-hexaoxaheneicosan-1-oyl)-2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, trifluoroacetate (#9) According to general procedure A above, #9 (400 mg, 54%) was synthesized as an amorphous solid from #8 (800 mg, 0.595 mmol), dichloromethane (20 mL) and diethylamine (2 mL, 20 mmol) after purification by reverse phase chromatography using a modified version of purification method A (utilizing gradient elution of 20-75% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min). LCMS: m / z 1122.1 [M+H] + , retention time = 0.81 min; HPLC (protocol B): retention time = 6.282 min.

[0243] Step 3. Synthesis of 2-methyl-1-{23-[2-(methylsulfonyl)pyrimidin-5-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#10) To a suspension of white 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (25.0 mg, 0.124 mmol) in tetrahydrofuran (0.5 mL) at room temperature was added 2-chloro-4,6-dimethoxy-1,3,5-triazine (22 mg, 0.12 mmol) followed by N-methylmorpholine (18 μL, 0.165 mmol). The suspension became thick and slightly yellow. N,N-dimethylacetamide (0.2 mL) was added and the reaction mixture was stirred at room temperature. After 2 hours, LCMS showed complete activation. A colorless solution of #9 (44 mg, 0.033 mmol) in tetrahydrofuran (0.5 mL) was added to the previous reaction mixture. After 40 minutes, LCMS analysis showed the reaction was complete. The reaction mixture was concentrated under a strong stream of nitrogen, diluted with dichloromethane (0.5 mL) and trifluoroacetic acid was added (1.5 mL). After 2 hours, LCMS analysis indicated that the Boc-deprotection reaction was complete. The mixture was diluted with DMSO and purified by reverse-phase chromatography using a modified version of purification method A (utilizing a gradient elution of 20-80% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #10 (18.2 mg, 44%) as a solid. LCMS (Protocol C): m / z 1250.6 [M+H] + , retention time = 8.252 minutes.

[0244] Preparation of 2-methyl-1-{23-[5-(methylsulfonyl)pyrazin-2-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#11)

[0245] [ka] To a suspension of white 5-(methylsulfonyl)pyrazine-2-carboxylic acid, #1 (24.1 mg, 0.119 mmol) in tetrahydrofuran (0.5 mL) at room temperature was added 2-chloro-4,6-dimethoxy-1,3,5-triazine (21 mg, 0.12 mmol) followed by N-methylmorpholine (24.8 μL, 0.225 mmol). The suspension became thick and slightly yellow. N,N-dimethylacetamide (0.2 mL) was added to aid solubility. The reaction mixture was stirred at room temperature. After 2 hours, LCMS indicated complete activation. A solution of colorless compound #9 (66 mg, 0.045 mmol) in tetrahydrofuran (0.5 mL) was added to the previous reaction mixture. After 40 minutes, LCMS analysis indicated the reaction was complete. The reaction mixture was concentrated under a strong stream of nitrogen, diluted with dichloromethane (0.5 mL), and trifluoroacetic acid was added (1.5 mL). After 1 h, LCMS analysis indicated that the Boc-deprotection reaction was complete. The mixture was diluted with DMSO and purified by reverse-phase chromatography using a modified version of purification method A (utilizing a gradient elution of 20-80% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #11 (12.9 mg, 23%) as a solid. LCMS: m / z 1249.5 [M+H] + , retention time = 0.92 min; HPLCMS (protocol C): m / z 1250.6 [M+H] + , retention time = 8.384 minutes.

[0246] Preparation of 2-methyl-1-{23-[2-(methylsulfonyl)-1,3-benzothiazol-6-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#14)

[0247] [ka] Step 1. Synthesis of 1-[1-(9H-fluoren-9-yl)-3,25-dioxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-yl]-2-methyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#12) According to general procedure B, after silica gel chromatography (gradient: 10-100% acetone in heptane), 2-methyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (US2013 / 0 #12 (164 mg, 86%) was synthesized as a white foam from Example #117 in 129753A1 (115 mg, 0.146 mmol), 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosane-25-oic acid (97.6 mg, 0.161 mmol), N,N-diisopropylethylamine (102 μL, 0.584 mmol), N,N-dimethylformamide and HATU (64.4 mg, 0.161 mmol). LCMS: m / z 1302.1 [M+H] + , retention time = 2.17 min; HPLC (Protocol G): retention time = 4.158 min; 1 H NMR (400 MHz, CDCl3): δ 7.77 (d, 2H), 7.61 (d, 2H), 7.39 (m, 2H), 7.33 (m, 2H), 7.22-6.95 (m, 7H), 5.44 (br, 1H), 4.86-4.70 (m, 3H), 4.42 (d, 2H), 4.24 (m, 3H), 3.87-3.55 (m, 29H), 3.38-3.32 (m, 9H), 3.10 (m, 4H), 2.61 (m, 2H), 2.41-2.39 (m, 3H), 1.91-1.62 (m, 13H), 1.30 (br 1H), 1.20 (d, 3H), 1.00-0.84 (m, 12H).

[0248] Step 2. Synthesis of 1-(21-amino-4,7,10,13,16,19-hexaoxaheneicosan-1-oyl)-2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, trifluoroacetate (#13) To a solution of #12 (88 mg, 0.068 mmol, 1 equiv) in N,N-dimethylformamide (1 mL) in an ice-cooled water bath was added a solution of lithium hydroxide (36 mg, 1.5 mmol) in water (0.4 mL). After 20 min, LCMS analysis indicated the reaction was complete and it was concentrated under a strong stream of nitrogen and the residue was purified by reverse phase chromatography by method B to give compound #13 (189 mg, 71%) as a white solid. LCMS (Protocol C): m / z 1065.7 [M+H] + , retention time = 6.959 minutes.

[0249] Step 3: 2-Methyl-1-{23-[2-(methylsulfonyl)-1,3-benzothiazol-6-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#14) To a suspension of white 2-(methylsulfonyl)-1,3-benzothiazole-6-carboxylic acid, #2 (5 mg, 0.02 mmol) in tetrahydrofuran (0.1 mL) was added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (5 mg, 0.018 mmol) followed by one drop of N-methylmorpholine at room temperature. The reaction mixture was stirred at room temperature. After 2 hours, LCMS showed complete activation. A colorless solution of #13 (10 mg, 0.009 mmol) in N,N-dimethylacetamide (0.1 mL) was added to the previous reaction mixture. After stirring overnight at room temperature, LCMS analysis indicated the reaction was complete and was then diluted with DMSO and purified by reverse phase chromatography via Method A (modifier: trifluoroacetic acid; gradient: 20-90% acetonitrile in water) to give the title compound #14 (0.9 mg, 8%) as a colorless glass. LCMS: m / z 1304.4 [M+H] + , retention time = 1.70 min; HPLC (protocol B): retention time = 6.412 min.

[0250] N-[6-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)hexanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Preparation of -carbamoyl-L-ornithineamide (#19)

[0251] [ka] Step 1: Synthesis of Fmoc-AmCapValCitPABC-OH (#15): A solution of Fmoc-6-aminohexanoic acid (4.2 g, 11.40 mmol) in anhydrous N,N-dimethylformamide (50 mL) was treated with N,N-diisopropylethylamine (3.8 mL, 2.82 g, 21.8 mmol, 1.8 equiv.) and HATU (5.6 g, 14.75 mmol, 1.25 equiv.) and the mixture was stirred at room temperature for 10 min. 5 -Carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithineamide 2 [ValCitPABA] (Dubowchik, GM et al., Bioconjug Chem, 13, 855–69 (2002) 5 Compound 64) (5.6 g, 14.75 mmol, 1.25 equiv.) in the above formula was then added to the mixture and stirring was continued at room temperature for 15 h. Dichloromethane was added to precipitate the product, which was filtered and air-dried to give 5.9 g (100%) of the desired material as an off-white solid. This material was used directly in the subsequent reaction without further purification. MS (ES) m / z: 715.6 [M+H] + .

[0252] Step 2: Synthesis of FmocAmCapValCitPABC-PNP (#16): A solution of FmocAmCapValCitPABA-OH, #15 (500 mg, 0.7 mmol), and bis-(4-nitrophenyl)carbonate (638 mg, 2.1 mmol, 3 equiv.) in anhydrous N,N-dimethylformamide (3 mL) was treated with N,N-diisopropylethylamine (365 μL, 270 mg, 2.1 mmol, 3 equiv.) and the reaction was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the resulting residue was purified by silica gel chromatography using a gradient elution of 0% to 25% methanol in dichloromethane over 15 min, followed by an isocratic elution of 25% methanol in dichloromethane. Pooled product-containing fractions were evaporated to give 402 mg (68%) of the desired product as an off-white solid. MS(ES)m / z:880.3[M+H] + .

[0253] Step 3: N-(6-aminohexanoyl)-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#18) 2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (US2013 / 012 A solution of 9753A1 Example #54) (105 mg, 0.141 mmol), FmocAmCapValCitPABC-PNP, #16 (138 mg, 0.157 mmol), HOAt (6.40 mg, 0.047 mmol), and 2,6-lutidine (91 uL, 84 mg, 0.783 mmol) in N,N-dimethylformamide (1.4 mL) was heated for 21 hours at 45° C. Additional FmocAmCapValCitPABC-PNP, #16 (31 mg, 0.035 mmol), HOAt (3.83 mg, 0.028 mmol), and 2,6-lutidine (16 uL, 15 mg, 0.141 mmol) was then added and the reaction was heated at 40° C. for an additional 6 hours and then held at room temperature overnight. LCMS analysis indicated the reaction contained a mixture of compounds #17 and #18. Piperidine (0.5 mL) was added and the reaction was stirred at room temperature for 1 h. LCMS analysis indicated the Fmoc-deprotection reaction was complete, so the reaction was concentrated and the residue was purified by reverse phase chromatography by a modified version of purification method A (utilizing a gradient elution of 10-85% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give 97 mg (54%) of the desired product (#18) as a white solid. LCMS (Protocol I): m / z 1062.3 [M+H] + , retention time = 2.36 minutes.

[0254] Step 4: N-[6-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)hexanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#19) N-(6-aminohexanoyl)-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 To a solution of 2-carbamoyl-L-ornithinamide, #18 (100 mg, 0.061 mmol) in tetrahydrofuran (1 mL) was added 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (41 mg, 0.2 mmol), followed by 2-chloro-4,6-dimethoxy-1,3,5-triazine (35 mg, 0.20 mmol) and N-methylmorpholine (50 μL, 0.46 mmol). The reaction mixture was stirred at room temperature for 2 days, then diluted with DMSO and purified by reverse phase chromatography by a modified version of purification method A (utilizing a gradient elution of 20-90% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #19 (52 mg, 59%) as a white solid. LCMS (Protocol C): m / z 1445.6 [M+H] + , 723.5[M+2H] / 2 +, retention time = 8.721 min.

[0255] N-[6-({[5-(methylsulfonyl)pyrazin-2-yl]carbonyl}amino)hexanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 -Carbamoyl-L-ornithineamide (#26)

[0256] [ka] Step 1. Synthesis of methyl 6-{[(5-chloropyrazin-2-yl)carbonyl]amino}hexanoate (#20) To a solution of 5-chloropyrazine-2-carboxylic acid (500 mg, 3.1 mmol) and methyl-6-aminohexanoate hydrochloride (688 mg, 3.71 mmol) in dichloromethane (15 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (711 mg, 3.71 mmol, 1.2 equiv). The reaction was stirred at room temperature for 18 hours, then diluted with dichloromethane (75 mL) and washed with 1N hydrochloric acid (15 mL). The layers were separated, the organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (gradient elution with 20% to 70% ethyl acetate in heptane) to give methyl 6-{[(5-chloropyrazine-2-yl)carbonyl]-amino}hexanoate, #20 (378 mg, 43%) as a white solid. LCMS (Protocol E): m / z 308.2 [M+Na+], retention time = 1.25 min; 1 H NMR (500 MHz, DMSO-d6): δ ppm 9.00 (d, J=1.22 Hz, 1 H), 8.96 (t, J=5.99 Hz, 1 H), 8.87 (d, J=1.47 Hz, 1 H), 3.58 (s, 3 H), 3.29 (q, J=6.85 Hz, 2 H), 2.30 (t, J=7.46 Hz, 2 H), 1.50 - 1.59 (m, 4 H), 1.25 - 1.33 (m, 2 H).

[0257] Step 2. Synthesis of methyl 6-({[5-(methylsulfanyl)pyrazin-2-yl]carbonyl}amino)hexanoate (#21) To a solution of methyl 6-{[(5-chloropyrazin-2-yl)carbonyl]amino}hexanoate, #20 (374 mg, 1.31 mmol) in dimethylsulfoxide (8 mL) was added sodium thiomethoxide (184 mg, 2.62 mmol). The reaction was stirred at room temperature for 18 h, then diluted with dichloromethane (50 mL) and washed with saturated ammonium chloride (10 mL). The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give methyl 6-({[5-(methylsulfanyl)pyrazin-2-yl]carbonyl}amino)hexanoate, #21 (262 mg, 67%) as a white solid. LCMS (Protocol E): m / z 298.2 [M+H] + , retention time = 1.35 minutes; 1 H NMR (500 MHz, DMSO-d6): δ 8.96 (d, J=1.47 Hz, 1 H), 8.77 (t, J=5.87 Hz, 1 H), 8.64 (d, J=1.71 Hz, 1 H), 3.58 (s, 3 H), 3.27 (q, J=6.77 Hz, 2 H), 2.62 (s, 3 H), 2.30 (t, J=7.34 Hz, 2 H), 1.49 - 1.59 (m, 4 H), 1.23 - 1.33 (m, 2 H).

[0258] Step 3. Synthesis of 6-({[5-(methylsulfanyl)pyrazin-2-yl]carbonyl}amino)hexanoic acid (#22) To a solution of methyl 6-({[5-(methylsulfanyl)pyrazin-2-yl]carbonyl}amino)hexanoate, #21 (50 mg, 0.17 mmol) in tetrahydrofuran (1 mL) was added a solution of lithium hydroxide (8 mg, 0.3 mmol) in water (0.3 mL). The reaction was stirred at room temperature for 3 h. The reaction was diluted with dichloromethane (25 mL) and 1N hydrochloric acid (6 mL) was added. The layers were separated and the organic layer was washed with brine (5 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give 6-({[5-(methylsulfanyl)pyrazin-2-yl]carbonyl}amino)hexanoic acid, #22 (43 mg, 90%) as a white solid. LCMS (Protocol E): m / z 284.2 [M+H] + , retention time = 1.07 min; 1 H NMR (500 MHz, DMSO-d6): δ 11.98 (br. s., 1 H), 8.97 (d, J=1.47 Hz, 1 H), 8.77 (t, J=5.87 Hz, 1 H), 8.64 (d, J=1.47 Hz, 1 H), 3.28 (q, J=6.85 Hz, 2 H), 2.62 (s, 3 H), 2.20 (t, J=7.34 Hz, 2 H), 1.48 - 1.57 (m, 4 H), 1.25 - 1.33 (m, 2 H).

[0259] Step 4. Synthesis of 6-({[5-(methylsulfonyl)pyrazin-2-yl]carbonyl}amino)hexanoic acid (#23) To a suspension of 6-({[5-(methylsulfanyl)pyrazin-2-yl]carbonyl}amino)hexanoic acid, #22 (40 mg, 0.14 mmol) in dichloromethane (2 mL) was added m-chloroperbenzoic acid (126 mg, 0.56 mmol). The reaction was stirred at room temperature for 18 h. The reaction was filtered and the solid collected to give 6-({[5-(methylsulfonyl)pyrazin-2-yl]carbonyl}amino)hexanoic acid, #23 (33 mg, 74%) as a white solid. LCMS (Protocol A): m / z 316.2 [M+H] + , retention time = 0.54 min; 1 H NMR (500 MHz, DMSO-d6): δ 11.97 (br. s., 1 H), 9.34 (d, J=1.47 Hz, 1 H), 9.25 (d, J=1.22 Hz, 1 H), 9.18 (t, J=5.99 Hz, 1 H), 3.40 (s, 3 H), 3.29 - 3.36 (m, 2 H), 2.21 (t, J=7.34 Hz, 2 H), 1.49 - 1.60 (m, 4 H), 1.31 (quintet, J=7.58 Hz, 2 H).

[0260] Step 5. N-(tert-butoxycarbonyl)-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#24) To a solution of 2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Example #54 in US2013 / 0129753A1) (50 mg, 0.067 mmol) in N,N-dimethylacetamide (1 mL) was added N-(tert-butoxycarbonyl)-L-valyl-N 5 -carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide (compound 10 in US8399403B2) (52 mg, 0.081 mmol), N,N-diisopropylethylamine (0.05 mL, 0.3 mmol), 2,6-lutidine (0.03 mL, 0.3 mmol), and 1-hydroxy-7-azabenzotriazole (11 mg, 0.081 mmol) were added. The reaction was stirred at 45° C. for 24 hours, then cooled to room temperature and stirred for 2 days. The crude reaction was filtered and the filtrate was purified by reverse phase HPLC by method C to give #24 (46 mg, 55%) as a white solid. LCMS (Protocol E): m / z 1248.8 [M+H] + , retention time = 1.83 min.

[0261] Step 6. L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -Carbamoyl-L-ornithineamide, trifluoroacetate (#25) Trifluoroacetic acid (0.1 mL, 1 mmol) was added to a solution of #24 (45 mg, 0.036 mmol) in dichloromethane (1 mL) and the reaction was stirred at room temperature for 20 minutes. The reaction was concentrated under reduced pressure to give L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 -Carbamoyl-L-ornithineamide, #25 (50 mg) was obtained as a white solid, which was used directly without further purification. LCMS (Protocol E): m / z 1149.2 [M+H] + , retention time = 1.50 min.

[0262] Step 7. N-[6-({[5-(methylsulfonyl)pyrazin-2-yl]carbonyl}amino)hexanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide, trifluoroacetate (#26) 6-({[5-(methylsulfonyl)pyrazin-2-yl]carbonyl}amino)hexanoic acid, #23 (5 mg, 0.02 mmol), and L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 To a solution of -carbamoyl-L-ornithineamide, #25 (10 mg, 0.007 mmol) in N,N-dimethylformamide (0.2 mL, 0.04 mmol) was added (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) (3 mg, 0.008 mmol) and N,N-diisopropylethylamine (0.002 mL, 0.014 mmol). The reaction was stirred at room temperature for 24 h. The crude reaction was filtered and the filtrate was purified directly by reverse phase HPLC by method C to give the title compound, #26 (3.7 mg, 37%) as a white solid. LCMS (Protocol E): m / z 1491.8 [M+HCO2 - ], retention time = 1.74 min.

[0263] N-[6-({[2-(methylsulfonyl)-1,3-benzothiazol-6-yl]carbonyl}amino)hexanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5Preparation of -carbamoyl-L-ornithineamide (#30)

[0264] [ka] Step 1. Synthesis of methyl 6-({[2-(methylsulfanyl)-1,3-benzothiazol-6-yl]carbonyl}amino)-hexanoate (#27) 2-(Methylsulfanyl)-1,3-benzothiazole-6-carboxylic acid 4 To a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (510 mg, 2.66 mmol) (500 mg, 2.22 mmol) and methyl 6-aminohexanoate hydrochloride (494 mg, 2.66 mmol) in dichloromethane (10 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (510 mg, 2.66 mmol). The reaction was stirred at room temperature for 18 h. The reaction was diluted with water (15 mL) and dichloromethane (75 mL). The aqueous layer was back-extracted with dichloromethane (50 mL). The combined organic layers were washed with brine (15 mL), dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and purified by silica gel chromatography (gradient: 50% to 100% ethyl acetate in heptane) to give methyl 6-({[2-(methylsulfanyl)-1,3-benzothiazol-6-yl]carbonyl}amino)-hexanoate, #27 (189 mg, 24%) as a yellow solid. LCMS (Protocol E): m / z 353.2 [M+H] + , retention time = 1.45 minutes; 1 H NMR (500 MHz, DMSO-d6): δ 8.53 (t, J=5.62 Hz, 1 H), 8.48 (d, J=1.22 Hz, 1 H), 7.87 - 7.94 (m, 2 H), 3.58 (s, 3 H), 3.24 - 3.31 (m, 2 H), 2.82 (s, 3 H), 2.32 (t, J=7.34 Hz, 2 H), 1.56 (tt, J=14.58, 7.31 Hz, 4 H), 1.29 - 1.38 (m, 2 H).

[0265] Step 2. Synthesis of 6-({[2-(methylsulfanyl)-1,3-benzothiazol-6-yl]carbonyl}amino)hexanoic acid (#28) To a solution of methyl 6-({[2-(methylsulfanyl)-1,3-benzothiazol-6-yl]carbonyl}amino)-hexanoate, #27 (50 mg, 0.14 mmol) in tetrahydrofuran (1 mL) was added a solution of lithium hydroxide (15 mg, 0.61 mmol) in water (0.3 mL). After 2 h, the reaction was diluted with dichloromethane (30 mL) and acidified to pH=3 with 1N hydrochloric acid. The aqueous layer was back-extracted with dichloromethane (15 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give 6-({[2-(methylsulfanyl)-1,3-benzothiazol-6-yl]carbonyl}amino)hexanoic acid, #28 (40 mg, 83%) as a yellow solid, which was used in the next step without further purification. LCMS (Protocol A): m / z 339.2 [M+H] + , retention time = 0.71 min; 1 H NMR (500 MHz, DMSO-d6): δ 8.65 (d, J=1.5 Hz, 1H), 8.53 (t, J=5.5 Hz, 1H), 8.48 (d, J=1.2 Hz, 1H), 8.02 (dd, J=8.3, 1.7 Hz, 1H), 7.87-7.95 (m, 2H), 3.27 (q, J=6.7 Hz, 2H), 2.82 (s, 3H), 2.22 (t, J=7.3 Hz, 2H), 1.54 (quintet, J=7.5 Hz, 4H), 1.29-1.38 (m, 2H).

[0266] Step 3. Synthesis of 6-({[2-(methylsulfonyl)-1,3-benzothiazol-6-yl]carbonyl}amino)hexanoic acid (#29) To a suspension of 6-({[2-(methylsulfanyl)-1,3-benzothiazol-6-yl]carbonyl}amino)hexanoic acid, #28 (39 mg, 0.12 mmol, 1 equiv) in dichloroethane (2 mL) was added m-chloroperbenzoic acid (103 mg, 0.46 mmol, 4 equiv). The reaction was stirred at room temperature for 24 h. The reaction was filtered and the solid collected to give 6-({[2-(methylsulfonyl)-1,3-benzothiazol-6-yl]carbonyl}amino)hexanoic acid, #29 (27 mg, 63%) as a yellow solid which was used in the next step without further purification. LCMS (Protocol E): m / z 371.2 [M+H] + , retention time = 0.98 min; 1 H NMR (500 MHz, DMSO-d6) δ: 11.96 (br. s., 1 H), 8.80 (s, 1 H), 8.70 - 8.76 (m, 1 H), 8.33 (d, J=8.80 Hz, 1 H), 8.11 - 8.15 (m, 1 H), 3.62 (s, 3 H), 3.27 - 3.32 (m, 2 H), 2.23 (t, J=7.34 Hz, 2 H), 1.56 (dq, J=14.82, 7.45 Hz, 4 H), 1.31 - 1.39 (m, 2 H).

[0267] Step 4. N-[6-({[2-(methylsulfonyl)-1,3-benzothiazol-6-yl]carbonyl}-amino)hexanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5Synthesis of -carbamoyl-L-ornithineamide (#30) 6-({[2-(methylsulfonyl)-1,3-benzothiazol-6-yl]carbonyl}amino)hexanoic acid, #29 (6 mg, 0.02 mmol), and L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 To a solution of 1-carbamoyl-L-ornithineamide, #25 (9.6 mg, 0.007 mmol) in N,N-dimethylformamide (0.2 mL, 0.04 mmol) was added (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) (3 mg, 0.008 mmol), and N,N-diisopropylethylamine (0.002 mL, 0.014 mmol). The reaction was stirred at room temperature for 24 h. The crude reaction mixture was filtered and the filtrate was purified by reverse phase HPLC by method C to give the title compound, #30 (2.3 mg, 22%) as a white solid. LCMS (Protocol E): m / z 1502.3 [M+2H + ], retention time = 1.79 min.

[0268] N-{23-[5-(methylsulfonyl)pyrazin-2-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl] ethyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Preparation of -carbamoyl-L-ornithineamide (#35)

[0269] [ka] Step 1. N-[1-(9H-fluoren-9-yl)-3,25-dioxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-yl]-L-valyl-N 5 Synthesis of -carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithineamide (#31) According to general procedure B, ice-cold, stirred L-valyl-N 5 -Carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithineamide [ValCitPABA] (compound 64, Dubowchik, GM et al., Bioconjug Chem, 13, 855-69 (2002) 5To a solution of 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosane-25-oic acid (1800 mg, 4.744 mmol), 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosane-25-oic acid (2.73 g, 4.74 mmol), and N,N-diisopropylethylamine (736 mg, 5.69 mmol) in N,N-dimethylformamide (12 mL) was added HATU (2160 mg, 5.69 mmol). The reaction was stirred at room temperature until complete by LCMS. The mixture was then diluted in ethyl acetate and the resulting solid was collected by filtration to give the title compound, #31 (1.9 g, 43%) as a yellow solid. LCMS (Protocol D): m / z 937.2 [M+H] + , retention time = 0.785 minutes; 1 H NMR (400 MHz, DMSO): δ 9.89 (m, 1H), 8.10-8.08 (d, 1H), 7.89-7.86 (m, 2H), 7.70-7.68 (d, 2H), 7.55-7.53 (d, 2H), 7.43-7.41 (m, 2H), 7.34-7.31 (m, 2H), 7.24-7.22 (d, 1H), 5.97 (br, 1H), 5.41 (s, 2H), 5.11-5.08 (m, 1H), 4.43-4.20 (m, 5H), 3.59-3.58 (d, 2H), 3.49-3.48 (m, 18H), 3.41-3.38 (m, 4H), 2.40-2.33 (m, 2H), 1.99-1.94 (m, 1H), 1.69-1.17 (m, 6H), 0.86-0.82 (m, 6H).

[0270] Step 2. N-[1-(9H-fluoren-9-yl)-3,25-dioxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-yl]-L-valyl-N 5 Synthesis of -carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithineamide (#32) Yellow N-[1-(9H-fluoren-9-yl)-3,25-dioxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-yl]-L-valyl-N 5 To a solution of -carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithinamide, #31 (9.35 g, 9.978 mmol) in anhydrous N,N-dimethylformamide (56 mL) was added bis(4-nitrophenyl)carbonate (9.11 g, 29.9 mmol) and N,N-diisopropylethylamine (1550 mg, 12.0 mmol). After stirring at room temperature for 1.5 h, TLC (dichloromethane:methanol:ammonium hydroxide 10:1:0.1) indicated completion of the reaction. The reaction mixture was added dropwise to tert-butyl methyl ether (2 L) and stirred for 30 min. A white precipitate formed which was filtered to give the title compound #32 (7.2 g, 65.5%) as a white solid. LCMS (Protocol D): m / z 1102.2 [M+H] + , 1124.2 [M+Na + ] retention time = 0.882 min; 1 H NMR (400 MHz, DMSO): δ 10.07 (s, 1H), 8.32-8.30 (d, 2H), 8.15-8.14 (d, 1H), 7.89-7.87 (d, 3H), 7.70-7.64 (m, 4H), 7.58-7.55 (d, 2H), 7.42-7.40 (m, 4H), 7.34-7.30 (m, 3H), 5.98 (br, 1H), 5.42 (s, 2H), 5.24 (s, 2H), 4.39-4.20 (m, 5H), 3.61-3.59 (d, 2H), 3.49-3.47 (m, 18H), 3.15-2.93 (m, 6H), 2.40-2.33 (m, 2H), 1.99-1.94 (m, 1H), 1.69-1.25 (m, 5H), 0.87-0.82 (m, 6H).

[0271] Step 3. N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#33) 2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- To a solution of valinamide (Example #54 in US 2013 / 0129753A1) (189 mg, 0.254 mmol) in N,N-dimethylacetamide (1 mL) was added N-[1-(9H-fluoren-9-yl)-3,25-dioxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-yl]-L-valyl-N 5-Carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide, #32 [PF-06452175] (336 mg, 0.305), N,N-diisopropylethylamine (0.2 mL, 1 mmol), 2,6-lutidine (0.1 mL, 1 mmol) and 1-hydroxy-7-azabenzotriazole (42 mg, 0.31 mmol) were added. The reaction was stirred at 45° C. overnight, following which LCMS analysis indicated the reaction was complete. The reaction mixture was directly purified by reverse phase chromatography on a 50 g C18 column using a gradient elution of 30-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min to afford the title compound, #33 (156 mg, 36%) as a gum. LCMS: m / z 1705.8 [M+H] + , retention time = 1.01 min; HPLC (protocol B): retention time = 6.225 min.

[0272] Step 4. N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide, trifluoroacetate (#34) N-(21-amino-4,7,10,13,16,19-hexaoxaheneicosan-1-oyl)-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidine To a solution of N,N-dimethylformamide (1 mL) of N,N-dimethylformamide, #33 (150 mg, 0.088 mmol) was added diethylamine (1 mL, 10 mmol). The reaction was stirred overnight at room temperature and then directly purified by reverse phase chromatography on a 50 g C18 column using a gradient elution of 20-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min to give the title compound #34 (60 mg, 46%, purity >99%) as a white solid. LCMS: m / z 1483.9 [M+H] + , retention time = 1.39 min; HPLC (protocol B): retention time = 5.282 min.

[0273] Step 5. N-{23-[5-(methylsulfonyl)pyrazin-2-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl] ethyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#35) N-(21-amino-4,7,10,13,16,19-hexaoxaheneicosan-1-oyl)-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4 To a solution of {N,7,10-triazatetradec-1-yl]phenyl}-N5-carbamoyl-L-ornithinamide, trifluoroacetate, #34 (24.3 mg, 0.015 mmol) and 5-(methylsulfonyl)pyrazine-2-carboxylic acid, #1 (8.3 mg, 0.041 mmol) in tetrahydrofuran (0.5 mL) was added N-methylmorpholine (1 drop, ca. 20 uL) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM, 12.5 mg, 0.045 mmol) at room temperature. The resulting orange suspension became more cloudy over time. After 30 min, LCMS analysis indicated the reaction was complete and the reaction mixture was diluted with DMSO and purified directly by reverse phase chromatography using a modified version of purification method A (utilizing a gradient elution of 20-90% acetonitrile in water containing 0.02% acetic acid over 20 min) to afford the title compound #35 (19.7 mg, 79%) as a glass. LCMS: m / z 1668.7 [M+H] + , retention time = 1.70 min; HPLCMS (protocol C): m / z 1669.9 [M+H] + , 835.5[M / 2+H + ]Retention time = 8.627 minutes.

[0274] N-{23-[2-(methylsulfonyl)pyrimidin-5-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-valyl-N 5Preparation of -carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(3-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide (#39)

[0275] [ka] Step 1. N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)-L-valyl-N 5 Synthesis of -carbamoyl-N-[4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl)phenyl]-L-ornithinamide, acetate (#36) To a solution of N-[1-(9H-fluoren-9-yl)-3,25-dioxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-yl]-L-valyl-N5-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide, #32 (308.0 mg, 0.279 mmol) in N,N-dimethylacetamide (2 mL), tert-butyl methyl[2-(methylamino)ethyl]carbamate was added. 6(56.0 mg, 0.30 mmol), 1-hydroxy-7-azabenzotriazole (49 mg, 0.36 mmol), N,N-diisopropylethylamine (0.2 mL, 1 mmol) and 2,6-lutidine (0.1 mL, 0.8 mmol) were added. The reaction was stirred at 45° C. overnight, following which LCMS analysis indicated the reaction was complete. The mixture was cooled to room temperature and piperidine (1 mL, 10 mmol) was added. After 15 min, LCMS indicated complete loss of Fmoc protection with conversion to the amine. The mixture was concentrated under a strong stream of nitrogen and the resulting residue was purified by reverse phase chromatography on a 50 g C18 column using a gradient elution of 10-70% acetonitrile in water containing 0.02% acetic acid over 20 min to give the title compound #36 (290 mg, quantitative) as a gum. LCMS: m / z 1014.8 [M+H] + , retention time = 0.61 min; HPLC (protocol B): retention time = 5.179 min.

[0276] Step 2. N-{23-[2-(methylsulfonyl)pyrimidin-5-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-valyl-N 5 Synthesis of -carbamoyl-N-[4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl)phenyl]-L-ornithineamide (#37) Acid 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (281.0 mg, 1.39 mmol), and N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)-L-valyl-N 5To a solution of -carbamoyl-N-[4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl)phenyl]-L-ornithinamide acetate, #36 (290.0 mg, 0.312 mmol) in N,N-dimethylacetamide (2 mL) was added 2-chloro-4,6-dimethoxy-1,3,5-triazine (241 mg, 1.37 mmol) followed by N-methylmorpholine (200 μL, 1.8 mmol). The reaction mixture was stirred at room temperature overnight following which LCMS analysis indicated the reaction was complete. The mixture was directly purified by reverse phase chromatography on a 50 g C18 column using a gradient elution of 10-90% acetonitrile in water containing 0.02% acetic acid over 20 min to give the title compound #37 (138.6 mg, 40%) as a white solid. LCMS: m / z 1113.5 [M+H] + , retention time = 0.81 min; HPLC (Protocol B): retention time = 5.774 min; 1 H NMR (400 MHz, methanol-d4) d ppm 9.39 (s, 2 H) 7.65 (d, J=4.7 Hz, 2 H) 7.36 (d, J=8.2 Hz, 2 H) 5.10 (s, 2 H) 4.55 (dd, J=8.8, 4.9 Hz, 1 H) 4.17 - 4.28 (m, 1 H) 3.76 - 3.83 (m, 2 H) 3.70 - 3.76 (m, 2 H) 3.58 - 3.70 (m, 22 H) 3.30 - 3.37 (m, 8 H) 3.09 - 3.28 (m, 2 H) 2.95 - 3.03 (m, 3 H) 2.90 (s, 1 H) 2.80 (s, 2 H) 2.59 (t, J=6.0 Hz, 2 H) 2.36 (s, 3 H) 2.20 (s, 1 H) 2.09 - 2.19 (m, 1 H) 1.89 - 2.01 (m, 1 H) 1.72 - 1.86 (m, 1 H) 1.54 - 1.71 (m, 2 H) 1.47 (s, 9 H) 1.02 (t, J=6.2 Hz, 6 H).

[0277] Step 3. N-{23-[2-(methylsulfonyl)pyrimidin-5-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-valyl-N 5 Synthesis of -carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-ornithineamide, trifluoroacetate salt #38 N-{23-[2-(methylsulfonyl)pyrimidin-5-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-valyl-N 5 To a solution of -carbamoyl-N-[4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl)phenyl]-L-ornithinamide, #37 (136.0 mg, 0.122 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.5 mL, 7 mmol). After 20 min, LCMS analysis indicated the reaction was complete, and the reaction mixture was concentrated under a stream of nitrogen, diluted with DMSO, and purified by reverse phase chromatography by a modified version of purification method A (utilizing a gradient elution of 5-60% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #38 (126 mg, 91.5%) as a gum. LCMS: m / z 1014.8 [M+H] + , retention time = 0.61 min; HPLC (protocol B): retention time = 4.280 min.

[0278] Step 4: N-{23-[2-(methylsulfonyl)pyrimidin-5-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-valyl-N 5Synthesis of -carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(3-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide (#39) N-{23-[2-(methylsulfonyl)pyrimidin-5-yl]-23-oxo-4,7,10,13,16,19-hexaoxa-22-azatricosane-1-oyl}-L-valyl-N 5 A solution of the trifluoroacetate salt of -carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-ornithinamide, #38 (37.9 mg, 0.0336 mmol) in N,N-dimethylacetamide (1 mL), and a solution of (1S)-1-(chloromethyl)-3-[(3-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl 4-nitrophenyl carbonate (28 mg, 0.034 mmol) were combined. 1-Hydroxy-7-azabenzotriazole (4.58 mg, 0.0336 mmol) was added followed by 2,6-lutidine (15.7 mL, 0.135 mmol) and N,N-diisopropylethylamine (23.7 mL, 0.135 mmol). The reaction mixture immediately turned yellow and the reaction was left at room temperature overnight. The crude reaction mixture was purified directly by a modified version of purification method A utilizing a gradient elution of 0-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min to give the title compound #39 (21.3 mg, 37%) as a glass. LCMS: m / z 1707.7 [M+H] + , retention time 0.82 min.

[0279] N-acetyl-3-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)-L-alanyl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Preparation of -carbamoyl-L-ornithineamide (#43)

[0280] [ka] Step 1: Synthesis of N-acetyl-3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-L-alanine (#40) To a suspension of Boc-N-β-Fmoc-L-diaminopropionic acid (346 mg, 0.811 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (2 mL), resulting in a colorless solution. After 30 min, the thick white reaction mixture was concentrated under reduced pressure. The residue was diluted with pyridine and cooled in an ice-water bath, then acetic anhydride (250 uL, 2.68 mmol) was added dropwise. After 20 min, the reaction mixture was concentrated under reduced pressure, diluted with dimethylsulfoxide, and purified directly by reverse phase chromatography using a modified version of purification method A (utilizing a gradient elution of 10-80% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #40 (169 mg, 56%) as a glass. LCMS (Protocol A): m / z 369.3 [M+H] + , retention time = 0.92 min; HPLC (Protocol B): retention time = 6.065 min.1H NMR (400 MHz, methanol-d4) d ppm 7.79 (d, J=7.8 Hz, 2 H) 7.64 (d, J=7.4 Hz, 2 H) 7.38 (t, J=7.4 Hz, 2 H) 7.31 (t, J=7.4 Hz, 2 H) 4.55 (t, J=5.9 Hz, 1 H) 4.33 (d, J=6.6 Hz, 2 H) 4.20 (t, J=6.8 Hz, 1 H) 3.60 (dd, J=14.2, 4.5 Hz, 1 H) 3.45 (dd, J=14.0, 7.4 Hz, 1 H) 1.97 (s, 3 H)

[0281] Step 2: N-acetyl-3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-L-alanyl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#41) In an ice-cold water bath, L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5To a solution of N,N-carbamoyl-L-ornithineamide, trifluoroacetate, #25 (40.0 mg, 0.032 mmol) in N,N-dimethylacetamide (400 μL) was added N-acetyl-3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-L-alanine, #40 (11.7 mg, 0.0317 mmol), HATU (25.0 mg, 0.066 mmol) and N,N-diisopropylethylamine (81.9 μL, 0.475 mmol). The reaction was stirred for 15 min, then diluted with dimethylsulfoxide and purified directly by reverse phase chromatography via a modified version of purification method A (utilizing a gradient elution of 20-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #41 (9.7 mg, 20%) as a white solid. LCMS (Protocol A): m / z 1500.0 [M+H] + , retention time = 1.11 min; HPLC (protocol B): retention time = 7.449 min.

[0282] Step 3. N-acetyl-3-amino-L-alanyl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -Carbamoyl-L-ornithineamide, trifluoroacetate (#42) To a solution of #41 (9.0 mg, 0.006 mmol) in acetonitrile (1 mL) in an ice-cooled water bath was added a solution of lithium hydroxide (10 mg, 0.2 mmol) in water (0.1 mL). After 45 min, LCMS analysis indicated the reaction was complete, and it was then neutralized with acetic acid, diluted with dimethylsulfoxide, and purified directly by reverse-phase chromatography by a modified version of purification method A (utilizing a gradient elution of 15-90% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #42 (2.8 mg, 30%) as a white solid. LCMS (Protocol A): m / z 1298.8 [M+H] + , retention time = 0.80 min; HPLC (protocol B): retention time = 5.717 min.

[0283] Step 4. N-acetyl-3-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)-L-alanyl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#43) Acid 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (20.0 mg, 0.099 mmol) and N-acetyl-3-amino-L-alanyl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 A mixture of the trifluoroacetate salt of -carbamoyl-L-ornithinamide, #43 (2.8 mg, 0.0022 mmol), was diluted with methanol and toluene and concentrated to dryness. This was repeated two more times. To the resulting mixture in N,N-dimethylacetamide (0.5 mL) was added 2-chloro-4,6-dimethoxy-1,3,5-triazine (5 mg, 0.03 mmol), followed by N-methylmorpholine (50 μL, 0.45 mmol). The reaction mixture was stirred at room temperature for 1 h, following which LCMS analysis indicated the reaction was complete. The mixture was diluted with dimethylsulfoxide and purified directly by reverse phase chromatography by a modified version of purification method A (utilizing a gradient elution of 10-80% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #43 (1.0 mg, 31%) as a white solid. LCMS (Protocol A): m / z 1461.8 [M+H] + , retention time = 0.93 min; HPLC (protocol B): retention time = 6.362 min.

[0284] N-[(2S)-2-(acetylamino)-4-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)butanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Preparation of -carbamoyl-L-ornithineamide (#46)

[0285] [ka] Step 1. N-{(2S)-2-(acetylamino)-4-[(tert-butoxycarbonyl)amino]butanoyl}-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#44) In an ice-cold water bath, L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 To a solution of 10-carbamoyl-L-ornithineamide, trifluoroacetate, #25 (60.0 mg, 0.048 mmol) in N,N-dimethylacetamide (600 μL) was added Ac-Dab(Boc)-OH (23.2 mg, 0.0891 mmol), HATU (33.4 mg, 0.0878 mmol) and N,N-diisopropylethylamine (23.0 μL, 0.13 mmol). The reaction was stirred at room temperature overnight. The reaction mixture was then diluted with dimethylsulfoxide and purified directly by reverse phase chromatography via a modified version of purification method A (utilizing a gradient elution of 20-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #44 (18.7 mg, 29%) as a white solid. LCMS (Protocol A): m / z 1391.9 [M+H] + , retention time = 0.98 min; HPLC (protocol B): retention time = 6.643 min.

[0286] Step 2. N-[(2S)-2-(acetylamino)-4-aminobutanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5Synthesis of -carbamoyl-L-ornithineamide, trifluoroacetate (#45) Trifluoroacetic acid (1 mL, 10 mmol) was added to a solution of #44 (13.6 mg, 0.0098 mmol) in acetonitrile (2 mL) and the reaction was stirred at room temperature for 20 min. The reaction was concentrated under reduced pressure to give the title compound #45 (13.7 mg) as a white solid, which was used directly without further purification. LCMS (Protocol E): m / z 1290.9 [M+H] + , retention time = 0.84 min.

[0287] Step 3. N-[(2S)-2-(acetylamino)-4-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)butanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#46) N-[(2S)-2-(acetylamino)-4-aminobutanoyl]-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5To a solution of the trifluoroacetate salt of -carbamoyl-L-ornithinamide, #45 (13.7 mg, 0.00975 mmol) in N,N-dimethylacetamide (1 mL) was added 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (27.9 mg, 0.138 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (24.0 mg, 0.137 mmol), followed by N-methylmorpholine (200 μL, 1.8 mmol). The reaction mixture was stirred at room temperature for 10 min, following which LCMS analysis indicated the reaction was complete. The mixture was diluted with dimethylsulfoxide and purified directly by reverse phase chromatography via a modified version of purification method A (utilizing a gradient elution of 10-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to afford the title compound #46 (8.0 mg, 56%) as a white solid. LCMS (Protocol A): m / z 1475.0 [M+H] + , retention time = 0.94 min; HPLC (protocol B): retention time = 6.344 min.

[0288] N 2 -Acetyl-N 5 -{[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}-L-ornityl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Preparation of -carbamoyl-L-ornithineamide (#52)

[0289] [ka] Step 1. N 2 -Acetyl-N 5Synthesis of -(tert-butoxycarbonyl)-L-ornithine (#47) [Ac-Orn(Boc)-OH] N 5 To a solution of -(tert-butoxycarbonyl)-L-ornithine, [H-Orn(Boc)-OH] (5.0 g, 21.5 mmol) in THF:water (80:20 mL) was slowly added an aqueous solution of potassium carbonate (11.9 g, 86.1 mmol in 50 mL water). The mixture was stirred for 5 min and then cooled in an ice-water bath, after which acetyl chloride (3.38 g, 43.1 mmol) was added dropwise. The mixture was stirred at room temperature for 2.5 h, whereby TLC (dichloromethane:methanol:acetic acid=100:10:) showed the reaction was complete. The mixture was concentrated under reduced pressure, and the resulting aqueous mixture was acidified to pH=2.0 with a 3M aqueous solution of hydrochloric acid, and then extracted with ethyl acetate (200 mL×3). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to give the title compound #47 (5.8 g, 98%) as a white solid. HPLC (Protocol J): Retention time = 2.94 minutes. 1 H NMR (400MHz, DMSO-d6) δ = 12.51 (br. s., 1H), 8.10 (d, J=7.5 Hz, 1H), 6.82 (br. s., 1H), 4.13 (d, J=5.0 Hz, 1H), 2.90 (d, J=6.0 Hz, 2H), 1.84 (s, 3H), 1.65 (br. s., 1H), 1.51 (d, J=9.0 Hz, 1H), 1.43-1.35 (m, 11H).

[0290] Step 2:N 2 -Acetyl-N 5 -(tert-Butoxycarbonyl)-L-ornithine-L-valyl-N 5 Synthesis of -carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithineamide (#48) N 2 -Acetyl-N 5A solution of -(tert-butoxycarbonyl)-L-ornithine #47 [Ac-Orn(Boc)-OH] (875 mg, 3.19 mmol) in anhydrous N,N-dimethylformamide (30 mL) was treated with HATU (1210 mg, 3.19 mmol). After 30 min, the mixture was cooled in an ice-water bath and L-valyl-N 5 -Carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithineamide 2 [ValCitPABA] (compound 64, Dubowchik, GM et al., Bioconjug Chem, 13, 855-69 (2002) 5 (1100 mg, 2.899 mmol) was added followed by N,N-diisopropylethylamine (1.5 mL, 1113 mg, 8.61 mmol). The mixture was stirred and allowed to reach room temperature. After 2 h, MTBE was added to precipitate the product, which was filtered and dispersed in dichloromethane (150 mL) for 20 min. The mixture was filtered, washed with dichloromethane:methanol (10:1), then air-dried to give the title compound #48 (1400 mg, 76%) as a grey solid. This material was used directly in the subsequent reaction without further purification. HPLC (Protocol J): Retention time = 3.16 minutes. 1H NMR (400MHz, DMSO-d6) δ = 9.96 (s, 1H), 8.12 (d, J=1.0 Hz, 1H), 8.05 (d, J=8.0 Hz, 1H), 7.73 (d, J=8.5 Hz, 1H), 7.55 (d, J=8.5 Hz, 2H), 7.25 (d, J=8.0 Hz, 2H), 6.80 (t, J=5.3 Hz, 1H), 6.00 (t, J=5.3 Hz, 1H), 5.78 (s, 1H), 5.44 (s, 2H), 5.11 (t, J=5.8 Hz, 1H), 4.45 (d, J=1.0Hz, 1H), 4.42 - 4.34 (m, J=1.0, 1.0, 1.0 Hz, 1H), 4.32 - 4.23 (m, 1H), 4.21 (t, J=8.5 Hz, 1H), 3.09 - 2.85 (m, 4H), 1.99 (qd, J=6.4, 13.4 Hz, 1H), 1.86 (s, 3H), 1.76 - 1.65 (m, J=9.5 Hz, 1H), 1.64 - 1.53 (m, J=9.0 Hz, 2H), 1.51 - 1.43 (m, 2H), 1.43 - 1.35 (m, 11H), 0.87 (d, J=7.0 Hz, 3H), 0.84 (d, J=6.5 Hz, 3H).

[0291] Step 3: N 2 -Acetyl-N 5 -(tert-Butoxycarbonyl)-L-ornithine-L-valyl-N 5 Synthesis of -carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithineamide (#49) In an ice-cold water bath, 2 -Acetyl-N 5 -(tert-Butoxycarbonyl)-L-ornithine-L-valyl-N 5A solution of -carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithinamide, #48 (1400 mg, 2.202 mmol) in anhydrous N,N-dimethylformamide (25 mL) was treated with bis-(4-nitrophenyl)carbonate (1.34 g, 4.40 mmol) with N,N-diisopropylethylamine (767 μL, 569 mg, 4.40 mmol) and the reaction was stirred at room temperature for 6 h. The reaction mixture was poured into MTBE and after 20 min the solid was isolated by filtration, washed with dichloromethane (150 mL) and then air-dried to give the title compound #49 (1.3 g, 74%) as a white solid. This material was used directly in the subsequent reaction without further purification. LCMS (Protocol K): m / z 801.3 [M+H] + , m / z 823.3 [M+Na] + Retention time = 4.066 min; HPLC (Protocol J): retention time = 2.63 min. 1 H NMR (400MHz, DMSO-d6) d = 10.21 - 10.02 (m, 1H), 8.39 - 8.27 (m, 2H), 8.20 - 7.95 (m, 2H), 7.76 - 7.53 (m, 5H), 7.42 (d, J=8.0 Hz, 2H), 6.84 - 6.72 (m, 1H), 6.07 - 5.92 (m, 1H), 5.48 - 5.39 (m, 2H), 5.29 - 5.20 (m, 2H), 4.47 - 4.10 (m, 3H), 3.06 - 2.81 (m, 4H), 2.10 - 1.92 (m, 1H), 1.91 - 1.79 (m, 3H), 1.76 - 1.53 (m, 3H), 1.37 (br. s., 14H), 0.94 - 0.74 (m, 6H)

[0292] Step 4: N 2 -Acetyl-N 5 -(tert-Butoxycarbonyl)-L-Ornityl-N 5-Carbamoyl-L-ornityl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#50) To a solution of 2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Example #54 in US2013 / 0129753A1) [Aur-0101] (113 mg, 0.152 mmol) in N,N-dimethylacetamide (4 mL), was added N 2 -Acetyl-N 5 -(tert-Butoxycarbonyl)-L-ornithine-L-valyl-N 5N,N-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide, #49 (124.5 mg, 0.155 mmol), HOAt (24 mg, 0.18 mmol), 2,6-lutidine (44.3 μL, 41 mg, 0.38 mmol), and N,N-diisopropylethylamine (66.9 μL, 50 mg, 0.38 mmol) were added. The orange reaction mixture was heated at 45 °C overnight. LCMS analysis indicated the reaction was complete. The mixture was diluted with dimethyl sulfoxide and purified by reverse phase chromatography by a modified version of purification method A (utilizing a gradient elution of 20-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the desired product #50 (82.2 mg, 38%) as a white solid. LCMS (Protocol C): m / z 1405.9 [M+H] + , retention time = 0.95 min. HPLC (Protocol B): retention time = 6.735 min.

[0293] Step 5: N 2 -Acetyl-L-ornithine-N 5 -Carbamoyl-L-ornityl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -Carbamoyl-L-ornithineamide, trifluoroacetate (#51) Trifluoroacetic acid (1 mL, 10 mmol) was dissolved in N 2 -Acetyl-N 5 -(tert-Butoxycarbonyl)-L-Ornityl-N 5-Carbamoyl-L-ornityl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 -carbamoyl-L-ornithineamide, #50 (82.2 mg, 0.0585 mmol) in acetonitrile (2 mL) was added and the reaction was stirred at room temperature for 15 min. The reaction was concentrated under reduced pressure to give the title compound #51 (90 mg) as a white solid, which was used directly without further purification. LCMS (Protocol E): m / z 1304.9 [M+H] + , retention time = 0.81 min; HPLC (protocol B): retention time = 5.804 min.

[0294] Step 6: N 2 -Acetyl-N 5 -{[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}-L-ornityl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#52) N 2 -Acetyl-L-ornithine-N 5-Carbamoyl-L-ornityl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 To a solution of the trifluoroacetate salt of -carbamoyl-L-ornithineamide, #51 (88.0 mg, 0.067 mmol) in N,N-dimethylacetamide (0.5 mL) was added 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (68.2 mg, 0.337 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (59.2 mg, 0.337 mmol), followed by N-methylmorpholine (74 μL, 68.2 mg, 0.675 mmol). The orange reaction mixture was stirred at room temperature for 90 min, following which LCMS analysis indicated the reaction was complete. The mixture was diluted with dimethylsulfoxide and purified directly by reverse-phase chromatography using a modified version of purification method A (utilizing a gradient elution of 10-80% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #52 (98.4 mg, 98%) as a white solid. LCMS (Protocol A): m / z 1489.8 [M+H] + , retention time = 0.92 min; HPLC (protocol B): retention time = 6.342 min.

[0295] N 2 -Acetyl-N 6-{[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}-L-lysyl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Preparation of -carbamoyl-L-ornithineamide (#58)

[0296] [ka] Step 1: 2 -Acetyl-N 6 -(tert-Butoxycarbonyl)-L-lysyl-L-valyl-N 5 Synthesis of -carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithineamide (#53) A solution of Ac-Lys(Boc)-OH (15.2 g, 52.7 mmol) in anhydrous N,N-dimethylformamide (250 mL) was treated with HATU (20.0 g, 52.7 mmol) in an ice-water bath. After 30 min, L-valyl-N 5 -Carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithineamide 2 (Dubowchik, GM et al., Bioconjug Chem, 13, 855-69 (2002) 5Compound 64) (20 g, 52.7 mmol) in 10 ml was added followed by N,N-diisopropylethylamine (12.8 mL, 9.54 g, 73.8 mmol). The mixture was allowed to reach room temperature. After 2 h, the reaction mixture was poured into MTBE to precipitate the product, which was filtered and dispersed in dichloromethane:methanol (10:1, 800 mL) for 20 min. The mixture was filtered, air-dried and purified by prep-HPLC by method D. After concentration under reduced pressure, the residue was poured into chloroform:isopropyl alcohol 4:1. The solid was collected by filtration and air-dried to give the title compound #53 (15.0 g, 44%) as a white solid. HPLC (Protocol J): Retention time = 3.63 minutes. 1 H NMR (400MHz, DMSO-d6) δ = 9.95 (s, 1H), 8.32 (s, 1H), 8.09 (d, J=7.5 Hz, 1H), 8.03 (d, J=8.0 Hz, 1H), 7.72 (d, J=9.0 Hz, 1H), 7.57 - 7.51 (m, J=8.5 Hz, 2H), 7.27 - 7.20 (m, J=8.5 Hz, 2H), 6.77 (t, J=5.5 Hz, 1H), 5.98 (t, J=5.8 Hz, 1H), 5.43 (s, 2H), 5.11 (t, J=5.5 Hz, 1H), 4.43 (d, J=5.5 Hz, 2H), 4.41 - 4.35 (m, 1H), 4.29 - 4.21 (m, 1H), 4.19 (dd, J=7.0, 8.5 Hz, 1H), 3.07 - 2.92 (m, 2H), 2.91 - 2.84 (m, 2H), 2.34 (s, 1H), 1.98 (qd, J=6.7, 13.6 Hz, 1H), 1.84 (s, 3H), 1.72 - 1.50 (m, 3H), 1.43 - 1.33 (m, 12H), 1.25 (dd, J=10.3, 15.8 Hz, 3H), 0.86 (d, J=6.5 Hz, 3H), 0.83 (d, J=6.5 Hz, 3H).

[0297] Step 2:N 2 -Acetyl-N 6 -(tert-Butoxycarbonyl)-L-lysyl-L-valyl-N 5 Synthesis of -carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithineamide (#55). 2 -Acetyl-N 6 -(tert-Butoxycarbonyl)-L-lysyl-L-valyl-N 5 A solution of -carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithinamide, #53 (12.0 g, 18.5 mmol) in anhydrous N,N-dimethylformamide (150 mL) was treated with bis-(4-nitrophenyl)carbonate (11.2 g, 36.9 mmol) with N,N-diisopropylethylamine (6.42 mL, 4.77 g, 36.9 mmol) and the reaction was stirred at room temperature for 3 h. The reaction mixture was poured into MTBE and after 20 min the solid was isolated by filtration, washed with dichloromethane (1 L) and then air-dried to give the title compound #55 (12.5 g, 83.1%) as a yellow solid. This material was used directly in the subsequent reaction without further purification. LCMS (Protocol K): m / z 815.2 [M+H] + , Retention time = 4.101 min; HPLC (Protocol J): Retention time = 4.58 min. 1 H NMR (400MHz, DMSO-d6) δ = 10.12 (s, 1H), 8.35 - 8.29 (m, J=9.0 Hz, 2H), 8.13 (d, J=7.0 Hz, 1H), 8.03 (d, J=7.5 Hz, 1H), 7.74 - 7.62 (m, 3H), 7.60 - 7.55 (m, J=9.0 Hz, 2H), 7.42 (d, J=8.5 Hz, 2H), 6.77 (br. s., 1H), 6.03 - 5.96 (m, 1H), 5.44 (s, 2H), 5.25 (s, 2H), 4.44 - 4.35 (m, 1H), 4.29 - 4.16 (m, 2H), 3.09 - 2.84 (m, 5H), 2.74 (s, 1H), 2.04 - 1.91 (m, 1H), 1.85 (s, 3H), 1.69 (br. s., 1H), 1.60 (d, J=9.0 Hz, 2H), 1.46 (br. s., 2H), 1.37 (s, 12H), 1.24 (br. s., 2H), 1.11 (s, 2H), 0.86 (d, J=7.0 Hz, 3H), 0.83 (d, J=6.5 Hz, 3H).

[0298] Step 3: N 2 -Acetyl-N 6 -(tert-Butoxycarbonyl)-L-lysyl-N 5 -Carbamoyl-L-ornityl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1i)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 Synthesis of -carbamoyl-L-ornithineamide (#56) 2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Example # in US2013 / 0129753A1) To a solution of 54 (88 mg, 0.12 mmol) in N,N-dimethylacetamide (1 mL) was added AcLys(Boc)ValCitPABC-PNP, #55 (100 mg, 0.123 mmol), HOAt (17.2 mg, 0.126 mmol), 2,6-lutidine (34.8 μL, 32 mg, 0.30 mmol), and N,N-diisopropylethylamine (52.6 μL, 39 mg, 0.30 mmol). The orange reaction mixture was heated at 45 °C overnight. LCMS analysis indicated the reaction was complete. The mixture was diluted with dimethyl sulfoxide and purified by reverse phase chromatography using a modified version of purification method A (utilizing a gradient elution of 20-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the desired product #56 (85.2 mg, 51%) as a white solid. LCMS (Protocol C): m / z 1419.9 [M+H] + , 1442.9[M+Na] + Retention time = 0.97 min. HPLC (Protocol B): Retention time = 6.836 min.

[0299] Step 4: N 2 -Acetyl-L-lysyl-N 5 -Carbamoyl-L-ornityl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N5 Synthesis of -Carbamoyl-L-ornithineamide, trifluoroacetate (#57) Trifluoroacetic acid (1 mL, 10 mmol) was dissolved in N 2 -Acetyl-N 6 -(tert-Butoxycarbonyl)-L-lysyl-N 5 -Carbamoyl-L-ornityl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1i)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 -carbamoyl-L-ornithineamide, #56 (85.2 mg, 0.0601 mmol) was added to a solution in acetonitrile (2 mL) and the reaction was stirred at room temperature for 15 min. The reaction was concentrated under reduced pressure to give the title compound, #57 (90 mg) as a white solid, which was used directly without further purification. LCMS (Protocol E): m / z 1318.9 [M+H] + , retention time = 0.81 min; HPLC (protocol B): retention time = 5.813 min.

[0300] Step 5: N 2 -Acetyl-N 6 -{[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}-L-lysyl-L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5Synthesis of -carbamoyl-L-ornithineamide (#58) N 2 -Acetyl-L-lysyl-N 5 -Carbamoyl-L-ornityl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N 5 To a solution of the trifluoroacetate salt of -carbamoyl-L-ornithineamide, #57 (90.0 mg, 0.068 mmol) in N,N-dimethylacetamide (0.5 mL) was added 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (69.0 mg, 0.341 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (59.9 mg, 0.341 mmol), followed by N-methylmorpholine (200 μL, 180 mg, 1.8 mmol). The orange reaction mixture was stirred at room temperature for 90 min, following which LCMS analysis indicated the reaction was complete. The mixture was diluted with dimethylsulfoxide and purified directly by reverse-phase chromatography using a modified version of purification method A (utilizing a modified gradient elution of 10-80% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the title compound #58 (91.7 mg, 89%) as a white solid. LCMS (Protocol A): m / z 1503.8 [M+H] + , retention time = 0.92 min; HPLC (protocol B): retention time = 6.360 min.

[0301] Preparation of N-({[4-(beta-D-glucopyranuronosyloxy)-3-({N-[6-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)hexanoyl]-beta-alanyl}amino)benzyl]oxy}carbonyl)-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#66)

[0302] [ka] Step 1. Synthesis of methyl 6-({[2-(methylsulfanyl)pyrimidin-5-yl]carbonyl}amino)hexanoate (#59) To a solution of 2-(methylsulfanyl)pyrimidine-5-carboxylic acid (Example 29 in US2010 / 210593A1; Yu-Xiu, Liu; Ming-Bo, Cui; Qi-Qi, Zhao; Qing-Min, Wang; Ying, Liu; Run-Qiu, Huang, Reduction of pyrimidine derivatives by LiAlH4, Journal of Chemical Research, 2007, #8, p.490-493) (228 mg, 1.34 mmol) and methyl 6-aminohexanoate hydrochloride (497 mg, 2.68 mmol) in dichloromethane (6 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (68 mg, 0.36 mmol) and N,N-diisopropylethylamine (0.5 mL, 3 mmol). The reaction was stirred at room temperature for 18 hours. The reaction was concentrated under reduced pressure and purified by silica gel chromatography (gradient: 20% to 100% ethyl acetate in heptane) to give methyl 6-({[2-(methylsulfanyl)pyrimidin-5-yl]carbonyl}amino)hexanoate, #59 (131 mg, 33%) as a white solid. LCMS (Protocol E): m / z 298.4 [M+H] + , retention time = 1.21 min; 1 H NMR(500 MHz, DMSO-d6): δ 1 H NMR (500MHz, DMSO-d6) δ = 8.97 (s, 2H), 8.67 (t, J=5.4 Hz, 1H), 3.58 (s, 3H), 3.33 (s, 2H), 3.29 - 3.23 (m, 2H), 2.57 (s, 3H), 2.32 (t, J=7.5 Hz, 2H), 1.54 (d quintet, 4H), 1.36 - 1.28 (m, 2H).

[0303] Step 2. Synthesis of 6-({[2-(methylsulfanyl)pyrimidin-5-yl]carbonyl}amino)hexanoic acid (#60) To a solution of methyl 6-({[2-(methylsulfanyl)pyrimidin-5-yl]carbonyl}amino)hexanoate, #59 (63 mg, 0.21 mmol) in tetrahydrofuran (1 mL) was added a solution of lithium hydroxide (21 mg, 0.86 mmol) in water (0.3 mL). After 3 h, the reaction was diluted with dichloromethane and acidified to pH=2 with 1N hydrochloric acid. The aqueous layer was back-extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give 6-({[2-(methylsulfanyl)pyrimidin-5-yl]carbonyl}amino)hexanoic acid, #60 (53 mg, 88%) as a white solid, which was used in the next step without further purification. LCMS (Protocol A): m / z 284.19 [M+H] + , retention time = 0.93 min; 1 H NMR (500 MHz, DMSO-d6): δ 11.99 (s, 1H), 8.97 (s, 2H), 8.67 (t, J=5.5 Hz, 1H), 3.29 - 3.17 (m, 2H), 2.57 (s, 3H), 2.22 (t, J=7.3 Hz, 2H), 1.53 (quintet, J=7.4 Hz, 4H), 1.39 - 1.24 (m, 2H).

[0304] Step 3. Synthesis of 6-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)hexanoic acid (#61) To a suspension of 6-({[2-(methylsulfanyl)pyrimidin-5-yl]carbonyl}amino)hexanoic acid, #60 (146 mg, 0.515 mmol) in dichloromethane (2 mL) was added m-chloroperbenzoic acid (272 mg, 1.55 mmol). The reaction was stirred at room temperature for 3 h. The reaction was filtered and the solid was collected to give 6-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)hexanoic acid, #61 (58 mg, 36%) as a yellow solid which was used in the next step without further purification. LCMS (Protocol E): m / z 338.3 [M+Na]+ , 314.4 [MH] + , retention time = 0.57 min; 1 H NMR (500 MHz, DMSO-d6) δ 9.36 (d, J=1.6 Hz, 1 H) 9.25 (d, J=1.6 Hz, 1 H) 9.13 (br. s., 1 H) 3.43 - 3.50 (m, 2 H) 3.33 (s, 3 H) 2.31 (t, J=7.4 Hz, 2 H) 1.62 - 1.73 (m, 4 H) 1.38 - 1.49 (m, 2 H).

[0305] Step 4. Synthesis of N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-2-(methylsulfonyl)pyrimidine-5-carboxamide (#62) To a solution of 6-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)hexanoic acid, #61 (14.2 mg, 0.045 mmol) and N-hydroxysuccinimide (5.6 mg, 0.049 mmol) in dichloromethane (1 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (8.6 mg, 0.045 mmol) and N,N-diisopropylethylamine (0.5 mL, 3 mmol). The reaction was stirred at room temperature for 72 h. The mixture was diluted with dimethylsulfoxide and purified directly by reverse phase chromatography using a modified version of purification method A (utilizing a gradient elution of 10-90% acetonitrile in water containing 0.02% acetic acid over 20 min) to give the title compound (9.2 mg, 50%) as a white solid. LCMS (Protocol E): m / z 413.3 [M+H] + , retention time = 0.65 min.

[0306] Step 5. Synthesis of N-[({3-({N-[(9H-fluoren-9-ylmethoxy)carbonyl]-beta-alanyl}amino)-4-[(2,3,4-tri-O-acetyl-6-methyl-beta-D-glucopyranuronosyl)oxy]benzyl}oxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#64) A solution of 2-({N-[(9H-fluoren-9-ylmethoxy)carbonyl]-beta-alanyl}amino)-4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenylmethyl-2,3,4-tri-O-acetyl-beta-D-glucopyranosiduronic acid, #63 (compound 7 in WO2007 / 11968A2) (585 mg, 0.54 mmol) and 2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{ To a mixture of [(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Example #54 in US2013 / 0129753A1) (105 mg, 0.141 mmol) (412.0 mg, 0.554 mmol), HOAt (85 mg, 0.62 mmol), 2,6-lutidine (216 μL, 200 mg, 0.1.9 mmol), and N,N-diisopropylethylamine (327 μL, 250 mg, 1.9 mmol) were added. The orange reaction mixture was heated at 45° C. for 1.5 hours and then stirred at room temperature overnight. LCMS analysis indicated the reaction was complete. The mixture was diluted with dimethylsulfoxide and purified by reverse-phase chromatography using a modified version of purification method A (utilizing a gradient elution of 30-100% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to give the desired product #64 (307.9 mg, 37%) as a white solid. LCMS (Protocol C): m / z 1519.0 [M+H] + , retention time = 1.09 min. HPLC (Protocol B): retention time = 8.559 min.

[0307] Step 6. Synthesis of N-({[3-(beta-alanylamino)-4-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, acetate (#65) In an ice-cold water bath, N-[({3-({N-[(9H-fluoren-9-ylmethoxy)carbonyl]-beta-alanyl}amino)-4-[(2,3,4-tri-O-acetyl-6-methyl-beta-D-glucopyranuronosyl)oxy]benzyl}oxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl- To a solution of 3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, #64 (119.0 mg, 0.0784 mmol) in N,N-dimethylacetamide (2 mL) was added an aqueous solution of lithium hydroxide (461 μL, 46.1 mg, 1.10 mmol). After 10 min, LCMS analysis indicated the reaction was complete, and it was then neutralized with acetic acid, diluted with dimethylsulfoxide and methanol, and purified directly by reverse phase chromatography by a modified version of purification method A (utilizing a gradient elution of 15-70% acetonitrile in water containing 0.02% acetic acid over 20 min) to give the title compound #65 (34.8 mg, 36.5%) as a white solid. LCMS (Protocol A): m / z 1155.9 [M+H] + , retention time = 0.73 min; HPLC (protocol B): retention time = 5.532 min.

[0308] Step 7. Synthesis of N-({[4-(beta-D-glucopyranuronosyloxy)-3-({N-[6-({[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}amino)hexanoyl]-beta-alanyl}amino)benzyl]oxy}carbonyl)-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#66) N-({[3-(beta-alanylamino)-4-(beta-D-glucopyranuronosyloxy)benzyl]oxy}carbonyl)-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptane-4 To a solution of [6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-2-(methylsulfonyl)pyrimidine-5-carboxamide, #62 (9.2 mg, 0.022 mmol) in N,N-dimethylformamide (1 mL) was added N,N-diisopropylethylamine (30 μL, 22 mg, 0.17 mmol). The reaction was stirred at room temperature for 1.5 h. The crude reaction mixture was diluted with dimethylsulfoxide and purified directly by reverse-phase HPLC using a modified version of purification method A (utilizing a gradient elution of 15-90% acetonitrile in water containing 0.02% trifluoroacetic acid over 20 min) to afford the title compound #66 (3.7 mg, 13.5%) as a white solid. LCMS (Protocol E): m / z 1453.4 [M+H + ], retention time = 0.84 min; HPLC (protocol B): retention time = 6.225 min.

[0309] [ka]

[0310] Step 1. Synthesis of tert-butyl [16-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-13,16-dioxo-3,6,9-trioxa-12-azahexadec-1-yl]carbamate (#67) A solution of 1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (0.10 g, 0.25 mmol) and tert-butyl(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate (0.073 g, 0.25 mmol) in dichloromethane (5 mL) was treated with N,N-diisopropylethylamine (0.05 mL, 0.3 mmol) and the solution was stirred at room temperature. After approximately 1 h, the reaction was concentrated and purified on 12 g of silica gel, eluting with 0-20% methanol in dichloromethane over 25 min. Product-containing fractions were combined and concentrated to give the desired compound, #67, (0.12 g, 83%) as a colorless oil. LCMS (Protocol P): m / z 580.5 [M+H] + , retention time=1.75 minutes

[0311] Step 2. Synthesis of N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)-4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanamide (#68) A solution of tert-butyl [16-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-13,16-dioxo-3,6,9-trioxa-12-azahexadec-1-yl]carbamate (0.119 g, 0.205 mmol) in dichloromethane (5 mL) was treated with 4 M HCl / dioxane solution (1 mL, 4 mmol) and the reaction was stirred at room temperature. After approximately 30 min, the reaction was concentrated and the residue was purified on 4 g of silica gel, eluting with 0-100% methanol in dichloromethane over 25 min. Product-containing fractions were combined and concentrated to give the desired compound, #68, (0.084 g, 79%) as a viscous oil. LCMS (Protocol O): m / z 480.4 [M+H] + , retention time=0.63 min

[0312] Step 3. Synthesis of N-[16-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-13,16-dioxo-3,6,9-trioxa-12-azahexadec-1-yl]-2-(methylsulfonyl)pyrimidine-5-carboxamide (#69) A solution of 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (0.032 g, 0.16 mmol) in dichloromethane (3 mL) was treated with HATU (0.060 g, 0.16 mmol) and N,N-diisopropylethylamine DIEA (0.05 mL, 0.31 mmol). After approximately 3 minutes, a solution of N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)-4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanamide (0.080 g, 0.16 mmol) in dichloromethane (2 mL) was added and the reaction was stirred at room temperature. After approximately 1.5-2 hours, the reaction was concentrated and the residue was purified on 12 g of silica gel, eluting with 0-20% methanol in dichloromethane over 25 minutes. The product-containing fractions were combined and concentrated to give the desired compound, #69 (0.070 g, 68%) as an oil. LCMS (Protocol P): m / z 664.5 [M+H] + , retention time = 1.46 min.

[0313] [ka]

[0314] Step 1. 6-Azide-N 2 Synthesis of -(tert-butoxycarbonyl)-N-[2-(dimethylamino)ethyl]-L-norleucine amide (#70) To a solution of 6-azido-N-(tert-butoxycarbonyl)-L-norleucine (4.9 g, 18 mmol) in anhydrous dichloromethane (80 mL) was added triethylamine (17.2 mL, 126 mmol) followed by T3P (16.6 mL, 27.9 mmol, 50% in EtOAc) at 0° C. and the mixture was stirred at 0° C. for 30 min. A solution of N,N-dimethylethane-1,2-diamine (2.38 g, 27.0 mmol) in anhydrous dichloromethane (20 mL) was added and the mixture was stirred at 0° C. and warmed to 15° C. overnight. The mixture was then quenched with saturated aqueous NaHCO3 and extracted with dichloromethane (3×100 mL). The combined organic extracts were washed with saturated aqueous NaHCO3 (100 mL), water (100 mL) and then concentrated. The resulting residue was purified on 40 g of silica gel and eluted with a gradient of 1:1 EtOAc:dichloromethane to 10:1 EtOAc:methanol. Product-containing fractions were pooled and concentrated to give the desired compound, #70 (3.15 g, 51% yield) as a yellow oil. LCMS (Protocol O): m / z 342.9 [M+H] + , retention time = 0.536 min.

[0315] Step 2. Synthesis of 3-[(2-{[6-azido-N-(tert-butoxycarbonyl)-L-norleucyl]amino}ethyl)(dimethyl)ammonio]propane-1-sulfonate (#71) 6-Azido-N 2A solution of -(tert-butoxycarbonyl)-N-[2-(dimethylamino)ethyl]-L-norleucine amide (3.98 g, 11.6 mmol) and 1,3-propane sultone (2.24 g, 18.4 mmol) in anhydrous EtOAc (70 mL) was added and the reaction mixture was stirred at 90° C. for 16 h under an atmosphere of nitrogen. Following this period, some white precipitate appeared and the mixture was allowed to cool to room temperature and stirred overnight. The white precipitate was then filtered, washed with anhydrous EtOAc (3×10 mL) and dried to give the desired product, #71 (4.31 g, 79.8%) as a white solid. LCMS (Protocol Q): m / z 466.3 [M+H] + , retention time=2.354 minutes

[0316] Step 3. Synthesis of 3-[{2-[(6-azido-L-norleucyl)amino]ethyl}(dimethyl)ammonio]-propane-1-sulfonate (#72) A solution of 3-[(2-{[6-azido-N-(tert-butoxycarbonyl)-L-norleucyl]amino}ethyl)(dimethyl)ammonio]propane-1-sulfonate (0.2 g, 0.4 mmol) in methanol (10.8 mL) and dichloromethane (2 mL) was treated with 4 M HCl / dioxane solution (1 mL, 4 mmol) and the solution was stirred overnight at room temperature under nitrogen atmosphere. The reaction was concentrated and used directly in the next step without purification. LCMS (Protocol O): m / z 365.2 [M+H] + , retention time=0.61 min

[0317] Step 4. Synthesis of 3-[{2-[(6-azido-N-{[2-(methylsulfonyl)pyrimidin-5-yl]carbonyl}-L-norleucyl)amino]ethyl}(dimethyl)ammonio]propane-1-sulfonate (#73) A solution of 3-[{2-[(6-azido-L-norleucyl)amino]ethyl}(dimethyl)ammonio]-propane-1-sulfonate hydrochloride (0.2 g, 0.5 mmol) in anhydrous dimethylsulfoxide (1.66 mL) was treated successively with N,N-diisopropylethylamine (0.193 g, 1.50 mmol, 0.3 mL), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (0.111 g, 0.55 mmol), and HATU (0.190 g, 0.50 mmol), and the mixture was stirred at room temperature for about 1 h. The mixture was then concentrated under vacuum without heating, and the resulting residue was purified on 12 g of silica gel, eluted with dichloromethane followed by a gradient of 0-100% methanol in dichloromethane over 20 min. Product-containing fractions were pooled and concentrated under a stream of nitrogen (without heat) to give the desired product, #73, as a yellow foam. LCMS (Protocol O): m / z 549.3 [M+H] + , retention time=0.49 min

[0318] Examples of antibody drug conjugates Conjugation Procedure A: Representative procedure for conjugation of a linker payload to an internal disulfide (natural cysteine ​​residue) of an antibody. Synthesis of H-(C)-#10. A commercially available Herceptin antibody (Genentech) solution in Dulbecco's phosphate buffered saline (DPBS, Lonza, pH 7.4) was reduced by adding 2.6 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 mM solution in DPBS). The reaction was incubated at 37° C. for 1 hour and then cooled to ambient temperature. Conjugation was performed by adding 7 equivalents of linker payload [compound #10, 10 mM solution in N,N-dimethylacetamide (DMA)]. Additional DMA was added to the reaction mixture to achieve a total organic solvent component of 15% (v / v) in the final reaction mixture. The reaction was incubated at ambient temperature for 1 hour. The reaction mixture was then desalted by GE Sephadex gel desalting column and DPBS (pH 7.4) eluent. The crude conjugate was purified by size exclusion chromatography (SEC) using a GE AKTA Explorer system with a GE Superdex200 (10 / 300GL) column and DPBS (pH 7.4) eluent.

[0319] Conjugation Procedure B: Representative example of conjugation of an antibody to an engineered cysteine ​​residue: Preparation of H-(E380C)-#30. A 20 mM tris(2-carboxyethyl)phosphine (TCEP) solution (133 μL, 100 equivalents) was added to the antibody (4.2 mg) such that the final antibody concentration was 5 mg / mL (0.84 mL final volume) in PBS buffer containing 5 mM EDTA (507 μL PBS / EDTA). The reaction was left at 37° C. for 1.5 hours, after which the antibody was buffer exchanged into PBS containing 5 mM EDTA using a 50 kDMW cut-off spin concentrator (3×2 mL washes per cycle, 10× concentration). The antibody thus obtained was diluted to 824 μL using PBS containing 5 mM EDTA, treated with a freshly prepared 50 mM solution of dehydroascorbic acid (DHA) in 1:1 PBS / ethanol (final DHA concentration, approx. 1 mM) and left at 4° C. overnight. The antibody / DHA mixture was buffer exchanged into PBS containing 5 mM EDTA using a 50 kDMW cut-off spin concentrator (3 x 2 mL washes per cycle, 10x concentration). The antibody thus obtained (90 μL, 1.5 mg Ab) was resuspended in PBS (204.3 μL) and N,N-dimethylacetamide (30 μL) and treated with 5.7 μL (5x linker payload relative to Ab) of 10 mM benzothiazole payload (compound 30) in DMA. After standing for 24 hours at 37°C, the material was buffer exchanged (as above) into PBS (3 x 2 mL washes per cycle, 10x concentration) to a final volume of 0.4 mL, DAR=1.6 and a concentration of 2.02 mg / mL.

[0320] Illustrative ADCs: As outlined in the tables below (Tables 1 and 2), conjugation of the 114th amino acid residue (H-(A114C)) was performed using the anti-CD33 mAb, CD33-11A1-v1417-hG1, and the anti-Her2 mAb, Her2-PT, using conjugation procedure A above and the linker payloads disclosed herein, and using conjugation procedure B above and the linker payloads disclosed herein. The above procedure was also performed for various anti-Her2 mAbs bearing engineered cysteine ​​residues at positions 290 and 334 (CD33-11A1-v1417-(K290C)-(K334C)) and positions 334 and 392 (CD33-11A1-v1417-(K334C)-(K392C)).

[0321] [Table 1-1]

[0322] [Table 1-2]

[0323] [Table 1-3]

[0324] [Table 1-4]

[0325] [Table 2-1]

[0326] [Table 2-2]

[0327] [Table 2-3]

[0328] Representative examples of GSH reversibility studies: To a solution of H-(E380C)-#30 (2.02 mg / ml in 50 mM pH 7.4 PBS buffer, 99 μL, 0.2 mg Ab) was added an additional 50 mM pH 7.4 PBS buffer (31.3 μL), followed by 25 mM aqueous glutathione (GSH) solution (2.7 μL) to yield a final protein concentration of 10 μM. A control sample (no GSH) was similarly prepared from 99 μL of ADC diluted to 10 μM in PBS. GSH-treated and control ADC samples were incubated at 37° C. and sampled at days 0, 4, and 6. Aliquots were reduced with excess TCEP and analyzed for loading by LC / MS. The results of this study, and further studies of other ADCs described herein, with site-specifically conjugated mc-MMAD (H-(E380C)-mcMMAD) and vc-0101 (H-(E380C-mcValCitPABC-Aur0101) as comparators, are shown in the table below (Table 3).

[0329] [Table 3]

[0330] In vitro cell assay procedure Cells expressing Her2-targets (BT474 (breast cancer), N87 (gastric cancer), MDA-MB-361-DYT2 (breast cancer)) or not expressing Her2 (MDA-MB-468, HT29), or cells expressing CD33-targets HL60, HEL92.1.7, NB4, or not expressing CD33 (raji) were added to 96-well cell culture plates 24 hours prior to treatment. Cells were treated in duplicate with 3-fold serial dilutions of antibody drug conjugates or free compound (i.e., antibody not conjugated to drug) at 10 concentrations. Cell viability was assessed 96 hours after treatment using CellTiter96® AQ ueous Cell proliferation was determined by One Solution cell proliferation MTS assay (Promega, Madison WI). Relative cell viability was determined as a percentage of untreated controls. IC50 values ​​were calculated with XLfit v4.2 (IDBS, Guildford, Surry, UK) using the four-parameter logistic model #203. Results are shown below in Table 4 (for Herceptin conjugates) or Table 5 (for CD33 conjugates).

[0331] [Table 4-1]

[0332] [Table 4-2]

[0333] [Table 5]

Claims

1. Compounds of formula (I) 【Chemistry 1】 or a pharma- ceutically acceptable salt or solvate thereof. [In the formula, Het is a monocyclic, bicyclic or polycyclic heteroaryl ring system having 1 to 4 heteroatoms, wherein the carbon atom on said ring system bonded to -SO 2 - is adjacent to at least one heteroatom on said ring system, said heteroatoms being independently selected from the group consisting of N, O and S; However, Het is 【Chemistry 2】 Neither of the above. m is 0, 1, 2, 3, 4, or 5; n is 0, 1, 2, 3, 4, or 5; p is 0, 1, or 2; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; r is 0, 1, or 2; s is 0, 1, or 2; t is 0 or 1; w is 1, E is defined as follows: When r is 2 and s is 1 or 2, each E is -C(R １ ) ２ -, -O-C(R １ ) ２ -C(R １ ) ２ -, and -C(R １ ) ２ -C(R １ ) ２ -O-, When r is 2 and s is 0, each E is -C(R １ ) ２ -, and -O-C(R １ ) ２ -C(R １ ) ２ - independently selected from the group consisting of When r is 0 or 1 and s is 1 or 2, each E is -C(R １ ) ２ -, and -C(R １ ) ２ -C(R １ ) ２ -O-, When r is 0 or 1 and s is 0, each E is independently -C(R １ ) ２ - is; Each R １ is H, C １ ~C ６ Linear or branched alkyl, C ２ ~C ６ Linear or branched alkenyl, and C ２ ~C ６ independently selected from the group consisting of linear or branched alkynyl; R ２ is C optionally substituted with halogen or haloalkyl １ ~C １０ C optionally substituted with alkyl, halogen or haloalkyl ５ ~C １２ is aryl, Each X is independently an amino acid, and each amino acid X is the same or different; each Y is independently an amino acid, and each amino acid Y is the same or different; each Z is independently a spacer element, each spacer element being the same or different; D is a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, -(OCH ２ CH ２ ) pH, -CO ２ H, -NH ２ , OH, -N ３ , 【Chemistry 3】 selected from the group consisting of a cytotoxic agent, a peptide, a protein, a nucleic acid, a growth factor, an antiviral agent, an immunological agent, and a fluorophore or other imaging agent; R is independently H or C １ ~C ６ is alkyl.

2. Compound of formula (II): 【Chemistry 4】 or a pharma- ceutically acceptable salt or solvate thereof. [In the formula, P is a peptide sequence containing at least one natural or engineered cysteine ​​residue; S is a sulfur atom in P; Het is a monocyclic, bicyclic or polycyclic heteroaryl ring system having 1 to 4 heteroatoms, and the carbon atom on said ring system bonded to -S(O 2 )- is adjacent to at least one heteroatom on said ring system, said heteroatoms being independently selected from the group consisting of N, O and S; However, Het is 【Chemistry 5】 Neither of the above. m is 0, 1, 2, 3, 4, or 5; n is 0, 1, 2, 3, 4, or 5; p is 0, 1, or 2; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; r is 0, 1, or 2; s is 0, 1, or 2; t is 0 or 1; w is 1, E is defined as follows: When r is 2 and s is 1 or 2, each E is -C(R １ ) ２ -, -O-C(R １ ) ２ -C(R １ ) ２ -, and -C(R １ ) ２ -C(R １ ) ２ -O-, When r is 2 and s is 0, each E is -C(R １ ) ２ -, and -O-C(R １ ) ２ -C(R １ ) ２ - independently selected from the group consisting of When r is 0 or 1 and s is 1 or 2, each E is -C(R １ ) ２ -, and -C(R １ ) ２ -C(R １ ) ２ -O-, When r is 0 or 1 and s is 0, each E is independently -C(R １ ) ２ - is; Each R １ is H, C １ ~C ６ Linear or branched alkyl, C ２ ~C ６ Linear or branched alkenyl, and C ２ ~C ６ independently selected from the group consisting of linear or branched alkynyl; Each X is independently an amino acid, and each amino acid X is the same or different; each Y is independently an amino acid, and each amino acid Y is the same or different; each Z is independently a spacer element, each spacer element being the same or different; D is a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, -(OCH ２ CH ２ ) pH, -CO ２ H, -NH ２ , OH, -N ３ , 【Chemistry 6】 selected from the group consisting of a cytotoxic agent, a peptide, a protein, a nucleic acid, a growth factor, an antiviral agent, an immunological agent, and a fluorophore or other imaging agent; R is independently H or C １ ~C ６ is alkyl, R ２ is C optionally substituted with halogen or haloalkyl １ ~C １０ C optionally substituted with alkyl, halogen or haloalkyl ５ ~C １２ is aryl.

3. Het is 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 3. The compound of claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof, selected from:

4. Het is 【Chemistry 10】 3. The compound of claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof, selected from:

5. D is a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, -(OCH ２ CH ２ ) p-, -CO ２ H, -NH ２ , OH, -N ３ , 【Chemistry 11】 is selected from the group consisting of R is independently H or C １ ~C ６ is alkyl, R ２ is C optionally substituted with halogen or haloalkyl １ ~C １０ C optionally substituted with alkyl, halogen or haloalkyl ５ ~C １２ is aryl, 3. A compound according to claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof.

6. 3. The compound of claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof, wherein D is a cytotoxic agent.

7. 7. The compound of claim 6, or a pharmaceutically acceptable salt or solvate thereof, wherein D is selected from the group consisting of anthracyclines, auristatins, splicostatins, CBI dimers, CPI dimers, CTI dimers; dimers comprising two different monomers selected from CBI, CPI and CTI; enediynes which may be selected from calicheamicins, esperamicins and neocarzinostatins; duocarmycins, geldanamycins, maytansines, puromycins, taxanes, vinca alkaloids, SN38, tubulysin, hemiasterin, camptothecins, combretastatins, dolastatins, indolinobenzodiazepine dimers, pyrrolobenzodiazepine dimers and pladienolides, and stereoisomers or isosteres thereof.

8. 7. The compound of claim 6, or a pharma- ceutically acceptable salt or solvate thereof, wherein D is selected from the group consisting of dolastatin, MMAD, MMAE, MMAF, PF-06380101, PF-06463377, PF-06456780, PF-06843982, PF-06874082, PF-06852321, PF-06757725, PF-06859944, and PF-06698970.

9. Z, Z Ａ - (Z Ｂ ) １～３ Or (Z Ｂ ) １～３ -Z Ａ and Z Ａ But, Z Ａ１ -Z Ａ２ Or Z Ａ２ -Z Ａ１ and Z Ａ１ is absent or is —C(O)—, —C(S)—, —C(O)NR—, —C(O)C １ ~C ６ Alkyl-, -C(O)NRC １ ~C ６ Alkyl-, -C １ ~C ６ Alkyl (OCH ２ CH ２ ) １～６ -, -C(O)C １ ~C ６ AlkylNRC(O)-, -C(O)C １ ~C ６ Alkyl (OCH ２ CH ２ ) １～６ -, -C １ ~C ６ Alkyl (OCH ２ CH ２ ) １～６ -C(O)-, -C １～ C ６ Alkyl-S-S-C １～ C ６ AlkylNRC(O)CH ２ -, -C １～ C ６ Alkyl (OCH ２ CH ２ ) １～６ NRC(O)CH ２ -, -C(O)C １～ C ６ Alkyl-NRC(O)C １～６ Alkyl-, -N=CR-phenyl-O-C １～ C ６ Alkyl-, -N=CR-phenyl-O-C １～ C ６ Alkyl-C(O)-, -C(O)-C １～ C ６ Alkyl (OCH ２ CH ２ ) １～６ NRC(O)-, -C(O)C １～ C ６ Alkyl-phenyl (NR-C(O)C １～ C ６ Alkyl) １～４ -, -C(O)C １～ C ６ Alkyl (OCH ２ CH ２ ) １～６ -NRC(O)C １～ C ６ Alkyl-, -C １～ C ６ Alkyl-, -S-, -C(O)-CH(NR-C(O)C １ ~C ６ Alkyl)-C １ ~C ６ Alkyl- and (-CH ２ -CH ２ -O-) １～２０ and one or more components selected from the group consisting of: Z Ａ２ but, 【Chemistry 12】 -C(O)(CH ２ ) ｎ C(O)— or absent, Z Ｂ does not exist or -C １～ C ６ Alkylene-, -NRC ３ ~C ８ -heterocyclylNR-, -NRC ３ ~C ８ -CarbocyclylNR-, -NRC １ ~C ６ AlkylNR-, -NR １ ~C ６ Alkylene-, -S-, -NR-, -NRNR-, -O(CR ２ ) １～４ S-S (CR ２ ) １～４ N(R)-,-NRC １ ~C ６ -AlkylenephenyleneNR-, -NRC １ ~C ６ Alkylenephenylene SO ２ NR-, -OC １～ C ６ Alkyl S-SC １～ C ６ AlkylC(COOR)NR-, -NRC(COOR)C １～ C ６ Alkyl S-SC １～ C ６ independently selected from the group consisting of alkylO-, R is independently H or C １ ~C ６ is alkyl, 3. A compound according to claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof.

10. Z, 【Chemistry 13】 3. The compound of claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof, wherein:

11. 11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10, or a pharma- ceutically acceptable salt or solvate thereof, and a pharma- ceutically acceptable excipient.

12. The pharmaceutical composition of claim 11 for use in treating cancer.

13. 13. The pharmaceutical composition of claim 12, wherein the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric (stomach) cancer, testicular cancer, leukemia and lymphoma.

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