Antibody drug conjugates

Abstract

The present invention provides antibody drug conjugates, and specifically provides antibody drug conjugates comprising a STING modulator of the following formula (I). Also provided are compositions comprising the antibody drug conjugates. The compounds and compositions are useful for stimulating an immune response in a subject in need thereof. # imgabs0 #

Description

[0001] This application is a divisional application of the patent application with application number 202080034546.2, application date May 8, 2020, and invention name “Antibody Drug Conjugate”.

[0002] References to sequence listings submitted electronically

[0003] The contents of the electronically submitted Sequence Listing (Name: 3817_053PC03_Seqlisting_ST25; Size: 7,104 bytes, and Creation Date: April 22, 2020) are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention provides antibody-drug conjugates comprising STING modulators. Also provided are compositions comprising the antibody-drug conjugates. The compounds and compositions can be used to stimulate an immune response in a subject in need thereof. Background Art

[0005] Antibody drug conjugates (ADCs) are a class of rapidly developing targeted therapeutics that represent a new approach that is expected to improve the selectivity and cytotoxic activity of drugs. These therapeutic agents comprise antibodies (or antibody fragments) that can be linked to a payload drug to form an immunoconjugate. The antibody directs the ADC to bind to the target cell. The ADC can then internalize and release its payload, thereby providing treatment for the cell. When the ADC is directed to its target cell, the side effects of the conjugated drug may be lower than those encountered when the same agent is administered systemically.

[0006] The adaptor protein STING (stimulator of interferon genes) has been shown to play a role in the innate immune system. Activation of the STING pathway triggers an immune response that produces specific killer T cells that shrink tumors and can provide long-lasting immunity, preventing tumor recurrence. The activated STING pathway also promotes an antiviral response by producing antiviral and proinflammatory cytokines that fight viruses and mobilize the innate and adaptive immune systems, ultimately producing long-lasting immunity against pathogenic viruses. The potential therapeutic benefits of enhancing the innate and adaptive immune responses make STING an attractive target for drug discovery. Cyclic dinucleotides may act as STING agonists and are being tested in clinical trials. However, their anionic nature makes them poorly membrane permeable, which may limit their ability to engage STING within cells, often resulting in undesirable distribution of these compounds in the bloodstream.

[0007] There remains a need for new STING agonists and improved methods of delivering them to targeted cells. Summary of the Invention

[0008] In a first aspect, the present invention provides a compound of formula (I):

[0009]

[0010] or a pharmaceutically acceptable salt thereof, wherein:

[0011] a is an integer from 1 to 20;

[0012] b is an integer from 1 to 20;

[0013] m is 0, 1, 2, 3, or 4;

[0014] n is 0 or 1;

[0015] D-NH- is part of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2;

[0016] Each R 1 independently selected from C1-C4 alkyl, O-C1-C4 alkyl and halogen;

[0017] R 2 Selected from C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10;

[0018] R 3 and R 3 ' are each independently selected from hydrogen and C1-C3 alkyl;

[0019] L is a cleavable linker; and

[0020] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0021] In a first embodiment of the first aspect, a is an integer from 1 to 4; b is an integer from 1 to 10; and m is 0.

[0022] In a second embodiment of the first aspect, a is an integer from 1 to 4; m is 0; n is 0; and R 3 and R 3 'Each is hydrogen.

[0023] In a third embodiment of the first aspect, L is

[0024]

[0025] in:

[0026] It is the point of attachment to the nitrogen atom;

[0027] It is the connection point with Ab;

[0028] t is an integer from 1 to 10;

[0029] W does not exist or is a self-immolative group;

[0030] Z is absent or is a peptide of 2 to 5 amino acids;

[0031] U and U' are independently absent or spacers; and

[0032] Q is a heterobifunctional group;

[0033] The condition is that W and Z do not exist at the same time.

[0034] In a fourth embodiment of the first aspect, W is a self-immolative group selected from:

[0035]

[0036]

[0037] in:

[0038] is the point of attachment to the carbonyl group; and

[0039] It is the connection point with Z.

[0040] In a fifth embodiment of the first aspect, W is selected from

[0041]

[0042] In a sixth embodiment of the first aspect, W is

[0043] In a seventh embodiment of the first aspect, Z is a peptide that can be enzymatically cleaved. In an eighth embodiment, Z is cleavable by a cathepsin. In a ninth embodiment, Z is a diamino acid peptide selected from the group consisting of Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys, and Lys-Phe. In a tenth embodiment of the first aspect, Z is Val-Ala or Ala-Val.

[0044] In an eleventh embodiment of the first aspect, U and U' are independently absent or selected from

[0045]

[0046] in:

[0047] is the connection point with Z;

[0048] It is the connection point with Q;

[0049] p is an integer from 1 to 6;

[0050] q is an integer from 1 to 20;

[0051] X is O or -CH2-; and

[0052] Each r is independently 0 or 1.

[0053] In a twelfth embodiment of the first aspect, U' is absent and U is

[0054]

[0055] In a thirteenth embodiment of the first aspect, Q is a heterobifunctional group capable of being linked to Ab via chemical or enzyme-mediated conjugation. In a fourteenth embodiment, Q is selected from

[0056]

[0057]

[0058] in

[0059] is a point of attachment to U, or, if U is absent, to Z; and

[0060] is the point of attachment to U', or when U' is absent, to Ab.

[0061] In a fifteenth embodiment of the first aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is:

[0062]

[0063] and wherein t is 1; W is absent or is a self-immolative group; and Z is absent or is a diamino acid peptide.

[0064] In a sixteenth embodiment of the first aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 2 is -CH3 or -(CH2CH2O) s -CH3 and s is an integer from 1 to 10.

[0065] In a seventeenth embodiment of the first aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D-NH2 is an amino-substituted compound that modulates STING activity. In an eighteenth embodiment of the first aspect, the amino-substituted compound that modulates STING activity comprises a guanine or adenine derivative.

[0066] In a nineteenth embodiment, the amino-substituted compound that modulates STING activity is a compound of formula (II):

[0067]

[0068] or a pharmaceutically acceptable salt thereof, wherein:

[0069] X 10 is SH or OH;

[0070] X 20 is SH or OH;

[0071] Y a is O, S or CH2;

[0072] Y b is O, S, NH, or NR a , where R a is a C1-C4 alkyl group;

[0073] R 10 It is hydrogen, fluorine, OH, NH2, OR b or NHR b ;

[0074] R 20 is hydrogen or fluorine;

[0075] R 30 is hydrogen; R 40 It is hydrogen, fluorine, OH, NH2, OR b or NHR b ; or R 30 and R 40 Together they form CH2O;

[0076] R 50 is hydrogen or fluorine;

[0077] R b is C1-C6 alkyl, halo(C1-C6)alkyl or C3-C6 cycloalkyl;

[0078] Ring A 10 is an optionally substituted 5-membered or 6-membered monocyclic heteroaryl ring containing 1-4 heteroatoms selected from N, O or S, or an optionally substituted 9-membered or 10-membered bicyclic heteroaryl ring containing 1-5 heteroatoms selected from N, O or S; wherein Ring A10 contains at least one N atom in the ring, and wherein Y b Connect to Ring A 10 of carbon atoms; and

[0079] Ring B 10 is an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 2 to 5 heteroatoms selected from N, O or S; wherein Ring B 10 Contains at least two N atoms in the ring;

[0080] The condition is ring A 10 or Ring B 10 The bond to the carbonyl group of formula (I) is via the -NH- group.

[0081] In a twentieth embodiment, the amino-substituted compound that modulates STING activity is

[0082]

[0083] or a pharmaceutically acceptable salt thereof, wherein is the point of attachment to the carbonyl group of formula (I).

[0084] In a twenty-first embodiment of the first aspect, the amino-substituted compound that modulates STING activity is a compound of formula (III):

[0085]

[0086] or a pharmaceutically acceptable salt thereof; wherein

[0087] X 10 is SH or OH;

[0088] X 20 is SH or OH;

[0089] Y c is O, S or CH2;

[0090] Y d is O, S or CH2;

[0091] B 100 It is given by the formula (B 1 -A) or formula (B 1 -B) represented by:

[0092]

[0093] R 13 、R 14 、R 15 、R 16 and R 17 are each independently a hydrogen atom or a substituent;

[0094] R 1000 is hydrogen or a bond to the carbonyl group of formula (I);

[0095] Y 11 、Y 12 、Y 13 、Y 14 、Y 15 and Y 16 Each independently is N or CR 1a , where R 1a is hydrogen or a substituent;

[0096] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 and Z 16 are each independently N or C;

[0097] R 105 is a hydrogen atom or a substituent;

[0098] B 200 It is given by the formula (B 2 -A) or formula (B 2 -B) represented by:

[0099]

[0100] R 23 、R 24 、R 25 、R 26 and R 27 are each independently a hydrogen atom or a substituent;

[0101] R 100 ' is hydrogen or a bond to the carbonyl group of formula (I);

[0102] Y 21 、Y 22 、Y 23 、Y 24 、Y 25 and Y 26 Each independently is N or CR 2a , where R 2a is hydrogen or a substituent;

[0103] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 and Z 26 are each independently N or C; and

[0104] R 205 is a hydrogen atom or a substituent; wherein R 105 and R 205 Each is independently attached to the 2-position or 3-position of the 5-membered ring to which it is attached;

[0105] The conditions are:

[0106] B 100 or B 200 One of them is connected to the carbonyl group of formula (I) via a -NH- group.

[0107] In a twenty-second embodiment of the first aspect, the amino-substituted compound that modulates STING activity is a compound of formula (IIIa),

[0108]

[0109] or a pharmaceutically acceptable salt thereof; wherein

[0110] B 100 It is given by the formula (B 1 -A) or formula (B 1 -B) represented by:

[0111]

[0112] R 13 、R 14 、R 15 、R 16 and R 17 are each independently a hydrogen atom or a substituent;

[0113] R 1000 is hydrogen or a bond to the carbonyl group of formula (I);

[0114] Y 11 、Y 12 、Y 13 、Y 14 、Y 15 and Y 16 Each independently is N or CR 1a , where R 1a is hydrogen or a substituent;

[0115] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 and Z 16 are each independently N or C;

[0116] R 105 is a hydrogen atom or a substituent;

[0117] B200 It is given by the formula (B 2 -A) or formula (B 2 -B) represented by:

[0118]

[0119] R 23 、R 24 、R 25 、R 26 and R 27 are each independently a hydrogen atom or a substituent;

[0120] R 100 ' is hydrogen or a bond to the carbonyl group of formula (I);

[0121] Y 21 、Y 22 、Y 23 、Y 24 、Y 25 and Y 26 Each independently is N or CR 2a , where R 2a is hydrogen or a substituent;

[0122] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 and Z 26 are each independently N or C; and

[0123] R 205 is a hydrogen atom or a substituent; wherein R 105 and R 205 Each is independently attached to the 2-position or 3-position of the 5-membered ring to which it is attached;

[0124] The conditions are:

[0125] B 100 or B 200 One of them is:

[0126]

[0127] in:

[0128] R 18 is hydrogen or C 1-6 alkyl; and

[0129] R 19 is a halogen atom;

[0130] And the other is linked to the carbonyl group of formula (I) via a -NH- group.

[0131] In a twenty-third embodiment of the first aspect, the amino-substituted compound that modulates STING activity is:

[0132]

[0133] or a pharmaceutically acceptable salt thereof, wherein is the point of attachment to the carbonyl group of formula (I).

[0134] In a second aspect, the present invention provides a compound of formula (IV):

[0135]

[0136] or a pharmaceutically acceptable salt thereof, wherein:

[0137] a is an integer from 1 to 20;

[0138] b is an integer from 1 to 20;

[0139] k is 0, 1, 2, or 3;

[0140] m is 0, 1, 2, 3, or 4;

[0141] D-NH- is part of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2;

[0142] R 2 Selected from H, C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10;

[0143] R 4 is selected from hydrogen and any naturally occurring amino acid side chain;

[0144] R 5 Selected from C1-C4 alkyl and O-C1-C4 alkyl;

[0145] L is a cleavable linker; and

[0146] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0147] In a first embodiment of the second aspect, L is

[0148]

[0149] in:

[0150] is the point of attachment to the carbonyl group;

[0151] It is the connection point with Ab;

[0152] W is a self-immolative group;

[0153] Z is absent or is a peptide of 2 to 5 amino acids; and

[0154] U and U' are independently absent or spacers; and

[0155] Q is a heterobifunctional group.

[0156] In a third aspect, the present invention provides a compound of formula (V):

[0157]

[0158] or a pharmaceutically acceptable salt thereof, wherein:

[0159] D-NH-Yes

[0160] X is O or CH2;

[0161] f is an integer from 1 to 10;

[0162] g is an integer from 1 to 20;

[0163] U and U' are independently absent or spacers;

[0164] Q is a heterobifunctional group; and

[0165] Ab is antibody, Ab is antibody, antibody fragment or antigen-binding fragment.

[0166] In a first embodiment of the third aspect, X is O.

[0167] In a fourth aspect, the present invention provides a compound of formula (VI):

[0168]

[0169] or a pharmaceutically acceptable salt thereof, wherein:

[0170] a is an integer from 1 to 20;

[0171] m is 0, 1, 2, 3, or 4;

[0172] n is 0 or 1;

[0173] D-NH- is part of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2;

[0174] Each R 1 independently selected from C1-C4 alkyl, O-C1-C4 alkyl and halogen;

[0175] R 2Selected from C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10;

[0176] R 3 and R 3 ' are each independently selected from hydrogen and C1-C3 alkyl;

[0177] L is a cleavable linker; and

[0178] LP is lipid.

[0179] In a first embodiment of the fourth aspect, the lipid is cholesterol or a phospholipid. In a second embodiment of the fourth aspect, the lipid is a phospholipid. In a third embodiment of the fourth aspect, the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean lecithin, and egg yolk lecithin. In a fifth embodiment of the fourth aspect, the phospholipid is phosphatidylethanolamine, wherein the phosphatidylethanolamine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0180] In a sixth embodiment of the fourth aspect, the present invention provides a lipoplex comprising a compound of formula (VI) or a pharmaceutically acceptable salt thereof. In a seventh embodiment, the lipoplex is in the form of a liposome.

[0181] In an eighth embodiment of the fourth aspect, the lipid complex further comprises one or more phospholipids. In a ninth embodiment, the one or more phospholipids are selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean lecithin, and egg yolk lecithin. In a tenth embodiment, the phospholipid is phosphatidylcholine, wherein the phosphatidylcholine is 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0182] In an eleventh embodiment of the fourth aspect, the lipid complex further comprises a fatty alcohol and / or cholesterol.

[0183] In a twelfth embodiment of the fourth aspect, the lipid complex further comprises at least one PEGylated lipid. In a thirteenth embodiment, the PEGylated lipid is selected from PEGylated phospholipids and PEGylated diacylglycerols. In a fourteenth embodiment, the PEGylated lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0184] In a fifth aspect, the present invention provides a pharmaceutical composition comprising a compound or complex as described herein or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

[0185] In a sixth aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable amount of a compound or complex described herein.

[0186] In a seventh aspect, the present invention provides a method of stimulating an immune response in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable amount of a compound or complex as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0187] Figure 1 Illustration of the preparation of Ab-STING agonist conjugates via random cysteine ​​conjugation.

[0188] Figure 2 Illustration of the preparation of Ab-STING agonist conjugates via transglutaminase conjugation.

[0189] Figure 3 Illustration of the preparation of Ab-STING agonist conjugates via transglutaminase conjugation.

[0190] Figure 4 Illustration of the preparation of Ab-STING agonist conjugates via sortase conjugation. (SEQ ID 3 and SEQ ID 4)

[0191] Figure 5 Depicted is the payload release of ADC-1 in a tritosomal assay.

[0192] Figure 6 Depicted is the payload release of ADC-3 in a decontaminated lysosome assay.

[0193] Figure 7 Depicted is the payload release of ADC-9 in a decontaminated lysosome assay.

[0194] Figure 8 Depicted are the plasma PK of ADC-1 in Balb / C mice bearing GCC-expressing CT26 (0.1 mg / kg payload dose).

[0195] Figure 9 Depicted are the plasma PK of ADC-8 in Balb / C mice bearing GCC-expressing CT26 (0.055 mg / kg payload dose, released CDN BLQ).

[0196] Figure 10 Depicted are the plasma PK of ADC-9 in Balb / C mice bearing GCC-expressing CT26 (0.05 mg / kg payload dose, released CDN BLQ).

[0197] Figure 11Depicted is the efficacy of ADC-1 in Balb / C mice bearing GCC-expressing CT26 (payload doses indicated).

[0198] Figure 12 Depicted is the efficacy of ADC-5 in Balb / C mice bearing GCC-expressing CT26 (payload dose indicated).

[0199] Figure 13 Depicted is the efficacy of ADC-8 in Balb / C mice bearing GCC-expressing CT26 (payload dose indicated).

[0200] Figure 14 Depicted is the efficacy of ADC-9 in Balb / C mice bearing GCC-expressing CT26 (payload dose indicated). DETAILED DESCRIPTION

[0201] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0202]

[0026] The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise.

[0203] Unless expressly indicated otherwise by terms such as "either," "unless," "alternatively," and words of similar effect, the term "or" as used herein is a logical disjunction (ie, and / or) and does not indicate an exclusive disjunction.

[0204] As used herein, the term "about" refers to ±10%.

[0205] Antibody-drug conjugates

[0206] In some embodiments, the present invention provides a compound of formula (I),

[0207]

[0208] or a pharmaceutically acceptable salt thereof, wherein:

[0209] a is an integer from 1 to 20;

[0210] b is an integer from 1 to 20;

[0211] m is 0, 1, 2, 3, or 4;

[0212] n is 0 or 1;

[0213] D-NH- is part of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2;

[0214] Each R 1 independently selected from C1-C4 alkyl, O-C1-C4 alkyl and halogen;

[0215] R 2 Selected from C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10;

[0216] R 3 and R 3 ' are each independently selected from hydrogen and C1-C3 alkyl;

[0217] L is a cleavable linker; and

[0218] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0219] In some embodiments, the present invention provides a compound of formula (IV),

[0220]

[0221] or a pharmaceutically acceptable salt thereof, wherein:

[0222] a is an integer from 1 to 20;

[0223] b is an integer from 1 to 20;

[0224] k is 0, 1, 2, or 3;

[0225] m is 0, 1, 2, 3, or 4;

[0226] D-NH- is part of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2;

[0227] R 2 Selected from H, C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10;

[0228] R 4 is selected from hydrogen and any naturally occurring amino acid side chain;

[0229] R 5 Selected from C1-C4 alkyl and O-C1-C4 alkyl;

[0230] L is a cleavable linker; and

[0231] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0232] In some embodiments, the present invention provides compounds of formula (V):

[0233]

[0234] or a pharmaceutically acceptable salt thereof, wherein:

[0235] D-NH is

[0236] X is O or CH2;

[0237] f is an integer from 1 to 10;

[0238] g is an integer from 1 to 20;

[0239] U and U' are independently absent or spacers;

[0240] Q is a heterobifunctional group; and

[0241] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0242] In the compounds of formula (I) and (IV), the amino-substituted compound D-NH2 can be any amino-containing drug. In certain embodiments, D-NH2 is an immunomodulator. As used herein, the term "immunomodulator" refers to a compound that produces an immune response in a subject in need thereof. Examples of immunomodulators include (but are not limited to) IDO inhibitors, MEK inhibitors, STING modulators, CCR4 antagonists, PD-1 / PD-L1 inhibitors, TLR7 / 8 agonists, etc. In some embodiments, D-NH2 is a STING modulator. In some embodiments, D-NH2 comprises a guanine or adenine derivative. In some embodiments, the STING modulator is a cyclic dinucleotide or a cyclic dinucleotide-like compound (each a CDN) of the following formula:

[0243]

[0244] wherein A' and A" are each independently a nucleoside or a synthetic analog thereof; and Q, Q', Q 2 and Q 2 Each of Q' is independently oxygen or sulfur. In some embodiments, Q' and Q 2Examples of CDNs (including cyclic dinucleotides and cyclic dinucleotide-like compounds) within the scope of the present invention include, but are not limited to, US 9,724,408, US 10,106,574, US 9,549,944, US 9,695,212, US 9,718,848, US CDNs disclosed in US Pat. Nos. 9,994,607, 10,047,115, US20180230178, US20180064745, US2018028132, US20180002369, US20180186828, US20190016750, US20180162899, US20180369268, US2018273578, US20180092937, WO2017 / 027646, and WO2018 / 100558; and compounds such as c-di-GMP, 2'3'-cGAMP, and SB 11285 (Spring Bank Pharmaceuticals).

[0245] In certain embodiments, the STING modulator is a compound of formula (II):

[0246]

[0247] or a pharmaceutically acceptable salt thereof, wherein:

[0248] X 10 is -SH or -OH;

[0249] X 20 is -SH or -OH;

[0250] Y a is -O-, -S- or -CH2-;

[0251] Y b Is -O-, -S-, -NH-, or -NR a -, where R a is a C1-C4 alkyl group;

[0252] R 10 It is hydrogen, fluorine, -OH, -NH2, -OR b or -NHR b ;

[0253] R 20 is hydrogen or fluorine;

[0254] R 30 is hydrogen; R 40 It is hydrogen, fluorine, -OH, -NH2, -OR b or -NHR b ; or R30 and R 40 Together they form -CH2O-;

[0255] R 50 is hydrogen or fluorine;

[0256] R b is C1-C6 alkyl, halo(C1-C6)alkyl or C3-C6 cycloalkyl;

[0257] Ring A 10 is an optionally substituted 5-membered or 6-membered monocyclic heteroaryl ring containing 1-4 heteroatoms selected from N, O or S, or an optionally substituted 9-membered or 10-membered bicyclic heteroaryl ring containing 1-5 heteroatoms selected from N, O or S; wherein Ring A 10 contains at least one N atom in the ring, and wherein Y b Connect to Ring A 10 of carbon atoms; and

[0258] Ring B 10 is an optionally substituted 9-membered or 10-membered bicyclic heteroaryl ring containing 2-5 heteroatoms selected from N, O or S; wherein Ring B 10 Contains at least two N atoms in the ring.

[0259] As described herein, Ring A 10 and Ring B 10 Can contain one or more substituents and thus can be optionally substituted. Suitable substituents on the unsaturated carbon atoms of the heteroaryl group include and are typically selected from -halo, -NO2, -CN, -R + 、-C(R + )=C(R + )2. -C≡CR + 、-OR + 、-SR o 、-S(O)R o 、-SO2R o 、-SO3R + 、-SO2N(R + )2、-N(R + )2、-NR + C(O)R + 、-NR + C(S)R + 、-NR + C(O)N(R + )2、-NR + C(S)N(R + )2、-N(R + )C(=NR + )-N(R + )2、-N(R+ )C(=NR + )-R o 、-NR + CO2R + 、-NR + SO2R o 、-NR + SO2N(R + )2、-OC(O)R + 、-O-CO2R + 、-OC(O)N(R + )2、-C(O)R + 、-C(S)R o 、-CO2R + 、-C(O)-C(O)R + 、-C(O)N(R + )2、-C(S)N(R + )2、-C(O)N(R + )-OR + 、-C(O)N(R + )C(=NR + )-N(R + )2、-N(R + )C(=NR + )-N(R + )-C(O)R + 、-C(=NR + )-N(R + )2、-C(=NR + )-OR + 、-N(R + )-N(R + )2、-C(=NR + )-N(R + )-OR + 、-C(R o )=N-OR + 、-P(O)(R + )2、-P(O)(OR + )2. -OP(O)-OR + and -P(O)(NR + )-N(R + )2, where R + is independently hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic or heterocyclyl, or two independent occurrences of R + Together with its intermediate atoms, it forms an optionally substituted 5-7 membered aryl, heteroaryl, cycloaliphatic or heterocyclic group. + are independently hydrogen, C 1-6Aliphatic group or C 3-6 Cycloaliphatic group. Each R o is independently an optionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic, or heterocyclyl.

[0260] As detailed above, in some embodiments, two independent occurrences of R + (or any other variable similarly defined in the specification and claims herein) together with its middle atoms form a monocyclic or bicyclic ring selected from: a 3-13 membered cycloaliphatic group, a 3-12 membered heterocyclyl group having 1-5 heteroatoms independently selected from nitrogen, oxygen or sulfur, a 6-10 membered aryl group, or a 5-10 membered heteroaryl group having 1-5 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0261] In some embodiments, the STING modulator is a compound of formula (IIA):

[0262]

[0263] or a pharmaceutically acceptable salt thereof, wherein R 10 and R 40 are each independently hydrogen, fluorine, -OH or -OCH2CF3 and Ring A 10 and B 10 is as defined for compounds of formula (II), provided that ring A 10 or Ring B 10 The carbonyl group of the parent molecular moiety of compound (I), (IV) or (V) is connected through the -NH- group.

[0264] In some embodiments, Ring A 10 is an optionally substituted 6-membered monocyclic heteroaryl ring containing 1, 2 or 3 nitrogen atoms.

[0265] In some embodiments, Ring B 10 yes:

[0266]

[0267] in:

[0268] Z 10 , Z 20 , Z 30 and Z 40 Each independently is N or CR 200 ;

[0269] R 210 is hydrogen or C1-C6 alkyl, halo(C1-C6)alkyl or C3-C6 cycloalkyl;

[0270] R 230 is hydrogen or -NH2; and

[0271] R 200 、R 220 and R 240 Each is independently hydrogen, halogen, -OH, -NH2, -CN, C1-C6 alkyl, halo(C1-C6)alkyl or C3-C6 cycloalkyl.

[0272] In some embodiments, the STING modulator is:

[0273]

[0274] or a pharmaceutically acceptable salt thereof, wherein It is the point of attachment to the carbonyl group of the parent molecular moiety.

[0275] In certain embodiments, the STING modulator is a cyclic dinucleotide of formula (X):

[0276]

[0277] or a pharmaceutically acceptable salt thereof, wherein:

[0278] Partial structure represented by formula (A-1):

[0279]

[0280] is a partial structure represented by formula (ZA) or formula (ZB):

[0281]

[0282] R 105 and R 205 are each independently a hydroxyl group or a halogen atom;

[0283] B 100 It is given by the formula (B 1 -A) or formula (B 1 -B) represented by:

[0284]

[0285] R 13 、R 14 、R 15 、R 16 and R 17 are each independently a hydrogen atom or a substituent; Y 11 、Y 12 、Y 13 、Y 14 、Y 15 and Y 16 Each independently is N or CR 1a ; Z11 , Z 12 , Z 13 , Z 14 , Z 15 and Z 16 are each independently N or C;

[0286] R 1a is a hydrogen atom or a substituent;

[0287] B 200 It is given by the formula (B 2 -A) or formula (B 2 -B) represented by:

[0288]

[0289] R 23 、R 24 、R 25 、R 26 and R 27 are each independently a hydrogen atom or a substituent; Y 21 、Y 22 、Y 23 、Y 24 、Y 25 and Y 26 Each independently is N or CR 2a ; Z 21 , Z 22 , Z 23 , Z 24 , Z 25 and Z 26 are each independently N or C;

[0290] R 2a is a hydrogen atom or a substituent;

[0291] X 1 and X 2 are each independently an oxygen atom or a sulfur atom; and

[0292] Q 1a , Q 2a , Q 3a and Q 4a are each independently an oxygen atom or a sulfur atom; and

[0293] B 100 or B 200 One of them is connected to the 5-membered ring of the parent structure via a -NH- group.

[0294] In another embodiment, B 100 or B 200 One of them is:

[0295]

[0296] in:

[0297] R 18 is hydrogen or C 1-6 alkyl; and the other is connected to the 5-membered ring of the parent structure via an -NH- group; and

[0298] R 19 is a halogen atom; and the other is linked to the carbonyl group of formula (I) via a -NH- group.

[0299] In another aspect, the present invention provides an antibody drug conjugate as described herein.

[0300] As described herein, certain R groups are selected from hydrogen and substituents. Examples of substituents include halogen atoms, cyano groups, nitro groups, optionally substituted hydrocarbon groups, optionally substituted heterocyclic groups, acyl groups, optionally substituted amino groups, optionally substituted carbamoyl groups, optionally substituted thiocarbamoyl groups, optionally substituted sulfamoyl groups, optionally substituted hydroxyl groups, optionally substituted sulfhydryl groups (SH), and optionally substituted silyl groups.

[0301] As used herein, in the present specification, examples of the “hydrocarbon group” (including the “hydrocarbon group” of “optionally substituted hydrocarbyl group”) include C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkenyl, C 6-14 Aryl and C 7-16 Aralkyl.

[0302] In the present specification, examples of the “optionally substituted hydrocarbon group” include hydrocarbon groups optionally having a substituent selected from the following Substituent Group A including:

[0303] (1) halogen atoms,

[0304] (2) nitro,

[0305] (3) cyano group,

[0306] (4) oxo group,

[0307] (5) hydroxyl groups,

[0308] (6) optionally halogenated C 1-6 Alkoxy,

[0309] (7)C 6-14 Aryloxy (e.g., phenoxy, naphthoxy),

[0310] (8)C7-16 aralkyloxy (e.g., benzyloxy),

[0311] (9) a 5- to 14-membered aromatic heterocyclic group (e.g., a pyridyloxy group),

[0312] (10) 3- to 14-membered non-aromatic heterocyclyloxy (e.g., morpholinyloxy, piperidinyloxy),

[0313] (11)C 1-6 alkyl-carbonyloxy (e.g., acetoxy, propionyloxy),

[0314] (12)C 6-14 aryl-carbonyloxy (e.g., benzoyloxy, 1-naphthoyloxy, 2-naphthoyloxy),

[0315] (13)C 1-6 Alkoxy-carbonyloxy (e.g., methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy),

[0316] (14) Single or Double C 1-6 alkyl-carbamoyloxy (e.g., methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy, diethylcarbamoyloxy),

[0317] (15)C 6-14 aryl-carbamoyloxy (e.g. phenylcarbamoyloxy, naphthylcarbamoyloxy),

[0318] (16) a 5- to 14-membered aromatic heterocyclic carbonyloxy group (e.g., a nicotinyloxy group),

[0319] (17) a 3- to 14-membered non-aromatic heterocyclic carbonyloxy group (e.g., morpholinylcarbonyloxy, piperidinylcarbonyloxy),

[0320] (18) optionally halogenated C 1-6 alkylsulfonyloxy (e.g., methylsulfonyloxy, trifluoromethylsulfonyloxy),

[0321] (19) optionally C 1-6 Alkyl substituted C 6-14 arylsulfonyloxy (e.g., phenylsulfonyloxy, toluenesulfonyloxy),

[0322] (20) optionally halogenated C 1-6 Alkylthio,

[0323] (21) a 5- to 14-membered aromatic heterocyclic group,

[0324] (22) a 3- to 14-membered non-aromatic heterocyclic group,

[0325] (23) formyl group,

[0326] (24) carboxyl group,

[0327] (25) optionally halogenated C 1-6 Alkyl-carbonyl,

[0328] (26)C 6-14 Aryl-carbonyl,

[0329] (27) a 5- to 14-membered aromatic heterocyclic carbonyl group,

[0330] (28) a 3- to 14-membered non-aromatic heterocyclic carbonyl group,

[0331] (29)C 1-6 Alkoxy-carbonyl,

[0332] (30)C 6-14 aryloxy-carbonyl (e.g. phenyloxycarbonyl, 1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl),

[0333] (31)C 7-16 Aralkyloxy-carbonyl (e.g., benzyloxycarbonyl, phenethyloxycarbonyl),

[0334] (32) carbamoyl,

[0335] (33) thiocarbamoyl,

[0336] (34) Single or Double C 1-6 Alkyl-carbamyl,

[0337] (35)C 6-14 aryl-carbamoyl (e.g. phenylcarbamoyl),

[0338] (36) a 5- to 14-membered aromatic heterocyclic carbamoyl group (e.g., a pyridylcarbamoyl group, a thienylcarbamoyl group),

[0339] (37) 3- to 14-membered non-aromatic heterocyclic carbamoyl (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl),

[0340] (38) optionally halogenated C 1-6 Alkylsulfonyl,

[0341] (39)C 6-14 Arylsulfonyl,

[0342] (40) a 5- to 14-membered aromatic heterocyclic sulfonyl group (e.g., a pyridylsulfonyl group, a thienylsulfonyl group),

[0343] (41) optionally halogenated C 1-6 Alkylsulfinyl,

[0344] (42)C 6-14 arylsulfinyl (e.g., phenylsulfinyl, 1-naphthylsulfinyl, 2-naphthylsulfinyl),

[0345] (43) a 5- to 14-membered aromatic heterocyclic sulfinyl group (e.g., a pyridylsulfinyl group, a thienylsulfinyl group),

[0346] (44) amino group,

[0347] (45) Single or Double C 1-6 Alkylamino (e.g., methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, N-ethyl-N-methylamino),

[0348] (46) Single or Double C 6-14 arylamino (e.g. phenylamino),

[0349] (47) a 5- to 14-membered aromatic heterocyclic amino group (e.g., a pyridylamino group),

[0350] (48)C 7-16 Aralkylamino (e.g., benzylamino),

[0351] (49) formylamino group,

[0352] (50)C 1-6 Alkyl-carbonylamino (e.g., acetylamino, propionylamino, butyrylamino),

[0353] (51)(C 1-6 Alkyl)(C 1-6 alkyl-carbonyl)amino (e.g. N-acetyl-N-methylamino),

[0354] (52)C 6-14 aryl-carbonylamino (e.g. phenylcarbonylamino, naphthylcarbonylamino),

[0355] (53)C 1-6 Alkoxy-carbonylamino (e.g., methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, tert-butoxycarbonylamino),

[0356] (54)C 7-16 aralkyloxy-carbonylamino (e.g., benzyloxycarbonylamino),

[0357] (55)C 1-6 Alkylsulfonylamino (e.g., methylsulfonylamino, ethylsulfonylamino),

[0358] (56) optionally C 1-6Alkyl substituted C 6-14 arylsulfonylamino (e.g., phenylsulfonylamino, toluenesulfonylamino),

[0359] (57) optionally halogenated C 1-6 alkyl,

[0360] (58)C 2-6 alkenyl,

[0361] (59)C 2-6 Alkynyl,

[0362] (60)C 3-10 Cycloalkyl,

[0363] (61)C 3-10 cycloalkenyl, and

[0364] (62)C 6-14 Aryl.

[0365] The number of the substituents mentioned above in the "optionally substituted hydrocarbon group" is, for example, 1 to 5, typically 1 to 3. When the number of the substituents is 2 or more, each substituent may be the same or different.

[0366] In the present specification, examples of the “heterocyclic group” (including the “heterocyclic group” of the “optionally substituted heterocyclic group”) include (i) an aromatic heterocyclic group, (ii) a non-aromatic heterocyclic group and (iii) a 7- to 10-membered bridged heterocyclic group each containing 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom and an oxygen atom as atoms constituting the ring other than carbon atoms.

[0367] In the present specification, examples of the “aromatic heterocyclic group” (including a “5- to 14-membered aromatic heterocyclic group”) include a 5- to 14-membered (typically 5- to 10-membered) aromatic heterocyclic group containing 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom as atoms constituting the ring other than carbon atoms.

[0368] Suitable examples of the “aromatic heterocyclic group” include 5-membered or 6-membered monocyclic aromatic heterocyclic groups such as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl and the like; and

[0369] 8- to 14-membered fused polycyclic (usually bicyclic or tricyclic) aromatic heterocyclic groups, for example, benzothienyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl, imidazopyrimidinyl, thienopyrimidinyl , furanopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl, phenoxathiyl, indolyl, isoindolyl, 1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, etc.

[0370] In the present specification, examples of the “non-aromatic heterocyclic group” (including a “3- to 14-membered non-aromatic heterocyclic group”) include a 3- to 14-membered (generally 4- to 10-membered) non-aromatic heterocyclic group containing 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom as atoms constituting the ring other than carbon atoms.

[0371] Suitable examples of the “non-aromatic heterocyclic group” include 3- to 8-membered monocyclic non-aromatic heterocyclic groups such as aziridinyl, oxiranyl, thirranyl, azetidinyl, oxetanyl, thietanyl, tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl , thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisoxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, azepanyl, oxepinyl, azocanyl, diazacyclocanyl and the like; and

[0372] 9- to 14-membered fused polycyclic (usually bicyclic or tricyclic) non-aromatic heterocyclic groups, for example, dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl, dihydrobenzothiazolyl, dihydrobenzisothiazolyl, dihydronaphtho[2,3-b]thienyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 4H-quinolizinyl, indolyl, isoindolyl, tetrahydrothieno[2,3-c]pyridinyl, tetrahydrobenzazepinyl, tetrahydroquinoxalinyl, tetrahydrophenanthridinyl, hexahydrophenothiazinyl, hexahydrophenoxazinyl, tetrahydrophthalazinyl, tetrahydronaphthyridinyl, tetrahydroquinazolinyl, tetrahydrocinnolinyl, tetrahydrocarbazolyl, tetrahydro-β-carbolinyl, tetrahydroacridinyl, tetrahydrophenazinyl, tetrahydrothioxanthenyl, octahydroisoquinolinyl, and the like.

[0373] In the present specification, suitable examples of the "7- to 10-membered bridged heterocyclic group" include quinuclidinyl and 7-azabicyclo[2.2.1]heptyl.

[0374] In the present specification, examples of the “nitrogen-containing heterocyclic group” include a “heterocyclic group” containing at least one nitrogen atom as an atom constituting a ring.

[0375] In the present specification, examples of the “optionally substituted heterocyclic group” include a heterocyclic group optionally having a substituent selected from the above-mentioned Substituent Group A.

[0376] The number of substituents in the "optionally substituted heterocyclic group" is, for example, 1 to 3. When the number of substituents is 2 or more, each substituent may be the same or different.

[0377] In the present specification, examples of the "acyl group" include formyl, carboxyl, carbamoyl, thiocarbamoyl, sulfinyl, sulfo, sulfamoyl and phosphono groups, each of which optionally has 1 or 2 groups selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkenyl, C 6-14 Aryl, C 7-16 arylalkyl, 5- to 14-membered aromatic heterocyclic group and 3- to 14-membered non-aromatic heterocyclic group, each of which optionally has 1 to 3 substituents selected from halogen atoms, optionally halogenated C 1-6 Substituents of alkoxy, hydroxy, nitro, cyano, amino and carbamoyl groups.

[0378] Examples of the "acyl group" also include a hydrocarbon-sulfonyl group, a heterocyclylsulfonyl group, a hydrocarbon-sulfinyl group, and a heterocyclylsulfinyl group.

[0379] Here, the hydrocarbon-sulfonyl group means a hydrocarbon-bonded sulfonyl group, the heterocyclylsulfonyl group means a heterocyclyl-bonded sulfonyl group, the hydrocarbon-sulfinyl group means a hydrocarbon-bonded sulfinyl group, and the heterocyclylsulfinyl group means a heterocyclyl-bonded sulfinyl group.

[0380] Suitable examples of "acyl" include formyl, carboxyl, C 1-6 Alkyl-carbonyl, C 2-6 Alkenyl-carbonyl (e.g. crotonyl), C 3-10 Cycloalkyl-carbonyl (e.g., cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl), C 3-10 Cycloalkenyl-carbonyl (e.g. 2-cyclohexenecarbonyl), C 6-14 Aryl-carbonyl, C 7-16 Aralkyl-carbonyl, 5- to 14-membered aromatic heterocyclylcarbonyl, 3- to 14-membered non-aromatic heterocyclylcarbonyl, C 1-6 Alkoxy-carbonyl, C6-14 Aryloxy-carbonyl (e.g., phenyloxycarbonyl, naphthyloxycarbonyl), C 7-16 Aralkyloxy-carbonyl (e.g., benzyloxycarbonyl, phenethyloxycarbonyl), carbamoyl, mono- or di-C 1-6 Alkyl-carbamoyl, mono- or di-C 2-6 Alkenyl-carbamoyl (e.g. diallylcarbamoyl), mono- or di-C 3-10 Cycloalkyl-carbamoyl (e.g. cyclopropylcarbamoyl), mono- or di-C 6-14 Aryl-carbamoyl (e.g. phenylcarbamoyl), mono- or di-C 7-16 Aralkyl-carbamoyl, 5- to 14-membered aromatic heterocyclic carbamoyl (e.g., pyridylcarbamoyl), thiocarbamoyl, mono- or di-C 1-6 Alkyl-thiocarbamoyl (e.g., methylthiocarbamoyl, N-ethyl-N-methylthiocarbamoyl), mono- or di-C 2-6 Alkenyl-thiocarbamoyl (e.g. diallylthiocarbamoyl), mono- or di-C 3-10 Cycloalkyl-thiocarbamoyl (e.g., cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), mono- or di-C 6-14 Aryl-thiocarbamoyl (e.g. phenylthiocarbamoyl), mono- or di-C 7-16 Aralkyl-thiocarbamoyl (e.g., benzylthiocarbamoyl, phenethylthiocarbamoyl), 5- to 14-membered aromatic heterocyclic thiocarbamoyl (e.g., pyridylthiocarbamoyl), sulfinyl, C 1-6 Alkylsulfinyl (e.g., methylsulfinyl, ethylsulfinyl), sulfo, C 1-6 Alkylsulfonyl, C 6-14 Arylsulfonyl, phosphonyl and mono- or di-C 1-6 Alkylphosphono (eg, dimethylphosphono, diethylphosphono, diisopropylphosphono, dibutylphosphono).

[0381] In the present specification, examples of the "optionally substituted amino group" include amino groups optionally having 1 or 2 groups selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 3-10 Cycloalkyl, C 6-14 Aryl, C 7-16 Aralkyl, C 1-6 Alkyl-carbonyl, C 6-14 Aryl-carbonyl, C 7-16 Aralkyl-carbonyl, 5- to 14-membered aromatic heterocyclylcarbonyl, 3- to 14-membered non-aromatic heterocyclylcarbonyl, C 1-6 Alkoxy-carbonyl, 5- to 14-membered aromatic heterocyclic group, carbamoyl, mono- or di-C 1-6Alkyl-carbamoyl, mono- or di-C 7-16 Aralkyl-carbamoyl, C 1-6 Alkylsulfonyl and C 6-14 The amino group of the substituent of the arylsulfonyl group each optionally has 1 to 3 substituents selected from Substituent Group A.

[0382] Suitable examples of optionally substituted amino groups include amino, mono- or di-(optionally halogenated C 1-6 alkyl)amino (e.g., methylamino, trifluoromethylamino, dimethylamino, ethylamino, diethylamino, propylamino, dibutylamino), mono- or di-C 2-6 Alkenylamino (e.g. diallylamino), mono- or di-C 3-10 Cycloalkylamino (e.g. cyclopropylamino, cyclohexylamino), mono- or di-C 6-14 Arylamino (e.g. phenylamino), mono- or di-C 7-16 Aralkylamino (eg benzylamino, dibenzylamino), mono- or di- (optionally halogenated C 1-6 Alkyl)-carbonylamino (e.g. acetylamino, propionylamino), mono- or di-C 6-14 Aryl-carbonylamino (e.g. benzoylamino), mono- or di-C 7-16 Aralkyl-carbonylamino (e.g., benzylcarbonylamino), mono- or di-5- to 14-membered aromatic heterocyclic carbonylamino (e.g., nicotinylamino, isonicotinylamino), mono- or di-3- to 14-membered non-aromatic heterocyclic carbonylamino (e.g., piperidinylcarbonylamino), mono- or di-C 1-6 Alkoxy-carbonylamino (e.g., tert-butoxycarbonylamino), 5- to 14-membered aromatic heterocyclic amino (e.g., pyridylamino), carbamoylamino, (mono- or di-C 1-6 Alkyl-carbamoyl)amino (e.g. methylcarbamoylamino), (mono- or di-C 7-16 Aralkyl-carbamoyl)amino (e.g., benzylcarbamoylamino), C 1-6 Alkylsulfonylamino (e.g., methylsulfonylamino, ethylsulfonylamino), C 6-14 Arylsulfonylamino (e.g., phenylsulfonylamino), (C 1-6 Alkyl)(C 1-6 alkyl-carbonyl)amino (e.g. N-acetyl-N-methylamino) and (C 1-6 Alkyl)(C 6-14 aryl-carbonyl)amino (e.g., N-benzoyl-N-methylamino).

[0383] In the present specification, examples of the "optionally substituted carbamoyl group" include optionally having 1 or 2 groups selected from C 1-6 Alkyl, C2-6 Alkenyl, C 3-10 Cycloalkyl, C 6-14 Aryl, C 7-16 Aralkyl, C 1-6 Alkyl-carbonyl, C 6-14 Aryl-carbonyl, C 7-16 Aralkyl-carbonyl, 5- to 14-membered aromatic heterocyclylcarbonyl, 3- to 14-membered non-aromatic heterocyclylcarbonyl, C 1-6 Alkoxy-carbonyl, 5- to 14-membered aromatic heterocyclic group, carbamoyl, mono- or di-C 1-6 Alkyl-carbamoyl and mono- or di-C 7-16 The carbamoyl group of the substituent of the aralkyl-carbamoyl group each optionally has 1 to 3 substituents selected from Substituent Group A.

[0384] Suitable examples of optionally substituted carbamoyl groups include carbamoyl, mono- or di-C 1-6 Alkyl-carbamoyl, mono- or di-C 2-6 Alkenyl-carbamoyl (e.g. diallylcarbamoyl), mono- or di-C 3-10 Cycloalkyl-carbamoyl (e.g. cyclopropylcarbamoyl, cyclohexylcarbamoyl), mono- or di-C 6-14 Aryl-carbamoyl (e.g. phenylcarbamoyl), mono- or di-C 7-16 Aralkyl-carbamoyl, mono- or di-C 1-6 Alkyl-carbonyl-carbamoyl (e.g., acetylcarbamoyl, propionylcarbamoyl), mono- or di-C 6-14 Aryl-carbonyl-carbamoyl (e.g., benzoylcarbamoyl) and 5- to 14-membered aromatic heterocyclylcarbamoyl (e.g., pyridylcarbamoyl).

[0385] In the present specification, examples of the "optionally substituted thiocarbamoyl group" include optionally having 1 or 2 groups selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 3-10 Cycloalkyl, C 6-14 Aryl, C 7-16 Aralkyl, C 1-6 Alkyl-carbonyl, C 6-14 Aryl-carbonyl, C 7-16 Aralkyl-carbonyl, 5- to 14-membered aromatic heterocyclylcarbonyl, 3- to 14-membered non-aromatic heterocyclylcarbonyl, C 1-6 Alkoxy-carbonyl, 5- to 14-membered aromatic heterocyclic group, carbamoyl, mono- or di-C 1-6 Alkyl-carbamoyl and mono- or di-C 7-16 The thiocarbamoyl group of the substituent of the aralkyl-carbamoyl group each optionally has 1 to 3 substituents selected from Substituent Group A.

[0386] Suitable examples of optionally substituted thiocarbamyl groups include thiocarbamyl, mono- or di-C 1-6 Alkyl-thiocarbamoyl (e.g., methylthiocarbamoyl, ethylthiocarbamoyl, dimethylthiocarbamoyl, diethylthiocarbamoyl, N-ethyl-N-methylthiocarbamoyl), mono- or di-C 2-6 Alkenyl-thiocarbamoyl (e.g. diallylthiocarbamoyl), mono- or di-C 3-10 Cycloalkyl-thiocarbamoyl (e.g., cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), mono- or di-C 6-14 Aryl-thiocarbamoyl (e.g. phenylthiocarbamoyl), mono- or di-C 7-16 Aralkyl-thiocarbamoyl (e.g., benzylthiocarbamoyl, phenethylthiocarbamoyl), mono- or di-C 1-6 Alkyl-carbonyl-thiocarbamoyl (e.g., acetylthiocarbamoyl, propionylthiocarbamoyl), mono- or di-C 6-14 Aryl-carbonyl-thiocarbamoyl (e.g., benzoylthiocarbamoyl) and 5- to 14-membered aromatic heterocyclylthiocarbamoyl (e.g., pyridylthiocarbamoyl).

[0387] In the present specification, examples of the "optionally substituted sulfamoyl group" include optionally having 1 or 2 groups selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 3-10 Cycloalkyl, C 6-14 Aryl, C 7-16 Aralkyl, C 1-6 Alkyl-carbonyl, C 6-14 Aryl-carbonyl, C 7-16 Aralkyl-carbonyl, 5- to 14-membered aromatic heterocyclylcarbonyl, 3- to 14-membered non-aromatic heterocyclylcarbonyl, C 1-6 Alkoxy-carbonyl, 5- to 14-membered aromatic heterocyclic group, carbamoyl, mono- or di-C 1-6 Alkyl-carbamoyl and mono- or di-C 7-16 The sulfamoyl group of the substituent of the aralkyl-carbamoyl group each optionally has 1 to 3 substituents selected from Substituent Group A.

[0388] Suitable examples of optionally substituted sulfamoyl groups include sulfamoyl, mono- or di-C 1-6 Alkyl-sulfamoyl (e.g., methylsulfamoyl, ethylsulfamoyl, dimethylsulfamoyl, diethylsulfamoyl, N-ethyl-N-methylsulfamoyl), mono- or di-C 2-6 Alkenyl-sulfamoyl (e.g. diallylsulfamoyl), mono- or di-C3-10 Cycloalkyl-sulfamoyl (e.g., cyclopropylsulfamoyl, cyclohexylsulfamoyl), mono- or di-C 6-14 Aryl-sulfamoyl (e.g. phenylsulfamoyl), mono- or di-C 7-16 Aralkyl-sulfamoyl (e.g., benzylsulfamoyl, phenethylsulfamoyl), mono- or di-C 1-6 Alkyl-carbonyl-sulfamoyl (e.g., acetylsulfamoyl, propionylsulfamoyl), mono- or di-C 6-14 Aryl-carbonyl-sulfamoyl (e.g., benzoylsulfamoyl) and 5- to 14-membered aromatic heterocyclylsulfamoyl (e.g., pyridylsulfamoyl).

[0389] In the present specification, examples of the "optionally substituted hydroxyl group" include a group optionally having a C 1-6 Alkyl, C 2-6 Alkenyl, C 3-10 Cycloalkyl, C 6-14 Aryl, C 7-16 Aralkyl, C 1-6 Alkyl-carbonyl, C 6-14 Aryl-carbonyl, C 7-16 Aralkyl-carbonyl, 5- to 14-membered aromatic heterocyclylcarbonyl, 3- to 14-membered non-aromatic heterocyclylcarbonyl, C 1-6 Alkoxy-carbonyl, 5- to 14-membered aromatic heterocyclic group, carbamoyl, mono- or di-C 1-6 Alkyl-carbamoyl, mono- or di-C 7-16 Aralkyl-carbamoyl, C 1-6 Alkylsulfonyl and C 6-14 The hydroxyl group of the substituent of the arylsulfonyl group each optionally has 1 to 3 substituents selected from Substituent Group A.

[0390] Suitable examples of optionally substituted hydroxy groups include hydroxy, C 1-6 Alkoxy, C 2-6 alkenyloxy (e.g., allyloxy, 2-butenyloxy, 2-pentenyloxy, 3-hexenyloxy), C 3-10 Cycloalkyloxy (e.g., cyclohexyloxy), C 6-14 Aryloxy (e.g. phenoxy, naphthyloxy), C 7-16 Aralkyloxy (e.g., benzyloxy, phenethyloxy), C 1-6 Alkyl-carbonyloxy (e.g., acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, valeryloxy), C 6-14 Aryl-carbonyloxy (e.g., benzoyloxy), C 7-16Aralkyl-carbonyloxy (e.g., benzylcarbonyloxy), 5- to 14-membered aromatic heterocyclylcarbonyloxy (e.g., nicotinyloxy), 3- to 14-membered non-aromatic heterocyclylcarbonyloxy (e.g., piperidinylcarbonyloxy), C 1-6 Alkoxy-carbonyloxy (e.g., tert-butoxycarbonyloxy), 5- to 14-membered aromatic heterocyclyloxy (e.g., pyridyloxy), carbamoyloxy, C 1-6 Alkyl-carbamoyloxy (e.g., methylcarbamoyloxy), C 7-16 Aralkyl-carbamoyloxy (e.g., benzylcarbamoyloxy), C 1-6 Alkylsulfonyloxy (e.g., methylsulfonyloxy, ethylsulfonyloxy) and C 6-14 Arylsulfonyloxy (eg, phenylsulfonyloxy).

[0391] In the present specification, examples of the "optionally substituted sulfhydryl group" include a group optionally having a C 1-6 Alkyl, C 2-6 Alkenyl, C 3-10 Cycloalkyl, C 6-14 Aryl, C 7-16 Aralkyl, C 1-6 Alkyl-carbonyl, C 6-14 A thiohydride group of a substituent of an aryl-carbonyl group and a 5- to 14-membered aromatic heterocyclic group, each of which optionally has 1 to 3 substituents selected from Substituent Group A, and a halogenated thiohydride group.

[0392] Suitable examples of optionally substituted sulfhydryl groups include sulfhydryl (-SH), C 1-6 Alkylthio, C 2-6 alkenylthio (e.g., allylthio, 2-butenylthio, 2-pentenylthio, 3-hexenylthio), C 3-10 Cycloalkylthio (e.g., cyclohexylthio), C 6-14 Arylthio (e.g., phenylthio, naphthylthio), C 7-16 Aralkylthio (e.g., benzylthio, phenethylthio), C 1-6 Alkyl-carbonylthio (e.g., acetylthio, propionylthio, butyrylthio, isobutyrylthio, valerylthio), C 6-14 an aryl-carbonylthio group (eg, benzoylthio), a 5- to 14-membered aromatic heterocyclic thio group (eg, pyridylthio), and a halogenated thio group (eg, pentafluorothio).

[0393] In the present specification, examples of the "optionally substituted silyl group" include silyl groups optionally having 1 to 3 groups selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 3-10 Cycloalkyl, C 6-14 Aryl and C7-16 A silyl group as a substituent of the aralkyl group, each of which optionally has 1 to 3 substituents selected from Substituent Group A. Examples of the optionally substituted silyl group include tri-C 1-6 Alkylsilyl (eg, trimethylsilyl, tert-butyl(dimethyl)silyl).

[0394] In some embodiments, the STING modulator is a compound of formula (III):

[0395]

[0396] or a pharmaceutically acceptable salt thereof, wherein:

[0397] R 105 and R 205 are each independently a hydroxyl group or a halogen atom;

[0398] B 100 It is given by the formula (B 1 -A) or formula (B 1 -B) represented by:

[0399]

[0400] R 13 、R 14 、R 15 、R 16 and R 17 are each independently a hydrogen atom or a substituent; R 1000 is hydrogen or a bond to the carbonyl group of formula (I);

[0401] Y 11 、Y 12 、Y 13 、Y 14 、Y 15 and Y 16 Each independently is N or CR 1a ; Z 11 , Z 12 , Z 13 , Z 14 , Z 15 and Z 16 are each independently N or C;

[0402] R 105 and R 205 are each independently a hydroxyl group or a halogen atom;

[0403] R 1a is a hydrogen atom or a substituent;

[0404] B 200 It is given by the formula (B 2-A) or formula (B 2 -B) represented by:

[0405]

[0406] R 23 、R 24 、R 25 、R 26 and R 27 are each independently a hydrogen atom or a substituent; R 100 ' is hydrogen or a bond to the carbonyl group of formula (I);

[0407] Y 21 、Y 22 、Y 23 、Y 24 、Y 25 and Y 26 Each independently is N or CR 2a ; Z 21 , Z 22 , Z 23 , Z 24 , Z 25 and Z 26 are each independently N or C;

[0408] R 2a is a hydrogen atom or a substituent;

[0409] X 1 and X 2 are each independently an oxygen atom or a sulfur atom; and Q 1 , Q 2 , Q3 and Q 4 are each independently an oxygen atom or a sulfur atom.

[0410] The conditions are:

[0411] B 100 or B 200 One of them is:

[0412]

[0413] in:

[0414] R 18 is hydrogen or C 1-6 alkyl; and

[0415] R 19 is a halogen atom, and B 100 or B 200 The other of the two is connected to the 5-membered ring of the parent structure via a -NH- group.

[0416] In some embodiments, the STING modulator is a compound of formula (III) or a pharmaceutically acceptable salt thereof, wherein R 205 It is F and B 100 yes

[0417]

[0418] In some embodiments, the cyclic dinucleotide is:

[0419]

[0420] or a pharmaceutically acceptable salt thereof, wherein It is the point of attachment to the carbonyl group of the parent molecular moiety.

[0421] The group "L" is a cleavable linker. As used herein, the term "linker" refers to any chemical moiety capable of connecting an antibody, antibody fragment, or antigen-binding fragment (Ab) to a drug-containing moiety within a compound of formula (I) and (IV). The linker may be branched and may be substituted with 1 to 20 drug-containing moieties. In some embodiments, the linker may be substituted with 1 to 10 drug-containing moieties. In some embodiments, the linker may be substituted with 1 to 5 drug-containing moieties. In some embodiments, the linker may be substituted with one or two drug-containing moieties. In some embodiments, the linker may be substituted with one drug-containing moiety.

[0422] The linker "L" is cleavable. In certain embodiments, under conditions where the drug and / or antibody can remain active, the linker may be susceptible to acid-induced cleavage, light-induced cleavage, enzymatic cleavage, and the like. In some embodiments, the cleavable linker may be enzymatically cleaved. In some embodiments, the cleavable linker may be cleaved by a protease, a peptidase, an esterase, a glycosidase, a phosphodiesterase, a phosphatase, or a lipase. In some embodiments, the cleavable linker may be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like. In certain embodiments, the linker may be any of those disclosed in PCT Publications WO 2018 / 200812 and WO 2018 / 100558, which are incorporated herein by reference in their entirety.

[0423] In certain embodiments, "L" has the formula:

[0424]

[0425] in:

[0426] is the point of attachment to the nitrogen atom; and

[0427] It is the connection point with Ab.

[0428] In some embodiments, "L" has the formula:

[0429]

[0430] in:

[0431] It is the point of attachment to the nitrogen atom;

[0432] It is the point of attachment to the antibody;

[0433] The group "W" is absent or is a self-immolative group. As used herein, the term "self-immolative" refers to a group that undergoes an electron cascade to release the group to which it is attached. In some embodiments, the self-immolative group includes one or more groups that can undergo 1,4-elimination, 1,6-elimination, 1,8-elimination, 1,6-cyclization elimination, 1,5-cyclization elimination, 1,3-cyclization elimination, intramolecular 5-exo-trig cyclization and / or 6-exo-trig cyclization. In certain embodiments, the self-immolative group can be any of those disclosed in PCT Publications WO 2018 / 200812, WO 2018 / 100558, which are incorporated herein by reference in their entirety.

[0434] The group "Z" is absent or is a peptide of 2 to 5 amino acids. In certain embodiments, the peptide is a cleavage site for the linker, thereby promoting drug release upon exposure to intracellular proteases (e.g., lysosomal enzymes) (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Examples of peptides with two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides with three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The above amino acid combinations can also be present in the reverse order (i.e., cit-val).

[0435] The peptides of the present invention may comprise naturally occurring and / or non-natural amino acid residues. The term "naturally occurring amino acids" refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. As non-limiting examples, "non-natural amino acids" (i.e., amino acids that do not occur naturally) include homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, seleno-cysteine, norleucine ("Nle"), norvaline ("Nva"), β-alanine, L- or D-naphthylalanine, ornithine ("Orn"), and the like. Peptides can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin.

[0436] Amino acids also include the D- forms of natural and non-natural amino acids. "D-" refers to an amino acid that has a "D" (dextrorotatory) configuration that is different from the configuration of naturally occurring ("L-") amino acids. Natural and non-natural amino acids can be purchased (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0437] The groups "U" and "U'" are independently absent or spacers. The term "spacer" as used herein refers to a chemical moiety used as a linker. In the present invention, the spacer can connect the antibody, antibody fragment or antigen fragment to the heterobifunctional group and / or connect the heterobifunctional group to the peptide "Z", or to the group "W" when "Z" is absent. Non-limiting exemplary spacers include -NH-, -S-, -O-, -NHC(=O)CH2CH2-, -S(=O)2-CH2CH2-, -C(=O)NHNH-, -C(=O)O-, -C(=O)NH-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2=CH2-, -C≡C-, -CH=NO-, polyethylene glycol (PEG),

[0438]

[0439] In the compounds of the present invention, when "U" is present, it may be a branched group substituted with 1 to 10 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 to 5 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 or 2 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 "-C(O)-WZ-" group. In certain embodiments, the spacer may be any of those disclosed in PCT Publications WO 2018 / 200812, WO 2018 / 100558, which are incorporated herein by reference in their entirety.

[0440] The group "Q" is a heterobifunctional group. In the present invention, the term "heterobifunctional group" refers to a chemical portion of which a linker of which it is a part is connected to an antibody, antibody fragment or antigen binding fragment. See, for example, WO2017 / 191579. A heterobifunctional group is characterized by having different reactive groups at either end of the chemical portion. A heterobifunctional group can be directly connected to "Ab", or alternatively can be connected via a linker "U'". Connection to "Ab" can be achieved via chemical or enzymatic conjugation or a combination of the two. Chemical conjugation involves a controlled reaction of accessible amino acid residues on the antibody surface with a reaction handle on "Q" or "U'". Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via non-natural amino acids incorporated by genetic engineering, wherein non-natural amino acid residues with desired reaction handles are installed on "Ab". In enzymatic conjugation, an enzyme mediates the coupling of a linker to accessible amino residues on an antibody, antibody fragment or antigen binding fragment. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation can also be used sequentially. For example, enzymatic conjugation can also be used to install a unique reaction handle on an "Ab" to be used for subsequent chemical conjugation. In certain embodiments, the heterobifunctional group may be any of those disclosed in PCT Publications WO 2018 / 200812 and WO 2018 / 100558, which are incorporated herein by reference in their entirety.

[0441] In some embodiments, "Q" is selected from

[0442] in

[0443] is a point of attachment to U, or, if U is absent, to Z; and

[0444] is the point of attachment to U', or when U' is absent, to Ab.

[0445] In certain embodiments, the present invention provides compounds of formula (XX):

[0446]

[0447] or a pharmaceutically acceptable salt thereof, wherein n, m, a, t, D-NH-, R 1 、R 2 、R 3 、R 3 ', W, Z and U are as described herein and wherein Q* is a reactive functional group capable of being conjugated to an antibody, antibody fragment or antigen-binding fragment. Examples of suitable Q* groups include, but are not limited to, activated carboxylic acid groups such as acyl chlorides -C(O)-Cl and anhydrides, haloacetamides, maleimides, alkynes, cycloalkynes (e.g., cyclooctyne), oxa-norbornadiene, norbornene, azide, diaryl tetrazine, monoaryl tetrazine, aldehyde, ketone, hydroxylamine, vinyl sulfone and aziridine. In certain embodiments, the reactive functional group may be any of those disclosed in PCT Publications WO 2018 / 200812 and WO 2018 / 100558, which are incorporated herein by reference in their entirety.

[0448] The group "Ab" is an antibody, antibody fragment or antigen binding fragment. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens typically have multiple binding sites, also referred to as epitopes, that are recognized by the CDRs on a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody. The term "antibody" is used in the broadest sense herein and specifically encompasses monoclonal antibodies, single domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric or derived from other species. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th edition, Garland Publishing, New York).

[0449] As used herein, the term "antibody" also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. The immunoglobulin can be derived from any species. However, in one aspect, the immunoglobulin is of human, murine, or rabbit origin.

[0450] The term "single domain antibody" (also known as nanobody) is an antibody fragment consisting of a single monomeric variable antibody domain with a molecular weight of about 12 kDa to about 15 kDa. Monomeric antibodies can be based on either a heavy chain variable domain or a light chain. Examples of single domain antibodies include, but are not limited to, V H H fragment and V NAR Fragments. See, for example, Harmsen MM et al., Applied Microbiology and Biotechnology 77(1):13-22.

[0451] "Antibody fragments" comprise a portion of an intact antibody, typically the antigen-binding region or its variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity-determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0452] An "intact antibody" is an antibody comprising an antigen-binding variable region and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0453] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homologous antibody population, i.e., the individual antibodies constituting the population are identical except for possible natural mutations, which may exist in small amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, unlike polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates that the characteristic of an antibody is obtained from a substantially homologous antibody population and should not be construed as requiring antibody production by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be obtained by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can be obtained by recombinant DNA methods (see U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597.

[0454] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, great apes, etc.) and human constant region sequences.

[0455] A variety of methods have been used to produce monoclonal antibodies (MAbs). Hybridoma technology refers to a clonal cell line that produces a single type of antibody, using cells from different species, including mice (murine), hamsters, rats, and humans. Another method for preparing MAbs uses genetic engineering, including recombinant DNA technology. Monoclonal antibodies made from these technologies include chimeric antibodies and humanized antibodies. Chimeric antibodies combine DNA coding regions from more than one type of species. For example, chimeric antibodies can obtain variable regions from mice and constant regions from humans. Humanized antibodies are mainly from humans, even if they contain non-human parts. Like chimeric antibodies, humanized antibodies can contain complete human constant regions. But unlike chimeric antibodies, variable regions can be partially derived from humans. The non-human synthetic parts of humanized antibodies are usually derived from CDRs in murine antibodies. In any case, these regions are crucial for making antibodies recognize and bind to specific antigens. Although they can be used for diagnostics and short-term therapies, murine antibodies cannot be administered to people for a long time without increasing the risk of harmful immunogenic reactions. This reaction, called human anti-mouse antibodies (HAMA), occurs when the human immune system recognizes mouse antibodies as foreign and attacks them. A HAMA reaction can cause toxic shock or even death.

[0456] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portion of the administered antibody. Additionally, chimeric and humanized antibodies may have the added benefit of activating secondary human immune responses such as antibody-dependent cellular cytotoxicity.

[0457] Intact antibodies may possess one or more "effector functions," which are those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

[0458] Intact antibodies can be assigned to different "classes" depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these antibodies can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0459] Useful non-immunoreactive protein, polypeptide or peptide antibodies include, but are not limited to, transferrin, epidermal growth factor ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factor ("TGF") (e.g., TGF-.α. and TGF-.β.), vaccinia growth factor ("VGF"), insulin and insulin-like growth factors I and II, lectins, and prosthetic group-deficient proteins from low-density lipoprotein.

[0460] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Various procedures well known in the art can be used to generate polyclonal antibodies against the target antigen. For example, to generate polyclonal antibodies, various host animals, including but not limited to rabbits, mice, rats, and guinea pigs, can be immunized by injection of the target antigen or its derivatives. Depending on the host species, various adjuvants can be used to increase the immune response, including but not limited to Freund's adjuvant (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin), pluronic polyols, polyanions, peptides, oily emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacillus Calmette-Guérin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

[0461] Useful monoclonal antibodies are homogeneous populations of antibodies directed against specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) directed against target antigens can be prepared using any technique known in the art that provides for the production of antibody molecules through continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), human B cell hybridoma technology (Kozbor et al., 1983, Immunology Today 4: 72), and EBV hybridoma technology (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas producing the mAbs used in the present invention can be cultured in vitro or in vivo.

[0462] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be prepared by any of a variety of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

[0463] Antibodies can also be bispecific antibodies. Methods for preparing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Due to the random grouping of immunoglobulin heavy chains and light chains, these hybridomas (quadroma) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Affinity chromatography steps are usually used to purify appropriate molecules, which is quite troublesome and has low product yields. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655-3659 (1991).

[0464] According to different approaches, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion can be performed using immunoglobulin heavy chain constant domains, including the hinge region, C H2 Area and C H3 At least a portion of the first heavy chain constant region (C H1 ) may contain a site required for light chain binding, present in at least one fusion. Nucleic acids having sequences encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments, when unequal ratios of the three polypeptide chains used for the construction provide optimal yields, this provides considerable flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, when expression of at least two polypeptide chains in equal ratios results in high yields, or when the ratios are not particularly important, the coding sequences for two or all three polypeptide chains may be inserted into one expression vector.

[0465] Bispecific antibodies can have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure helps separate the desired bispecific compound from undesirable immunoglobulin chain combinations because the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient separation method (WO 94 / 04690; Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigues et al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992, Bio / Technology 10:163-167; Carter et al., 1995, J. of Hematotherapy 4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681). Using such techniques, bispecific antibodies can be prepared for conjugation as ADCs to treat or prevent diseases as defined herein.

[0466] Hybrid or bifunctional antibodies may be derived biologically (ie by cell fusion techniques) or chemically (especially with cross-linking or disulfide bridge forming agents) and may comprise whole antibodies or fragments thereof (EP 105360; WO 83 / 03679; EP 217577).

[0467] The antibody can be an antibody that immunospecifically binds to a cancer cell antigen, a viral antigen, or a microbial antigen, or a functionally active fragment, derivative, or analog of other antibodies that bind to a tumor cell or matrix. In this regard, "functional activity" means that the fragment, derivative, or analog is capable of eliciting an anti-idiotype antibody that recognizes the same antigen as the antibody from which the fragment, derivative, or analog was derived. Specifically, in an exemplary embodiment, the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deleting the framework and CDR sequences, which are the C-terminal ends of the CDR sequences that specifically recognize the antigen. In order to determine the CDR sequence that binds to the antigen, a synthetic peptide containing the CDR sequence can be used for binding assays with the antigen by any binding assay method known in the art (e.g., BIA core assay) (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. of Immunology 125 (3): 961-969).

[0468] Other useful antibodies include antibody fragments, such as, but not limited to, F(ab')2 fragments, which contain the variable region, the light chain constant region, and the CH1 domain of the heavy chain (which can be generated by pepsin digestion of the antibody molecule), and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragment. Other useful antibodies are antibody heavy and light chain dimers, or any minimal fragment thereof, such as Fv or single-chain antibodies (SCAs) (e.g., as described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., (1989) Nature 334:544-54), or any other molecule with the same specificity as an antibody.

[0469] In addition, recombinant antibodies (e.g., chimeric and humanized monoclonal antibodies, comprising human and non-human parts, which can be prepared using standard recombinant DNA techniques) are useful antibodies. Chimeric antibodies are molecules in which different parts are derived from different animal species, such as those having variable regions derived from murine monoclonals and human immunoglobulin constant regions. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816,397). Humanized antibodies are antibody molecules from non-human species that have one or more complementary determining regions (CDRs) from non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Patent No. 5,585,089). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using the methods described in WO 87 / 02671; EP 184,187; EP 171496; EP 173494; WO 86 / 01533; U.S. Pat. No. 4,816,567; EP 12023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Patent No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060.

[0470] Using transgenic mice that cannot express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes, complete human antibodies can be produced. Transgenic mice are immunized in a normal manner with a selected antigen (e.g., all or part of a polypeptide of the present invention). Monoclonal antibodies against antigens can be obtained using conventional hybridoma technology. The human immunoglobulin transgene carried by the transgenic mice rearranges during B cell differentiation and subsequently undergoes class switching and somatic mutation. Therefore, using this technology, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced. For a review of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93). For a detailed discussion of the technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806. Other human antibodies are commercially available from, e.g., Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0471] A technique called "guided selection" can be used to generate complete human antibodies that recognize selected epitopes. In this method, a selected non-human monoclonal antibody (e.g., a mouse antibody) is used to guide the selection of complete human antibodies that recognize the same epitope. (Jespers et al. (1994) Biotechnology 12:899-903). Human antibodies can also be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).

[0472] The antibody may be a fusion protein of an antibody or a functionally active fragment thereof, for example, wherein the antibody is fused to an amino acid sequence of another protein (or a portion thereof, such as at least a 10, 20, or 50 amino acid portion of a protein) that is not an antibody via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus. The antibody or fragment thereof may be covalently linked to another protein at the N-terminus of the constant domain.

[0473] Antibodies include analogs and derivatives that have been modified, i.e., by covalent attachment of any type of molecule, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, and not by way of limitation, derivatives and analogs of antibodies include those that have been further modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular antibody units or other proteins, and the like. Any of a variety of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavages, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like. In addition, analogs or derivatives may contain one or more unnatural amino acids.

[0474] The antibody in the antibody drug conjugate includes an antibody having a modification (e.g., substitution, deletion, or addition) in the amino acid residues that interact with the Fc receptor. In particular, the antibody includes an antibody having a modification in the amino acid residues identified as participating in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., WO 97 / 34631). Antibodies that are immunospecific for cancer cell antigens can be purchased, for example, from Genentech (San Francisco, Calif.) or produced by any method known to those skilled in the art (e.g., chemical synthesis or recombinant expression technology). Nucleotide sequences encoding antibodies that are immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by conventional cloning and sequencing.

[0475] The antibody of the ADC can be a monoclonal antibody, such as a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. The antibody can be an antibody fragment, such as a Fab fragment.

[0476] Antibodies known to be used to treat or prevent cancer can be conjugated into ADCs. Antibodies immunospecific for cancer cell antigens can be purchased or produced by any method known to those skilled in the art (e.g., recombinant expression technology). Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by conventional cloning and sequencing. Examples of antibodies that can be used to treat cancer include, but are not limited to, humanized anti-HER2 monoclonal antibodies for the treatment of patients with metastatic breast cancer; RITUXAN.RTM. (rituximab; Genentech), which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA), which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC), which is a murine IgG for the treatment of colorectal cancer. 2aantibodies; Cetuximab Erbitux (Imclone Systems Inc., NY), an anti-EGFR IgG chimeric antibody used to treat epidermal growth factor-positive cancers (e.g., head and neck cancer); Vitaxin (MedImmune, Inc., MD), a humanized antibody used to treat sarcomas; Campath I / H (Leukosite, MA), a humanized IgG1 antibody used to treat chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA), a humanized anti-CD33 IgG antibody used to treat acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ), a humanized anti-CD22 IgG antibody used to treat non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA), a humanized anti-CD33 IgG antibody used to treat acute myeloid leukemia (AML); Design Labs, Inc., CA), a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA), a radiolabeled murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA), a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; Avastin (Genentech, Inc., CA), a humanized anti-VEGF antibody for the treatment of lung and colorectal cancer; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA), an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0477] Other antibodies that can be used to treat cancer include, but are not limited to, antibodies to the following antigens: CA125 (ovarian cancer), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha-fetoprotein (carcinoma), CA 242 (colorectal cancer), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate cancer), prostatic acid phosphatase (prostate cancer), epidermal growth factor (carcinoma), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal cancer), gp100 (melanoma), ), MART1 (melanoma), PSA (prostate cancer), IL-2 receptor (T-cell leukemia and lymphoma), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma), and Neu oncogene products (carcinoma). Some useful specific antibodies include, but are not limited to, BR96 mAb (Trail, PA et al., Science (1993) 261, 212-215), BR64 (Trail, PA et al., Cancer Research (1997) 57, 100-105), mAbs against the CD40 antigen (e.g., S2C6 mAb) (Francisco, JA et al., Cancer Res. (2000) 60:3225-3231), mAbs against the CD70 antigen (e.g., 1F6 mAb), and mAbs against the CD30 antigen (e.g., AC10) (Bowen, MA et al. (1993) J. Immunol., 151:5896-5906; Wahl et al., 2002 Cancer Res. 62(13):3736-42). Many other internalizing antibodies that bind to tumor-associated antigens are available and have been reviewed (Franke, AE et al., Cancer Biother Radiopharm. (2000) 15:459-76; Murray, JL, (2000) Semin Oncol., 27:64-70; Breitling, F. and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

[0478] Antibodies that bind to antigens associated with antigen-presenting cells (e.g., CD40, OX40L, endoglin, DEC-205, 4-1BBL, CD36, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-L1, BDCA-2, XCR-1, and CCR2) can also be conjugated as ADCs.

[0479] Antibodies known for treating or preventing autoimmune disorders can be conjugated to ADCs. Autoimmune disorders include systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjogren's syndrome, immune thrombocytopenia, and multiple sclerosis. Antibodies immunospecific for the antigens of cells responsible for producing autoimmune antibodies can be obtained by any method known to those skilled in the art (e.g., chemical synthesis or recombinant expression technology). SLE is marked by overexpression of interferon-α (IFN-α) cytokine genes (Bennett et al. (2003) Jour. Exp. Med. 197: 711-723). Type 1 interferons (IFN-α / β) play an important role in the pathogenesis of lupus (Santiago-Raber (2003) Jour. Exp. Med. 197: 777-788). Knockout mice (-IFN-.α. / .β.) showed significantly reduced anti-erythrocyte autoantibodies, erythroblastosis, hemolytic anemia, anti-DNA autoantibodies, nephropathy, and mortality. These results suggest that type 1 IFN mediates murine lupus and that reducing the activity of its human counterpart may be beneficial. Anti-IFN Abs conjugated to a bis-1,8-naphthalimide drug moiety may be effective therapeutics for SLE and other autoimmune disorders.

[0480] In another embodiment, antibodies useful in ADCs that are immunospecific for treating autoimmune diseases include, but are not limited to, antinuclear antibodies; anti-ds DNA; anti-ss DNA, anti-cardiolipin antibodies IgM, IgG; anti-phospholipid antibodies IgM, IgG; anti-SM antibodies; anti-mitochondrial antibodies; thyroid antibodies; microsomal antibodies; thyroglobulin antibodies; anti-SCL-70; anti-Jo; anti-U1 RNP; anti-La / SSB; anti-SSA; anti-SSB; anti-parietal cell antibodies; anti-histone; anti-RNP; C-ANCA; P-ANCA; anti-centromere; anti-nucleolar fiber and anti-GBM antibodies.

[0481] The antibody of ADC can be bound to the receptor or receptor complex expressed on activated lymphocytes. Receptor or receptor complex can include immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins or complement control proteins. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD 19, CD22, CD28, CD79, CD90, CD 152 / CTLA-4, PD-1 and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD 103, and CD 104. Non-limiting examples of suitable lectins are C-type, S-type, and I-type lectins.

[0482] As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide protein capable of eliciting an immune response (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoproteins (e.g., Gb, Gc, Gd, and Ge), and hepatitis B surface antigen). As used herein, the term "microbial antigen" includes, but is not limited to, any microbial peptide, polypeptide, protein, sugar, polysaccharide, or lipid molecule capable of eliciting an immune response (e.g., bacterial, fungal, pathogenic protozoan, or yeast polypeptides, including, for example, LPS and capsular polysaccharide 5 / 8).

[0483] Antibodies immunospecific for viral or microbial antigens can be purchased, for example, from BD Biosciences (San Francisco, Calif.), Chemicon International, Inc. (Temecula, Calif.), or Vector Laboratories, Inc. (Burlingame, Calif.) or produced by any method known to those skilled in the art (e.g., chemical synthesis or recombinant expression techniques). Nucleotide sequences encoding antibodies immunospecific for viral or microbial antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by conventional cloning and sequencing.

[0484] In some embodiments, the antibodies that can be used in the present ADC are those that treat or prevent viral or microbial infections according to the methods disclosed herein. Examples of antibodies that can be used to treat viral or microbial infections include, but are not limited to, SYNAGIS (MedImmune, Inc., MD), a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody that can be used to treat RSV-infected patients; PRO542 (Progenics), a CD4 fusion antibody that can be used to treat HIV infection; OSTAVIR (Protein Design Labs, Inc., CA), a human antibody that can be used to treat hepatitis B virus; PROTOVIR (Protein Design Labs, Inc., CA), a humanized IgG antibody that can be used to treat cytomegalovirus (CMV); and anti-LPS antibodies.

[0485] Other antibodies that can be used in ADCs to treat infectious diseases include, but are not limited to, antibodies against antigens from strains of pathogenic bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae, and Staphylococcus aureus. colerae), Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum), Borrelia vincentii, Borreliaburgdorferi), Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumoniae pneumonia), Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Enterobiusvermicularis, Trichuristrichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).

[0486] Other antibodies that can be used in ADCs to treat viral diseases include, but are not limited to, antibodies directed against antigens of pathogenic viruses, including, but not limited to, Poxviridae, Herpesviridae, Herpes simplex virus 1, Herpes simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, and Enteroviridae. viridae), Retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, non-A / non-B hepatitis viruses, Rhinoviridae, Coronaviridae, Rotaviridae, and human immunodeficiency virus.

[0487] The term "amino acid sequence variant" refers to a polypeptide whose amino acid sequence differs to some extent from that of a native sequence polypeptide. Typically, amino acid sequence variants will have at least about 70% sequence identity with at least one receptor binding domain of a native antibody or at least one ligand binding domain of a native receptor, and typically, they will be at least about 80%, more typically at least about 90%, homologous to the sequence of such receptor or ligand binding domain. Amino acid sequence variants have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by conventional names, single-letter and three-letter codes.

[0488] "Sequence identity" is defined as the percentage of identical residues in amino acid sequence variants after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity. Methods and computer programs for alignment are well known in the art. One such computer program is "Align 2" designed by Genentech, Inc., with a user file filed on December 10, 1991, in the United States Copyright Office, Washington, DC 20559.

[0489] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native sequence human FcR. In addition, FcRs can be receptors that bind IgG antibodies (gamma receptors) and include Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA ("activating receptor") and Fc.gamma.RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See review M. Daeron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those yet to be identified, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

[0490] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), can be performed.

[0491] "Natural antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain and the heavy chain variable domains.

[0492] The term "variable" refers to the fact that some parts of the variable domain are very different in sequence between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed in the variable domains of antibodies. It is concentrated in three segments called hypervariable regions of the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs, mainly adopting a β-sheet configuration, connected by three hypervariable regions, which form loops that connect the β-sheet structure and in some cases form a part of the β-sheet structure. The hypervariable regions in each chain are tightly connected by the FRs and, together with the hypervariable regions in the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institute of Health, Bethesda, Md.). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0493] The term "hypervariable region" as used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., supra) and / or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol., 196:901-917). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0494] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0495] "Fv" is the smallest antibody fragment that contains complete antigen recognition and antigen binding sites. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to confine the antigen binding site to the surface of the VH-VL dimer. The six hypervariable regions together give the antibody antigen binding specificity. However, even if a single variable domain (or only half of an Fv comprising three antigen-specific hypervariable regions) still has the ability to recognize and bind antigen, its affinity is lower than that of the entire binding site.

[0496] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that a small number of residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab', in which the cysteine ​​residues of the constant domains carry at least one free sulfhydryl group. F(ab')2 antibody fragments were initially produced as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0497] The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0498] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994). Anti-ErbB2 antibody scFv fragments are described in WO 93 / 16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.

[0499] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) connected to each other in the same polypeptide chain (VH-VL). By using a linker that is too short to pair between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

[0500] The " humanization " form of non-human (such as rodent) antibody is chimeric antibody, and it contains the minimum sequence derived from non-human immunoglobulin.Humanization is the method that murine antigen binding information is transferred to non-immunogenic human antibody receptor, and has produced many therapeutically useful drugs.Humanization method usually starts from transferring all six murine complementary determining regions (CDR) to human antibody framework (Jones et al., (1986) Nature 321:522-525). The antibody of these CDR transplants usually can not keep its original affinity for antigen binding, and in fact, affinity is usually seriously damaged.Except CDR, the non-human antibody framework residues of selection must also be incorporated into, to maintain appropriate CDR conformation (Chothia et al. (1989) Nature 342:877). Transferring key mouse framework residues to the human acceptor to support the structural conformation of the grafted CDRs has been shown to restore antigen binding and affinity (Riechmann et al., (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al., (1993) J. Immunol. 151, 2623-2632; Werther et al., (1996) J. Immunol. Methods 157:4986-4995; and Presta et al. (2001) Thromb. Haemost. 85:379-389). In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) with desired specificity, affinity and ability, wherein the residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of non-human species (donor antibody) (such as mouse, rat, rabbit or non-human primate). In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies can include residues not found in recipient antibody or donor antibody. These modifications are carried out to further refine antibody performance. In general, humanized antibodies will include substantially all at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulin, and all or substantially all of the FRs are the FRs of human immunoglobulin sequences. Humanized antibodies optionally also include at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. For more details, see U.S. Patent No. 6,407,213; Jones et al. (1986) Nature, 321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596.

[0501] A "parent antibody" is an antibody comprising an amino acid sequence in which one or more amino acid residues are replaced by one or more cysteine ​​residues. The parent antibody may comprise a native or wild-type sequence. The parent antibody may have pre-existing amino acid sequence modifications (e.g., additions, deletions, and / or substitutions) relative to other native, wild-type, or modified forms of the antibody. The parent antibody is directed against a relevant target antigen. Antibodies directed against non-polypeptide antigens (e.g., tumor-associated glycolipid antigens; see U.S. Patent No. 5,091,178) are also contemplated.

[0502] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody (as determined by the Lowry method), or greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues using a gas phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibodies include antibodies in situ within recombinant cells because at least one component of the antibody's natural environment is absent. However, typically, isolated antibodies will be prepared by at least one purification step.

[0503] An antibody that "binds" a molecular target or antigen of interest is one that is capable of binding the antigen with sufficient affinity such that the antibody can be used to target cells expressing the antigen.

[0504] The terms "treat" or "treatment" refer to both therapeutic treatment and protective or preventative measures, wherein the goal is to prevent or slow (lessen) an undesirable physiological change or condition, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, alleviation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and regression (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already suffering from the disease or condition, as well as those susceptible to the disease or condition, or those in need of prevention of the disease or condition.

[0505] "Phage display" is a technology by which variant polypeptides are displayed on the surface of phage (e.g., filamentous phage) particles as fusion proteins with coat proteins. One use of phage display is the fact that large libraries of randomized protein variants can be quickly and efficiently sorted for sequences that bind to target molecules with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to find polypeptides with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins, typically via fusion to PIII or PVIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol., 3: 355-362 (1992) and references cited therein. In monovalent phage display, a protein or peptide library is fused to a phage coat protein or a portion thereof and expressed at low levels in the presence of wild-type protein. Compared to multivalent phage, avidity effects are reduced so that scoring is based on intrinsic ligand affinity, and phagemid vectors are used, which simplifies DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3: 205-0216 (1991). Phage display includes technologies for producing antibody-like molecules (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th ed., Garland Publishing, New York, pp. 627-628).

[0506] "Phagemid" is a plasmid vector that has a bacterial origin of replication (e.g., Co1E1) and a copy of the intergenic region of the phage. Phagemids can be used for any known phage, including filamentous phage and herringbone phage. Plasmids also typically contain a selective marker for antibiotic resistance. The DNA segments cloned into these vectors can be propagated along with the plasmid. When the cells carrying these vectors are provided with all the genes necessary to produce phage particles, the replication mode of the plasmid becomes rolling circle replication to produce a copy of one chain of the plasmid DNA and package the phage particles. Phagemids can form infectious or non-infectious phage particles. The term includes phagemids containing a phage coat protein gene or a fragment thereof, which is connected to a heterologous polypeptide gene as a gene fusion so that the heterologous polypeptide is displayed on the surface of the phage particle. The compounds described herein may be in the form of a pharmaceutically or pharmaceutically acceptable salt. In some embodiments, such salts are derived from inorganic or organic acids or bases. For reviews of suitable salts, see, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19 and Remington: The Science and Practice of Pharmacy, 20th edition, A. Gennaro (ed.), Lippincott Williams & Wilkins (2000).

[0507] In the present invention, the group "Ab" (i.e., an antibody, antibody fragment, and / or antigen fragment) can be conjugated to more than one drug-containing moiety. In some embodiments, "Ab" can be conjugated to 1 to 20 drug-containing moieties. In some embodiments, "Ab" can be conjugated to 1 to 10 drug-containing moieties. In some embodiments, "Ab" can be conjugated to 1 to 5 drug-containing moieties. In some embodiments, "Ab" can be conjugated to 1 or 2 drug-containing moieties. In some embodiments, "Ab" can be conjugated to one drug-containing moiety.

[0508] The compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases. For a review of suitable salts, see, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19 and Remington: The Science and Practice of Pharmacy, 20th edition, A. Gennaro (ed.), Lippincott Williams & Wilkins (2000).

[0509] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, gluconoheptate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, and undecanoate.

[0510] Examples of suitable base addition salts include ammonium salts; alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; organic base salts such as dicyclohexylamine salts, N-methyl-D-glucamine; and amino acid salts such as arginine salts, lysine salts and the like.

[0511] For example, Berge lists the following commercially available salts that are approved by the FDA: anionic acetate, besylate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, edetate (EDTA), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (EDTA), edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycolyllarsanilate (glycollamidophenylarsonate), hexylresorcinol, amine (N,N'-di(dehydroabietic acid)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-hydroxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, methanesulfonate (mesylate, methanesulfonate), methyl bromide, methyl nitrate, methyl sulfate, mucate, naphthalenesulfonate (2-naphthalenesulfonate), nitrate, pamoate (pamoate, embona te), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theophylline (8-chlorophylline), and triethyl iodide; the organic cations benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and the metal cations aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0512] Berge additionally lists the following non-FDA approved commercially available (outside the U.S.) salts: anionic adipates, alginate, aminosalicylate, anhydrous methylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylenebis(salicylate), naphthalenedisulfonate (1,5-naphthalenedisulfonate), oxalate, pectinate, persulfate, phenylethylbarbiturate, picrate, propionate, thiocyanate, tosylate, and undecanoate salts; organic cations phenethidamine (N-benzylphenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and metal cations barium and bismuth.

[0513] The compounds described herein may also contain suitable carriers, excipients, and adjuvants, which may vary depending on the mode of administration.

[0514] In some embodiments, the pharmaceutical composition can be formulated into a suitable parenteral dosage form. The formulation can be prepared by various methods known in the art. The pharmaceutical composition can be administered directly into the bloodstream, into a muscle, or directly into an organ. Suitable modes for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle syringes, needle-free syringes, and infusion techniques.

[0515] Parenteral compositions are generally aqueous solutions which may contain excipients such as salts, carbohydrates and buffers. However, the compositions may also be formulated as sterile non-aqueous solutions or dry forms for use in combination with a suitable vehicle (eg, sterile pyrogen-free water).

[0516] Parenteral compositions may readily be prepared under sterile conditions using standard techniques well known to those skilled in the art, for example, by lyophilization.

[0517] Compositions for parenteral administration can be formulated to release immediately and / or modified. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release. Thus, the composition can be formulated as a solid, semisolid, or thixotropic liquid and administered as an implanted depot, thereby providing modified release of the active agent.

[0518] The parenteral formulation may be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms, such as, but not limited to, preservatives.

[0519] In another embodiment, the pharmaceutical composition can be formulated into a suitable oral dosage form, such as tablets, capsules, powders, pills, suspensions, solutions, emulsions, etc. Other suitable carriers may be present, such as disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, bulking agents, anti-adherents, etc.

[0520] Oral dosage formulations may also contain other suitable pharmaceutical excipients, such as sweeteners, vehicles / wetting agents, colorants, flavorings, preservatives, viscosity / thickening agents, and the like.

[0521] The dosage of the pharmaceutical composition of the present invention can be adjusted for individual patients.

[0522] Lipid conjugates

[0523] In certain embodiments, the present invention provides a compound of formula (VI),

[0524]

[0525] or a pharmaceutically acceptable salt thereof, wherein:

[0526] a is an integer from 1 to 20;

[0527] m is 0, 1, 2, 3, or 4;

[0528] n is 0 or 1;

[0529] D-NH- is part of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2;

[0530] Each R 1 independently selected from C1-C4 alkyl, O-C1-C4 alkyl and halogen;

[0531] R 2 Selected from C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10;

[0532] R 3 and R 3 ' are each independently selected from hydrogen and C1-C3 alkyl;

[0533] L is a cleavable linker; and

[0534] LP is lipid.

[0535] In some embodiments, D-NH2 is a STING modulator. In some embodiments, D-NH2 is a cyclic dinucleotide (CDN) or a CDN-like compound. In some embodiments, D-NH2 is one of the formulas described herein.

[0536] In certain embodiments, the present invention provides compounds of formula (VI), wherein L has the formula

[0537]

[0538] wherein W, Z, t, U, Q and U' are as described herein.

[0539] In some embodiments, the compound of formula (VI) is associated with a lipoplex. The term "lipoplex" is a generally accepted term in the preparation of pharmaceutical compounds. Lipoplexes are characterized by non-covalent bonding between the lipid and the compound of formula (VI). Without being bound by a particular theory, the compound of formula (VI) may associate with the lipoplex via electrostatic interactions, hydrophilic interactions, hydrophobic interactions, or any combination thereof.

[0540] In some embodiments, the lipid complexes described herein are in the form of lipid particles. In one embodiment, the complex is in the form of a lipid nanoparticle. In one embodiment, the complex is in the form of a liposome. In some embodiments, the liposomes of the present invention comprise a lipid monolayer or a lipid multilayer. In some embodiments, the liposomes comprise a lipid bilayer and an aqueous core. In some embodiments, the average particle size of the liposomes is from about 5 nm to about 1 μm. In some embodiments, the average particle size of the lipid nanoparticles or liposomes is from about 5 nm to about 500 nm, from about 10 nm to about 150 nm, and from about 30 nm to about 100 nm. In some embodiments, the compound of formula (V) resides in the aqueous core, the lipid bilayer, or a combination thereof.

[0541] In certain embodiments, the LP may be cholesterol or a phospholipid. In some embodiments, the LP is a phospholipid. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, soybean lecithin, and egg yolk lecithin. In some embodiments, the phospholipid is phosphatidylethanolamine. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0542] In some embodiments, the present invention provides a lipoplex comprising a compound of formula (VI). In some embodiments, the lipoplex can be a liposome.

[0543] In some embodiments, the lipoplex comprises from about 0.5 mol% to about 40 mol% of a compound of Formula (VI) or a pharmaceutically acceptable salt thereof. In some embodiments, the lipoplex comprises from about 1 mol% to about 35 mol% of a compound of Formula (VI) or a pharmaceutically acceptable salt thereof. In some embodiments, the lipoplex comprises from about 1 mol% to about 30 mol% of a compound of Formula (VI) or a pharmaceutically acceptable salt thereof. In some embodiments, the lipoplex comprises from about 1 mol% to about 20 mol% of a compound of Formula (VI) or a pharmaceutically acceptable salt thereof. In some embodiments, the lipoplex comprises from about 1 mol% to about 15 mol% of a compound of Formula (VI) or a pharmaceutically acceptable salt thereof.

[0544] In some embodiments, the lipid complex further comprises one or more additional phospholipids. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean lecithin, and egg yolk lecithin.

[0545] In some embodiments, the lipoplex comprises from about 30 mol% to about 90 mol% of one or more phospholipids. In some embodiments, the lipoplex comprises from about 40 mol% to about 85 mol% of one or more phospholipids. In some embodiments, the lipoplex comprises from about 50 mol% to about 80 mol% of one or more phospholipids.

[0546] In some embodiments, the lipoplex further comprises a fatty alcohol or cholesterol. In some embodiments, the lipoplex comprises from about 5 mol% to about 35 mol% cholesterol. In some embodiments, the lipoplex comprises from about 5 mol% to about 30 mol% cholesterol. In some embodiments, the lipoplex comprises from about 5 mol% to about 25 mol% cholesterol.

[0547] In some embodiments, the lipid complex further comprises at least one PEGylated phospholipid. In some embodiments, at least one PEGylated lipid is a PEGylated phospholipid. In some embodiments, the PEGylated phospholipid is selected from N-(carbonyl-methoxy polyethylene glycol 5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxy polyethylene glycol 5000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, -glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 5000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 2000)-distearoyl-rac-glycerol and N-(carbonyl-methoxypolyethylene glycol 5000)-distearoyl-rac-glycerol, pharmaceutically acceptable salts thereof, or mixtures thereof. In some embodiments, the PEGylated phospholipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0548] In some embodiments, the lipoplex comprises from about 0 to about 6 mol% of one or more PEGylated phospholipids. In some embodiments, the lipoplex comprises from about 0.5 mol% to 5 mol% of one or more PEGylated phospholipids. In some embodiments, the lipoplex comprises from about 1 mol% to 5 mol% of one or more PEGylated phospholipids. In some embodiments, the lipoplex comprises about 3 mol% of one or more PEGylated phospholipids. In some embodiments, the lipoplex comprises about 4 mol% of one or more PEGylated phospholipids. In some embodiments, the lipoplex comprises about 5 mol% of one or more PEGylated phospholipids.

[0549] Methods of using compounds and compositions

[0550] Certain compounds described herein are STING agonists and are therefore useful for stimulating an immune response in a subject thereof. The compositions can be used to treat viruses.

[0551] The compounds of the present invention exhibit STING modulatory / agonist activity. Certain compounds of the present invention may be excellent in efficacy expression, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water solubility), interactions with other drugs (e.g., drug metabolizing enzyme inhibition), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central nervous system toxicity), and / or stability (e.g., chemical stability, enzyme stability), and may be used as pharmaceuticals.

[0552] The compounds of the present invention can be used to increase STING activity in mammals (eg, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans).

[0553] The compounds of the present invention can be used as drugs, for example, agents for preventing or treating diseases that can be affected by STING (sometimes abbreviated as "STING-related diseases" in this specification), such as cancers, for example, colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumor), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestinal cancer, breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., ovarian epithelial cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, non-hormone-dependent prostate cancer, castration-resistant prostate cancer), liver cancer (e.g., hepatocellular carcinoma), thyroid cancer (e.g., medullary thyroid cancer), kidney cancer (e.g., renal cell carcinoma (e.g., clear cell renal cell carcinoma), transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (e.g., cervical cancer, uterine corpus cancer, uterine sarcoma), gestational choriocarcinoma, brain tumors (e.g., medulloblastoma, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., Such as basal cell tumor, malignant melanoma), sarcoma (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumor, bladder cancer, blood cancer (e.g., multiple myeloma, leukemia (e.g., acute myeloid leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disease), cancer of unknown primary; cancer growth inhibitors; cancer metastasis inhibitors; apoptosis promoters; agents for treating precancerous lesions (e.g., myelodysplastic syndrome); and the like.

[0554] In certain embodiments, the compounds of the present invention are useful as medicaments for colorectal cancer, breast cancer, skin cancer, malignant lymphoma, or lung cancer.

[0555] In addition, the compound of the present invention or the combination of the present invention can be used concurrently with non-drug therapies. Specifically, the compound of the present invention or the combination of the present invention can be used in combination with, for example, the following non-drug therapies: (1) surgery, (2) chemotherapy for hypertension using angiotensin II, etc., (3) gene therapy, (4) thermotherapy, (5) cryotherapy, (6) laser ablation, and (7) radiotherapy.

[0556] For example, by using the compound of the present invention before or after the above-mentioned surgery, etc., or before or after combination treatment of two or three thereof, effects such as prevention of the emergence of resistance, prolongation of disease-free survival, inhibition of cancer metastasis or recurrence, and prolongation of life can be provided.

[0557] In addition, treatment with the compound of the present invention or the combination agent of the present invention can be combined with supportive therapy: (i) administration of antibiotics (e.g., β-lactam type, such as pansporin, etc.; macrolide type, such as clarithromycin, etc.) for complications of various infectious diseases, (ii) administration of high-calorie infusions, amino acid preparations or general vitamin preparations to improve malnutrition, (iii) administration of morphine to relieve pain, (iv) administration of pharmaceutical agents for improving side effects (e.g., nausea, vomiting, anorexia, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, liver disease, kidney disease, DIC, fever, etc.), and (v) administration of pharmaceutical agents for suppressing multidrug resistance in cancer, etc.

[0558] Example

[0559] General synthetic methods and intermediates

[0560] The compounds of the present invention can be prepared by one of ordinary skill in the art based on the present invention and knowledge in the art, and / or by reference to the schemes and synthesis examples shown below. Exemplary synthetic routes are described in the schemes and examples below. It should be understood that the variables (e.g., "R" groups) appearing in the schemes and examples below should be understood independently of the variables appearing elsewhere in this application. One of ordinary skill in the art will readily appreciate the manner in which the schemes and examples shown below illustrate the preparation of the compounds described herein.

[0561] Scheme 1: General route for conversion of carboxylic acids to the corresponding acid chlorides or (isobutyl carbonate) propionic anhydride

[0562]

[0563] Scheme 1 shows a general route for activating carboxylic acids i to the corresponding acid chlorides or (isobutyl carbonate) propionic anhydride (eg ii), where Yc is an alkyl or aromatic chain and R 6 is a methyl group or a polyether-containing alkyl chain. ii (wherein E is OCO2CH2CH(CH3)2) can be prepared by treating i with isobutyl chloroformate in the presence of a base. When E is Cl, formation of ii is achieved by treating i with oxalyl chloride or with a Ghosez reagent (see α-Chloro enamines, reactive intermediates for synthesis: 1-Chloro-N,N,2-trimethylpropenylamine. Haveaux B. et al., Org. Synth. 1980, 59, 26).

[0564] Scheme 2: General route for attaching a spacer to an amino group in a drug moiety using a stepwise approach

[0565]

[0566] Scheme 2 shows a general method for attaching a spacer (e.g., ii) to an amino group in a drug moiety (e.g., iii). After compound iii is subjected to persilyl protection by treatment with trimethylsilyl chloride and pyridine, it is reacted with activated spacer ii. Removal of the silyl group with NEt3-3HF yields iv. Deprotection of the amine of iv is accomplished by treatment with an acid (e.g., TFA or HCl), and the resulting amine salt is treated with activated dipeptide carbonate v to provide vi.

[0567] Scheme 3: General route for attaching a spacer to an amino group in a drug moiety using a stepwise approach

[0568]

[0569] Scheme 3 shows a general method for attaching a spacer (e.g., ii) to an amino group in a drug moiety (e.g., iii-a), where X 1 and X 2 is oxygen, sulfur or CH2, X 3 is nitrogen or CH, R 3 is hydrogen or fluorine, R 4 is hydroxyl, hydrogen, fluorine or R 3 Together they form OCH2, R c is OH or SH, R dis methyl or (CH2)3NHC(O)NH2. After compound iii-a is subjected to persilyl protection by treatment with trimethylsilyl chloride and pyridine, it is reacted with activated spacer ii. Removal of the silyl group with NEt3-3HF yields iv-a. Deprotection of the amine of iv-a is accomplished by treatment with an acid (e.g., TFA or HCl), and the resulting amine salt is treated with activated dipeptide carbonate v to provide vi-a.

[0570] Scheme 4: General route for attaching a spacer to an amino group in a drug moiety using a stepwise approach

[0571]

[0572] Scheme 4 shows a general method for attaching a spacer group (eg, ii) to an amino group in a drug moiety (eg, iii-b) following the general method described in Scheme 3.

[0573] Scheme 5: General route to generate spacer peptide chlorides

[0574]

[0575] Scheme 5 shows a general method for preparing acid chlorides viii. After deprotection of Boc-amine i using an acid (such as TFA or HCl), treatment with activated carbonate v affords carboxylic acid vii. vii can be converted to acid chloride viii by treatment with oxalyl chloride or sulfonyl chloride.

[0576] Scheme 6: Alternative general route for attaching a spacer to an amino group in a drug moiety

[0577]

[0578] Scheme 6 shows an alternative general method for the direct conversion of payload iii to compound ix. After persilyl protection with trimethylsilyl chloride, iii is treated with acid chloride viii. Removal of the silyl group affords ix.

[0579] Scheme 7: Alternative general route for attaching a spacer to an amino group in a drug moiety

[0580]

[0581] Scheme 7 shows an alternative general method for converting payload iii-a directly to compound ix-a following the general method described in Scheme 6.

[0582] Scheme 8: Alternative general route for attaching a spacer to an amino group in a drug moiety

[0583]

[0584] Scheme 8 shows an alternative general method for attaching a spacer to an amino group in a drug moiety (eg, iii-b) following the general method described in Scheme 6.

[0585] Scheme 9: General route for preparing linker-payload

[0586]

[0587] Scheme 9 shows a general method for preparing linker-payload xi from vi, where Yd is a linear or branched alkyl chain or polyether chain, and Z is a conjugation handle such as maleimide, BCN, or DBCO. Deprotection of the amine of vi with an acid (e.g., TFA or HCl) followed by reaction with an activated ester x affords linker-payload xi.

[0588] Scheme 10: General route for preparing linker-payload

[0589]

[0590] Scheme 10 shows a general method for preparing linker-payload xi-a from vi-a following the general method described in Scheme 9.

[0591] Scheme 11: General route for preparing linker-payload

[0592]

[0593] Scheme 11 shows a general method for attaching a conjugation handle to a compound (eg, vi-b) following the general method described in Scheme 9.

[0594] Scheme 12: General route for preparing linker-payload

[0595]

[0596] An alternative method for preparing linker-payload xi from iv is shown in Scheme 12. Deprotection of the amine of iv with an acid (such as TFA or HCl) followed by reaction with xii provides xi.

[0597] Scheme 13: General route for preparing linker-payload

[0598]

[0599] Scheme 13 shows an alternative method for preparing linker-payload xi-a from iv-a following the general method described in Scheme 12.

[0600] Scheme 14: General route for preparing linker-payload

[0601]

[0602] Scheme 14 shows a general method for preparing glucuronate-containing linker payload xvi. Deprotection of the amine of iv with an acid (e.g., TFA or HCl) followed by reaction with an activated carbonate xiii yields xiv. Peracetate hydrolysis with a base (e.g., lithium hydroxide) and removal of the FMOC group in xiv yields xv. Treatment of xv with an activated conjugation handle x provides the glucuronate-containing linker payload xvi.

[0603] Scheme 15: General route for preparing linker-payload

[0604]

[0605] Scheme 15 shows a general method for preparing glucuronate-containing linker payload xvi-a following the general method described in Scheme 14.

[0606] Scheme 16: General route for preparing linker-payload

[0607]

[0608] Scheme 16 shows a general method for preparing terminal amine-containing linker payload xviii, where Ye is a linear alkyl chain or a polyether-containing alkyl chain. Deprotection of the amine of vi with an acid (e.g., TFA or HCl) is followed by reaction with an activated conjugation handle xvii. Removal of the terminal protecting group with an acid (e.g., TFA) provides terminal amine-containing linker payload xviii.

[0609] Scheme 17: General route for preparing linker-payload

[0610]

[0611] Scheme 17 shows a general method for preparing terminal amine containing linker payload xviii-a following the general method described in Scheme 16.

[0612] Scheme 18: General route for preparing linker-payload

[0613]

[0614] Scheme 18 shows a general method for preparing linker payloads (e.g., xxii) incorporating a dipeptide spacer, wherein R 7is a hydrogen, alkyl, or aromatic moiety. Compound iii is fully protected by treatment with trimethylsilyl chloride and pyridine, followed by treatment with Boc-proline succinimidyl ester. Removal of the silyl group with a fluoride source (e.g., NEt3-3HF) yields xix. Deprotection of the amine of xix with an acid (e.g., TFA or HCl) is followed by reaction with a succinimidyl ester-activated amino acid xx to yield xxi. Deprotection of the amine of xxi with an acid (e.g., TFA or HCl) is followed by installation of a maleimide handle to provide the linker payload xxii.

[0615] Scheme 19: General route for preparing linker-payload

[0616]

[0617] Scheme 19 shows a general method for preparing linker payloads (eg, xxii-a) following the general method described in Scheme 18.

[0618] Preparation of exemplary ADCs and intermediates

[0619] definition

[0620] AA LCMS method using ammonium acetate

[0621] Ac acetate

[0622] ACN acetonitrile

[0623] atm atmospheric pressure

[0624] aq water-based

[0625] BCN bicyclo[6.1.0]nonyne

[0626] BLQ below the limit of quantification

[0627] Bn benzyl

[0628] Boc tert-Butoxycarbonyl

[0629] tBu tert-butyl

[0630] Bz benzoyl

[0631] C degrees Celsius

[0632] calcd calculated value

[0633] Cbz benzyloxycarbonyl

[0634] DAR drug-antibody ratio

[0635] DBCO Dibenzocyclooctyne

[0636] DCC N,N-Dicyclohexylcarbodiimide

[0637] DCM dichloromethane

[0638] DIPEA N,N-Diisopropylethylamine

[0639] DMAP 4-dimethylaminopyridine

[0640] DMA N,N-dimethylacetamide

[0641] DMB 2,4-dimethoxybenzyl

[0642] DMF N,N-dimethylformamide

[0643] DMSO dimethyl sulfoxide

[0644] DTT dithiothreitol

[0645] ε extinction coefficient

[0646] E 0.1% Extinction coefficient of 0.1% solution

[0647] EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDTA Ethylenediaminetetraacetic acid

[0648] Et ethyl

[0649] EtOH

[0650] EtOAc

[0651] LCMS method using formic acid

[0652] Fmoc fluorenylmethyloxycarbonyl

[0653] h hour

[0654] HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HIC Hydrophobic Interaction Chromatography

[0656] HOAt 1-Hydroxy-7-azabenzotriazole

[0657] HPLC High-pressure liquid chromatography

[0658] HRMS High Resolution Mass Spectrometry

[0659] IC 50 50% inhibitory concentration

[0660] LC liquid chromatography

[0661] LCMS liquid chromatography mass spectrometry

[0662] m / z mass-to-charge ratio

[0663] MHz Megahertz

[0664] Me methyl

[0665] MeOH methanol

[0666] μM micrometer

[0667] min minutes

[0668] mL milliliters

[0669] MS

[0670] MWCO Molecular weight cutoff

[0671] NMR Nuclear Magnetic Resonance

[0672] PBS Phosphate-buffered saline

[0673] PE petroleum ether

[0674] Ph Phenyl

[0675] psi pounds per square inch

[0676] psig pounds per square inch gauge

[0677] Pyr Pyridine

[0678] QTOF Quadrupole Time of Flight

[0679] rt room temperature

[0680] SEC Size Exclusion Chromatography

[0681] SFC Supercritical Fluid Chromatography

[0682] TBS tert-Butyldimethylsilyl

[0683] TCEP (Tris(2-carboxyethyl)phosphine)

[0684] TEA triethylamine

[0685] TFA trifluoroacetic acid

[0686] THF Tetrahydrofuran

[0687] TIPS triisopropylsilyl

[0688] TMS trimethylsilyl

[0689] Tris(hydroxymethyl)aminomethane

[0690] UPLC Ultra-Performance Liquid Chromatography

[0691] Analytical methods

[0692] NMR conditions:

[0693] Unless otherwise stated, 1 H NMR spectra were acquired on a 400 MHz Bruker spectrometer. 31 P NMR spectra were recorded at 162 MHz on a 400 MHz Bruker spectrometer and 1 H decoupling is obtained. Unless otherwise specified, 19 F NMR spectra were recorded at 376 MHz on a 400 MHz Bruker spectrometer.

[0694] HRMS conditions:

[0695] HRMS spectra were acquired using an Agilent 1260 Infinity II bio-inert multisampler, a bio-inert column compartment, a bio-inert quadrupole pump, a DAD multi-wavelength detector, and an Agilent 6545 LC-QTOF mass spectrometer using an Osaka SodaCapcell PAK C1 UG120 (5 μm, 2.0 mm ID × 35 mm length) reversed-phase column. The LC solvent system consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B) or an equivalent solvent system. Typically, LC elution began with a 95% / 5% mobile phase A / B ratio. After an isocratic flow interval (typically 0.5 min), the mobile phase was changed to 100% B via a 1.5 min linear gradient. The flow rate was 0.7 mL / min for 5 min.

[0696] Typically, 2 μL of a 1 mM solution in DMSO was injected into the mass spectrometer. QTOF settings: gas temperature 35°C, drying gas flow rate = 10 L / min, nebulizer pressure 55 psig, sheath gas temperature and flow rate 35°C and 12 L / min, respectively. The following voltage settings were applied: capillary voltage 4500 V, nozzle voltage 2000 V, and disruptor voltage 75 V.

[0697] The data were analyzed using Agilent Mass Hunter qualitative analysis software version 8.07.00SP2. The mass error [ppm] was expressed as the difference between the accurate mass and the observed mass divided by the accurate mass multiplied by 1 × 10 6 .

[0698] LCMS conditions:

[0699] LCMS spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100 series LC system connected to a Micromass mass spectrometer using a reverse phase C18 column. Multiple gradients and run times were selected to best characterize the compounds. The mobile phases were based on ACN / water or MeOH / water gradients and contained 0.1% formic acid (method designation FA) or 10 mM ammonium acetate (method designation AA). An example of a solvent gradient used was 100% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid) at a flow rate of 1 mL / min over a 16.5 min run.

[0700] In some cases, LCMS spectra were recorded on an Agilent 1290 Infinity UPLC system connected to an Agilent 6130 mass spectrometer, a Waters Acquity UPLC system connected to a Waters Acquity SQ mass spectrometer, or an Agilent 1100 Series HPLC system connected to a Waters Micromass ZQ mass spectrometer using a reversed phase C18 column. Multiple gradients and run times were selected to best characterize the compounds. The mobile phase was based on an ACN / water or MeOH / water gradient and contained 0.1% formic acid (method designation FA) or 10 mM ammonium acetate (method designation AA). An example of a solvent gradient used was 95% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid) at a flow rate of 0.5 mL / min for 5 min.

[0701] Preparative HPLC:

[0702] Preparative HPLC separations were performed using an 18 x 150 mm Sunfire C-18 column, eluted with a water-ACN gradient using a Gilson instrument operated by a 322 pump, and fraction collection triggered by a UV / Visible 155 detector set between 200 nm and 400 nm. Mass-gated fraction collection was performed on an Agilent 1100 LC / MSD instrument.

[0703] Those skilled in the art will recognize that variations in gradients, column lengths, and flow rates are possible, and that some conditions may be more suitable for compound characterization than others, depending on the chemical being analyzed.

[0704] Preparative SFC:

[0705] Preparative SFC was performed using 10, 20 or 30 mm x 250 mm ChiralPak columns (typically IA, IB, IC, ID, IE and IF), 10 or 20 mm x 250 mm Phenomenex Lux Cellulose-4 or 2-ethylpyridine columns, eluting with appropriate percentages of supercritical carbon dioxide and alcohol containing 0.3% diethylamine, 0.3% TEA, 0.3% formic acid or without any acid or base additives. Typical isocratic conditions were a flow rate in the range of 10-100 mL / min and a column temperature of 40°C. Preparative SFC was performed on a Jasco SFC preparative purification system with UV / visible light triggered fraction collection set between 200 nm and 400 nm and back pressure regulation set to 10 MPa.

[0706] One of ordinary skill in the art will recognize that variations in gradients, column lengths, and flow rates are possible, and that some conditions may be more suitable for compound characterization than others, depending on the chemical being analyzed.

[0707] Analytical SEC conditions:

[0708] SEC spectra were recorded at 280 nm on a Hewlett-Packard HP1100 or Agilent 1100 Series LC system with a diode array detector using a SEC column (typically a Tosoh Biosep TSK Gel, G3000SWxl; P / N 8541; 250A; 5 μm; 7.8 mm × 300 mm). The mobile phase was 100 mM sodium phosphate, 300 mM sodium chloride, pH 6.8, 10% acetonitrile (v / v) or 1× PBS. A typical run was an isocratic run for 20 min at a flow rate of 1 mL / min.

[0709] Analytical HIC conditions:

[0710] HIC spectra were recorded at 280 nm on a Hewlett-Packard HP1100 or Agilent 1100 Series LC system with a diode array detector using an HIC column (typically Tosoh Butyl-NPR, 4.6×35 mm, 2.5 μm, P / N: 14947). Mobile phase A was 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7, and mobile phase B was 75% 25 mM sodium phosphate, pH 7, 25% isopropanol. For a typical 20-minute run, a 12-minute linear gradient from 95% / 5% A / B to 100% B was used between the initial and final intervals of the isocratic flow.

[0711] LC-QTOF conditions:

[0712] LCMS spectra were recorded on a reverse phase column (usually Agilent, PLRP-S, 5 μm, The HPLC-MS / MS was performed on an Agilent 1260 Bioinert Series LC system (2.1 mm x 50 mm) connected to an Agilent 6545QTOF mass spectrometer. Multiple gradients and run times were selected to best characterize the compounds. The mobile phase was based on an ACN / water gradient and contained 0.1% formic acid. An example of a solvent gradient used was 95% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid), where the conditions are shown in Table 1.

[0713] Table 1

[0714]

[0715]

[0716] Samples were either intact or reduced (20 μL of 1-5 mg / mL ADC solution treated with 4 μL of 0.5 M DTT solution at 37° C. for 30 min). Raw data were deconvoluted using Agilent BioConfirm software in the appropriate mass range to obtain protein molecular weight, and DAR was calculated using the Agilent DAR calculator.

[0717] LC / MS / MS conditions:

[0718] LC / MS / MS analyses were performed using a Shimadzu UFLC LC-20AD XR binary pump and SIL-30AC MP autosampler system and an ABSCIEX triple quadrupole 4500ESI mass spectrometer.

[0719] Typically, a 5 μL sample aliquot was injected into the LC / MS / MS after passing through a Waters Xselect C18 CSH 3.5 μL 2.1 mm ID x 30 mm column. Mobile phase A contained 0.1% formic acid in water, and mobile phase B contained 0.1% formic acid in 5% water and 95% acetonitrile. The total run time was 3 min, with a flow rate of 1.5 mL / min and a 1.5 min linear gradient from 100% A to 100% B. Initially, the instrument ran at 100% aqueous mobile phase solvent for 0.5 min, then increased to 100% organic solvent over the next 1.5 min.

[0720] Preparative SEC:

[0721] Preparative SEC purification was performed on a Gilson preparative HPLC system using a SEC column (typically a GE Superdex 200 Increase 10 / 300 GL) with a UV detector. The mobile phase was 1×PBS (pH 7.4). A typical run was an isocratic run for 30 min at a flow rate of 1 mL / min. Fraction collection was triggered based on UV thresholds (at 214 nm and 280 nm).

[0722] ADC concentration:

[0723] ADC concentrations were calculated based on the UV absorbance at 280 nm measured by NanoDrop (2000c; Fisher Scientific) minus the UV absorbance of the corresponding linker-payload construct.

[0724] Example 1

[0725] N-[(2-Chlorocarbonylphenyl)methyl]-N-methyl-carbamic acid tert-butyl ester (Intermediate 1)

[0726]

[0727] Step 1: 2-(((tert-Butoxycarbonyl)(methyl)amino)methyl)benzoic acid

[0728] To a solution of 2-[(methylamino)methyl]benzoic acid (7.50 g, 44 mmol) in 1N sodium hydroxide (182 mL) in 1,4-dioxane (136 mL) and water was added di-tert-butyl dicarbonate (19.2 g, 88 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated to remove the organic solvent and the pH was adjusted to pH 3 using 1N HCl. The crude product was extracted with EtOAc (3×). The combined organic phases were washed with brine, dried (Na2SO4), filtered and evaporated. The crude compound was purified by silica gel chromatography (0-50% EtOAc / DCM) to give 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid (8.88 g, 75%). LCMS (AA): m / z=264.1 (MH).

[0729] Step 2: N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamic acid tert-butyl ester, intermediate 1

[0730] 1-Chloro-N,N,2-trimethylpropenylamine (0.52 mL, 4.0 mmol) was slowly added to a solution of 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid (525 mg, 2.0 mmol) in DCM (16 mL) at 0°C. The reaction mixture was then warmed to room temperature and stirred for 1 h. The reaction mixture was then concentrated to dryness to afford crude tert-butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamate (Intermediate 1, 562 mg, 100%).

[0731] Example 2

[0732] N-[[2-(2-Chloro-2-oxo-ethyl)phenyl]methyl]-N-methyl-carbamic acid tert-butyl ester (Intermediate 2)

[0733]

[0734] Step 1: 2-[2-[[tert-Butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid

[0735] To a solution of 2-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)acetic acid (1.50 g, 5.7 mmol) in THF (20 mL) was added iodomethane (2.8 mL, 45.2 mmol) at room temperature. The reaction mixture was cooled to 0°C and sodium hydride (60% dispersion in mineral oil, 905 mg, 22.6 mmol) was added in three portions. The reaction mixture was then stirred at room temperature for 16 h. The reaction mixture was quenched with water and the organic solvent was removed by concentration. The aqueous phase was acidified to pH 3 using 1N HCl and the crude product was extracted with Et2O (2×). The combined organic phases were washed with brine, dried (Na2SO4), filtered and evaporated to give 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid (1.36 g, 86%). LCMS (AA): m / z=280.2 (M+H).

[0736] Step 2: N-[[2-(2-chloro-2-oxo-ethyl)phenyl]methyl]-N-methyl-carbamic acid tert-butyl ester, intermediate 2

[0737] The title compound was prepared following the procedure described in step 2 of example 1 starting with 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid (650 mg, 2.33 mmol) to give crude intermediate 2 (693 mg, 100%).

[0738] Example 3

[0739] Isobutoxycarbonyl 4-[tert-Butoxycarbonyl(methyl)amino]butyrate (Intermediate 3)

[0740]

[0741] Step 1: 4-((tert-Butoxycarbonyl)(methyl)amino)butanoic acid

[0742] The title compound was prepared following the procedure described in Step 1 of Example 2 using 4-(tert-butoxycarbonylamino)butanoic acid (2.92 g, 14.4 mmol) instead of 2-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)acetic acid to give 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (2.89 g, 93%). LCMS (AA): m / z = 216.1 (MH).

[0743] Step 2: Isobutoxycarbonyl 4-[tert-Butoxycarbonyl(methyl)amino]butyrate, Intermediate 3

[0744] To a solution of 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (360 mg, 1.57 mmol) and triethylamine (0.24 mL, 1.73 mmol) dissolved in DCM (7 mL) and cooled to 0°C was added isobutyl chloroformate (0.22 mL, 1.73 mmol) dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 2 h. The mixture was then concentrated to dryness to give crude isobutyloxycarbonyl 4-[tert-butoxycarbonyl(methyl)amino]butanoate (Intermediate 3, 499 mg, 100%).

[0745] Example 4

[0746] The compounds listed below were prepared as described in Example 3, using the starting materials shown in Table 2 instead of 4-(tert-butoxycarbonylamino)butanoic acid.

[0747] Table 2

[0748]

[0749] Example 5

[0750] N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-[(methylamino)methyl]benzamide (Intermediate 6)

[0751]

[0752] Step 1: tert-Butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate

[0753] 2-Amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Compound I-5c, see PCT / IB2018 / 058846, 160 mg, 0.19 mmol) as an N,N-diethylethylamine salt was dissolved in anhydrous pyridine and concentrated to dryness (3×2 mL), then placed under vacuum for 15 min. Under an argon atmosphere, the residue was dissolved in pyridine (3 mL) and chlorotrimethylsilane (0.15 mL, 1.13 mmol) was added. The reaction mixture was stirred at room temperature for 30 min. Intermediate 1 (800 mg, 2.82 mmol) dissolved in pyridine (3 mL) was then added via a syringe. The reaction mixture was stirred at room temperature for 16 h under an argon atmosphere. The reaction mixture was then concentrated to dryness and MeOH (10 mL) and ammonium hydroxide (28-30% aqueous solution, 10 mL) were added and stirred for 30 min. The reaction mixture was concentrated to dryness and the residue was dissolved in MeOH (15 mL). Triethylamine trihydrofluoride (0.12 mL, 0.75 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was concentrated to dryness and the crude residue was absorbed onto Celite and purified by reverse phase flash column chromatography (0-50% ACN in aqueous triethylammonium acetate (10 mM)) to give tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate as N,N-diethylethylamine salt (120 mg, 58%). LCMS (AA): m / z = 897.3 (M+H).1 HNMR(400MHz,CD3OD)δ8.70(s,1H),8.49(s,1H),8.41(d,J=6.0Hz,1H),7.70(d,J=7.5Hz,1H),7.58-7.5 2(m,1H),7.44-7.39(m,1H),7.33(d,J=7.8Hz,1H),6.85(d,J=6.0Hz,1H),6.17(d,J=8.3Hz,1H),5.64-5. 58(m,1H),5.49-5.44(m,1H),5.08-5.02(m,1H),4.88-4.78(m,1H),4.76-4.67(m,2H),4.37-4.21(m,3H ),4.06-4.00(m,1H),3.83-3.74(m,1H),2.82(s,3H),2.60-2.32(m,4H),1.54-1.46(m,1H),1.42(s,9H). 31 P NMR (162MHz, CD3OD) δ55.19(s,1P),53.20(s,1P).

[0754] Step 2: N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-[(methylamino)methyl]benzamide, Intermediate 6

[0755] Tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (50 mg, 0.045 mmol) as N,N-diethylethylamine salt was added to a round-bottom flask and cooled to 0° C. A solution of trifluoroacetic acid (0.24 mL, 3.2 mmol) and DCM (0.58 mL) was then added via syringe, and the reaction mixture was stirred at 0° C. for 30 min. The reaction mixture was then concentrated to dryness and placed under vacuum for 2 h to afford Intermediate 6 (41 mg, 100%) as a 2,2,2-trifluoroacetic acid salt. LCMS (AA): m / z = 797.1 (M+H).

[0756] Example 6

[0757] 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, Compound 1 (C-1)

[0758]

[0759] Step 1: 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, Intermediate 7

[0760] To a solution of tert-butyloxycarbonyl-valyl-alanyl-(4-aminobenzyl)-(4-nitrophenyl) carbonate (58 mg, 0.10 mmol) and 4-dimethylaminopyridine (12 mg, 0.10 mmol) in DMF (0.38 mL) and triethylamine (0.055 mL, 0.40 mmol) was added a solution of intermediate 6 (45 mg, 0.05 mmol) in DMF (1.5 mL) at room temperature. The reaction mixture was stirred at room temperature for 15 min. Celite was added and the mixture was concentrated to dryness. The product was purified by reverse phase flash column chromatography (0-40% The crude residue, which was adsorbed onto Celite, was purified by ACN / aqueous triethylammonium acetate solution (10 mM) to afford [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-dihydro-1,3-dihydro-2,10 ... 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl methylcyclopenta[1-][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (Intermediate 7, 10 mg, 16%). LCMS (AA): m / z = 1216.3 (M+H). 1H NMR (400MHz, CD3OD) δ8.70(s,1H),8.43(brs,1H),8.40(d,J=5.9Hz,1H),7.70(d,J=7.5Hz,1H),7.56-7.46(m,3H),7.42-7.38(m,1H),7.3 2-7.19(m,3H),6.84(d,J=5.5Hz,1H),6.15(d,J=7.3Hz,1H),5.62-5.56(m,1H),5.54-5.48(m,1H),5.08-5.02(m,1H),5.04(s,2H),4.88- 4.83(m,1H),4.81-4.76(m,2H),4.53-4.47(m,1H),4.37-4.22(m,3H),4.08-4.03(m,1H),3.92-3.89(m,1H),3.82-3.74(m,1H),2.88(s,3 H),2.58-2.30(m,4H),2.10-2.03(m,1H),1.54-1.47(m,1H),1.46-1.44(m,3H),1.44(s,9H),0.98(d,J=6.8Hz,3H),0.93(d,J=6.8Hz,3H). 31 P NMR (162MHz, CD3OD) δ55.01 (s, 1P), 53.03 (s, 1P).

[0761] Step 2: 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, Intermediate 8

[0762] The title compound was prepared by following the procedure described in step 2 of Example 5 using intermediate 7 (10 mg, 0.008 mmol) instead of tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate and stirring at room temperature for 10 min to give 2, 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate as 2,2-trifluoroacetate salt (Intermediate 8, 9 mg, 100%). LCMS (AA): m / z = 1116.3 (M+H).

[0763] Step 3: 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, Compound C-1

[0764] To a solution of intermediate 8 (8 mg, 0.007 mmol) and N-succinimidyl 6-maleimidocaproate (3.1 mg, 0.010 mmol) in THF (0.20 mL) and DMF (0.10 mL) was added N,N-diisopropylethylamine (2.5 uL, 0.014 mmol) dropwise. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated to dryness and purified by reverse phase flash column chromatography (0-100%). The crude residue was purified by ACN / aqueous ammonium acetate (10 mM) to afford [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]]] as an ammonium salt. 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl]methylcarbamate (C-1, 4.2 mg, 45%). LCMS (AA): m / z = 1309.4 (M+H). HRMS (m / z): C 55 H 66 N 12 O 18 P2S2's [M+H] + Calculated value 1309.3607; experimental value 1309.3639.

[0765] Example 7

[0766] 2-[tert-Butoxycarbonyl(methyl)amino]ethyl chloroformate (Intermediate 9)

[0767]

[0768] Step 1: 2-[tert-Butoxycarbonyl(methyl)amino]ethyl chloroformate, intermediate 9

[0769] To a solution of tert-butyl N-(2-hydroxyethyl)-N-methylcarbamate (296 mg, 1.69 mmol) in THF (4.2 mL) and N,N-diisopropylethylamine (1 mL, 5.9 mmol) cooled to 0°C was slowly added triphosgene (752 mg, 2.53 mmol). The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was filtered, the solid washed with THF, and the filtrate concentrated to dryness to give crude 2-[tert-butoxycarbonyl(methyl)amino]ethyl chloroformate (Intermediate 9, 402 mg, 100%).

[0770] Example 8

[0771] N-[(2-Chlorocarbonylphenyl)methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]phenyl]methyl ester (Intermediate 10)

[0772]

[0773] Step 1: 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid

[0774] The title compound was prepared following the procedure described in Step 1 of Example 6, starting with 2-[(methylamino)methyl]benzoic acid HCl (150 mg, 0.72 mmol) instead of Intermediate 6. After purification by silica gel chromatography (0-25% MeOH / DCM), 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid (306 mg, 67%) was obtained. LCMS (AA): m / z = 583.4 (MH).

[0775] Step 2: N-[(2-Chlorocarbonylphenyl)methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl ester, intermediate 10

[0776] To a solution of 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid (295 mg, 0.50 mmol) in THF (1.5 mL) cooled to 0° C. was added oxalyl chloride (2.0 M solution in DCM, 0.25 mL, 0.50 mmol) followed by 3 drops of DMF. The reaction mixture was stirred at 0° C. for 45 min. The mixture was then concentrated to dryness to give crude Intermediate 10 (304 mg, 100%).

[0777] Example 8A

[0778] 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl (4-chloro-4-oxobutyl)methylcarbamate (Intermediate 34)

[0779]

[0780] Step 1: Methyl 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoate

[0781] The title compound was prepared following the procedure described in Step 1 of Example 6, starting with methyl 4-(methylamino)butanoate HCl (1.44 g, 8.16 mmol) instead of intermediate 6. After purification by silica gel chromatography (0-70% EtOAc / DCM), methyl 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoate (3.45 g, 92%) was obtained. LCMS (AA): m / z = 549.3 (MH).

[0782] Step 2: 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid

[0783] To a solution of methyl 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoate (3.45 g, 6.27 mmol) in THF (40.7 mL) and water (20.3 mL) was added lithium hydroxide monohydrate (657 mg, 15.7 mmol) at 0°C. The homogeneous mixture was then warmed to room temperature and stirred for 2 h. The reaction mixture was cooled to 0°C and added hydrochloric acid (1.0 mol / L aqueous solution, 1.83 mL). The mixture was adjusted to pH <4 by the addition of additional 1N HCl. The solution was warmed to room temperature and diluted with EtOAc and water. The aqueous phase was further extracted with EtOAc. The combined organic phases were washed with brine and dried over magnesium sulfate. After filtration and solvent removal, the residue was purified by silica gel chromatography (0-10% MeOH / EtOAc). Removal of the solvent afforded 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid (3.17 g, 94%) as a white solid. LCMS (AA): m / z = 535.3 (MH).

[0784] Step 3: 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl (4-chloro-4-oxobutyl)methylcarbamate

[0785] The title compound was prepared following the procedure described in step 2 of Example 8, starting with 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid (387 mg, 0.721 mmol) instead of 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid to afford crude intermediate 34 (400 mg, 100%) after concentration of the reaction mixture.

[0786] Example 9

[0787] 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (Intermediate 7)

[0788]

[0789] 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, intermediate 7

[0790] The title compound was prepared following the procedure described in Step 1 of Example 5, substituting Intermediate 10 for tert-butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamate (Intermediate 1). The crude product was purified by reverse phase flash column chromatography (0-50% ACN / aqueous ammonium bicarbonate (5 mM)) to afford Intermediate 7 as an ammonium salt. This material was then dissolved in MeOH (2 mL) and triethylamine (5 mL) was added. The mixture was shaken for 1 min, the solvent was removed and lyophilized overnight to give 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate as N,N-diethylethylamine salt (147 mg, 48%). LCMS (AA): m / z = 1216.3 (M+H). 1H NMR was consistent with that of Example 6, Step 1.

[0791] Example 10

[0792] 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-(chlorocarbonyl)benzyl]2,5,8,11-tetraoxatridecan-13-ylcarbamate (Intermediate 11)

[0793]

[0794] Step 1: 2-(2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid

[0795] 2-carboxybenzaldehyde (1.10g, 7.3mmol) was dissolved in MeOH (15mL) and 2,5,8,11-tetraoxa-13-tridecan-13-amine (1.82g, 8.8mmol) was added and the reaction mixture was stirred at room temperature for 1h. Then sodium borohydride (139mg, 3.7mmol) was slowly added portion by portion. The reaction mixture was stirred at room temperature for 1h. The reaction mixture was quenched by adding acetone (0.65mL) and then concentrated to dryness. The crude residue was purified by silica gel chromatography (0-25% MeOH / DCM) to obtain 2-(2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (2.25g, 87%). LCMS (AA): m / z=342.3 (M+H).

[0796] Step 2: 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid

[0797] The title compound was prepared following the procedure described in Step 1 of Example 6, starting with 2-(2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (764 mg, 2.24 mmol) instead of Intermediate 6, using DCM as the solvent, and stirring for 16 h. After purification by silica gel chromatography (0-25% MeOH / DCM), 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (855 mg, 47%) was obtained. LCMS (AA): m / z = 759.4 (MH).

[0798] Step 3: 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl[2-(chlorocarbonyl)benzyl]2,5,8,11-tetraoxatridecan-13-ylcarbamate, intermediate 11

[0799] The title compound was prepared following the procedure described in step 2 of Example 8, starting with 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (591 mg, 0.777 mmol) instead of 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid to afford, after concentration of the reaction mixture, crude intermediate 11 (605 mg, 100%).

[0800] Example 11

[0801] The compounds listed in Table 3 below were prepared as described in Example 5, using the starting materials indicated in the table in place of tert-butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamate (Intermediate 1).

[0802] Table 3

[0803]

[0804]

[0805] *In step 2, 4 M HCl in 1,4-dioxane with methanol as solvent was used for the deprotection step.

[0806] Example 12

[0807] N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide (Intermediate 17)

[0808]

[0809] Step 1: tert-Butyl [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate

[0810] The title compound was prepared following the procedure described in step 1 of Example 5 using 7-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (compound 1) as its sodium salt. No.14, see WO2018100558A2, 200 mg, 0.265 mmol) and intermediate 3 (503 mg, 1.59 mmol) were used instead of 2-amino-9-[(2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyl-7-yl]-1,9-dihydro-6H-purin-6-one and intermediate 1 to prepare. Purification by reverse phase flash column chromatography (0-50% ACN in aqueous ammonium bicarbonate (5 mM)) gave tert-butyl [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate as an ammonium salt (148 mg, 59%). LCMS (AA): m / z = 910.5 (M+H). 1H NMR(400MHz,D2O)δ8.70(s,1H),8.34(s,1H),8.01(s,1H),7.28(s,1H),6.56(d,J=16. 1Hz,1H),6.43(d,J=8.4Hz,1H),5.67(dd,J=51.0,3.9Hz,1H),5.18-4.99(m,2H),4.82- 4.77(m,1H),4.63-4.58(m,1H),4.49-4.38(m,3H),4.31-4.25(m,1H),4.12-4.06(m,1 H),3.43-3.36(m,2H),2.88(s,3H),2.73-2.68(m,2H),2.04-1.97(m,2H),1.34(s,9H). 31 P NMR(162MHz,D2O)δ55.47(s,1P),52.07(s,1P). 19 FNMR(376MHz, CD3OD)δ-165.50(m,1F),-203.45(m,1F).

[0811] Step 2: N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide, Intermediate 17

[0812] The title compound was prepared following the procedure described in step 2 of Example 5 using [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfanyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-1-[4-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfanyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino]-1-[4-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1 Prepared by replacing tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate with tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate and stirring at room temperature for 30 min. N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfonyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide was obtained as a 2,2,2-trifluoroacetate salt (Intermediate 17, 41 mg, 100%). LCMS (AA): m / z = 810.2 (M+H).

[0813] Example 13

[0814] The compounds listed in Table 5 below were prepared as described in Example 12 using the starting materials shown in Table 4 instead of isobutoxycarbonyl 4-[tert-butoxycarbonyl(methyl)amino]butyrate (Intermediate 3).

[0815] Table 4

[0816]

[0817]

[0818] *In step 2, 4 M HCl in 1,4-dioxane was used for the deprotection step.

[0819] Table 5

[0820]

[0821]

[0822] Example 14

[0823] The compounds listed in Table 7 were prepared as described in Example 6, using the starting materials shown in Table 6 in place of Intermediate 6.

[0824] Table 6

[0825]

[0826]

[0827] *In step 2, p-cresol was added to the reaction mixture at a ratio of 2:1 (p-cresol:TFA).

[0828] Table 7

[0829]

[0830]

[0831] Example 15

[0832] The compounds listed in Table 8 (Intermediate 19) were prepared as described in Step 1 of Example 6, using the starting materials indicated in the table instead of tert-butyloxycarbonyl-valyl-alanyl-(4-aminobenzyl)-(4-nitrophenyl) carbonate.

[0833] Table 8

[0834]

[0835] Example 16

[0836] The compounds listed in Table 10 were prepared as described in Steps 2 and 3 of Example 6, using the starting materials shown in Table 9 instead of Intermediate 7.

[0837] Table 9

[0838]

[0839] Table 10

[0840]

[0841]

[0842] Example 17

[0843] The compounds listed in Table 12 were prepared as described in Step 3 of Example 6, using the starting materials shown in Table 11 instead of N-succinimidyl 6-maleimidocaproate.

[0844] Table 11

[0845]

[0846]

[0847]

[0848] *Triethylamine was used as the base instead of N,N-diisopropylethylamine.

[0849] Table 12

[0850]

[0851]

[0852] Example 18

[0853] The compounds listed in Table 14 were prepared as described in Steps 2 and 3 of Example 6, using the starting materials shown in Table 13 instead of Intermediate 7 and N-succinimidyl 6-maleimidocaproate, respectively.

[0854] Table 13

[0855]

[0856]

[0857]

[0858]

[0859]

[0860]

[0861] *Triethylamine was used as the base instead of N,N-diisopropylethylamine. **Step 2 in Example 6 was skipped.

[0862] Table 14

[0863]

[0864]

[0865]

[0866]

[0867] Example 19

[0868] [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo- 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl ester (compound 20 (C-20))

[0869]

[0870] Step 1: [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphatidyl]cyclotetradecin-7-yl] ]-6-oxo-6,9-dihydro-1H-purin-2-yl}amino)-2,2-dimethyl-4-oxobutyl]methylcarbamate, 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl ester, Compound 20

[0871] To a solution of [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (47.9 mg, 0.037 mmol) and 1-hydroxy-7-azabenzotriazole (4.08 mg, 0.03 mmol) in DMF (0.60 mL) and N,N-diisopropylethylamine (26 uL, 0.15 mmol) was added N-{9-[(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (47.9 mg, 0.037 mmol) and 1-hydroxy-7-azabenzotriazole (4.08 mg, 0.03 mmol) in DMF (0.60 mL) and N,N-diisopropylethylamine (26 uL, 0.15 mmol) at room temperature was added N-{9-[(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (47.9 mg, 0.037 mmol) and 1-hydroxy-7-azabenzotriazole (4.08 mg, 0.03 mmol) in DMF (0.60 mL) and N,N-diisopropylethylamine (26 uL, 0.15 mmol) as a 2,2,2-trifluoroacetate salt. A solution of (2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15aS, 16R) -16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy) -2,10-dihydrosulfanyl decahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -6-oxo-6,9-dihydro-1H-purin-2-yl} -3,3-dimethyl-4-(methylamino)butanamide (Intermediate 14, 20 mg, 0.02 mmol) in DMF (0.54 mL) was added. The reaction mixture was stirred at room temperature for 16 h. Celite was added and the mixture was concentrated to dryness. The product was purified by reverse phase flash column chromatography (0-100%). The crude residue adsorbed onto Celite was purified by ACN / aqueous ammonium acetate solution (10 mM) to afford [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}amino)-2,2-dimethyl-4-oxobutyl]methylcarbamate (C-20, 4 mg, 17%). LCMS (AA): m / z = 1287.4 (MH).

[0872] Example 20

[0873] The compounds listed in Table 16 were prepared as described in Example 19 using starting materials A and B shown in Table 15 instead of Intermediate 14 and [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate, respectively.

[0874] Table 15

[0875]

[0876]

[0877] *1-Hydroxybenzotriazole and triethylamine were used in the reaction instead of 1-hydroxy-7-azabenzotriazole and N,N-diisopropylethylamine.

[0878] **4-Dimethylaminopyridine and triethylamine were used in the reaction instead of 1-hydroxy-7-azabenzotriazole and N,N-diisopropylethylamine.

[0879] Table 16

[0880]

[0881]

[0882] Example 21

[0883] N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14-pentaoxaheptadecan-17-amide (Intermediate 20)

[0884]

[0885] Step 1: (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaeicosanoic acid

[0886] To a solution of 1-[(17-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl)oxy]pyrrolidine-2,5-dione (4.0 g, 10.5 mmol) in anhydrous DCM (10 mL) was added (S)-2-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid hydrochloride (3.4 g, 12.9 mmol) dissolved in DMF (40 mL), followed by N,N-diisopropylethylamine (6.9 mL, 42 mmol). The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was filtered and concentrated to dryness. The crude residue was purified by preparative HPLC to give (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaeicosanoic acid (1.26 g, 24%). LCMS (AA): m / z = 489.3 (M+H).

[0887] Step 2: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14-pentaoxaheptadecan-17-carboxamide, intermediate 20

[0888] To a solution of (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaeicosane-20-oic acid (65 mg, 0.133 mmol) and N,N'-dicyclohexylcarbodiimide (30 mg, 0.145 mmol) in 1,4-dioxane (0.68 mL) was added N-hydroxysuccinimide (15.3 mg, 0.133 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. Celite was added and the mixture was filtered, rinsed with 1,4-dioxane and concentrated to dryness. The crude residue adsorbed onto Celite was purified by silica gel chromatography (0-10% MeOH / DCM) to give Intermediate 20 (14.6 mg, 19%). LCMS (AA): m / z = 586.3 (M+H).

[0889] Example 22

[0890] The compounds listed in Table 17 were prepared as described in Example 9, substituting the starting materials indicated in the table for Intermediate 10.

[0891] Table 17

[0892]

[0893]

[0894] Example 23

[0895] (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yl)- [4-[( ...

[0896]

[0897] Step 1: (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propionyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo- 14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylic acid methyl ester

[0898] The title compound was prepared following the procedure described in Step 1 of Example 6 using intermediate 6 (104 mg, 0.094 mmol) and methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[2-[3-(9H-fluoren-9-ylmethoxycarbonylamino)propionylamino]-4-[(4-nitrophenoxy)carbonyloxymethyl]phenoxy]tetrahydropyran-2-carboxylate (150 mg, 0.16 mmol) (for synthesis, see Jeffrey, SC et al., Bioconjugate Chem. 2006, 17, 831-840), using N,N-diisopropylethylamine as a base to prepare (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16 [R]-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylic acid methyl ester (71 mg, 48%). LCMS (AA): m / z = 1570.9 (M+H).

[0899] Step 2: (2S,3S,4S,5R,6S)-6-{2-[(3-aminopropanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, Intermediate 22

[0900] At room temperature, (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[ To a solution of [1,3,6,9,11,2,10] pentaoxadiphosphatetradecyl ring-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylic acid methyl ester (66 mg, 0.042 mmol) dissolved in THF (13 mL) was added LiOH (0.5 M aqueous solution, 1.25 mL, 0.625 mmol) and the reaction mixture was stirred for 2.5 h. The reaction mixture was neutralized to pH 7 with 1 M HCl and the mixture was concentrated to dryness. The crude residue was purified by reverse phase flash column chromatography (0-30% ACN / ammonium bicarbonate aqueous solution (5 mM)) to obtain intermediate 22 (26 mg, 50%) in the form of an ammonium salt. This substance was then dissolved in MeOH (5 mL) and triethylamine (1 mL) was added. The mixture was shaken for 1 min. The solvent was removed and the resulting residue was lyophilized overnight to give (2S,3S,4S,5R,6S)-6-{2-[(3-aminopropanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)- [2,10-Dihydrothiodecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl]carbamoyl]benzyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (Intermediate 22, 30 mg, 100%). LCMS (AA): m / z = 1209.0 (M+H).

[0901] Step 3: (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin ... [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, Compound 24

[0902] The title compound was prepared following the procedure described in step 3 of Example 6 using intermediate 22 (24 mg, 0.02 mmol) instead of intermediate 8 to give (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS, [16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (C-24, 17 mg, 71%). LCMS (AA): m / z = 1402.4 (M+H). HRMS (m / z): C 56 H 65 N 11 O 24 P2S2's [M+H] + Calculated value 1402.3193; experimental value 1402.3232.

[0903] Example 24

[0904] The compounds listed in Table 18 (Compound 25) were prepared as described in Step 3 of Example 23, using the starting materials indicated in the table in place of N-succinimidyl-6-maleimidocaproate.

[0905] Table 18

[0906]

[0907] C-25 Structure:

[0908]

[0909] Example 25

[0910] {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl] 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate, Compound 26 (C-26)

[0911]

[0912] Step 1: 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methoxycarbonylamino]acetic acid

[0913] To a solution of glycine (24 mg, 0.32 mmol) in triethylamine (0.10 mL, 0.72 mmol), DMF (0.50 mL) and DMSO (0.50 mL) was added [4-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (220 mg, 0.34 mmol) at room temperature. The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was then cooled to 0 ° C. Water (1 mL) was added and the solution was acidified to pH 4 by adding formic acid. The mixture was concentrated to dryness, and the crude residue was purified by reverse phase flash column chromatography (0-100% ACN / aqueous formic acid (0.1%)) to give 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]acetic acid (87 mg, 44%). LCMS (AA): m / z = 586.3 (MH).

[0914] Step 2: {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphatadecadecyl]-1,6-dihydro-2,10-dioxo-1,4-dihydro ... 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl ester, Compound 26

[0915] To a solution of 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methoxycarbonylamino]acetic acid (40 mg, 0.068 mmol) and triethylamine (35 uL, 0.25 mmol) in DMF (1 mL) was added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (46 mg, 0.12 mmol) at room temperature, and the reaction mixture was stirred for 10 min. To this solution was then added dropwise a solution of (2R)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide (Intermediate 15, 50 mg, 0.047 mmol) in DMF (0.60 mL) as a 2,2,2-trifluoroacetate salt at room temperature. The reaction mixture was stirred at room temperature for 30 min. The mixture was then cooled to 0° C., water (1 mL) was added, and the solution was acidified to pH 4 by the addition of formic acid. The mixture was concentrated to dryness and the crude residue was purified by reverse phase flash column chromatography (0-100% ACN / aqueous formic acid (0.1%)) to afford {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphine]-1,4-dihydro-1,4-dihydro-2,10-dioxo ... 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate (C-26, 32 mg, 50%). LCMS (AA): m / z = 1316.4 (M+H). HRMS (m / z): C 53 H 67 N 13 O 19 P2S2's [M+H] + Calculated value 1316.3666; experimental value 1316.3705.

[0916] Example 26

[0917] (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}acetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)- 16-Hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide, Compound 27 (C-27)

[0918]

[0919] Step 1: tert-Butyl {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate

[0920] To a mixture of intermediate 15 (60 mg, 0.05 mmol) and (tert-butoxycarbonyl)glycine 2,5-dioxopyrrolidin-1-yl ester (19 mg, 0.07 mmol) in DMF (1.6 mL) was added triethylamine (50 uL, 0.355 mmol), and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with water, Celite was added, and the reaction mixture was concentrated to dryness. The crude residue adsorbed onto Celite was purified by reverse phase column chromatography (0-100% ACN / ammonium bicarbonate aqueous solution (5 mM)). Triethylamine (14 uL, 0.1 mmol) was added to the aqueous solution of the product. Lyophilization overnight provided tert-butyl {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate (49 mg, 87%) as an N,N-diethylethanamine salt. LCMS (AA): m / z=904.4 (M+H).

[0921] Step 2: (2R)-1-(Aminoacetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide

[0922] To a solution of tert-butyl {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate as N,N-diethylethanamine salt (37 mg, 0.033 mmol) in MeOH (1 mL) was added hydrochloric acid (4 M solution in dioxane, 1 mL, 4 mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated to dryness and placed under vacuum for 2 h to give (2R)-1-(aminoacetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide (37 mg, 100%) as the hydrochloride salt. LCMS (AA): m / z=804.2 (M+H).

[0923] Step 3: (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}acetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-ethylidenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide, Compound 27

[0924] The title compound was prepared following the procedure described in step 1 of Example 26 using (2R)-1-(aminoacetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphatadecadecyl]-1-[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[ 7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide (30 mg, 0.027 mmol) and (2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butyryl]amino]propionic acid (2,5-dioxopyrrolidin-1-yl) ester (24 mg, 0.04 mmol) (for synthesis, see US20180015176A1). The product was prepared by reverse phase flash column chromatography (0-100%). The product was purified by ACN / aqueous formic acid solution (0.1%) to give (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}acetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,1 5aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide (C-27, 20 mg, 64%). LCMS (AA): m / z = 1167.1 (M+H). HRMS (m / z): C 45 H 60 N 12 O 17 P2S2's [M+H] + Calculated value 1167.3189; experimental value 1167.3206.

[0925] Example 27

[0926] 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, Compound 28 (C-28)

[0927]

[0928] Step 1: (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1]] tert-Butyl]-1-oxopropyl-2-yl]amino]-3-methyl-1-oxobutyl-2-yl]amino]-2-oxoethyl]carbamate

[0929] The title compound was prepared following the procedure described in step 3 of Example 6, starting with intermediate 8 (23 mg, 0.016 mmol) and (tert-butoxycarbonyl)glycine 2,5-dioxopyrrolidin-1-yl ester (11 mg, 0.039 mmol) instead of N-succinimidyl 6-maleimidocaproate. The reaction mixture was stirred at room temperature for 30 min. The product was purified by reverse phase flash column chromatography (0-100%). The product was purified by ACN / triethylammonium acetate aqueous solution (10 mM) to afford (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5-nitropropane)]-1,4-dihydro-1,6-dihydro-2,10-dioxo ...hydro-1,6-dihydro-1,6-dihydro-2,10-dioxo-1,4-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-1,6-dihydro-2,10-dioxo-1,6-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-2,10-dioxo-1,6-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-2,10-dihydro-1,6-dihydro-2 tert-Butyl (1,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}amino)-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]amino}-2-oxoethyl)carbamate (13 mg, 57%). LCMS(AA): m / z=1273.1(M+H).

[0930] Step 2: 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, Compound 28

[0931] The title compound was prepared following the procedure described in step 2 of Example 5 using (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl} as its N,N-diethylethanamine salt. Prepared starting with tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate. The reaction mixture was stirred at room temperature for 5 min to give 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate as 2,2,2-trifluoroacetate salt (C-28, 5.6 mg, 91%). LCMS (AA): m / z = 1173.3 (M+H).

[0932] Example 28

[0933] The compounds listed in Table 20 were prepared as described in Example 27 using the starting materials shown in Table 19 instead of (tert-butoxycarbonyl)glycine 2,5-dioxopyrrolidin-1-yl ester.

[0934] Table 19

[0935]

[0936] Table 20

[0937]

[0938]

[0939] Example 29

[0940] tert-Butyl {14-[(2,5-dioxopyrrolidin-1-yl)oxy]-14-oxo-3,6,9,12-tetraoxatetradec-1-yl}carbamate (Intermediate 23)

[0941]

[0942] Step 1: tert-Butyl {14-[(2,5-dioxopyrrolidin-1-yl)oxy]-14-oxo-3,6,9,12-tetraoxatetradec-1-yl}carbamate, Intermediate 23

[0943] To a solution of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecan-19-oic acid (120 mg, 0.32 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (78 mg, 0.49 mmol) in DCM (6.4 mL) was added N-hydroxysuccinimide (46 mg, 0.39 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2.5 h. The reaction mixture was quenched with water and extracted with DCM (3×). The combined organic phases were washed with brine, dried (NaSO), filtered, and evaporated to afford crude tert-butyl {14-[(2,5-dioxopyrrolidin-1-yl)oxy]-14-oxo-3,6,9,12-tetraoxatetradec-1-yl}carbamate (Intermediate 23, 146 mg, 100%). LCMS (AA): m / z = 447.2 (MH).

[0944] Example 30

[0945] Tert-butyl N-[(1S)-2-[(2-chlorocarbonylphenyl)methyl-[(2,4-dimethoxyphenyl)methyl]amino]-1-methyl-2-oxoethyl]carbamate (Intermediate 24)

[0946]

[0947] Step 1: 2-[[(2,4-Dimethoxyphenyl)methylamino]methyl]benzoic acid

[0948] The title compound was prepared following the procedure described in Step 1 of Example 10 starting with 2-carboxybenzaldehyde (6.0 g, 40 mmol) and 2,4-dimethoxybenzylamine (12 mL, 80 mmol) to give 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid (7.6 g, 61%). LCMS (AA): m / z = 302.2 (M+H).

[0949] Step 2: 2-[[[(2S)-2-(tert-Butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid

[0950] To a solution of 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid (380 mg, 1.26 mmol) and triethylamine (0.5 mL, 3.55 mmol) in DMF (6 mL) was added (tert-butoxycarbonyl)-L-alanine 2,5-dioxopyrrolidin-1-yl ester (640 mg, 2.2 mmol). Water (2 mL) was added to dissolve the reaction mixture, which was stirred at room temperature for 1 day. The reaction mixture was then concentrated to dryness, and the crude residue was purified by reverse phase flash column chromatography (0-100% ACN / aqueous formic acid (0.1%)) to give 2-[[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid (322 mg, 51%). LCMS (AA): m / z = 473.3 (M+H).

[0951] Step 3: tert-Butyl N-[(1S)-2-[(2-chlorocarbonylphenyl)methyl-[(2,4-dimethoxyphenyl)methyl]amino]-1-methyl-2-oxo-ethyl]carbamate, intermediate 24

[0952] The title compound was prepared following the procedure described in step 2 of Example 1 starting with 2-[[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid (270 mg, 0.57 mmol) instead of 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid to give crude intermediate 24 (280 mg, 100%).

[0953] Example 31

[0954] 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide, Compound 31 (C-31)

[0955]

[0956] Step 1: tert-Butyl [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]amino}-1-oxopropyl-2-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamate

[0957] The title compound was prepared following the procedure described in Step 1 of Example 26, starting with Intermediate 16 (45 mg, 0.53 mmol) and (tert-butoxycarbonyl)-L-valine 2,5-dioxopyrrolidin-1-yl ester (100 mg, 0.32 mmol) instead of Intermediate 15 and (tert-butoxycarbonyl)glycine 2,5-dioxopyrrolidin-1-yl ester, respectively. The product was purified by reverse phase flash column chromatography (0-100%). The mixture was purified by ACN / aqueous ammonium acetate (10 mM) to afford tert-butyl [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamate as an ammonium salt (24 mg, 44%). LCMS (AA): m / z = 1053.0 (M+H).

[0958] Step 2: 2-({[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide

[0959] The title compound was prepared following the procedure described in step 2 of Example 5 using [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphine] as an ammonium salt. [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dihydro-14-(pyrimidine-4-yl)-1-oxo ... [(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}methyl]-1-[[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino} ... The product was prepared by mixing with 4-[[(4-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide (24 mg, 97%). LCMS (AA): m / z = 953.1 (M+H).

[0960] Step 3: 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide, Compound 31

[0961] The title compound was prepared following the procedure described in step 3 of Example 6 starting with 2-({[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide as the 2,2,2-trifluoroacetate salt (24 mg, 0.022 mmol) instead of intermediate 8 to obtain 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16 R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide (C-31, 6.2 mg, 23%). LCMS (AA): m / z = 1146.1 (M+H). HRMS (m / z): C 46 H 57 N 11 O 16 P2S2's [M+H] + Calculated value 1146.2974; experimental value 1146.2998.

[0962] Example 32

[0963] 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propionyl]amino}benzyl 4-nitrophenyl carbonate (Intermediate 25)

[0964]

[0965] Step 1: 6-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-N-[(2S)-1-{[(2S)-1-{[4-(hydroxymethyl)phenyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-oxohexanamide

[0966] To a solution of (2S)-2-amino-N-[(1S)-2-[4-(hydroxymethyl)phenylamino]-1-methyl-2-oxo-ethyl]-3-methyl-butyramide (240 mg, 0.82 mmol) dissolved in DMF (2 mL) and THF (5.5 mL) was added N,N-diisopropylethylamine (0.39 mL, 2.24 mmol). The reaction mixture was cooled to 0°C and 1-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]oxy}pyrrolidine-2,5-dione (320 mg, 0.75 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 30 min. The reaction mixture was diluted with water and extracted with EtOAc (3×). The combined organic phases were washed with saturated aqueous sodium bicarbonate solution and brine, dried (Na2SO4), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (50-100% EtOAc / DCM) to give 6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-N-[(2S)-1-{[(2S)-1-{[4-(hydroxymethyl)phenyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-oxohexanamide (347 mg, 76%). LCMS (AA): m / z = 609.4 (M+H).

[0967] Step 2: 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl 4-nitrophenyl carbonate, intermediate 25

[0968] To a solution of 6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-N-[(2S)-1-{[(2S)-1-{[4-(hydroxymethyl)phenyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-oxohexanamide (257 mg, 0.42 mmol) in THF (4.2 mL) and N,N-diisopropylethylamine (0.37 mL, 2.1 mmol) cooled to 0°C was added 4-nitrophenyl chloroformate (362 mg, 1.69 mmol) in one portion. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was diluted with water and extracted with DCM (3×). The combined organic phases were washed with saturated aqueous sodium bicarbonate solution and brine, dried (Na2SO4), filtered and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-100% EtOAc / DCM) to give 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl 4-nitrophenyl carbonate (Intermediate 25, 137 mg, 42%). LCMS (AA): m / z = 774.4 (M+H).

[0969] Example 33

[0970] The compounds listed in Table 21 (Intermediate 26) were prepared as described in Example 32, substituting the starting materials indicated in the table for 1-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]oxy}pyrrolidine-2,5-dione.

[0971] Table 21

[0972]

[0973]

[0974] Example 34

[0975] (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosano-22-yl)-4-oxobutanamide (Intermediate 27) and (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosanoic-27-oic acid (Intermediate 27A)

[0976]

[0977] Step 1: (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester

[0978] The title compound was prepared following the procedure described in Step 1 of Example 26 starting with tert-butyl (3S)-3-{[(benzyloxy)carbonyl]amino}-4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutanoate (2.2 g, 5.3 mmol) and 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (2.0 g, 5.9 mmol) instead of Intermediate 15 and (tert-butoxycarbonyl)glycine 2,5-dioxopyrrolidin-1-yl ester, respectively. Purification by silica gel chromatography (0-10% MeOH / DCM) gave tert-butyl (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (3.8 g, 100%).

[0979] Step 2: (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester

[0980] Tert-butyl (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (3.8 g, 5.9 mmol) was dissolved in ethanol (100 mL). Palladium (10% on carbon, 1.38 g, 1.17 mmol) was added and the mixture was stirred under hydrogen (15 psi) for 3 h. The reaction mixture was filtered, washed with methanol, and the filtrate was evaporated to dryness to yield tert-butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (2.56 g, 85%).

[0981] Step 3: (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester

[0982] To a solution of (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester (3.32 g, 6.5 mmol), 4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoic acid (1.5 g, 4.91 mmol) and 1-hydroxy-7-azabenzotriazole (884 mg, 6.57 mmol) in DCM (50 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1.25 g, 6.56 mmol) and triethylamine (1.26 mL, 9.1 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction was diluted with EtOAc and potassium carbonate (20 mL, 4.0 M in water) was added. The mixture was stirred for 15 min and the organic phase was separated, washed with saturated aqueous sodium bicarbonate, brine, dried (Na2SO4), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-10% MeOH / DCM) to give (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester (2.27 g, 44%).

[0983] Step 4: (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosa-27-oic acid

[0984] The title compound was prepared following the procedure described in step 2 of Example 5, substituting (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester (278 mg, 0.35 mmol) for [2-({9-[(2R,5R,7

[00145] The invention relates to the preparation of the present invention using tert-butyl 1,4-dihydro-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate. Purification by silica gel chromatography (0-20% MeOH / DCM) gave (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (Intermediate 27A, 180 mg, 70%). LCMS (AA): m / z = 742.4 (M+H).

[0985] Step 5: (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosan-22-yl)-4-oxobutanamide, Intermediate 27

[0986] The title compound was prepared following the procedure described in Example 29 starting with (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosacan-27-oic acid (60 mg, 0.08 mmol) instead of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecan-19-oic acid. The reaction mixture was stirred at room temperature for 30 min to give (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azenin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosan-22-yl)-4-oxobutanamide (Intermediate 27, 68 mg, 100%). LCMS (AA): m / z = 839.4 (M+H).

[0987] Example 35

[0988] The compounds listed in Table 23 were prepared as described in Step 3 of Example 6, using the starting materials shown in Table 22 instead of N-succinimidyl 6-maleimidocaproate.

[0989] Table 22

[0990]

[0991]

[0992] *Triethylamine was used as the base instead of N,N-diisopropylethylamine.

[0993] Table 23

[0994]

[0995] Example 36

[0996] Tert-butyl {(29S)-30-[(2,5-dioxopyrrolidin-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacont-29-yl}carbamate (Intermediate 30)

[0997]

[0998] Step 1: (29S)-29-[(tert-Butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacont-30-oic acid

[0999] The title compound was prepared following the procedure described in step 1 of Example 21 starting with (S)-3-amino-2-((tert-butoxycarbonyl)amino)propanoic acid (238 mg, 1.17 mmol) and 1-[(26-oxo-2,5,8,11,14,17,20,23-octaoxahexacosacontan-26-yl)oxy]pyrrolidine-2,5-dione (300 mg, 0.58 mmol) instead of (S)-2-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid hydrochloride and 1-[(17-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl)oxy]pyrrolidine-2,5-dione using DMF as solvent and stirring at room temperature for 2 h. Purification by reverse phase flash column chromatography (0-100% ACN / 10 mM aqueous triethylammonium acetate) gave (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontano-30-oic acid (344 mg, 99%). LCMS (AA): m / z = 599.4 (M+H).

[1000] Step 2: tert-Butyl {(29S)-30-[(2,5-dioxopyrrolidin-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacont-29-yl}carbamate, intermediate 30

[1001] The title compound was prepared following the procedure described in step 2 of Example 21 starting with (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacont-30-oic acid (100 mg, 0.166 mmol) instead of (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaeicosano-20-oic acid using DCM as solvent and stirring at room temperature for 1 h. The reaction mixture was filtered and concentrated to give crude tert-butyl {(29S)-30-[(2,5-dioxopyrrolidin-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacont-29-yl}carbamate (Intermediate 30, 116 mg, 100%).

[1002] Example 37

[1003] N-[[2-[[9-[(2R,3R,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]phenyl]methyl ester (Intermediate 32)

[1004]

[1005] Step 1: [4-[[(2S)-2-[[(2S)-2-(tert-Butyloxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-3-[tert-Butyl(dimethyl)silyl]oxy-5-[[tert-Butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]phenyl]methyl ester

[1006] 2-Amino-9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-1H-purin-6-one (1.62 g, 3.27 mmol) (for synthesis, see Prakash, TPJ Med. Chem. 2005, 48, 1199-1210) was dissolved in anhydrous pyridine and concentrated to dryness (3 x 3 mL), then placed under vacuum for 15 min. The residue was dissolved in pyridine (43 mL) under an argon atmosphere and a solution of Intermediate 10 (5.91 g, 9.80 mmol) in pyridine (32 mL) was added. The reaction mixture was stirred at room temperature for 16 h. MeOH (20 mL) was added and the reaction mixture was concentrated to dryness. MeOH (100 mL) and ammonium hydroxide (28-30% aqueous solution, 50 mL) were added and the resulting mixture was stirred for 60 min. The reaction mixture was concentrated to dryness and the crude residue was adsorbed onto Celite and purified by silica gel chromatography (0-100% EtOAc / hexanes) to give [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl ester (2.17 g, 63%). LCMS (AA): m / z = 1062.0 (M+H).

[1007] Step 2: N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl ester

[1008] To a polypropylene tube was added N-[[2-[[9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl ester (2.17 g, 2.04 mmol) dissolved in THF (27 mL). Triethylamine (2.26 mL, 16.1 mmol) was added followed by triethylamine trihydrofluoride (1.19 mL, 7.33 mmol) and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was slowly quenched by the addition of saturated aqueous sodium bicarbonate and then diluted with EtOAc. The organic phase was separated and washed with brine, dried (Na2SO4), filtered and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-20% MeOH / DCM) to give [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl ester (1.18 g, 69%). LCMS (AA): m / z=834.4 (M+H).

[1009] Step 3: N-[[2-[[9-[(2R,3R,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl ester, intermediate 32

[1010] N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl ester (1.13 g, 1.36 mmol) was dissolved in anhydrous pyridine and concentrated to dryness (3×15 mL). The residue was dissolved in pyridine (13.3 mL) and 4,4′-dimethoxytrityl chloride (650 mg, 1.9 mmol) was added, followed by 4-dimethylaminopyridine (8 mg, 0.068 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated to dryness and adsorbed onto Celite and purified by silica gel chromatography (0-5% MeOH / DCM with 0.5% NEt3) to give N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl ester (Intermediate 32, 1.12 g, 73%). LCMS (AA): m / z = 1136.5 (M+H).

[1011] Example 38

[1012] 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxo-14-(9H-purin-9-yl)-2-hydrosulfenyldecahydro-5,8-methylenefuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate or [2- 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl ({9-[(2R,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxo-14-(9H-purin-9-yl)-2-hydrosulfenyldecahydro-5,8-methylenefuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (Intermediate 33)

[1013]

[1014] Step 1: N-[[2-[[9-[(2R,3R,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl ester

[1015] N-[[2-[[9-[(2R,3R,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl ester (Intermediate 32,1 .1 g, 0.97 mmol), N-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-purin-9-yl-tetrahydrofuran-2-yl]methyl]aminosulfonic acid (4-nitrophenyl) ester (667 mg, 1.17 mmol) (for synthesis, see PCT Publication No. PCT / IB2019 / 052364), 4-dimethylaminopyridine (122 mg, 0.97 mmol) and A mixture of molecular sieves (1.74 g) in anhydrous DCM (13.6 mL) was stirred at room temperature under argon for 10 min. Triethylamine (0.60 mL, 4.3 mmol) was then added and the reaction mixture was stirred at 40 ° C for 16 h. The reaction mixture was filtered and concentrated, and the product was purified by reverse phase column chromatography (10-100%). The resulting residue was purified by ACN / aqueous ammonium bicarbonate solution (5 mM) to give [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl ester (0.94 g, 62%). LCMS (AA): m / z = 1565.0 (M+H).

[1016] Step 2: N-[[2-[[9-[(2R,3R,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl ester

[1017] To N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl To a solution of [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl]-N-methylcarbamate (939 mg, 0.60 mmol) in THF (12 mL) in a polypropylene tube was added triethylamine trihydrofluoride (0.52 mL, 3.17 mmol) and triethylamine (0.98 mL, 6.95 mmol). The reaction solution was stirred at room temperature overnight, then diluted with dichloromethane and washed sequentially with saturated aqueous NaHCO3 and brine. The organic phase was dried over anhydrous MgSO4, filtered, and concentrated. The obtained residue was purified by reverse phase column chromatography (10-100% ACN / aqueous ammonium bicarbonate solution (5 mM)) to give [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl ester (795 mg, 89%). LCMS (AA): m / z = 1452.2 (M+H).

[1018] Step 3: [(2R,3R,4R,5R)-2-[[[(2R,3R,5S)-2-[2-[[2-[[[4-[[(2S)-2-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoyl]amino]-6-oxo-1H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid

[1019] Diphenyl phosphite (0.225 mL, 1.17 mmol) was added to a 0° C. solution of N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propionyl]amino]phenyl]methyl ester (792 mg, 0.55 mmol) in pyridine (2 mL). After complete addition, the reaction mixture was stirred at room temperature for 1 h. Triethylamine (0.62 mL, 4.37 mmol) and water (0.51 mL, 28.3 mmol) were then added, and the reaction mixture was stirred at room temperature for another 60 minutes and then concentrated to dryness. The residue was then dissolved in acetic acid (1.25 mL, 21.8 mmol) and water (0.33 mL, 18.6 mmol) and stirred at room temperature for 1 h. The reaction mixture was quenched by adding triethylsilane (4.3 mL, 26.2 mmol) and stirred at room temperature for 1 h. The reaction mixture was then concentrated to dryness, and the residue was purified by reverse phase column chromatography (10-100% ACN in aqueous ammonium bicarbonate (5 mM)) to give [(2R,3R,4R,5R)-2-[[[(2R,3R,5S)-2-[2-[[2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoyl]amino]-6-oxo-1H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid as an ammonium salt (396 mg, 59%). LCMS (AA): m / z = 1213.4 (M+H).

[1020] Step 4: 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxo-14-(9H-purin-9-yl)-2-hydrosulfenyldecahydro-5,8-methylenefuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate or 4-{[(2S)-2-({(2S)-2-[(tert-Butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxo-14-(9H-purin-9-yl)-2-hydrosulfenyldecahydro-5,8-methylenefuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, intermediate 33

[1021] Diphenyl chlorophosphate (0.055 mL, 0.26 mmol) was added to pyridine (1.6 mL) and the solution was cooled to -35 °C. To the cooled reaction mixture was added a solution of [(2R,3R,4R,5R)-2-[[[(2R,3R,5S)-2-[2-[[2-[[[4-[[(2S)-2-[[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoyl]amino]-6-oxo-1H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid (80 mg, 0.065 mmol) as an ammonium salt in DCM (2.5 mL) and pyridine (3.2 mL) dropwise over 15 min. The resulting solution was stirred at -35 °C for 30 min. Solid 3H-1,2-benzodithiol-3-one (25 mg, 0.15 mmol) and water (0.056 mL) were added to the cooled reaction mixture, which was stirred at room temperature for another 1 h. The reaction solution was then cooled to 0 ° C in an ice bath and quenched by adding aqueous sodium thiosulfate solution (0.2 M aqueous solution, 0.14 mL, 0.70 mmol). The mixture was stirred at room temperature for 5 min and concentrated under reduced pressure. The product was purified by reverse phase column chromatography (0-50% The resulting residue was purified with ACN / aqueous triethylammonium acetate (10 mM) to afford 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl]-2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxo-14-(9H-purin-9-yl)-2-hydrosulfenyldecahydro-5,8-methylenefuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate as N,N-diethylethanamine salt.}amino)propanoyl]amino}benzyl [2-({9-[(2R,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxo-14-(9H-purin-9-yl)-2-hydrosulfenyldecahydro-5,8-methylenefuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (Intermediate 33, 27 mg, 31%).LCMS (AA): m / z = 1227.9 (M+H). 1 HNMR(400MHz,CD3OD)δ9.14(s,1H),8.75(s,1H),8.63(s,1H),8.30(s,1H),7.53-7.36(m,5H),7.34-7.17(m,3H),6.47(d,J=19.3Hz, 1H),6.16(d,J=5.1Hz,1H),5.87-5.82(m,1H),5.76-5.55(m,1H),5.50-5.40(m,1H),5.00(s,2H),4.79-4.75(m,2H),4.68-4.61(m,1 H),4.58-4.47(m,2H),4.44-4.39(m,1H),4.09-4.02(m,1H),3.92-3.89(m,1H),3.67-3.62(m,1H),3.57-3.53(m,1H),3.01-2.94(m, 1H),2.87(s,3H),2.74-2.68(m,1H),2.11-2.03(m,1H),1.45-1.43(m,3H),1.44(s,9H),0.98(d,J=6.7Hz,3H),0.93(d,J=6.7Hz,3H). 31 P NMR (162MHz, CD3OD) δ57.35 (s, 1P).

[1022] Example 38A

[1023] [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H 4-{[(25S,29S,32S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-29-isopropyl-32-methyl-24,27,30,33-tetraoxo-2,5,8,11,14,17,20-heptaoxa-23,28,31-triazatricarboxan-33-yl]amino}benzyl ester (C-34)

[1024]

[1025] to 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}-[4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamic acid To a suspension of the benzyl ester (Int-35, 17.4 mg, 0.0154 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.49 mg, 0.0182 mmol), and (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (Int-27A) (10.4 mg, 0.0140 mmol) was added triethylamine (0.004 mL, 0.0266 mmol) in DCM (0.37 mL). The mixture was stirred at room temperature for 20 min, then DMF (0.15 mL, 2.23 mmol) was added. After this mixture was stirred at room temperature for 2 h, the solvent was concentrated. The crude residue was purified by reverse phase flash column chromatography (0-50% ACN / triethylammonium acetate aqueous solution (10 mM)) to give [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfanyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiazole] as N,N-diethylethanamine salt. 4-{[(25S,29S,32S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-29-isopropyl-32-methyl-24,27,30,33-tetraoxo-2,5,8,11,14,17,20-heptaoxa-23,28,31-triazatricarboxan-33-yl]amino}benzyl phosphatetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate (C-34) (2.0 mg, 6.9%).LCMS (AA): m / z=926.9 (M / 2+H). HRMS(m / z):C. 80 H 101 F2N 15 O 26 P2S2's [M+H] + Calculated value 1852.6000; experimental value 1852.6006.

[1026] Example 38B

[1027] (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl {(25S)-27-[(2,5-dioxopyrrolidin-1-yl)oxy]-24,27-dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosa-25-yl}carbamate (Intermediate 36)

[1028]

[1029] Step 1: (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester

[1030] The title compound was prepared following the procedure described in Step 1 of Example 26 starting with tert-butyl (3S)-3-{[(benzyloxy)carbonyl]amino}-4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutanoate (2.2 g, 5.3 mmol) and 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (2.0 g, 5.9 mmol) instead of Intermediate 15 and (tert-butoxycarbonyl)glycine 2,5-dioxopyrrolidin-1-yl ester, respectively. Purification by silica gel chromatography (0-10% MeOH / DCM) gave tert-butyl (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (3.8 g, 100%).

[1031] Step 2: (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester

[1032] Tert-butyl (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (3.8 g, 5.9 mmol) was dissolved in ethanol (100 mL). Palladium (10% on carbon, 1.38 g, 1.17 mmol) was added and the mixture was stirred under hydrogen (15 psi) for 3 h. The reaction mixture was filtered, washed with methanol, and the filtrate was evaporated to dryness to yield tert-butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (2.56 g, 85%).

[1033] Step 3: (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid

[1034] To a solution of (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-seven oxa-23-azaheptacosane-27-acid (587mg, 1.15mmol) in DCM (11.3mL) was added TFA (11.3mL). The mixture was stirred at room temperature for 1h. The reactant was concentrated and azeotroped with toluene, and further dried on high vacuum to obtain an oily crude intermediate. 1-({[(1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}oxy)pyrrolidine-2,5-dione (279mg, 0.959mmol) and DCM (9.8mL), triethylamine (1.34mL, 9.59mmol) were added to the crude oil intermediate at room temperature. The mixture was stirred at room temperature for 2h. The reaction mixture was diluted with EtOAc (50 mL) and potassium carbonate solution (10 mL, 4.0 M in water). The separated organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by reverse phase column chromatography (0-100% ACN / ammonium bicarbonate aqueous solution (5 mM)). The desired fraction was concentrated in vacuo to obtain (25S)-25-({[(1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosa-27-oic acid (19 mg, 3.1% yield). LCMS (AA): m / z=629.2 (MH); 1H NMR (400MHz, methanol-d4) δ = 4.46 (dd, J = 5.7, 7.4Hz, 1H), 4.25-4.12 (m, 2H), 3.68-3.51 (m, 28H), 3.41-3.34 (m, 5 H), 2.80-2.60 (m, 2H), 2.33-2.12 (m, 4H), 1.69-1.54 (m, 2H), 1.40 (quin, J = 8.7Hz, 1H), 1.02-0.89 (m, 2H).

[1035] Step 4: (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl {(25S)-27-[(2,5-dioxopyrrolidin-1-yl)oxy]-24,27-dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosa-25-yl}carbamate (Intermediate 36)

[1036] The title compound was prepared following the procedure described in Example 29 starting with (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosacan-27-oic acid instead of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecan-19-oic acid. The reaction mixture was stirred at room temperature for 30 minutes to give (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl {(25S)-27-[(2,5-dioxopyrrolidin-1-yl)oxy]-24,27-dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosa-25-yl}carbamate (Intermediate 36, 22 mg, 100%). LCMS (AA): m / z = 728.4 (M+H).

[1037] Example 38C

[1038] The compounds listed in Table 22A (Intermediate 37 and Intermediate 42) were prepared as described in Example 10, substituting the starting materials shown in Step 1 and Step 2 for 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (Intermediate 37 and Intermediate 42) and (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenoxy}tetrahydro-2H-pyran-2-carboxylic acid methyl ester (Intermediate 42).

[1039] Table 22A

[1040]

[1041] *Not using DMAP in step 2

[1042] Example 38D

[1043] [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro- 4-{[(2S,5S)-5,7-amino-5-isopropyl-2-methyl-4,7-dioxo-10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecano-3,6-diazaheptadecanoyl)benzyl]methylcarbamate (C-37)

[1044]

[1045] Step 1: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro 4-{[(53S,56S)-1-azido-53-isopropyl-56-methyl-51,54,57-trioxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-hexadecano-52,55-diazaheptadec-57-yl]amino}benzyl]methylcarbamate

[1046] To a solution of intermediate 8 (18.68 mg, 0.01674 mmol), 1-[(1-azido-51-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-hexadecane-51-yl)oxy]pyrrolidine-2,5-dione (32.3 mg, 0.03348 mmol), THF (0.6 mL) and DMF (0.2 mL) was added DIEA (0.044 mL, 0.2511 mmol). The mixture was stirred at room temperature for 3 h and then quenched with MeOH (5 mL). After evaporation of most of the solvent, the residue was purified by reverse phase column chromatography (0-50% ACN / aqueous ammonium bicarbonate solution (5 mM)). The desired fractions were concentrated and lyophilized to give the title compound (17.8 mg, 55%) as a white powder. LCMS (AA): m / z = 1915 (MH).

[1047] Step 2: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro- 4-{[(2S,5S)-5,7-amino-5-isopropyl-2-methyl-4,7-dioxo-10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecano-3,6-diazaheptadecanoyl]amino}benzyl]methylcarbamate (C-37)

[1048] To a 50-mL round-bottom flask was added [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylamino Benzyl formate (17.7 mg, 0.00923 mmol), THF (1.0 mL), and triphenylphosphine (24.2 mg, 0.0923 mmol) were added to 4-{[(53S,56S)-1-azido-53-isopropyl-56-methyl-51,54,57-trioxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-hexadecano-52,55-diazaheptadecane-57-yl]amino}benzyl formate (17.7 mg, 0.00923 mmol), THF (1.0 mL), and triphenylphosphine (24.2 mg, 0.0923 mmol). This mixture was stirred at room temperature overnight. Sodium hydroxide (0.0554 mL, 1.0 mol / L aqueous solution) was added to the reaction mixture. After stirring the mixture at room temperature for 1 hour, it was concentrated. The residue was purified by reverse phase column chromatography (0-50% ACN / ammonium bicarbonate aqueous solution (5 mM)). The desired fractions were collected and lyophilized to give [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfanyldecahydro-5,8-methylenecyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1,4-dihydro-2,10-dioxo-1,4-dihydro-2,10-dioxo-1,4-dihydro-2,10-dihydro ...2,10-dihydro-1,4-dihydro-2,10-dihydro-2,10-dihydro-2,10-dihydro-1,4-dihydro-2,10-dihydro-2,10-dihydro-2,10-dihydro-1 4-{[(2S,5S)-5,7-amino-5-isopropyl-2-methyl-4,7-dioxo-10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecano-3,6-diazaheptadecanoyl]amino}benzyl]methylcarbamate (C-37) (5.7 mg, 32%). LCMS (AA): m / z = 1889 (MH). HRMS (m / z): C 80 H 124 N 12 O 32 P2S2's [M+H] + Calculated value 1891.7434; experimental value 1891.7433.

[1049] Example 38E

[1050] N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosa-26-carboxamide (Intermediate 39)

[1051]

[1052] Step 1: (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-oic acid

[1053] To a mixture of (2S)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride (1.05 g, 4.00 mmol) and 1-[(26-oxo-2,5,8,11,14,17,20,23-octaoxahexacosa-26-yl)oxy]pyrrolidine-2,5-dione (2.08 g, 4.04 mmol) in pyridine (21.0 mL) was added DIEA (1.43 mL, 8.20 mmol) and the mixture was stirred at room temperature for 30 min, then cooled to 0°C and stirred overnight. The volatile solvents were removed, and the residue was azeotroped with toluene (3×20 mL) and acetonitrile (10 mL) to afford (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-oic acid (4.00 g, 100% yield) as a colorless oil. LCMS (AA): m / z = 619.3 (MH).

[1054] Step 2: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosa-26-amide (Intermediate 39)

[1055] A mixture of N-hydroxysuccinimide (469 mg, 4.08 mmol), N,N'-dicyclohexylcarbodiimide (824 mg, 4.00 mmol) and crude (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-oic acid (4.00 g, 4.00 mmol) from step 1 in DCM (76.7 mL, 1200 mmol) was stirred at room temperature for 16 h. The precipitated solid from the reaction was filtered and the filtrate was concentrated to give a crude oil. Purification by silica gel chromatography (0-20% IPA / DCM) gave N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosa-26-amide (Intermediate 39) as a colorless oil (1.70 g, 52%). 1 H NMR (400MHz, methanol-d4) δppm 1.42-1.55(m,2H)1.64(m,2H)1.81-1.93(m,1H)1.94-2.01(m,1H)2.52(m,2H)2.81-2.86(m,4H)3.36(s,3H)3 .51-3.57(m,4H)3.60-3.66(m,26H)3.68-3.76(m,2H)4.03-4.17(m,2H)4.74-4.80(m,1H)6.74-6.86(s,2H).

[1056] Example 38F

[1057] N-{(2S)-6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosa-26-carboxamide (Intermediate 40)

[1058]

[1059] Step 1: (28S)-28-{4-[(tert-Butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-oic acid

[1060] The title compound was prepared following the procedure described in Step 1 of Example 38E, starting with N6-(tert-butoxycarbonyl)-L-lysine (0.49 g, 1.99 mmol) instead of (2S)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride. Purification by reverse phase column chromatography (0-60% ACN / aqueous formic acid (0.1%)) afforded (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-oic acid (1.14 g, 90%) as a colorless oil. LCMS (AA): m / z = 639.4 (MH).

[1061] Step 2: (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-oic acid

[1062] To a solution of (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-oic acid (148 mg, 0.2310 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at room temperature. The mixture was stirred at room temperature for 15 min, evaporated, and then co-evaporated with toluene (×3) to give the crude TFA salt. The crude product was dried under high vacuum for an additional 2 h before maleimidoacetic acid NHS ester (69.89 mg, 0.2772 mmol) and DIEA (0.121 mL, 0.6929 mmol) in DCM (2650 mg, 2.00 mL, 31.2 mmol) were added and the reaction was stirred at room temperature for 16 h. The reaction mixture was evaporated. Purification by reverse-phase column chromatography (10-30% ACN / 10 mM aqueous ammonium acetate) gave (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-oic acid (120 mg, 38%) as an oil. LCMS (AA): m / z = 676.3 (MH).

[1063] Step 3: N-{(2S)-6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosa-26-amide (Intermediate 40)

[1064] To a flask containing (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosanetaconic acid (311 mg, 0.459 mmol) and DCM (2.94 mL) was added N,N'-dicyclohexylcarbodiimide (94.7 mg, 0.459 mmol) and N-hydroxysuccinimide (53.9 mg, 0.459 mmol). The suspension was stirred at room temperature under an argon atmosphere for 1 hour. The reaction mixture was then cooled to 0°C, filtered through Celite and rinsed with MeCN. The filtrate was evaporated. The residue was redissolved in DCM, washed with water (×2), dried over MgSO 4 , and concentrated to give N-{(2S)-6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosa-26-amide (Intermediate 40) (300 mg, 84.4%) as a viscous oil. LCMS (AA): m / z=773.2 (MH).

[1065] Example 38G

[1066] Compound (C-43) listed in Table 22C was prepared as described in Step 2 and Step 3 of Example 23, except that the starting materials indicated in the table were substituted for (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS ,16R)-16-hydroxy-2,10-dioxo-14-(pyrimidin-4-yloxy)-2,10-dihydrosulfenyldecahydro-5,8-methylenecyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylic acid methyl ester.

[1067] Table 22B

[1068]

[1069]

[1070] Table 22C

[1071]

[1072] Example 38H

[1073] Compound Intermediate-44 listed in Table 22D was prepared as described in Example 38E using the starting material (2R)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride as shown in Table 22D instead of (2S)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride in Step 1.

[1074] Table 22D

[1075]

[1076] Example 39

[1077] The procedure for preparing Ab-STING agonist conjugates via random cysteine ​​conjugation (shown in Figure 1 middle)

[1078] To a solution of anti-GCC antibody (clone 5F9, see PCT Publication No. WO 2011 / 050242, 60 mg / mL) in 50 mM histidine, 100 mM arginine, pH 6.0 buffer, 0.5 M Tris / 25 mM EDTA pH 8.0 buffer was added until the pH of the solution was 7.5-8.0. TCEP (10 mM solution in H2O, 3-5 equivalents) was added to the above solution. The reaction mixture was purged with argon and incubated at room temperature or 37°C with gentle shaking for 2 h. The desired linker-payload construct (10 mM solution in DMA, 5-9 equivalents) was then slowly added to the above mixture. The reaction was purged with argon and incubated at 37°C with gentle shaking for another 1.5-2 h. The reaction mixture was purified following the preparative SEC method described herein to obtain ADC. ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the Analytical Methods.

[1079] The above procedure can be used to prepare other antibody conjugates.

[1080] Example 40

[1081] The antibody drug conjugates listed in Table 24 were prepared as described in Example 39 using the starting linker-payload constructs shown as starting materials.

[1082] Table 24

[1083]

[1084]

[1085] Example 41

[1086] The procedure for preparing Ab-STING agonist conjugates via transglutaminase conjugation (shown in Figure 2 middle)

[1087] Deglycosylation: A solution of anti-GCC antibody (clone 5F9, see PCT Publication No. WO2011 / 050242, 60 mg / mL) in 50 mM histidine, 100 mM arginine, pH 6.0 buffer was diluted with an equal volume of pH 7.2 PBS. N-Glycosidase F (New England Biolabs, P0704S, 500,000 units / mL, 300 units / 1 mg antibody) was added to the solution, and the reaction mixture was heated to 37°C overnight with gentle mixing. The resulting deglycosylated 5F9 was buffer exchanged with PBS, pH 7.2.

[1088] Transglutaminase conjugation: To the deglycosylated 5F9 solution (10-20 mg / mL) in PBS prepared above, a 0.1 M DMSO solution of 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-1-amine (azido-PEG9-amine, BroadPharm, BP-23556, 40 equivalents) was added, followed by the addition of transglutaminase (ACTIVA TM The reaction mixture was heated to 37°C overnight with gentle mixing. The product was purified using the preparative SEC method described herein to yield 5F9-NH-PEG-azide.

[1089] Strain-promoted azide-alkyne cycloaddition: A 4-10 mM DMSO solution (3-5 equivalents, with DMSO <10% of the total solvent volume) of the linker-payload construct containing the strained alkyne was added to a solution of the 5F9-NH-PEG-azide conjugate prepared above (2-15 mg / mL in PBS). The resulting solution was gently stirred at room temperature overnight. The product was purified using the preparative SEC method described herein to obtain the ADC. ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[1090] The above procedure can be used to prepare other antibody conjugates.

[1091] Example 42

[1092] The antibody drug conjugates listed in Table 25 were prepared as described in Example 41 using the starting linker-payload constructs shown in the table as starting materials.

[1093] Table 25

[1094]

[1095]

[1096] Example 43

[1097] Ab-STING agonist conjugates were prepared via transglutaminase conjugation (shown in Figure 3 middle)

[1098] To a solution of deglycosylated 5F9 (10-20 mg / mL, prepared following the deglycosylation procedure described in Example 41) in PBS was added 1 M Tris, 5 M NaCl, pH 8.0 buffer (10-20% of the total volume) to adjust the pH to 8.0. A 10 mM DMSO solution of a primary amine-containing linker-payload construct (20 equivalents) was added to the solution, followed by the addition of transglutaminase (ACTIVA TM The ADC was purified using a HiTrap Protein A HP column (GE Healthcare, 17-0402-01) by first washing with 20 mM phosphate, pH 7.0, and then eluting with 0.1 M citric acid, pH 4.0.

[1099] The above procedure can be used to prepare other antibody conjugates.

[1100] Example 44

[1101] The antibody drug conjugates listed in Table 26 were prepared as described in Example 43 using the starting linker-payload constructs shown in the table as starting materials.

[1102] Table 26

[1103]

[1104] Example 45

[1105] 2-Amino-N-(2-((2-(2-azidoacetamido)ethyl)amino)-2-oxoethyl)acetamide

[1106]

[1107] Step 1: Benzyl (2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-yl)carbamate

[1108] To a solution of Boc-Gly-Gly-OH (550 mg, 2.32 mmol), EDC·HCl (437 mg, 2.28 mmol), and HOAt (317 mg, 2.33 mmol) in DMF (8.0 mL) at 0°C was added triethylamine (0.340 mL, 2.44 mmol). The resulting yellow heterogeneous mixture was stirred for 15 min. A solution of benzyl (2-aminoethyl)carbamate (500 mg, 2.44 mmol) in DMF (6.0 mL) was slowly added. The mixture was warmed to room temperature over 10 min and stirred for more than 3 days. The reaction mixture was partitioned between water (100 mL) and EtOAc (50 mL). The separated aqueous layer was extracted with EtOAc (50 mL). The combined organic layers were washed with 50 mL of water and then brine, dried over Na2SO4, and concentrated. The crude product was purified by silica gel chromatography (0-6% MeOH / CH2Cl2) to give tert-butyl N-[2-[[2-[2-(benzyloxycarbonylamino)ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (617 mg, 1.51 mmol, 65% yield) as a white solid. LCMS (FA): m / z = 431.2 (M+Na). 1H NMR(400MHz,DMSO-d6)δ7.97-7.97(m,1H),7.86-7.84(m,1H),7.39-7.29(m,5H),7.27-7.24(m,1H),7.01- 6.98(m,1H),5.02(s,2H),3.67(d,J=5.62Hz,2H),3.58(d,J=5.87Hz,2H),3.03-3.18(m,4H),1.39(s,9H).

[1109] Step 2: tert-Butyl (2-((2-((2-aminoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate

[1110] To a solution of tert-butyl N-[2-[[2-[2-(benzyloxycarbonylamino)ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (615 mg, 1.51 mmol) in ethanol (20.0 mL) was added 10% Pd / C (62.0 mg, 0.0583 mmol). The mixture was purged with H2 (1 atm) and stirred at room temperature for 3 h. Celite was added to the reaction mixture and the slurry was filtered through a short bed of Celite. The filtrate was concentrated and the residue was dried by co-evaporation with toluene and then high vacuum to give tert-butyl N-[2-[[2-(2-aminoethylamino)-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (407 mg, 1.48 mmol, 98.5% yield) as an off-white solid. LCMS (FA): m / z=275.2 (M+H). 1 H NMR (400MHz, DMSO-d6) δ8.05-7.89(m,1H),7.80-7.63(m,1H),7.08-6.96(m,1H),3.67(d,J=5.75Hz,2 H), 3.56 (d, J = 5.87Hz, 2H), 3.06 (q, J = 6.24Hz, 2H), 2.59-2.53 (m, 2H), 1.87-1.61 (m, 2H), 1.39 (s, 9H).

[1111] Step 3: tert-Butyl N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate

[1112] A mixture of EDC·HCl (57.6 mg, 0.300 mmol), HOAt (43.6 mg, 0.320 mmol) and 2-azidoacetic acid (27.0 μL, 0.315 mmol) in dichloromethane (2.70 mL) was stirred for 10 minutes. The mixture was cooled in an ice bath, and then tert-butyl N-[2-[[2-(2-aminoethylamino)-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (75.0 mg, 0.273 mmol) was added, followed by N,N-diisopropylethylamine (55.8 μL, 0.320 mmol) over a few minutes. The mixture was stirred at 0°C for 5 minutes and then stirred at room temperature overnight. Water (2 mL) and EtOAc (5 mL) were added, and the mixture was stirred vigorously for 20 minutes. The mixture was diluted with EtOAc (25 mL) and then washed with 50% brine / water (20 mL) and brine. The separated organic layer was dried over NaSO and concentrated. Purification by silica gel chromatography (0-8% MeOH / CHCl) afforded tert-butyl N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (88.0 mg, 0.246 mmol, 90.1% yield) as a glassy solid. LCMS (FA): m / z = 356.2 (MH). 1 H NMR (400MHz, DMSO-d6) δ8.16-8.08(m,1H),8.04-7.92(m,1H),7.92-7.81(m,1H),7.05-6.94(m,1 H), 3.82 (s, 2H), 3.67 (d, J = 5.62Hz, 2H), 3.58 (d, J = 5.87Hz, 2H), 3.10-3.19 (m, 4H), 1.39 (s, 9H).

[1113] Step 4: 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]acetamide (Intermediate 34)

[1114] To a solution of tert-butyl N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (86.0 mg, 0.241 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (1.00 mL, 8.77 mmol). The mixture was stirred at room temperature for 30 min. The mixture was diluted with 5 mL of toluene and concentrated. The residue was dissolved in 5 mL of water and lyophilized to give 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]acetamide 2,2,2-trifluoroacetate (102 mg, 0.275 mmol, quantitative) as a colorless syrup. LCMS (FA): m / z=258.1 (M+H). 1 H NMR(400MHz,DMSO-d6)δ8.55-8.62(m,1H),8.12-8.20(m,1H),8.02-8.09(m,1H),7.9 0-8.02(m,3H),3.74-3.82(s,2H),3.80(m,2H),3.55-3.68(m,2H),3.11-3.19(m,4H).

[1115] Example 46

[1116] Production and purification of 5F9-LPETGG-His6

[1117] 5F9-LPETGG-His6 protein was produced using the transient Expi293F mammalian protein expression system kit from Thermo Fisher catalog number A14635, which includes Expi293F™ cells, Expi293™ expression growth medium, and Expifectamine 293™ transfection kit. 5F9-LPETGG-His6 protein was produced using 0.666 mg of light chain DNA plus 0.334 mg of heavy chain DNA mixed with 5.4 mL of Expifectamine 293™ transfection reagent in a 5 L open-well shake flask at a density of 2.8 × 10 61.8 liters of Expi293FTM cells at 100 cells / mL were added and incubated overnight at 37°C, 8% CO2 in an incubator with shaking at 100 rpm. The next day, 10 mL of Enhancer 1 and 100 mL of Enhancer 2 from the test kit were added to the flask. The flask was incubated under the above conditions and harvested on day 5 after transfection. The cells were spun down at 9000 rpm for 10 minutes at 4°C, then sterile filtered through a 0.22 μM filter unit and stored at 4°C until purified using a freshly filled 30 mL MabSelect SuRe LX Protein A column (GE Healthcare, catalog number 17-5474-03). After using peristaltic pump to load supernatant on post, resin is washed with 60mL [2 column volumes (CV]) of low salt buffer (25mM trisodium citrate pH 7.6,125mM NaCl), then washed with 60mL (2CV) of high salt buffer (25mM trisodium citrate pH 7.6,2MNaCl), and then washed with 60mL (2CV) of low salt buffer. Protein is eluted by carrying out pH gradient elution with 25mM citric acid, 125mM NaCl pH 8 to pH 2.2, and peak fractions are collected. Each fraction is neutralized with 1M trisodium citrate pH 8.2, to 70mM, about pH 6. The fractions in the main peak (2A5-2C5) are pooled. The fractions pooled are concentrated, and exchanged into 25mM sodium citrate pH 5.5, 125mM NaCl buffer using a Sartorius VIVAFLOW200 column buffer with a molecular weight cutoff of 50kDa. The sample was then filtered through a 0.22 μM syringe filter unit, aliquoted and stored at −80° C. The final yield was 623 mg of purified 5F9-LPETGG-His6 antibody with a purity of >95% and an endotoxin level of 0.64 EU / mL.

[1118] Heavy chain sequence:

[1119] MGWSCIILFLVATATGVHSQVQLQQWGAGLLKPSETLSLTCAVFGGSFSGYYWSWIRQPPGKGLEWIGEINHRGNTNDNPSLKSRVTISSVDTSKNQFALKLSSVTAADTAVYYCARERGYTYGNFDHWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAGGGSLPETGGHHHHHH (the underlined part is the signal sequence) (SEQ ID NO.1)

[1120] Light chain sequence:

[1121] MGWSCIILFLVATATGVHS EIVMTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTIGSLQSEDFAVYYCQQYKTWPRTFGQGTNVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (The underlined part is the signal sequence) (SEQ ID NO.2)

[1122] Example 47

[1123] The procedure for preparing Ab-STING agonist conjugates via sortase conjugation (shown in Figure 4 middle)

[1124] Sortase conjugation: The 5F9 antibody containing LPETGG-His6 at the C-terminus of the heavy chain was generated according to the procedure described in Example 46. A solution of 5F9 containing a LPETG-His6 tag at the C-terminus of the heavy chain (5F9-LPETGG-His6) in 25 mM citrate, 125 mM NaCl, pH 5.5 buffer was diluted with 50 mM HEPES, 150 mM NaCl, pH 7.45 buffer to a protein concentration of 48 μM. To the solution were added CaCl2 (4 mM aqueous solution, 80-100 equiv) and 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]acetamide (Intermediate 34, 800 μM solution in 50 mM HEPES, 150 mM NaCl, pH 7.45 buffer containing 0.1% DMSO, 20 equiv), followed by the addition of sortase AQ60-K206-P94S-D160N-D165A-K196His6 (as described in Antos, JM, Ingram, J., Fang, T., Pishesha, N., Truttmann, M.C., Ploegh, H.L. (2017). Site-specific protein labeling via sortase-mediated transpeptidation. Current Protocols in Protein The product was expressed as described in Science, 89, 15.3.1-15.3.19, 2.0 μM solution in 50 mM HEPES, 150 mM NaCl, pH 7.45 buffer, 0.03 equivalents). The reaction mixture was stirred at room temperature for 4 h. The product was purified by passing through a Capturem nickel column (from Takara, washed with PBS) followed by dialysis or by following the preparative SEC method described herein to provide 5F9-LPETG-azide.

[1125] Strain-promoted azide-alkyne cycloaddition: To a solution of the 5F9-LPETGG-NH(CH2)2NHCOCH2-azide conjugate prepared above (2-15 mg / mL in PBS), a 4-10 mM DMSO solution (3-5 equivalents, with DMSO <10% of the total solvent volume) of the strained alkyne-containing linker-payload construct was added. The resulting solution was gently stirred overnight at room temperature. The product was purified using the preparative SEC method described herein to obtain the ADC. ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[1126] The above procedure can be used to prepare other antibody conjugates.

[1127] Example 48

[1128] The antibody drug conjugates listed in Table 27 were prepared as described in Example 47 using the starting linker-payload constructs shown in the table as starting materials.

[1129] Table 27

[1130]

[1131] Example 49

[1132] Biological protocols and STING cell data

[1133] Decontaminated lysosome assay conditions

[1134] The test compound (121 uM linker-payload construct solution in DMSO or ADC solution in PBS, 4.65 uL) was added to a mixture of 125 uL rat liver decontaminated lysosomes (purchased from XenoTech) and 433 uL of aqueous buffer (54.7 mg / mL phosphate, 1.7 mg / mL EDTA, pH 6.0), and the solution was incubated at 37 ° C. 40 uL samples were taken at 10 min and 30 min, 1 h, 3 h, 5 h and 24 h and treated with 160 uL of 0.1% formic acid in methanol in a 96-well plate and then stored at -80 ° C. After collecting the last time point, each sample was thawed and treated with 200 uL of 0.1% formic acid in methanol containing 150 nM sulfabutamide (internal standard). The samples were centrifuged at 4000 g for 10 min and analyzed according to the LC / MS / MS method described herein. Calculate t using the Excel-Fit program 1 / 2 Calculated t 1 / 2 Reported in Table 28. As shown in Table 28, no free payload release from ADC-3 was observed in this decontaminated lysosome assay within 24 h, while ADC-9 with the same payload successfully released the payload and t 1 / 2 The PCR reaction time was 7 hours, indicating that the linker of ADC-9 can significantly improve the rate of payload dissociation under alternative cell release conditions compared to ADC-3.

[1135] Table 28

[1136] ADC Payload <![CDATA[t 1 / 2 (hours)]]> ADC-1 Compound No.14 2.4 ADC-3 Compound I-5c -* ADC-9 Compound I-5c 7.0 ADC-15 Compound No.14 2.9 ADC-16 Compound No.14 3.7

[1137] *No free payload release observed within 24 hours

[1138] A graphical representation of the payload release over time for ADC-1, ADC-3, and ADC-9 in Table 28 is shown in Figure 5 、 Figure 6 and Figure 7 middle.

[1139] Plasma stability test conditions

[1140] Test compound is mixed in 1mL blood plasma with the concentration of 10 μ g / mL, and then 5 equal volume aliquots are distributed in 2mL Eppendorf microcentrifuge tubes (labeled as 0 hour, 24 hour, 48 hour, 72 hour and 96 hour). Time 0 sample tube is stored at -80 ℃ immediately, and the remaining tube is incubated under 37 ℃ of appropriate shaking. Take out aliquots from incubator and store at -80 ℃ at its corresponding time point. After collecting all samples, it is thawed at room temperature and placed on wet ice. Each sample of 50 μ L is distributed in 96-well microtiter plates in triplicate. Contain the ice-cold methanol quenching sample of 50nM internal standard with 200 μ L. Sample is vortexed for 2min, then centrifuged for 10min with 3000rpm. 185 μ L supernatants are transferred to clean injection plates, then at 40 ℃ under N2 gas drying. The dried sample extracts were reconstituted with 100 μL of LCMS-grade water and vortexed for 1 min in preparation for LC-MS / MS analysis.

[1141] The HPLC was carried out at 40 °C using a Synergi 2.5μ polar-RP 100A C18 column (2.0 mm x 30 mm). Each sample was separated using a gradient consisting of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Analytes were detected using a SCIEX API 4500QTRAP instrument in multiple reaction monitoring (MRM) mode by cation spray. The percentage loss of payload in human, primate, and mouse plasma at different time points is reported in Table 29.

[1142] Table 29

[1143]

[1144]

[1145] THP1 Dual Lucia Reporter Gene Assay Conditions

[1146] THP1-Dual was derived from the human THP-1 monocytic cell line by stable biallelic knockout of the endogenous human HAQ STING gene and knockin of the R232 variant of human STING. TMKI-hSTING-R232 cells (InvivoGen #thpd-r232). These cells also stably express an inducible secreted Lucia luciferase reporter gene under the control of the ISG54 (interferon-stimulated gene) minimal promoter combined with five IFN-stimulated response elements (ISREs). Expression of the reporter gene allows the study of the IFN regulatory factor (IRF) pathway by evaluating the activity of Lucia luciferase. In addition to human STING and luciferase, these cells are engineered to stably express human guanylate cyclase C (GCC) to allow the study of target-mediated IRF pathway activation. Non-GCC expression vector cells were used as a control.

[1147] On the day of the experiment, cells were plated at a density of 15,000 cells / 25 μL / well in a white 384-well plate (Corning 356661) in growth medium (RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg / mL Normocin). TM , 100U / mL-100μg / mL penicillin-streptomycin, 10μg / mL blasticidin, 100μg / mL Zeocin, and 1μg / mL Puromycin. 5μL of GCC-targeted ADC sample was added to the cell plate and then incubated at 37°C for 20 hours. At the end of the incubation, 10μL / well of QUANTI-Luc TM (InvivoGen #rep-qlc1), and luminescence was immediately measured using LeadSeeker.

[1148] For the above assay, the percentage of luminescent signal induction was calculated for each tested ADC at different concentrations relative to untreated and control treated samples. Compound concentration versus percentage signal induction curves were fitted to generate EC 50 Value. Those skilled in the art will understand that as EC 50 The values ​​produced are subject to experimental variation. The observed EC 50 and Emax are reported in Table 30. As shown in Table 30 below, ADC-16 was approximately 21-fold more potent than ADC-15 in vector THP1 cells, which may be attributed to the more rapid release of the intracellular payload from ADC-16 compared to ADC-15.

[1149] Table 30

[1150]

[1151]

[1152] Example 50

[1153] Pharmacokinetic evaluation in mice

[1154] To evaluate the ADC in vivo in Balb / C mice bearing GCC-expressing CT26 colon cancer mouse tumors, 6-8 week-old female Balb / C mice (purchased from Jackson Laboratory) were used. CT26.WT parental cells were obtained from ATCC catalog number CRL-2638. GCC-expressing CT26 cells were constructed at Takeda by transducing CT26.WT with the lentiviral vector pLenti6.3 containing the human GCC gene to obtain CT26 pLenti6.3 GCC clone A5. CT26 pLenti6.3 GCC clone A5 cells were grown under sterile conditions in a 37°C incubator with 5% CO2 for 12-14 days. Cells were grown in RPMI-1640 medium containing 10% fetal bovine serum. Cells were passaged every 3-4 days using 0.05% trypsin / EDTA to detach the cells. On the day of implantation, cells were aspirated and resuspended in RPM-1640 serum-free medium at a concentration of 0.5×10 6 cells / 100 μL. CT26 pLenti6.3 GCC clone A5 cells (0.5×10 6 cells / 100 μL) were implanted by subcutaneous injection into the lower flank of mice using a 27-gauge needle. 6 cells / 100 μl injection volume / mouse). After cell implantation, tumors were measured by caliper and mice were weighed twice weekly as soon as tumors became palpable. Tumors were measured in two dimensions. (L×W 2 ) / 2. Mice were fed a normal diet and housed in an SPF animal facility in accordance with the Guide for the Care and Use of Laboratory Animals and the regulations of the Institutional Animal Care and Use Committee. Animals were maintained at a temperature of 18-26°C, a relative humidity of 50±20%, and a 12-hour intermittent light and dark cycle with ad libitum access to food and water.

[1155] When the mean tumor volume (MTV) reaches 500-800 mm 3 The pharmacokinetics of the ADC were studied after injection of the ADC into Balb / C mice bearing CT26-GCC tumors (n=3 / treatment group) at 4 hr. Serum samples were collected at multiple time points and stored frozen for analysis.

[1156] The mouse plasma levels of total antibody and conjugated effective load were measured by LC / MS determination based on 2-in-1 immune capture on a Shimadzu UHPLC system interfaced with a Sciex 6500QTRAP mass spectrometer. Briefly, mouse plasma samples were incubated at room temperature for 45 min with anti-human IgG coated magnetic beads, and non-specifically bound proteins were then removed by washing the magnetic beads with PBST (PBS buffer, pH 7.4, containing 0.05% tween 20) and PBS buffer continuously. Thereafter, naked antibody (DAR=0) and ADC (DAR≥1) were eluted from the magnetic beads into 0.1% trifluoroacetic acid. After neutralizing the eluent and incorporating a stable isotope-labeled internal standard, an aliquot of the sample was removed and digested with papain at 37°C for 1 hour, then used for LC / MS analysis of conjugated effective load. The remaining sample was subjected to trypsin / lys-C digestion at 70°C for 1 hour, then used for LC / MS analysis of total antibody.

[1157] Free payload in the circulation was also measured by LC / MS after plasma protein precipitation. Briefly, mouse plasma was mixed with 8 volumes of methanol containing a stable isotope-labeled internal standard, and the supernatant was evaporated to dryness at 40°C under a gentle stream of nitrogen. Finally, the residue was reconstituted in LC / MS grade water and then subjected to LC / MS analysis. The PK profiles of ADC-1, ADC-8, and ADC-9 are summarized in Table 31. A graphical representation of plasma PK is shown in Figure 8 、 Figure 9 and Figure 10 As shown in Table 24, the DAR values ​​of ADC-1, ADC-8, and ADC-9 were 4.5, 3.9, and 4.3, respectively, which resulted in a higher molar concentration of conjugated CDN at time 0 than Figures 8-10 The molar concentration of total Ab is shown in each of the . Figure 8 As shown in , the molar concentration of conjugated CDN of ADC-1 decreased faster than the molar concentration of total Ab and both concentrations were combined on day 7 (resulting in a DAR of approximately 1 on that day), indicating that the DAR of ADC-1 decreased over time due to the decreased stability of its linker. Figure 9 and Figure 10 As shown in Table 31, the molar concentration of the conjugated CDN for ADC-8 and ADC-9 was consistently higher than the molar concentration of their respective total Abs throughout the experiment. Furthermore, as shown in Table 31, the half-life of the conjugated payload / CDN for ADC-8 and ADC-9 was longer than that for ADC-1, likely a result of enhanced linker stability in ADC-8 and ADC-9. Overall, this experiment indicates that the linkers of ADC-8 and ADC-9 have improved stability in mouse plasma relative to the linker of ADC-1.

[1158] Table 31

[1159]

[1160]

[1161] Example 51

[1162] Evaluation of antitumor activity in mice

[1163] Balb / C mice bearing GCC-expressing CT26 colon carcinoma mouse tumors were generated as described in Example 50.

[1164] When the mean tumor volume (MTV) reaches approximately 75 mm 3 At 4 hr, animals were randomly divided into several treatment groups (n=8 / group) and administered once with different doses of ADC via intravenous route. Tumors were measured twice weekly. The effect of ADC treatment on tumor growth was assessed by measuring tumor volume and comparing it with vehicle-treated tumors.

[1165] The endpoint of the efficacy study was reached when the tumor size reached 10% of the animal's body weight in volume or 2 cm in either direction. After injection, the mice were also closely monitored for signs of clinical decline. If for any reason the mice showed any signs of morbidity, including respiratory distress, hunched posture, decreased mobility, hind leg paralysis, tachypnea as a sign of pleural effusion, weight loss approaching 20% ​​or 15% plus other signs, or if their ability to perform normal activities (eating, motility) was impaired, the mice were euthanized.

[1166] Graphical representations of the observed antitumor activities of ADC-1, ADC-5, ADC-8, and ADC-9 are shown in Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 and Figure 14 middle.

[1167] Lipid conjugates:

[1168] abbreviation:

[1169] AA LCMS method using ammonium acetate

[1170] Ac acetate

[1171] ACN acetonitrile

[1172] atm atmospheric pressure

[1173] aq water-based

[1174] BBN Borahertabicyclo (3.3.1) nonane

[1175] Bn benzyl

[1176] Boc tert-Butoxycarbonyl

[1177] (Bpin)2 Bis(pinacol)diboron

[1178] tBu tert-butyl

[1179] Bz benzoyl

[1180] C degrees Celsius

[1181] CAD-charged aerosol detection

[1182] cGAMP cyclic guanosine monophosphate-adenosine monophosphate

[1183] DBAD Di-tert-butyl azodicarboxylate

[1184] DBU 1,8-diazabicyclo[5.4.0]undec-7-ene dichloroacetic acid

[1185] DCA dichloroacetic acid

[1186] DCE dichloroethane

[1187] DCM dichloromethane

[1188] DEAD Diethyl azodicarboxylate

[1189] DIAD Diisopropyl azodicarboxylate

[1190] DIPEA N,N-Diisopropylethylamine

[1191] DMAP 4-dimethylaminopyridine

[1192] DMF N,N-dimethylformamide

[1193] DMSO dimethyl sulfoxide

[1194] DMTr 4,4'-dimethoxytrityl

[1195] DOTMA 1,2-di-O-octadecenyl-3-trimethylammonium propane

[1196] DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

[1197] DSPC 1,2-Distearoyl-sn-glycero-3-phosphocholine

[1198] Et ethyl

[1199] EtOH

[1200] EtOAc

[1201] LCMS method using formic acid

[1202] h hour

[1203] Int intermediate

[1204] HPLC High-pressure liquid chromatography

[1205] HRMS High Resolution Mass Spectrometry

[1206] IC 50 50% inhibitory concentration

[1207] IPA Isopropyl alcohol

[1208] IPC Diisopinene

[1209] LCMS liquid chromatography mass spectrometry

[1210] LC / MS / MS liquid chromatography / tandem mass spectrometry

[1211] LDA lithium diisopropylamide

[1212] LHMDS Lithium bis(trimethylsilyl)amide

[1213] m / z mass-to-charge ratio

[1214] MHz Megahertz

[1215] Me methyl

[1216] MeOH methanol

[1217] min minutes

[1218] mL milliliters

[1219] MMP methylmorpholine

[1220] MS

[1221] nBu n-butane

[1222] NMP 1-Methyl-2-pyrrolidone

[1223] NMR Nuclear Magnetic Resonance

[1224] PE petroleum ether

[1225] Ph Phenyl

[1226] psi pounds per square inch

[1227] pyr pyridine

[1228] rt room temperature

[1229] SFC Supercritical Fluid Chromatography

[1230] T3P 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphanin-2,4,6-trioxide

[1232] TDA-1 Tris[2-(2-methoxyethoxy)ethyl]amine

[1233] TBAF Tetrabutylammonium fluoride

[1234] TBS tert-Butyldimethylsilyl

[1235] TBTU O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate

[1236] TEA triethylamine

[1237] TFA trifluoroacetic acid

[1238] TIDPSi 1,1,3,3-Tetraisopropyldisiloxane

[1239] TIPS triisopropylsilyl

[1240] THF Tetrahydrofuran

[1241] UPLC Ultra-Performance Liquid Chromatography

[1242] Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

[1243] Analytical methods:

[1244] NMR conditions:

[1245] Unless otherwise stated, NMR spectra were acquired on a Bruker Avance III 400 spectrometer at 400 MHz. 1 Recorded at H frequencies. Operating at 162 MHz and 376 MHz on the same spectrometer 31 P NMR and 19 F NMR spectra. Unless otherwise stated, all heteronuclear experiments were performed using 1 H decoupling acquisition.

[1246] LCMS conditions:

[1247] LCMS spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100 series LC system connected to a Micromass mass spectrometer using a reversed phase C18 column. Multiple gradients and run times were selected to best characterize the compounds. The mobile phase was based on an ACN / water or MeOH / water gradient and contained 0.1% formic acid (method indicated as FA) or 10 mM ammonium acetate (method indicated as AA). An example of a solvent gradient used was 100% mobile phase A (mobile phase A=99% water+1% ACN+0.1% formic acid) to 100% mobile phase B (mobile phase B=95% ACN+5% water+0.1% formic acid) at a flow rate of 1 mL / min for 16.5 min.

[1248] In some cases, LCMS spectra were recorded on an Agilent 1290 Infinity UPLC system connected to an Agilent 6130 mass spectrometer, a Waters Acquity UPLC system connected to a Waters Acquity SQ mass spectrometer, or an Agilent 1100 Series HPLC system connected to a Waters Micromass ZQ mass spectrometer using a reversed phase C18 column. Multiple gradients and run times were selected to best characterize the compounds. The mobile phase was based on an ACN / water or MeOH / water gradient and contained 0.1% formic acid (method indicated as FA) or 10 mM ammonium acetate (method indicated as AA). An example of a solvent gradient used was 95% mobile phase A (mobile phase A=99% water+1% ACN+0.1% formic acid) to 100% mobile phase B (mobile phase B=95% ACN+5% water+0.1% formic acid), running for 5 min at a flow rate of 0.5 mL / min.

[1249] Preparative HPLC:

[1250] Preparative HPLC was performed using a Phenomenex 5 micron C5 21.2 x 250 mm instrument operated by a Gilson 322 pump using a gradient elution with solvent A (99% 10 mM ammonium acetate / 1% acetonitrile) / solvent B (5% 10 mM ammonium acetate / 95% acetonitrile) with UV / Vis 155 detector triggered fraction collection set from 200 nm to 400 nm. Mass gated fraction collection was performed on an Agilent 1100 LC / MSD instrument.

[1251] One of ordinary skill in the art will recognize that gradients, column lengths, and flow rates can be varied and that some conditions may be more suitable for compound characterization than others, depending on the chemical being analyzed.

[1252] Preparative SFC:

[1253] Preparative SFC was performed using 10 mm, 20 mm, or 30 mm x 250 mm ChiralPak columns (typically IA, IB, IC, ID, IE, and IF), 10 mm or 20 mm x 250 mm Phenomenex Lux Cellulose-4, or 2-ethylpyridine columns, eluting with appropriate percentages of supercritical carbon dioxide and alcohol containing 0.3% diethylamine, 0.3% TEA, or 0.3% formic acid, or without any acid or base additives. Typical isocratic conditions were a flow rate in the range of 10-100 mL / min and a column temperature of 40°C. Preparative SFC was performed on a Jasco SFC preparative purification system with UV / visible light triggered fraction collection set to 200 nm to 400 nm and back pressure regulation set to 10 MPa.

[1254] One of ordinary skill in the art will recognize that gradients, column lengths, and flow rates can be varied and that some conditions may be more suitable for compound characterization than others, depending on the chemical being analyzed.

[1255] HPLC conditions of Example B:

[1256] HPLC analysis was performed using a SHIMADZU LC-2010C with a SHISEIDO 3 micron C18 4.6×75 mm, with a gradient elution of solvent A (80% water / 10% 50 mM ammonium acetate / 10% acetonitrile) / solvent B (10% 50 mM ammonium acetate / 90% acetonitrile) at a flow rate of 1 mL / min, a column temperature of 40° C., and UV detection at 254 nm.

[1257] LC / MS / MS conditions for Example B:

[1258] LC / MS / MS analysis was performed using a Shimadzu UltraFast liquid chromatograph interfaced with an AB SCIEX 4500 or AB SCIEX 5500 Triple Quad mass spectrometer and autosampler, or equivalent system.

[1259] For the analysis of Compound No. 14 and Compound I-5c, mobile phase A was prepared with 10 mM ammonium acetate in 99% water and 1% acetonitrile or 0.1% formic acid in water, and mobile phase B was prepared with 10 mM ammonium acetate in 5% water and 95% acetonitrile or 0.1% formic acid in 5% water and 95% acetonitrile. A liquid chromatography gradient was run at 1.5 mL / min for 3 minutes. The HPLC column was a Waters Xselect C18 CSH 3.5-μm column with a 2.1 mm ID x 30 mm length, and the column temperature was room temperature.

[1260] For analysis of compound CP-1, 10 mM ammonium acetate in 99% water and 1% acetonitrile was prepared as mobile phase A solvent, and 10 mM ammonium acetate in 5% water and 95% acetonitrile was used as mobile phase B solvent. The liquid chromatography gradient was run at 1.5 ml / min for 3.5 minutes. The HPLC column was an Osaka Soda Capcell PAK C1 UG120 5 micron, 2.0 mm ID x 35 mm length, and the column temperature was room temperature.

[1261] Analyst software 1.7 was used to analyze the MS / MS peak areas and calculate the concentrations of the samples.

[1262] UPLC-CAD conditions:

[1263] Using Thermo Scientific TM Dionex TM Corona TM Veo TM UPLC-CAD analysis was performed using an RS (Rapid Separation) charged aerosol detector and a Waters ACQUITY UPLC system with an ACE Excel C4 2 micron 2.1 x 100 mm or similar column, eluting with a gradient method using solvent A (10 mM ammonium acetate in water, 2.6 mL ammonium hydroxide in 4 L) / solvent B (10 mM ammonium acetate in methanol, 2.6 mL 95% ammonium hydroxide in 4 L) using a flow rate of 0.8 mL / min and a column temperature of 35° C. Up to ±20% variation can result from UPLC-CAD analysis.

[1264] Example A1

[1265] [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamic acid tert-butyl ester TEA salt, intermediate-1

[1266]

[1267] Compound No. 14 (300 mg, 0.40 mmol) was dissolved in anhydrous pyridine and concentrated to dryness (5 mL x 3). Under an argon atmosphere, the residue was dissolved in pyridine (6.41 mL) and treated with chlorotrimethylsilane (0.30 mL, 2.38 mmol) cooled in an ice-water bath. The reaction was allowed to warm to room temperature and stirred for 2 h to produce TMS-protected Compound No. 14. Separately (after stirring the reaction at room temperature for approximately 30 min), 4-methylmorpholine (0.28 mL, 2.50 mmol) was added to an ice-water-cooled solution of 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (520.0 mg, 2.27 mmol) in tetrahydrofuran (10.7 mL), followed by the slow addition of isobutyl chloroformate (0.29 mL, 2.27 mmol) in THF. The turbid suspension was warmed to room temperature and stirred for 1 h before being slowly added to the TMS-protected intermediate via syringe. The reaction mixture was stirred at room temperature overnight. MeOH (10 mL) was added to the above reaction mixture and the reaction mixture was concentrated. The residue was redissolved in MeOH (3.8 mL). Triethylamine trihydrofluoride (0.26 mL, 1.59 mmol) was added to the reaction mixture cooled in an ice-water bath and warmed to room temperature and stirred for 30 min. The reaction mixture was adsorbed on diatomaceous earth. (1 g) and purified by reverse phase flash column chromatography (0-50% ACN in aqueous triethylammonium acetate (10 mM)) to afford Intermediate-1 as a TEA salt (214.0 mg, 48.5%). LCMS (AA): m / z = 910.2 (M+H). 1 H NMR(D2O)δ8.55(s,1H),8.20(s,1H),7.87(s,1H),7.14(d,,J=4.0Hz,1H),6.42(d,J=16.0Hz,1H),6.29(d,J=8 .0Hz,1H),5.5d(dd,J=4.0,52.0Hz,1H),5.04-4.93(m,1H),4.91-4.86(m,1H),4.66(d,J=4.0Hz,1H),4.46(d, J=8.0Hz,1H),4.33-4.25(m,3H),4.14(dd,J=4.0,12.0Hz,1H),3.94(dd,J=4.0,8.0Hz,1H),3.25(br,m,2H),3 .05(q,J=8.0Hz,15H),2.73(s,3H),2.59-2.54(m,1H),1.90-1.82(m,1H),1.20(s,9H),1.13(t,J=8.0Hz,22H). 31P NMR(D2O)δ55.46(s,1P),52.06(s,1P). 19 F NMR(D2O)δ-164.19(s,br,1F),-200.75 to -200.99(m,1F).

[1268] Example A2

[1269] Octadecanoic acid [(2R)-3-[hydroxy-[2-[[5-[[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)phenylamino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-5-oxo-pentanoyloxy]amino]ethoxy]phosphoryl]oxy-2-octadecanoyloxy-propyl] ester, intermediate-2

[1270]

[1271] (2S)-2-Amino-N-[(1S)-2-[4-(hydroxymethyl)phenylamino]-1-methyl-2-oxo-ethyl]-3-methyl-butyramide (112.1 mg, 0.38 mmol) was suspended in toluene in a vial and evaporated to dryness (1 mL x 3). The residue was dissolved in DMF (1.5 mL) and treated with DIPEA (88.9 uL, 0.51 mmol). The DMF solution was then added to sodium [(2R)-2,3-dioctadecanoyloxypropyl] 2-[[5-(2,5-dioxopyrrolidin-1-yl)oxy-5-oxo-pentanoyloxy]amino]ethyl phosphate (250.0 mg, 0.25 mmol) in DCM (15 mL). The reaction mixture was stirred at room temperature for 4 h. Most of the solvent (DCM) was evaporated and the remaining solution (DMF) was poured into ice-cold aqueous HCl (0.1 N, 5 mL). The solid was collected by filtration and washed with water (3 mL x 3) and DCM (3 mL x 3) and dried under vacuum to give Intermediate-2 (247.0 mg, 85.2%). LCMS (AA): m / z = 1137.8 (M+H). 1H NMR (MeOD / CDCl3) δ7.55 (masked in CDCl3 peak, 2H), 7.29 (d,, J = 8.0 Hz, 1H), 5.25-5.22 (m, 1H), 4.53-4.49 (masked in MeOD residual water peak, 3H), 4.40 (dd, J = 4.0, 12.0 Hz, 1H), 4.19-4.00 (m, 6H), 3.49-3.39 (m, 2H ),2.35-2.30(m,6H),2.22(t,J=8.0Hz,2H),2.17-2.09(m,1H),1.97-1.89(m,2H),1.65-1.5 8(m,4H),1.45(d,J=8.0Hz,3H),1.35-1.28(br,m,56H),0.99-0.97(m,6H),0.89-0.86(6H). 31 P NMR(MeOD / CDCl3)δ0.77(s,1P).

[1272] Example A3

[1273] Octadecanoic acid [(2R)-3-[hydroxy-[2-[[5-[[(1S)-2-methyl-1-[[(1S)-1-methyl-2-[4-[(4-nitrophenoxy)carbonyloxymethyl]phenylamino]-2-oxo-ethyl]carbamoyl]propyl]amino]-5-oxo-pentanoyloxy]

[1274] 2-octadecanoyloxy-propyl] ester, intermediate-3

[1275]

[1276] To a vial was added Intermediate-2 (247.0 mg, 0.22 mmol), THF (4.0 mL), and DIPEA (83.3 μL, 0.48 mmol). The solution was cooled with an ice-water bath under an N2 atmosphere and then bis(4-nitrophenyl) carbonate (97%, 74.9 mg, 0.24 mmol) was added in one portion. The pale yellow solution was stirred at 50°C for 1 hour. Bis(4-nitrophenyl) carbonate (97%, 145.3 mg, 0.48 mmol) was further added and stirred at 50°C for 1 hour and then at room temperature overnight. The reaction mixture was poured into an ice-cold aqueous HCl solution (0.05N, 30 mL). The solid was collected by filtration and washed with water (10 mL × 3) and EtOAc (8 mL × 6) to give solid Intermediate-3 (282.8 mg, 79.5%). LCMS (FA): m / z = 1302.8 (M+H). 1H NMR (MeOD / CDCl3) δ8.28 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.42-7.38 (m, 4H), 5.25-5.22 (m, 3H), 4.53-4.49 (masked in the MeOD residual water peak, 1H), 4.38 (dd, J = 4.0, 12.0 Hz, 1H), 4.19-4.02 (m, 6H), 3.61-3 .387(m,2H),2.35-2.29(m,6H),2.22-2.20(m,2H),2.17-2.08(m,1H),1.98-1.90(m,2H),1.64- 1.56(m,4H),1.46(d,J=8.0Hz,3H),1.35-1.28(br,m,56H),0.99-0.97(m,6H),0.89-0.85(6H). 31 P NMR(MeOD / CDCl3)δ0.74(s,1P).

[1277] Example A4

[1278] Octadecanoic acid (2S,5S,19R)-2-({4-[({[4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxo-2,10-dihydrosulfenyloctahydro-12H-5,8-methylenefuro[3,2-l [1,3,6,9,11,2,10]pentaoxadiphosphatetradecin-14-yl]-9H-purin-6-yl]amino)-4-oxobutyl](methyl)carbamoyl]oxy)methyl]phenyl]carbamoyl)-16-hydroxy-5-isopropyl-16-oxo-4,7,11-trioxo-19-(stearoyloxy)-15,17-dioxa-3,6,12-triaza-16λ~5~-phosphaicosane-20-yl ester, CP-1

[1279]

[1280] Intermediate-1 (15.0 mg, 0.013 mmol) was dissolved in 1,4-dioxane (0.29 mL) and treated at room temperature for 1 h with 4M HCl (0.15 ml, 0.6 mml) in 1,4-dioxane. Volatiles were removed and dried under vacuum for 1 h to obtain 4-A, which was directly used in the next step as a crude product. Separately, intermediate-3 (17.6 mg, 0.013 mmol), DMAP (3.3 mg, 0.027 mmol), TEA (0.015 ml, 0.11 mmol) and DCM (0.35 mL) were loaded into a vial. A solution of 4-A in DMF (0.42 mL) was slowly added to this mixture. The reactant was stirred at room temperature for 15 min and concentrated to a small volume (approximately 0.5 mL). The crude compound was purified by preparative HPLC (Phenomenex 5 micron C5 21.2×250 mm, eluted with solvent A [99% 10 mM ammonium acetate / 1% acetonitrile] / solvent B [5% 10 mM ammonium acetate / 95% acetonitrile] from 60% B to 100% B in 16 min at a flow rate of 20 mL / min) to give CP-1 (11.0 mg, 40.3%) as an ammonium salt. LCMS (AA): m / z=1972.8 (M+H). 1 HNMR (MeOD) δ8.55 (br, s, 1H), 8.28 (br, s, 1H), 7.75 (br, s, 1H) 7.52-7.42 (m, 2H), 7.24-7.17 (m, 3H), 6.43-6.36 (m, 2H), 5.60 (dd, J = 4.0 and 52.0 Hz, 1H), 5.17-5.12 (m, 1H), 5.10-5.00 (m, 1H), 4.99 (s, 2H), 4.95-4.89 (m, 1H), 4.78-4.76 (hidden in the MeOD residual water peak, 1H), 4.48-4.28 (m, 6H), 4.23 (m,1H),4.13-4.09(m,2H),3.96-3.82(m,5H),3.42-3.33(m,4H),2.91(s,3H),2.73-2.64(m,2H),2.31-2.16(m,8H),2.11-2.03(m,1H),1 .99-1.92(m,2H),1.89-1.82(m,2H),1.57-1...

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein: a is an integer from 1 to 20; b is an integer from 1 to 20; m is 0, 1, 2, 3, or 4; n is 0 or 1; D-NH- is a portion of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2; Each R 1 Independently selected from C1-C4 alkyl, O-C1-C4 alkyl and halogen; R 2 Selected from C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10; R 3 and R 3 ' are each independently selected from hydrogen and C1-C3 alkyl; L is a cleavable linker; and Ab is an antibody, antibody fragment or antigen-binding fragment.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: a is an integer from 1 to 4; b is an integer from 1 to 10; and m is 0.

3. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein: m is 0; n is 0; and R 3 and R 3 'Each is hydrogen.

4. The compound or pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3, wherein L is in: It is the point of attachment to the nitrogen atom; It is the connection point with Ab; t is an integer from 1 to 10; W does not exist or is a self-immolative group; Z is absent or is a peptide of 2 to 5 amino acids; U and U' are independently absent or spacers; and Q is a heterobifunctional group; The condition is that W and Z do not exist at the same time.

5. The compound of claim 4 or a pharmaceutically acceptable salt thereof, wherein W is a self-immolative group selected from the following: in: is the point of attachment to the carbonyl group; and It is the connection point with Z.

6. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein W is selected from 7. The compound according to any one of claims 4 to 6, or a pharmaceutically acceptable salt thereof, wherein Z is a peptide capable of being cleaved enzymatically.

8. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein Z is cleavable by cathepsin.

9. The compound or pharmaceutically acceptable salt thereof as claimed in any one of claims 4 to 8, wherein Z is a diamino acid peptide selected from Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys and Lys-Phe.

10. The compound or pharmaceutically acceptable salt thereof according to any one of claims 4 to 9, wherein U and U' are independently selected from in: is the connection point with Z; is the connection point with Q; p is an integer from 1 to 6; q is an integer from 1 to 20; X is O or -CH2-; and Each r is independently 0 or 1.

11. The compound of any one of claims 4 to 10, or a pharmaceutically acceptable salt thereof, wherein Q is a heterobifunctional group linked to Ab via chemical or enzyme-mediated conjugation.

12. The compound of claim 11 or a pharmaceutically acceptable salt thereof, wherein Q is selected from in is a point of attachment to U, or, if U is absent, to Z; and is the point of attachment to U', or when U' is absent, to Ab.

13. The compound of claim 4, wherein t is 1; W is absent or is a self-immolative group; and Z is absent or is a diamino acid peptide.

14. A compound as claimed in any one of claims 1 to 13, wherein R 2 is -CH3 or -(CH2CH2O) s -CH3 and s is an integer from 1 to 10.

15. The compound of any one of claims 1 to 14, wherein D-NH2 is an amino-substituted compound that modulates STING activity.

16. The compound of claim 15, wherein the amino-substituted compound that modulates STING activity comprises a guanine or adenine derivative.

17. The compound of claim 15, wherein the amino-substituted compound that modulates STING activity is a compound of formula (II): (II) or a pharmaceutically acceptable salt thereof, wherein: X 10 is SH or OH; X 20 is SH or OH; Y a is O, S or CH2; Y b is O, S, NH or NR a , where R a is a C1-C4 alkyl group; R 10 It is hydrogen, fluorine, OH, NH2, OR b or NHR b ; R 20 is hydrogen or fluorine; R 30 is hydrogen; R 40 It is hydrogen, fluorine, OH, NH2, OR b or NHR b ; or R 30 and R 40 Together they form CH2O; R 50 is hydrogen or fluorine; R b is C1-C6 alkyl, halo(C1-C6)alkyl or C3-C6 cycloalkyl; Ring A 10 is an optionally substituted 5- or 6-membered monocyclic heteroaryl ring containing 1-4 heteroatoms selected from N, O or S, or an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 1-5 heteroatoms selected from N, O or S; wherein Ring A 10 The ring contains at least one N atom, and wherein Y b Connect to Ring A 10 of carbon atoms; and Ring B 10 is an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 2 to 5 heteroatoms selected from N, O or S; wherein Ring B 10 comprising at least two N atoms in the ring; The condition is that ring A 10 or Ring B 10 The bond to the carbonyl group of formula (I) is via the -NH- group.

18. The compound of any one of claims 15 to 17, wherein the amino-substituted compound that modulates STING activity is or a pharmaceutically acceptable salt thereof, wherein is the point of attachment to the carbonyl group of formula (I).

19. The compound of claim 15, wherein the amino-substituted compound that modulates STING activity is a compound of formula (III): or a pharmaceutically acceptable salt thereof; wherein X 10 is SH or OH; X 20 is SH or OH; Y c is O, S or CH2; Y d is O, S or CH2; B 100 It is given by the formula (B 1 -A) or formula (B 1 -B) represents a group: R 13 , R 14 , R 15 , R 16 and R 17 are each independently a hydrogen atom or a substituent; R 1000 is hydrogen or a bond to the carbonyl group of formula (I); Y 11 , Y 12 , Y 13 , Y 14 , Y 15 and Y 16 Each independently is N or CR 1a , where R 1a is hydrogen or a substituent; Z 11 , Z 12 , Z 13 , Z 14 , Z 15 and Z 16 each independently is N or C; R 105 is a hydrogen atom or a substituent; B 200 It is given by the formula (B 2 -A) or formula (B 2 -B) represents a group: R 23 , R 24 , R 25 , R 26 and R 27 are each independently a hydrogen atom or a substituent; R 100 ' is hydrogen or a bond to the carbonyl group of formula (I); Y 21 , Y 22 , Y 23 , Y 24 , Y 25 and Y 26 Each independently is N or CR 2a , where R 2a is hydrogen or a substituent; Z 21 , Z 22 , Z 23 , Z 24 , Z 25 and Z 26 are each independently N or C; and R 205 is a hydrogen atom or a substituent; wherein R 105 and R 205 Each is independently connected to the 2-position or 3-position of the 5-membered ring to which it is respectively connected; The conditions are: B 100 or B 200 One of them is connected to the carbonyl group of formula (I) via a -NH- group.

20. The compound of claim 19 or a pharmaceutically acceptable salt thereof, which has formula (IIIa): or a pharmaceutically acceptable salt thereof; wherein B 100 It is given by the formula (B 1 -A) or formula (B 1 -B) represents a group: R 13 , R 14 , R 15 , R 16 and R 17 are each independently a hydrogen atom or a substituent; R 1000 is hydrogen or a bond to the carbonyl group of formula (I); Y 11 , Y 12 , Y 13 , Y 14 , Y 15 and Y 16 Each independently is N or CR 1a , where R 1a is hydrogen or a substituent; Z 11 , Z 12 , Z 13 , Z 14 , Z 15 and Z 16 each independently is N or C; R 105 is a hydrogen atom or a substituent; B 200 It is given by the formula (B 2 -A) or formula (B 2 -B) represents a group: R 23 , R 24 , R 25 , R 26 and R 27 are each independently a hydrogen atom or a substituent; R 100 ' is hydrogen or a bond to the carbonyl group of formula (I); Y 21 , Y 22 , Y 23 , Y 24 , Y 25 and Y 26 Each independently is N or CR 2a , where R 2a is hydrogen or a substituent; Z 21 , Z 22 , Z 23 , Z 24 , Z 25 and Z 26 are each independently N or C; and R 205 is a hydrogen atom or a substituent; wherein R 105 and R 205 Each is independently connected to the 2-position or 3-position of the 5-membered ring to which it is respectively connected; The conditions are: B 100 or B 200 One of them is: in: R 18 is hydrogen or C 1-6 Alkyl; and R 19 is a halogen atom; And the other is linked to the carbonyl group of formula (I) via a -NH- group.

21. The compound of claim 19 or 20, wherein the amino-substituted compound that modulates STING activity is: or a pharmaceutically acceptable salt thereof, wherein is the point of attachment to the carbonyl group of formula (I).

22. A compound of formula (IV), or a pharmaceutically acceptable salt thereof, wherein: a is an integer from 1 to 20; b is an integer from 1 to 20; k is 0, 1, 2, or 3; m is 0, 1, 2, 3, or 4; D-NH- is a portion of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2; R 2 Selected from H, C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10; R 4 is selected from hydrogen and any naturally occurring amino acid side chain; R 5 Selected from C1-C4 alkyl and O-C1-C4 alkyl; L is a cleavable linker; and Ab is an antibody, antibody fragment or antigen-binding fragment.

23. The compound of claim 22 or a pharmaceutically acceptable salt thereof, wherein L is in: is the point of attachment to the carbonyl group; It is the connection point with Ab; W is a self-immolative group; Z is absent or is a peptide of 2 to 5 amino acids; and U and U' are independently absent or spacers; and Q is a heterobifunctional group.

24. A compound of formula (V), or a pharmaceutically acceptable salt thereof, wherein: D-NH-Yes X is O or CH2; f is an integer from 1 to 10; g is an integer from 1 to 20; U and U' are independently absent or are spacers; Q is a heterobifunctional group; and Ab is an antibody, antibody fragment or antigen-binding fragment.

25. The compound of claim 24, wherein X is O.

26. A compound of formula (VI), or a pharmaceutically acceptable salt thereof, wherein: a is an integer from 1 to 20; m is 0, 1, 2, 3, or 4; n is 0 or 1; D-NH- is a portion of an amino-substituted compound, wherein the amino-substituted compound has the formula D-NH2; Each R 1 Independently selected from C1-C4 alkyl, O-C1-C4 alkyl and halogen; R 2 Selected from C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10; R 3 and R 3 ' are each independently selected from hydrogen and C1-C3 alkyl; L is a cleavable linker; and LP is lipid.

27. The compound of claim 26, wherein the lipid is cholesterol or a phospholipid.

28. The compound of claim 27, wherein the lipid is a phospholipid.

29. The compound of claim 28, wherein the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean lecithin, and egg yolk lecithin.

30. The compound of claim 28, wherein the phospholipid is phosphatidylethanolamine, wherein the phosphatidylethanolamine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

31. A lipoplex comprising the compound of any one of claims 26 to 30.

32. The lipid complex of claim 31, wherein the lipid complex is in the form of a liposome.

33. The lipoplex of claim 31 or 32, further comprising one or more phospholipids.

34. The lipid complex of claim 33, wherein the one or more phospholipids are selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean lecithin, and egg yolk lecithin.

35. The lipid complex of claim 34, wherein the phospholipid is phosphatidylcholine, wherein the phosphatidylcholine is 1,2-distearoyl-sn-glycero-3-phosphocholine.

36. The lipoplex of any one of claims 31 to 35, further comprising a fatty alcohol.

37. The lipid complex of any one of claims 31 to 36, further comprising cholesterol.

38. The lipoplex of any one of claims 31 to 37, further comprising at least one pegylated lipid.

39. The lipid complex of claim 38, wherein the PEGylated lipid is selected from the group consisting of PEGylated phospholipids and PEGylated diacylglycerols.

40. The liposome of claim 39, wherein the PEGylated lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

41. A pharmaceutical composition comprising a compound according to any one of claims 1 to 40 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

42. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound of any one of claims 1 to 40.

43. A method of stimulating an immune response in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of the compound, lipoplex or composition of any one of claims 1 to 41.

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