Novel auristatin analogues and immunoconjugates thereof

Abstract

The present invention provides novel auristatin analogs and immunoconjugates thereof, as well as pharmaceutical compositions and methods of preparation and use for treating various diseases and disorders, such as cancer.

Description

[Technical field]

[0001] Priority claims and related patent applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 275,177, filed November 3, 2021, and U.S. Provisional Application No. 63 / 295,476, filed December 30, 2021, the entire contents of each of which are incorporated herein by reference.

[0002] TECHNICAL FIELD OF THEINVENTION The present invention relates generally to novel compounds and their therapeutic uses. More specifically, the present invention provides novel auristatin analogs and immunoconjugates thereof, as well as pharmaceutical compositions and methods of manufacture and use for treating various diseases and disorders, such as cancer. [Background technology]

[0003] 2. Background of the Invention Cytotoxic agents are commonly used chemotherapeutic agents due to their high cytotoxicity, often suffering from fast plasma clearance and low selectivity for cancer cells. Monoclonal antibody therapy is characterized by high selectivity and long plasma half-life, but often has limited cytotoxicity. Antibody-drug conjugates (ADCs) are a type of therapy with high cytotoxicity and long plasma half-life, and represent a promising therapeutic approach in cancer treatment. Gemtuzumab ozogamicin (Mylotarg), the first ADC approved by the FDA in 2000, has been shown to be effective in treating cancer. TMTo date, 11 ADCs have been approved by the FDA, including the 1990-1995 ADC (see, e.g., Drago et al. 2021 Nature Reviews 18, 327-344; Mckertish et al. 2021 Biomedicines 9, 872; Khongorzui et al. 2020 Molecular Cancer Res. 18:3-19; Bross et al. 2001 Clin. Cancer Res. 7, 1490-1496; Hamann et al. 2002 Bioconjug. Chem. 13, 47-58; Lamb, 2017 Drugs 77, 1603-1610.).

[0004] Auristatins are a family of complex analogues to the natural antitumor agent dolastatin 10. These cytotoxic agents are 100 to 1000 times more toxic than the traditional cancer chemotherapy drug doxorubicin. [ka]

[0005] Auristatins are believed to arrest cancer cells in mitosis and ultimately induce apoptosis. Auristatin-based ADCs have been the subject of clinical investigations in recent years, and some of them, such as brentuximab vedotin (Adcetris), the first approved in 2011. TM ) have been approved by the FDA (see, e.g., McGinn et al. 2012 Clin. Cancer Res. 18, 5845-5849; Deng et al. 2013 Clin. Cancer Res. 19, 22-27; U.S. Patent No. 6,884,869 B2; U.S. Patent No. 7,498,298 B2; WO 2015 / 095301 A2; WO 2015 / 151079 A2; WO 2015 / 151081 A2; WO 2016 / 123412 A1; WO 2011 / 097627 A1; WO 2001 / 018032 A2).

[0006] Despite significant progress in the clinical development of ADCs in recent years, their design and development present many challenges, including lack of stability, high tendency for aggregation and limited bioavailability, as well as the limited number of potent cytotoxic agents amenable for development.

[0007] There is a great need for novel auristatin analogs and novel auristatin-based immunoconjugates that are potent and amenable to development. Summary of the Invention

[0008] The present invention provides novel auristatin analogs with high cytotoxicity and favorable stability as well as other properties suitable for use in immunoconjugates. The auristatin analogs disclosed herein are characterized by a synergistic combination of a unique N-substitution at position 5 (P5) with a nearby anilino group for linking with a linker, and have additional variations at position 1 (P1) to fine-tune the payload to suit different ADC constructs and applications. These compounds are ideally suited as cytotoxic agents for the development of immunoconjugates as novel therapeutic agents against cancer due to their high potency, high stability, low immunogenicity, as well as enhanced permeability and sufficient solubility.

[0009] In one aspect, the present invention generally provides a compound of formula (I): [ka] (I) [During the ceremony, R 1 teeth [ka] where R 2 is unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R a , R b and R c are H and NR x R yis selected from, with the proviso that R a , R b and R c Only one of the following is NR x R y and each of the others is H; R x and R y Each of these is independently R, R r and L-R z is selected from, with the proviso that R x and R y One of them is LR z or R r if , then the other is R; R 5 is CR'3, where each R' is independently H or F; L is a linker; R r is (C=O)-O-(CH2) p -R v or (C=O)-(CH2) q -R v and; R v is R, OR, NHR, NR2, an aryl group or an amino acid; p is 0, 1, 2, 3, 4, 5 or 6; q is 0, 1, 2, 3, 4, 5 or 6; R z contains a functional or reactive group; and R is H or C1-C3 alkyl. or a pharma- ceutically acceptable salt thereof.

[0010] In another aspect, the invention generally relates to compounds disclosed herein, such as those of formula (I)-(V) herein. 6 ) and in Table 1 herein, or a pharma- ceutically acceptable salt thereof, and optionally a pharma- ceutically acceptable excipient, carrier, or diluent.

[0011] In yet another aspect, the present invention generally provides a compound of formula (VI): [ka] (VI) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth [ka] where R 2 is unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and R y Each of R and LR is independently z is selected from, with the proviso that R x and R y One of them is NR z if , then the other is R; R 5 is CR'3, where each R' is independently H or F; L is a linker; R is H or C1-C3 alkyl; and and i is an integer ranging from 1 to about 20. or a pharma- ceutically acceptable salt thereof.

[0012] In yet another aspect, the present invention generally provides a compound of formula (VII): [ka] (VII) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth [ka] where R 2 is unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and Ry Each of R and LR is independently z is selected from, with the proviso that R x and R y One of them is NR z if , then the other is R; R 5 is CR'3, where each R' is independently H or F; L is a linker; and R is H or C1-C3 alkyl; and j is an integer ranging from 1 to about 20. or a pharma- ceutically acceptable salt thereof.

[0013] In yet another aspect, the present invention generally provides a compound of formula (VIII): [ka] (VIII) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth [ka] where R 2 is unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl or cycloheteroalkyl; R x and R y Each of R and LR is independently z is selected from, with the proviso that R x and R y One of them is NR z if , then the other is R; R 5 is CR'3, where each R' is independently H or F; L is a linker; and R is H or C1-C3 alkyl; and k is an integer ranging from 1 to about 20. or a pharma- ceutically acceptable salt thereof.

[0014] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an immunoconjugate as disclosed herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0015] In yet another aspect, the invention generally relates to combinations comprising a therapeutically effective amount of an immunoconjugate disclosed herein and one or more therapeutically active co-agents and / or adjuvants.

[0016] In yet another aspect, the present invention generally relates to a method for treating or ameliorating a disease or condition comprising administering to a subject in need thereof a therapeutically effective amount of the immunoconjugates disclosed herein.

[0017] In yet another aspect, the invention generally relates to the use of the immunoconjugates disclosed herein for the manufacture of a medicament.

[0018] In yet another aspect, the present invention generally relates to the use of the immunoconjugates disclosed herein for use in the treatment of a disease or condition (e.g., cancer). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention The present invention is based in part on the discovery of novel auristatin analogs with favorable potency, stability and other profiles as payloads for immunoconjugates. Key structural improvements to existing auristatins include the synergistic combination of N-methyl substitution at P5 with a nearby anilino group for linker attachment. These modifications improve payload permeability and allow linker introduction via the C-terminus. Further fine-tuning of the payload molecule can be achieved by modifications at P1 to accommodate a wide range of ADC constructs and applications. For example, the anilino group allows for ADC constructs that are more prone to retain payload potency. Furthermore, the highly potent and stable cytotoxic agents also combine sufficient solubility and low immunogenicity, making them suitable for development as novel therapeutic agents for immunoconjugates and cancer.

[0020] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. General principles of organic chemistry and specific functional moieties and reactivities are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 2006.

[0021] The following terms are intended to have the following meanings, unless otherwise indicated according to the context in which the term is found.

[0022] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 16 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0023] As used herein, "at least" a particular value is understood to refer to that value and all values ​​greater than that value.

[0024] As used herein, "greater than 1" is understood to mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, etc., or any value therebetween.

[0025] In this specification and the appended claims, the singular terms "a," "an," and similar terms include plural referents unless the context clearly indicates otherwise.

[0026] Unless otherwise specified or clear from the context, the term "about" used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean value. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein can be modified by the term "about".

[0027] Unless otherwise stated or clear from the context, the term "or" as used herein is understood to be inclusive.

[0028] Any composition or method disclosed herein can be combined with any one or more of the other compositions and methods provided herein.

[0029] The reference to a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of groups listed in the list. The reference to an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0030] When used to define compositions and methods, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. When used to define compositions and methods, the term "consisting essentially of" is intended to mean that the compositions and methods include the recited elements and exclude other elements of essential importance to the compositions and methods. For example, "consisting essentially of" refers to the administration of a pharmacologically active substance that is expressly stated and excludes pharmacologically active substances that are not expressly stated. The term "consisting essentially of" does not exclude pharmacologically inactive or inactive agents, such as pharma- ceutical acceptable excipients, carriers, or diluents. When used to define compositions and methods, the term "consisting of" is intended to mean that it excludes trace elements and substantial method steps of other components. Embodiments defined by each of these transitional phrases are within the scope of the present invention.

[0031] Certain compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, atropisomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are intended to be included in the present invention. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess of either the R- or S-configuration. Of optically active compounds, it is often preferable to use one enantiomer to the substantial exclusion of the other enantiomer.

[0032] Isomeric mixtures containing any of a variety of isomeric ratios can be utilized according to the present invention. For example, when only two isomers are combined, mixtures containing isomeric ratios of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 are contemplated by the present invention. Those skilled in the art can readily appreciate that similar ratios are contemplated for more complex isomeric mixtures.

[0033] For example, if a particular enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, in which case the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group, such as an amino group, or an acidic functional group, such as a carboxyl group, a diastereomeric salt can be formed with an appropriate optically active acid or base, and the diastereomers thus formed can then be separated by fractional crystallization or chromatographic methods well known in the art, followed by recovery of the pure enantiomer.

[0034] Mixtures of isomers can be separated on the basis of the physical chemical differences of the components, into pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and / or fractional crystallization.

[0035] Definitions of certain functional groups and chemical terms are set forth in more detail below. When a range of values ​​is listed, it is intended to encompass each value and subrange within the range. For example, "C 1-6 Alkyl" is C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C4-6 , C 4-5 , and C 5-6 Alkyl is intended to be included.

[0036] Where substituents are defined by a conventional chemical formula written from left to right, they equally encompass the chemically identical substituents which would result if the structure were written from right to left, e.g., -C(=O)-O- is equivalent to -OC(=O)-.

[0037] The structure of the compound of the present invention is restricted by the principles of chemical bonding known to those skilled in the art.Therefore, when a group can be substituted with one or more of a large number of substituents, such substitutions are selected to comply with the principles of chemical bonding and to obtain a compound known to those skilled in the art that is not inherently unstable and / or likely to be unstable under ambient conditions (e.g., aqueous, neutral, and some known physiological conditions).

[0038] As used herein, the term "alkyl" refers to a straight or branched hydrocarbon chain radical, consisting solely of carbon and hydrogen atoms, containing no unsaturation, and having from 1 to 10 carbon atoms (e.g., C 1-10 In this specification, whenever a numerical range such as "1 to 10" appears, it refers to each integer within the given range; for example, "1 to 10 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, but this definition also extends to the occurrence of the term "alkyl" without a numerical range specified. In some embodiments, "alkyl" refers to C 1-6It can be an alkyl group. In some embodiments, the alkyl group has 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, and the like. The alkyl is attached to the parent molecule by a single bond. Unless stated otherwise in the specification, an alkyl group can be acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R a )3, -OR a , -SR a , -OC(O)-R a , -N(R a )2, -C(O)R a , -C(O)OR a , -OC(O)N(R a )2, -C(O)N(R a )2, -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a )2, -N(R a )C(NRa )N(R a )2, -N(R a )S(O) t N(R a )2 (where t is 1 or 2), -P(=O)(R a )(R a ), or -OP(=O)(OR a )2(where each R a is independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein. In non-limiting embodiments, the substituted alkyl can be selected from fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, and phenethyl.

[0039] As used herein, the term "alkoxy" refers to an alkoxy group having 1 to 10 carbon atoms (C 1-10 "O-alkyl" refers to an -O-alkyl group, including any straight chain, branched, saturated cyclic form, and combinations thereof, attached to the parent molecular structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy, cyclopropyloxy, cyclohexyloxy, and the like. "Lower alkoxy" refers to an alkoxy group containing from 1 to 6 carbons. In some embodiments, C 1-3Alkoxy is an alkoxy group that includes both straight and branched chain alkyls of 1 to 3 carbon atoms. Unless otherwise stated in the specification, alkoxy groups include acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R a )3, -OR a , -SR a , -OC(O)-R a , -N(R a )2, -C(O)R a , -C(O)OR a , -OC(O)N(R a )2, -C(O)N(R a )2, -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a )2, -N(R a )C(NR a )N(R a )2, -N(R a )S(O) t N(R a )2 (where t is 1 or 2), -P(=O)(R a )(R a ), or -OP(=O)(OR a )2(where each R ais independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein.

[0040] As used herein, the term "aromatic" or "aryl" refers to a ring system having 6 to 14 ring atoms (e.g., C 6-14 Aromatic or C 6-14 In some embodiments, aryl refers to a radical having C 6-10It is an aryl radical. For example, a divalent radical formed from a substituted benzene derivative and having a free valence at a ring atom is named a substituted phenylene radical. In another embodiment, a divalent radical derived from a monovalent polycyclic hydrocarbon radical whose name ends with "-yl" by removing one hydrogen atom from a carbon atom having a free valence is named by adding "-idene" to the name of the corresponding monovalent radical, for example, a naphthyl group with two attachment points is called naphthylidene. Whenever a numerical range such as "6 to 14 aryl" appears in this specification, it means each integer within the given range; for example, "6 to 14 ring atoms" means that the aryl group can consist of 6 ring atoms, 7 ring atoms, etc. (up to and including 14 ring atoms). The term includes monocyclic or fused ring polycyclic (i.e., rings sharing adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicyclic, tricyclic, tetracyclic, etc. In polycyclic groups, only one ring is required to be aromatic, thus groups such as indanyl are included in the definition of aryl. Non-limiting examples of aryl groups include phenyl, phenalenyl, naphthalenyl, tetrahydronaphthyl, phenanthrenyl, anthracenyl, fluorenyl, indolyl, indanyl, and the like. Unless otherwise stated in the specification, an aryl moiety can be acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R a )3, -OR a , -SR a , -OC(O)-R a , -N(R a )2, -C(O)R a , -C(O)OR a, -OC(O)N(R a )2, -C(O)N(R a )2, -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a )2, -N(R a )C(NR a )N(R a )2, -N(R a )S(O) t N(R a )2 (where t is 1 or 2), -P(=O)(R a )(R a ), or -OP(=O)(OR a )2(where each R a is independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein.

[0041] As used herein, the terms "cycloalkyl" and "carbocyclyl" refer to monocyclic or polycyclic radicals, respectively, that contain only carbon and hydrogen and may be saturated or partially unsaturated. Unless otherwise stated in the specification, the terms are intended to include both substituted and unsubstituted cycloalkyl groups. Partially unsaturated cycloalkyl groups may be referred to as "cycloalkenyl" if the carbocyclic ring contains at least one double bond, or "cycloalkynyl" if the carbocyclic ring contains at least one triple bond. Cycloalkyl groups are groups having from 3 to 13 ring atoms (i.e., C 3-13In this specification, whenever a numerical range such as "3 to 10" appears, it refers to each integer within the stated range; for example, "3 to 13 carbon atoms" means that the cycloalkyl group can consist of 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, etc. up to and including 13 carbon atoms. The term "cycloalkyl" also includes bridged and spiro-fused ring structures that do not contain heteroatoms. The term also includes monocyclic or fused ring polycyclic (i.e., rings that share adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicyclic, tricyclic, tetracyclic, etc. In some embodiments, "cycloalkyl" refers to C 3-8 Specific examples of cycloalkyl groups include, but are not limited to, the following moieties: 3-6 Carbocyclyl groups include, but are not limited to, cyclopropyl (C3), cyclobutyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. 3-7 Examples of carbocyclyl groups include norbornyl (C7). 3-8 Examples of the carbocyclyl group are those shown in the above C 3-7 Examples include, but are not limited to, carbocyclyl groups, as well as cycloheptyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, and the like. 3-13 Examples of carbocyclyl groups are the aforementioned C 3-8Unless stated otherwise in the specification, cycloalkyl groups include acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R a )3, -OR a , -SR a , -OC(O)-R a , -N(R a )2, -C(O)R a , -C(O)OR a , -OC(O)N(R a )2, -C(O)N(R a )2, -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a )2, -N(R a )C(NR a )N(R a )2, -N(R a )S(O) t N(R a )2 (where t is 1 or 2), -P(=O)(R a )(R a ), or -OP(=O)(OR a )2(where each R aare independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which may be optionally substituted as defined herein. The terms "cycloalkenyl" and "cycloalkynyl" reflect the above description of "cycloalkyl", where the prefix "alk" is replaced with "alkene" or "alkyne", respectively, and the parent "alkenyl" or "alkynyl" terms are as described herein. For example, cycloalkenyl groups can have 3 to 13 ring atoms, for example 5 to 8 ring atoms. In some embodiments, cycloalkynyl groups can have 5 to 13 ring atoms.

[0042] The term "heterocycloalkyl" as used herein refers to a cycloalkyl radical having one or more skeletal atoms selected from atoms other than carbon, such as O, N, S, P, or combinations thereof. Unless otherwise stated in the specification, the term is intended to include both substituted and unsubstituted heterocycloalkyl groups. Specific examples of heterocycloalkyl include 2-hydroxy-aziridin-1-yl, 3-oxo-1-oxacyclobutan-2-yl, 2,2-dimethyl-tetrahydrofuran-3-yl, 3-carboxy-morpholin-4-yl, 1-cyclopropyl-4-methyl-piperazin-2-yl, 2-pyrrolinyl, 3-pyrrolinyl, dihydro-2H-pyranyl, 1,2,3,4-tetrahydropyridine, 3,4-dihydro-2H-[1,4]oxazine, and the like.

[0043] The term "halogen" as used herein refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). The term "halide" or "halo" as used herein means fluoro, chloro, bromo, or iodo. The terms "haloalkyl", "haloalkenyl", "haloalkynyl" and "haloalkoxy" include alkyl, alkenyl, alkynyl and alkoxy structures substituted with one or more halo groups or combinations thereof. For example, the terms "fluoroalkyl" and "fluoroalkoxy" each include haloalkyl and haloalkoxy groups in which the halo is fluorine, such as, but not limited to, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. Each of the alkyl, alkenyl, alkynyl and alkoxy groups is as defined herein and can be optionally further substituted as defined herein.

[0044] The term "heteroatom" as used herein refers to oxygen (O), nitrogen (N), sulfur (S) and phosphorus (P).

[0045] The term "heteroalkyl" as used herein refers to an alkyl radical having one or more skeletal atoms selected from atoms other than carbon, such as oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), or combinations thereof. For example, C 1-4Numerical ranges may be given to refer to the overall chain length of a heteroalkyl, such as 4 atoms long in this example. For example, the -CH2OCH2CH3 radical is referred to as a "C4" heteroalkyl, and includes the heteroatom center in the description of the atom chain length. The bond to the parent molecular structure may be through either a heteroatom or a carbon in the heteroalkyl chain. For example, an N-containing heteroalkyl moiety refers to a group in which at least one of the backbone atoms is a nitrogen atom. One or more heteroatoms in a heteroalkyl radical may be optionally oxidized. If one or more nitrogen atoms are present, they may also be optionally quaternized. For example, heteroalkyl also includes a backbone chain substituted with one or more nitrogen oxide (-O-) substituents. Exemplary heteroalkyl groups include, but are not limited to, ethers such as methoxyethanyl (-CH2CH2OCH3), ethoxymethanyl (-CH2OCH2CH3), (methoxymethoxy)ethanyl (-CH2CH2OCH2OCH3), (methoxymethoxy)methanyl (-CH2OCH2OCH3), and (methoxyethoxy)methanyl (-CH2OCH2CH2OCH3); amines such as CH2CH2NHCH3, -CH2CH2N(CH3)2, -CH2NHCH2CH3, -CH2N(CH2CH3)(CH3).

[0046] The term "heteroaryl" or "heteroaromatic" as used herein refers to a radical of a 5-18 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic, tetracyclic, etc.) aromatic ring system (e.g., having 6, 10 or 14 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-6 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, phosphorus and sulfur ("5-18 membered heteroaryl"). A heteroaryl polycyclic ring system may contain one or more heteroatoms in one or both rings. Whenever a numerical range such as "5 to 18" appears herein, it refers to each integer within the given range; for example, "5 to 18 ring atoms" means that the heteroaryl group may consist of 5 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms. In some instances, a heteroaryl may have 5 to 14 ring atoms. In some embodiments, heteroaryl has a divalent radical, e.g., derived from a monovalent heteroaryl radical whose name ends in "-yl" by removing a hydrogen atom from the atom having the free valence, and named by adding "-ene" to the name of the corresponding monovalent radical (e.g., a pyridyl group having two points of attachment is a pyridylene).

[0047] For example, an N-containing "heteroaromatic" or "heteroaryl" moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom. One or more heteroatoms in a heteroaryl radical can be optionally oxidized. One or more nitrogen atoms, if present, can also be optionally quaternized. Heteroaryl also includes ring systems substituted with one or more nitrogen oxide (-O-) substituents, such as pyridinyl N-oxide. Heteroaryl is attached to the parent molecular structure through any atom of the ring.

[0048] "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more aryl groups, where the point of attachment to the parent molecular structure is on either the aryl or heteroaryl ring, or a heteroaryl ring, as defined above, is fused to one or more cycloalkyl or heterocyclyl groups, where the point of attachment to the parent molecular structure is on the heteroaryl ring. In the case of polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment to the parent molecular structure can be on either ring, i.e., on either ring with a heteroatom (e.g., 2-indolyl) or on the ring without a heteroatom (e.g., 5-indolyl). In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, phosphorus, and sulfur ("5-10 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, phosphorus, and sulfur ("5-8 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, phosphorus, and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur.

[0049] Examples of heteroaryl are azepinyl, acridinyl, benzimidazolyl, benzoindolyl, 1,3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzopyranonyl, and benzofuraza. nyl, benzothiazolyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzo[6,7]cyclohepta[1,2-c]pyridazinyl, Zofuranil, dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8- Methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2 ,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e., thienyl). Unless otherwise stated in the specification, a heteroaryl moiety can be any of acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R, a )3, -OR a , -SR a , -OC(O)-R a , -N(R a )2, -C(O)R a , -C(O)OR a , -OC(O)N(R a )2, -C(O)N(R a )2, -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a )2, -N(R a )C(NR a )N(Ra )2, -N(R a )S(O) t N(R a )2 (where t is 1 or 2), -P(=O)(R a )(R a ), or -OP(=O)(OR a )2(where each R a is independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein.

[0050] The terms "administer" and "administration" as used herein refer to oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intracranial administration, inhalation administration, intraocular administration, intranasal administration, subcutaneous administration, or implantation of a sustained release device (e.g., a mini-osmotic pump) to a subject. The route of administration suitable for a particular patient will depend on the nature and severity of the disease or condition being treated, or the nature of the therapy being used and the nature of the active compound.

[0051] Administration can be by any suitable route, including parenteral and transmucosal (e.g., buccal, sublingual, palate, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0052] As used herein, the term "co-administration" refers to the simultaneous presence of two agents in the blood. The two agents may be administered simultaneously or sequentially.

[0053] As used herein, the term "affinity" refers to the strength of the interaction between an antigen-binding moiety (eg, an antibody) and an antigen at a single antigenic site.

[0054] The term "agonist" as used herein refers to a compound that can combine with a receptor to produce a cellular response. An agonist can be a ligand that directly binds to a receptor. Alternatively, an agonist can bind to a receptor indirectly, for example, (a) by forming a complex with another molecule that directly binds to the receptor, or (b) by modifying another compound so that the other compound directly binds to the receptor.

[0055] The term "antagonist" as used herein refers to a compound that competes with an agonist or inverse agonist for binding to a receptor, thereby blocking the action of the agonist or inverse agonist on the receptor. However, antagonists do not affect constitutive receptor activity.

[0056] The term "amino acid" as used herein refers to a molecule of the general formula NH2-CHR-COOH, where "R" is one of a number of different side chains, or a residue in a peptide having a parent amino acid. Amino acids include naturally occurring amino acids where "R" is a substituent found in naturally occurring amino acids. "R" can also be a substituent not found in naturally occurring amino acids. The term "amino acid residue" refers to the portion of an amino acid that remains after the amino acid loses a water molecule when it combines with another amino acid. The term "modified amino acid" refers to an amino acid having an "R" substituent that does not correspond to one of the 20 genetically encoded amino acids.

[0057] The term "antigen" as used herein means any substance that causes the immune system to produce antibodies or a specific cell-mediated immune response against it. Disease-associated antigen means any substance associated with any disease that causes the immune system to produce antibodies or a specific cell-mediated immune response against it. An antigen can be recognized by the immune system and / or can induce a humoral and / or cellular immune response that activates B and / or T lymphocytes. An antigen can have one or more epitopes (B cell epitopes and / or T cell epitopes). An antigen preferably reacts, typically highly selectively, with a corresponding antibody or TCR and not with a large number of other antibodies or TCRs that may be elicited by other antigens. An antigen as used herein may also be a mixture of several individual antigens.

[0058] As used herein, the term "antigen-binding portion" refers to a portion capable of specifically binding to an antigen, including, but not limited to, antibodies and antibody fragments, peptides and small molecule ligands.

[0059] The term "antibody" as used herein refers to a molecule capable of binding an epitope or antigenic determinant. The term is meant to include whole antibodies and antigen-binding fragments thereof. The term encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, Fabs antibodies, Fvs antibodies, single chain antibodies, single or multiple immunoglobulin variable chain or CDR domain designs, as well as bispecific and multispecific antibodies. The antibody may be from any animal. Preferably, the antibody is mammalian, such as human, mouse, rabbit, goat, guinea pig, camel, horse, etc., or other suitable animal. The antibody may recognize a polypeptide or polynucleotide antigen. The term includes, for example, antigen-binding fragments of immunoglobulins, active fragments including the variable and / or constant regions of the heavy chain, the variable and / or constant regions of the light chain, the complementarity determining regions (cdrs), and the framework regions. The term includes polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, modified antibodies, chimeric antibodies, hybrid antibody molecules, F(ab)2 and F(ab) fragments; Fv molecules (e.g., non-covalent heterodimers), dimeric and trimeric antibody fragment constructs; minibodies, humanized antibody molecules, and any functional fragments derived from such molecules, which fragments retain specific binding.

[0060] The term "antigen-binding fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen, e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution.

[0061] Examples of binding fragments are single chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, V L , V H , C L and C H a monovalent fragment consisting of one domain; F(ab)2 fragment, a bivalent fragment containing two Fab fragments disulfide-bonded at the hinge region; V H and an Fd fragment consisting of the CH1 domain; L and VH Fv fragment consisting of domains; V H These include, but are not limited to, dAb fragments consisting of domains (Ward et al. 1989 Nature 341:544- 546); as well as isolated complementarity determining regions (CDRs) or other epitope-binding fragments of antibodies.

[0062] In addition, the two domains of the Fv fragment, V L and V H Using recombinant methods, V L and V H The domains can be linked by a synthetic linker that allows them to be produced as a single protein chain that pairs to form a monovalent molecule (known as single-chain Fv ("scFv"); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al. 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment." These antigen-binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0063] Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, 2005 Nature Biotechnology 23:1 126-1136.). Antigen-binding fragments can be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see, e.g., U.S. Patent U.S. Pat. No. 6,703,199, which describes monobodies ... H -C H 1-V H -C H1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8:1057-1062; U.S. Patent No. 5,641,870).

[0064] The term "bispecific antibody" or "bispecific" as used herein refers to an antibody, typically a monoclonal antibody, that has binding specificities for at least two different antigenic epitopes. The epitopes may be from the same antigen or from two different antigens. Methods for producing bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using coexpression of two immunoglobulin heavy / light chain pairs. Alternatively, bispecific antibodies can be prepared using chemical conjugation. Bispecific antibodies also include bispecific antibody fragments (see, for example, Milstein et al. 1983 Nature 305:537-39; Brennan et al. 1985 Science 229:81; Hollinger et al. 1994 Proc. Natl. Acad. Sci. USA 90:6444-48; Gruber et al. 1994 J. Immunol. 152:5368-74).

[0065] The term "chimeric antibody" or "chimera" as used herein refers to antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies so long as they specifically bind to a target antigen and / or exhibit the desired biological activity.

[0066] The term "human antibody" as used herein refers to an antibody having variable regions in which both framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a human sequence, for example, an antibody containing a human germline sequence, or a mutated version of a human germline sequence or a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al. 2000 J. Mol. Biol.296:57-86). A human antibody may contain amino acid residues that are not encoded by human sequences, for example, mutations introduced by random or site-specific mutagenesis in vitro, or mutations introduced by somatic mutation in vivo, or substitutions to promote stability or production.

[0067] The term "humanized antibody" as used herein refers to an antibody that contains non-human (e.g., murine) and human antibody sequences. Such antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In general, a humanized antibody contains 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 FR regions being of human immunoglobulin sequences. A humanized antibody also optionally contains at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region (Fc) (see, e.g., Cabilly US Patent 4,816,567; Queen et al. 1989 Proc. Nat'l Acad. Sci. USA 86:10029-10033; ANTIBODY ENGINEERING: A PRACTICAL APPROACH, Oxford University Press 1996).

[0068] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies (i.e., the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in minor amounts). Monoclonal antibodies are highly specific and directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of antibodies directed against (or specific for) different epitopes. "Monoclonal" refers to the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by a particular method. For example, monoclonal antibodies used according to the present invention can be produced by various methods known in the art, including the hybridoma method first described by Kohler et al. 1975 Nature 256: 495, or can be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries, for example, using the techniques described in Clackson et al. 1991 Nature 352: 624-628 and Marks et al. 1991 J. Mol. Biol.222:581-597. These monoclonal antibodies generally bind with a Kd of at least about 1 μM, more generally at least about 300 nM, typically at least about 30 nM, and preferably at least about 10 nM.

[0069] As used herein, the term "biologically active" or "biologically active" refers to those that have a structural, regulatory, or biochemical function of a naturally occurring molecule, or any function related to or associated with a metabolic or physiological process. Biologically active polypeptides or fragments thereof include those that can participate in a biological process or reaction and / or produce a desired effect. Biological activity can include improved desired activity or decreased undesirable activity. For example, an entity exhibits biological activity if it participates in a molecular interaction with another molecule, has therapeutic value in alleviating a disease state, has preventive value in inducing an immune response, or has diagnostic and / or prognostic value in determining the presence of a molecule. Biologically active proteins or polypeptides can be naturally occurring, can be synthesized (e.g., by recombinant or chemical synthesis) from known components, and can include heterologous components.

[0070] The terms "cancer" and "cancerous" as used herein refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, sarcoma, blastoma, and leukemia. More specific examples of such cancer include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

[0071] The term "cleavable" linker as used herein refers to a linker or linker component that covalently links two moieties but degrades under physiologically relevant conditions to cleave the covalent bond between the moieties. Typically, cleavable linkers are cleaved more quickly in an intracellular environment than in an extracellular environment in vivo, and release of the payload occurs preferentially within the target cell. Cleavage may be enzymatic or non-enzymatic. The payload is typically released from the antibody without degrading the antibody. Cleavage may leave a portion of the linker or linker component attached to the payload, or may release the payload without any remaining linker portion or component (i.e., traceless release).

[0072] The term "non-cleavable" linker as used herein refers to a linker or linker component that is not particularly susceptible to degradation under physiological conditions, i.e., a linker that is at least as stable as the antibody or antigen-binding fragment portion of the immunoconjugate. Such linkers are sometimes referred to as "stable", meaning that they are sufficiently resistant to degradation to keep the payload attached to the antigen-binding moiety until the antigen-binding moiety itself is at least partially degraded. In such cases, in vivo degradation of the Ab precedes cleavage of the linker. Degradation of the antibody portion of an immunoconjugate with a stable or non-cleavable linker may leave some or all of the linker and one or more amino acid groups from the antibody attached to the payload or drug moiety to be delivered in vivo.

[0073] The term "cell" as used herein refers to any group of such cells in a prokaryotic, eukaryotic, primary or immortalized cell line, tissue or organ, etc. Preferably, the cells are of mammalian (e.g., human) origin and can be infected by one or more pathogens.

[0074] The terms "cytotoxic agent" and "payload" are used interchangeably herein and refer to a compound or substance that inhibits or blocks or stops the expression activity of a cell, the function of a cell, and / or causes the destruction of a cell. The terms are intended to include radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof.

[0075] As used herein, the terms "disease," "condition," or "disorder" are used interchangeably herein and refer to a pathological condition, e.g., a condition that can be identified by symptoms or other distinguishing factors as deviating from a healthy or normal state. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative diseases, inflammatory diseases, immune diseases, metabolic diseases, infectious diseases, and ischemic diseases.

[0076] The term "homology" or "homology" as used herein refers to sequence similarity between two polypeptides or two polynucleotides. Similarity can be determined by comparing positions of each sequence that can be aligned for purposes of comparison. If a given position of two polypeptide sequences is not identical, the similarity or conservation of that position can be determined by evaluating the similarity of the amino acids at that position. The degree of similarity between sequences is a function of the number of matching or homologous positions that the sequences share. Alignment of two sequences to determine percent sequence similarity can be performed using software programs known in the art, for example, as described in Ausubel et al. 1999 Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD. The term "homolog" to a given amino acid or nucleic acid sequence is intended to indicate that the corresponding sequence of the "homolog" has substantial identity or homology with the given amino acid or nucleic acid sequence.

[0077] In sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0078] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al. 1977 Nuc. Acids Res. 25:3389-3402 and Altschul et al. 1990 J. Mol. Biol. 215:403-410, respectively. The BLAST software is publicly available through the National Center for Biotechnology Information on the World Wide Web at ncbi.nlm.nih.gov / . Both default and non-default parameters can be used. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) uses as defaults an alignment (B) of 50, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0079] The term "identical" or percent "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 70% identity over a particular region, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity when compared and aligned for maximum correspondence over a comparison window or designated region) as determined by the BLAST or BLAST 2.0 sequence comparison algorithms using default parameters as described below, or by manual alignment and visual inspection. Such sequences are said to be "substantially identical". This definition also refers to or can be applied to the complement of a test sequence. This definition also includes sequences that have deletions and / or additions as well as sequences that have substitutions. As described below, preferred algorithms can take into account gaps, etc. Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and often over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length, or over the entire length of the amino acid or nucleic acid sequence.

[0080] The compound of the present invention can be administered alone or co-administered to a patient. Co-administration means to include simultaneous or sequential administration alone or in combination (of more than one compound or agent).Therefore, if desired, the formulation can also be combined with other active substances (e.g., to reduce metabolic degradation).

[0081] The compositions of the present invention can be delivered by topical route transdermally by being formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powders, dragees, capsules, liquids, troches, cachets, gels, syrups, slurries, suspensions, and the like, suitable for ingestion by the patient. Solid preparations include powders, tablets, pills, capsules, cachets, suppositories, and granules. Liquid preparations include solutions, suspensions, and emulsions, gels, such as water or water / propylene glycol solutions.

[0082] The compositions of the present invention may further comprise components for providing sustained release and / or comfort. Such components include high molecular weight anionic mucus mimetic polymers, gelling polysaccharides and finely divided drug carrier substrates. These components are discussed in more detail in U.S. Patent Nos. 4,911,920, 5,403,841, 5,212,162 and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention may also be delivered as microspheres for sustained release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres for slow subcutaneous release (see Rao, 1995 J. Biomater Sci. Polym. Ed. 7:623-645); as biodegradable and injectable gel formulations (see, e.g., Gao 1995 Pharm. Res. 12:857-863); or as microspheres for oral administration (see, e.g., Eyles 1997 J. Pharm. Pharmacol. 49:669-674).

[0083] As used herein, the term "in need of" a treatment refers to a subject who will benefit biologically, medically or in quality of life from such treatment.

[0084] As used herein, the term "specifically binds" or "selectively binds", when used in the context of describing the interaction between an antigen (e.g., a protein or glycan) and an antibody, antibody fragment, or antibody-derived binder, refers to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biologics, such as a biological sample (e.g., blood, serum, plasma, or tissue sample). Thus, under a specified immunoassay condition, an antibody or binder with a particular binding specificity binds to a particular antigen at least twice as much as background and does not bind substantially significantly to other antigens present in the sample. In certain embodiments, under a specified immunoassay condition, an antibody or binder with a particular binding specificity binds to a particular antigen at least 10 times as much as background and does not bind substantially significantly to other antigens present in the sample. In order to specifically bind to an antibody or binder under such conditions, the antibody or agent may need to be selected for its specificity for a particular protein. If desired or appropriate, this selection can be achieved by excluding antibodies that cross-react with molecules of other species (e.g., mouse or rat) or other subtypes. Alternatively, in some embodiments, an antibody or antibody fragment is selected that cross-reacts with a desired molecule.

[0085] A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein. (See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to measure specific immunoreactivity.) Typically, a specific or selective binding reaction produces a signal that is at least two times the background signal, and more typically at least 10 to 100 times the background signal.

[0086] The term "therapeutically effective amount" as used herein refers to a dose of a therapeutic agent or drug sufficient to achieve the intended therapeutic effect with minimal or no undesirable side effects. A therapeutically effective amount can be easily determined by a skilled physician, for example, by administering a low dose of the drug initially, and then gradually increasing the dose until the desired therapeutic effect is achieved with minimal or no undesirable side effects.

[0087] The terms "immunoconjugate" and "antibody-drug conjugate" are used interchangeably herein and refer to a compound having an antigen-binding moiety (e.g., an antibody or antigen-binding fragment thereof, a peptide or a small molecule ligand) linked to a cytotoxic agent or payload. The linkage may be covalent or non-covalent, and may include chelation. Thus, the terms "immunoconjugate" and "antibody-drug conjugate" include peptide-drug conjugates and small molecule-drug conjugates. A variety of linkers and linking strategies are known in the art and can be used to form immunoconjugates.

[0088] The terms "inhibition", "inhibit" and "inhibitory" as used herein with respect to biological target-inhibitor interactions refer to negatively affecting (e.g., decreasing) the activity or function of a protein compared to the activity or function of the protein in the absence of an inhibitor. In certain embodiments, inhibition means negatively affecting (e.g., decreasing) the concentration or level of a protein compared to the concentration or level of the protein in the absence of an inhibitor. In certain embodiments, suppression refers to the alleviation of a disease or a symptom of a disease. In certain embodiments, inhibition refers to a decrease in the activity of a particular protein target. Inhibition includes at least partially, partially or totally blocking a stimulus, reducing, preventing or delaying activation, or inactivating, desensitizing or downregulating signal transduction or enzyme activity or the amount of a protein. In certain embodiments, inhibition refers to a decrease in the activity of a target protein due to a direct interaction (e.g., an inhibitor binds to a target protein). In certain embodiments, inhibition refers to a decrease in the activity of a target protein resulting from an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing activation of the target protein).

[0089] The terms "isolated" or "purified" as used herein refer to a material that is substantially or essentially free from components that normally accompany it in its natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis and high performance liquid chromatography. The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. However, an isolated antibody that specifically binds to an antigen may have cross-reactivity to other antigens. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0090] The term "modulate" as used herein refers to directly or indirectly increasing or decreasing, stimulating, inhibiting, interfering, or blocking measured activity when compared to a suitable control. A "modulator" of a polypeptide or polynucleotide refers to a substance that affects, e.g., increases, decreases, stimulates, inhibits, interferes, or blocks, the measured activity of a polypeptide or polynucleotide when compared to a suitable control. For example, a "modulator" can bind to a target with measurable affinity and / or activate or inhibit a target, or directly or indirectly affect the normal control of receptor activity.

[0091] As used herein, a "pharmaceutically acceptable form" of a disclosed compound includes, but is not limited to, its pharmaceutically acceptable salts, esters, hydrates, solvates, isomers, prodrugs, and isotopically labeled derivatives. In some embodiments, a "pharmaceutically acceptable form" includes, but is not limited to, its pharmaceutically acceptable salts, esters, prodrugs, and isotopically labeled derivatives. In some embodiments, a "pharmaceutically acceptable form" includes, but is not limited to, its pharmaceutically acceptable isomers and stereoisomers, prodrugs, and isotopically labeled derivatives.

[0092] In certain embodiments, the pharma- ceutically acceptable form is a pharma- ceutically acceptable salt.

[0093] The term "pharmaceutically acceptable salts" as used herein refers to salts that are suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic reaction, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts include salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid, and the like, or organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, and the like, or by other methods used in the art, such as ion exchange. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfonate, heptanoate, hexanoate, hydroiodide, dihydrogen phosphate, and the like. -hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, p-toluenesulfonate, undecanoate, valerate, and the like.In some embodiments, organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0094] Such salts can be prepared in situ during the isolation and purification of the disclosed compounds, or can be prepared separately, such as by reacting the free base or free acid of the parent compound with a suitable base or acid, respectively. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N + (C 1-4 Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Pharmaceutically acceptable salts further include non-toxic ammonium salts, quaternary ammonium salts, and amine cation salts, formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, where appropriate. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt can be selected from ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts.

[0095] In certain embodiments, the pharma- ceutically acceptable form is a "solvate" (e.g., a hydrate). The term "solvate" as used herein refers to a compound that further comprises a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. The solvate may be of the disclosed compound or a pharma- ceutically acceptable salt thereof. When the solvent is water, the solvate is a "hydrate". Pharmaceutically acceptable solvates and hydrates are complexes that may contain, for example, 1 to about 100, or 1 to about 10, or 1 to about 2, about 3, or about 4 solvent or water molecules. It is understood that the term "compound" as used herein includes compounds and solvates of compounds, as well as mixtures thereof.

[0096] In certain embodiments, the pharma- ceutically acceptable form is a prodrug. The term "prodrug" as used herein refers to a compound that is converted in vivo to produce the disclosed compound or a pharma- ceutically acceptable form of the compound. A prodrug may be inactive when administered to a subject, but is converted to an active compound in vivo, for example, by hydrolysis (e.g., hydrolysis in blood). In certain cases, a prodrug has improved physical properties and / or delivery properties compared to the parent compound. A prodrug can improve the bioavailability of a compound when administered to a subject (e.g., promote absorption into the blood after oral administration) or promote delivery to a biological compartment of interest (e.g., the brain or lymphatic system) compared to the parent compound. Exemplary prodrugs include derivatives of the disclosed compounds that have enhanced water solubility or active transport across the intestinal membrane relative to the parent compound.

[0097] Prodrug compounds often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism. (See, e.g., Bundgard, H. 1985 Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam; Higuchi et al. 1987 "Pro-drugs as Novel Delivery Systems" ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.)

[0098] Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in mammalian organisms. (See, for example, Bundgard, Design of Prodrugs, pp. 7-9,21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, Calif., 1992.) Prodrugs generally known in the art include well-known acid derivatives, such as, for example, esters prepared by reacting the parent acid with an appropriate alcohol, amides prepared by reacting the parent acid compound with an amine, and base groups reacted to form acylated base derivatives. Other prodrug derivatives can be combined with other features disclosed herein to enhance bioavailability. Thus, one skilled in the art will appreciate that certain of the compounds disclosed herein that have free amino, amide, hydroxy, or carboxyl groups can be converted into prodrugs. Prodrugs include compounds having a carbonate, carbamate, amide or alkyl ester moiety covalently bonded to any of the above substituents disclosed herein.

[0099] Exemplary advantages of a prodrug include, but are not limited to, physical properties such as improved water solubility for parenteral administration at physiological pH, improved absorption from the gastrointestinal tract, or improved drug stability for long-term storage compared to the parent compound.

[0100] The term "pharmacologically acceptable" excipient, carrier, or diluent as used herein refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting the agent of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of substances which may function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances used in pharmaceutical preparations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate, magnesium stearate, polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0101] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. In addition, a polypeptide may refer to a protein that contains modifications (generally conservative in nature), such as deletions, additions, and substitutions to the native sequence, so long as the protein maintains the desired activity. These modifications may be intentional or accidental. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0102] The term "receptor" as used herein refers to a protein, including a glycoprotein or a fragment thereof, that can interact with another molecule, called a ligand. A ligand is usually an extracellular molecule, which upon binding to a receptor usually initiates a cellular response, such as the initiation of a signal transduction pathway. A receptor is not necessarily a membrane-bound protein. A ligand may belong to any class of biochemical or chemical compound.

[0103] The term "sample" as used herein refers to a sample of human, animal origin, or a research sample, such as a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulated material. A "sample" may be tested in vivo, e.g., without removal from a human or animal, or in vitro. A sample may also be tested after processing, e.g., by histological methods. A "sample" may also refer to, for example, cells constituting a fluid or tissue sample, or cells separated from a fluid or tissue sample. A "sample" may also refer to fresh cells, tissues, organs, or fluids taken from a human or animal, or cells, tissues, organs, or fluids that have been processed or stored.

[0104] As used herein, the term "stimulate" or "stimulatory" refers to increasing, amplifying, enhancing, or enhancing a physiological activity, such as an immune response. Stimulation can be a positive change. For example, the increase can be a 5%, 10%, 25%, 50%, 75%, or 90 to 100% increase. Other exemplary increases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or 100-fold.

[0105] The term "subject" as used herein refers to any animal (e.g., mammal) that will be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, and the like. Subjects contemplated for administration include, but are not limited to, humans (e.g., male or female of any age, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)), and / or other non-human animals, e.g., non-human mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats, and / or dogs), rodents (e.g., rats and / or mice), and the like. In certain embodiments, the non-human animal is a mammal. The non-human animal may be male or female at any stage of development. The non-human animal may be a transgenic animal. Typically, the terms "subject" and "patient" are used interchangeably herein with respect to human subjects.

[0106] The term "suppress" or "inhibitory" as used herein refers to decreasing, attenuating, decreasing, stopping, or stabilizing a physiological activity, such as an immune response. The suppression can be a negative change. For example, the decrease can be a 5%, 10%, 25%, 50%, 75%, or a 90 to 100% decrease. Exemplary decreases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or 100-fold.

[0107] As used herein, the term "treatment" or "treating" of a disease or disorder refers to a method for alleviating, delaying or improving such a condition before or after it occurs. Treatment can be directed at one or more effects or symptoms of the disease and / or underlying pathology. Treatment can be any alleviation, including but not limited to complete elimination of the disease or symptoms of the disease. Thus, "treating" or "treatment" refers to any indicator of success in the treatment or improvement of an injury, disease, pathology or condition, including any objective or subjective parameter, such as attenuation, remission, alleviation of symptoms or making the injury, pathology or condition more tolerable to the patient, slowing the rate of degeneration or debilitation, reducing the degree of debilitation of the degenerative end point, improving the physical or mental well-being of the patient. Treatment or improvement of symptoms can be based on objective or subjective parameters, such as the results of a physical examination, a psychoneurological examination, and / or a psychiatric evaluation. The extent of such reduction or improvement, as compared to comparable untreated controls, can be at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, as measured by any standard technique.

[0108] The treatment method includes administering a therapeutically effective amount of the compound described herein to the subject. The administration step may be a single administration or may include a series of administrations. The length of the treatment period depends on various factors, such as the severity of the condition, the age of the patient, the concentration of the compound, the activity of the composition used for treatment, or a combination thereof. It is also understood that the effective amount of the agent used for treatment may increase or decrease during the course of a particular treatment regimen. Changes in dosage may be guided and revealed by standard diagnostic assays known in the art. In some cases, chronic administration may be required. For example, the composition is administered to the subject in an amount and for a period sufficient to treat the patient.

[0109] Auristatin Analogs and Cytotoxins Disclosed herein are a variety of novel auristatin analogs and cytotoxic agents.

[0110] In one aspect, the present invention generally relates to a compound represented by structural formula (I): [ka] (I) [During the ceremony, R 1 teeth [ka] where R 2 is unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R a , R b and R c are H and NR x R y is selected from, with the proviso that R a , R b and R c Only one of the following is NR x R y and each of the others is H; R x and R y Each of these is independently R, R r and L-R z is selected from, with the proviso that R x and R y One of them is LR z or R r if , then the other is R; R 5 is CR'3, where each R' is independently H or F; L is a linker; R r is (C=O)-O-(CH2) p -R v or (C=O)-(CH2) q -R v and; R v is R, OR, NHR, NR2, an aryl group or an amino acid; p is 0, 1, 2, 3, 4, 5 or 6; q is 0, 1, 2, 3, 4, 5 or 6; R z contains a functional or reactive group; and R is H or C1-C3 alkyl. or a pharma- ceutically acceptable salt thereof.

[0111] In certain embodiments, R 5 is CH3. In certain embodiments, R 5 is CF3. In certain embodiments, R 5 is CHF2. In certain embodiments, R 5 is CH2F.

[0112] In certain embodiments, R a is NR x R y and R b is H, and R c is H. In certain embodiments, R a is H and R b is NR x R y and R c is H. In certain embodiments, R a is H and R b is H, and R c is NR x R y It is.

[0113] In certain embodiments, R 5 is CH3, while R a is NR x R y and R b is H, and R c is H. In certain embodiments, R 5 is CH3, while R a is H and R b is H, and R c is NR x R y In certain embodiments, R 5is CH3, while R a is H and R b is H, and R c is NR x R y It is.

[0114] In certain embodiments, R 5 is CF3, while R a is NR x R y and R b is H, and R c is H. In certain embodiments, R 5 is CF3, while R a is H and R b is H, and R c is NR x R y In certain embodiments, R 5 is CF3, while R a is H and R b is H, and R c is NR x R y It is.

[0115] In certain embodiments, R 5 is CH3, and R c is H, and the structural formula (II) [ka] (II) has.

[0116] In certain embodiments of (11), R a is H, and R b is NR x R y and the compound has the structural formula (III) [ka] (III) has.

[0117] In certain (III) embodiments, R x is H, and R y is H, and the compound has the structural formula (III 1 ) [ka] (III 1 ) has.

[0118] In certain (III) embodiments, R x is H or CH3, and R y is (C=O)-O-(CH2) p -R v where R v is R, OR, NHR, NR2, an aryl group or an amino acid; and p is 0, 1, 2 or 3.

[0119] In certain (III) embodiments, R x is H or CH3, and R y is (C=O)-(CH2) q -R v where R v is R, OR, NHR, NR2, an aryl group or an amino acid; and q is 0, 1, 2 or 3.

[0120] In certain (III) embodiments, R y is LR z and the compound has the structural formula (III 2 ) [ka] (III 2 ) has.

[0121] Specific (III 2 In an embodiment of the present invention, R x is H, and the compound has the structural formula (III 3 ) [ka] (III 3 ) has.

[0122] In certain (II) embodiments, R a is NR x R y and R b is H, and the compound has the structural formula (IV) [ka] (IV) has.

[0123] In certain (IV) embodiments, R x is H, and R y is H, and the compound has the structural formula (IV 1 ) [ka] (IV 1 ) has.

[0124] In certain (IV) embodiments, R x is H or CH3, and R y is (C=O)-O-(CH2) p -R v where R v is R, OR, NHR, NR2, an aryl group or an amino acid; and p is 0, 1, 2 or 3.

[0125] In certain (IV) embodiments, R x is H or CH3, and R y is (C=O)-(CH2) q -R v where R v is R, OR, NHR, NR2, an aryl group or an amino acid; and q is 0, 1, 2 or 3.

[0126] In certain (IV) embodiments, R y is LRz and the compound has the structural formula (IV 2 ) [ka] (IV 2 ) has.

[0127] Specific (IV 2 In an embodiment of the present invention, R x is H, and the compound has the structural formula (IV 3 ) [ka] (IV 3 ) has.

[0128] In certain embodiments of (I), R 5 is CH3 and R a is H and R b is H, and R c is NR x R y and structural formula (V) [ka] (V) has.

[0129] In certain embodiments of (V), R x is H, and R y is H, and the structural formula (V 1 ) [ka] (V 1 ) has.

[0130] In certain embodiments of (V), R x is H or CH3, and R y is (C=O)-O-(CH2) p -R v where R vis R, OR, NHR, NR2, an aryl group or an amino acid; and p is 0, 1, 2 or 3.

[0131] In certain embodiments of (V), R x is H or CH3, and R y is (C=O)-(CH2) q -R v where R v is R, OR, NHR, NR2, an aryl group or an amino acid; and q is 0, 1, 2 or 3.

[0132] In certain embodiments of (V), R y is LR z and the structural formula (V 2 ) [ka] (V 2 ) has.

[0133] Specific (V 2 In an embodiment of the present invention, R x is H, and the structural formula (V 3 ) [ka] (V 3 ) has.

[0134] In certain embodiments of (I), R 5 is CF3, R a is H and R b is H, and R c is NR x R y The compound has the structural formula (V 4 ) [ka] (V 4 ) has.

[0135] Specific (V 4 In an embodiment of the present invention, R x is H, and R y is H, and the structural formula (V 5 ) [ka] (V 5 ) has.

[0136] Specific (V 4 In an embodiment of the present invention, R x is H or CH3, and R y is (C=O)-O-(CH2) p -R v where R v is R, OR, NHR, NR2, an aryl group or an amino acid; and p is 0, 1, 2 or 3.

[0137] Specific (V 4 In an embodiment of the present invention, R x is H or CH3, and R y is (C=O)-(CH2) q -R v where R v is R, OR, NHR, NR2, an aryl group or an amino acid; and q is 0, 1, 2 or 3.

[0138] Specific (V 4 In an embodiment of the present invention, R x is H, and R y is LR z The compound has the structural formula (V 6 ) [ka] (V 6 ) has.

[0139] The above formulas (I) to (V 6 In certain embodiments of any one of the above, R 1 teeth [ka] where R 3 and R 4 are each independently H or unsubstituted or substituted C1-C5 alkyl, or together with the N and C atoms to which they are attached form a 5-7 membered heterocycloalkyl containing one or more of O, N and S optionally substituted with one or more halogen atoms or C1-C3 alkyl.

[0140] The above formulas (I) to (V 6 In certain embodiments of any one of the above, R 1 teeth [ka] where R 3 and R 4 are each independently H or unsubstituted or substituted C1-C5 alkyl, or together with the N and C atoms to which they are attached form a 5-7 membered heterocycloalkyl containing one or more of O, N and S optionally substituted with one or more halogen atoms or C1-C3 alkyl.

[0141] In certain embodiments, R 3 is H, and R 4 is H or unsubstituted or substituted C1-C5 alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl).

[0142] In certain embodiments, R 3 is methyl optionally substituted with one or more halogen atoms (e.g., F, Cl), and R 4 is H or unsubstituted or substituted C1-C5 alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl).

[0143] In certain embodiments, R 3is ethyl optionally substituted with one or more halogen atoms (e.g., F, Cl), and R 4 is H or unsubstituted or substituted C1-C5 alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl).

[0144] In certain embodiments, R 3 is propyl or isopropyl, optionally substituted with one or more halogen atoms (e.g., F, Cl), and R 4 is H or unsubstituted or substituted C1-C5 alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl).

[0145] In certain embodiments, R 4 is H. In certain embodiments, R 4 is methyl. In certain embodiments, R 4 is isopropyl.

[0146] In certain embodiments, R 3 and R 4 together with the N and C atoms to which they are respectively attached form a 5-membered heterocycloalkyl optionally substituted with one or more of F, Cl and Br. 3 and R 4 together with the N and C atoms to which they are respectively attached form a 6-membered heterocycloalkyl optionally substituted with one or more of F, Cl and Br. 3 and R 4 together with the N and C atoms to which they are respectively attached form a 7-membered heterocycloalkyl optionally substituted with one or more of F, Cl and Br.

[0147] The above formulas (I) to (V 6 In certain embodiments of any one of the above, R 1 teeth, [ka] is selected from.

[0148] Formula (I)~~(V 6 In certain embodiments of any one of claims 1 to 5, L is a non-cleavable linker.

[0149] Formula (I)~(V 6 In certain embodiments of any one of claims 1 to 5, L is a cleavable linker.

[0150] In certain embodiments, L is an acid labile or acid sensitive linker. In certain embodiments, L is a protease sensitive linker. In certain embodiments, L is a lysosomal protease sensitive linker. In certain embodiments, L is a β-glucuronide sensitive linker. In certain embodiments, L is a glutathione sensitive disulfide linker.

[0151] In certain embodiments, L is an unbranched linker, ie, suitable for conjugation to a single cytotoxic agent or payload per linker.

[0152] In certain embodiments, L is a branched linker, e.g., having 2, 3, 4, 5, 6, 7, 8 or more branches, where each branch is suitable for conjugation to a cytotoxic agent or payload, thereby making it suitable for conjugation to more than one cytotoxic agent or payload per linker.

[0153] Formula (I)~(V 6 In certain embodiments of any one of the above, R z When present, it comprises a functional or reactive group suitable for conjugation with an antigen-binding moiety, e.g., the functional or reactive group is -N3, -NR u C(=O)CH=CH2, -SH, -SSR t , -S(=O)2(CH=CH2), -(CH2)2S(=O)2(CH=CH2), -NR u S(=O2)(CH=CH2), -NR uC(=O)CH2R w , -NR u C(=O)CH2Br, -NR u C(=O)CH2I, -NHC(=O)CH2Br, NHC(=O)CH2I, -ONH2, -C(=O)NHNH2, -CO2H, -NH2, -NCO, -NCS, [ka] [ka] where: R u is H or a C1-C6 alkyl group, R t is 2-pyridyl or 4-pyridyl, and R w teeth, [ka] It is.

[0154] R Z Further disclosure regarding linkers and reactive or functional groups that may be used in the components of and / or L is provided in the "Linkers and Linking Techniques" and "Linker-Antibody and Linker-Payload Conjugations" sections and the references cited therein, each of which is incorporated herein by reference.

[0155] The present invention also includes methods for synthesizing auristatin analogs, including their intermediates or precursors.

[0156] Non-limiting examples of auristatin analogs of the present invention include the following: [Table 1-1] [Table 1-2]

[0157] In another aspect, the invention generally relates to a drug-linker conjugate formed by coupling a compound disclosed herein with a linker.

[0158] Non-limiting examples of linker-linked auristatin analogs include the following: [Table 2]

[0159] Methods for determining the binding affinity of a compound to tubulin are known in the art. (See, e.g., Muller et al. 2006 Anal. Chem. 78, 4390-4397; Hamel et al. 1995 Molecular Pharmacology 47: 965-976; Hamel et al. 1990 J. Biological Chemistry 265:28, 17141-17149.)

[0160] In some embodiments, the auristatin analogs disclosed herein bind to tubulin with an affinity ranging from 10-fold lower (weak) than the binding affinity of monomethylauristatin E (MMAE) to tubulin to 5-fold, 10-fold, 20-fold, 30-fold, 50-fold or 100-fold higher (strong) than the binding affinity of MMAE to tubulin.

[0161] immune complex A typical ADC is composed of an antigen-binding moiety (Ab), e.g., a monoclonal antibody, a linker (L) and a cytotoxic agent or payload (D), as depicted below: (D m -L) n -Ab where each of m and n is an integer. Payload D (e.g., an auristatin analog disclosed herein) can be attached to a different portion of Ab, typically via a cysteine ​​or lysine residue. Generally, more than one payload D molecule can be attached to each Ab. If a branched linker is used, more than one payload D moiety can be attached to each linker L. In some embodiments, n ranges from 1 to 16, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, n ranges from 2 to 10, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 2, 3, or 4. In some embodiments, L is an unbranched linker and m is 1. In some embodiments, L is a branched linker and m can range from 2 to 10, 2 to 8, 2 to 6, or 2 to 4. In some embodiments, m is 2, 3, or 4.

[0162] The drug-to-antibody ratio (DAR) or drug loading can be characterized by conventional means such as UV, mass spectrometry, ELISA assay, HIC, HPLC or electrophoresis. In exemplary embodiments, the DAR is in the range of 1 to 16, 2 to 8, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or about 1.

[0163] The DAR of the immunoconjugate can be controlled by a variety of methods, including limiting the molar excess of the payload-linker intermediate or linker reagent relative to the antigen-binding moiety, limiting the conjugation reaction time or temperature, varying the reductive conditions for cysteine ​​thiol modification, and altering the number and position of cysteine ​​residues and the position of the linker-payload attachment (see, e.g., WO 2006 / 034488 A2).

[0164] In one aspect, the present invention generally relates to an immunoconjugate formed by the conjugation of a compound disclosed herein with an antigen-binding moiety via a linker.

[0165] In another aspect, the present invention generally provides a compound of formula (VI): [ka] (VI) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth [ka] where R 2 is unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and R y Each of R and LR is independently z is selected from, with the proviso that R x and R y One of them is NR z if , then the other is R; R 5 is CR'3, where each R' is independently H or F; L is a linker; R is H or C1-C3 alkyl; and and i is an integer ranging from 1 to about 20. or a pharma- ceutically acceptable salt thereof.

[0166] In certain embodiments of the immunoconjugate of formula (VI), R 5 is CH3, and R x is H, and the immune complex has the structural formula (VI 1 ) [ka] (VI 1 ) has.

[0167] Formula (VI) to (VI 1 In certain embodiments of i, i is an integer ranging from 1 to 20. In certain embodiments, i is an integer ranging from 1 to 16. In certain embodiments, i is an integer ranging from 1 to 12. In certain embodiments, i is an integer ranging from 1 to 10. In certain embodiments, i is an integer ranging from 1 to 8. In certain embodiments, i is an integer ranging from 1 to 6. In certain embodiments, i is an integer ranging from 1 to 5. In certain embodiments, i is an integer ranging from 1 to about 4. In certain embodiments, i is an integer ranging from 1 to 3. In certain embodiments, i is an integer ranging from 1 to 2. In certain embodiments, i is 1.

[0168] In another aspect, the present invention generally provides a compound of formula (VII): [ka] (VII) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth [ka] where R 2 is unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and R y Each of R and LR is independently z is selected from, with the proviso that R x and R y One of them is NR z if , then the other is R; R 5 is CR'3, where each R' is independently H or F; L is a linker; R is H or C1-C3 alkyl; and j is an integer ranging from 1 to about 20. or a pharma- ceutically acceptable salt thereof.

[0169] In certain embodiments of the immunoconjugate of formula (VII), R 5 is CH3, and R x is H, and the structural formula (VII 1 ) [ka] (VII 1 ) has.

[0170] Formula (VII) to (VII 1 In certain embodiments of (a), j is an integer ranging from 1 to 20. In certain embodiments, j is an integer ranging from 1 to 16. In certain embodiments, j is an integer ranging from 1 to 12. In certain embodiments, j is an integer ranging from 1 to 10. In certain embodiments, j is an integer ranging from 1 to 8. In certain embodiments, j is an integer ranging from 1 to 6. In certain embodiments, j is an integer ranging from 1 to 5. In certain embodiments, j is an integer ranging from 1 to about 4. In certain embodiments, j is an integer ranging from 1 to 3. In certain embodiments, j is an integer ranging from 1 to 2. In certain embodiments, j is 1.

[0171] In yet another aspect, the present invention generally provides a compound represented by structural formula (VIII): [ka] (VIII) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth [ka] where R 2 is unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and R y Each of R and LR is independently z is selected from, with the proviso that R x and R y One of them is NR z if , then the other is R; R 5 is CR'3, where each R' is independently H or F; L is a linker; R is H or C1-C3 alkyl; and k is an integer ranging from 1 to about 20. or a pharma- ceutically acceptable salt thereof.

[0172] In certain embodiments of the immunoconjugate of Formula (VIII), R 5 is CF3 and R x is H, and the structural formula (VIII 1 ). TIFF2024540402000045.tif28131(VIII 1 )

[0173] Formula (VIII) to (VIII 1 In certain embodiments of the above, k is an integer ranging from 1 to 20. In certain embodiments, k is an integer ranging from 1 to 16. In certain embodiments, k is an integer ranging from 1 to 12. In certain embodiments, k is an integer ranging from 1 to 10. In certain embodiments, k is an integer ranging from 1 to 8. In certain embodiments, k is an integer ranging from 1 to 6. In certain embodiments, k is an integer ranging from 1 to 5. In certain embodiments, k is an integer ranging from 1 to about 4. In certain embodiments, k is an integer ranging from 1 to 3. In certain embodiments, k is an integer ranging from 1 to 2. In certain embodiments, k is 1.

[0174] Formulas (VI)~(VIII 1 ) and all the substituents found in 1 , R2 , R 3 , R 4 , R 5 , R x , R y , R, R', L) are represented by the formulas (I) to (V 6 ), may be selected as described in the section entitled "Auristatin Analogs and Cytotoxins" in connection with R 1 , R 2 , R 3 , R 4 , R 5 , R x , R y , R, R', L and R z Each and every combination and resulting compound is incorporated herein in its entirety. Thus, the present invention relates to Ab binding formulas (I)-(V 6 ) including immune complexes corresponding to

[0175] In addition to immunoconjugates in which the antigen-binding moiety is an antibody or antibody fragment, the present invention also includes immunoconjugates in which the antigen-binding moiety is a peptide and immunoconjugates in which the antigen-binding moiety is a small molecule ligand. (See, e.g., Zhuang et al. 2019 Eur. J. Med. Chem. 163, 883-895; Patel et al. 2021 New J. Chem. 45, 5291-5321.)

[0176] The present invention also includes methods for the synthesis of immunoconjugates, including intermediates or precursors thereof.The present invention further includes compositions comprising the immunoconjugates, intermediates or precursors thereof.

[0177] antigen binding part To date, numerous unique antigens have been identified and can be used as targets in antibody-based therapy. Several factors are generally considered when selecting an antigen. First, the target antigen should be highly expressed in tumors and absent or low expressed in healthy cells. For example, the HER2 receptor is nearly 100-fold more highly expressed in tumor cells compared to healthy cells. Second, the target antigen should be expressed on the tumor cell surface such that circulating monoclonal antibodies are available. In addition, the target antigen should have internalizing properties to facilitate the trafficking of ADC into cells and enhance the efficacy of cytotoxic agents. However, several studies have shown that non-internalizing ADC formulations against components of the tumor microenvironment can efficiently detach the drug in the extracellular space and exert potent therapeutic activity in some cases, and that ADCs often induce a strong "bystander effect." (Strohl WR 2018 Protein & Cell. 9(1):86-120; Damelin et al. 2015 Pharma. Res. 32(11):3494-507; Diamantis et al. 2016 British J. Cancer114(4):362-7; Tipton et al. 2015 Blood 125(12):1901-9; Donaghy et al. 2016 mAbs. 8(4):659-71; Casi et al. 2015 Molecular Pharmaceutics 12(6):1880-4).

[0178] The antigen-binding moiety can be any moiety that selectively binds to a cell surface marker found on the targeted cell type. In general, the antibody should preferably have target specificity, deliver the cytotoxic agent to the tumor cell, and have target binding affinity, i.e., high binding affinity to the tumor cell surface antigen. More preferably, the antibody should have good retention, low immunogenicity, low cross-reactivity, and suitable tethering binding properties. (Peters et al. 2015 Bioscience Reports 35(4); Hughes B 2010 Nature Reviews Drug Discovery 9(9):665-7.)

[0179] In certain embodiments, the Ab is an antibody.

[0180] In certain embodiments, the Ab is a monoclonal antibody.

[0181] In certain embodiments, the Ab is a chimeric antibody.

[0182] In certain embodiments, the Ab is a humanized antibody.

[0183] In certain embodiments, the Ab is a bispecific antibody.

[0184] In certain embodiments, the Ab is an antibody fragment.

[0185] In certain embodiments, the Ab is a Fab fragment.

[0186] In certain embodiments, the Ab is a peptide.

[0187] In certain embodiments, the Ab is a small molecule ligand.

[0188] In some embodiments, the Ab is an antibody or antibody fragment (e.g., an antigen-binding fragment of an antibody) that specifically binds to an antigen that is predominantly or preferentially found on the surface of cancer cells, e.g., a tumor-associated antigen.

[0189] In some embodiments, an Ab is an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to a cell surface receptor protein or other cell surface molecule, a cell survival regulator, a cell proliferation regulator, a molecule associated with tissue development or differentiation, a molecule known or suspected to contribute functionally to tissue development or differentiation, a lymphokine, a cytokine, a molecule involved in cell cycle regulation, a molecule involved in vasculogenesis, a molecule associated with angiogenesis, or a molecule known or suspected to contribute functionally to angiogenesis.

[0190] Thus, antigen-binding moieties useful in the immunoconjugates of the invention include, but are not limited to, antibodies against cell surface receptors and tumor-associated or tumor-specific antigens, which are well known in the art and can be prepared for use in generating antibodies using methods and information known in the art.

[0191] In an attempt to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or other tumor-associated or tumor-specific polypeptides that are differentially expressed on the surface of one or more particular types of cancer cells compared to one or more normal non-cancerous cells. Tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells, whereas tumor-specific polypeptides are differentially expressed on the surface of one or more particular types of cancer cells but not on non-cancerous cells. The identification of such cell surface antigen polypeptides has made it possible to specifically target and destroy cancer cells by antibody-based therapy. (See, e.g., Liu et al. 2017 Eur. J. Cancer Care(Engl). 2017 Sep; 26(5), doi: 10.1111 / ecc.12446; WO 2016 / 192527 A1.)

[0192] The tumor-associated antigen may be a cluster of differentiation factor (e.g., CD protein). In some embodiments of the invention, the antigen-binding moiety of the invention specifically binds to one antigen. In some embodiments of the invention, the antigen-binding moiety of the invention specifically binds to two or more antigens described herein, for example, the antigen-binding moiety of the invention is a bispecific or multispecific antibody or antigen-binding fragment thereof.

[0193] Non-limiting examples of antibodies or antigen-binding portions include anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2 antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9 / p24 antibodies, anti-CD1 antibodies, anti-CD1-c antibodies, anti-CD13 antibodies, anti-CD14 antibodies, anti-CD15 antibodies Antibodies include anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD23 antibodies, anti-CD30 antibodies, anti-CD31 antibodies, anti-CD33 antibodies, anti-CD34 antibodies, anti-CD35 antibodies, anti-CD38 antibodies, anti-CD39 antibodies, anti-CD41 antibodies, anti-LCA / CD45 antibodies, anti-CD45RO antibodies, anti-CD45RA antibodies, anti-CD71 antibodies, anti-CD95 / Fas antibodies, anti-CD99 antibodies, anti-CD100 antibodies, anti-S-100 antibodies, anti-CD106 antibodies, anti-ubiquitin antibodies, anti-c-myc antibodies, anti-cytokeratin antibodies, anti-lambda light chain antibodies, anti-melanosome antibodies, anti-prostate specific antigen antibodies, anti-tau antigen antibodies, anti-fibrin antibodies, anti-keratin antibodies, and anti-Tn antigen antibodies.

[0194] Antibodies and antibody fragments useful in the immunoconjugates of the invention include modified or engineered antibodies, such as antibodies modified to introduce a cysteine ​​residue or other reactive amino acid, including Pel, pyrrolysine, peptide tags and unnatural amino acids, in place of at least one amino acid of the native sequence, thus providing a reactive site on the antibody or antigen-binding fragment for attachment of a cytotoxic agent.

[0195] The location of the drug moiety can be designed, controlled and known. For example, cysteine ​​amino acids can be engineered at the reactive sites of the antibody, which do not form intrachain or intermolecular disulfide bonds. (Junutula, et al. 2008 Nature Biotech. 26(8):925-932; Dornan et al. 2009 Blood 114(13):2721-2729; U.S. Patent No. 7,521,541 B2; U.S. Patent No. 7,723,485 B2; WO 2009 / 052249 A2.) The engineered cysteine ​​thiols can react with linker reagents or drug-linker reagents of the invention that have thiol-reactive electrophilic groups, such as maleimides or alpha-haloamides, to form ADCs with cysteine ​​engineered antibodies and drug moieties.

[0196] Additionally, antibodies or antibody fragments can be modified to incorporate Pel or pyrrolysine or unnatural amino acids as drug attachment sites. Peptide tags for enzymatic conjugation can be introduced into the antibodies. (Junutula et al. 2008 Nat. Biotechnol. 26:925-932; Ou et al. 2011 PNAS 108(26), 10437-10442; Axup et al. 2012 Proc. Natl. Acad. Sci. USA, 109, 16101-16106; Liu et al. 2010 Annu. Rev. Biochem. 79, 413-444; Kim et al. 2013 Curr. Opin. Chem. Biol. 17, 412-419; Strop et al. 2013 Chem. Biol. 20(2):161-7; Rabuka 2010 Curr. Opin. Chem. Biol. 14(6):790-6; Rabuka et al. 2012 Nat. Protoc. 7(6): 1052-67; WO 2015 / 095301 A2; WO 2013 / 184514 A2. )

[0197] Antibodies and antibody fragments can be readily produced by any method known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc. (e.g. Carvalho et al. 2016 “Production Processes for Monoclonal Antibodies”, DOI: 10.5772 / 64263(https: / / www.intechopen.com / chapters / 51512); Monoclonal Antibody Production, Committee on Methods of Producing Monoclonal Antibodies, Institute for Laboratory Animal Research, National Research Council, NATIONAL ACADEMY PRESS Washington, DC 1999; Jakobovits 1998 Adv. Drug Del. Rev. 31:33-42; Marks et al. 1991 J. Mol. Biol. 222:581; Cole et al. 1985 Monoclonal Antibodies And Cancer Therapy 77-96; Teng et al. 1983 Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., (See 1983 Immunology Today 4:72-79; Olsson et al. 1982 Meth. Enzymol. 92:3-16; U.S. Patent No. 6,657,103 B2.)

[0198] Linkers and Linking Technologies The cytotoxic agent disclosed herein is suitable for use as a payload in immunoconjugates.The auristatin analog of the present invention can be linked to a linker or directly linked to an antigen-binding moiety.The linker of ADC is typically designed to achieve high stability in circulation and, in the case of cleavable linker, to specifically release the payload in target tissue.

[0199] Linkers and linking techniques suitable for use in constructing immunoconjugates are well known in the art and can be used in producing the immunoconjugates of the present invention. In general, the linker can be attached to the antigen-binding moiety at any suitable available position on the antigen-binding moiety, for example, to an available amino nitrogen atom (e.g., a primary or secondary amine) or a hydroxyl oxygen atom or to an available sulfhydryl, such as on a cysteine. The attachment of the linker to the cytotoxic auristatin analog disclosed herein can be at the N-terminus or C-terminus of the cytotoxic agent.

[0200] A variety of linkers and conjugation strategies are known and can be used in preparing the immunoconjugates of the invention. (For example, Kang et al. 2021 “Recent developments in chemical conjugation strategies targeting native amino acids in proteins and their applications in antibody-drug conjugates” Chemical Science Royal Soc. of Chem., DOI: 10.1039 / d1sc02973h; Su et al. 2021 “Antibody-drug conjugates: Recent advances in linker chemistry” Acta Pharmaceutica Sinica B, https: / / doi.org / 10.1016 / j.apsb.2021.03.042; Drago et al. 2021 Nature Reviews 18, 327-344; Mckertish et al. 2021 Biomedicines 9, 872; Bargh et al. 2019 “Cleavable linkers in antibody-drug conjugates” Chem. Soc. Rev. 48, 4361, DOI: 10.1039 / c8cs00676h; Lash 2011 “Antibody-Drug Conjugates: the Next Generation of Moving Parts” Start-Up, Dec. 2011, 1-6; WO 2021 / 055865 A1; WO 2016 / 192527 A1; WO 2015 / 095301 A2; WO 2011 / 097627 A1, WO 2004 / 010957 A1, U.S. Patent Publication No. 20060074008 A2, U.S. Patent Publication No. 20050238649 A2 and U.S. Patent Publication No. 20060024317 A2.)

[0201] Linkers are classified as either cleavable or non-cleavable. For ADCs with non-cleavable linkers, release typically occurs via internalization of the ADC followed by degradation of the antibody in lysosomes, resulting in the release of the payload that remains attached to the amino acid residue of the antibody via the linker. Examples of non-cleavable linkers include maleimidoca-proyl (MC) and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC) linkers. Examples of cleavable linkers include Val-Cit, N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP) and hydrazide.

[0202] In the case of immunoconjugates that include a cleavable linker, the linker is substantially stable in vivo until the immunoconjugate binds to or enters a cell, at which point either intracellular enzymes or intracellular chemical conditions (pH, reducing potential) cleave the linker and liberate the cytotoxic peptide.

[0203] Cleavable linkers can be further classified into chemically cleavable linkers (such as acid-cleavable linkers, reducible disulfide linkers, and exogenous stimulus-induced linkers) and enzyme-cleavable linkers (such as dipeptide Val-Cit-containing linkers, glycosidase-cleavable linkers, phosphatase-cleavable linkers) based on the cleavage mechanism. Acid-cleavable linkers (also known as pH-sensitive linkers) are designed to take advantage of the acidity of endosomes (pH 5.5-6.2) and lysosomes (pH 4.5-5.0), yet maintain stability in the blood circulation at pH 7.4. An example of an acid-cleavable linker is the acid-sensitive N-acylhydrazine bond, which hydrolyzes to ketone and hydrazide payloads upon acid catalysis. Acid-cleavable linkers containing other functional groups have also been reported, such as carbonate linkers. Glycosidase cleavable linkers include β-glucuronidase cleavable linkers, β-galactosidase cleavable linkers, and phosphatase cleavable linkers.(For example, Bargh et al. 2019 “Cleavable linkers in antibody-drug conjugates” Chem. Soc. Rev. 48, 4361, DOI: 10.1039 / c8cs00676h; Ducry, et al. 2010 Bioconiuqate Chem., vol. 21 , 5-13; Jeffrey et al. 2006 Bioconjugate Chem. 17, 831-840; Burke et al. 2009 Bioconjugate Chem. 20, 1242-1250; Kolodych et al. 2017 J. Med. Chem. 142, 376-382; Kern et al. 2016 Bioconjugate Chem. 27, 2081-2088; Stenton et al. al. 2018 Chem. Sci. 9, 4185-4189; Pillow et al. 2017 Mol. Cancer Ther. 16, 871-878; Dubowchik et al. 1998 Bioorg. Med. Chem. Lett. 8, 3341-3346; Dubowchik et al. 1998 Bioorg. Med. Chem. Lett. 8, See WO 2021 / 055865 A1; WO 2016 / 192527 A1; WO 2015 / 095301 A2; US 2021 / 0138077 A1; WO 2013 / 173393 A1; WO 2011 / 097627 A1. ).

[0204] Linker-Antibody and Linker-Payload Conjugations A variety of conjugation strategies have been developed over the years, including site-specific conjugation techniques, antibody engineering, and chemical modification.

[0205] The main linking techniques include maleimide linkages (eg, N-alkylmaleimides, N-phenylmaleimides), bis(vinylsulfonyl)piperazine linkages, N-methyl-N-phenylvinylsulfonamide linkages, and Pt(II)-based linkages. (For example, Su et al. 2021 “Antibody-drug conjugates: Recent advances in linker chemistry” Acta Pharmaceutica Sinica B, https: / / doi.org / 10.1016 / j.apsb.2021.03.042; Mckertish et al. 2021 Biomedicines 9, 872; Patterson et al. 2015 Bioconjug. Chem. 26:2243e8; Lyu et al. 2018 ACS Chem. Biol. 13:958e64; Zhou 2017 Biomedicines 5:64; Christie et al. 2017 Antibodies(Basel)6:20; Sun et al. 2019 Org. Biomol. Chem. 17: 2005e12; Huang et al. 2018 Org. Lett. 20: 6526e9; Sijbrandi et al. 2017 Cancer Res. 77: 257e67; Merkul et al. 2020 Angew Chem. Int. Ed. Engl. 60:3008e15; Merkul et al. 2019 Expert Opin. Drug Deliv. 16:783e93; WO See 2015 / 095301 A2; US 2021 / 0138077 A1; WO 2013 / 173393 A1; WO 2016 / 192527 A1; WO 2021 / 055865 A1. )

[0206] Various linker-payload conjugation strategies have been reported, including carbamate and carbonate conjugations (see, e.g., Wahby et al. 2020 Clin. Cancer Res. Available from: https: / / doi.10.1158 / 1078-0432.CCR-20-3119; Perini et al. 2013 Biol. Ther. 3:15e23; Burke et al. 2016 Mol. Cancer Ther. 15:938e45; WO 2015 / 095301 A2; US 2021 / 0138077 A1; WO 2013 / 173393 A1; WO 2016 / 192527 A1; WO 2021 / 055865 A1).

[0207] Non-limiting examples of conjugation strategies and reactive groups are provided in Table 3. (See, e.g., WO 2015 / 095301 A2; U.S. Patent No. 9,988,420 B2.) [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0208] Pharmaceutical Compositions and Methods of Use Pharmaceutical Compositions In another aspect, the present invention generally provides compounds of formula (I)-(V 6 ) and any one of Tables 1 and 2, or a pharma- ceutically acceptable salt thereof, and optionally a pharma- ceutically acceptable excipient, carrier, or diluent.

[0209] In yet another aspect, the invention generally comprises: 1The present invention relates to a pharmaceutical composition comprising an immunoconjugate as disclosed herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable excipient, carrier, or diluent, such as according to any one of claims 1 to 5, wherein the immunoconjugate is a medicament for administering a therapeutically effective amount of the ...

[0210] Thus, the present invention provides a pharmaceutical formulation comprising a therapeutically effective amount of a compound or immunoconjugate according to the invention.

[0211] Examples of excipients that may be useful include, but are not limited to, water, saline, glucose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine ​​hydrochloride, starch, cellulose and gums. In a preferred embodiment, the pharmaceutical composition of the present invention is formulated in a pharmaceutical form for administration as a solid (e.g., tablet, capsule, lozenge, granule, suppository, crystalline or amorphous sterile solid that can be reconstituted to provide a liquid form, etc.), liquid (e.g., solution, suspension, emulsion, elixir, lotion, ointment, etc.) or semi-solid (gel, ointment, cream, etc.). The pharmaceutical composition of the present invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal. A review of the various dosage forms of active ingredients, the excipients to be used, and their manufacturing procedures can be found in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000). Examples of pharma-ceutically acceptable vehicles are known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of moistening agents, sterile solutions, and the like. Compositions containing said vehicles can be prepared by conventional procedures known in the art. Preservatives, stabilizers, dyes, as well as flavoring agents, antioxidants, and / or suspending agents can be provided in the pharmaceutical composition. For example, sodium benzoate, esters of ascorbic acid and p-hydroxybenzoic acid can be added as preservatives.

[0212] The present invention also contemplates kits comprising at least the immunoconjugates disclosed herein and syringes and / or vials or ampoules into which the immunoconjugates and / or pharmaceutical compositions are disposed.

[0213] How to use In yet another aspect, the present disclosure generally relates to a method of treating or ameliorating a disease or condition comprising administering to a subject in need thereof a therapeutically effective amount of an immunoconjugate disclosed herein.

[0214] In certain embodiments, the disease or condition is cancer.

[0215] In certain embodiments, the method further comprises administering to the subject one or more of chemotherapy and radiation therapy.

[0216] In yet another aspect, the invention generally relates to the use of the immunoconjugates disclosed herein for the manufacture of a medicament.

[0217] In certain embodiments, the immunoconjugates disclosed herein are used to treat a disease or condition, wherein the disease or condition is cancer.

[0218] In yet another aspect, the present invention generally relates to the use of the immunoconjugates disclosed herein for use in the treatment of cancer.

[0219] Exemplary cancers include carcinomas, sarcomas, leukemias, and lymphomas. A comprehensive list of cancer types and cancers by body site can be found on the National Cancer Institute's website, e.g., https: / / www.cancer.gov / types and https: / / www.cancer.gov / types / by-body-location and IEEE Transactions on Clinical Chemistry, vol. 11, no. 10, pp. 1111-1115, 2002, each of which is incorporated herein by reference in its entirety.

[0220] In certain embodiments, the disease or disorder is one or more cancers selected from gastric cancer, myeloid cancer, colon cancer, nasopharyngeal cancer, esophageal cancer, and prostate cancer, glioma, neuroblastoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (e.g., myeloid leukemia, lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, T-lineage acute lymphoblastic leukemia or T-ALL chronic lymphocytic leukemia, myelodysplastic syndrome, hairy cell leukemia), lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma), multiple myeloma, bladder cancer, renal cancer, gastric cancer (e.g., gastrointestinal stromal tumor), liver cancer, melanoma, and pancreatic cancer, and sarcoma.

[0221] The immunoconjugate can generally be administered by systemic route, particularly intravenous, intramuscular, intradermal, intraperitoneal or subcutaneous route, or oral route.The immunoconjugate is typically administered intravenously into the subject's bloodstream to avoid antibody degradation by stomach acid or proteolytic enzymes.In some embodiments, the composition comprising the immunoconjugate disclosed herein is administered several times sequentially.

[0222] Combination Therapy In yet another aspect, the invention generally relates to combinations comprising a therapeutically effective amount of an immunoconjugate disclosed herein and one or more therapeutically active co-agents and / or adjuvants.

[0223] Co-agents include, but are not limited to, chemotherapeutic agents, growth factor inhibitors, biological response modifiers, anti-hormonal therapies, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors and anti-androgens.

[0224] Adjuvants include, but are not limited to, those known in the art. (See, e.g., Temizoz et al. 2016 Int. Immunol. 28(7): 329-338.)

[0225] The term "chemotherapeutic agent" as used herein refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith). Kline), lonafarnib (SCH66336), sorafenib (BAY43-9006, Bayer Labs), and gefitinib (IRESSA®, AstraZeneca), alkylating agents such as AG1478, AG1571 (SU 5271; Sugen), thiotepa, and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and urdopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine. ) class agents; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatins; calistatins; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatins;nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics, such as the calicheamicins, especially calicheamicin gammall and calicheamicin omegall (1994 Angew Chem. Intl. Ed. Engl. 33: 183-186); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and antibiotics, such as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores); aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodeoxyglucose, 2-pyrrolino-doxorubicin ... xorubicin and deoxydoxorubicin, mitomycins such as epirubicin, ethonibicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamnipurine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone;antiadrenergic agents such as aminoglutethimide, mitotane, and trilostane;folic acid supplements such as floric acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;Bestra Bucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diazicon; Elformitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol; Nitraerin; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as TAXOL® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (without cremophor), albumin nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE® (doxetaxel; Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate;Platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylomitin (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0226] In certain embodiments, the treatment methods disclosed herein allow for reduced dosage and / or less frequent administration of chemotherapy (or other treatments), which is beneficial for all patients, particularly those who cannot tolerate the toxicity of chemotherapeutic agents.

[0227] In addition, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and antiandrogens can be used. For example, antihormones, such as antiestrogens (e.g., Nolvadex (tamoxifen)) or antiandrogens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide) can be used.

[0228] Further examples of second, third or additional agents or therapies include immunotherapies, such as PD-1 inhibitors (pembrolizumab, nivolumab, cempirimab), PD-L1 inhibitors (atezolizumab, avelumab, durvalumab), CTLA4 antagonists, cell signaling inhibitors (e.g., imatinib, gefitinib, bortezomib, erlotinib, sorafenib, sunitinib, dasatinib, vorinostat, lapatinib, temsirolimus, nilotinib, everolimus, pazopanib, trastuzumab, bevacizumab, cetuximab, ranibizumab, pegaptanib, panitumumab, etc.), mitotic inhibitors (e.g., paclitaxel, vincristine, vinblastine, etc.), alkylating agents (e.g., cisplatin, cyclophosphamide, clomabucil, carmustine, etc.), antimetabolites (e.g., methotrexate, 5-FU, etc.), intercalating anticancer agents (e.g., actinomycin, anthracyclines, bleomycin, mitomycin-C, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, teniposide, etc.), immunotherapeutic agents (e.g., interleukins, interferons, etc.), and antihormonal agents (e.g., tamoxifen, raloxifene, etc.).

[0229] Isotopically labeled compounds are also within the scope of this disclosure.As used herein, "isotopically labeled compounds" refers to compounds disclosed herein, including pharmaceutical salts and prodrugs thereof, each of which is described herein, in which one or more atoms are replaced with atoms having different atomic masses or mass numbers from those normally found in nature.Examples of isotopes that can be incorporated into compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as isotopes of each of 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F and 36 Contains Cl.

[0230] By isotopically labeling the compounds disclosed herein, the compounds may be useful in drug and / or substrate tissue distribution assays. 3 H) and carbon-14 ( 14 C) labeled compounds are particularly preferred because they are easy to prepare and detect. 2 Substitution with heavier isotopes, such as H, can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced required dosage, and therefore may be preferred in some circumstances. The isotopically-labeled compounds disclosed herein (including pharmaceutical salts, esters and prodrugs thereof) can be prepared by any means known in the art.

[0231] In addition, hydrogen ( 1 Substitution of hydrogen (H) with heavier isotopes, such as deuterium, can provide certain therapeutic advantages, for example, improved absorption, distribution, metabolism and / or excretion (ADME) properties, resulting in drugs with improved efficacy, safety and / or tolerability. 12 C 13 Advantages may also be obtained by replacing with C. (WO 2007 / 005643, WO 2007 / 005644, WO 2007 / 016361, and WO 2007 / 016431.)

[0232] Thus, isotopically derivatized compounds having one or more hydrogen atoms (e.g., 1, 2, 4, 5, 6, 7, 8, 9, 10, etc.) replaced with a deuterium atom are contemplated in the present invention. In certain embodiments, an isotopically derivatized compound of the present invention has one hydrogen atom replaced with a deuterium atom.

[0233] All stereoisomers (e.g., cis and trans isomers) and all optical isomers (e.g., R and S enantiomers) of the compounds disclosed herein, as well as racemic, diastereomeric and other mixtures of such isomers, are within the scope of the disclosure.

[0234] After preparation, the compounds of the present invention are preferably isolated and purified to obtain compositions containing an amount equal to or greater than 95% by weight ("substantially pure"), which are then used or formulated as described herein. In certain embodiments, the compounds of the present invention are greater than 99% pure.

[0235] Solvates and polymorphs of the compounds of the invention are also contemplated herein. Solvates of the compounds of the invention include, for example, hydrates.

[0236] The following examples are intended to illustrate the practice of the present invention and are not intended to be limiting in any way. EXAMPLES

[0237] synthesis [ka] (S)-tert-Butyl 2-((1R,2R)-3-(benzyloxy)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate INT-2 To a solution of (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid INT-1 (19 g, 66.12 mmol; Leyan) in 190 mL of dry THF was added Cs2CO3 (28 g, 85.96 mmol) followed by BnBr (7.27 mL, 78.02 mmol). The resulting mixture was stirred at room temperature for 16 h. LCMS showed completion. The mixture was directly filtered and the filter cake was washed with TNF (30 mL*3). The filtrate was collected and concentrated to give crude INT-2 (ca. 33 g, yield >100%, containing BnBr) as a yellow liquid, which was used directly without further purification.

[0238] [ka] (2R,3R)-Benzyl 3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanoate hydrochloride INT-3 The above INT-2 (66.12 mmol) was dissolved in 160 mL DCM, then 4 M HCl / dioxane (80 mL, 320 mmol) was added. The resulting mixture was stirred at room temperature for 3 h. TCL showed completion. The reaction was concentrated and directly dried, then redissolved in DCM (30 mL), then MTBE (300 mL) was added. The mixture was stirred at 0° C. for 0.5 h, during which time a white solid precipitated. The white solid was collected by filtration and washed with MTBE / DCM = 10:1 (20 mL*3) to give INT-3 (19.5 g, 94% yield): LCMS (ESI): m / z 278.2 [M + H] + ; 1 H NMR (400 MHz, CDCl3)δ 10.27(s, 1H), 9.08(s, 1H), 7.44 - 7.26(m, 5H), 5.13(q, J = 12.3 Hz, 2H), 4.11-4.01(m, 1H), 3.75-3.63(m, 1H), 3.58(s, 3H), 3.37-3.21(m, 2H), 2.88 - 2.78(m, 1H), 1.99 - 1.82(m, 4H), 1.28(d, J = 7.0 Hz, 3H).

[0239] [ka] (3R,4S,5S)-tert-Butyl 4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoate INT-5 (3R,4S,5S)-tert-Butyl 3-methoxy-5-methyl-4-(methylamino)heptanoic acid hydrochloride. To a solution of INT-4 (60 g, 185.9 mmol; Leyan) in 1 L of dry DCM was added DIEA (141.3 mL, 743.6 mmol). The mixture was stirred at room temperature for 10 min and then cooled to 0°C. o C. Cbz-Val-OH (61.2 g, 223.1 mmol; Leyan) and BEP (74.98 g, 250.9 mmol) were added. The resulting mixture was allowed to warm to room temperature naturally and stirred for 16 h. LCMS showed completion. The reaction was washed with H2O (1 L*2) and brine, dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by flash chromatography (petroleum ether: EtOAc = 4:1 to 2:1, v / v) to give INT-5 (79.5 g, 86% yield) as a yellow oil. LCMS(ESI): m / z 493.0 [M + H] + .

[0240] [ka] (3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoic acid INT-6 To a solution of INT-5 (79.5 g, 161.5 mmol) in 320 mL of DCM was added 20 mL of 4 M HCl / dioxane (480 mL, 1.9 mmol). oC or less for 15 min. The reaction was then stirred at room temperature for 16 h. LCMS showed completion. The mixture was concentrated to dryness and the residue was purified by reverse phase column (H2O / CH3CN) to give INT-6 (55.4 g, 78.6% yield) as a white solid. LCMS (ESI): m / z 437.1 [M + H] + .

[0241] [ka] (2R,3R)-Benzyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid INT-7 To a solution of INT-6 (9.7 g, 22.22 mmol) and INT-3 (7.32 g, 23.33 mmol) in 200 mL DMF was added HATU (16.9 g, 44.44 mmol) at room temperature. o C and DIEA (16.5 mL, 0.1 mmol) was added. The resulting mixture was allowed to warm to room temperature naturally and stirred for 3 h. LCMS showed completion. The reaction was diluted with DCM (500 mL), washed with H2O (1 L*2), dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by reverse phase column (H2O / CH3CN) to give INT-7 (12.5 g, 81% yield) as a yellow oil. LCMS (ESI): m / z 696.3 [M + H] + .

[0242] [ka] (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-amino-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid INT-8 To a solution of INT-7 (12.5 g, 17.96 mmol) in 125 mL MeOH was added 10% Pd / C (3.75 g). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 4 h. LCMS showed completion. The mixture was filtered and the filter cake was washed with DCM / MeOH = 1:1 (v / v) (50 mL*3). The filtrates were combined and concentrated to give INT-8 (8 g, 94% yield) as a white solid. LCMS(ESI): m / z 472.1 [M + H] + ; HPLC (NH2 column): 99.7% @210 nm, R t = 6.59 minutes.

[0243] [ka] (3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoic acid INT-10 To a solution of INT-9 (8 g, 16.47 mmol) in 100 mL DCM was added TFA (34.5 mL, 461.2 mmol). The mixture was stirred at room temperature for 6 h. HPLC showed completion. The reaction was directly concentrated to give crude INT-10 (9.5 g, 100% yield) as a yellow oil, which was used directly without further purification. LCMS (ESI): m / z 430.2 [M + H] + .

[0244] [ka] (2R,3R)-Benzyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid INT-11 To a solution of crude INT-10 (9.5 g, 16.47 mmol; see above) in 50 mL DMF was added HATU (12.39 g, 32.6 mmol) and DIEA (12.1 mL, 73.33 mmol). The reaction was stirred at room temperature for 0.5 h, after which INT-3 (6.9 g, 22 mmol) was added. The resulting mixture was stirred at room temperature for 20 h. LCMS showed completion. The reaction was concentrated and the residue was purified by reverse phase column (H2O / CH3CN) to give INT-11 (6.57 g, 8.5% yield for two steps) as a white solid. LCMS (ESI): m / z 689.5 [M + H] + .

[0245] [ka] (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid INT-12 To a solution of INT-11 (6.4 g, 9.29 mmol) in 315 mL MeOH / DCM (v / v = 20:1) was added 10% Pd / C (1.6 g). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 16 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated to give INT-12 (5.61 g, 100% yield) as a white solid. LCMS (ESI): m / z 599.3 [M + H] + HPLC: 97.8% @210 nm, R t = 8.61 minutes; 1H NMR (400 MHz, DMSO) δ 12.29(s, 1H), 9.51(s, 1H), 8.91(s, 1H), 4.66(bs, 1H), 4.61 - 4.50(m, 1H), 4.03 - 3.95(m, 2H), 3.92 - 3.76(m, 1H), 3.74 - 3.61 (m, 1H), 3.56 - 3.48 (m, 1H), 3.29 (s, 3H), 3.22 - 3.14 (m, 4H), [3.04 (s, 0.6H), 3.01 (s, 2.4H)], 2.82 - 2.68 (m, 6H), 2.48 - 2.43(m, 1H), 2.39 - 2.26(m, 3H), 2.08 - 1.83(m, 4H), 1.83 - 1.68(m, 3H), 1.35 - 1.27(m, 1H), [1.18(d, J = 6.9 Hz, 0.6H), 1.11(d, J = 6.8 Hz, 2.4H)], 0.97 - 0.84(m, 15H), 0.77(t, J = 7.1 Hz, 3H).

[0246] [ka] tert-Butyl 3-nitrophenethylcarbamate INT-14 2-(3-Nitrophenyl)ethanamine hydrochloride. To a solution of INT-13 (5.5 g, 27.14 mmol) in 110 mL dry DCM was added Et3N (11.32 mL / 8.24 g, 81.43 mmol) at room temperature under N2 atmosphere. The mixture was cooled to 0 °C using an ice bath, and then Boc2O (2.37 g, 10.86 mmol) was added. The reaction was allowed to warm to room temperature and stirred for 16 h. TLC showed completion (petroleum ether: EtOAc = 1:1, R f = 0.75). The mixture was quenched by adding 120 mL H2O, then extracted with DCM (70 mL*3). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by flash chromatography (petroleum ether: EtOAc = 10:1~5:1, v / v) to give INT-14 (7.23 g, 100% yield) as a pale yellow oil. 1H NMR (400 MHz, CDCl3)δ 8.11-8.04(m, 2H), 7.55-7.51(m, 1H), 7.50-7.44(m, 1H), 4.60(bs, 1H), 3.41(q, J = 6.7 Hz, 2H), 2.92(t, J = 7.0 Hz, 2H), 1.42(s, 9H).

[0247] [ka] tert-Butyl methyl(3-nitrophenethyl)carbamate INT-15 To a solution of tert-butyl 3-nitrophenethylcarbamate INT-14 (7.23 g, 27.13 mmol) in 80 mL dry DMF was added 60% NaH (1.63 g, 40.7 mmol) in three portions over 20 min at 0 °C under N2 atmosphere. The mixture was stirred at 0 °C for 10 min, after which CHI (2.87 mL, 46.12 mmol) was added. The reaction was allowed to warm to room temperature and stirred for 3 h. TLC showed completion (petroleum ether: EtOAc = 5:1, R f = 0.6). 0 o The mixture was quenched by slowly adding 100 mL H2O at C and extracted with EtOAc (60 mL*3). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by flash chromatography (petroleum ether: EtOAc = 50:1 ~ 40:1 ~ 30:1, v / v) to give INT-15 (6.56 g, 86% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3)δ 8.12-8.03(m, 2H), 7.60-7.41(m, 2H), 3.48(t, J = 7.2 Hz, 2H), 2.99-2.88(m, 2H), 2.84(s, 3H), 1.37(s, 9H).

[0248] [ka] N-Methyl-2-(3-nitrophenyl)ethanamine hydrochloride INT-16 tert-Butyl methyl(3-nitrophenethyl)carbamate. To a solution of INT-15 (6.56 g, 23.40 mmol) in 60 mL DCM was added 4 M HCl / dioxane (30 mL, 120 mmol). The reaction was stirred at room temperature for 2 h during which time a lot of white solid precipitated. TCL showed completion. The mixture was concentrated and dried. The residue was suspended three times with MTBE (40 mL) to give INT-16 (4.92 g, 97% yield) as a pale yellow solid: LCMS (ESI): m / z 181.1 [M + H] + HPLC: 99.2% @210 nm, R t = 11.90 minutes; 1 H NMR (400 MHz, DMSO-d6)δ 9.22(bs, 2H), 8.17(t, J = 1.8 Hz, 1H), 8.15-8.09(m, 1H), 7.77(d, J = 7.7 Hz, 1H), 7.64(t, J = 7.9 Hz, 1H), 3.25-3.08(m, 4H), 2.54(s, 3H).

[0249] [ka] (2R,3R)-Methyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-amino-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid INT-17 Acetyl chloride (1.16 mL, 16.22 mmol) was added dropwise to dry MeOH (17 mL) at 0° C. under N2 atmosphere. The solution was stirred at 0° C. for 1 h, then INT-8 (1.5 g, 3.18 mmol) was added in one portion. The reaction was allowed to warm to room temperature and stirred for 16 h. LCMS showed completion. The mixture was stirred for 35 h. o C and concentrated to dryness under reduced pressure. The residue was suspended in MTBE (15 mL*2) to give INT-17 (1.55 g, 100% yield) as a yellow foamy solid: LCMS(ESI): m / z 486.1 [M + H] + ; HPLC (NH2 column): 96.1% @210 nm, Rt = 11.30 minutes; 1 H NMR (400 MHz, CDCl3)δ 8.67-8.07(m, 2H), 4.90-4.51(m, 1H), 4.50-4.30(m, 1H), 4.22-4.09(m, 1H), 4.02-3.88(m, 1H), 3.87-3.73(m, 1H), 3.71(s, 3H), 3.60 - 3.37(m, 6H), 3.32(s, 3H), 3.21-3.04(m, 2H), 3.03-2.89(m, 1H), 2.62 - 2.54(m, 1H), 2.53-2.42(m, 2H), 2.36-2.22(m, 1H), 2.13-2.01(m, 2H), 1.96-1.81( m, 2H), 1.60-1.47(m, 1H), 1.36-1.21(m, 7H), 1.15-1.00(m, 7H), 0.95-0.82(m, 3H).

[0250] [ka] (2R,3R)-Methyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid INT-18 To a solution of INT-17 (3.96 g, 7.58 mmol) in 40 mL dry THF was added Et3N (3.15 mL / 2.30 g, 22.73 mmol) at room temperature under N2 atmosphere. The mixture was cooled to 0°C by ice bath, then Boc2O (1.82 g, 8.33 mmol) was added. The reaction was allowed to warm to room temperature naturally and stirred for 16 h. LCMS showed completion. The solvent of THF was removed by concentration, then 100 mL EtOAc was added. The resulting mixture was washed with H2O (30 mL*2) and brine (30 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (petroleum ether: EtOAc = 5:1~3:1~1:1, v / v) to give INT-18 (4.55 g, 76% yield) as a colorless oil. LCMS(ESI): m / z 586.3 [M+H]+ .

[0251] [ka] (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid INT-19 To a solution of INT-18 (1.61 g, 2.75 mmol) in 20 mL THF was added 10 mL LiOH (330 mg, 13.78 mmol) in water. The mixture was stirred at room temperature for 16 h. HPLC showed completion (<5% de-Boc by-product was detected). 4 M HCl / dioxane (~4 mL) was added slowly to adjust the PH to ~2. The resulting mixture was diluted with H2O (15 mL) and then extracted with DCM (30 mL*3). The combined organic layers were washed with H2O (15 mL) and brine (15 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give INT-19 (1.06 g, 67% yield) as a colorless oil. LCMS(ESI): m / z 572.3 [M + H] +

[0252] [ka] tert-Butyl ((S)-2-(((2S,3R)-5-(ethyl((2S,3R)-3-methoxy-5-((3-nitrophenethyl)amino)-5-oxopentan-2-yl)amino)-3-methoxy-5-oxopentan-2-yl)(methyl)amino)-4-methylpent-1-en-3-yl)carbamate INT-20 Tube A: To a solution of INT-16 (524 mg, 2.42 mmol) in DMF (3 mL) was added DIEA (0.8 mL, 4.7 mmol). The mixture was stirred at room temperature for 0.5 h to form solution A.

[0253] Tube B: To another solution of INT-19 (1.06 g, 1.86 mmol) in 10 mL DMF was added HATU (1.42 g, 3.72 mmol) at room temperature. The mixture was stirred at room temperature for 0.5 h, solution A was added, followed by DIEA (0.6 mL, 3.75 mmol). The resulting mixture was stirred at room temperature for 2 h. LCMS showed completion. The reaction was directly purified by reverse phase column (H2O / CH3CN) to give INT-20 (1.16 g, 85% yield) as a yellow oil. LCMS (ESI): m / z 734.1 [M + H] + .

[0254] [ka] (3R,4S)-4-((S)-2-amino-N,3-dimethylbutanamido)-N-ethyl-3-methoxy-N-((2S,3R)-3-methoxy-5-((3-nitrophenethyl)amino)-5-oxopentan-2-yl)pentanamide hydrochloride INT-21 To a solution of INT-20 (1.16 g, 1.58 mmol) in 12 mL DCM was added 4 M HCl / dioxane (6 mL, 24 mmol). The reaction was stirred at room temperature for 2 h. TCL showed completion. The mixture was concentrated and dried. The residue was suspended in MTBE (10 mL*3) and then lyophilized to give INT-21 (1.1 g, 100% yield) as a yellow solid. LCMS(ESI): m / z 634.2 [M + H] + .

[0255] [ka] (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-22 To a solution of INT-16 (178 mg, 0.818 mmol) in 7 mL DMF, DIEA (227 mg, 1.753 mmol) was added dropwise, followed by HATU (334 mg, 0.877 mmol), INT-12 (350 mg, 0.584 mmol) and DMF (7 mL). The mixture was stirred at room temperature for 2 h, and HPLC showed completion. EtOAc (100 mL) was added to the reaction mixture, which was then washed with brine (50 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column (DCM: MeOH = 100:1 to 10:1, v / v), followed by Prep-TLC (DCM: MeOH = 12:1, v / v; R f = 0.6) to give INT-22 (277 mg, 62% yield) as a yellow oil.

[0256] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide 1 To a solution of INT-22 (277 mg, 0.364 mmol) in 25 mL MeOH was added 10% Pd / C (50 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 16 h. TLC (DCM: MeOH = 12:1, v / v, R f = 0.6) indicated completion. The mixture was filtered and the filtrate was concentrated. The residue was analyzed by prep-TLC (DCM:MeOH = 12:1, v / v, R f = 0.6) to give 1 (122 mg, 46% yield) as an off-white solid. LCMS (ESI): m / z 731.0 [M + H] + ; HPLC: 98.6% @210 nm, R t = 8.10 minutes; 1H NMR (400 MHz, CDCl3)δ 7.15 - 7.03(m, 1H), 7.03 - 6.94(m, 1H), 6.61 - 6.53(m, 1H), 6.53 - 6.41(m, 2H), 4.97 - 4.89(m, 1H), 4.89 - 4.66 (m, 1H), 4.27 - 4.14 (m, 1H), 4.01 - 3.88 (m, 1H), 3.86 - 3.68 (m, 2H), 3.60 - 3.53 (m, 1H), 3.45 - 3.37 (m, 4H), 3.37 - 3.29 (m, 4H), 3.27 - 3.22 (m, 1H), 3.17 - 3.14 (m, 1H), 3.04 - 2.98 (m, 1H), 2.94 - 2.85 (m, 3H), 2.80 - 2.68 (m, 3H), 2.62 - 2.45 (m, 3H), 2.38 - 2.27 (m, 6H), 2.12 - 1.20 - 1.12(m, 3H), 1.06 - 0.96(m, 9H), 0.96 - 0.90(m, 6H), 0.85 - 0.78(m, 3H).

[0257] [ka] (S)-2-((S)-2-(dimethylamino)propanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-23 To a solution of INT-21 (200 mg, 0.32 mmol) and (S)-2-(dimethylamino)propanoic acid (44 mg, 0.38 mmol) in 5 mL DMF was added HATU (1.42 g, 3.72 mmol) followed by DIEA (124 mg, 0.96 mmol). The mixture was stirred at room temperature for 1 h and LCMS showed completion (LCMS(ESI): m / z 733.1 [M + H] + ). The reaction was quenched with 20 mL H2O and extracted with DCM (15 mL*3). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered and concentrated. The residue was analyzed by Prep-TLC (DCM: MeOH = 12:1, v / v; R f = 0.7) to afford INT-23 (210 mg, 91% yield) as a pale yellow oil (87% purity @ 210 nm on HPLC) which was used as is without further purification.

[0258] [ka] Step 2: (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)propanamide)-N,3-dimethylbutanamide 2 To a solution of INT-23 (210 mg, 0.287 mmol) in 5 mL MeOH was added 10% Pd / C (42 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 3 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 2 (180 mg, 94% yield) as an off-white solid. LCMS (ESI): m / z 703.4 [M + H] + HPLC: 95.1% @210 nm, R t = 9.32 minutes; 1H NMR (400 MHz, DMSO) δ 7.73(t, J = 10.0 Hz, 1H), 6.95-6.85(m, 1H), 6.45-6.31(m, 3H), 5.01-4.84(m, 2H), 4.79-4.51(m, 2H), 4.05-3.93(m, 1H), 3.93- 3.76(m, 1H), 3.75-3.67(m, 1H), 3.67 - 3.55(m, 1H), 3.54-3.41(m, 2H), 3.40-3.35(m, 1H), 3.30-3.24(m, 2H), 3.24-3.05( m, 5H), 2.94(s, 1H), 2.91-2.88(m, 2H), 2.88-2.74(m, 2H), 2.68-2.54(m, 3H), 2.49- 2.38(m, 1H), 2.26-2.14(m, 6H), 2.07-1.78(m, 4H), 1.76-1.55(m, 2H), 1.33-1.21( m, 2H), 1.12-1.05(m, 4H), 1.04-0.95(m, 2H), 0.94-0.81(m, 9H), 0.80-0.73(m, 3H).

[0259] [ka] (S)-2-((R)-2-(dimethylamino)propanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-24 To a solution of INT-21 (165 mg, 0.246 mmol) and N,N-dimethyl-L-alanine (38 mg, 0.32 mmol) in 3 mL DMF, HATU (187 mg, 0.492 mmol) was added followed by DIEA (0.16 mL, 0.985 mmol). The mixture was stirred at room temperature under N2 atmosphere for 2 h, and LCMS showed completion. The reaction mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-24 (130 mg, 72% yield) as a colorless oil. LCMS (ESI): m / z 733.1 [M + H] + .

[0260] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((R)-2-(dimethylamino)propanamide)-N,3-dimethylbutanamide 3 To a solution of INT-24 (130 mg, 0.177 mmol) in 3 mL MeOH was added 10% Pd / C (40 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 3 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was lyophilized to give 3 (115 mg, 92% yield) as an off-white solid. LCMS (ESI): m / z 703.2 [M + H] + ; HPLC: 99.4% @210 nm, R t = 7.94 minutes; 1 H NMR (400 MHz, DMSO) δ 7.85-7.74(m, 1H), 6.96-6.86(m, 1H), 6.53(bs, 1H), 6.47-6.31(m, 3H), 4.93(dd, J1= 29.9 Hz, J2= 10.8 Hz, 2H), 4.79-4.62(m, 1H), 4.60 - 4.49(m, 1H), 4.07-3.95(m, 1H), 3.93-3.67(m, 2H), 3.66 - 3.43(m, 3H), 3.40-3.34(m, 1H), 3.30-3.24(m, 2H), 3.21-3.05(m, 5H), 2.97-2.94(m, 1H), 2.93-2.87(m, 2H), 2.86-2.73(m, 2H), 2.68- 2.54(m, 3H), 2.49-2.37(m, 1H), 2.18-2.12(m, 6H), 2.01-1.78(m, 4H), 1.72-1.54(m, 2H), 1.35-1.26(m, 1H), 1.10-0.96(m, 6H), 0.94- 0.82(m, 10H), 0.81- 0.73(m, 3H).

[0261] [ka] (S)-2-(2-(dimethylamino)acetamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-21 To a solution of INT-21 (210 mg, 0.332 mmol) and 2-(dimethylamino)acetic acid (41 mg, 0.398 mmol) in 5 mL DMF was added HATU (189 mg, 0.497 mmol) followed by DIEA (128 mg, 0.992 mmol). The mixture was stirred at room temperature for 1 h and LCMS showed completion (LCMS(ESI): m / z 719.1 [M + H] + ). The reaction was diluted with 20 mL H2O and then extracted with DCM (15 mL*3). The combined organic layers were washed with H2O (10 mL) and brine (10 mL*3), dried over Na2SO4, filtered and concentrated. The residue was analyzed by Prep-TLC (DCM: MeOH = 13:1, v / v; R f = 0.7) to give INT-25 (190 mg, yield 79.8%) as a colorless oil.

[0262] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(2-(dimethylamino)acetamide)-N,3-dimethylbutanamide 4 To a solution of INT-25 (190 mg, 0.264 mmol) in 5 mL MeOH was added 10% Pd / C (38 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 3 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 4 (103 mg, 56.6% yield) as a white solid. LCMS (ESI): m / z 689.2 [M + H] + HPLC: 99.2% @210 nm, R t = 11.16 minutes; 1 H NMR (400 MHz, DMSO) δ 7.69-7.60(m, 1H), 6.96-6.86(m, 1H), 6.45-6.31(m, 3H), 4.93(dd, J1= 31.4 Hz, J2= 12.4 Hz, 2H), 4.82-4.55(m, 2H), 4.06-3.94(m, 1H), 3.93-3.63(m, 2H), 3.60 -3.42(m, 2H), 3.39-3.35(m, 1H), 3.30-3.24(m, 2H), 3.22-3.15(m, 3H) , 3.15-3.04(m, 2H), 3.01-2.92(m, 2H), 2.91-2.85(m, 2H), 2.85-2.77( m, 2H), 2.67-2.54(m, 3H), 2.48-2.38(m, 1H), 2.25-2.15(m, 6H), 2.03- 1.77(m, 4H), 1.74-1.56(m, 2H), 1.33-1.21(m, 2H), 1.09-0.99(m, 2H), 0.97- 0.70(m, 14H).

[0263] [ka] (S)-2-(3-(dimethylamino)-2,2-dimethylpropanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-26 To a solution of INT-21 (200 mg, 0.30 mmol) in 4 mL DMF was added DIEA (174 mg, 1.34 mmol). The mixture was stirred at room temperature for 5 min, then 3-(dimethylamino)-2,2-dimethylpropanoic acid (52 mg, 0.36 mmol) and HATU (170 mg, 0.448 mmol) were added. The resulting mixture was stirred at room temperature for 1 h, and LCMS showed completion. The reaction was directly concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give crude INT-26 (223 mg, 98% yield) as a yellow oil, which was used directly without further purification. LCMS (ESI): m / z 761.0 [M + H] + .

[0264] [ka] Step 2: (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(3-(dimethylamino)-2,2-dimethylpropanamide)-N,3-dimethylbutanamide 5 To a solution of INT-26 (223 mg, 0.293 mmol) in 4 mL MeOH was added 10% Pd / C (45 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 2 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 5 (184 mg, 85.6% yield) as an off-white solid. LCMS (ESI): m / z 731.2 [M + H] + HPLC: 96.7% @210 nm, R t = 12.84 minutes; 1H NMR (400 MHz, DMSO) δ 7.93(s, 1H), 6.98 - 6.86(m, 1H), 6.48 - 6.31(m, 3H), 5.16 - 4.79(m, 1H), 4.78 - 4.59(m, 1H), 4.58 - 4.39(m, 1H), 4.09 - 3.96 (m, 1H), 3.95 - 3.57 (m, 2H), 3.56 - 3.39 (m, 2H), 3.31 - 3.24 (m, 4H), 3.23 - 3.12 (m, 5H), 3.10 - 2.95 (m, 3H), 2.91 - 2.86 (m, 2H), 2.85 - 2.75 (m, 2H), 2.72 - 2.54 (m, 7H), 2.47 - 2.39 (m, 1H), 2.36 - 2.20 (m, 1H), 2.14 - 2.03 (m, 1H), 1.96 - 1.75 (m, 3H), 1.74 - 1.56 (m, 2H), 1.31 - 1.14 (m, 6H), 1.12 - 0.96 (m, 3H), 0.96 - 0.82 (m, 10H), 0.82 - 0.59 (m, 5H).

[0265] [ka] (R)-N-((S)-1-(((3R,4S,5S)-3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-3-carboxamide INT-27 To a solution of INT-21 (200 mg, 0.30 mmol) in 4 mL DMF was added DIEA (174 mg, 1.34 mmol). The mixture was stirred at room temperature for 5 min, then (R)-1-methylpiperidine-3-carboxylic acid (51 mg, 0.36 mmol) and HATU (170 mg, 0.448 mmol) were added. The resulting mixture was stirred at room temperature for 1 h, and LCMS showed completion. The reaction was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give INT-27 (232 mg, 100% yield) as a pale yellow oil. LCMS (ESI): m / z 759.0 [M + H] + .

[0266] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-3-carboxamide 6 To a solution of INT-27 (232 mg, 0.293 mmol) in 4 mL MeOH was added 10% Pd / C (46 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 3 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 6 (191 mg, 85.7% yield) as an off-white solid. LCMS (ESI): m / z 729.1 [M + H] + HPLC: 97.7% @210 nm, R t = 12.00 minutes; 1H NMR (400 MHz, DMSO) δ 8.46 - 8.25 (m, 1H), 7.02 - 6.82 (m, 1H), 6.44 - 6.29 (m, 3H), 5.07 - 4.83 (m, 1H), 4.79 - 4.59 (m, 1H), 4.56 - 4.42 (m, 1H), 4.07 - 3.97 (m, 1H), 3.96 - 3.69 (m, 2H), 3.65 - 3.56 (m, 1H), 3.54 - 3.45 (m, 2H), 3.29 - 3.26 (m, 2H), 3.23 - 3.12 (m, 6H), 3.08 - 2.93(m, 3H), 2.92 - 2.87 (m, 2H), 2.85 - 2.78 (m, 2H), 2.77 - 2.69 (m, 2H), 2.65 - 2.57 (m, 5H), 2.47 - 2.38 (m, 1H), 2.04 - 1.82 (m, 4H), 1.81 - 1.69(m, 3H), 1.67 - 1.56(m, 2H), 1.47 - 1.36(m, 1H), 1.32 - 1.25(m, 1H), 1.12 - 0.95(m, 4H), 0.95 - 0.81(m, 11H), 0.81 - 0.73(m, 3H).

[0267] [ka] (S)-2-(2-(dimethylamino)-2-methylpropanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-28 To a solution of 2-(dimethylamino)-2-methylpropanoic acid (78 mg, 0.597 mmol) and HATU (227 mg, 0.597 mmol) in 3 mL DMF was added DIEA (0.2 mL, 1.2 mmol). The mixture was stirred at room temperature under N2 atmosphere for 30 min, after which INT-21 (200 mg, 0.298 mmol) was added. The resulting mixture was stirred at room temperature for 4 h. LCMS showed completion. The reaction mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-28 (140 mg, 63% yield) as a pale yellow solid. LCMS(ESI): m / z 747.0 [M + H] + .

[0268] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(2-(dimethylamino)-2-methylpropanamide)-N,3-dimethylbutanamide 7 To a solution of INT-28 (140 mg, 0.187 mmol) in 3 mL MeOH was added 10% Pd / C (40 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 4 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was lyophilized to give 7 (125 mg, 93% yield) as a yellow solid. LCMS (ESI): m / z 717.1 [M + H] + HPLC: 97.7% @210 nm, R t = 12.62 minutes; 1H NMR (400 MHz, DMSO) δ 7.71-7.55 (m, 1H), 6.96 - 6.85(m, 1H), 6.96-6.85(m, 1H), 6.48-6.31(m, 3H), 5.04-4.85(m, 2H), 4.78-4.49(m, 2H), 4.04 -3.96(m, 1H), 3.95-3.68(m, 2H), 3.66-3.55(m, 1H), 3.54-3.42(m, 2H), 3.41-3.36(m, 1H), 3.34 -3.25(m, 2H), 3.24-3.10(m, 4H), 3.10-2.92(m, 2H), 2.92-2.84(m, 2H), 2.81(d, J = 11.2 Hz, 1H), 2.69-2.53(m, 4H), 2.48-2.38(m, 1H), 2.31-2.05(m, 6H), 2.03-1.76(m, 4H), 1.76 -1.55(m, 2H), 1.32-1.22(m, 2H), 117-1.02(m, 5H), 1.01-0.95(m, 3H), 0.94-0.72(m, 12H).

[0269] [ka] (S)-N-((S)-1-(((3R,4S,5S)-3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-2-carboxamide INT-29 To a solution of N-methyl-L-proline monohydrate (50 mg, 0.388 mmol) and HATU (227 mg, 0.597 mmol) in 3 mL DMF, DIEA (0.22 mL, 1.34 mmol) was added. The mixture was stirred at room temperature under N2 atmosphere for 30 min. INT-21 (200 mg, 0.298 mmol) was then added. The resulting mixture was stirred at room temperature for 2 h. LCMS showed completion. The reaction mixture was concentrated and the residue was redissolved in 70 mL EtOAc. The organic layer was washed with H2O (15 mL*2) and brine (10 mL), dried over Na2SO4, filtered and concentrated to give crude INT-29 (260 mg, yield >100%) as a yellow foamy solid, which was used as is without further purification. LCMS(ESI): m / z 745.1 [M + H] + .

[0270] [ka] (S)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-2-carboxamide 8 To a solution of INT-29 (220 mg, 0.295 mmol; see above) in 5 mL MeOH was added 10% Pd / C (60 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 5 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 8 (150 mg, 71% yield) as an off-white solid. LCMS (ESI): m / z 715.1 [M + H] + ; HPLC: 98.2% @210 nm, R t = 11.96 minutes; 1H NMR (400 MHz, DMSO) δ 9.06 - 8.95 (m, 1H), 7.08 - 6.96 (m, 1H), 6.68 - 6.49 (m, 3H), 4.80 - 4.50 (m, 2H), 4.19 - 3.81 (m, 3H), 3.79 - 3.71 (m, 1H), 3.69 - 3.43 (m, 5H), 3.29 - 3.25 (m, 2H), 3.22 - 3.09 (m, 6H), 3.01 - 2.86 (m, 4H), 2.84 - 2.76 (m, 4H), 2.72 - 2.59 (m, 3H), 2.47 - 2.40(m, 1H), 2.40 - 2.17(m, 1H), 2.12 - 1.78(m, 6H), 1.78 - 1.56(m, 3H), 1.36 - 1.15(m, 2H), 1.12 - 1.01(m, 2H), 1.01 - 0.84(m, 11H), 0.84 - 0.70(m, 3H).

[0271] [ka] (R)-N-((S)-1-(((3R,4S,5S)-3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-2-carboxamide INT-30 To a solution of INT-21 (200 mg, 0.3 mmol) and (R)-1-methylpyrrolidine-2-carboxylic acid (50 mg, 0.387 mmol) in 5 mL DMF, HATU (182 mg, 0.48 mmol) was added followed by DIEA (124 mg, 0.96 mmol). The mixture was stirred at room temperature for 1 h and LCMS showed completion (LCMS(ESI): m / z 745.0 [M+ H] + The reaction was quenched with 0.5 mL H2O and concentrated directly. The residue was analyzed by prep-TLC (DCM: MeOH = 12:1, v / v; f= 0.7) to give crude INT-30 (220 mg, yield 93.6%) as a colorless oil.

[0272] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-2-carboxamide 9 To a solution of INT-30 (220 mg, 0.296 mmol; see above) in 5 mL MeOH was added 10% Pd / C (50 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 3 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 9 (168 mg, 79.6% yield) as a white solid. LCMS (ESI): m / z 715.3 [M+ H] + ; HPLC: 98.2% @210 nm, R t = 11.88 minutes; 1H NMR (400 MHz, DMSO)δ 7.71 - 7.59(m, 1H), 6.96 - 6.85(m, 1H), 6.46 - 6.30(m, 3H), 4.99 - 4.86(m, 2H), 4.76 - 4.50(m, 2H), 4.13 - 3.87 (m, 2H), 3.78 - 3.60 (m, 2H), 3.59 - 3.41 (m, 3H), 3.30 - 3.23 (m, 2H), 3.22 - 3.10 (m, 4H), 3.09 - 2.92 (m, 3H), 2.91 - 2.83 (m, 2H), 2.83 - 2.76(m, 2H), 2.68 - 2.55 (m, 3H), 2.49 - 2.37 (m, 1H), 2.34 - 2.22 (m, 4H), 2.11 - 1.95 (m, 2H), 1.95 - 1.77 (m, 3H), 1.75 - 1.48 (m, 5H), 1.33 - 1.18 (m, 2H), 1.11 - 1.01 (m, 2H), 1.00 - 0.82 (m, 10H), 0.80 - 0.71 (m, 3H).

[0273] [ka] (R)-N-((S)-1-(((3R,4S,5S)-3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-3-carboxamide INT-31 To a solution of (R)-1-methylpyrrolidine-3-carboxylic acid (50 mg, 0.39 mmol) and HATU (227 mg, 0.6 mmol) in 3 mL DMF was added DIEA (0.22 mL, 1.34 mmol). The mixture was stirred at room temperature under N2 atmosphere for 30 min, then INT-21 (200 mg, 0.3 mmol) was added. The resulting mixture was stirred at room temperature for 2 h. LCMS showed completion. The reaction was directly concentrated and the residue was redissolved in 70 mL EtOAc. The organic layer was washed with H2O (15 mL*2) and brine (10 mL), dried over Na2SO4, filtered and concentrated to give crude INT-31 (260 mg, LCMS purity approx. 76%) as a yellow foamy solid, which was used as is without further purification. LCMS(ESI): m / z 745.0 [M + H] + .

[0274] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-3-carboxamide 10 To a solution of crude INT-31 (150 mg, 0.2 mmol; see above) in 5 mL MeOH was added 10% Pd / C (40 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 4 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 10 (130 mg, 90.3% yield) as a yellow solid. LCMS (ESI): m / z 715.1 [M + H] + HPLC: 96.2% @210 nm, R t = 7.87 minutes; 1H NMR (400 MHz, DMSO) δ 8.33 - 8.21 (m, 1H), 6.95 - 6.86 (m, 1H), 6.46 - 6.31 (m, 3H), 5.08 - 4.88 (m, 1H), 4.76 - 4.59 (m, 1H), 4.57 - 4.42 (m, 1H), 4.10 - 3.89 (m, 2H), 3.82 - 3.68 (m, 2H), 3.65 - 3.53 (m, 2H), 3.51 - 3.46 (m, 2H), 3.45 - 3.43 (m, 1H), 3.42 - 3.40 (m, 1H), 3.20 - 3.13(m, 5H), 3.09 - 2.94 (m, 4H), 2.91 - 2.86 (m, 2H), 2.84 - 2.74 (m, 2H), 2.68 - 2.54 (m, 6H), 2.49 - 2.19 (m, 2H), 2.13 - 1.96 (m, 2H), 1.95 - 1.90 (m, 1H), 1.89 - 1.75 (m, 3H), 1.74 - 1.57 (m, 2H), 1.35 - 1.23 (m, 2H), 1.11 - 1.01 (m, 2H), 1.00 - 0.92 (m, 2H), 0.91 - 0.82 (m, 8H), 0.81 - 0.74(m, 3H).

[0275] [ka] (R)-N-((S)-1-(((3R,4S,5S)-3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-2-carboxamide INT-32 To a solution of INT-21 (200 mg, 0.3 mmol) in 4 mL DMF was added DIEA (174 mg, 1.34 mmol). The mixture was stirred at room temperature for 5 min, after which (R)-1-methylpiperidine-2-carboxylic acid (51 mg, 0.36 mmol) was added, followed by HATU (170 mg, 0.448 mmol). The resulting mixture was stirred at room temperature for 1 h, and LCMS showed completion. The reaction was concentrated. The residue was purified by reverse phase column (H2O: CH3CN) to give INT-32 (206 mg, 86% yield) as a pale yellow oil, which was used as is without further purification. LCMS (ESI): m / z 759.0 [M + H] + .

[0276] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-2-carboxamide 11 To a solution of INT-32 (156 mg, 0.206 mmol) in 5 mL MeOH was added 10% Pd / C (30 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 16 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 11 (85 mg, 55.7% yield) as an off-white solid. LCMS (ESI): m / z 365.2 [M / 2 + H] + HPLC: 96.6% @210 nm, R t = 11.81 minutes; 1H NMR (400 MHz, DMSO) δ 7.41 (t, J = 8.8 Hz, 1H), 7.02 - 6.83 (m, 1H), 6.46 - 6.30 (m, 3H), 5.14 - 4.92 (m, 1H), 4.92 - 4.53 (m, 2H), 4.39 - 4.27 (m, 1H), 4.09 - 3.87 (m, 2H), 3.83 - 3.69 (m, 1H), 3.68 - 3.57 (m, 1H), 3.55 - 3.44 (m, 2H), 3.43 - 3.35 (m, 1H), 3.30 - 3.25 (m, 2H), 3.24 - 3.16 (m, 4H), 3.14 - 3.07 (m, 3H), 3.05 - 2.95 (m, 4H), 2.93 - 2.89 (m, 3H), 2.87 - 2.76 (m, 7H), 2.69 - 2.53 (m, 4H), 2.49 - 2.40 (m, 1H), 2.34 - 2.21 (m, 1H), 2.04 - 1.88 (m, 2H), 1.87 - 1.75 (m, 2H), 1.73 - 1.45 (m, 2H), 1.33 - 1.17 (m, 2H), 1.16 - 1.00 (m, 4H), 0.99 - 0.87(m, 10H), 0.85 - 0.77(m, 3H).

[0277] [ka] 1-(Dimethylamino)-N-((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)cyclobutanecarboxamide INT-33 To a solution of 1-(dimethylamino)cyclobutanecarboxylic acid (85 mg, 0.597 mmol) in 3 mL DMF was added DIEA (0.2 mL, 1.19 mmol) and HATU (227 mg, 0.597 mmol). The mixture was stirred at room temperature for 0.5 h, after which INT-21 (200 mg, 0.298 mmol) was added. The resulting mixture was stirred at room temperature for 1.5 h. LCMS showed completion. The reaction was directly purified by reverse phase column (H2O / CH3CN) to give INT-33 (178 mg, 78.6% yield) as a white solid. LCMS (ESI): m / z 759.2 [M + H] + .

[0278] [ka] N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-(dimethylamino)cyclobutanecarboxamide 12 To a solution of INT-33 (178 mg, 0.235 mmol) in 5 mL MeOH was added 10% Pd / C (36 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 2 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was analyzed by prep-TLC (DCM:MeOH= 14:1, v / v, R f = 0.5) and then purified by reverse phase column (HO:CHCN) to give 12 (97 mg, 56.6% yield) as an off-white solid. LCMS (ESI): m / z 729.5 [M + H] + ; HPLC: 99.9% @210 nm, R t = 8.13 minutes; 1H NMR (400 MHz, DMSO) δ 7.43(m, 1H), 6.91(m, 1H), 6.46 - 6.31(m, 3H), 4.98(d, J = 6.4 Hz, 1H), 4.90(d, J = 13.8 Hz, 1H), 4.77 - 4.50 (m, 2H), 4.00 (m, 1H), 3.95 - 3.75 (m, 1H), 3.75 - 3.66 (m, 1H), 3.66 - 3.54 (m, 1H), 3.54 - 3.40 (m, 2H), 3.39 - 3.33 (m, 1H), 3.30 - 3.25(m, 2H), 3.24 - 3.07(m, 5H), 2.98(bs, 1H), 2.89(d, J = 3.1 Hz, 2H), 2.84 - 2.79(m, 1H), 2.79 - 2.65(m, 1H), 2.65 - 2.51(m, 3H), 2.49 - 2.38(m, 1H), 2.31 - 2.14 (m, 2H), 2.14 - 2.08 (m, 7H), 2.08 - 1.99 (m, 2H), 1.99 - 1.70 (m, 4H), 1.68 - 1.57 (m, 3H), 1.36 - 1.25 (m, 1H), 1.08 (d, J = 6.7 Hz, 1H), 1.04(d, J = 6.7 Hz, 1H), 0.98(d, J = 6.6 Hz, 1H), 0.95 - 0.82(m, 9H), 0.81 - 0.72(m, 3H).

[0279] [ka] (S)-N-((S)-1-(((3R,4S,5S)-3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(3-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-2-carboxamide INT-34 To a solution of (S)-1-methylpiperidine-2-carboxylic acid (54 mg, 0.38 mmol) in 5 mL DMF was added HATU (182 mg, 0.48 mmol) followed by DIEA (124 mg, 0.96 mmol). The reaction mixture was stirred at room temperature for 0.5 h, after which INT-21 (200 mg, 0.3 mmol) was added. The resulting mixture was stirred at room temperature for 1 h. LCMS showed completion (LCMS(ESI): m / z 759.2 [M+ H] + ). The reaction was quenched with 50 mL H2O and extracted with DCM (20 mL*3). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered and concentrated to give the crude product INT-34 (350 mg, yield>100%) as a yellow oil.

[0280] [ka] (S)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-2-carboxamide 13 To a solution of INT-34 (270 mg, 0.356 mmol) in 10 mL MeOH was added 10% Pd / C (54 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 2 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) to give 13 (224.22 mg, 86.6% yield) as an off-white solid. LCMS (ESI): m / z 729.5 [M+ H] + HPLC: 96.8% @210 nm, R t = 12.03 minutes. 1H NMR (400 MHz, DMSO)δ 7.54 - 7.43(m, 1H), 6.95 - 6.85(m, 1H), 6.47 - 6.30(m, 3H), 4.97(d, J = 5.7 Hz, 1H), 4.90(d, J = 17.0 Hz, 1H), 4.79 - 4.48 (m, 2H), 4.04 - 3.95 (m, 1H), 3.95 - 3.66 (m, 2H), 3.66 - 3.54 (m, 1H), 3.54 - 3.41 (m, 2H), 3.40 - 3.35 (m, 1H), 3.30 - 3.25 (m, 2H), 3.23 - 3.05 (m, 5H), 2.94 (bs, 1H), 2.89 (d, J = 5.0 Hz, 2H), 2.87 - 2.70 (m, 3H), 2.69 - 2.52 (m, 4H), 2.47 - 2.20 (m, 2H), 2.07 - 2.01 (m, 3H), 2.01 - 1.88(m, 3H), 1.88 - 1.75(m, 2H), 1.72 - 1.55(m, 4H), 1.53 - 1.38(m, 2H), 1.33 - 1.25(m, 1H), 1.22 - 1.12(m, 1H), 1.08(d, J = 6.7 Hz, 1H), 1.04(d, J = 6.7 Hz, 1H), 1.00 - 0.92 (m, 2H), 0.91 - 0.80 (m, 8H), 0.80 - 0.72 (m, 3H).

[0281] [ka] (1S,2R)-2-(Methylamino)-1-phenylpropan-1-ol INT-XX A solution of (4R,5S)-4-methyl-5-phenyl-1,3-oxazolidin-2-one INT-35 (100 mg, 0.56 mmol) in THF (4 mL) was stirred for 25 min under nitrogen. o At C, LiAlH4 (43 mg, 1.13 mmol) was added. The reaction mixture was stirred for 60 oC for 4 h. It was quenched with Na2SO4*10 H2O (1 g), filtered and concentrated. The residue was purified by Combi-Flash (petroleum ether: EtOAc = 3 / 1) to give INT-36 (100 mg, 90.47%) as a white solid. LCMS (ESI): m / z 166.1 (M + H) + .

[0282] [ka] (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide 23 To a solution of INT-12 (54 mg, 0.09 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uranium (41 mg, 0.10 mmol) and 2,4,6-collidine (22 mg, 0.18 mmol) in DMAc (0.5 mL) was added a solution of INT-36 (15 mg, 0.09 mmol) in DMAc (0.5 mL) at 25 °C under nitrogen. The reaction mixture was stirred at 25 °C for 30 min. Filtration and direct purification by prep-HPLC (ACN-H20 (0.1% TFA)) gave 23 (45.1 mg, 63.55%) as a white solid. LCMS(ESI): m / z 746.3(M + H)+; 1H NMR(400 MHz, DMSO)δ 9.57(s, 1H), 8.92(d, J = 8.3 Hz, 1H), 7.32 - 7.28(m, 2H), 7.24 - 7.19(m, 2H), 4.91 - 4.42(m, 6H), 4.09(d, J = 4.3 Hz, 1H), 3.99(s, 1H), 3.76(s, 1H), 3.71(s, 1H), 3.65 - 3.57(m, 2H), 3.37(t, J = 9.4 Hz, 1H), 3.29 - 3.21 (m, 6H), 3.20 - 3.06 (m, 3H), 3.04 - 2.98 (m, 1H), 2.95 - 2.89 (m, 1H), 2.81 - 2.74 (m, 6H), 2.72 (d, J = 4.5 Hz, 2H), 2.42 (d, J = 4.4 Hz, 1H), 2.34 - 2.14 (m, 3H), 1.94 - 1.83 (m, 1H), 1.72 - 1.60 (m, 1H), 1.55 - 1.44 (m, 1H), 1.41 - 1.29 (m, 2H), 1.26 - 1.09 (m, 3H), 1.08 - 0.97 (m, 6H), 0.97 - 0.93(m, 6H), 0.92 - 0.85 (m, 6H), 0.78 (dd, J = 15.0, 7.4 Hz, 3H).

[0283] [ka] tert-Butyl (S)-(2-phenyl-1-(thiazol-2-yl)ethyl)carbamate INT-38 To a stirred solution of (1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethanamine INT-37 (204 mg, 1.00 mmol) and Et3N (202 mg, 2.00 mmol) in DCM (5 mL) was added (Boc)2O (327 mg, 1.50 mmol). The mixture was stirred at 25° C. for 16 h. It was diluted with water (10 mL) and extracted with DCM (2*10 mL), the organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuum. The residue was purified by Combi-Flash (petroleum ether: EtOAc = 5 / 1) to give INT-38 (250 mg, 82.2%) as a white solid. LCMS (ESI): m / z 305 (M + H) + .

[0284] [ka] tert-Butyl (S)-methyl(2-phenyl-1-(thiazol-2-yl)ethyl)carbamate INT-39 To a solution of INT-38 (100 mg, 0.328 mmol) in THF (2 mL) was added sodium hydride (27 mg, 0.657 mmol, 60% in mineral oil) at 0° C. The reaction was stirred at 0° C. for 10 min, then MeI (93 mg, 0.657 mmol) was added at 0° C. It was stirred at 25° C. for 2 h, quenched with water (10 mL), extracted with EtOAc (10 mL*3), the organic layers were combined, dried over sodium sulfate, filtered and concentrated. Purification by Combi-Flash (petroleum ether:EtOAc=5:1) gave the methylated product (60 mg). LCMS (ESI): m / z 319.2(M + H) + The methylated product was dissolved in HCl / dioxane (3 mL, 1N), stirred at 25° C. for 1 h, and concentrated to give INT-39 (48 mg, crude) as a white solid.

[0285] [ka] (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide 24 To a solution of INT-12 (30 mg, 0.05 mmol), INT-39 (11 mg, 0.05 mmol) and HATU (23 mg, 0.06 mmol) in DMAc (1.5 mL) was added DIEA (13 mg, 0.10 mmol) and the reaction was stirred at 25 °C for 2 h. Purification directly by prep-HPLC (ACN-H20 (0.1% TFA)) gave 24 (21 mg, 52.3%) as a white solid. LCMS (ESI): m / z 799.5 (M + H). + ; 1 H NMR (400 MHz, DMSO) δ 9.64(s, 1H), 9.00-8.85(m, 1H), 7.90(m, 2H), 7.31 - 7.19(m, 4H), 7.18 - 7.00(m, 1H), 6.50-6.30(m, 1H), 4.90-4.50 m, 3H), 3.66 - 3.40(m, 4H), 3.37-3.29(m, 1H), 3.28(s, 3H), 3.23 - 3.12(m, 3H), 3.11 - 2.91(m, 3H), 2.84- 2.70(m, 8H), 2.46-2.41(m, 1H), 2.34 - 2.23(m, 1H), 2.19 - 2.09(m, 1H), 2.08(m, 6H), 1.94-1.64(m, 2H), 1.59- 1.16(m, 3H), 1.12 - 0.69(m, 21H).

[0286] [ka] Methyl N-(tert-butoxycarbonyl)-N-methyl-L-phenylalaninate INT-41 (4R,5S)-4-Methyl-5-phenyloxazolidin-2-one To a mixture of INT-40 (100 mg, 0.36 mmol) and Ag2O (413 mg, 1.78 mmol) in DMF (4 mL) under nitrogen at 25 °C, a solution of MeI (101 mg, 0.71 mmol) in DMF (1 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 12 h. It was diluted with water (20 mL) and extracted with EtOAc (10 mL*3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi-Flash (petroleum ether: EtOAc = 3 / 1) to give INT-41 (100 mg, 90.5%) as a white solid. LCMS (ESI): m / z 316 (M+Na)+.

[0287] [ka] Methyl Methyl-L-phenylalaninate hydrochloride INT-42 twenty five o To a solution of INT-41 (100 mg, 0.34 mmol) in DCM (4 mL) stirred at 37° C., 1,4-dioxane / HCl (1 mL, 4N) was added. o C for 2 h. Concentration gave INT-42 (60 mg, crude) as a white solid. LCMS (ESI): m / z 194.2 (M+H)+.

[0288] [ka] Methyl N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-N-methyl-L-phenylalaninate 25 To a solution of INT-12 (30 mg, 0.05 mmol), DIEA (18 mg, 0.15 mmol) and HATU (29 mg, 0.07 mmol) in DMAc (1.5 mL) under nitrogen and stirring at 25 °C, a solution of INT-42 (17 mg, 0.07 mmol) in DMAc (0.5 mL) was added. The reaction mixture was stirred at 25 °C for 2 h. Direct purification by prep-HPLC (ACN-H20 (0.1% TFA)) gave 25 (13.3 mg, 32.9%) as a white solid. LCMS(ESI): m / z 774.2(M+H)+; 1H NMR(400 MHz, DMSO)δ 9.59(s, 1H), 8.93(t, J = 8.1 Hz, 1H), 7.28(dd, J = 15.0, 6.7 Hz, 1H), 7.20(s, 4H), 5.43(d, J = 7.2 Hz, 1H), 5.08(ddd, J = 38.3, 11.0, 5.0 Hz, 1H), 4.80 - 4.55(m, 2H), 4.02(d, J = 41.8 Hz, 1H), 3.79 - 3.56(m, 6H), 3.45(dd, J = 17.3, 8.2 Hz, 1H), 3.24(t, J = 10.9 Hz, 5H), 3.17(d, J = 8.8 Hz, 4H), 3.10(d, J = 11.0 Hz, 2H), 3.04 - 2.95(m, 2H), 2.85 - 2.68(m, 10H), 2.48 - 2.39(m, 2H), 2.29(dt, J = 16.0, 8.0 Hz, 1H), 2.18 - 1.96(m, 1H), 1.88(s, 1H), 1.79 - 1.68(m, 1H), 1.61(dd, J = 12.1, 6.3 Hz, 1H), 1.47 - 1.13(m, 2H), 1.05(d, J = 6.6 Hz, 3H), 1.02 - 0.92(m, 9H), 0.92 - 0.89(m, 3H), 0.88 - 0.84(m, 3H), 0.79(t, J = 7.4 Hz, 3H).

[0289] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide 20 To a solution of 3-(2-amino-ethyl)-phenylamine dihydrochloride (1.67 g, 8.01 mmol) in DMF (100 mL) was added DIEA (4.6 mL, 26.72 mmol). The mixture was stirred at room temperature for 0.5 h, then INT-12 (4 g, 6.68 mmol) was added, followed by HATU (3.3 g, 8.68 mmol). The resulting mixture was stirred at room temperature for 2 h. LCMS showed completion. The reaction was quenched with H2O (150 mL) and then extracted with EtOAc (100 mL*3). The combined organic layers were washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by reverse phase column (H2O / CH3CN) to give 20 (3.4 g, 71% yield) as a white solid. LCMS(ESI): m / z 717.2 [M + H] + HPLC: 99.48% @210 nm, R t = 10.72 minutes; 1H NMR (400 MHz, DMSO-d6)δ 8.09 - 7.96(m, 1H), 7.82(t, J = 5.6 Hz, 1H), 6.89(t, J = 8.0 Hz, 1H), 6.42 - 6.36(m, 2H), 6.33(t, J = 8.2 Hz, 1H), 4.90(d, J = 14.7 Hz, 2H), 4.79 - 4.61(m, 1H), 4.61 - 4.48(m, 1H), 4.04 - 3.94(m, 1H), 3.88 - 3.80(m, 1H), 3.77 - 3.70(m, 1H), 3.61 - 3.48(m, 1H), 3.46 - 3.36(m, 1H), 3.29(d, 3H), 3.27 - 3.22(m, 1H), 3.18(d, 3H), [3.15(s, 1.5H); 3.00(s, 1.5H)], 3.14 - 3.09(m, 1H), 2.68 - 2.53 (m, 3H), 2.46 - 2.40 (m, 1H), 2.34 - 2.22 (m, 1H), 2.22 - 2.13 (m, 7H), 1.97 - 1.82 (m, 4H), 1.73 - 1.56 (m, 2H), 1.36 - 1.25 (m, 1H), 1.10 - 1.03(m, 3H), 0.94 - 0.82(m, 13H), 0.78 - 0.67(m, 6H).

[0290] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-aminophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide 21 To a solution of 2-(2-amino-ethyl)-phenylamine dihydrochloride (20.9 mg, 100.2 umol) in DMF (2 mL) was added DIEA (53.9 mg, 417.5 umol). The mixture was stirred at room temperature for 0.5 h. Then EDCI (24 mg, 125.2 umol) and HOBt (22.6 mg, 167 umol) were added, followed by dropwise addition of INT-12 (50 mg, 83.5 umol) in DMF (0.5 mL). The resulting mixture was stirred at room temperature for 3 h. LCMS showed completion. The reaction was directly purified by reverse phase column (CH3CN / H2O) to give 21 (50 mg, 83.5% yield) as an off-white solid. LCMS (ESI): m / z 717.0 [M + H] + ; HPLC: 99.6% @210 nm, R t = 11.04 minutes; 1 H NMR(400 MHz, CDCl3)δ 7.11 - 6.94(m, 3H), 6.94 - 6.82(m, 1H), 6.75 - 6.62(m, 2H), 4.93 - 4.69(m, 2H), 4.39 - 4.05(m, 4H), 4.03 - 3.67(m, 2H), 3.56 - 3.44(m, 2H), 3.43 - 3.25(m, 8H), [3.15(s, 0.8H); 3.03(s, 2.2H)], 2.79 - 2.70(m, 2H), 2.46 - 2.32(m, 3H), 2.30 - 2.18 (m, 6H), 2.12 - 1.94(m, 4H), 1.87 - 1.77(m, 2H), 1.41 - 1.30(m, 1H), 1.28 - 1.20(m, 3H), 1.10 - 0.88(m, 16H), 0.82(t, J = 6.9 Hz, 3H).

[0291] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-aminophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide 20 To a solution of 4-(2-aminoethyl)aniline (13.7 mg, 100.2 umol) in DMF (2 mL) were added EDCI (24 mg, 0.125 mmol), HOBt (16.9 mg, 0.125 mmol) and DIEA (21.6 mg, 0.167 mmol) successively. INT-12 (50 mg, 83.5 umol) in DMF (0.5 mL) was then added dropwise over 2 min. The reaction was stirred at room temperature for 4 h. LCMS showed completion. The mixture was directly purified by prep-HPLC (H2O / CH3CN) to give 22 (7 mg, 12% yield) as an off-white solid. LCMS (ESI): m / z 717.1 [M + H] + ; HPLC: 96.4% @210 nm, R t = 10.29 minutes; 1H NMR(400 MHz, CDCl3)δ 6.98(d, J = 8.3 Hz, 2H), 6.92(dd, J = 21.8, 8.7 Hz, 2H), 6.64(d, J = 8.5 Hz, 1H), 6.61(d, J = 8.3 Hz, 2H), 6.40(s, 1H), 4.96 - 4.83 (m, 1H), 4.83 - 4.70 (m, 2H), 4.16 - 4.07 (m, 2H), 3.88 - 3.84 (m, 1H), 3.83 - 3.74 (m, 1H), 3.62 - 3.56 (m, 1H), 3.46 - 3.43 (m, 2H), 3.37 - 3.31(m, 8H), [3.14(s, 1H); 3.02(s, 2H)], 2.71(t, J = 7.0 Hz, 2H), 2.46 - 2.41(m, 2H), 2.37 - 2.34(m, 1H), 2.26 - 2.23(m, 6H), 2.08 - 1.93 (m, 5H), 1.38 - 1.28 (m, 2H), 1.23 - 1.20 (m, 3H), 1.01 - 0.93 (m, 15H), 0.83 - 0.79 (m, 3H).

[0292] [ka] 2-(2-nitrophenyl)ethanamine INT-43 To a solution of 2-(2-nitrophenyl)acetonitrile (3 g, 18.5 mmol) in 50 mL of dry THF, pre-cooled to 0 °C in an ice bath, was added 2 M BH3-THF (21.3 mL, 42.6 mmol) under N2 atmosphere. The mixture was allowed to warm to room temperature and stirred for 9 h. TLC showed completion (DCM:MeOH = 10:1, R f = 0.4). The mixture was again cooled to 0 °C and then quenched by slow addition of 30 mL MeOH, then concentrated to dryness to give crude INT-43 (3.07 g, 100% yield) as a brown solid: LCMS (ESI): m / z 167.1 [M + H] + which was used as is without further purification.

[0293] [ka] tert-Butyl 2-nitrophenethylcarbamate INT-44 2-(2-Nitrophenyl)ethanamine To a solution of INT-43 (3.07 g crude, 18.5 mmol) in 40 mL dry DCM was added Et3N (7.8 mL, 56.0 mmol) at room temperature under N2 atmosphere. The mixture was cooled to 0 °C in an ice bath, and then a solution of Boc2O (4.7 mL, 20.52 mmol) in 20 mL DCM was added dropwise. The reaction was allowed to warm to room temperature and stirred for 16 h. TLC showed completion (DCM : MeOH = 10:1, R f = 0.95). The mixture was quenched by adding 100 mL H2O, then extracted with DCM (50 mL*3). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by flash chromatography (petroleum ether: EtOAc = 40:1~30:1~20:1, v / v) to give INT-44 (3.85 g, 78% yield for two steps) as a pale yellow oil. LCMS(ESI): m / z 167.1 [M - t-Bu].

[0294] [ka] tert-Butyl methyl(2-nitrophenethyl)carbamate INT-45 To a solution of tert-butyl 2-nitrophenethylcarbamate INT-44 (2 g, 7.51 mmol) in 40 mL of dry DMF at 0 °C under N2 atmosphere was added 60% NaH (601 mg, 15 mmol). The mixture was stirred at 0 °C for 30 min, after which CHI (1.1 mL, 17.3 mmol) was added. The reaction was allowed to warm to room temperature and stirred overnight. TLC showed completion (petroleum ether: EtOAc = 5:1, R f = 0.7). 0 oThe mixture was quenched by slowly adding 80 mL H2O at C, then extracted with EtOAc (30 mL*3). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by flash chromatography (petroleum ether: EtOAc = 40:1 ~ 30:1 ~ 25:1, v / v) to give INT-45 (1.44 g, 62% yield). LCMS(ESI): m / z 181.1 [M - t-Bu].

[0295] [ka] N-Methyl-2-(2-nitrophenyl)ethanamine hydrochloride INT-46 tert-Butyl methyl(2-nitrophenethyl)carbamate. To a solution of INT-45 (1.34 g, 4.78 mmol) in 15 mL DCM was added 4 M HCl / dioxane (10 mL, 40 mmol). The reaction was stirred at room temperature for 2.5 h. TCL showed completion. The mixture was concentrated and dried. The residue was suspended in MTBE (20 mL) three times to give INT-46 (920 mg, 64% yield) as a pale yellow solid: LCMS (ESI): m / z 181.1 [M + H] + ; 1 H NMR (400 MHz, DMSO-d6)δ 9.14(s, 2H), 8.02(dd, J = 8.2, 1.2 Hz, 1H), 7.73(td, J = 7.6, 1.3 Hz, 1H), 7.63 - 7.52(m, 2H), 3.25 - 3.15(m, 4H), 2.57(s, 3H).

[0296] [ka] tert-Butyl ((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(2-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate INT-47 Tube A: To a solution of INT-46 (870 mg, 4.02 mmol) in DMF (30 mL) was added DIEA (2.3 mL, 13.9 mmol). The mixture was stirred at room temperature for 20 min to form solution A.

[0297] Tube B: To another solution of INT-19 (1.77 g, 3.09 mmol) in 20 mL DMF, HATU (2.35 g, 6.18 mmol) and DIEA (2.3 mL, 13.9 mmol) were added at room temperature. The mixture was stirred at room temperature for 0.5 h, after which solution A was added. The resulting mixture was stirred at room temperature for 3.5 h. LCMS showed completion. The reaction was directly purified by reverse phase column (H2O / CH3CN) to give INT-47 (1.74 g, 73% yield) as a yellow oil. LCMS (ESI): m / z 734.2 [M + H] + .

[0298] [ka] (S)-2-Amino-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(2-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide hydrochloride INT-48 To a solution of INT-47 (1.54 g, 2.1 mmol) in 20 mL DCM was added 4 M HCl / dioxane (5 mL, 20 mmol). The reaction was stirred at room temperature for 3 h. TCL showed completion. The mixture was concentrated and dried. The residue was suspended in MTBE (30 mL*3) and then lyophilized to give INT-48 (1.27 g, 90% yield) as a yellow solid. LCMS(ESI): m / z 634.2 [M + H] + ; HPLC: 98.2% @210 nm, R t = 11.74 minutes.

[0299] [ka] (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(2-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-49 To a solution of INT-48 (100 mg, 0.149 mmol) and (S)-2-(dimethylamino)-3-methylbutanoic acid (26 mg, 0.179 mmol) in 2 mL DMF, EDCI (43 mg, 0.224 mmol) and HOBt (40 mg, 0.298 mmol) were added, followed by DIEA (87 mg, 0.671 mmol). The mixture was stirred at room temperature under N2 atmosphere for 2 h, LCMS showed completion. The reaction mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-49 (96 mg, 85% yield) as a colorless oil. LCMS (ESI): m / z 760.9 [M + H] + .

[0300] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide 14 To a solution of INT-49 (96 mg, 0.126 mmol) in 4 mL MeOH was added 10% Pd / C (20 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 2.5 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was lyophilized to give 14 (33 mg, 36% yield) as a white solid. LCMS (ESI): m / z 730.9 [M + H] + ; HPLC: 95.5% @210 nm, R t = 13.51 minutes; 1 H NMR (400 MHz, DMSO)δ 8.14(d, J = 8.9 Hz, 1H), 8.01(d, J = 8.3 Hz, 1H), 6.96 - 6.79(m, 2H), 6.68 - 6.58(m, 1H), 6.54 - 6.41(m, 1H), 5.14 - 4.91(m, 2H), 4.75 - 4.47(m, 2H), 4.14 - 3.73(m, 3H), 3.70 - 3.39(m, 3H), 3.34(s, 3H), 3.30 - 3.23(m, 2H), 3.23 - 3.19(m, 1H), 3.19 - 3.07(m, 4H), 3.01 - 2.92 (m, 3H), 2.86 - 2.81 (m, 1H), 2.76 - 2.52 (m, 4H), 2.41 (d, J = 20.7 Hz, 1H), 2.30 - 2.14 (m, 6H), 2.03 - 1.84 (m, 4H), 1.84 - 1.55 (m, 3H), 1.36 - 1.25 (m, 1H), 1.10 (dd, J = 21.8, 6.7 Hz, 2H), 0.95 - 0.80 (m, 12H), 0.79 - 0.69 (m, 6H).

[0301] [ka] (S)-2-((R)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(2-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-50 To a solution of INT-48 (100 mg, 0.149 mmol) and (R)-2-(dimethylamino)-3-methylbutanoic acid (32 mg, 0.179 mmol) in 2 mL DMF, EDCI (43 mg, 0.224 mmol) and HOBt (40 mg, 0.298 mmol) were added, followed by DIEA (87 mg, 0.671 mmol). The mixture was stirred at room temperature under N2 atmosphere for 3 days, and LCMS showed completion. The reaction mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-50 (100 mg, 88% yield) as a colorless oil. LCMS (ESI): m / z 761.1 [M + H] + .

[0302] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((R)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide 15 To a solution of INT-50 (100 mg, 0.131 mmol) in 3 mL MeOH was added 10% Pd / C (20 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 2 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was lyophilized to give 15 (62 mg, 64% yield) as an off-white solid. LCMS (ESI): m / z 731.1 [M + H] +HPLC: 97.4% @210 nm, R t = 8.67 minutes; 1 H NMR (400 MHz, DMSO-d6)δ 8.14(d, J = 9.0 Hz, 1H), 8.01(d, J = 8.7 Hz, 1H), 6.98 - 6.79(m, 2H), 6.66 - 6.57(m, 1H), 6.51 - 6.41(m, 1H), 5.14 - 4.91 (m, 2H), 4.78 - 4.60 (m, 1H), 4.59 - 4.44 (m, 1H), 4.10 - 3.81 (m, 2H), 3.81 - 3.46 (m, 3H), 3.46 - 3.35 (m, 1H), 3.34 - 3.32 (m, 3H), 3.31 - 3.30 (m, 1H), 3.30 - 3.23 (m, 2H), 3.23 - 3.10 (m, 5H), 3.03 - 2.95 (m, 3H), 2.85 - 2.81 (m, 1H), 2.78 - 2.53 (m, 4H), 2.41 (d, J = 20.1 Hz, 1H), 2.34 - 2.18(m, 1H), 2.17 - 2.11(m, 5H), 2.00 - 1.84(m, 4H), 1.78 - 1.60(m, 2H), 1.41 - 1.29(m, 1H), 1.10(dd, J = 22.2, 6.7 Hz, 2H), 0.96 - 0.83(m, 11H), 0.82 - 0.73(m, 7H).

[0303] [ka] (S)-2-((S)-2-(dimethylamino)propanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(2-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-51 To a solution of INT-48 (100 mg, 0.149 mmol) and (S)-2-(dimethylamino)propanoic acid (22 mg, 0.188 mmol) in 3 mL DMF, EDCI (45 mg, 0.235 mmol) and HOBt (32 mg, 0.237 mmol) were added, followed by DIEA (71 mg, 0.55 mmol). The mixture was stirred at room temperature under N2 atmosphere for 3 h, and LCMS showed completion. The mixture was quenched by adding 15 mL H2O, then extracted with DCM (20 mL*3). The organic layers were combined, washed with H2O and brine, dried over Na2SO4, filtered and concentrated. The residue was analyzed by Prep-TLC (DCM : MeOH = 10:1, R f = 0.7) to give INT-51 (100 mg, 91% yield) as a pale yellow oil. LCMS (ESI): m / z 733.5 [M + H] + .

[0304] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)propanamide)-N,3-dimethylbutanamide 16 To a solution of INT-51 (110 mg, 0.15 mmol) in 5 mL MeOH was added 10% Pd / C (22 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 1.5 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was lyophilized to give 16 (90 mg, 86% yield) as an off-white solid. LCMS (ESI): m / z 703.3 [M + H] + HPLC: 96.6% @210 nm, R t = 8.52 minutes; 1H NMR (400 MHz, DMSO-d6)δ 7.78 (dd, J = 25.1, 8.5 Hz, 1H), 6.94 - 6.76 (m, 2H), 6.69 - 6.57 (m, 1H), 6.51 - 6.41 (m, 1H), 5.20 - 4.87 (m, 2H), 4.76 - 4.50 (m, 2H), 4.14 - 3.84 (m, 2H), 3.84 - 3.72 (m, 1H), 3.71 - 3.60 (m, 1H), 3.59 - 3.49 (m, 1H), 3.49 - 3.37 (m, 2H), 3.31 - 3.27 (m, 2H), 3.27 - 3.22 (m, 1H), 3.22 - 3.19 (m, 1H), 3.19 - 3.15 (m, 2H), 3.15 - 3.02 (m, 2H), 3.00 - 2.89 (m, 4H), 2.89 - 2.74 (m, 2H), 2.73 - 2.60 (m, 2H), 2.59 - 2.52 (m, 1H), 2.48 - 2.35 (m, 1H), 2.24 - 2.15 (m, 6H), 2.03 - 1.86 (m, 3H), 1.83 - 1.55 (m, 3H), 1.34 - 1.26 (m, 1H), 1.14 - 1.02(m, 5H), 0.95 - 0.80(m, 9H), 0.80 - 0.66(m, 5H).

[0305] [ka] (S)-2-((R)-2-(dimethylamino)propanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(2-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-52 To a solution of INT-48 (100 mg, 0.149 mmol) and (R)-2-(dimethylamino)propanoic acid (21 mg, 0.174 mmol) in 4 mL DMF was added HATU (78 mg, 0.205 mmol) followed by DIEA (51 mg, 0.395 mmol). The mixture was stirred at room temperature under N2 atmosphere for 4 h and LCMS showed completion. The mixture was directly concentrated to dryness to give crude. The crude residue was diluted with 15 mL H2O and then extracted with EtoAc (10 mL*4). The organic layers were combined, washed with H2O and brine, dried over Na2SO4, filtered and concentrated to give crude INT-52 (180 mg) as a yellow oil. LCMS(ESI): m / z 733.1 [M + H] + .

[0306] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((R)-2-(dimethylamino)propanamide)-N,3-dimethylbutanamide 17 To a solution of crude INT-52 (180 mg) in 5 mL MeOH was added 10% Pd / C (20 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 4 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) and then lyophilized to give 17 (60 mg, 54% yield for two steps) as a white solid. LCMS (ESI): m / z 703.4 [M + H] + ; HPLC: 99.4% @210 nm, R t = 12.52 minutes; 1H NMR (400 MHz, DMSO-d6)δ 9.04 - 8.90 (m, 1H), 7.00 - 6.81 (m, 2H), 6.70 - 6.58 (m, 1H), 6.58 - 6.43 (m, 1H), 4.79 - 4.58 (m, 1H), 4.57 - 4.42 (m, 1H), 4.06 - 3.97 (m, 1H), 3.96 - 3.89 (m, 1H), 3.89 - 3.73 (m, 1H), 3.73 - 3.56 (m, 1H), 3.56 - 3.39 (m, 2H), 3.33 - 3.28 (m, 5H), 3.27 - 3.10(m, 6H), 3.09 - 2.85 (m, 4H), 2.84 - 2.79 (m, 1H), 2.79 - 2.52 (m, 9H), 2.41 (d, J = 20.2 Hz, 1H), 2.37 - 2.17 (m, 1H), 2.09 - 1.85 (m, 3H), 1.85 - 1.55(m, 3H), 1.49 - 1.34(m, 3H), 1.34 - 1.25(m, 1H), 1.09(dd, J = 17.8, 6.7 Hz, 2H), 0.97 - 0.80(m, 9H), 0.80 - 0.62(m, 5H).

[0307] [ka] (S)-2-(2-(dimethylamino)acetamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(2-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-53 To a solution of INT-48 (100 mg, 0.149 mmol) and 2-(dimethylamino)acetic acid (20 mg, 0.189 mmol) in 5 mL DMF, EDCI (45 mg, 0.235 mmol) and HOBt (32 mg, 0.237 mmol) were added, followed by DIEA (71 mg, 0.55 mmol). The mixture was stirred at room temperature under N2 atmosphere for 3 h, and LCMS showed completion. The mixture was directly concentrated under reduced pressure and dried. The residue was purified by Prep-TLC (DCM : MeOH = 10:1, Rf = 0.7) to give crude INT-53 (117 mg, 100% yield) as a yellow oil. LCMS (ESI): m / z 719.0 [M + H] + .

[0308] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(2-(dimethylamino)acetamide)-N,3-dimethylbutanamide 18 To a solution of INT-53 (117 mg, 0.163 mmol) in 5 mL MeOH was added 10% Pd / C (23 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 2 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated to give 18 (103 mg, 92% yield) as a white foamy solid. LCMS (ESI): m / z 690.2 [M + H] + , 345.2 [M + 2H] 2+ ; HPLC: 98.6% @210 nm, R t = 13.22 minutes; 1H NMR (400 MHz, DMSO-d6)δ 7.73 - 7.61 (m, 1H), 6.95 - 6.79 (m, 2H), 6.66 - 6.57 (m, 1H), 6.52 - 6.41 (m, 1H), 5.15 - 4.91 (m, 2H), 4.80 - 4.55 (m, 2H), 4.08 - 3.83 (m, 2H), 3.82 - 3.57 (m, 2H), 3.56 - 3.40 (m, 2H), 3.39 - 3.33 (m, 2H), 3.31 - 3.26 (m, 2H), 3.26 - 3.13 (m, 4H), 3.13 - 3.06(m, 1H), 3.06 - 2.99 (m, 1H), 2.99 - 2.97 (m, 2H), 2.95 - 2.92 (m, 3H), 2.91 - 2.77 (m, 1H), 2.73 - 2.66 (m, 2H), 2.66 - 2.52 (m, 1H), 2.47 - 2.34 (m, 1H), 2.22 - 2.19 (m, 5H), 2.00 - 1.84 (m, 3H), 1.84 - 1.56 (m, 3H), 1.35 - 1.18 (m, 2H), 1.09 (dd, J = 15.8, 6.7 Hz, 2H), 1.01 - 0.84(m, 7H), 0.84 - 0.79(m, 3H), 0.79 - 0.68(m,4H).

[0309] [ka] tert-Butyl 4-nitrophenethylcarbamate INT-54 To a solution of 2-(4-nitrophenyl)ethanamine hydrochloride (3 g, 14.8 mmol) in 50 mL of dry DCM at room temperature under N2 atmosphere was added Et3N (4.49 g, 44.41 mmol). The mixture was cooled to 0 °C in an ice bath, and then Boc2O (4.85 g, 22.21 mmol) was added. The reaction was allowed to warm to room temperature and stirred for 16 h. TLC showed completion (petroleum ether: EtOAc = 3:1, R f= 0.45). The mixture was quenched by adding 30 mL H2O, then extracted with EtOAc (30 mL*3). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by flash chromatography (petroleum ether: EtOAc = 5:1, v / v) to give INT-54 (3.8 g, 96% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 1 H NMR(400 MHz, CDCl3)δ 8.19 - 8.14(m, 2H), 7.36(d, J = 8.6 Hz, 2H), 4.57(s, 1H), 3.41(dd, J = 13.2, 6.6 Hz, 2H), 2.92(t, J = 7.0 Hz, 2H), 1.43(s, 9H).

[0310] [ka] tert-Butyl methyl(4-nitrophenethyl)carbamate INT-55 A solution of tert-butyl methyl(4-nitrophenethyl)carbamate INT-54 (3.2 g, 12 mmol) in 60 mL of dry DMF was cooled to 0 °C by ice bath under N2 atmosphere for 20 min. To this solution was added 60% NaH (0.72 g, 18 mmol) in three portions over 5 min at 0 °C, followed immediately by CHI (2.87 mL, 46.12 mmol). The reaction was allowed to warm to room temperature and stirred for 3 h. TLC showed completion (petroleum ether: EtOAc = 5:1, R f = 0.75). 0 o The mixture was quenched by slowly adding 150 mL H2O at C, then extracted with EtOAc (60 mL*3). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered and concentrated to give INT-55 (2.4 g, 71% yield) as a yellow oil. LCMS (ESI): m / z 181.1 [M - t-Bu].

[0311] [ka] N-Methyl-2-(4-nitrophenyl)ethanamine hydrochloride INT-56 tert-Butyl methyl(4-nitrophenethyl)carbamate. To a solution of INT-55 (2.4 g, 9 mmol) in 40 mL DCM was added 4 M HCl / dioxane (20 mL, 80 mmol). The reaction was stirred at room temperature for 2 h. TCL showed completion. The mixture was concentrated and dried. The residue was suspended three times with MTBE (20 mL) to give INT-56 (1.69 g, 91% yield) as a pale yellow solid: LCMS (ESI): m / z 181.1 [M + H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 1 H NMR (400 MHz, DMSO) δ 9.28 (s, 2H), 8.27 - 8.14 (m, 2H), 7.57 (m, 2H), 3.13 (m, 4H), 2.54 (s, 3H).

[0312] [ka] tert-Butyl ((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(4-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate INT-57 Tube A: To a solution of INT-56 (500 mg, 2.3 mmol) in DMF (10 mL) was added DIEA (640 mg, 3.99 mmol). The mixture was stirred at room temperature for 0.5 h to form solution A.

[0313] Tube B: To another solution of INT-19 (1.02 g, 1.78 mmol) in 30 mL DMF, HATU (1.35 g, 3.55 mmol) and DIEA (640 mg, 3.99 mmol) were added at room temperature. The mixture was stirred at room temperature for 0.5 h, then solution A was added. The resulting mixture was stirred at room temperature for 4 h. LCMS showed completion. The reaction was directly purified by reverse phase column (H2O / CH3CN) to give INT-57 (1.01 g, 75% yield) as a yellow oil. LCMS (ESI): m / z 734.1 [M + H] + .

[0314] [ka] (S)-2-Amino-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(4-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide hydrochloride INT-58 To a solution of INT-57 (1.0 g, 1.36 mmol) in 10 mL DCM was added 4 M HCl / dioxane (5 mL, 20 mmol). The reaction was stirred at room temperature for 3 h. TCL showed completion. The mixture was concentrated and dried. The residue was suspended in MTBE (10 mL*3) and then lyophilized to give INT-58 (0.76 g, 86% yield) as a yellow solid. LCMS(ESI): m / z 634.2 [M + H] + ; HPLC: 97.1% @210 nm, R t = 9.36 minutes.

[0315] [ka] (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(4-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-59 To a solution of INT-58 (100 mg, 0.149 mmol) and (S)-2-(dimethylamino)-3-methylbutanoic acid (33 mg, 0.224 mmol) in 2 mL DMF, EDCI (46 mg, 0.235 mmol) and HOBt (32 mg, 0.328 mmol) were added followed by DIEA (124 mg, 0.969 mmol). The mixture was stirred at room temperature under N2 atmosphere for 2 h, and LCMS showed completion. The reaction mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-59 (120 mg, 100% yield) as a colorless oil. LCMS (ESI): m / z 761.1[M+ H] + .

[0316] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide 19 To a solution of INT-59 (120 mg, 0.149 mmol) in 3 mL MeOH was added 10% Pd / C (24 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 3 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by Prep-TLC (DCM : MeOH = 12:1, R f= 0.5) followed by lyophilization to give 19 (45 mg, 41% yield) as an off-white solid. LCMS (ESI): m / z 366.2 [M + 2H] 2+ HPLC: 95.6% @210 nm, R t = 12.36 minutes; 1 H NMR (400 MHz, DMSO-d6)δ 8.01(d, J = 8.7 Hz, 1H), 6.90 - 6.81(m, 2H), 6.53 - 6.42(m, 2H), 4.90 - 4.75(m, 2H), 4.73 - 4.47(m, 2H), 4.17 - 3.79 (m, 2H), 3.79 - 3.59 (m, 2H), 3.55 - 3.41 (m, 2H), 3.40 - 3.33 (m, 2H), 3.29 - 3.25 (m, 2H), 3.24 - 3.16 (m, 4H), 3.15 - 2.93 (m, 3H), 2.91 - 2.82(m, 2H), 2.82 - 2.77 (m, 1H), 2.67 - 2.58 (m, 3H), 2.57 - 2.52 (m, 1H), 2.49 - 2.39 (m, 1H), 2.24 - 2.17 (m, 6H), 1.99 - 1.83 (m, 4H), 1.82 - 1.56 (m, 3H), 1.34 - 1.25 (m, 1H), 1.11 - 1.00 (m, 2H), 0.99 - 0.94 (m, 1H), 0.93 - 0.84 (m, 13H), 0.79 - 0.68 (m, 6H).

[0317] [ka] (S)-2-((R)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(4-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-60 To a solution of INT-58 (100 mg, 0.149 mmol) and (R)-2-(dimethylamino)-3-methylbutanoic acid (49 mg, 0.268 mmol) in 3 mL DMF, EDCI (68 mg, 0.355 mmol) and HOBt (64 mg, 0.473 mmol) were added, followed by DIEA (0.19 mL, 1.06 mmol). The mixture was stirred at room temperature under N2 atmosphere for 2 days, and LCMS showed near completion. The reaction mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-60 (60 mg, 53% yield) as a colorless oil. LCMS (ESI): m / z 761.1 [M+ H] + .

[0318] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((R)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide 23 To a solution of INT-60 (60 mg, 0.788 mmol) in 2 mL MeOH was added 10% Pd / C (18 mg). The reaction was then stirred at room temperature overnight under H2 atmosphere (1 atm). LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was lyophilized to give 23 (45 mg, 78% yield) as a white solid. LCMS (ESI): m / z 731.4 [M + H] + HPLC: 95.9% @210 nm, R t = 12.13 minutes; 1H NMR (400 MHz, DMSO-d6)δ 8.01(d, J = 8.7 Hz, 1H), 6.91 - 6.79(m, 2H), 6.53 - 6.41(m, 2H), 4.89 - 4.74(m, 2H), 4.73 - 4.44(m, 2H), 4.15 - 3.84 (m, 2H), 3.77 - 3.58 (m, 2H), 3.57 - 3.38 (m, 3H), 3.30 - 3.23 (m, 3H), 3.23 - 3.16 (m, 4H), 3.16 - 2.95 (m, 3H), 2.89 - 2.82 (m, 2H), 2.82 - 2.77(m, 1H), 2.69 - 2.54(m, 4H), 2.43(d, J = 15.3 Hz, 1H), 2.28 - 2.12(m, 6H), 2.01 - 1.84(m, 4H), 1.81 - 1.57(m, 3H), 1.40 - 1.29(m, 1H), 1.05(dd, J = 17.7, 6.6 Hz, 2H), 0.98 - 0.94(m, 1H), 0.93 - 0.83(m, 13H), 0.82 - 0.70(m, 6H).

[0319] [ka] (S)-2-((R)-2-(dimethylamino)propanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(4-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-61 To a solution of INT-58 (100 mg, 0.149 mmol) and (R)-2-(dimethylamino)propanoic acid (24 mg, 0.194 mmol) in 2 mL DMF, EDCI (46 mg, 0.239 mmol) and HOBt (43 mg, 0.313 mmol) were added, followed by DIEA (0.12 mL, 0.744 mmol). The mixture was stirred at room temperature under N2 atmosphere for 2 days, and LCMS showed 30% STM remaining. The reaction was stopped and directly purified by reverse phase column (H2O:CH3CN) to give crude INT-61 (120 mg) as a yellow oil. LCMS (ESI): m / z 733 [M + H] + .

[0320] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((R)-2-(dimethylamino)propanamide)-N,3-dimethylbutanamide 24 To a solution of INT-61 (120 mg crude) in 3 mL MeOH was added 10% Pd / C (24 mg). The reaction was then stirred at room temperature under H2 atmosphere (1 atm) for 2 h. LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by Prep-TLC (DCM : MeOH = 13:1, R f = 0.5) to give 24 (37 mg, 35% yield over two steps) as an off-white solid. LCMS (ESI): m / z 703.8 [M + H] +; HPLC: 97.7% @210 nm, Rt = 11.99 min; 1H NMR (400 MHz, DMSO-d6)δ 7.80(d, J = 9.2 Hz, 1H), 6.90 - 6.79(m, 2H), 6.54 - 6.39(m, 2H), 4.93 - 4.78 (m, 2H), 4.74 - 4.47 (m, 2H), 4.05 - 3.75 (m, 2H), 3.74 - 3.56 (m, 2H), 3.55 - 3.41 (m, 2H), 3.30 - 3.23 (m, 3H), 3.19 - 3.14 (m, 3H), 3.13 - 2.91(m, 4H), 2.86 - 2.73 (m, 3H), 2.65 - 2.53 (m, 3H), 2.48 - 2.37 (m, 1H), 2.34 - 2.17 (m, 1H), 2.17 - 2.10 (m, 6H), 2.02 - 1.79 (m, 4H), 1.76 - 1.59 (m, 2H), 1.34 - 1.26 (m, 1H), 1.08 - 1.00 (m, 5H), 0.97 - 0.71 (m, 15H).

[0321] [ka] (S)-2-((S)-2-(dimethylamino)propanamide)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(4-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-62 To a solution of INT-58 (100 mg, 0.149 mmol) and (S)-2-(dimethylamino)propanoic acid (21 mg, 0.174 mmol) in 3 mL DMF was added HATU (78 mg, 0.205 mmol) followed by DIEA (51 mg, 0.395 mmol). The mixture was stirred at room temperature under N2 atmosphere for 3 h and LCMS showed completion. The mixture was directly concentrated and dried to give crude. The crude residue was diluted with 10 mL H2O and then extracted with EtoAc (10 mL*4). The organic layers were combined, washed with H2O and brine, dried over Na2SO4, filtered and concentrated to give crude INT-62 (185 mg) as a yellow oil. LCMS(ESI): m / z 733.3 [M + H] + .

[0322] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)propanamide)-N,3-dimethylbutanamide 26 To a solution of INT-62 (185 mg crude) in 5 mL MeOH was added 10% Pd / C (30 mg). The reaction was then stirred at room temperature overnight under H2 atmosphere (1 atm). LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) and then lyophilized to give 26 (30 mg, 28% yield for two steps) as a pale pink solid. LCMS (ESI): m / z 725.8 [M + Na] + , 352.6 [M + 2H] 2+ HPLC: 92.9% @210 nm, R t = 11.53 minutes

[0323] [ka] (S)-2-(2-(dimethylamino)acetamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-(methyl(4-nitrophenethyl)amino)-3-oxopropyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide INT-63 To a solution of INT-58 (100 mg, 0.149 mmol) and 2-(dimethylamino)acetic acid (18 mg, 0.174 mmol) in 4 mL DMF was added HATU (78 mg, 0.205 mmol) followed by DIEA (51 mg, 0.395 mmol). The mixture was stirred at room temperature under N2 atmosphere for 3 h and LCMS showed completion. The mixture was directly concentrated to dryness to give crude. The crude residue was diluted with 15 mL H2O and then extracted with EtoAc (10 mL*4). The organic layers were combined, washed with H2O and brine, dried over Na2SO4, filtered and concentrated to give crude INT-63 (190 mg) as a yellow oil. LCMS(ESI): m / z 719.3 [M + H] + .

[0324] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-aminophenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(2-(dimethylamino)acetamide)-N,3-dimethylbutanamide 27 To a solution of crude INT-63 (190 mg) in 5 mL MeOH was added 10% Pd / C (30 mg). The reaction was then stirred at room temperature overnight under H2 atmosphere (1 atm). LCMS showed completion. The mixture was filtered and the filtrate was concentrated. The residue was purified by reverse phase column (H2O:CH3CN) followed by prep-TLC (DCM:MeOH = 13:1, R f= 0.5) to give 27 (23 mg, 22% yield over two steps) as an off-white solid. LCMS (ESI): m / z 345.3 [M + 2H] 2+ HPLC: 98.0% @210 nm, R t = 12.07 minutes; 1 H NMR (400 MHz, DMSO-d6) δ 7.65 (d, J = 6.0 Hz, 1H), 6.93 - 6.80 (m, 2H), 6.57 - 6.43 (m, 2H), 4.94 - 4.78 (m, 2H), 4.77 - 4.57 (m, 2H), 4.12 - 3.90 (m, 2H), 3.90 - 3.69 (m, 2H), 3.66 - 3.58 (m, 1H), 3.53 - 3.48 (m, 1H), 3.47 - 3.43 (m, 1H), 3.31 - 3.25 (m, 2H), 3.24 - 3.04 (m, 4H), 3.01 - 2.91(m, 2H), 2.88 - 2.82 (m, 2H), 2.82 - 2.69 (m, 2H), 2.68 - 2.53 (m, 3H), 2.48 - 2.35 (m, 1H), 2.25 - 2.16 (m, 5H), 2.07 - 1.91 (m, 2H), 1.90 - 1.77(m, 2H), 1.76 - 1.57(m, 2H), 1.51 - 1.34(m, 1H), 1.33 - 1.17(m, 4H), 1.04(dd, J = 15.9, 6.7 Hz, 2H), 0.97 - 0.73(m, 12H).

[0325] [ka] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate GINT-64 To a solution of 1 (110 mg, 150 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (47 mg, 181 umol) in 3 mL CH3CN was added a mixture of EDCI (43 mg, 226 umol) and HOPO (25 mg, 226 mmol) followed by 2,6-lutidine (53 uL, 451 umol). The reaction was stirred at room temperature under N2 atmosphere for 16 h and LCMS showed completion. The mixture was directly concentrated to dryness and the residue was purified by Prep-TLC (DCM:MeOH = 13:1, R f = 0.65) to give INT-64 (70 mg, 49% yield) as a yellow oil. LCMS (ESI): m / z 972.8 [M + H] + , 995.7 [M + Na + ].

[0326] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido bis(2,2,2-trifluoroacetate) INT-65 To a mixture of INT-64 (80 mg, 82 umol) and anisole (45 uL, 411 umol) was added TFA (0.7 mL). The reaction was then stirred at room temperature for 10 min and quenched by the addition of an additional 350 mL MTBE, during which time much white solid precipitated. The resulting mixture was filtered and the filter cake was collected and dried under vacuum to give INT-65 (70 mg, 76% yield) as an off-white solid. LCMS (ESI): m / z 873.8 [M + H] + .

[0327] [ka] (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamide)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide 2,2,2-trifluoroacetate 28 To a solution of INT-65 (70 mg, 64 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (26 mg, 104 umol) in 2 mL CH3CN was added DIEA (27 uL, 160 umol). The reaction was then stirred at room temperature for 45 min. LCMS showed completion. The mixture was quenched by direct addition of TFA (0.02 mL) and stirred for 5 min. The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 28 (45 mg, 63% yield) as a white solid. LCMS (ESI): m / z 505.5 [M + 2H] 2+ HPLC: 99.7% @210 nm, R t = 8.87 minutes; 11H NMR (400 MHz, DMSO) δ 9.84 - 9.75 (m, 1H), 9.52 (s, 1H), 8.91 (d, J = 8.1 Hz, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.19 - 8.11 (m, 1H), 7.49 (d, J = 7.4 Hz, 1H), 7.45 - 7.35 (m, 1H), 7.23 - 7.15 (m, 1H), 7.09 (s, 2H), 6.95 - 6.87 (m, 1H), 4.77 - 4.64 (m, 1H), 4.63 - 4.55 (m, 1H), 4.40 - 4.28 (m, 2H), 4.13 - 4.04 (m, 2H), 4.03 - 3.96 (m, 1H), 3.92 (m, 1H), 3.78 - 3.69 (m, 3H), 3.51 - 3.49 (m, 1H), 3.48 - 3.46 (m, 1H), 3.45 - 3.42 (m, 1H), 3.35 - 3.24 (m, 4H), 3.22 - 3.16 (m, 3H), [3.13 (s, 1.5H), 2.99 (s, 1.5H)], 2.90 (d, J = 2.2 Hz, 2H), 2.84 - 2.79 (m, 2H), 2.79 - 2.70 (m, 6H), 2.70 - 2.58 (m, 2H), 2.48 - 2.40 (m, 1H), 2.36 - 2.18 (m, 2H), 2.06 - 1.92 (m, 2H), 1.89 - 1.84 (m, 1H), 1.81 - 1.56 (m, 3H), 1.30 (dd, J = 7.0, 3.2 Hz, 4H), 1.25 - 1.18 (m, 4H), 1.04 (dd, J = 14.9, 6.7 Hz, 2H), 0.98 - 0.82 (m, 15H), 0.81 - 0.72 (m, 3H).

[0328]

Chem.

[0329] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)propanamido)-N,3-dimethylbutanamide bis(2,2,2-trifluoroacetate) INT-67 To a mixture of INT-66 (120 mg, 127 umol) and anisole (69 mg, 638 umol) was added TFA (3 mL). The reaction was then stirred at room temperature for 5 min. TLC showed completion (DCM / MeOH = 10:1, v / v; R as INT-66). f = 0.75). The mixture was diluted with 150 mL MTBE, during which a lot of white solid precipitated. The resulting mixture was filtered, and the filter cake was collected and dried under reduced pressure to give INT-67 (148 mg, 100% yield) as a colorless oil. LCMS (ESI): m / z 846.1 [M + H] + .

[0330] [ka] (S)-2-((S)-2-(dimethylamino)propanamide)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide)propanamide)propanamide)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide 2,2,2-trifluoroacetate 29 To a solution of INT-67 (148 mg, 129 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (47 mg, 186 umol) in 5 mL CH3CN was added DIEA (30 mg, 232 umol). The reaction was then stirred at room temperature for 30 min. LCMS showed completion. The mixture was quenched by direct addition of TFA (0.12 mL). The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 29 (40 mg, 28% yield) as a white solid. LCMS (ESI): m / z 982.9 [M + H] + , 491.6 [M + 2H]2+ ; HPLC: 98.5% @210 nm, R t = 8.65 min; 1 H NMR (400 MHz, DMSO) δ 9.90 - 9.70 (m, 2H), 8.97 (d, J = 8.4 Hz, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.22 - 8.10 (m, 1H), 7.54 - 7.48 (m, 1H), 7.48 - 7.36 (m, 1H), 7.27 - 7.17 (m, 1H), 7.09 (s, 2H), 6.98 - 6.87 (m, 1H), 4.79 - 4.60 (m, 1H), 4.61 - 4.49 (m, 1H), 4.43 - 4.28 (m, 2H), 4.17 - 4.04 (m, 2H), 4.02 - 3.89 (m, 2H), 3.80 - 3.63 (m, 2H), 3.33 - 3.25 (m, 4H), 3.23 - 3.13 (m, 4H), [3.11 (s, 1.2H), 2.98 (s, 1.8H)], 2.94 - 2.87 (m, 2H), 2.86 - 2.81 (m, 1H), 2.81 - 2.72 (m, 7H), 2.72 - 2.60 (m, 2H), 2.45 - 2.40 (m, 1H), 2.40 - 2.20 (m, 2H), 2.11 - 1.93 (m, 2H), 1.93 - 1.76 (m, 3H), 1.71 - 1.55 (m, 2H), 1.39 - 1.28 (m, 6H), 1.22 (d, J = 6.3 Hz, 4H), 1.05 (dd, J = 13.4, 6.7 Hz, 2H), 1.01 - 0.82 (m, 11H), 0.81 - 0.74 (m, 3H).

[0331]

Chem.

[0332] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((R)-2-(dimethylamino)propanamido)-N,3-dimethylbutanamide bis(2,2,2-trifluoroacetate) INT-69 To a mixture of INT-68 (75 mg, 79 umol) and anisole (43 uL, 400 umol) was added TFA (0.75 mL). The reaction was then stirred at room temperature for 20 minutes and then quenched by adding 40 mL MTBE, during which time a lot of white solid precipitated. The resulting mixture was filtered and the filter cake was collected and dried under vacuum to give INT-69 (60 mg, 70% yield) as a yellow solid. LCMS (ESI): m / z 845.1 [M + H] + , 423.1 [M + 2H] 2+ .

[0333] [ka] (S)-2-((R)-2-(dimethylamino)propanamide)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide)propanamide)propanamide)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide 2,2,2-trifluoroacetate 30 To a solution of INT-69 (60 mg, 56 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (21 mg, 81 umol) in 2 mL CH3CN was added DIEA (20 uL, 125 umol). The reaction was then stirred at room temperature for 45 min. LCMS showed completion. The mixture was quenched by direct addition of TFA (30 uL). The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 30 (45 mg, 73% yield) as a white solid. LCMS (ESI): m / z 982.6 [M + H] + , 492.0 [M + 2H] 2+ HPLC: 98.8% @210 nm, Rt = 8.63 points; 1 1H NMR (400 MHz, DMSO) δ 9.90 - 9.71 (m, 2H), 9.01 - 8.90 (m, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.20 - 8.11 (m, 1H), 7.53 - 7.46 (m, 1H), 7.45 - 7.33 (m, 1H), 7.25 - 7.14 (m, 1H), 7.09 (s, 2H), 6.97 - 6.87 (m, 1H), 4.78 - 4.59 (m, 1H), 4.56 - 4.44 (m, 1H), 4.41 - 4.27 (m, 2H), 4.14 - 4.04 (m, 2H), 4.00 - 3.89 (m, 2H), 3.80 - 3.71 (m, 1H), 3.69 - 3.63 (m, 1H), 3.44 - 3.41 (m, 1H), 3.34 - 3.24 (m, 4H), 3.22 - 3.15 (m, 4H), [3.12 (s, 1.3H), 2.99 (s, 1.7H)], 2.94 - 2.86 (m, 2H), 2.84 - 2.74 (m, 5H), 2.73 - 2.61 (m, 5H), 2.46 - 2.39 (m, 1H), 2.31 - 2.18 (m, 1H), 2.08 - 1.91 (m, 2H), 1.89 - 1.76 (m, 2H), 1.72 - 1.55 (m, 2H), 1.46 - 1.38 (m, 3H), 1.35 - 1.26 (m, 4H), 1.21 (d, J = 7.1 Hz, 4H), 1.04 (dd, J = 14.2, 6.7 Hz, 2H), 0.99 - 0.82 (m, 11H), 0.77 (q, J = 7.2 Hz, 3H).

[0334] [Chemical formula] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(2-(dimethylamino)acetamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-70 To a solution of 4 (80 mg, 116 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (36 mg, 139 umol) in 5 mL CH3CN was added a mixture of EDCI (33 mg, 172 umol) and HOPO (20 mg, 180 umol) followed by 2,6-lutidine (37 mg, 345 umol). The reaction was stirred at room temperature under a N2 atmosphere for 16 h and LCMS showed completion. The mixture was directly concentrated to dryness and the residue was purified by Prep-TLC (DCM:MeOH = 10:1, R f = 0.75) to give INT-70 (100 mg, 93% yield) as a yellow oil. LCMS (ESI): m / z 931.0 [M + H] + .

[0335] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(2-(dimethylamino)acetamido)-N,3-dimethylbutanamide bis(2,2,2-trifluoroacetate) GEF2101-46-3(1550-26) To a mixture of INT-70 (100 mg, 107 umol) and anisole (60 mg, 555 umol) was added TFA (3 mL). The reaction was then stirred at room temperature for 5 min. TLC showed completion (DCM / MeOH = 10:1, v / v; R as INT-70). f = 0.75). The mixture was diluted with 100 mL MTBE, during which much white solid precipitated. The resulting mixture was filtered, and the filter cake was collected and dried under reduced pressure to give INT-71 (160 mg, >100% yield) as a yellow oil, which was used as is without further characterization.

[0336] [ka] (S)-2-(2-(dimethylamino)acetamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide 2,2,2-trifluoroacetate 31 To a solution of INT-71 (160 mg crude, 107 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (52 mg, 206 umol) in 5 mL CH3CN was added DIEA (33 mg, 256 umol). The reaction was then stirred at room temperature for 80 min. HPLC showed completion. The mixture was quenched by direct addition of TFA (0.14 mL). The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 31 (65 mg, 56% yield) as a white solid. LCMS (ESI): m / z 484.6 [M + 2H] 2+ HPLC: 99.5% @210 nm, R t= 8.61 minutes; 1 H NMR (400 MHz, DMSO)δ 9.90 - 9.65(m, 2H), 8.86(d, J = 8.3 Hz, 1H), 8.43(d, J = 7.2 Hz, 1H), 8.21 - 8.11(m, 1H), 7.49(s, 1H), 7.46 - 7.34 (m, 1H), 7.24 - 7.15 (m, 1H), 7.08 (s, 2H), 6.97 - 6.87 (m, 1H), 4.75 - 4.52 (m, 2H), 4.41 - 4.28 (m, 2H), 4.14 - 4.04 (m, 2H), 4.04 - 3.95(m, 2H), 3.94 - 3.88 (m, 1H), 3.79 - 3.71 (m, 1H), 3.70 - 3.62 (m, 1H), 3.61 - 3.54 (m, 1H), 3.52 - 3.46 (m, 2H), 3.33 - 3.23 (m, 4H), 3.21 - 3.09(m, 3H), [3.14(s, 1.5H), 2.97(s, 1.5H)], 2.93 - 2.87(m, 2H), 2.83 - 2.73(m, 8H), 2.71 - 2.66(m, 1H), 2.64 - 2.57(m, 1H), 2.49 - 2.18(m, 2H), 2.10 - 1.97(m, 1H), 1.95 - 1.76 (m, 3H), 1.75 - 1.54 (m, 2H), 1.38 - 1.27 (m, 4H), 1.22 (d, J = 6.8 Hz, 3H), 1.04 (dd, J = 13.5, 6.7 Hz, 2H), 1.01 - 0.83(m, 11H), 0.79(q, J = 7.5 Hz, 3H).

[0337] [ka] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(2-(dimethylamino)-2-methylpropanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-72 To a solution of 7 (110 mg, 153 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (48 mg, 184 umol) in 3 mL CH3CN was added a mixture of EDCI (44 mg, 230 umol) and HOPO (26 mg, 230 umol) followed by 2,6-lutidine (54 uL, 460 umol). The reaction was stirred at room temperature under N2 atmosphere for 16 hours and LCMS showed completion. The mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-72 (130 mg, 88% yield) as a pale yellow oil. LCMS (ESI): m / z 959.2 [M + H] + .

[0338] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(2-(dimethylamino)-2-methylpropanamido)-N,3-dimethylbutanamide bis(2,2,2-trifluoroacetate) INT-73 To a mixture of INT-72 (130 mg, 136 umol) and anisole (75 uL, 691 umol) was added TFA (1.2 mL). The reaction was then stirred at room temperature for 10 minutes and then quenched by adding 60 mL MTBE, during which time a lot of white solid precipitated. The resulting mixture was filtered and the filter cake was collected and dried under vacuum to give INT-73 (115 mg, 77% yield) as a pale yellow solid. LCMS (ESI): m / z 859.3 [M + H] + .

[0339] [ka] (S)-2-(2-(dimethylamino)-2-methylpropanamide)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide)propanamide)propanamide)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide 2,2,2-trifluoroacetate 32 To a solution of INT-73 (110 mg, 101 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (37 mg, 147 umol) in 2.5 mL CH3CN was added DIEA (38 uL, 226 umol). The reaction was then stirred at room temperature for 50 min and quenched by the direct addition of TFA (70 uL). The resulting mixture was immediately submitted to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 32 (55 mg, 49% yield) as a white solid. LCMS (ESI): m / z 997.2 [M + H] + , 498.8 [M + 2H] 2+ HPLC: 96.3% @210 nm, R t = 8.69 minutes; 1H NMR (400 MHz, DMSO)δ 9.91 - 9.75(m, 1H), 9.68(s, 1H), 8.56 - 8.35(m, 2H), 8.27 - 8.08(m, 1H), 7.50(s, 1H), 7.48 - 7.33(m, 1H), 7.29 - 7.14 (m, 1H), 7.10 (s, 2H), 6.99 - 6.85 (m, 1H), 4.82 - 4.58 (m, 1H), 4.57 - 4.44 (m, 1H), 4.44 - 4.23 (m, 2H), 4.17 - 4.04 (m, 2H), 4.02 - 3.88(m, 1H), 3.79 - 3.65 (m, 1H), 3.62 - 3.36 (m, 3H), 3.33 - 3.24 (m, 3H), 3.22 - 3.16 (m, 3H), 3.16 - 2.96 (m, 3H), 2.96 - 2.86 (m, 2H), 2.86 - 2.79 (m, 1H), 2.79 - 2.72 (m, 1H), 2.72 - 2.59 (m, 7H), 2.43 (d, J = 14.5 Hz, 1H), 2.35 - 2.20 (m, 1H), 2.20 - 2.04 (m, 1H), 2.03 - 1.77(m, 3H), 1.77 - 1.58(m, 2H), 1.54 - 1.43(m, 5H), 1.35 - 1.27(m, 3H), 1.27 - 1.14(m, 4H), 1.05(dd, J = 14.4, 6.7 Hz, 2H), 0.98 - 0.85(m, 9H), 0.81 - 0.68(m, 3H).

[0340] [ka] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(1-(dimethylamino)cyclobutanecarboxamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-74 To a solution of 12 (75 mg, 103 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid 1 (31 mg, 118 umol) in 2 mL CH3CN was added a mixture of EDCI (30 mg, 154 umol) and HOPO (17 mg, 154 mmol) followed by 2,6-lutidine (33 mg, 309 umol). The reaction was stirred at room temperature under N2 atmosphere for 16 h and LCMS showed completion. The mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-74 (74 mg, 74% yield) as a colorless oil. LCMS (ESI): m / z 971.6 [M + H] + .

[0341] [ka] N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-(dimethylamino)cyclobutanecarboxamide bis(2,2,2-trifluoroacetate) INT-75 To a mixture of INT-74 (72 mg, 75 umol) and anisole (41 mg, 376 umol) was added TFA (0.7 mL). The reaction was then stirred at room temperature for 30 min. TLC showed completion (DCM / MeOH = 13:1, v / v; INT-74 as R f = ca. 0.6). The mixture was diluted with 35 mL MTBE, during which a lot of white solid precipitated. The resulting mixture was filtered, and the filter cake was collected and dried under reduced pressure to give INT-75 (56 mg, 68% yield) as a pale yellow solid. LCMS (ESI): m / z 436.4 [M + 2H] 2+ .

[0342] [ka] 1-(Dimethylamino)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)cyclobutanecarboxamide 2,2,2-trifluoroacetate 33 To a solution of INT-75 (56 mg, 51 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (19 mg, 76 umol) in 2 mL CH3CN was added DIEA (17 uL, 102 umol). The reaction was then stirred at room temperature for 50 min. TLC showed completion (DCM / MeOH = 7:1, v / v; INT-75 as R f = ca. 0.15). The mixture was quenched by direct addition of TFA (50 uL) and stirred for 5 min. The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 33 (26 mg, 45% yield) as an off-white solid. LCMS (ESI): m / z 505.0 [M + 2H] 2+ ; HPLC: 99.9% @210 nm, R t = 8.92 minutes; 1H NMR (400 MHz, DMSO-d6)δ 10.46(s, 1H), 9.88 - 9.74(m, 1H), 8.64(s, 1H), 8.42(d, J = 7.2 Hz, 1H), 8.20 - 8.08(m, 1H), 7.49(d, J = 7.4 Hz, 1H), 7.47 - 7.34(m, 1H), 7.26 - 7.13(m, 1H), 7.08(s, 2H), 6.96 - 6.87(m, 1H), 4.81 - 4.64(m, 1H), 4.53 - 4.43(m, 1H), 4.40 - 4.29(m, 2H), 4.11 - 4.07(m, 2H), 3.96 - 3.93(m, 2H), 3.91 - 3.86(m, 2H)], 3.77 - 3.71(m, 1H), 3.68 - 3.61(m, 1H), 3.58 - 3.40(m, 3H), 3.35 - 3.21 (m, 4H), 3.20 - 3.05 (m, 5H), [3.03 (s, 1H), 2.91 (s, 2H)], 2.84 - 2.80 (m, 1H), 2.79 - 2.56 (m, 10H), 2.48 - 2.40 (m, 2H), 2.38 - 2.04(m, 2H), 1.99 - 1.77(m, 4H), 1.75 - 1.54(m, 3H), 1.38 - 1.26(m, 4H), 1.25 - 1.20(m, 3H), 1.05(dd, J = 16.6, 6.7 Hz, 2H), 1.00 - 0.84(m, 10H), 0.77(q, J = 7.5 Hz, 3H).

[0343] [ka] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-1-methylpiperidine-2-carboxamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-76 To a solution of INT-76 (150 mg, 206 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (64 mg, 247 umol) in 3 mL CH3CN was added a mixture of EDCI (59 mg, 309 umol) and HOPO (34 mg, 309 umol) followed by 2,6-lutidine (72 uL, 617 umol). The reaction was stirred at room temperature under N2 atmosphere for 16 hours and LCMS showed completion. The mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-76 (120 mg, 60% yield) as a yellow solid. LCMS (ESI): m / z 972.0 [M + H] + .

[0344] [ka] (S)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-2-carboxamide bis(2,2,2-trifluoroacetate) INT-77 To a mixture of INT-76 (90 mg, 93 umol) and anisole (50 uL, 463 umol) was added TFA (0.9 mL). The reaction was then stirred at room temperature for 10 min and quenched by the addition of an additional 45 mL MTBE, during which time much white solid precipitated. The resulting mixture was filtered and the filter cake was collected and dried under vacuum to give INT-77 (85 mg, 82% yield) as an off-white solid. LCMS (ESI): m / z 436.2 [M + 2H] 2+ .

[0345] [ka] (S)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-2-carboxamide 2,2,2-trifluoroacetate 34 To a solution of INT-77 (80 mg, 73 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (27 mg, 106 umol) in 3 mL CH3CN was added DIEA (21 mg, 162 umol). The reaction was then stirred at room temperature for 35 min and quenched by the addition of additional TFA (60 uL). LCMS showed completion. The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 34 (16 mg, 20% yield) as a white solid. LCMS (ESI): m / z 505.3 [M + 2H] 2+ HPLC: 97.4% @210 nm, R t = 14.41 minutes; 1H NMR (400 MHz, DMSO)δ 9.91 - 9.78(m, 1H), 9.70(s, 1H), 9.00(d, J = 8.4 Hz, 1H), 8.45(d, J = 7.1 Hz, 1H), 7.49(s, 1H), 7.46 - 7.34(m, 1H), 7.26 - 7.16 (m, 1H), 7.09 (s, 2H), 6.98 - 6.86 (m, 1H), 4.78 - 4.61 (m, 1H), 4.61 - 4.50 (m, 1H), 4.40 - 4.27 (m, 2H), 4.13 - 4.04 (m, 2H), 4.04 - 3.84(m, 2H), 3.81 - 3.69 (m, 2H), 3.69 - 3.62 (m, 1H), 3.44 - 3.35 (m, 2H), 3.34 - 3.23 (m, 4H), 3.23 - 3.15 (m, 3H), 3.14 - 2.94 (m, 4H), 2.93 - 2.87(m, 2H), 2.84 - 2.79(m, 1H), 2.78 - 2.58(m, 6H), 2.49 - 2.18(m, 2H), 2.08 - 1.85(m, 4H), 1.84 - 1.74(m, 3H), 1.73 - 1.53(m, 3H), 1.51 - 1.37(m, 2H), 1.35 - 1.25(m, 4H), 1.24 - 1.17(m, 4H), 1.04(dd, J = 13.6, 6.6 Hz, 2H), 0.98 - 0.81(m, 11H), 0.76(q, J = 7.0 Hz, 3H).

[0346] [ka] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-78 To a solution of 11 (100 mg, 137 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (43 mg, 165 umol) in 5 mL CH3CN was added a mixture of EDCI (40 mg, 209 umol) and HOPO (23 mg, 207 umol) followed by 2,6-lutidine (44 mg, 410 umol). The reaction was stirred at room temperature under N2 atmosphere for 2.5 h and LCMS showed completion. The mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-78 (120 mg, 90% yield) as a yellow oil. LCMS (ESI): m / z 971.2 [M + H] + .

[0347] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-2-carboxamide bis(2,2,2-trifluoroacetate) INT-79 To a mixture of INT-78 (120 mg, 124 umol) and anisole (67 mg, 620 umol) was added TFA (3 mL). The reaction was then stirred at room temperature for 5 min. TLC showed completion (DCM / MeOH = 8:1, v / v; INT-78 as R f = ca. 0.55). The mixture was diluted with 200 mL MTBE, during which time a lot of white solid precipitated. The resulting mixture was filtered, and the filter cake was collected and dried under reduced pressure to give INT-79 (110 mg, 80% yield) as an off-white solid. LCMS (ESI): m / z 436.2 [M + 2H] 2+ .

[0348] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-2-carboxamide 2,2,2-trifluoroacetate 35 To a solution of INT-79 (110 mg, 100 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (43 mg, 170 umol) in 6 mL CH3CN was added DIEA (29 mg, 225 umol). The reaction was then stirred at room temperature for 30 min. LCMS showed completion. The mixture was quenched by adding TFA (0.2 mL). The resulting mixture was purified twice by Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 35 (26 mg, 23% yield) as a white solid. LCMS (ESI): m / z 1030.3 [M + Na + ], 504.8 [M + 2H] 2+ HPLC: 97.5% @210 nm, R t = 19.05 minutes; 11H NMR (400 MHz, DMSO) δ 9.85 - 9.77 (m, 1H), 9.68 (s, 1H), 8.95 (d, J = 7.8 Hz, 1H), 8.43 (d, J = 7.3 Hz, 1H), 8.20 - 8.11 (m, 1H), 7.49 (s, 1H), 7.40 (dd, J = 24.0, 15.8 Hz, 1H), 7.20 (dd, J = 19.6, 11.4 Hz, 1H), 7.09 (s, 2H), 6.96 - 6.87 (m, 1H), 4.79 - 4.58 (m, 1H), 4.56 - 4.45 (m, 1H), 4.43 - 4.28 (m, 2H), 4.15 - 4.04 (m, 2H), 4.04 - 3.84 (m, 2H), 3.81 - 3.72 (m, 2H), 3.51 (s, 1H), 3.46 (s, 1H), 3.43 (d, J = 7.2 Hz, 1H), 3.36 - 3.23 (m, 5H), 3.20 - 3.18 (m, 2H), 3.17 - 2.98 (m, 5H), 2.93 - 2.87 (m, 2H), 2.83 - 2.79 (m, 1H), 2.77 - 2.72 (m, 1H), 2.71 - 2.66 (m, 1H), 2.64 - 2.59 (m, 3H), 2.45 - 2.36 (m, 1H), 2.32 - 2.18 (m, 1H), 2.07 - 1.96 (m, 2H), 1.94 - 1.75 (m, 5H), 1.71 - 1.54 (m, 4H), 1.45 - 1.36 (m, 1H), 1.34 - 1.26 (m, 4H), 1.25 - 1.19 (m, 4H), 1.04 (dd, J = 13.8, 6.7 Hz, 2H), 0.99 - 0.82 (m, 11H), 0.80 - 0.74 (m, 3H).

[0349]

Chem.

[0350] [ka] (S)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-2-carboxamide bis(2,2,2-trifluoroacetate) INT-81 To a mixture of INT-80 (110 mg, 115 umol) and anisole (65 uL, 598 umol) was added TFA (1 mL). The reaction was then stirred at room temperature for 10 min and quenched by the addition of an additional 50 mL MTBE, during which time much white solid precipitated. The resulting mixture was filtered and the filter cake was collected and dried under vacuum to give INT-81 (110 mg, 87% yield) as an off-white solid. LCMS (ESI): m / z 857.2 [M + H] + .

[0351] [ka] (S)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-2-carboxamide 2,2,2-trifluoroacetate 36 To a solution of INT-81 (110 mg, 101 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (36 mg, 142 umol) in 2 mL CH3CN was added DIEA (38 uL, 227 umol). The reaction was then stirred at room temperature for 45 min and quenched by the addition of additional TFA (70 uL). LCMS showed completion. The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 36 (60 mg, 53% yield) as an off-white solid. LCMS (ESI): m / z 994.1 [M + H] + , 497.8 [M + 2H] 2+ HPLC: 98.8% @210 nm, R t= 8.62 minutes; 1 H NMR (400 MHz, DMSO)δ 9.86 - 9.75(m, 1H), 9.62(s, 1H), 9.00(d, J = 8.6 Hz, 1H), 8.43(d, J = 7.2 Hz, 1H), 8.22 - 8.12(m, 1H), 7.49(s, 1H), 7.46 - 7.34(m, 1H), 7.26 - 7.16(m, 1H), 7.09(s, 2H), 6.97 - 6.87(m, 1H), 4.81 - 4.65(m, 2H), 4.65 - 4.47(m, 2H), 4.41 - 4.28(m, 2H), 4.15 - 4.01(m, 3H), 3.99 - 3.88 (m, 1H), 3.78 - 3.66 (m, 1H), 3.65 - 3.51 (m, 2H), 3.51 - 3.34 (m, 2H), 3.33 - 3.25 (m, 3H), 3.24 - 3.16 (m, 3H), 3.16 - 2.94 (m, 4H), 2.94 - 2.85 (m, 2H), 2.84 - 2.72 (m, 5H), 2.71 - 2.54 (m, 2H), 2.47 - 2.37 (m, 2H), 2.34 - 2.19 (m, 1H), 2.10 - 1.97 (m, 2H), 1.96 - 1.75(m, 4H), 1.75 - 1.56(m, 3H), 1.33 - 1.26(m, 3H), 1.21(d, J = 7.0 Hz, 4H), 1.04(dd, J = 12.9, 6.7 Hz, 2H), 0.99 - 0.69(m, 14H).

[0352] [ka] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((R)-1-methylpyrrolidine-2-carboxamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-82 To a solution of 9 (90 mg, 126 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (38 mg, 145 umol) in 3 mL CH3CN was added a mixture of EDCI (36 mg, 189 mmol) and HOPO (21 mg, 189 umol) followed by 2,6-lutidine (40 mg, 378 umol). The reaction was stirred at room temperature under N2 atmosphere for 16 h and LCMS showed completion. The mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-82 (110 mg, 91% yield) as an off-white solid. LCMS (ESI): m / z 957.7 [M + H] + .

[0353] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-2-carboxamide bis(2,2,2-trifluoroacetate) INT-83 To a mixture of INT-82 (110 mg, 117 umol) and anisole (63 mg, 583 umol) was added TFA (1.1 mL). The reaction was then stirred at room temperature for 30 min. TLC showed completion (DCM / MeOH = 13:1, v / v; R f = 0.4). The mixture was diluted with 50 mL MTBE, during which time a lot of white solid precipitated. The resulting mixture was filtered, and the filter cake was collected and dried under reduced pressure to give INT-83 (127 mg, 100% yield) as an off-white solid, which was used directly in the next step.

[0354] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-2-carboxamide 2,2,2-trifluoroacetate 37 To a solution of INT-83 (127 mg, 117 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (44 mg, 176 umol) in 2 mL CH3CN was added DIEA (39 uL, 234 umol). The reaction was then stirred at room temperature for 30 minutes. TLC & LCMS showed completion (DCM / MeOH = 6:1, v / v; INT-83 as R f = ca. 0.15). The mixture was quenched by direct addition of TFA (0.1 mL) and stirred for 5 min. The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 37 (29 mg, 22% yield) as an off-white solid. LCMS (ESI): m / z 1016.0 [M + Na + ]; HPLC: 99.2% @210 nm, R t = 8.70 minutes; 1H NMR (400 MHz, DMSO-d6)δ 9.87 - 9.76(m, 1H), 9.69(s, 1H), 9.02(d, J = 8.1 Hz, 1H), 8.43(d, J = 7.1 Hz, 1H), 8.21 - 8.11 (m, 1H), 7.49 (s, 1H), 7.47 - 7.34 (m, 1H), 7.25 - 7.15 (m, 1H), 7.09 (s, 2H), 6.96 - 6.86 (m, 1H), 4.74 - 4.64 (m, 1H), 4.58 - 4.53(m, 1H), 4.38 - 4.36(m, 1H), 4.33 - 4.30 (m, 1H), 4.11 - 4.07 (m, 2H), 4.04 - 4.00 (m, 1H), 3.97 - 3.88 (m, 1H), 3.78 - 3.61 (m, 2H), 3.60 - 3.51 (m, 2H), 3.51 - 3.39(m, 2H), 3.36 - 3.24(m, 4H), 3.23 - 3.16(m, 4H), [3.12(s, 1.3H), 2.99(s, 1.7H)], 2.93 - 2.87(m, 2H), 2.83 - 2.80(m, 1H), 2.79 - 2.60(m, 6H), 2.49 - 2.19(m, 2H), 2.13 - 2.00 (m, 2H), 1.99 - 1.71 (m, 6H), 1.70 - 1.49 (m, 2H), 1.32 - 1.28 (m, 3H), 1.24 - 1.20 (m, 4H), 1.04 (dd, J = 13.6, 6.6 Hz, 2H), 1.00 - 0.85(m, 10H), 0.84 - 0.74(m, 4H).

[0355] [ka] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(3-(dimethylamino)-2,2-dimethylpropanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-84 To a solution of 5 (60 mg, 82 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (26 mg, 98.5 umol) in 3 mL CH3CN was added a mixture of EDCI (24 mg, 123 umol) and HOPO (14 mg, 123 umol) followed by 2,6-lutidine (29 uL, 246 umol). The reaction was stirred at room temperature under N2 atmosphere for 16 hours and LCMS showed completion. The mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-84 (50 mg, 63% yield) as a white solid. LCMS (ESI): m / z 974.2 [M + H] + .

[0356] [ka] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(3-(dimethylamino)-2,2-dimethylpropanamido)-N,3-dimethylbutanamide 2,2,2-trifluoroacetate INT-85 To a mixture of INT-84 (50 mg, 51 umol) and anisole (28 mg, 257 umol) was added TFA (0.5 mL). The reaction was then stirred at room temperature for 10 min and quenched by the addition of an additional 25 mL MTBE, during which time much white solid precipitated. The resulting mixture was filtered and the filter cake was collected and dried under vacuum to give INT-85 (50 mg, 88% yield) as a pale yellow solid. LCMS (ESI): m / z 873.6 [M + H] + , 437.5 [M + 2H] 2+ .

[0357] [ka] (S)-2-(3-(dimethylamino)-2,2-dimethylpropanamide)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamide)propanamide)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide 2,2,2-trifluoroacetate 38 To a solution of INT-85 (50 mg, 45 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (17 mg, 66 umol) in 2 mL CH3CN was added DIEA (17 uL, 101 umol). The reaction was then stirred at room temperature for 45 min and quenched by direct addition of additional TFA (0.04 mL). LCMS showed completion. The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 38 (23 mg, 45% yield) as a white solid. LCMS (ESI): m / z 506.0 [M + 2H] 2+ ; HPLC: 99.9% @210 nm, R t = 8.77 minutes; 1H NMR (400 MHz, DMSO)δ 9.90 - 9.73(m, 1H), 8.91(s, 1H), 8.43(d, J = 7.1 Hz, 1H), 8.24 - 8.10(m, 1H), 7.99 - 7.84(m, 1H), 7.49(s, 1H), 7.47 - 7.34 (m, 1H), 7.25 - 7.16 (m, 1H), 7.09 (s, 2H), 6.97 - 6.87 (m, 1H), 4.76 - 4.58 (m, 1H), 4.55 - 4.44 (m, 1H), 4.40 - 4.31 (m, 2H), 4.11 - 4.08(m, 2H), 3.97 - 3.94 (m, 1H), 3.75 - 3.74 (m, 1H), 3.60 - 3.54 (m, 1H), 3.50 - 3.42 (m, 2H), 3.36 - 3.23 (m, 6H), 3.22 - 3.17 (m, 3H), 3.15 - 2.95 (m, 4H), 2.92 - 2.88 (m, 2H), 2.82 - 2.72 (m, 8H), 2.69 - 2.64 (m, 1H), 2.49 - 2.19 (m, 2H), 2.14 - 2.04 (m, 1H), 2.02 - 1.82 (m, 3H), 1.80 - 1.72(m, 1H), 1.71 - 1.56(m, 2H), 1.31 - 1.26(m, 6H), 1.24 - 1.19(m, 7H), 1.04(dd, J = 13.2, 6.7 Hz, 2H), 0.98 - 0.83(m, 11H), 0.77(q, J = 7.2 Hz, 3H).

[0358] [ka] tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((R)-1-methylpiperidine-3-carboxamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-86 To a solution of 6 (50 mg, 69 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (22 mg, 82 umol) in 2 mL CH3CN was added EDCI (20 mg, 103 umol) and HOPO (12 mg, 103 umol) followed by 2,6-lutidine (24 uL, 206 umol). The reaction was stirred at room temperature under N2 atmosphere for 16 h and LCMS showed completion. The mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-86 (35 mg, 53% yield) as a white solid. LCMS (ESI): m / z 971.2 [M + H] + .

[0359] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-3-carboxamide bis(2,2,2-trifluoroacetate) INT-87 To a mixture of INT-86 (35 mg, 36 umol) and anisole (20 uL, 180 umol) was added TFA (0.35 mL). The reaction was then stirred at room temperature for 10 minutes and quenched by the addition of an additional 18 mL MTBE, during which time much white solid precipitated. The resulting mixture was filtered and the filter cake was collected and dried under vacuum to give INT-87 (30 mg, 75% yield) as a pale yellow solid, which was used as is without further characterization.

[0360] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpiperidine-3-carboxamide 2,2,2-trifluoroacetate 39 To a solution of INT-87 (35 mg, 36 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (12 mg, 46 umol) in 6 mL CH3CN was added DIEA (12 uL, 71 umol). The reaction was then stirred at room temperature for 30 min. LCMS showed completion. The mixture was quenched by adding TFA (20 uL). The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 39 (5 ​​mg, 14% yield) as a white solid. LCMS (ESI): m / z 505.0 [M + 2H] 2+ ; HPLC: 99.8% @210 nm, R t = 8.62 minutes.

[0361] [ka] Step 1: tert-Butyl ((S)-1-(((S)-1-((3-(2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((R)-1-methylpyrrolidine-3-carboxamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-N,2-dimethylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate INT-88 To a solution of 10 (110 mg, 154 umol) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid (48 mg, 185 umol) in 3 mL CH3CN was added a mixture of EDCI (44 mg, 231 mmol) and HOPO (26 mg, 231 umol) followed by 2,6-lutidine (54 uL, 462 umol). The reaction was stirred at room temperature under N2 atmosphere for 16 hours and LCMS showed completion. The mixture was directly purified by reverse phase column (H2O:CH3CN) to give INT-88 (105 mg, 71% yield) as a yellow foamy solid. LCMS (ESI): m / z 957.1 [M + H] + .

[0362] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-aminopropanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-3-carboxamide bis(2,2,2-trifluoroacetate) INT-89 To a mixture of INT-88 (100 mg, 104 umol) and anisole (57 uL, 522 umol) was added TFA (1 mL). The reaction was then stirred at room temperature for 10 min and quenched by the addition of an additional 50 mL MTBE, during which time much white solid precipitated. The resulting mixture was filtered and the filter cake was collected and dried under vacuum to give INT-89 (90 mg, 78% yield) as a pale yellow solid. LCMS (ESI): m / z 880.3 [M + Na + ], 429.1 [M + 2H] 2+ .

[0363] [ka] (R)-N-((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)phenethyl)(methyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-1-methylpyrrolidine-3-carboxamide 2,2,2-trifluoroacetate 40 To a solution of INT-89 (90 mg, 83 umol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (30 mg, 116 umol) in 2 mL CH3CN was added DIEA (30 uL, 185 umol). The reaction was then stirred at room temperature for 50 min. LCMS showed completion. The mixture was quenched by adding TFA (60 uL). The resulting mixture was immediately sent to Prep-HPLC (0.1% TFA in H2O / CH3CN) and then lyophilized to give 40 (65 mg, 71% yield) as a white solid. LCMS (ESI): m / z 995.2 [M + H] + , 497.7 [M + 2H] 2+ ; HPLC: 98.6% @210 nm, R t = 8.57 minutes; 1H NMR (400 MHz, DMSO) δ 9.92 - 9.67 (m, 2H), 8.54 - 8.39 (m, 2H), 8.21 - 8.10 (m, 1H), 7.49 (s, 1H), 7.46 - 7.34 (m, 1H), 7.25 - 7.16 (m, 1H), 7.09 (s, 2H), 6.97 - 6.87 (m, 1H), 4.75 - 4.59 (m, 2H), 4.55 - 4.44 (m, 2H), 4.38 - 4.31 (m, 2H), 4.13 - 4.05 (m, 2H), 4.03 - 3.85(m, 2H), 3.79 - 3.57(m, 3H), 3.55 - 3.38 (m, 3H), 3.29 (dd, J = 13.5, 5.7 Hz, 4H), 3.21 - 3.16 (m, 3H), 3.13 - 2.95 (m, 4H), 2.91 - 2.86 (m, 2H), 2.85 - 2.78 (m, 3H), 2.77 - 2.73 (m, 1H), 2.72 - 2.58 (m, 2H), 2.46 - 2.32 (m, 2H), 2.30 - 2.04 (m, 2H), 2.00 - 1.73 (m, 5H), 1.67 - 1.42 (m, 2H), 1.33 - 1.26(m, 3H), 1.21(d, J = 6.8 Hz, 4H), 1.04(dd, J = 12.4, 6.7 Hz, 2H), 0.98 - 0.70(m, 14H).

[0364] biological activity Assay protocol HCC1954 breast ductal carcinoma cells and T47D cells (ATCC, Manassas, VA, USA) were seeded in 384-well white-walled culture plates and allowed to adhere for 2–4 h. Cells were then treated in at least duplicates by adding 5-fold serially diluted test articles prepared at twice the final concentration and incubated at 37°C for 120 h. Cell viability after treatment was measured by Cell Titer Glo 2.0 Assay (Promega, Madison, WI, USA) and normalized to untreated controls. Dose-response relationships were analyzed using GraphPad Prism (La Jolla, CA, USA), and IC50 values ​​were derived from nonlinear regression analysis using a 4-parameter logistic equation. [Table 4] +++: <5 nM; ++: 5-10 nM; +: >10 nM [Table 5]

[0365] Compared with the 1,4-NH2 configuration of the P5 moiety in 19 and 26, the 1,2-NH2 configuration (14 and 16) and the 1,3-NH2 configuration (1 and 2) were surprisingly more potent. [Table 6] [ka]

[0366] These results showed that methyl substitution on the N between P4 and P5 retained potency. This was unexpected and in contrast to other well-known auristatin derivatives where methylation occurred. For example, N-methylation of the amide between P4 and P5 of auristatin E, auristatin PHE, and dolastatin 10 molecules resulted in a 23- to 240-fold decrease in potency. This highlights the unique and unexpected properties of the ethylene functionality of the P5 moiety in compound 1. [Table 7]

[0367] Exemplary compounds of the invention were conjugated to certain antibodies (e.g., trastuzumab) and tested for efficacy. These conjugates showed a favorable DAR of about 8 and an aggregation (SEC) of about 1%.

[0368] These complexes showed excellent IC 50 showed. [Table 8] +++: <5 nM; ++: 5-10 nM; +: >10 nM

[0369] Applicant's disclosure is described herein in preferred embodiments with reference to the figures, in which like numbers represent the same or similar elements. References throughout this specification to "an embodiment" or similar terms mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the appearances of "in an embodiment" and similar terms throughout this specification do not necessarily all refer to the same embodiment.

[0370] The described features, structures, or characteristics of the applicant's disclosure can be combined in any suitable manner in one or more embodiments. Numerous specific details are described herein to provide a thorough understanding of the embodiments of the present invention. However, those skilled in the relevant art will recognize that the applicant's compositions and / or methods can be implemented without one or more of the specific details, or with other methods, components, materials, etc. In other embodiments, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the present disclosure.

[0371] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Any method and material similar or equivalent to those described herein can also be used in the implementation or testing of this disclosure, but preferred methods and materials are described herein.The methods described herein can be carried out in any order that is logically possible, in addition to the specific order disclosed.

[0372] Citation by reference In this disclosure, references and citations are made to other documents, such as patents, patent applications, patent publications, journals, books, papers, manuscripts, web content, etc. All such documents are incorporated herein by reference in their entirety for all purposes. Any material or portion thereof that is incorporated herein by reference that conflicts with existing definitions, descriptions, or other disclosure materials expressly set forth herein is incorporated only to the extent that no conflict occurs between the incorporated material and this disclosure material. In case of conflict, the conflict is resolved with this disclosure as the preferred disclosure.

[0373] Equivalent The representative examples are intended to be illustrative of the invention and are not intended to, and should not be construed as, limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this specification, including the examples contained herein and references to the scientific and patent literature. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of the invention in its various embodiments and equivalents thereof.

Claims

1. Structural formula (I) 【Chemistry 1】 (I) [During the ceremony, R 1 teeth 【Chemistry 2】 where R 2 is unsubstituted or substituted C 1 -C 6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R a , R b and R c are H and NR x R y selected from, with the proviso that R a , R b and R c Only one of the x R y and each of the others is H; R x and R y are independently R, R r and L.R. z selected from, with the proviso that R x and R y One of them is LR z or R r when the other is R; R 5 is CR' 3 wherein each R' is independently H or F; L is a linker; R r is (C=O)-O-(CH 2 ) p -R v or (C=O)-(CH 2 ) q -R v and R v are R, OR, NHR, and NR 2 , an aryl group, or an amino acid; p is 0, 1, 2, 3, 4, 5 or 6; q is 0, 1, 2, 3, 4, 5 or 6; R z comprises a functional or reactive group; and R is H or C 1 -C 3 It is alkyl. or a pharmaceutically acceptable salt thereof.

2. R 5 is CH 3 2. The compound of claim 1, wherein:

3. R 5 CF 3 2. The compound of claim 1, wherein:

4. R 5 is CH 3 and R a is H, R c is H, and R b NR x R y and structural formula (III) 【Transformation 3】 (III) 2. The compound of claim 1, wherein:

5. R x is H and R y is H, and the structural formula (III 1 ) 【Chemistry 4】 (III 1 ) 5. The compound of claim 4, wherein:

6. R x is H or CH 3 and R y (C=O)-O-(CH 2 ) p -R v (where R v R, OR, NHR, NR 2 , an aryl group or an amino acid, and p is 0, 1, 2 or 3), or a pharmaceutically acceptable salt thereof.

7. R x is H or CH 3 and R y (C=O)-(CH 2 ) q -R v (where R v R, OR, NHR, NR 2 , an aryl group or an amino acid, and q is 0, 1, 2 or 3), or a pharmaceutically acceptable salt thereof.

8. R y is LR z and the structural formula (III 2 ) 【Transformation 5】 (III 2 ) 5. The compound of claim 4, wherein:

9. R x is H, and the structural formula (III 3 ) 【Transformation 6】 (III 3 ) 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein:

10. R 5 is CH 3 and R a is H and R b is H and R c NR x R y and structural formula (V) 【Transformation 7】 (V) 2. The compound of claim 1, wherein:

11. R x is H and R y is H and the structural formula (V 1 ) 【Transformation 8】 (V 1 ) 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein:

12. R x is H or CH 3 and R y (C=O)-O-(CH 2 ) p -R v (where R v R, OR, NHR, NR 2 , an aryl group or an amino acid, and p is 0, 1, 2 or 3), or a pharmaceutically acceptable salt thereof.

13. R x is H or CH 3 and R y (C=O)-(CH 2 ) q -R v (where R v R, OR, NHR, NR 2 , an aryl group or an amino acid, and q is 0, 1, 2 or 3), or a pharmaceutically acceptable salt thereof.

14. R y is LR z and the structural formula (V 2 ) 【Chemistry 9】 (V 2 ) 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein:

15. R x is H and the structural formula (V 3 ) 【Chemistry 10】 (V 3 ) 15. The compound of claim 14, or a pharmaceutically acceptable salt thereof, wherein:

16. R 5 CF 3 and R a is H and R b is H and R c NR x R y and the structural formula (V 4 ) 【Chemistry 11】 (V 4 ) 2. The compound of claim 1, wherein:

17. R 1 but 【Chemistry 12】 or 【Chemistry 13】 where R 3 and R 4 each independently represents H or unsubstituted or substituted C 1 -C 5 alkyl or optionally halogen atoms or C together with the N and C atoms to which they are attached 1 -C 3 17. The compound of any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, which forms a 5- to 7-membered heterocycloalkyl containing one or more of O, N, and S substituted with one or more of alkyl.

18. R 4 18. The compound of claim 17, or a pharmaceutically acceptable salt thereof, wherein is isopropyl.

19. R 4 18. The compound of claim 17, or a pharmaceutically acceptable salt thereof, wherein is methyl.

20. R 3 and R 4 together with the N and C atoms to which they are attached, optionally a halogen atom or C 1 -C 3 18. The compound of claim 17, or a pharmaceutically acceptable salt thereof, which forms a 5- or 6-membered heterocycloalkyl containing N substituted with one or more alkyl.

21. R 1 but 【Chemistry 14】 17. The compound according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, selected from:

22. 17. The compound of any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein L is a non-cleavable linker.

23. 17. The compound of any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein L is a cleavable linker.

24. R z が、-N 3 、-NR u C(=O)CH=CH 2 、-SH、-SSR t 、-S(=O) 2 (CH=CH 2 )、-(CH 2 ) 2 S(=O) 2 (CH=CH 2 )、-NR u S(=O 2 )(CH=CH 2 )、-NR u C(=O)CH 2 R w 、-NR u C(=O)CH 2 Br、-NR u C(=O)CH 2 I、-NHC(=O)CH 2 Br、NHC(=O)CH 2 I、-ONH 2 、-C(=O)NHNH 2 、-CO 2 H、-NH 2 、-NCO、-NCS、 【Chemistry 15】 【Chemistry 16】 [In the formula, R u is H or C 1 -C 6 is an alkyl group, and R t is 2-pyridyl or 4-pyridyl, and R w but 【Chemistry 17】 [Chemistry 18] It is.

17. The compound of any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, comprising a functional or reactive group selected from: 【Request Item 25】 【Table 1-1】 Table 1-2 or a pharmaceutically acceptable salt thereof.

26. A drug-linker conjugate or a pharmaceutically acceptable salt thereof formed by bonding the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16 and 25 to a linker.

27. An immunoconjugate or a pharmaceutically acceptable salt thereof formed by binding the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16 and 25 to an antigen-binding moiety via a linker.

28. Structural formula (VI) 【Chemistry 19】 (VI) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth 【Chemistry 20】 where R 2 is unsubstituted or substituted C 1 -C 6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and R y Each of these is independently R and LR z selected from, with the proviso that R x and R y One of them is NR z when the other is R; R 5 is CR' 3 where each R' is independently H or F; L is a linker; R is H or C 1 -C 3 is alkyl; and and i is an integer ranging from 1 to about 20. or a pharmaceutically acceptable salt thereof.

29. R 5 is CH 3 and R x is H, and the structural formula (VI 1 ) 【Chemistry 21】 (WE 1 ) 29. The immunoconjugate of claim 28, or a pharmaceutically acceptable salt thereof, having the formula:

30. Structural formula (VII) 【Chemistry 22】 (VII) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth 【Chemistry 23】 where R 2 is unsubstituted or substituted C 1 -C 6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and R y Each of these is independently R and LR z selected from, with the proviso that R x and R y One of them is NR z when the other is R; R 5 is CR' 3 where each R' is independently H or F; L is a linker; R is H or C 1 -C 3 is alkyl; and j is an integer ranging from 1 to about 20. or a pharmaceutically acceptable salt thereof.

31. Structural formula (VIII) 【Chemistry 24】 (VIII) [During the ceremony, Ab stands for antigen-binding moiety; R 1 teeth 【Chemistry 25】 where R 2 is unsubstituted or substituted C 1 -C 6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and R y Each of these is independently R and LR z selected from, with the proviso that R x and R y One of them is NR z when the other is R; R 5 is CR' 3 where each R' is independently H or F; L is a linker; R is H or C 1 -C 3 is alkyl; and k is an integer ranging from 1 to about 20. or a pharmaceutically acceptable salt thereof.

32. R 5 CF 3 and R x is H, and the structural formula (VIII 1 ) 【Chemistry 26】 (VIII 1 ) 32. The immunoconjugate of claim 31, or a pharmaceutically acceptable salt thereof, having the formula:

33. R 1 but 【Chemistry 27】 or 【Chemistry 28】 where R 3 and R 4 each independently represents H or unsubstituted or substituted C 1 -C 5 alkyl or optionally halogen atoms or C together with the N and C atoms to which they are attached 1 -C 3 33. The immunoconjugate of any one of claims 28 to 32, or a pharmaceutically acceptable salt thereof, which forms a 5-7 membered heterocycloalkyl containing one or more of O, N, and S substituted with one or more of alkyl.

34. R 4 34. The immunoconjugate or a pharmaceutically acceptable salt thereof of claim 33, wherein is isopropyl or methyl.

35. R 3 and R 4 together with the N and C atoms to which they are attached, optionally a halogen atom or C 1 -C 3 34. The immunoconjugate of claim 33, or a pharmaceutically acceptable salt thereof, which forms a 5- or 6-membered heterocycloalkyl containing N substituted with one or more alkyl.

36. R 1 but 【Chemistry 29】 33. The immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28 to 32, selected from:

37. 33. The immunoconjugate or pharmaceutically acceptable salt thereof according to any one of claims 28 to 32, wherein L is a non-cleavable linker.

38. 33. The immunoconjugate or pharmaceutically acceptable salt thereof according to any one of claims 28 to 32, wherein L is a cleavable linker.

39. 33. The immunoconjugate or pharmaceutically acceptable salt thereof according to claims 28 to 32, wherein Ab is an antibody or an antibody fragment.

40. 33. The immunoconjugate or pharmaceutically acceptable salt thereof according to any one of claims 28 to 32, wherein Ab is a peptide.

41. A pharmaceutical composition comprising the immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28 to 32 and a pharmaceutically acceptable excipient, carrier or diluent.

42. A combination comprising a therapeutically effective amount of the immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28 to 32, and one or more therapeutically active co-agents and / or adjuvants.

43. A pharmaceutical composition for treating cancer, comprising the immunoconjugate of any one of claims 28 to 32 or a pharmaceutically acceptable salt thereof.

44. Use of the immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 28 to 32 for the manufacture of a medicament for treating cancer.

45. A medicament for treating cancer, comprising the immunoconjugate of any one of claims 28 to 32 or a pharmaceutically acceptable salt thereof.

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