SAFINA BRIAN (US)
ZHOU MATTHEW (US)
WO2018140831A2 | 2018-08-02 | |||
WO2018170179A1 | 2018-09-20 | |||
WO2019084060A1 | 2019-05-02 | |||
WO2020056194A1 | 2020-03-19 | |||
WO2020056008A1 | 2020-03-19 | |||
WO2011096519A1 | 2011-08-11 | |||
WO2013125654A1 | 2013-08-29 | |||
WO2013125636A1 | 2013-08-29 | |||
WO2013125640A1 | 2013-08-29 | |||
WO2013125630A1 | 2013-08-29 | |||
WO2013018889A1 | 2013-02-07 | |||
WO2013018891A1 | 2013-02-07 | |||
WO2013018883A1 | 2013-02-07 | |||
WO2013018892A1 | 2013-02-07 | |||
WO2014014082A1 | 2014-01-23 | |||
WO2014014086A1 | 2014-01-23 | |||
WO2015020212A1 | 2015-02-12 | |||
WO2018079740A1 | 2018-05-03 | |||
WO2009052249A1 | 2009-04-23 |
US20160145350A1 | 2016-05-26 | |||
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US6054297A | 2000-04-25 | |||
US6165464A | 2000-12-26 | |||
US6339142B1 | 2002-01-15 | |||
US6407213B1 | 2002-06-18 | |||
US6639055B1 | 2003-10-28 | |||
US6719971B1 | 2004-04-13 | |||
US6800738B1 | 2004-10-05 | |||
US7074404B2 | 2006-07-11 | |||
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US7723485B2 | 2010-05-25 | |||
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CLAIMS: 1. An immunoconjugate comprising an antibody covalently attached to one or more 8-amido-2-aminobenzazepine moieties by a linker, and having Formula I: or a pharmaceutically acceptable salt thereof, wherein: Ab is the antibody; p is an integer from 1 to 8; 8AmBza is the 8-amido-2-aminobenzazepine moiety having the formula: y is 0 or 1; Het is selected from the group consisting of heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl; Ra is H or forms Het with the nitrogen atom it is bound to; R1, R2, R3, and R4 are independently selected from the group consisting of H, C1-C12 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 carbocyclyl, C6-C20 aryl, C2-C9 heterocyclyl, and C1-C20 heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally substituted with one or more groups selected from: −(C1-C12 alkyldiyl)−N(R5)−*; −(C1-C12 alkyldiyl)−N(R5)2; −(C1-C12 alkyldiyl)−OR5; −(C3-C12 carbocyclyl); −(C3-C12 carbocyclyl)−*; −(C3-C12 carbocyclyl)−(C1-C12 alkyldiyl)−NR5−*; −(C3-C12 carbocyclyl)−(C1-C12 alkyldiyl)−N(R5)2; −(C3-C12 carbocyclyl)−NR5−C(=NR5)NR5−*; −(C6-C20 aryl); −(C6-C20 aryl)−*; −(C6-C20 aryldiyl)−N(R5)−*; −(C6-C20 aryldiyl)−(C1-C12 alkyldiyl)−N(R5)−*; −(C6-C20 aryldiyl)−(C1-C12 alkyldiyl)−(C2-C20 heterocyclyldiyl)−*; −(C6-C20 aryldiyl)−(C1-C12 alkyldiyl)−N(R5)2; −(C6-C20 aryldiyl)−(C1-C12 alkyldiyl)−NR5−C(=NR5a)N(R5)−*; −(C2-C20 heterocyclyl); −(C2-C20 heterocyclyl)−*; −(C2-C9 heterocyclyl)−(C1-C12 alkyldiyl)−NR5−*; −(C2-C9 heterocyclyl)−(C1-C12 alkyldiyl)−N(R5)2; −(C2-C9 heterocyclyl)−NR5−C(=NR5a)NR5−*; −(C1-C20 heteroaryl); −(C1-C20 heteroaryl)−*; −(C1-C20 heteroaryl)−(C1-C12 alkyldiyl)−N(R5)−*; −(C1-C20 heteroaryl)−(C1-C12 alkyldiyl)−N(R5)2; −(C1-C20 heteroaryl)−NR5−C(=NR5a)N(R5)−*; −C(=O)−*; −C(=O)−(C1-C12 alkyldiyl)−N(R5)−*; −C(=O)−(C2-C20 heterocyclyldiyl)−*; −C(=O)N(R5)2; −C(=O)N(R5)−*; −C(=O)N(R5)−(C1-C12 alkyldiyl)−N(R5)C(=O)R5; −C(=O)N(R5)−(C1-C12 alkyldiyl)−N(R5)C(=O)N(R5)2; −C(=O)NR5−(C1-C12 alkyldiyl)−N(R5)CO2R5; −C(=O)NR5−(C1-C12 alkyldiyl)−N(R5)C(=NR5a)N(R5)2; −C(=O)NR5−(C1-C12 alkyldiyl)−NR5C(=NR5a)R5; −C(=O)NR5−(C1-C8 alkyldiyl)−NR5(C2-C5 heteroaryl); −C(=O)NR5−(C1-C20 heteroaryldiyl)−N(R5)−*; −C(=O)NR5−(C1-C20 heteroaryldiyl)−*; −C(=O)NR5−(C1-C20 heteroaryldiyl)−(C1-C12 alkyldiyl)−N(R5)2; −C(=O)NR5−(C1-C20 heteroaryldiyl)−(C2-C20 heterocyclyldiyl)−C(=O)NR5−(C1-C12 alkyldiyl)−NR5−*; −N(R5)2; −N(R5)−*; −N(R5)C(=O)R5; −N(R5)C(=O)−*; −N(R5)C(=O)N(R5)2; −N(R5)C(=O)N(R5)−*; −N(R5)CO2R5; −NR5C(=NR5a)N(R5)2; −NR5C(=NR5a)N(R5)−*; −NR5C(=NR5a)R5; −N(R5)−(C2-C5 heteroaryl); −O−(C1-C12 alkyl); −O−(C1-C12 alkyldiyl)−N(R5)2; −O−(C1-C12 alkyldiyl)−N(R5)−*; −S(=O)2−(C2-C20 heterocyclyldiyl)−*; −S(=O)2−(C2-C20 heterocyclyldiyl)−(C1-C12 alkyldiyl)−N(R5)2; −S(=O)2−(C2-C20 heterocyclyldiyl)−(C1-C12 alkyldiyl)−NR5−*; and −S(=O)2−(C2-C20 heterocyclyldiyl)−(C1-C12 alkyldiyl)−OH; or R2 and R3 together form a 5- or 6-membered heterocyclyl ring; X1, X2, X3, and X4 are independently selected from the group consisting of a bond, C(=O), C(=O)N(R5), O, N(R5), S, S(O)2, and S(O)2N(R5); R5 is selected from the group consisting of H, C6-C20 aryl, C6-C20 aryldiyl, C1-C12 alkyl, and C1-C12 alkyldiyl, or two R5 groups together form a 5- or 6-membered heterocyclyl ring; R5a is selected from the group consisting of C6-C20 aryl and C1-C20 heteroaryl; where the asterisk * indicates the attachment site of L, and where one of R1, R2, R3 and R4 is attached to L; L is the linker selected from the group consisting of: −C(=O)−(PEG)−C(=O)−(PEP)−; −C(=O)−(PEG)−NR5−; −C(=O)−(PEG)−NR5−(PEG)−C(=O)−(PEP)−; −C(=O)−(PEG)−N+(R5)2−(PEG)−C(=O)−(PEP)−; −C(=O)−(PEG)−C(=O)−; −C(=O)−(PEG)−NR5CH(AA1)C(=O)−(PEG)−C(=O)−(PEP)−; −C(=O)−(PEG)−SS−(C1-C12 alkyldiyl)−OC(=O)−; −C(=O)−(PEG)−SS−(C1-C12 alkyldiyl)−C(=O)−; −C(=O)−(PEG)−; −C(=O)−(PEG)−C(=O)NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)NR5(C1-C12 alkyldiyl)−; −C(=O)−(C1-C12 alkyldiyl)−C(=O)−(PEP)−; −C(=O)−(C1-C12 alkyldiyl)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)−; −C(=O)−(C1-C12 alkyldiyl)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5−C(=O); −C(=O)−(C1-C12 alkyldiyl)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)− (C2-C5 monoheterocyclyldiyl)−; −C(=O)−CH2CH2OCH2CH2−(C1-C20 heteroaryldiyl)−CH2O−(PEG)−C(=O)− (MCgluc)−; −C(=O)−CH2CH2OCH2CH2−(C1-C20 heteroaryldiyl)−CH2O−(PEG)−C(=O)− (MCgluc)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)−NR5(C1-C12 alkyldiyl)−; −C(=O)−(PEG)−C(=O)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)−; −C(=O)−(PEG)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; and −(succinimidyl)−(CH2)m−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2- C5 monoheterocyclyldiyl)−; PEG has the formula: −(CH2CH2O)n−(CH2)m−; m is an integer from 1 to 5, and n is an integer from 2 to 50; PEP has the formula: where AA1 and AA2 are independently selected from an amino acid side chain, or AA1 or AA2 and an adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates a point of attachment and; R6 is selected from the group consisting of C6-C20 aryldiyl and C1-C20 heteroaryldiyl, substituted with −CH2O−C(=O)− and optionally with: MCgluc is selected from the groups: where q is 1 to 8, and AA is an amino acid side chain; where alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are optionally substituted with one or more groups independently selected from F, Cl, Br, I, −CN, − CH3, −CH2CH3, −CH=CH2, −C ≡CH, −C ≡CCH3, −CH2CH2CH3, −CH(CH3)2, −CH2CH(CH3)2, −CH2OH, −CH2OCH3, −CH2CH2OH, −C(CH3)2OH, −CH(OH)CH(CH3)2, −C(CH3)2CH2OH, − CH2CH2SO2CH3, −CH2OP(O)(OH)2, −CH2F, −CHF2, −CF3, −CH2CF3, −CH2CHF2, − CH(CH3)CN, −C(CH3)2CN, −CH2CN, −CH2NH2, −CH2NHSO2CH3, −CH2NHCH3, − CH2N(CH3)2, −CO2H, −COCH3, −CO2CH3, −CO2C(CH3)3, −COCH(OH)CH3, −CONH2, − CONHCH3, −CON(CH3)2, −C(CH3)2CONH2, −NH2, −NHCH3, −N(CH3)2, −NHCOCH3, − N(CH3)COCH3, −NHS(O)2CH3, −N(CH3)C(CH3)2CONH2, −N(CH3)CH2CH2S(O)2CH3, −NO2, =O, −OH, −OCH3, −OCH2CH3, −OCH2CH2OCH3, −OCH2CH2OH, −OCH2CH2N(CH3)2, − O(CH2CH2O)n−(CH2)mCO2H, −O(CH2CH2O)nH, −OP(O)(OH)2, −S(O)2N(CH3)2, −SCH3, − S(O)2CH3, and −S(O)3H. 2. The immunoconjugate of claim 1 wherein the antibody is an antibody construct that has an antigen binding domain that binds PD-L1. 3. The immunoconjugate of claim 2 wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, and avelumab, or a biosimilar or a biobetter thereof. 4. The immunoconjugate of claim 1 wherein the antibody is an antibody construct that has an antigen binding domain that binds HER2. 5. The immunoconjugate of claim 4 wherein the antibody is selected from the group consisting of trastuzumab and pertuzumab, or a biosimilar or a biobetter thereof. 6. The immunoconjugate of claim 1 wherein the antibody is an antibody construct that has an antigen binding domain that binds CEA. 7. The immunoconjugate of claim 6 wherein the antibody is labetuzumab, or a biosimilar or a biobetter thereof. 8. The immunoconjugate of any one of claims 1 to 7 wherein y is 0. 9. The immunoconjugate of any one of claims 1 to 7 wherein y is 1. 10. The immunoconjugate of any one of claims 1 to 7 wherein PEP has the formula: wherein AA1 and AA2 are independently selected from a side chain of a naturally- occurring amino acid. 11. The immunoconjugate of claim 10 wherein AA1 or AA2 with an adjacent nitrogen atom form a 5-membered ring proline amino acid. 12. The immunoconjugate of claim 11 wherein PEP has the formula: . 13. The immunoconjugate of any one of claims 1 to 7 wherein MCgluc has the formula: 14. The immunoconjugate of claim 10 wherein AA1 and AA2 are independently selected from H, −CH3, −CH(CH3)2, −CH2(C6H5), −CH2CH2CH2CH2NH2, −CH2CH2CH2NHC(NH)NH2, −CHCH(CH3)CH3, −CH2SO3H, and −CH2CH2CH2NHC(O)NH2. 15. The immunoconjugate of claim 10 wherein AA1 is −CH(CH3)2, and AA2 is −CH2CH2CH2NHC(O)NH2. 16. The immunoconjugate of any one of claims 1 to 7 wherein X1 is a bond, and R1 is H. 17. The immunoconjugate of any one of claims 1 to 7 wherein X2 is a bond, and R2 is C1-C8 alkyl. 18. The immunoconjugate of any one of claims 1 to 7 wherein X2 and X3 are each a bond, and R2 and R3 are independently selected from C1-C8 alkyl, −O−(C1-C12 alkyl), −(C1-C12 alkyldiyl)−OR5, −(C1-C8 alkyldiyl)−N(R5)CO2R5, and −O−(C1-C12 alkyl)−N(R5)CO2R5. 19. The immunoconjugate of claim 18 wherein R2 and R3 are each independently selected from −CH2CH2CH3, −OCH2CH3, −CH2CH2CF3, and −CH2CH2CH2OH. 20. The immunoconjugate of claim 18 wherein R2 is C1-C8 alkyl and R3 is −(C1-C8 alkyldiyl)−N(R5)CO2R4. 21. The immunoconjugate of claim 20 wherein R2 is −CH2CH2CH3 and R3 is − CH2CH2CH2NHCO2(t-Bu). 22. The immunoconjugate of claim 23 wherein R2 and R3 are each −CH2CH2CH3. 23. The immunoconjugate of claim 17 wherein X3-R3 is selected from the group consisting of: 24. The immunoconjugate of any one of claims 1 to 7 wherein Het is a 5- or 6- membered monocyclic heteroaryldiyl selected from the group consisting of pyridyldiyl, imidazolyldiyl, pyrimidinyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyldiyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl. 25. The immunoconjugate of any one of claims 1 to 7 wherein Het is a 5- or 6- membered monocyclic heterocyclyldiyl selected from the group consisting of morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl,thiomorpholinyldiyl, and S- dioxothiomorpholinyldiyl. 26. The immunoconjugate of any one of claims 1 to 7 wherein Het is 1,6- naphthyridyl or 1,6-naphthyridiyl. 27. The immunoconjugate of any one of claims 1 to 7 wherein L is selected from the group consisting of: −C(=O)−CH2CH2OCH2CH2−(C1-C20 heteroaryldiyl)−CH2O−(PEG)−C(=O)− (MCgluc)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)−NR5(C1-C12 alkyldiyl)−; −C(=O)−(PEG)−C(=O)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)−; −C(=O)−(PEG)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; and −(succinimidyl)−(CH2)m−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2- C5 monoheterocyclyldiyl)−. 28. The immunoconjugate of any one of claims 1 to 7 selected from Formulae Ia-d: y is 0 or 1; Het is selected from the group consisting of heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl; Ra is H or forms Het with the nitrogen atom it is bound to; R1, R2, R3, and R4 are independently selected from the group consisting of H, C1-C12 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C12 carbocyclyl, C6-C20 aryl, C2-C9 heterocyclyl, and C1-C20 heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally substituted with one or more groups selected from: −(C1-C12 alkyldiyl)−N(R5)−*; −(C1-C12 alkyldiyl)−N(R5)2; −(C1-C12 alkyldiyl)−OR5; −(C3-C12 carbocyclyl); −(C3-C12 carbocyclyl)−*; −(C3-C12 carbocyclyl)−(C1-C12 alkyldiyl)−NR5−*; −(C3-C12 carbocyclyl)−(C1-C12 alkyldiyl)−N(R5)2; −(C3-C12 carbocyclyl)−NR5−C(=NR5)NR5−*; −(C6-C20 aryl); −(C6-C20 aryl)−*; −(C6-C20 aryldiyl)−N(R5)−*; −(C6-C20 aryldiyl)−(C1-C12 alkyldiyl)−N(R5)−*; −(C6-C20 aryldiyl)−(C1-C12 alkyldiyl)−(C2-C20 heterocyclyldiyl)−*; −(C6-C20 aryldiyl)−(C1-C12 alkyldiyl)−N(R5)2; −(C6-C20 aryldiyl)−(C1-C12 alkyldiyl)−NR5−C(=NR5a)N(R5)−*; −(C2-C20 heterocyclyl); −(C2-C20 heterocyclyl)−*; −(C2-C9 heterocyclyl)−(C1-C12 alkyldiyl)−NR5−*; −(C2-C9 heterocyclyl)−(C1-C12 alkyldiyl)−N(R5)2; −(C2-C9 heterocyclyl)−NR5−C(=NR5a)NR5−*; −(C1-C20 heteroaryl); −(C1-C20 heteroaryl)−*; −(C1-C20 heteroaryl)−(C1-C12 alkyldiyl)−N(R5)−*; −(C1-C20 heteroaryl)−(C1-C12 alkyldiyl)−N(R5)2; −(C1-C20 heteroaryl)−NR5−C(=NR5a)N(R5)−*; −C(=O)−*; −C(=O)−(C1-C12 alkyldiyl)−N(R5)−*; −C(=O)−(C2-C20 heterocyclyldiyl)−*; −C(=O)N(R5)2; −C(=O)N(R5)−*; −C(=O)N(R5)−(C1-C12 alkyldiyl)−N(R5)C(=O)R5; −C(=O)N(R5)−(C1-C12 alkyldiyl)−N(R5)C(=O)N(R5)2; −C(=O)NR5−(C1-C12 alkyldiyl)−N(R5)CO2R5; −C(=O)NR5−(C1-C12 alkyldiyl)−N(R5)C(=NR5a)N(R5)2; −C(=O)NR5−(C1-C12 alkyldiyl)−NR5C(=NR5a)R5; −C(=O)NR5−(C1-C8 alkyldiyl)−NR5(C2-C5 heteroaryl); −C(=O)NR5−(C1-C20 heteroaryldiyl)−N(R5)−*; −C(=O)NR5−(C1-C20 heteroaryldiyl)−*; −C(=O)NR5−(C1-C20 heteroaryldiyl)−(C1-C12 alkyldiyl)−N(R5)2; −C(=O)NR5−(C1-C20 heteroaryldiyl)−(C2-C20 heterocyclyldiyl)−C(=O)NR5−(C1-C12 alkyldiyl)−NR5−*; −N(R5)2; −N(R5)−*; −N(R5)C(=O)R5; −N(R5)C(=O)−*; −N(R5)C(=O)N(R5)2; −N(R5)C(=O)N(R5)−*; −N(R5)CO2R5; −NR5C(=NR5a)N(R5)2; −NR5C(=NR5a)N(R5)−*; −NR5C(=NR5a)R5; −N(R5)−(C2-C5 heteroaryl); −O−(C1-C12 alkyl); −O−(C1-C12 alkyldiyl)−N(R5)2; −O−(C1-C12 alkyldiyl)−N(R5)−*; −S(=O)2−(C2-C20 heterocyclyldiyl)−*; −S(=O)2−(C2-C20 heterocyclyldiyl)−(C1-C12 alkyldiyl)−N(R5)2; −S(=O)2−(C2-C20 heterocyclyldiyl)−(C1-C12 alkyldiyl)−NR5−*; and −S(=O)2−(C2-C20 heterocyclyldiyl)−(C1-C12 alkyldiyl)−OH; or R2 and R3 together form a 5- or 6-membered heterocyclyl ring; X1, X2, X3, and X4 are independently selected from the group consisting of a bond, C(=O), C(=O)N(R5), O, N(R5), S, S(O)2, and S(O)2N(R5); R5 is selected from the group consisting of H, C6-C20 aryl, C6-C20 aryldiyl, C1-C12 alkyl, and C1-C12 alkyldiyl, or two R5 groups together form a 5- or 6-membered heterocyclyl ring; R5a is selected from the group consisting of C6-C20 aryl and C1-C20 heteroaryl; where the asterisk * indicates the attachment site of L, and where one of R1, R2, R3 and R4 is attached to L; L is the linker selected from the group consisting of: Q−C(=O)−(PEG)−C(=O)−(PEP)−; Q−C(=O)−(PEG)−NR5−; Q−C(=O)−(PEG)−NR5−(PEG)−C(=O)−(PEP)−; Q−C(=O)−(PEG)−N+(R5)2−(PEG)−C(=O)−(PEP)−; Q−C(=O)−(PEG)−C(=O)−; Q−C(=O)−(PEG)−NR5CH(AA1)C(=O)−(PEG)−C(=O)−(PEP)−; Q−C(=O)−(PEG)−SS−(C1-C12 alkyldiyl)−OC(=O)−; Q−C(=O)−(PEG)−SS−(C1-C12 alkyldiyl)−C(=O)−; Q−C(=O)−(PEG)−; Q−C(=O)−(PEG)−C(=O)NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)NR5(C1-C12 alkyldiyl)−; Q−C(=O)−(C1-C12 alkyldiyl)−C(=O)−(PEP)−; Q−C(=O)−(C1-C12 alkyldiyl)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)−; Q−C(=O)−(C1-C12 alkyldiyl)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5−C(=O); Q−C(=O)−(C1-C12 alkyldiyl)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)− (C2-C5 monoheterocyclyldiyl)−; Q−C(=O)−CH2CH2OCH2CH2−(C1-C20 heteroaryldiyl)−CH2O−(PEG)−C(=O)− (MCgluc)−; Q−C(=O)−CH2CH2OCH2CH2−(C1-C20 heteroaryldiyl)−CH2O−(PEG)−C(=O)− (MCgluc)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)−NR5(C1-C12 alkyldiyl)−; Q−C(=O)−(PEG)−C(=O)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)−; Q−C(=O)−(PEG)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; and Q−(CH2)m−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; where PEG has the formula: −(CH2CH2O)n−(CH2)m−; m is an integer from 1 to 5, and n is an integer from 2 to 50; PEP has the formula: where AA1 and AA2 are independently selected from an amino acid side chain, or AA1 or AA2 and an adjacent nitrogen atom form a 5-membered ring proline amino acid,, and the wavy line indicates a point of attachment and; R6 is selected from the group consisting of C6-C20 aryldiyl and C1-C20 heteroaryldiyl, substituted with −CH2O−C(=O)− and optionally with: MCgluc is selected from the groups: where q is 1 to 8, and AA is an amino acid side chain; and Q is selected from the group consisting of N-hydroxysuccinimidyl, N- hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with one or more groups independently selected from F, Cl, NO2, and SO3-; where alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are optionally substituted with one or more groups independently selected from F, Cl, Br, I, −CN, − CH3, −CH2CH3, −CH=CH2, −C ≡CH, −C ≡CCH3, −CH2CH2CH3, −CH(CH3)2, −CH2CH(CH3)2, −CH2OH, −CH2OCH3, −CH2CH2OH, −C(CH3)2OH, −CH(OH)CH(CH3)2, −C(CH3)2CH2OH, − CH2CH2SO2CH3, −CH2OP(O)(OH)2, −CH2F, −CHF2, −CF3, −CH2CF3, −CH2CHF2, − CH(CH3)CN, −C(CH3)2CN, −CH2CN, −CH2NH2, −CH2NHSO2CH3, −CH2NHCH3, − CH2N(CH3)2, −CO2H, −COCH3, −CO2CH3, −CO2C(CH3)3, −COCH(OH)CH3, −CONH2, − CONHCH3, −CON(CH3)2, −C(CH3)2CONH2, −NH2, −NHCH3, −N(CH3)2, −NHCOCH3, − N(CH3)COCH3, −NHS(O)2CH3, −N(CH3)C(CH3)2CONH2, −N(CH3)CH2CH2S(O)2CH3, −NO2, =O, −OH, −OCH3, −OCH2CH3, −OCH2CH2OCH3, −OCH2CH2OH, −OCH2CH2N(CH3)2, − O(CH2CH2O)n−(CH2)mCO2H, −O(CH2CH2O)nH, −OP(O)(OH)2, −S(O)2N(CH3)2, −SCH3, − S(O)2CH3, and −S(O)3H. 30. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein y is 0. 31. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein y is 1. 32. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein PEP has the formula: wherein AA1 and AA2 are independently selected from a side chain of a naturally- occurring amino acid. 33. The 8-amido-2-aminobenzazepine-linker compound of claim 32 wherein AA1 or AA2 with an adjacent nitrogen atom form a 5-membered ring to form a proline amino acid. 34. The 8-amido-2-aminobenzazepine-linker compound of claim 33 wherein PEP has the formula: . 35. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein MCgluc has the formula: . 36. The 8-amido-2-aminobenzazepine-linker compound of claim 32 wherein AA1 and AA2 are independently selected from H, −CH3, −CH(CH3)2, −CH2(C6H5), −CH2CH2CH2CH2NH2, −CH2CH2CH2NHC(NH)NH2, −CHCH(CH3)CH3, −CH2SO3H, and −CH2CH2CH2NHC(O)NH2. 37. The 8-amido-2-aminobenzazepine-linker compound of claim 32 wherein AA1 is −CH(CH3)2, and AA2 is −CH2CH2CH2NHC(O)NH2. 38. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein X1 is a bond, and R1 is H. 39. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein X2 is a bond, and R2 is C1-C8 alkyl. 40. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein X2 and X3 are each a bond, and R2 and R3 are independently selected from C1-C8 alkyl, −O−(C1-C12 alkyl), −(C1-C12 alkyldiyl)−OR5, −(C1-C8 alkyldiyl)−N(R5)CO2R5, and −O−(C1-C12 alkyl)− N(R5)CO2R5. 41. The 8-amido-2-aminobenzazepine-linker compound of claim 40 wherein R2 and R3 are each independently selected from −CH2CH2CH3, −OCH2CH3, −CH2CH2CF3, and − CH2CH2CH2OH. 42. The 8-amido-2-aminobenzazepine-linker compound of claim 40 wherein R2 is C1-C8 alkyl and R3 is −(C1-C8 alkyldiyl)−N(R5)CO2R4. 43. The 8-amido-2-aminobenzazepine-linker compound of claim 42 wherein R2 is − CH2CH2CH3 and R3 is −CH2CH2CH2NHCO2(t-Bu). 44. The 8-amido-2-aminobenzazepine-linker compound of claim 40 wherein R2 and R3 are each −CH2CH2CH3. 45. The 5-amino-pyrazoloazepine-linker compound of claim 39 wherein X3-R3 is selected from the group consisting of: . 46. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein Het is a 5- or 6-membered monocyclic heteroaryldiyl selected from the group consisting of pyridyldiyl, imidazolyldiyl, pyrimidinyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyldiyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl. 47. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein Het is a 5- or 6-membered monocyclic heterocyclyldiyl selected from the group consisting of morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl,thiomorpholinyldiyl, and S-dioxothiomorpholinyldiyl. 48. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein Het is 1,6-naphthyridyl or 1,6-naphthyridiyl. 49. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein L is selected from the group consisting of: Q−C(=O)−CH2CH2OCH2CH2−(C1-C20 heteroaryldiyl)−CH2O−(PEG)−C(=O)− (MCgluc)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)−NR5(C1-C12 alkyldiyl)−; Q−C(=O)−(PEG)−C(=O)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)−; Q−C(=O)−(PEG)−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−; and Q−(CH2)m−C(=O)−(PEP)−NR5(C1-C12 alkyldiyl)NR5C(=O)−(C2-C5 monoheterocyclyldiyl)−. 50. The 8-amido-2-aminobenzazepine-linker compound of claim 29 wherein Q is selected from: . 51. The aminoquinoline-linker compound of claim 29 wherein Q is phenoxy substituted with one or more F. 52. The aminoquinoline-linker compound of claim 51 wherein Q is 2,3,5,6- tetrafluorophenoxy. 53. The 8-amido-2-aminobenzazepine-linker compound of claim 29 selected from Formulae IIa-d: . 54. The 8-amido-2-aminobenzazepine-linker compound of claim 29 selected from Table 2a. 55. The 8-amido-2-aminobenzazepine-linker compound of claim 29 selected from Table 2b. 56. A method for treating cancer comprising administering a therapeutically effective amount of an immunoconjugate according to any one of claims 1 to 7, to a patient in need thereof. 57. The method of claim 56, wherein the cancer is susceptible to a pro-inflammatory response induced by TLR7 and/or TLR8 agonism. 58. The method of claim 56, wherein the cancer is a PD-L1-expressing cancer. 59. The method of claim 56, wherein the cancer is a HER2-expressing cancer. 60. The method of claim 56 wherein the cancer is a CEA-expressing cancer. 61. The method of claim 56 wherein the cancer is a Caprin-1-expressing cancer. 62. The method of any one of claims 56-61, wherein the cancer is selected from bladder cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer. 63. The method of claim 62, wherein the breast cancer is triple-negative breast cancer. 64. The method of claim 62, wherein the Merkel cell carcinoma cancer is metastatic Merkel cell carcinoma. 65. The method of claim 62, wherein the gastric cancer is HER2 overexpressing gastric cancer. 66. The method of claim 62, wherein the cancer is gastroesophageal junction adenocarcinoma. 67. Use of an immunoconjugate according to any one of claims 1 to 7 for treating cancer. 68. A method of preparing an immunoconjugate of Formula I of claim 1 wherein an 8-amido-2-aminobenzazepine-linker compound of Formula II of claim 29 is conjugated with the antibody. 69. The method of claim 68 wherein the 8-amido-2-aminobenzazepine-linker compound is selected from Table 2a or Table 2b. |
Exemplary 8-amido-2-aminobenzazepine compounds (8AmBza) of the invention are shown in Tables 1a and 1b. Each compound was characterized by mass spectrometry and shown to have the mass indicated. Activity against HEK293 NFKB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 30. Table 1a 8-Amido-2-aminobenzazepine compounds (8AmBza)
Table 1b 8-Amido-2-aminobenzazepine compounds (8AmBza)
8-AMIDO-2-AMINOBENZAZEPINE-LINKER COMPOUNDS The immunoconjugates of the invention are prepared by conjugation of an antibody with an 8-amido-2-aminobenzazepine-linker compound. The 8-amido-2-aminobenzazepine-linker compounds comprise an 8-amido-2-aminobenzazepine (8AmBza) moiety covalently attached to a linker unit, L. The linker units comprise functional groups and subunits which affect stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugates. The linker unit includes a reactive functional group which reacts, i.e. conjugates, with a reactive functional group of the antibody. For example, a nucleophilic group such as a lysine side chain amino of the antibody reacts with an electrophilic reactive functional group of the 8AmBza-linker compound to form the immunoconjugate. Also, for example, a cysteine thiol of the antibody reacts with a maleimide or bromoacetamide group of the 8AmBza- linker compound to form the immunoconjugate. Electrophilic reactive functional group suitable for the 8AmBza-linker compounds include, but are not limited to, N-hydroxysuccinimidyl (NHS) esters and N- hydroxysulfosuccinimidyl (sulfo-NHS) esters (amine reactive); carbodiimides (amine and carboxyl reactive); hydroxymethyl phosphines (amine reactive); maleimides (thiol reactive); halogenated acetamides such as N-iodoacetamides (thiol reactive); aryl azides (primary amine reactive); fluorinated aryl azides (reactive via carbon-hydrogen (C-H) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) esters (amine reactive); imidoesters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiol reactive); and benzophenone derivatives (reactive via C-H bond insertion). Further reagents include, but are not limited, to those described in Hermanson, Bioconjugate Techniques 2nd Edition, Academic Press, 2008. The invention provides solutions to the limitations and challenges to the design, preparation and use of immunoconjugates. Some linkers may be labile in the blood stream, thereby releasing unacceptable amounts of the adjuvant/drug prior to internalization in a target cell (Khot, A. et al (2015) Bioanalysis 7(13):1633–1648). Other linkers may provide stability in the bloodstream, but intracellular release effectiveness may be negatively impacted. Linkers that provide for desired intracellular release typically have poor stability in the bloodstream. Alternatively stated, bloodstream stability and intracellular release are typically inversely related. In addition, in standard conjugation processes, the amount of adjuvant/drug moiety loaded on the antibody, i.e. drug loading, the amount of aggregate that is formed in the conjugation reaction, and the yield of final purified conjugate that can be obtained are interrelated. For example, aggregate formation is generally positively correlated to the number of equivalents of adjuvant/drug moiety and derivatives thereof conjugated to the antibody. Under high drug loading, formed aggregates must be removed for therapeutic applications. As a result, drug loading-mediated aggregate formation decreases immunoconjugate yield and can render process scale-up difficult. Exemplary embodiments include an 8-amido-2-aminobenzazepine-linker compound of Formula II: wherein y is 0 or 1; Het is selected from the group consisting of heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl; R a is H or forms Het with the nitrogen atom it is bound to; R 1 , R 2 , R 3 , and R 4 are independently selected from the group consisting of H, C 1 -C 12 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl, C 2 -C 9 heterocyclyl, and C 1 -C 20 heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally substituted with one or more groups selected from: −(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 1 -C 12 alkyldiyl)−OR 5 ; −(C 3 -C 12 carbocyclyl); −(C 3 -C 12 carbocyclyl)−*; −(C 3 -C 12 carbocyclyl)−(C 1 -C 12 alkyldiyl)−NR 5 −*; −(C 3 -C 12 carbocyclyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 3 -C 12 carbocyclyl)−NR 5 −C(=NR 5 )NR 5 −*; −(C 6 -C 20 aryl); −(C 6 -C 20 aryl)−*; −(C 6 -C 20 aryldiyl)−N(R 5 )−*; −(C 6 -C 20 aryldiyl)−(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −(C 6 -C 20 aryldiyl)−(C 1 -C 12 alkyldiyl)−(C 2 -C 20 heterocyclyldiyl)−*; −(C 6 -C 20 aryldiyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 6 -C 20 aryldiyl)−(C 1 -C 12 alkyldiyl)−NR 5 −C(=NR 5a )N(R 5 )−*; −(C 2 -C 20 heterocyclyl); −(C 2 -C 20 heterocyclyl)−*; −(C 2 -C 9 heterocyclyl)−(C 1 -C 12 alkyldiyl)−NR 5 −*; −(C 2 -C 9 heterocyclyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 2 -C 9 heterocyclyl)−NR 5 −C(=NR 5a )NR 5 −*; −(C 1 -C 20 heteroaryl); −(C 1 -C 20 heteroaryl)−*; −(C 1 -C 20 heteroaryl)−(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −(C 1 -C 20 heteroaryl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 1 -C 20 heteroaryl)−NR 5 −C(=NR 5a )N(R 5 )−*; −C(=O)−*; −C(=O)−(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −C(=O)−(C 2 -C 20 heterocyclyldiyl)−*; −C(=O)N(R 5 ) 2 ; −C(=O)N(R 5 )−*; −C(=O)N(R 5 )−(C 1 -C 12 alkyldiyl)−N(R 5 )C(=O)R 5 ; −C(=O)N(R 5 )−(C 1 -C 12 alkyldiyl)−N(R 5 )C(=O)N(R 5 ) 2 ; −C(=O)NR 5 −(C 1 -C 12 alkyldiyl)−N(R 5 )CO 2 R 5 ; −C(=O)NR 5 −(C 1 -C 12 alkyldiyl)−N(R 5 )C(=NR 5a )N(R 5 ) 2 ; −C(=O)NR 5 −(C 1 -C 12 alkyldiyl)−NR 5 C(=NR 5a )R 5 ; −C(=O)NR 5 −(C1-C8 alkyldiyl)−NR 5 (C 2 -C 5 heteroaryl); −C(=O)NR 5 −(C 1 -C 20 heteroaryldiyl)−N(R 5 )−*; −C(=O)NR 5 −(C 1 -C 20 heteroaryldiyl)−*; −C(=O)NR 5 −(C 1 -C 20 heteroaryldiyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −C(=O)NR 5 −(C 1 -C 20 heteroaryldiyl)−(C 2 -C 20 heterocyclyldiyl)−C(=O)NR 5 −(C 1 -C 12 alkyldiyl)−NR 5 −*; −N(R 5 ) 2 ; −N(R 5 )−*; −N(R 5 )C(=O)R 5 ; −N(R 5 )C(=O)−*; −N(R 5 )C(=O)N(R 5 ) 2 ; −N(R 5 )C(=O)N(R 5 )−*; −N(R 5 )CO 2 R 5 ; −NR 5 C(=NR 5a )N(R 5 ) 2 ; −NR 5 C(=NR 5a )N(R 5 )−*; −NR 5 C(=NR 5a )R 5 ; −N(R 5 )−(C 2 -C 5 heteroaryl); −O−(C 1 -C 12 alkyl); −O−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −O−(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −S(=O) 2 −(C 2 -C 20 heterocyclyldiyl)−*; −S(=O) 2 −(C 2 -C 20 heterocyclyldiyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −S(=O) 2 −(C 2 -C 20 heterocyclyldiyl)−(C 1 -C 12 alkyldiyl)−NR 5 −*; and −S(=O) 2 −(C 2 -C 20 heterocyclyldiyl)−(C 1 -C 12 alkyldiyl)−OH; or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring; X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of a bond, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O) 2 , and S(O) 2 N(R 5 ); R 5 is selected from the group consisting of H, C 6 -C 20 aryl, C 6 -C 20 aryldiyl, C 1 -C 12 alkyl, and C 1 -C 12 alkyldiyl, or two R 5 groups together form a 5- or 6-membered heterocyclyl ring; R 5a is selected from the group consisting of C 6 -C 20 aryl and C 1 -C 20 heteroaryl; where the asterisk * indicates the attachment site of L, and where one of R 1 , R 2 , R 3 and R 4 is attached to L; L is the linker selected from the group consisting of: Q−C(=O)−(PEG)−C(=O)−(PEP)−; Q−C(=O)−(PEG)−NR 5 −; Q−C(=O)−(PEG)−NR 5 −(PEG)−C(=O)−(PEP)−; Q−C(=O)−(PEG)−N + (R 5 ) 2 −(PEG)−C(=O)−(PEP)−; Q−C(=O)−(PEG)−C(=O)−; Q−C(=O)−(PEG)−NR 5 CH(AA1)C(=O)−(PEG)−C(=O)−(PEP)−; Q−C(=O)−(PEG)−SS−(C 1 -C 12 alkyldiyl)−OC(=O)−; Q−C(=O)−(PEG)−SS−(C 1 -C 12 alkyldiyl)−C(=O)−; Q−C(=O)−(PEG)−; Q−C(=O)−(PEG)−C(=O)NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)NR 5 (C 1 -C 12 alkyldiyl)−; Q−C(=O)−(C 1 -C 12 alkyldiyl)−C(=O)−(PEP)−; Q−C(=O)−(C 1 -C 12 alkyldiyl)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)−; Q−C(=O)−(C 1 -C 12 alkyldiyl)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 −C(=O); Q−C(=O)−(C 1 -C 12 alkyldiyl)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)− (C 2 -C 5 monoheterocyclyldiyl)−; Q−C(=O)−CH 2 CH 2 OCH 2 CH 2 −(C 1 -C 20 heteroaryldiyl)−CH 2 O−(PEG)−C(=O)− (MCgluc)−; Q−C(=O)−CH 2 CH 2 OCH 2 CH 2 −(C 1 -C 20 heteroaryldiyl)−CH 2 O−(PEG)−C(=O)− (MCgluc)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)−NR 5 (C 1 -C 12 alkyldiyl)−; Q−C(=O)−(PEG)−C(=O)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)−; Q−C(=O)−(PEG)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; and Q−(CH 2 )m−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; where PEG has the formula: −(CH 2 CH 2 O)n−(CH 2 )m−; m is an integer from 1 to 5, and n is an integer from 2 to 50; PEP has the formula: where AA 1 and AA 2 are independently selected from an amino acid side chain, or AA 1 or AA 2 and an adjacent nitrogen atom form a 5-membered ring proline amino acid,, and the wavy line indicates a point of attachment and; R 6 is selected from the group consisting of C 6 -C 20 aryldiyl and C 1 -C 20 heteroaryldiyl, substituted with −CH 2 O−C(=O)− and optionally with: MCgluc is selected from the groups:
where q is 1 to 8, and AA is an amino acid side chain; and Q is selected from the group consisting of N-hydroxysuccinimidyl, N- hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with one or more groups independently selected from F, Cl, NO 2 , and SO 3 ; , where alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are optionally substituted with one or more groups independently selected from F, Cl, Br, I, −CN, − CH 3 , −CH 2 CH 3 , −CH=CH 2 , −C ≡CH, −C ≡CCH 3 , −CH 2 CH 2 CH 3 , −CH(CH 3 ) 2 , −CH 2 CH(CH 3 ) 2 , −CH 2 OH, −CH 2 OCH 3 , −CH 2 CH 2 OH, −C(CH 3 ) 2 OH, −CH(OH)CH(CH 3 ) 2 , −C(CH 3 ) 2 CH 2 OH, − CH 2 CH 2 SO 2 CH 3 , −CH 2 OP(O)(OH) 2 , −CH 2 F, −CHF2, −CF3, −CH 2 CF3, −CH 2 CHF2, − CH(CH 3 )CN, −C(CH 3 ) 2 CN, −CH 2 CN, −CH 2 NH 2 , −CH 2 NHSO 2 CH 3 , −CH 2 NHCH 3 , − CH 2 N(CH 3 ) 2 , −CO 2 H, −COCH 3 , −CO 2 CH 3 , −CO 2 C(CH 3 )3, −COCH(OH)CH 3 , −CONH 2 , − CONHCH 3 , −CON(CH 3 ) 2 , −C(CH 3 ) 2 CONH 2 , −NH 2 , −NHCH 3 , −N(CH 3 ) 2 , −NHCOCH 3 , − N(CH 3 )COCH 3 , −NHS(O) 2 CH 3 , −N(CH 3 )C(CH 3 ) 2 CONH 2 , −N(CH 3 )CH 2 CH 2 S(O) 2 CH 3 , −NO 2 , =O, −OH, −OCH 3 , −OCH 2 CH 3 , −OCH 2 CH 2 OCH 3 , −OCH 2 CH 2 OH, −OCH 2 CH 2 N(CH 3 ) 2 , − O(CH 2 CH 2 O) n −(CH 2 ) m CO 2 H, −O(CH 2 CH 2 O) n H, −OP(O)(OH) 2 , −S(O) 2 N(CH 3 ) 2 , −SCH 3 , − S(O) 2 CH 3 , and −S(O) 3 H. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein y is 0. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein y is 1. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein PEP has the formula: wherein AA 1 and AA 2 are independently selected from a side chain of a naturally- occurring amino acid. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein AA1 or AA2 with an adjacent nitrogen atom form a 5-membered ring to form a proline amino acid. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein PEP has the formula: . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein MCgluc has the formula: . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein AA1 and AA2 are independently selected from H, −CH 3 , − CH(CH 3 ) 2 , −CH 2 (C6H5), −CH 2 CH 2 CH 2 CH 2 NH 2 , −CH 2 CH 2 CH 2 NHC(NH)NH 2 , −CHCH(CH 3 )CH 3 , −CH 2 SO3H, and −CH 2 CH 2 CH 2 NHC(O)NH 2 . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein AA1 is −CH(CH 3 ) 2 , and AA2 is −CH 2 CH 2 CH 2 NHC(O)NH 2 . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein AA 1 and AA 2 are independently selected from GlcNAc aspartic acid, −CH 2 SO 3 H, and −CH 2 OPO 3 H. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein X 1 is a bond, and R 1 is H. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein X 2 is a bond, and R 2 is C 1 -C 8 alkyl. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein X 2 and X 3 are each a bond, and R 2 and R 3 are independently selected from C 1 -C 8 alkyl, −O−(C 1 -C 12 alkyl), −(C 1 -C 12 alkyldiyl)−OR 5 , −(C 1 -C 8 alkyldiyl)− N(R 5 )CO 2 R 5 , and −O−(C 1 -C 12 alkyl)−N(R 5 )CO 2 R 5 . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein R 2 and R 3 are each independently selected from −CH 2 CH 2 CH 3 , − OCH 2 CH 3 , −CH 2 CH 2 CF 3 , and −CH 2 CH 2 CH 2 OH. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein R 2 is C 1 -C 8 alkyl and R 3 is −(C 1 -C 8 alkyldiyl)−N(R 5 )CO 2 R 4 . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein R 2 is −CH 2 CH 2 CH 3 and R 3 is −CH 2 CH 2 CH 2 NHCO 2 (t-Bu). An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein R 2 and R 3 are each −CH 2 CH 2 CH 3 . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein NR 5 (C 2 -C 5 heteroaryl) of R 1 or R 3 is selected from: . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein X 3 -R 3 is selected from the group consisting of:
. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein Het is a 5- or 6-membered monocyclic heteroaryldiyl selected from the group consisting of pyridyldiyl, imidazolyldiyl, pyrimidinyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyldiyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein Het is a 5- or 6-membered monocyclic heterocyclyldiyl selected from the group consisting of morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl,thiomorpholinyldiyl, and S-dioxothiomorpholinyldiyl. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein Het is 1,6-naphthyridyl or 1,6-naphthyridiyl. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II includes wherein L is selected from the group consisting of: Q−C(=O)−CH 2 CH 2 OCH 2 CH 2 −(C 1 -C 20 heteroaryldiyl)−CH 2 O−(PEG)−C(=O)− (MCgluc)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)−NR 5 (C 1 -C 12 alkyldiyl)−; Q−C(=O)−(PEG)−C(=O)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; Q−C(=O)−(PEG)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)−; Q−C(=O)−(PEG)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; and Q−(CH 2 ) m −C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula IIa includes wherein Q is selected from: . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula IIa includes wherein Q is phenoxy substituted with one or more F. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula IIa includes wherein Q is 2,3,5,6-tetrafluorophenoxy. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formula II selected from Formulae IIa-d:
. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formulae IIa-d includes wherein R 2 is C 1 -C 8 alkyl and R 3 is −(C 1 -C 8 alkyldiyl)−N(R 5 )CO 2 R 4 . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formulae IIa-d includes wherein R 2 is −CH 2 CH 2 CH 3 and R 3 is −CH 2 CH 2 CH 2 NHCO 2 (t-Bu). An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formulae IIa-d includes wherein R 2 and R 3 are −CH 2 CH 2 CH 3 . An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound of Formulae IIa-d includes wherein Q is tetrafluorophenyl. An exemplary embodiment of the 8-amido-2-aminobenzazepine-linker compound is selected from Table 2. Each compound was characterized by mass spectrometry and shown to have the mass indicated. Table 2a 8-Amido-2-aminobenzazepine-linker (8AmBza-L) Formula II compounds Table 2b 8-Amido-2-aminobenzazepine-linker (8AmBza-L) Formula II compounds
IMMUNOCONJUGATES Exemplary embodiments of immunoconjugates comprise an antibody covalently attached to one or more 8-amido-2-aminobenzazepine (8AmBza) moieties by a linker, and having Formula I: Ab−[L−8AmBza] p I or a pharmaceutically acceptable salt thereof, wherein: Ab is the antibody; p is an integer from 1 to 8; 8AmBza is the 8-amido-2-aminobenzazepine moiety having the formula: y is 0 or 1; Het is selected from the group consisting of heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl; R a is H or forms Het with the nitrogen atom it is bound to; R 1 , R 2 , R 3 , and R 4 are independently selected from the group consisting of H, C 1 -C 12 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 12 carbocyclyl, C 6 -C 20 aryl, C 2 -C 9 heterocyclyl, and C 1 -C 20 heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally substituted with one or more groups selected from: −(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 1 -C 12 alkyldiyl)−OR 5 ; −(C 3 -C 12 carbocyclyl); −(C 3 -C 12 carbocyclyl)−*; −(C 3 -C 12 carbocyclyl)−(C 1 -C 12 alkyldiyl)−NR 5 −*; −(C 3 -C 12 carbocyclyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 3 -C 12 carbocyclyl)−NR 5 −C(=NR 5 )NR 5 −*; −(C 6 -C 20 aryl); −(C 6 -C 20 aryl)−*; −(C 6 -C 20 aryldiyl)−N(R 5 )−*; −(C 6 -C 20 aryldiyl)−(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −(C 6 -C 20 aryldiyl)−(C 1 -C 12 alkyldiyl)−(C 2 -C 20 heterocyclyldiyl)−*; −(C 6 -C 20 aryldiyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 6 -C 20 aryldiyl)−(C 1 -C 12 alkyldiyl)−NR 5 −C(=NR 5a )N(R 5 )−*; −(C 2 -C 20 heterocyclyl); −(C 2 -C 20 heterocyclyl)−*; −(C 2 -C 9 heterocyclyl)−(C 1 -C 12 alkyldiyl)−NR 5 −*; −(C 2 -C 9 heterocyclyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 2 -C 9 heterocyclyl)−NR 5 −C(=NR 5a )NR 5 −*; −(C 1 -C 20 heteroaryl); −(C 1 -C 20 heteroaryl)−*; −(C 1 -C 20 heteroaryl)−(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −(C 1 -C 20 heteroaryl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −(C 1 -C 20 heteroaryl)−NR 5 −C(=NR 5a )N(R 5 )−*; −C(=O)−*; −C(=O)−(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −C(=O)−(C 2 -C 20 heterocyclyldiyl)−*; −C(=O)N(R 5 ) 2 ; −C(=O)N(R 5 )−*; −C(=O)N(R 5 )−(C 1 -C 12 alkyldiyl)−N(R 5 )C(=O)R 5 ; −C(=O)N(R 5 )−(C 1 -C 12 alkyldiyl)−N(R 5 )C(=O)N(R 5 ) 2 ; −C(=O)NR 5 −(C 1 -C 12 alkyldiyl)−N(R 5 )CO 2 R 5 ; −C(=O)NR 5 −(C 1 -C 12 alkyldiyl)−N(R 5 )C(=NR 5a )N(R 5 ) 2 ; −C(=O)NR 5 −(C 1 -C 12 alkyldiyl)−NR 5 C(=NR 5a )R 5 ; −C(=O)NR 5 −(C 1 -C 8 alkyldiyl)−NR 5 (C 2 -C 5 heteroaryl); −C(=O)NR 5 −(C 1 -C 20 heteroaryldiyl)−N(R 5 )−*; −C(=O)NR 5 −(C 1 -C 20 heteroaryldiyl)−*; −C(=O)NR 5 −(C 1 -C 20 heteroaryldiyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −C(=O)NR 5 −(C 1 -C 20 heteroaryldiyl)−(C 2 -C 20 heterocyclyldiyl)−C(=O)NR 5 −(C 1 -C 12 alkyldiyl)−NR 5 −*; −N(R 5 ) 2 ; −N(R 5 )−*; −N(R 5 )C(=O)R 5 ; −N(R 5 )C(=O)−*; −N(R 5 )C(=O)N(R 5 ) 2 ; −N(R 5 )C(=O)N(R 5 )−*; −N(R 5 )CO 2 R 5 ; −NR 5 C(=NR 5a )N(R 5 ) 2 ; −NR 5 C(=NR 5a )N(R 5 )−*; −NR 5 C(=NR 5a )R 5 ; −N(R 5 )−(C 2 -C 5 heteroaryl); −O−(C 1 -C 12 alkyl); −O−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −O−(C 1 -C 12 alkyldiyl)−N(R 5 )−*; −S(=O) 2 −(C 2 -C 20 heterocyclyldiyl)−*; −S(=O) 2 −(C 2 -C 20 heterocyclyldiyl)−(C 1 -C 12 alkyldiyl)−N(R 5 ) 2 ; −S(=O) 2 −(C 2 -C 20 heterocyclyldiyl)−(C 1 -C 12 alkyldiyl)−NR 5 −*; and −S(=O) 2 −(C 2 -C 20 heterocyclyldiyl)−(C 1 -C 12 alkyldiyl)−OH; or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring; X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of a bond, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O) 2 , and S(O) 2 N(R 5 ); R 5 is selected from the group consisting of H, C 6 -C 20 aryl, C 6 -C 20 aryldiyl, C 1 -C 12 alkyl, and C 1 -C 12 alkyldiyl, or two R 5 groups together form a 5- or 6-membered heterocyclyl ring; R 5a is selected from the group consisting of C 6 -C 20 aryl and C 1 -C 20 heteroaryl; where the asterisk * indicates the attachment site of L, and where one of R 1 , R 2 , R 3 and R 4 is attached to L; L is the linker selected from the group consisting of: −C(=O)−(PEG)−C(=O)−(PEP)−; −C(=O)−(PEG)−NR 5 −; −C(=O)−(PEG)−NR 5 −(PEG)−C(=O)−(PEP)−; −C(=O)−(PEG)−N + (R 5 ) 2 −(PEG)−C(=O)−(PEP)−; −C(=O)−(PEG)−C(=O)−; −C(=O)−(PEG)−NR 5 CH(AA 1 )C(=O)−(PEG)−C(=O)−(PEP)−; −C(=O)−(PEG)−SS−(C 1 -C 12 alkyldiyl)−OC(=O)−; −C(=O)−(PEG)−SS−(C 1 -C 12 alkyldiyl)−C(=O)−; −C(=O)−(PEG)−; −C(=O)−(PEG)−C(=O)NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)NR 5 (C 1 -C 12 alkyldiyl)−; −C(=O)−(C 1 -C 12 alkyldiyl)−C(=O)−(PEP)−; −C(=O)−(C 1 -C 12 alkyldiyl)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)−; −C(=O)−(C 1 -C 12 alkyldiyl)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 −C(=O); −C(=O)−(C 1 -C 12 alkyldiyl)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)− (C 2 -C 5 monoheterocyclyldiyl)−; −C(=O)−CH 2 CH 2 OCH 2 CH 2 −(C 1 -C 20 heteroaryldiyl)−CH 2 O−(PEG)−C(=O)− (MCgluc)−; −C(=O)−CH 2 CH 2 OCH 2 CH 2 −(C 1 -C 20 heteroaryldiyl)−CH 2 O−(PEG)−C(=O)− (MCgluc)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)−NR 5 (C 1 -C 12 alkyldiyl)−; −C(=O)−(PEG)−C(=O)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)−; −C(=O)−(PEG)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; and −(succinimidyl)−(CH 2 )m−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 - C 5 monoheterocyclyldiyl)−; PEG has the formula: −(CH 2 CH 2 O)n−(CH 2 )m−; m is an integer from 1 to 5, and n is an integer from 2 to 50; PEP has the formula: where AA1 and AA2 are independently selected from an amino acid side chain, or AA1 or AA 2 and an adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates a point of attachment and; R 6 is selected from the group consisting of C 6 -C 20 aryldiyl and C 1 -C 20 heteroaryldiyl, substituted with −CH 2 O−C(=O)− and optionally with: MCgluc is selected from the groups:
where q is 1 to 8, and AA is an amino acid side chain; where alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are optionally substituted with one or more groups independently selected from F, Cl, Br, I, −CN, − CH 3 , −CH 2 CH 3 , −CH=CH 2 , −C ≡CH, −C ≡CCH 3 , −CH 2 CH 2 CH 3 , −CH(CH 3 ) 2 , −CH 2 CH(CH 3 ) 2 , −CH 2 OH, −CH 2 OCH 3 , −CH 2 CH 2 OH, −C(CH 3 ) 2 OH, −CH(OH)CH(CH 3 ) 2 , −C(CH 3 ) 2 CH 2 OH, − CH 2 CH 2 SO 2 CH 3 , −CH 2 OP(O)(OH) 2 , −CH 2 F, −CHF 2 , −CF 3 , −CH 2 CF 3 , −CH 2 CHF 2 , − CH(CH 3 )CN, −C(CH 3 ) 2 CN, −CH 2 CN, −CH 2 NH 2 , −CH 2 NHSO 2 CH 3 , −CH 2 NHCH 3 , − CH 2 N(CH 3 ) 2 , −CO 2 H, −COCH 3 , −CO 2 CH 3 , −CO 2 C(CH 3 ) 3 , −COCH(OH)CH 3 , −CONH 2 , − CONHCH 3 , −CON(CH 3 ) 2 , −C(CH 3 ) 2 CONH 2 , −NH 2 , −NHCH 3 , −N(CH 3 ) 2 , −NHCOCH 3 , − N(CH 3 )COCH 3 , −NHS(O) 2 CH 3 , −N(CH 3 )C(CH 3 ) 2 CONH 2 , −N(CH 3 )CH 2 CH 2 S(O) 2 CH 3 , −NO 2 , =O, −OH, −OCH 3 , −OCH 2 CH 3 , −OCH 2 CH 2 OCH 3 , −OCH 2 CH 2 OH, −OCH 2 CH 2 N(CH 3 ) 2 , − O(CH 2 CH 2 O)n−(CH 2 )mCO 2 H, −O(CH 2 CH 2 O)nH, −OP(O)(OH) 2 , −S(O) 2 N(CH 3 ) 2 , −SCH 3 , − S(O) 2 CH 3 , and −S(O)3H. An exemplary embodiment of the immunoconjugate of Formula I includes wherein y is 0. An exemplary embodiment of the immunoconjugate of Formula I includes wherein y is 1. An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is an antibody construct that has an antigen binding domain that binds PD-L1. An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, and avelumab, or a biosimilar or a biobetter thereof. An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is an antibody construct that has an antigen binding domain that binds HER2. An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is selected from the group consisting of trastuzumab and pertuzumab, or a biosimilar or a biobetter thereof. An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is an antibody construct that has an antigen binding domain that binds CEA. An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is labetuzumab, or a biosimilar or a biobetter thereof. An exemplary embodiment of the immunoconjugate of Formula I includes wherein PEP has the formula: wherein AA1 and AA2 are independently selected from a side chain of a naturally- occurring amino acid. An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA 1 or AA2 with an adjacent nitrogen atom form a 5-membered ring proline amino acid. An exemplary embodiment of the immunoconjugate of Formula I includes wherein PEP has the formula: . An exemplary embodiment of the immunoconjugate of Formula I includes wherein MCgluc has the formula: . An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA1 and AA 2 are independently selected from H, −CH 3 , −CH(CH 3 ) 2 , −CH 2 (C 6 H 5 ), −CH 2 CH 2 CH 2 CH 2 NH 2 , −CH 2 CH 2 CH 2 NHC(NH)NH 2 , −CHCH(CH 3 )CH 3 , −CH 2 SO 3 H, and −CH 2 CH 2 CH 2 NHC(O)NH 2 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA1 is −CH(CH 3 ) 2 , and AA 2 is −CH 2 CH 2 CH 2 NHC(O)NH 2 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA 1 and AA 2 are independently selected from GlcNAc aspartic acid, −CH 2 SO 3 H, and −CH 2 OPO 3 H. An exemplary embodiment of the immunoconjugate of Formula I includes wherein X 1 is a bond, and R 1 is H. An exemplary embodiment of the immunoconjugate of Formula I includes wherein X 2 is a bond, and R 2 is C 1 -C 8 alkyl. An exemplary embodiment of the immunoconjugate of Formula I includes wherein X 2 and X 3 are each a bond, and R 2 and R 3 are independently selected from C 1 -C 8 alkyl, −O−(C 1 -C 12 alkyl), −C 1 -C 12 alkyldiyl)−OR 5 , −(C 1 -C 8 alkyldiyl)−N(R 5 )CO 2 R 5 , and −O−C 1 -C 12 alkyl)−N(R 5 )CO 2 R 5 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 2 and R 3 are each independently selected from −CH 2 CH 2 CH 3 , −OCH 2 CH 3 , −CH 2 CH 2 CF 3 , and − CH 2 CH 2 CH 2 OH. An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 2 is C 1 -C 8 alkyl and R 3 is −(C 1 -C 8 alkyldiyl)−N(R 5 )CO 2 R 4 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 2 is −CH 2 CH 2 CH 3 and R 3 is −CH 2 CH 2 CH 2 NHCO 2 (t-Bu). An exemplary embodiment of the immunoconjugate of Formula I includes wherein R 2 and R 3 are each −CH 2 CH 2 CH 3 . An exemplary embodiment of the immunoconjugate of Formula I includes wherein X 3 - R 3 is selected from the group consisting of:
. An exemplary embodiment of the immunoconjugate of Formula I includes wherein NR 5 (C 2 -C 5 heteroaryl) of R 1 or R 3 is selected from: . An exemplary embodiment of the immunoconjugate of Formula I includes wherein Het is a 5- or 6-membered monocyclic heteroaryldiyl selected from the group consisting of pyridyldiyl, imidazolyldiyl, pyrimidinyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyldiyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl. An exemplary embodiment of the immunoconjugate of Formula I includes wherein Het is a 5- or 6-membered monocyclic heterocyclyldiyl selected from the group consisting of morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl,thiomorpholinyldiyl, and S-dioxothiomorpholinyldiyl. An exemplary embodiment of the immunoconjugate of Formula I includes wherein Het is 1,6-naphthyridyl or 1,6-naphthyridiyl. An exemplary embodiment of the immunoconjugate of Formula I includes wherein L is selected from the group consisting of: −C(=O)−CH 2 CH 2 OCH 2 CH 2 −(C 1 -C 20 heteroaryldiyl)−CH 2 O−(PEG)−C(=O)− (MCgluc)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)−NR 5 (C 1 -C 12 alkyldiyl)−; −C(=O)−(PEG)−C(=O)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; −C(=O)−(PEG)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)−; −C(=O)−(PEG)−C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 -C 5 monoheterocyclyldiyl)−; and −(succinimidyl)−(CH 2 ) m −C(=O)−(PEP)−NR 5 (C 1 -C 12 alkyldiyl)NR 5 C(=O)−(C 2 - C5 monoheterocyclyldiyl)−. An exemplary embodiment of the immunoconjugate of Formula I selected from Formulae Ia-d:
. The invention includes all reasonable combinations, and permutations of the features, of the Formula I embodiments. In certain embodiments, the immunoconjugate compounds of the invention include those with immunostimulatory activity. The antibody-drug conjugates of the invention selectively deliver an effective dose of an 8-amido-2-aminobenzazepine drug to tumor tissue, whereby greater selectivity (i.e., a lower efficacious dose) may be achieved while increasing the therapeutic index (“therapeutic window”) relative to unconjugated 8-amido-2- aminobenzazepine. Drug loading is represented by p, the number of 8AmBza moieties per antibody in an immunoconjugate of Formula I. Drug (8AmBza) loading may range from 1 to about 8 drug moieties (D) per antibody. Immunoconjugates of Formula I include mixtures or collections of antibodies conjugated with a range of drug moieties, from 1 to about 8. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues such as lysine and cysteine. In some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. In such aspects, p may be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges thereof, such as from 1 to 8 or from 2 to 5. In any such aspect, p and n are equal (i.e., p = n = 1, 2, 3, 4, 5, 6, 7, or 8, or some range there between). Exemplary antibody-drug conjugates of Formula I include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al. (2012) Methods in Enzym.502:123-138). In some embodiments, one or more free cysteine residues are already present in an antibody forming intrachain disulfide bonds, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody to a drug. In some embodiments, an antibody is exposed to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues. For some immunoconjugates, p may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in certain exemplary embodiments described herein, an antibody may have only one or a limited number of cysteine thiol groups, or may have only one or a limited number of sufficiently reactive thiol groups, to which the drug may be attached. In other embodiments, one or more lysine amino groups in the antibody may be available and reactive for conjugation with an 8AmBza-linker compound of Formula II. In certain embodiments, higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an immunoconjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. The loading (drug/antibody ratio) of an immunoconjugate may be controlled in different ways, and for example, by: (i) limiting the molar excess of the 8AmBza-linker intermediate compound relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive denaturing conditions for optimized antibody reactivity. It is to be understood that where more than one nucleophilic group of the antibody reacts with a drug, then the resulting product is a mixture of antibody-drug conjugate compounds with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug. Individual immunoconjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography (see, e.g., McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K.J., et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No.624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, March 27- 31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, a homogeneous immunoconjugate with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography. An exemplary embodiment of the immunoconjugate of Formula I is selected from the Table 3a and 3b Immunoconjugates. Table 3a Immunoconjugates (IC) Table 3b Immunoconjugates (IC) COMPOSITIONS OF IMMUNOCONJUGATES The invention provides a composition, e.g., a pharmaceutically or pharmacologically acceptable composition or formulation, comprising a plurality of immunoconjugates as described herein and optionally a carrier therefor, e.g., a pharmaceutically or pharmacologically acceptable carrier. The immunoconjugates can be the same or different in the composition, i.e., the composition can comprise immunoconjugates that have the same number of adjuvants linked to the same positions on the antibody construct and/or immunoconjugates that have the same number of 8AmBza adjuvants linked to different positions on the antibody construct, that have different numbers of adjuvants linked to the same positions on the antibody construct, or that have different numbers of adjuvants linked to different positions on the antibody construct. In an exemplary embodiment, a composition comprising the immunoconjugate compounds comprises a mixture of the immunoconjugate compounds, wherein the average drug (8AmBza) loading per antibody in the mixture of immunoconjugate compounds is about 2 to about 5. A composition of immunoconjugates of the invention can have an average adjuvant to antibody construct ratio of about 0.4 to about 10. A skilled artisan will recognize that the number of 8AmBza adjuvants conjugated to the antibody construct may vary from immunoconjugate to immunoconjugate in a composition comprising multiple immunoconjugates of the invention, and, thus, the adjuvant to antibody construct (e.g., antibody) ratio can be measured as an average, which may be referred to as the drug to antibody ratio (DAR). The adjuvant to antibody construct (e.g., antibody) ratio can be assessed by any suitable means, many of which are known in the art. The average number of adjuvant moieties per antibody (DAR) in preparations of immunoconjugates from conjugation reactions may be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of immunoconjugates in a composition in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous immunoconjugates where p is a certain value from immunoconjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. In some embodiments, the composition further comprises one or more pharmaceutically or pharmacologically acceptable excipients. For example, the immunoconjugates of the invention can be formulated for parenteral administration, such as IV administration or administration into a body cavity or lumen of an organ. Alternatively, the immunoconjugates can be injected intra-tumorally. Compositions for injection will commonly comprise a solution of the immunoconjugate dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and an isotonic solution of one or more salts such as sodium chloride, e.g., Ringer's solution. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These compositions desirably are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The composition can contain any suitable concentration of the immunoconjugate. The concentration of the immunoconjugate in the composition can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. In certain embodiments, the concentration of an immunoconjugate in a solution formulation for injection will range from about 0.1% (w/w) to about 10% (w/w). METHOD OF TREATING CANCER WITH IMMUNOCONJUGATES The invention provides a method for treating cancer. The method includes administering a therapeutically effective amount of an immunoconjugate as described herein (e.g., as a composition as described herein) to a subject in need thereof, e.g., a subject that has cancer and is in need of treatment for the cancer. The method includes administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 3. It is contemplated that the immunoconjugate of the present invention may be used to treat various hyperproliferative diseases or disorders, e.g. characterized by the overexpression of a tumor antigen. Exemplary hyperproliferative disorders include benign or malignant solid tumors and hematological disorders such as leukemia and lymphoid malignancies. In another aspect, an immunoconjugate for use as a medicament is provided. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual comprising administering to the individual an effective amount of the immunoconjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein. In a further aspect, the invention provides for the use of an immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of cancer, the method comprising administering to an individual having cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein. Carcinomas are malignancies that originate in the epithelial tissues. Epithelial cells cover the external surface of the body, line the internal cavities, and form the lining of glandular tissues. Examples of carcinomas include, but are not limited to, adenocarcinoma (cancer that begins in glandular (secretory) cells such as cancers of the breast, pancreas, lung, prostate, stomach, gastroesophageal junction, and colon) adrenocortical carcinoma; hepatocellular carcinoma; renal cell carcinoma; ovarian carcinoma; carcinoma in situ; ductal carcinoma; carcinoma of the breast; basal cell carcinoma; squamous cell carcinoma; transitional cell carcinoma; colon carcinoma; nasopharyngeal carcinoma; multilocular cystic renal cell carcinoma; oat cell carcinoma; large cell lung carcinoma; small cell lung carcinoma; non-small cell lung carcinoma; and the like. Carcinomas may be found in prostrate, pancreas, colon, brain (usually as secondary metastases), lung, breast, and skin. In some embodiments, methods for treating non-small cell lung carcinoma include administering an immunoconjugate containing an antibody construct that is capable of binding PD-L1 (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or biobetters thereof). In some embodiments, methods for treating breast cancer include administering an immunoconjugate containing an antibody construct that is capable of binding PD-L1 (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or biobetters thereof). In some embodiments, methods for treating triple-negative breast cancer include administering an immunoconjugate containing an antibody construct that is capable of binding PD-L1 (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or biobetters thereof). Soft tissue tumors are a highly diverse group of rare tumors that are derived from connective tissue. Examples of soft tissue tumors include, but are not limited to, alveolar soft part sarcoma; angiomatoid fibrous histiocytoma; chondromyoxid fibroma; skeletal chondrosarcoma; extraskeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplastic small round-cell tumor; dermatofibrosarcoma protuberans; endometrial stromal tumor; Ewing’s sarcoma; fibromatosis (Desmoid); fibrosarcoma, infantile; gastrointestinal stromal tumor; bone giant cell tumor; tenosynovial giant cell tumor; inflammatory myofibroblastic tumor; uterine leiomyoma; leiomyosarcoma; lipoblastoma; typical lipoma; spindle cell or pleomorphic lipoma; atypical lipoma; chondroid lipoma; well-differentiated liposarcoma; myxoid/round cell liposarcoma; pleomorphic liposarcoma; myxoid malignant fibrous histiocytoma; high-grade malignant fibrous histiocytoma; myxofibrosarcoma; malignant peripheral nerve sheath tumor; mesothelioma; neuroblastoma; osteochondroma; osteosarcoma; primitive neuroectodermal tumor; alveolar rhabdomyosarcoma; embryonal rhabdomyosarcoma; benign or malignant schwannoma; synovial sarcoma; Evan’s tumor; nodular fasciitis; desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcoma protuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma; tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis (PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovial sarcoma; malignant peripheral nerve sheath tumor; neurofibroma; pleomorphic adenoma of soft tissue; and neoplasias derived from fibroblasts, myofibroblasts, histiocytes, vascular cells/endothelial cells, and nerve sheath cells. A sarcoma is a rare type of cancer that arises in cells of mesenchymal origin, e.g., in bone or in the soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissue. Different types of sarcoma are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examples of sarcomas include, but are not limited to, askin's tumor; sarcoma botryoides; chondrosarcoma; ewing's sarcoma; malignant hemangioendothelioma; malignant schwannoma; osteosarcoma; and soft tissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma; cystosarcoma phyllodesdermatofibrosarcoma protuberans (DFSP); desmoid tumor; desmoplastic small round cell tumor; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); hemangiopericytoma; hemangiosarcoma (more commonly referred to as “angiosarcoma”); kaposi’s sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma). A teratoma is a type of germ cell tumor that may contain several different types of tissue (e.g., can include tissues derived from any and/or all of the three germ layers: endoderm, mesoderm, and ectoderm), including, for example, hair, muscle, and bone. Teratomas occur most often in the ovaries in women, the testicles in men, and the tailbone in children. Melanoma is a form of cancer that begins in melanocytes (cells that make the pigment melanin). Melanoma may begin in a mole (skin melanoma), but can also begin in other pigmented tissues, such as in the eye or in the intestines. Merkel cell carcinoma is a rare type of skin cancer that usually appears as a flesh-colored or bluish-red nodule on the face, head or neck. Merkel cell carcinoma is also called neuroendocrine carcinoma of the skin. In some embodiments, methods for treating Merkel cell carcinoma include administering an immunoconjugate containing an antibody construct that is capable of binding PD-L1 (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or biobetters thereof). In some embodiments, the Merkel cell carcinoma has metastasized when administration occurs. Leukemias are cancers that start in blood-forming tissue, such as the bone marrow, and cause large numbers of abnormal blood cells to be produced and enter the bloodstream. For example, leukemias can originate in bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named for how quickly the disease develops and progresses (e.g., acute versus chronic) and for the type of white blood cell that is affected (e.g., myeloid versus lymphoid). Myeloid leukemias are also called myelogenous or myeloblastic leukemias. Lymphoid leukemias are also called lymphoblastic or lymphocytic leukemia. Lymphoid leukemia cells may collect in the lymph nodes, which can become swollen. Examples of leukemias include, but are not limited to, Acute myeloid leukemia (AML), Acute lymphoblastic leukemia (ALL), Chronic myeloid leukemia (CML), and Chronic lymphocytic leukemia (CLL). Lymphomas are cancers that begin in cells of the immune system. For example, lymphomas can originate in bone marrow-derived cells that normally mature in the lymphatic system. There are two basic categories of lymphomas. One category of lymphoma is Hodgkin lymphoma (HL), which is marked by the presence of a type of cell called the Reed-Sternberg cell. There are currently 6 recognized types of HL. Examples of Hodgkin lymphomas include nodular sclerosis classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte- depletion CHL, lymphocyte-rich CHL, and nodular lymphocyte predominant HL. The other category of lymphoma is non-Hodgkin lymphomas (NHL), which includes a large, diverse group of cancers of immune system cells. Non-Hodgkin lymphomas can be further divided into cancers that have an indolent (slow-growing) course and those that have an aggressive (fast-growing) course. There are currently 61 recognized types of NHL. Examples of non-Hodgkin lymphomas include, but are not limited to, AIDS-related Lymphomas, anaplastic large-cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt’s lymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma), chronic lymphocytic leukemia/small lymphocytic lymphoma, cutaneous T-Cell lymphoma, diffuse large B-Cell lymphoma, enteropathy-type T-Cell lymphoma, follicular lymphoma, hepatosplenic gamma- delta T-Cell lymphomas, T-Cell leukemias, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-Cell lymphoma, pediatric lymphoma, peripheral T-Cell lymphomas, primary central nervous system lymphoma, transformed lymphomas, treatment- related T-Cell lymphomas, and Waldenstrom's macroglobulinemia. Brain cancers include any cancer of the brain tissues. Examples of brain cancers include, but are not limited to, gliomas (e.g., glioblastomas, astrocytomas, oligodendrogliomas, ependymomas, and the like), meningiomas, pituitary adenomas, and vestibular schwannomas, primitive neuroectodermal tumors (medulloblastomas). Immunoconjugates of the invention can be used either alone or in combination with other agents in a therapy. For instance, an immunoconjugate may be co-administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. Such combination therapies encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the immunoconjugate can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Immunoconjugates can also be used in combination with radiation therapy. The immunoconjugates of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. Atezolizumab, durvalumab, avelumab, biosimilars thereof, and biobetters thereof are known to be useful in the treatment of cancer, particularly breast cancer, especially triple negative (test negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma. The immunoconjugate described herein can be used to treat the same types of cancers as atezolizumab, durvalumab, avelumab, biosimilars thereof, and biobetters thereof, particularly breast cancer, especially triple negative (test negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma. The immunoconjugate is administered to a subject in need thereof in any therapeutically effective amount using any suitable dosing regimen, such as the dosing regimens utilized for atezolizumab, durvalumab, avelumab, biosimilars thereof, and biobetters thereof. For example, the methods can include administering the immunoconjugate to provide a dose of from about 100 ng/kg to about 50 mg/kg to the subject. The immunoconjugate dose can range from about 5 mg/kg to about 50 mg/kg, from about 10 µg/kg to about 5 mg/kg, or from about 100 µg/kg to about 1 mg/kg. The immunoconjugate dose can be about 100, 200, 300, 400, or 500 µg/kg. The immunoconjugate dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. The immunoconjugate dose can also be outside of these ranges, depending on the particular conjugate as well as the type and severity of the cancer being treated. Frequency of administration can range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week. In another aspect, the invention provides a method for preventing cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate (e.g., as a composition as described above) to a subject. In certain embodiments, the subject is susceptible to a certain cancer to be prevented. For example, the methods can include administering the immunoconjugate to provide a dose of from about 100 ng/kg to about 50 mg/kg to the subject. The immunoconjugate dose can range from about 5 mg/kg to about 50 mg/kg, from about 10 µg/kg to about 5 mg/kg, or from about 100 µg/kg to about 1 mg/kg. The immunoconjugate dose can be about 100, 200, 300, 400, or 500 µg/kg. The immunoconjugate dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. The immunoconjugate dose can also be outside of these ranges, depending on the particular conjugate as well as the type and severity of the cancer being treated. Frequency of administration can range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week. Some embodiments of the invention provide methods for treating cancer as described above, wherein the cancer is breast cancer. Breast cancer can originate from different areas in the breast, and a number of different types of breast cancer have been characterized. For example, the immunoconjugates of the invention can be used for treating ductal carcinoma in situ; invasive ductal carcinoma (e.g., tubular carcinoma; medullary carcinoma; mucinous carcinoma; papillary carcinoma; or cribriform carcinoma of the breast); lobular carcinoma in situ; invasive lobular carcinoma; inflammatory breast cancer; and other forms of breast cancer such as triple negative (test negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer. In some embodiments, methods for treating breast cancer include administering an immunoconjugate containing an antibody construct that is capable of binding HER2 (e.g. trastuzumab, pertuzumab, biosimilars, or biobetters thereof ) and PD-L1 (e.g., atezolizumab, durvalumab, avelumab, biosimilars, or biobetters thereof). In some embodiments, methods for treating colon cancer lung cancer, renal cancer, pancreatic cancer, gastric cancer, and esophageal cancer include administering an immunoconjugate containing an antibody construct that is capable of binding CEA, or tumors over-expressing CEA (e.g. labetuzumab, biosimilars, or biobetters thereof). In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by TLR7 and/or TLR8. EXAMPLES Preparation of 8-amido-2-aminobenzazepine compounds (8AmBza) and intermediates Example 1 Synthesis of tert-butyl ((5-(2-amino-4-(dipropylcarbamoyl)-3H- benzo[b]azepine-8-carboxamido)pyridin-3-yl)methyl)carbamate, 8AmBza-1 8AmBza-1 was prepared and characterized according to the procedures described herein. Example 2 Synthesis of tert-butyl (3-(8-((6-(4-((2- acetamidoethyl)carbamoyl)piperidin-1-yl)pyridin-3-yl)carbamo yl)-2-amino-N-propyl-3H- benzo[b]azepine-4-carboxamido)propyl)carbamate, 8AmBza-2 Preparation of N-(2-acetamidoethyl)-1-(5-nitropyridin-2-yl) piperidine-4-carboxamide, 8AmBza-2b To a mixture of acetyl chloride (142.82 mg, 1.82 mmol, 129.83 µL, 3 eq) and N-(2- aminoethyl)-1-(5-nitro-2-pyridyl)piperidine-4-carboxamide, 8AmBza-2a (0.2 g, 606.46 µmol, 1 eq, HCl) in THF (10 mL) was added Et3N (245.47 mg, 2.43 mmol, 337.65 µL, 4 eq) at 25°C under N 2 . The mixture was stirred at 25°C for 1 hour. LCMS showed the reaction was completed. The mixture was pour into water (20 mL). The mixture was filtered to give 8AmBza-2b (0.2 g, 596.38 µmol, 98.34% yield) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.95 (d, J = 2.4 Hz, 1H), 8.19 (dd, J = 9.6, 2.4 Hz, 1H), 7.78-7.98 (m, 2H), 6.95 (d, J = 9.6 Hz, 1H), 4.50 (d, J = 9.6 Hz, 2H), 2.93-3.15 (m, 7H), 1.73-1.80 (m, 5H), 1.43-1.62 (m, 2H), 1.07-1.28 (m, 3H). Preparation of N-(2-acetamidoethyl)-1-(5-aminopyridin-2-yl) piperidine-4-carboxamide, 8AmBza-2c To a solution of N-(2-acetamidoethyl)-1-(5-nitro-2-pyridyl)piperidine-4-carbo xamide, 8AmBza-2b (0.2, 596.38 µmol, 1 eq) in MeOH (20 mL) was added Pd/C (0.2 g, 5% purity) under N 2 . The suspension was degassed under vacuum and purged with H 2 several times. The mixture was stirred under H2 (15psi) at 25°C for 4 hours. LCMS showed the reaction was completed. The mixture was filtered and concentrated to give 8AmBza-2c (0.18 g, 589.44 µmol, 98.84% yield) as yellow solid. Preparation of tert-butyl (3-(8-((6-(4-((2-acetamidoethyl)carbamoyl)piperidin-1- yl)pyridin-3-yl)carbamoyl)-2-amino-N-propyl-3H-benzo[b]azepi ne-4- carboxamido)propyl)carbamate, 8AmBza-2 To a mixture of 2-amino-4-[3-(tert-butoxycarbonylamino) propyl-propyl-carbamoyl]- 3H-1-benzazepine-8-carboxylic acid, 8AmBza-2d (0.22 g, 494.91 µmol, 1 eq) 1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridi nium 3-oxide hexafluorophosphate, Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium, HATU, CAS Reg. No.148893-10-1 (225.82 mg, 593.90 µmol, 1.2 eq) in DMF (5 mL) was added Et3N (150.24 mg, 1.48 mmol, 206.66 µL, 3 eq) at 25°C. The mixture was stirred at 25 °C for 5 min, then N-(2-acetamidoethyl)-1-(5-amino-2-pyridyl)piperidine-4-carbo xamide, 8AmBza-2c (151.13 mg, 494.91 µmol, 1 eq) was added to the mixture, stirred for 30 min. The mixture was poured into water (50mL). The aqueous phase was extracted with ethyl acetate (50 mL*1). The combined organic phase was washed with brine (50 mL*1), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by prep-HPLC column: Welch Xtimate C18150*25mm*5um;mobile phase: [water(10mM NH4HCO3)-ACN];B%: 30%- 50%,10.5min to afford 8AmBza-2 (96 mg, 131.17 µmol, 26.50% yield) as an off-white solid. 1 H NMR (MeOD, 400 MHz) δ 8.39 (d, J = 2.6 Hz, 1H), 7.90 (dd, J = 9.2, 2.6 Hz, 1H), 7.69 (d, J = 1.2 Hz, 1H), 7.54-7.60 (m, 1H), 7.46 (br d, J = 8.0 Hz, 1H), 6.85-6.95 (m, 2H), 4.30 (d, J = 13.6 Hz, 2H), 3.39-3.53 (m, 4H), 3.28 (s, 2H), 3.08-3.12 (m, 2H), 2.83-2.93 (m, 2H), 2.37-2.47 (m, 1H), 1.94 (s, 3H), 1.60-1.90 (m, 8H), 1.24-1.50 (m, 9H). LC/MS [M+H] 732.42 (calculated); LC/MS [M+H] 732.40 (observed). Example 3 Synthesis of 2-amino-N8-(6-(4-((2-aminoethyl)carbamoyl)piperidin-1- yl)pyridin-3-yl)-N4,N4-dipropyl-3H-benzo[b]azepine-4,8-dicar boxamide, 8AmBza-3 8AmBza-3 was prepared and characterized according to the procedures described herein. Example 4 Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5- ureidopentanamido)benzyl ((5-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8- carboxamido)pyridin-3-yl)methyl)carbamate, 8AmBza-4 8AmBza-4 was prepared and characterized according to the procedures described herein. Example 5 Synthesis of tert-butyl (3-(2-amino-8-((6-(4-((2- aminoethyl)carbamoyl)piperidin-1-yl)pyridin-3-yl)carbamoyl)- N-propyl-3H-benzo[b]azepine-4- carboxamido)propyl)carbamate, 8AmBza-5 Preparation of N-(2-aminoethyl)-1-(5-nitropyridin-2-yl)piperidine-4-carboxa mide, 8AmBza-5b To a mixture of tert-butyl N-[2-[[1-(5-nitro-2-pyridyl)piperidine-4-carbonyl] amino]ethyl]carbamate, 8AmBza-5a (0.5 g, 1.27 mmol, 1 eq) in EtOAc (10 mL) was added HCl/EtOAc (4 M, 3.18 mL, 10 eq) at 25°C. The mixture was stirred at 25°C for 2 hours. LCMS showed the reaction was completed. The reaction was concentrated in vacuum to give 8AmBza-5b (0.4 g, 1.21 mmol, 95.44% yield, HCl) as a yellow solid. Preparation of 1-(5-nitropyridin-2-yl)-N-(2-(2,2,2-trifluoroacetamido) ethyl)piperidine-4- carboxamide, 8AmBza-5c To a mixture of N-(2-aminoethyl)-1-(5-nitro-2-pyridyl)piperidine-4-carboxami de, 8AmBza-5b (0.4 g, 1.21 mmol, 1 eq, HCl) in THF (10 mL) was added Et 3 N (368.21 mg, 3.64 mmol, 506.47 µL, 3 eq) and (2,2,2-trifluoroacetyl) 2,2,2-trifluoroacetate (382.13 mg, 1.82 mmol, 253.06 µL, 1.5 eq) at 25°C. The mixture was stirred at 25°C for 1 hours. LCMS showed major as desired. The mixture was poured into water (50 mL). The aqueous phase was extracted with ethyl acetate (30 mL*3). The combined organic phase was washed with brine (30 mL*1), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was used to next step directly, containing 8AmBza-5c (0.4 g, 1.03 mmol, 84.71% yield) as a yellow solid. 1 H NMR (DMSO-d 6 , 400 MHz) δ 9.37-9.45 (m, 1H), 8.95 (d, J = 2.8 Hz, 1H), 8.19 (dd, J = 9.6, 2.8 Hz, 1H), 8.03 (br t, J = 5.2 Hz, 1H), 6.96 (d, J = 9.6 Hz, 1H), 4.47-4.53 (m, 2H), 2.99- 3.25 (m, 6H), 2.38-2.47 (m, 3H), 1.73-1.80 (m, 2H), 1.41-1.58 (m, 2H) Preparation of 1-(5-aminopyridin-2-yl)-N-(2-(2,2,2-trifluoroacetamido) ethyl)piperidine- 4-carboxamide, 8AmBza-5d To a solution of 1-(5-nitro-2-pyridyl)-N-[2-[(2,2,2-trifluoroacetyl)amino]eth yl] piperidine-4-carboxamide, 8AmBza-5c (0.4 g, 1.03 mmol, 1 eq) in MeOH (30 mL) was added Pd/C (0.5 g, 5% purity) under N 2 . The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (50 psi) at 25°C for 2 hours. TLC showed the reaction was completed. The mixture was filtered and concentrated in vacuum to give 8AmBza-5d (0.3 g, 834.85 µmol, 81.26% yield) as a gray solid. 1 H NMR (DMSO-d 6 , 400 MHz) δ 9.39-9.46 (m, 1H), 7.97 (t, J = 5.2 Hz, 1H), 7.59 (d, J = 2.8 Hz, 1H), 6.90 (dd, J = 8.8, 2.8 Hz, 1H), 6.64 (d, J = 8.8 Hz, 1H), 3.99 (d, J = 12.8 Hz, 2H), 3.15-3.26 (m, 6H), 2.54-2.63 (m, 2H), 2.16-2.26 (m, 1H), 1.65-1.71 (m, 2H), 1.48-1.60 (m, 2H)
Preparation of tert-butyl (3-(2-amino-8-bromo-N-propyl-3H-benzo[b]azepine-4- carboxamido)propyl)carbamate, 8AmBza-5g To a mixture of 2-amino-8-bromo-3H-1-benzazepine-4-carboxylic acid, 8AmBza-5f (4.09 g, 14.56 mmol, 1 eq) and tert-butyl N-[3-(propylamino)propyl]carbamate (3.78 g, 17.47 mmol, 1.2 eq) in DMF (10 mL) was added HATU (6.64 g, 17.47 mmol, 1.2 eq) and Et 3 N (2.95 g, 29.12 mmol, 4.05 mL, 2 eq) in one portion at 25 C. The mixture was stirred at 25°C for 1 h. LCMS showed the reaction was finished. The mixture was diluted with water and extracted with EtOAc (50 mL x 3). The organic layer was washed with brine, dried over Na 2 SO 4 , filtered and concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=1/0, 0/1) to afford 8AmBza-5g (6 g, 12.52 mmol, 85.95% yield) as a yellow oil. Preparation of methyl 2-amino-4-[3-(tert-butoxycarbonylamino)propyl-propyl – carbamoyl]-3H-1-benzazepine-8-carboxylate, 8AmBza-5h To a solution of tert-butyl N-[3-[(2-amino-8-bromo-3H-1-benzazepine-4-carbonyl)– propyl -amino]propyl] carbamate, Bz-39g (5 g, 10.43 mmol, 1 eq) in MeOH (50 mL) was added Et3N (3.17 g, 31.29 mmol, 4.35 mL, 3 eq) and [1,1′- bis(diphenylphosphino)ferrocene]dichloropalladium(II), Pd(dppf)Cl2, CAS Reg. No.72287-26-4 (763.13 mg, 1.04 mmol, 0.1 eq) under N 2 . The suspension was degassed under vacuum and purged with CO (10.43 mmol, 1 eq) several times. The mixture was stirred under CO (50psi) at 80°C for 12 hours. LCMS showed the reaction was finished. The mixture was filtered and concentrated to give 8AmBza-5h (7 g, crude) as yellow oil. Preparation of 2-amino-4-((3-((tert-butoxycarbonyl)amino)propyl)(propyl)car bamoyl)- 3H-benzo[b]azepine-8-carboxylic acid, 8AmBza-5e To a mixture of methyl 2-amino-4-[3-(tert-butoxycarbonylamino)propyl-propyl- carbamoyl] -3H-1-benzazepine-8-carboxylate, Bz-39h (6 g, 13.08 mmol, 1 eq) in MeOH (80 mL) was added LiOH (1.25 g, 52.34 mmol, 4 eq) in one portion at 30°C. The mixture was stirred at 30°C for 12 h. LCMS showed the reaction was finished. The mixture was adjusted pH 6 with aq (aqueous) HCl (1 M) at 25 °C. The mixture was concentrated. The mixture was further purification by pre-HPLC(column: Phenomenex luna® C18250*50mm*10 um;mobile phase: [water(0.1%TFA)-ACN];B%: 10%-40%,20min) to give 8AmBza-5e (1.4 g, 3.09 mmol, 23.64% yield, 98.23% purity) as yellow oil. 1 H NMR (MeOD, 400MHz) δ 8.06 (d, J =1.2 Hz, 1H), 8.02 (dd, J =1.6, 8.0 Hz, 1H), 7.68 (s, 1H), 7.14 (s, 1H), 3.58-3.44 (m, 4H), 3.37 (s, 2H), 3.10 (m, 2H), 1.85 (m, 2H), 1.71 (m, 2H), 1.51-1.33 (m, 9H), 0.92-0.98 (m, 3H). LC/MS [M+H] 445.25 (calculated); LC/MS [M+H] 445.10 (observed). Preparation of tert-butyl (3-(2-amino-N-propyl-8-((6-(4-((2-(2,2,2- trifluoroacetamido)ethyl)carbamoyl)piperidin-1-yl)pyridin-3- yl)carbamoyl)-3H- benzo[b]azepine-4-carboxamido)propyl)carbamate, 8AmBza-5i To a mixture of 2-amino-4-[3-(tert-butoxycarbonylamino)propyl-propyl-carbamo yl] - 3H-1-benzazepine-8-carboxylic acid, 8AmBza-5e (200 mg, 449.92 µmol, 1 eq) HATU (205.29 mg, 539.90 µmol, 1.2 eq) in DMF (3 mL) was added Et 3 N (136.58 mg, 1.35 mmol, 187.87 µL, 3 eq) at 25°C. The mixture was stirred at 25°C for 5 min, then 1-(5-amino-2-pyridyl)-N-[2- [(2,2,2-trifluoroacetyl)amino]ethyl]piperidine-4-carboxamide , 8AmBza-5d (161.68 mg, 449.92 µmol, 1 eq) was added to the mixture, stirred for 30 min. LCMS showed major as desired. The mixture was poured into water (50mL). The aqueous phase was extracted with ethyl acetate (50 mL*1). The combined organic phase was washed with brine (50 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give 8AmBza-5i (0.3 g, 381.75 µmol, 84.85% yield) as yellow oil. Preparation of tert-butyl (3-(2-amino-8-((6-(4-((2-aminoethyl)carbamoyl)piperidin-1- yl)pyridin-3-yl)carbamoyl)-N-propyl-3H-benzo[b]azepine-4-car boxamido)propyl)carbamate, 8AmBza-5 To a mixture of tert-butyl N-[3-[[2-amino-8-[[6-[4-[2-[(2,2,2-trifluoroacetyl) amino]ethylcarbamoyl]-1-piperidyl]-3-pyridyl]carbamoyl]-3H-1 -benzazepine-4-carbonyl]- propyl-amino]propyl]carbamate, 8AmBza-5i (0.25 g, 318.13 µmol, 1 eq) in MeOH (10 mL) was added LiOH.H 2 O (40.05 mg, 954.38 µmol, 3 eq) in H 2 O (1 mL) at 25°C. The mixture was stirred at 40°C for 12 hours. LCMS showed major as desired. The mixture was concentrated in vacuum. The residue was purified by prep-HPLC column: Nano-micro Kromasil C18 100*30mm 5um;mobile phase: [water(0.1%TFA)-ACN];B%: 15%-45%,10min to give 8AmBza-5 (45 mg, 65.23 µmol, 20.51% yield) as a white solid. 1 H NMR (MeOD, 400 MHz) δ 8.73 (d, J = 2.4 Hz, 1H), 8.24 (dd, J = 9.8, 2.4 Hz, 1H), 7.75 (br s, 1H), 7.45 (d, J = 9.8 Hz, 1H), 7.15 (br s, 1H), 4.24 (br d, J = 13.6 Hz, 2H), 3.35-3.62 (m, 9H), 3.05-3.12 (m, 4H), 2.59-2.72 (m, 1H), 1.99-2.09 (m, 2H), 1.65-1.94 (m, 6H), 1.45 (s, 9H), 0.90-0.98 (m, 3H). LC/MS [M+H] 690.41 (calculated); LC/MS [M+H] 690.40 (observed). Example 6 Synthesis of tert-butyl N-[3-[[2-amino-8- [[6-[4-[2- [(2,2,2- trifluoroacetyl)amino]ethylcarbamoyl]-1-piperidyl]-3-pyridyl ] carbamoyl] -3H-1-benzazepine- 4-carbonyl]-propyl-amino]propyl]carbamate, 8AmBza-6 To a mixture of 2-amino-4- [3-(tert-butoxycarbonylamino) propyl-propyl-carbamoyl] - 3H-1-benzazepine-8-carboxylic acid (0.43 g, 976 µmol, 1.0 eq) and 1-(5-amino-2-pyridyl) -N- [2-[(2,2,2-trifluoroacetyl)amino]ethyl]piperidine-4-carboxam ide (526.26 mg, 1.46 mmol, 1.5 eq) in MeOH (2 mL) and DCM (4 mL) was added N-Ethoxycarbonyl-2-ethoxy-1,2- dihydroquinoline, EEDQ (362 mg, 1.46 mmol, 1.5 eq) at 25°C and stirred for 12 hours at this temperature. The mixture was then concentrated under reduced pressure, and the residue was purified by column chromatography (SiO 2 , Petroleum ether/Ethyl acetate=30/1 to 0:1). 8AmBza-6 (0.58 g, 687 µmol, 70.4% yield, 93.14% purity) was obtained as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ8.70 (d, J = 2.4 Hz, 1H), 8.19 (dd, J = 2.4, 9.8 Hz, 1H), 8.05-7.89 (m, 2H), 7.74 (s, 1H), 7.42 (d, J = 9.8 Hz, 1H), 7.14 (s, 1H), 4.21 (d, J = 13.6 Hz, 1H), 3.59-3.32 (m, 10H), 3.28-3.24 (m, 2H), 3.16-3.11 (m, 2H), 2.63-2.53 (m, 1H), 2.06-1.90 (m, 2H), 1.89- 1.78 (m, 3H), 1.74-1.61 (m, 2H), 1.53-1.25 (m, 9H), 1.06-0.84 (m, 3H). LC/MS [M+H] 785.38 (calculated); LC/MS [M+H] 786.0 (observed). Example 7 Synthesis of tert-butyl N-[3-[[2-amino-8-[[2-[2-(tert- butoxycarbonylamino)ethylamino]pyrimidin-5-yl]carbamoyl]-3H- 1-benzazepine-4-carbonyl]- propyl-amino]propyl]carbamate, 8AmBza-7 To a mixture of 2-chloro-5-nitro-pyrimidine (2.9 g, 18.2 mmol, 1.0 eq) and tert-butyl N- (2-aminoethyl)carbamate (3.2 g, 20.0 mmol, 3.14 mL, 1.1 eq) in THF (50 mL) was added DIEA (4.7 g, 36.4 mmol, 6.33 mL, 2.0 eq) at 25°C and it was stirred for 2 hours at this temperature. The mixture was added water (100 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with brine (50 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. Compound tert-butyl N-[2-[(5-nitropyrimidin-2- yl)amino]ethyl]carbamate, 8AmBza-7a (5.7 g, crude) was obtained as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ9.11 (d, J = 2.8 Hz, 1H), 9.05 (d, J = 2.8 Hz, 1H), 6.59 (s, 1H), 4.85 (s, 1H), 3.66 (q, J = 5.6 Hz, 2H), 3.44-3.41 (m, 2H), 1.45 (s, 9H). To a solution of 8AmBza-7a (1.0 g, 3.53 mmol, 1.0 eq) in MeOH (30 mL) was added Pd/C (0.5 g, 10% purity) under N2. The suspension was degassed under vacuum and purged with H 2 several times. The mixture was stirred under H 2 (15 psi) at 25°C for 12 hours, then filtered and the filtrate was concentrated in vacuum. 8AmBza-7b (0.8 g, crude) was obtained as a yellow solid. To a mixture of 2-amino-4- [3-(tert-butoxycarbonylamino) propyl-propyl-carbamoyl] - 3H-1-benzazepine-8-carboxylic acid, 8AmBza-7c (60 mg g, 135 µmol, 1.0 eq) and 8AmBza-7b (103 mg, 405 µmol, 3 eq) in MeOH (5 mL) and DCM (10 mL) was added EEDQ (50 mg, 202 µmol, 1.5 eq) at 25°C and it was stirred for 12 hours at this temperature. The mixture was concentrated under reduced pressure, and then the residue was purified by prep-HPLC (column: Welch Xtimate C18100 x 25mm x 3um; mobile phase: [water (0.1%TFA)-ACN]; B%: 25%- 45%, 12 min). 8AmBza-7 (13 mg, 16.8 µmol, 12.4% yield, 87.7% purity) was obtained as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ8.64 (s, 2H), 8.05-7.90 (m, 2H), 7.73 (s, 1H), 7.14 (s, 1H), 3.53-3.48 (m, 6H), 3.37-3.34 (m, 2H), 3.31 (s, 2H), 3.29-3.13 (m, 2H), 1.90-1.78 (m, 2H), 1.75-1.64 (m, 2H), 1.56-1.40 (m, 18H), 1.02-0.87 (m, 3H). LC/MS [M+H] 680.4 (calculated); LC/MS [M+H] 680.3 (observed). Example 8 Synthesis of tert-butyl N-[3-[[2-amino-8-[[3-[2- [2-(tert- butoxycarbonylamino)ethoxy]ethoxymethyl]phenyl]carbamoyl]-3H -1-benzazepine-4-carbonyl]- propyl-amino]propyl]carbamate, 8AmBza-8
To a mixture of tert-butyl N-[2-(2-hydroxyethoxy)ethyl]carbamate (2.9 g, 14.1 mmol, 1.0 eq) in DMF (10 mL) was added sodium hydride, NaH (565 mg, 14.1 mmol, 60% purity, 1.0 eq) slowly at 0°C and it was stirred for 1h at this temperature, then 1-(bromomethyl)-3-nitro- benzene (3.05 g, 14.13 mmol, 1.0 eq) was added to the mixture and stirred for 0.5 h. The mixture was diluted with water (30 ml) and extracted with ethylacetate, EtOAc (30 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=10/1 to 1/1) to afford tert-butyl N-[2-[2-[(3- nitrophenyl)methoxy]ethoxy]ethyl]carbamate, 8AmBza-8a (2.2 g, 6.46 mmol, 45.75% yield) as yellow oil. 1 H NMR (CDCl3, 400MHz) δ8.24 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 4.96 (s, 1H), 4.67 (s, 2H), 3.71-3.64 (m, 4H), 3.59-3.52 (m, 2H), 3.37-3.28 (m, 2H), 1.43 (s, 9H). To a solution of 8AmBza-8a (400 mg, 1.18 mmol, 1.0 eq) in EtOAc (10 mL) was added Pd/C (0.3 g, 10% purity) under N 2 . The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25°C for 3 hours, then filtered and concentrated in vacuum to afford tert-butyl N-[2-[2-[(3- aminophenyl)methoxy]ethoxy]ethyl]carbamate, 8AmBza-8b (0.35 g, crude) as yellow oil. To a mixture of 8AmBza-8b (42 mg, 135 µmol, 1.2 eq) and 2-amino-4-[3-(tert- butoxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepin e-8-carboxylic acid, 8AmBza-8c (50 mg, 112 µmol, 1.0 eq) in MeOH (0.5 mL) and DCM (1 mL) was added EEDQ (42 mg, 168 µmol, 1.5 eq) at 25°C. The mixture was stirred at 25°C for 12 h, and then concentrated in vacuum. The residue was purified by prep-HPLC (column: Welch Xtimate C18 100*25mm*3um; mobile phase: [water (0.1% TFA) - ACN]; B%: 30%-50%, 12min) to give 8AmBza-8 (8 mg, 10.9 µmol, 9.6% yield) as white solid. 1 H NMR (MeOD, 400MHz) δ8.02- 7.95 (m, 2H), 7.80-7.71 (m, 2H), 7.68 (d, J = 8.8 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.16 (s, 1H), 4.62 (s, 2H), 3.73-3.65 (m, 4H), 3.55 (t, J = 5.6 Hz, 4H), 3.50 (s, 2H), 3.39 (s, 2H), 3.25 (t, J = 5.6 Hz, 2H), 3.12 (d, J = 18.4 Hz, 2H), 1.92-1.81 (m, 2H), 1.77-1.64 (m, 2H), 1.43 (s, 18H), 0.94 (s, 3H). LC/MS [M+H] 737.4 (calculated); LC/MS [M+H] 737.4 (observed). Example 9 Synthesis of tert-butyl (3-(2-amino-8-(phenylcarbamoyl)-N-propyl-3H- benzo [b]azepine-4-carboxamido)propyl)carbamate, 8AmBza-9 To a mixture of aniline (25 mg, 270 µmol, 2.0 eq) and 2-amino-4-[3-(tert- butoxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepin e-8-carboxylic acid (60 mg, 135 µmol, 1.0 eq) in DCM (2 mL) and MeOH (0.5 mL) was added EEDQ (50 mg, 202 µmol, 1.5 eq) at 25 under N 2 . The mixture was stirred at 25°C for 2 hours and then concentrated in vacuum. The residue was purified by prep-HPLC (column: Welch Xtimate C18 150*25mm*5um;mobile phase: [water(10mM NH4HCO3)-ACN];B%: 40%-70%,10.5min) to afford 8AmBza-9 (10 mg, 19.2 µmol, 14.26% yield) as a white solid. 1 H NMR (MeOD, 400MHz) δ 7.73-7.66 (m, 3H), 7.57 (dd, J = 1.6, 8.0 Hz, 1H), 7.47 (br d, J = 8.0 Hz, 1H), 7.37 (t, J = 8.0 Hz, 2H), 7.20-7.12 (m, 1H), 6.93 (s, 1H), 3.50 (br t, J = 7.2 Hz, 2H), 3.45-3.38 (m, 2H), 3.21-2.96 (m, 2H), 2.85 (s, 2H), 1.89-1.77 (m, 2H), 1.70-1.62 (m, 2H), 1.44 (s, 9H), 1.05- 0.8 (m, 3H). LC/MS [M+H] 520.3 (calculated); LC/MS [M+H] 520.3 (observed). Example 10 Synthesis of 2-amino-N4 -(3-aminopropyl)-N8-phenyl-N4-propyl-3H-1- benzazepine-4,8-dicarboxamide, 8AmBza-10
Preparation of ethyl 2-amino-8-formyl-3H-1-benzazepine-4-carboxylate, 8AmBza-10b To a solution of ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylate, 8AmBza-10a (10 g, 32.4 mmol, 1 eq) in DMF (100 mL) was added Et3SiH (72.8 g, 626.09 mmol, 100 mL, 19.36 eq), Et3N (6.5 g, 64.69 mmol, 9.00 mL, 2 eq) and Pd(dppf)Cl2 (1.18 g, 1.62 mmol, 0.05 eq) under N 2 . The suspension was degassed under vacuum and purged with CO several times and it was stirred under CO (50 psi) at 80 °C for 12 h (hours). The mixture was diluted with water (300 mL) and extracted with EtOAc (80 mL x 3). The organic layer was washed with brine (50 mL), dried over Na 2 SO 4 , filtered and concentrated, and the residue was purified by flash silica gel chromatography (ISCO®; 15 g SepaFlash® Silica Flash Column, Eluent of 0~100% Ethyl acetate/Petroleum ethergradient at 65 mL/min) to give 8AmBza-10b (3 g, 11.6 mmol, 35.9% yield) as yellow solid. 1 H NMR (DMSO-d 6 , 400 MHz) δ10.00 (s, 1H) 7.79 (s, 1H) 7.61 (d, J = 8.4 Hz, 1H) 7.55 (d, J = 1.2 Hz, 1H) 7.40 (dd, J = 8.0, 1.2 Hz, 1H) 7.07 (s, 2 H) 4.25 (q, J = 6.8 Hz, 2H) 2.91 (s, 2H) 1.31 (t, J = 6.8 Hz, 3H). Preparation of 2-amino-4-ethoxycarbonyl-3H-1-benzazepine-8-carboxylic acid, 8AmBza-10c To a solution of 8AmBza-10b (2.6 g, 10.1 mmol, 1.0 eq) in CH 3 CN (15 mL) was added NaH 2 PO 4 (362 mg, 3.02 mmol, 0.3 eq), H 2 O 2 (5.71 g, 50.33 mmol, 4.84 mL, 30% purity, 5 eq) and NaClO 2 (1.46 g, 16.1 mmol, 1.6 eq) at 0°C and it was stirred at 25°C for 5 h. The reaction mixture was quenched with Na2SO3 (aq) and diluted with H2O (30 mL) and EtOAc (30 ml), the pH of the mixture was adjusted to 4 with aq HCl (1 M), then filtered to give desired solid The solid was dried under vacuum to give 8AmBza-10c (2.1 g, 7.66 mmol, 76.1% yield) as white solid. 1 H NMR (DMSO-d6, 400 MHz) δ7.87 (s, 1H), 7.81 (s, 1H), 7.72-7.67 (m, 2H), 4.27 (q, J = 7.2 Hz, 2H), 3.28 (s, 2 H), 1.31 (t, J = 7.2 Hz, 3H). Preparation of ethyl 2-amino-8-(phenylcarbamoyl)-3H-1-benzazepine-4-carboxylate, 8AmBza-10d To a mixture of 8AmBza-10c (1.0 g, 3.65 mmol, 1.0 eq) in DMF (20 mL) was added (7- Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate), PyAOP (2.28 g, 4.38 mmol, 1.2 eq) and DIEA (2.36 g, 18.2 mmol, 3.18 mL, 5.0 eq) at 25°C and it was stirred at 25 °C for 10 min, then aniline (373 mg, 4.01 mmol, 366 µL, 1.1 eq) was then added and stirred for 1 hour at 25°C. The mixture was poured into ice water (50 mL) and stirred for 2 min. The aqueous phase was extracted with ethyl acetate (20 mL x 3). The combined organic phase was washed with brine (20 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum and the residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=0/1 to EtOAc/MeOH=2/1) to afford 8AmBza-10d (0.5 g, 1.43 mmol, 39.25% yield) as yellow solid. 1 H NMR (MeOD, 400 MHz) δ 7.89 (s, 1H), 7.76-7.65 (m, 3H), 7.62-7.56 (m, 1H), 7.37 (t, J = 8.0 Hz, 2H), 7.16 (t, J = 8.0 Hz, 1H), 4.35 (q, J = 7.2 Hz, 2H), 3.32 (s, 2H), 1.38 (t, J = 7.2 Hz, 3H). Preparation of 2-amino-8-(phenylcarbamoyl)-3H-1-benzazepine-4-carboxylic acid, 8AmBza-10e To a mixture of 8AmBza-10d (0.36 g, 1.03 mmol, 1.0 eq) in EtOH (10 mL) was added a solution of LiOH•H2O (216 mg, 5.15 mmol, 5.0 eq) in H2O (1 mL) at 25 °C and it was stirred for 16 hours at this temperature. The mixture was quenched with HCl (4M) until pH to 5 and concentrated under reduced pressure at 40 °C to remove EtOH. Water (10 mL) was added to the mixture and then filtered to give desired solid 8AmBza-10e (0.2 g, 622 µmol, 60.41% yield) was obtained as yellow solid which was used into the next step without further purification. 1 H NMR (DMSO-d 6 , 400 MHz) δ7.84-7.74 (m, 3H), 7.66 (s, 1H), 7.56-7.47 (m, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.09 (t, J = 7.2 Hz, 2H), 2.92 (s, 2H). Preparation of tert-butyl N-[3-[[2-amino-8-(phenylcarbamoyl)-3H-1-benzazepine-4- carbonyl]-propyl-amino]propyl]carbamate, 8AmBza-10f To a solution of 8AmBza-10e (0.2 g, 622 µmol, 1.0 eq) in DMF (5 mL) was added HATU (284 mg, 746 µmol, 1.2 eq) and DIEA (241 mg, 1.87 mmol, 325 µL, 3.0 eq) at 25 °C and it was stirred for 10 min at this temperature, then tert-butyl N-[3- (propylamino)propyl]carbamate (161 mg, 746 µmol, 1.2 eq) was added to the mixture and stirred at 25°C for 3 hours. The mixture was poured into ice water (30 mL) and stirred for 10 min. The aqueous phase was extracted with EtOAc (10 mL x 3), and the combined organic phase was washed with H2O (10 mL x 2) and brine (10 mL), dried by Na2SO4 and concentrated to give 8AmBza-10f (0.3 g, 577 µmol, 92.76% yield) as yellow oil. Preparation of 2-amino-N4 -(3-aminopropyl)-N8-phenyl-N4-propyl-3H-1-benzazepine- 4,8-dicarboxamide, 8AmBza-10 To a solution of 8AmBza-10f (0.4 g, 769 µmol, 1.0 eq) in MeOH (10 mL) was added HCl/MeOH (4 M, 9.62 mL, 50 eq) at 25 °C. The mixture was stirred at 25°C for 1 hour, and then concentrated under reduced pressure at 40°C. The residue was purified by prep-HPLC (column: Nano-micro Kromasil C18100*30mm 8um; mobile phase: [water (0.1%TFA) - ACN]; B%: 5% - 30%, 10min) to afford 8AmBza-10 (0.23 g, 431 µmol, 56.0% yield, TFA salt) as yellow solid. 1 H NMR (MeOD, 400 MHz) δ8.01-7.94 (m, 2H), 7.76-7.70 (m, 3H), 7.41 (t, J = 8.0 Hz, 2H), 7.21 (t, J = 7.6 Hz, 2H), 3.63 (t, J = 7.2 Hz, 2H), 3.58-3.49 (m, 2H), 3.41 (s, 2H), 3.10-2.95 (m, 2H), 2.12-1.99 (m, 2H), 1.82-1.68 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H). LC/MS [M+H] 420.2 (calculated); LC/MS [M+H] 420.2 (observed). Example 11 Synthesis of tert-butyl N-[4-[[2-amino-8-(phenylcarbamoyl)-3H-1- benzazepine-4-carbonyl]-propyl-amino]but-2-ynyl]carbamate, 8AmBza-11
Preparation of N-(4-chlorobut-2-ynyl)-4-nitro-N-propyl-benzenesulfonamide, 8AmBza- 11b To a solution of propan-1-amine (7 g, 118 mmol, 9.74 mL, 1.0 eq) and Et 3 N (24 g, 237 mmol, 33 mL, 2.0 eq) in DCM (50 mL) was added 4-nitrobenzenesulfonyl chloride (26.2 g, 118 mmol, 1.0 eq) and it was stirred at 25 °C for 0.5 h. The reaction mixture was poured into water (60 mL) and extracted with DCM (100 mL*3). The combined organic phases was washed with brine (50 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude product 4-nitro-N-propyl-benzenesulfonamide, 8AmBza-11a (28 g, 114.6 mmol, 96.8% yield) as yellow solid which was used into the next step without further purification. 1 H NMR (CDCl 3 , 400MHz) δ8.38 (d, J = 8.8 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H), 4.77 (s, 1H), 3.02-2.99 (m, 2H), 1.57-1.48 (m, 2H), 0.89 (t, J = 7.6 Hz, 3H) To a solution of 8AmBza-11a (28 g, 115 mmol, 1.0 eq) in DMF (300 mL) was added Cs 2 CO 3 (56 g, 172 mmol, 1.5 eq) and 1, 4-dichlorobut-2-yne (28.2 g, 229 mmol, 2.0 eq) and it was stirred at 25°C for 16 h. The reaction mixture was poured into water (300 mL) and extracted with MTBE (150 mL*3). The combined organic phases was washed with brine (150 mL), dried over Na2SO4, filtered and concentrated under reduced pressure, and the residue was purified by column chromatography (SiO 2 , Petroleum ether/Ethyl acetate=50/1 to 5/1) to give 8AmBza-11b (28 g, 84.6 mmol, 73.84% yield) as yellow oil. 1 H NMR (CDCl 3 , 400MHz) δ8.37 (d, J = 8.8 Hz, 2H), 8.05 (d, J = 8.8 Hz, 2H), 4.22 (t, J = 2.0 Hz, 2H), 3.85 (t, J = 2.0 Hz, 2H), 3.17 (t, J = 7.6 Hz, 2H), 1.65-1.56 (m, 2H), 0.94 (t, J = 7.6 Hz, 3H). Preparation of tert-butyl (tert-butoxycarbonyl)(4-((4-nitro-N- propylphenyl)sulfonamido)but-2-yn-1-yl)carbamate, 8AmBza-11c To a solution of 8AmBza-11b (23.5 g, 71.0 mmol, 1.0 eq) in DMF (250 mL) was added Cs 2 CO 3 (46.3 g, 142 mmol, 2.0 eq) and tert-butyl N-tert-butoxycarbonylcarbamate (23.1 g, 106 mmol, 1.5 eq). The mixture was stirred at 25°C for 16 h, and then poured into water (300 mL) and extracted with MTBE (150 mL*3). The combined organic phases was washed with brine (200 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , Petroleum ether/Ethyl acetate=50/1 to 5/1) to give 8AmBza-11c (32 g, crude) as yellow oil. 1 H NMR (CDCl3, 400MHz) δ 8.39 (d, J = 8.8 Hz, 2H), 8.05 (d, J = 8.8 Hz, 2H), 4.21 (s, 2H), 4.11(s, 2H), 3.14 (t, J = 7.2 Hz, 2H), 1.66- 1.54 (m, 2H), 1.49 (s, 18H), 0.93 (t, J = 7.2 Hz, 3H). Preparation of N-(4-aminobut-2-ynyl)-4-nitro-N-propyl -benzenesulfonamide, 8AmBza- 11d To a solution of 8AmBza-11c (32 g, 62.5 mmol, 1.0 eq) in EtOAc (50 mL) was added HCl/EtOAc (4 M, 60 mL, 3.8 eq). The mixture was stirred at 25°C for 1 h and then concentrated under reduced pressure to give 8AmBza-11d (27 g, crude, HCl salt) as yellow solid. Preparation of tert-butyl N-[4-[(4-nitrophenyl)sulfonyl-propyl-amino]but-2- ynyl]carbamate, 8AmBza-11e To a solution of 8AmBza-11d (27 g, 77.6 mmol, 1.0 eq, HCl) in THF (100 mL) and water (10 mL) was added Boc 2 O (13.5 g, 62.1 mmol, 14.3 mL, 0.8 eq) and K 2 CO 3 (21.5 g, 155 mmol, 2 eq). The mixture was stirred at 25°C for 1 hr and then poured into water (100 mL) and extracted with EtOAc (100 mL*3). The combined organic phases was washed with brine (100 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , Petroleum ether/Ethyl acetate=80/1 to 3/1) to give 8AmBza-11e (20 g, 48.6 mmol, 62.6% yield) as yellow solid. 1 H NMR (CDCl3, 400MHz) δ8.37 (d, J = 8.8 Hz, 2H), 8.05 (d, J = 8.8 Hz, 2H), 4.42 (s, 1H), 4.19 (s, 2H), 3.67 (d, J = 5.2 Hz, 2H), 3.17 (t, J = 7.2 Hz, 2H), 1.64-1.59 (m, 2H), 1.44 (s, 9H), 0.95 (t, J = 7.6 Hz, 3H). Preparation of tert-butyl N-[4-(propylamino)but-2-ynyl]carbamate, 8AmBza-11f To a solution of 8AmBza-11e (20 g, 48.6 mmol, 1.0 eq) and LiOH•H 2 O (12.2 g, 291 mmol, 6.0 eq) in MeCN (100 mL) was added methyl 2-mercaptoacetate (15.5 g, 146 mmol, 13.2 mL, 3.0 eq) at 0°C. The mixture was stirred at 25°C for 2 hr. Water (100 mL) was added to the mixture and adjusted the pH of aqueous phase to 2 with 1N HCl at 0°C. The mixture was extracted with MTBE (100 mL *2), the pH of aqueous phase was adjusted to 9 with sat. NaHCO3 and then extracted with EtOAc (50 mL x 3). The organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give crude product 8AmBza-11f (10 g, 44.2 mmol, 90.91% yield) as brown oil which was used into the next step without further purification. 1 H NMR (CDCl3, 400MHz) δ3.95 (s, 2H), 3.46 (s, 2H), 2.67 (t, J = 7.2 Hz, 2H), 1.59-1.50 (m, 2H), 1.47 (s, 9H), 0.96 (t, J = 7.2 Hz, 3H). Preparation of tert-butyl N-[4-[[2-amino-8-(phenylcarbamoyl)-3H-1-benzazepine-4- carbonyl]-propyl-amino]but-2-ynyl]carbamate, 8AmBza-11 To a mixture of 2-amino-8-(phenylcarbamoyl)-3H-benzo[b]azepine-4-carboxylic acid, 8AmBza-11g (0.1 g, 311 µmol, 1.0 eq) in DMF (3 mL) was added PYAOP (194 mg, 373 µmol, 1.2 eq) and DIEA (120 mg, 933 µmol, 162 µL, 3.0 eq) at 25°C. Then 8AmBza-11f (84 mg, 373 µmol, 1.2 eq) was added to the mixture and stirred at 25°C for 1 h. The mixture filtered and concentrated, the residue was purified by prep-HPLC (column: Xtimate C18100*30mm*3um; mobile phase: [water (0.1%TFA) - ACN]; B%: 25% - 55%, 10min) to give 8AmBza-11 (13 mg, 24.6 µmol, 7.89% yield) as white solid. 1 H NMR (MeOD, 400 MHz) δ7.98-7.93 (m, 2H), 7.71 (d, J = 8.0 Hz, 3H), 7.39 (t, J = 8.0 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 4.33 (s, 2H), 3.86 (s, 2H), 3.61-3.47 (m, 2H), 3.39 (s, 2H), 1.80-1.70 (m, 2H), 1.43 (s, Example 12 Synthesis of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(Z)-N'-[3-[[2-amino-8- (phenylcarbamoyl)-3H-1-benzazepine -4-carbonyl]-propyl-amino]propyl]-N-(3- cyanophenyl)carbamimidoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]ethoxy]propanoic acid, 8AmBza-12
Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[(3- cyanophenyl)carbamothioylamino]ethoxy]ethoxy]ethoxy]ethoxy]e thoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]propanoate, 8AmBza-12b To a mixture of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 8AmBza-12a (2.7 g, 4.61 mmol, 1.0 eq) in THF (20 mL) was added Et3N (700 mg, 6.91 mmol, 960 µL, 1.5 eq) and 3- isothiocyanatobenzonitrile (1.48 g, 9.22 mmol, 2.0 eq) at 25°C and it was stirred for 1 hour at this temperature. Then the mixture was diluted with water (30 mL) and extracted with EtOAc (50 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (MeOH/Ethyl acetate=0/1, 1/10) to afford 8AmBza-12b (0.5 g, 670 µmol, 14.54% yield) as yellow oil. 1 H NMR (CDCl3, 400MHz) δ7.99 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.44-7.39 (m, 2H), 3.76-3.58 (m, 42H), 2.55- 2.46 (m, 2H), 1.45 (s, 9H) Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(3- cyanophenyl)iminomethyleneamino]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethox y]ethoxy]ethoxy]propanoate, 8AmBza-12c To a mixture of 8AmBza-12b (0.4 g, 536 µmol, 1.0 eq) and Et3N (163 mg, 1.61 mmol, 223 µL, 3.0 eq) in DCM (10 mL) and DMF (0.4 mL) was added 2-chloro-1-methylpyridin-1- ium iodide (164 mg, 643 µmol, 1.2 eq) at 25°C under N 2 . The mixture was stirred at 25°C for 1 hour and then concentrated under reduce pressure. The residue was purified by silica gel chromatography (CH 3 CN/Ethyl acetate = 0/1 to 1/1) to afford 8AmBza-12c (0.29 g, 407 µmol, 75.9% yield) as yellow oil. 1 H NMR (CDCl 3 , 400MHz) δ7.43-7.33 (m, 4H), 3.70-3.62 (m, 42H), 2.51 (t, J = 6.4 Hz, 2H), 1.45 (s, 9H). Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(Z)-N'-[3- [[2-amino-8- (phenylcarbamoyl)-3H-1-benzazepine-4-carbonyl]-propyl-amino] propyl]-N-(3- cyanophenyl)carbamimidoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]ethoxy]propanoate 8AmBza-12e To a mixture of 2-amino-N4-(3-aminopropyl)-N8-phenyl-N4-propyl-3H-1-benzazep ine- 4,8-dicarboxamide, 8AmBza-12d (0.06 g, 112 µmol, 1.0 eq, TFA salt) in DMF (1 mL) was added Et 3 N (28 mg, 281 µmol, 2.5 eq) and 8AmBza-12c (88 mg, 123 µmol, 1.1 eq) at 25°C. The mixture was stirred at 25°C for 1 hour and then filtered and purified by prep-HPLC (column: Nano-micro Kromasil C18100*30mm 8um; mobile phase: [water (0.1%TFA) - ACN]; B%: 20% - 50%, 10min) to afford 8AmBza-12e (0.08 g, 70.7 µmol, 62.9% yield) as colorless oil. Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(Z)-N'-[3-[[2-amino-8- (phenylcarbamoyl)-3H-1-benzazepine -4-carbonyl]-propyl-amino]propyl]-N-(3- cyanophenyl)carbamimidoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy ]ethoxy]ethoxy]propanoic acid, 8AmBza-12 To a solution of 8AmBza-12e (0.07 g, 61 µmol, 1.0 eq) in H 2 O (5 mL) and CH 3 CN (1 mL) was added TFA (211 mg, 1.86 mmol, 30 eq) at 25°C. The mixture was stirred at 80°C for 2 hours and then concentrated under reduced pressure at 50°C. The residue was freeze-dried to give 8AmBza-12 (51 mg, 42.9 µmol, 69.3% yield, TFA salt) as light yellow oil. 1 H NMR (MeOD, 400 MHz) δ8.01-7.94 (m, 2H), 7.79-7.75 (m, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.66-7.64 (m, 4H), 7.39 (t, J = 7.6 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H), 7.13 (s, 1H), 3.76-3.52 (m, 46H), 3.42-3.40 (m, 4H), 2.53 (t, J = 6.4 Hz, 2H), 2.04 (m, 2H), 1.79-1.65 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H). LC/MS [M+H] 1075.6 (calculated); LC/MS [M+H] 1075.6 (observed). Example 18 Synthesis of 2-amino-N8-[6-[4-(2-aminoethylcarbamoyl)-1-piperidyl]-3- pyridyl]-N4-ethoxy-N4- propyl-3H-1-benzazepine-4,8-dicarboxamide, 8AmBza-18 Preparation of 2-amino-8-bromo-N-ethoxy-N-propyl -3H-1-benzazepine-4-carboxamide, 8AmBza-18b To a mixture of 2-amino-8-bromo-3H-1-benzazepine-4-carboxylic acid, 8AmBza-18a (9.00 g, 32.0 mmol, 1.0 eq) and N-ethoxypropan-1-amine (5.81 g, 41.6 mmol, 1.3 eq, HCl) in DCM (150 mL) and DMA (150 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDCI, CAS Reg. No.1892-57-5 (24.5 g, 128 mmol, 4.0 eq) in one portion at 20°C under N2, and then stirred at 20 °C for 10 hours. The mixture was concentrated in vacuum to remove DCM, then water (200 mL) was added and the aqueous phase was extracted with ethyl acetate (100 mL*4), the combined organic phase was washed with brine (200 mL*1), dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=10/1, 0/1) to afford 8AmBza-18b (6.00 g, 16.3 mmol, 51.1% yield) as white solid. 1 H NMR (400 MHz, MeOD) δ7.32 (d, J = 2.0 Hz, 1H), 7.27-7.23 (m, 1H), 7.20 (s, 1H), 7.19-7.16 (m, 1H), 3.94 (q, J = 7.2 Hz, 2H), 3.73 (t, J = 7.2 Hz, 2H), 3.33 (s, 2H), 1.82-1.72 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H), 0.99 (t, J = 7.2 Hz, 3H). Preparation of methyl 2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine -8- carboxylate, 8AmBza-18c To a solution of 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4- carboxamide (340 mg, 928 umol, 1.0 eq) in MeOH (10 mL) was added Pd(dppf)Cl 2 (34.0 mg, 46.4 umol, 0.05 eq) and Et3N (282 mg, 2.78 mmol, 388 uL, 3.0 eq) under N2, the suspension was degassed under vacuum and purged with CO several times, the mixture was stirred under CO (50psi) at 80 °C for 10 hours. The reaction mixture was concentrated in vacuum, then water (10 mL) was added and the aqueous phase was extracted with ethyl acetate (10 mL*3), the combined organic phase was washed with brine (10 mL*1), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=10/1, 0/1) to afford 8AmBza-18c (180 mg, 521 umol, 56.1% yield) as yellow solid. 1 H NMR (400 MHz, CDCl3) δ7.84 (d, J = 1.2 Hz, 1H), 7.69-7.65 (m, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.28 (s, 1H), 3.96 (t, J = 14.4 Hz, 2H), 3.93 (s, 3H), 3.74 (t, J = 7.2 Hz, 2H), 3.33 (s, 2H), 1.83- 1.72 (m, 2H), 1.18 (t, J = 7.2 Hz, 3H), 1.00 (t, J = 7.2 Hz, 3H). Preparation of 2-amino-4-[ethoxy(propyl)carbamoyl] -3H-1-benzazepine-8-carboxylic acid, 8AmBza-18d To a solution of 8AmBza-18c (180 mg, 521 umol, 1.0 eq) in MeOH (1 mL) and H 2 O (3 mL) was added LiOH•H2O (65.6 mg, 1.56 mmol, 3.0 eq) in one portion at 20°C under N2, the mixture was stirred at 20°C for 7 hours. The mixture was quenched with HCl (4M) until pH=7, desired solid precipitated from the mixture and then filtered to afford 8AmBza-18d (150 mg, 452 umol, 86.8% yield) as gray solid. Preparation of tert-butyl N-[2-[[1-[5-[[2-amino-4- [ethoxy(propyl)carbamoyl]-3H-1- benzazepine-8-carbonyl]amino]-2-pyridyl]piperidine-4-carbony l]amino]ethyl]carbamate, 8AmBza-18e To a solution of 8AmBza-18d (137 mg, 413 umol, 1.0 eq) in DMF (2 mL) was added HATU (141 mg, 372 umol, 0.9 eq) and DIEA (160 mg, 1.24 mmol, 216 uL, 3.0 eq) in one portion at 20 °C under N 2 . The mixture was stirred at 20 °C for 30 min, then tert-butyl N-[2-[[1- (5-amino-2-pyridyl)piperidine-4- carbonyl]amino]ethyl]carbamate (195 mg, 537 umol, 1.3 eq) was added and stirred at 20 °C for another 10 hours. The reaction mixture was filtered and the filtrate was purified by prep-HPLC (column: Phenomenex Synergi C18150*25*10um;mobile phase: [water(0.1%TFA)-ACN];B%: 10%-40%,8min) to afford 8AmBza-18e (20.0 mg, crude) as brown solid. Preparation of 8AmBza-18 To a solution of 8AmBza-18e (20 mg, 29.5 umol, 1.0 eq) in EtOAc (2 mL) was added HCl/EtOAc (4 M, 369 uL, 50 eq) in one portion at 20 °C under N 2 , and then stirred at 20°C for 3 hours. The reaction mixture was concentrated in vacuum and the residue was purified by prep- HPLC (column: Phenomenex Synergi C18150*25*10um; mobile phase: [water(0.1%TFA)- ACN];B%: 1%-25%,8min) to afford 8AmBza-18 (12.6 mg, 17.5 umol, 59.2% yield, 95.98% purity, TFA) as white solid. 1 H NMR (400 MHz, MeOD) δ8.57(d, J = 2.4Hz, 1H), 8.07 (dd, J = 2.4, 9.6 Hz, 1H), 8.00-7.96 (m, 2H), 7.74 (d, J = 8.4 Hz, 1H), 7.47 (s, 1H), 7.18 (d, J = 9.6 Hz, 1H), 4.30 (d, J = 13.6 Hz, 2H), 4.00 (q, J = 7.2 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 3.51-3.44 (m, 5H), 3.17-3.05 (m, 4H), 2.62-2.53 (m, 1H), 1.96 (d, J = 3.6 Hz, 2H), 1.87-1.75 (m, 4H), 1.22 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H). LC/MS [M+H] 577.3 (calculated); LC/MS [M+H] 577.2 (observed). Example L-1 Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzy l (2-(1-(5-(2-amino-4- (dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyridin -2-yl)piperidine-4- carboxamido)ethyl)carbamate, 8AmBza-L-1 8AmBza-L-1 was prepared and characterized according to the procedures described herein. Example L-2 Synthesis of rac-2,3,5,6-tetrafluorophenyl (6R,9R)-1-amino-6-((4-((((2- (1-(5-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-ca rboxamido)pyridin-2- yl)piperidine-4-carboxamido)ethyl)carbamoyl)oxy)methyl)pheny l)carbamoyl)-9-isopropyl- 1,8,11-trioxo-14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,5 9,62,65,68,71,74,77,80,83,86- pentacosaoxa-2,7,10-triazanonaoctacontan-89-oate, 8AmBza-L-2 Preparation of bis(2,3,5,6-tetrafluorophenyl) 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64 ,67,70,73,76- pentacosaoxanonaheptacontanedioate, TFP-PEG25-TFP
A vial was charged with 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64 ,67,70,73,76- pentacosaoxanonaheptacontanedioic acid (269 mg, 0.221 mmol), 2,3,5,6-tetrafluorophenol (110 mg, 0.662 mmol), collidine (176 µL, 1.33 mmol), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (127 mg, 0.221 mmol) and 3 mL DMF. The reaction was stirred for 16 h, then purified by reverse phase preparative HPLC utilizing a 25-75% gradient of acetonitrile:water containing 0.1% trifluoroacetic acid. The purified fractions were combined and lyophilized to afford 266 mg of TFP-PEG25-TFP in 79% yield. LC/MS [M+H] 1515.68 (calculated); LC/MS [M+H] 1516.00 (observed).
4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamid o)benzyl (2-(1-(5-(2- amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido )pyridin-2-yl)piperidine-4- carboxamido)ethyl)carbamate, 8AmBza-L-2a and TFP-PEG25-TFP were reacted in collidine and DMF, and purified by reverse phase preparative HPLC utilizing a 25-75% gradient of acetonitrile:water containing 0.1% trifluoroacetic acid. The purified fractions were combined and lyophilized to afford 8 AmBza-L-2. LC/MS [M+2H/2] 1165.10 (calculated); LC/MS [M+H] 1165.91 (observed). Example L-3 Synthesis of 2,3,5,6-Tetrafluorophenyl (6S,9S)-1-amino-6-((4-(((((6-(2- amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido )pyridin-3- yl)methyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-9-isopropyl -1,8,11-trioxo- 14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71, 74,77,80,83,86-pentacosaoxa- 2,7,10-triazanonaoctacontan-89-oate, 8AmBza-L-3
4-((S)-2-((S)-2-Amino-3-methylbutanamido)-5-ureidopentanamid o)benzyl ((5-(2-amino- 4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyrid in-3-yl)methyl)carbamate, 8AmBza-L-3 and TFP-PEG25-TFP were reacted in collidine and DMF, and purified by reverse phase preparative HPLC utilizing a 25-75% gradient of acetonitrile:water containing 0.1% trifluoroacetic acid. The purified fractions were combined and lyophilized to afford 8 AmBza- L-3. LC/MS [M+2H/2] 1095.06 (calculated); LC/MS [M+H] 1095.87 (observed). Example L-4 Synthesis of 2,3,5,6-tetrafluorophenyl 1-(1-(5-(2-amino-4- (dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyridin -2-yl)piperidin-4-yl)-1,6- dioxo-9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,6 3,66,69,72,75,78,81- pentacosaoxa-2,5-diazatetraoctacontan-84-oate, 8AmBza-L-4
2-Amino-N8-(6-(4-((2-aminoethyl)carbamoyl)piperidin-1-yl)pyr idin-3-yl)-N4,N4- dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide, 8 AmBza-L-4a and TFP-PEG25-TFP reacted in collidine and DMF, and purified by reverse phase preparative HPLC utilizing a 25-75% gradient of acetonitrile:water containing 0.1% trifluoroacetic acid. The purified fractions were combined and lyophilized to afford 8 AmBza-L-4. LC/MS [M+H] 1924.01 (calculated); LC/MS [M+H] 1925.23 (observed). Example L-5 Synthesis of 2,3,5,6-Tetrafluorophenyl 1-(6-(2-amino-4- (dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyridin -3-yl)-3-oxo- 6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66 ,69,72,75,78-pentacosaoxa-2- azahenoctacontan-81-oate, 8AmBza-L-5 2-Amino-N8-(5-(aminomethyl)pyridin-3-yl)-N4,N4-dipropyl-3H-b enzo[b]azepine-4,8- dicarboxamide, 8 AmBza-L-5a and TFP-PEG25-TFP reacted in collidine and DMF, and purified by reverse phase preparative HPLC utilizing a 25-75% gradient of acetonitrile:water containing 0.1% trifluoroacetic acid. The purified fractions were combined and lyophilized to afford 8 AmBza-L-5. LC/MS [M+H] 1783.92 (calculated); LC/MS [M+H] 1784.19 (observed). Example L-6 Synthesis of 2,3,5,6-Tetrafluorophenyl 1-(1-(5-(2-amino-4-((3-((tert- butoxycarbonyl)amino)propyl)(propyl)carbamoyl)-3H-benzo[b]az epine-8-carboxamido)pyridin- 2-yl)piperidin-4-yl)-1,6-dioxo- 9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,6 9,72,75,78,81-pentacosaoxa-2,5- diazatetraoctacontan-84-oate, 8AmBza-L-6. tert-Butyl (3-(2-amino-8-((6-(4-((2-aminoethyl)carbamoyl)piperidin-1-yl )pyridin-3- yl)carbamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)prop yl)carbamate, 8AmBza-5 from Example 5 and TFP-PEG25-TFP were reacted in collidine and DMF, and purified by reverse phase preparative HPLC utilizing a 25-75% gradient of acetonitrile:water containing 0.1% trifluoroacetic acid. The purified fractions were combined and lyophilized to afford 8 AmBza-L-6. LC/MS [M+H] 2039.07 (calculated); LC/MS [M+H] 2039.40 (observed). Example L-7 Synthesis of (2S,4S,6S)-6-(4-((((2-(1-(5-(2-amino-4-(dipropylcarbamoyl)- 3H-benzo[b]azepine-8-carboxamido)pyridin-2-yl)piperidine-4- carboxamido)ethyl)carbamoyl)oxy)methyl)-2-(20-oxo-1-(1-(2-(3 -oxo-3- (perfluorophenoxy)propoxy)ethyl)-1H-1,2,3-triazol-4-yl)-2,5, 8,11,14,17-hexaoxa-21- azatetracosan-24-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2 H-pyran-2-carboxylic acid, 8AmBza-L-7 8AmBza-L-7 was prepared and characterized according to the procedures described herein. Example L-8 Synthesis of 2,3,5,6-tetrafluorophenyl 1-(3-(2-amino-4- (dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)phenyl) -8-methyl- 2,5,11,14,17,20,23,26,29,32,35,38-dodecaoxa-8-azahentetracon tan-41-oate, 8AmBza-L-8 8AmBza-L-8 was prepared and characterized according to the procedures described herein. Example L-9 Synthesis of 2,3,5,6-tetrafluorophenyl 1-((5-(2-amino-4- (dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)pyrimid in-2-yl)amino)-3-methyl- 6,9,12,15,18,21,24,27,30,33-decaoxa-3-azahexatriacontan-36-o ate, 8AmBza-L-9 8AmBza-L-9 was prepared and characterized according to the procedures described herein. Example L-10 Synthesis of 2,3,5,6-tetrafluorophenyl (R)-1-(4-((3-(2-amino-4- (dipropylcarbamoyl)-3H-benzo[b]azepine-8-carboxamido)piperid in-1-yl)methyl)phenyl)-2- methyl-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azapentatriacon tan-35-oate, 8AmBza-L-10 8AmBza-L-10 was prepared and characterized according to the procedures described herein. Example L-11 Synthesis of 2,3,5,6-tetrafluorophenyl 1-(4-((4-(2-amino-4- (dipropylcarbamoyl)-3H-benzo[b]azepine-8-carbonyl)piperazin- 1-yl)methyl)phenyl)-2-methyl- 5,8,11,14,17,20,23,26,29,32-decaoxa-2-azapentatriacontan-35- oate, 8AmBza-L-11 8AmBza-L-11 was prepared and characterized according to the procedures described herein. Example L-16 Synthesis of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2- [2- [2-[2-[[1-[5-[[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benz azepine-8-carbonyl]amino]- 2-pyridyl]piperidine-4-carbonyl]amino]ethyl-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethox y]ethoxy]ethoxy]propanoate, 8AmBza-L-16 Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[[1-[5-[[2-amino-4- [ethoxy(propyl)carbamoyl]-3H-1-benzazepine-8-carbonyl]amino] -2-pyridyl]piperidine-4- carbonyl]amino]ethyl-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethox y]ethoxy]ethoxy]propanoate, 8AmBza-L-16a To a mixture of 2-amino-N8-[6-[4-(2-aminoethylcarbamoyl)-1-piperidyl]-3-pyri dyl]-N4- ethoxy-N4-propyl-3H-1-benzazepine-4,8-dicarboxamide, 8AmBza-18 (130 mg, 225 umol, 1.0 eq) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- oxoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]e thoxy]ethoxy]propanoate (395 mg, 676 umol, 3.0 eq) in MeOH (5 mL) was added NaBH3CN (42.5 mg, 676 umol, 3.0 eq) and Et3N (22.8 mg, 225 umol, 31.3 uL, 1.0 eq) in one portion at 20 °C under N2, the mixture was stirred at 20°C for 40 hours, then HCHO (91.4 mg, 1.13 mmol, 83.9 uL, 37% purity, 5.0 eq) was added and stirred for another 3 hours at 20°C. The reaction mixture was concentrated in vacuum and the residue was purified by prep-HPLC (column: Phenomenex Synergi C18 150*30mm*4um;mobile phase: [water(0.1%TFA)-ACN];B%: 20%-45%,8min) to afford 8AmBza-L-16a (50.0 mg, 43.1 umol, 19.1% yield) as brown oil. Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2- [[1-[5-[[2-amino-4- [ethoxy(propyl)carbamoyl]-3H- 1-benzazepine-8-carbonyl]amino]-2-pyridyl]piperidine-4- carbonyl]amino]ethyl-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethox y]ethoxy]ethoxy]propanoic acid, 8AmBza-L-16b To a solution of 8AmBza-L-16a (50.0 mg, 43.1 umol, 1.0 eq) in MeCN (0.5 mL) and H 2 O (2 mL) was added HCl (12 M, 107 uL, 30 eq) in one portion at 20°C under N 2 , the mixture was stirred at 80°C for 1 hour. The reaction mixture was concentrated in vacuum to afford 8AmBza-L-16b (45 mg, 40.79 umol, 94.6% yield) as colorless oil. Preparation of 8AmBza-L-16 To a mixture of 8AmBza-L-16b (45.0 mg, 40.7 umol, 1.0 eq) and 2,3,5,6- tetrafluorophenol (67.7 mg, 407 umol, 10 eq) in DCM (2 mL) and DMA (0.5 mL) was added EDCI (39.0 mg, 203 umol, 5.0 eq) in one portion at 20 °C under N 2 , the mixture was stirred at 20°C for 1 hour. DCM (2 mL) was removed in vacuum and the mixture was filtered, the filtrate was purified by prep-HPLC (column: Phenomenex Synergi C18150*30mm*4um;mobile phase: [water(0.1%TFA)- ACN];B%: 20%-45%,8min) to afford 8AmBza-L-16 (15.0 mg, 11.9 umol, 29.3% yield, 99.7% purity) as brown oil. 1 H NMR (400 MHz, MeOD) δ8.55 (d, J = 1.8 Hz, 1H), 8.03 (dd, J = 2.4, 9.2 Hz, 1H), 7.98 (s, 2H), 7.74 (d, J = 9.2 Hz, 1H), 7.47 (s, 1H), 7.16-7.09 (m, 1H), 4.34-4.28 (m, 2H), 4.00 (d, J = 7.0 Hz, 2H), 3.91-3.85 (m, 4H), 3.74-3.59 (m, 42H), 3.50 (s, 2H), 3.45 (s, 3H), 3.17-3.07 (m, 2H), 3.01 (s, 3H), 1.96 (d, J = 10.6 Hz, 2H), 1.86-1.75 (m, 4H), 1.22 (t, J = 7.2 Hz, 3H), 1.06-0.99 (m, 3H). LC/MS [M+H] 1251.6 (calculated); LC/MS [M+H] 1251.4 (observed). Example 201 Preparation of Immunoconjugates (IC) In an exemplary procedure, an antibody is buffer exchanged into a conjugation buffer containing 100 mM boric acid, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3, using G-25 SEPHADEX TM desalting columns (Sigma-Aldrich, St. Louis, MO). The eluates are then each adjusted to a concentration of about 1-10 mg/ml using the buffer and then sterile filtered. The antibody is pre-warmed to 20-30 °C and rapidly mixed with 2-20 (e.g., 7- 10) molar equivalents of 8AmBza-linker compound of Formula II. The reaction is allowed to proceed for about 16 hours at 30 °C and the immunoconjugate (IC) is separated from reactants by running over two successive G-25 desalting columns equilibrated in phosphate buffered saline (PBS) at pH 7.2 to provide the Immunoconjugate (IC) of Table 3. Adjuvant-antibody ratio (DAR) is determined by liquid chromatography mass spectrometry analysis using a C4 reverse phase column on an ACQUITY TM UPLC H-class (Waters Corporation, Milford, Massachusetts) connected to a XEVO TM G2-XS TOF mass spectrometer (Waters Corporation). For conjugation, the antibody may be dissolved in a aqueous buffer system known in the art that will not adversely impact the stability or antigen-binding specificity of the antibody. Phosphate buffered saline may be used. The 8AmBza-linker intermediate compound is dissolved in a solvent system comprising at least one polar aprotic solvent as described elsewhere herein. In some such aspects, 8AmBza-linker intermediate is dissolved to a concentration of about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM or about 50 mM, and ranges thereof such as from about 5 mM to about 50mM or from about 10 mM to about 30 mM in pH 8 Tris buffer (e.g., 50 mM Tris). In some aspects, the 8AmBza-linker intermediate is dissolved in DMSO (dimethylsulfoxide), DMA (dimethylacetamide) or acetonitrile, or another suitable dipolar aprotic solvent. Alternatively in the conjugation reaction, an equivalent excess of 8AmBza-linker intermediate solution may be diluted and combined with antibody solution. The 8AmBza-linker intermediate solution may suitably be diluted with at least one polar aprotic solvent and at least one polar protic solvent, examples of which include water, methanol, ethanol, n-propanol, and acetic acid. The molar equivalents of 8AmBza-linker intermediate to antibody may be about 1.5:1, about 3:1, about 5:1, about 10:1, about 15:1, or about 20:1, and ranges thereof, such as from about 1.5:1 to about 20:1 from about 1.5:1 to about 15:1, from about 1.5:1 to about 10:1,from about 3:1 to about 15:1, from about 3:1 to about 10:1, from about 5:1 to about 15:1 or from about 5:1 to about 10:1. The reaction may suitably be monitored for completion by methods known in the art, such as LC-MS. The conjugation reaction is typically complete in a range from about 1 hour to about 16 hours. After the reaction is complete, a reagent may be added to the reaction mixture to quench the reaction. If antibody thiol groups are reacting with a thiol-reactive group such as maleimide of the 8AmBza-linker intermediate, unreacted antibody thiol groups may be reacted with a capping reagent. An example of a suitable capping reagent is ethylmaleimide. Following conjugation, the immunoconjugates may be purified and separated from unconjugated reactants and/or conjugate aggregates by purification methods known in the art such as, for example and not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, tangential flow filtration, and combinations thereof. For instance, purification may be preceded by diluting the immunoconjugate, such in 20 mM sodium succinate, pH 5. The diluted solution is applied to a cation exchange column followed by washing with, e.g., at least 10 column volumes of 20 mM sodium succinate, pH 5. The conjugate may be suitably eluted with a buffer such as PBS. Example 202 HEK Reporter Assay HEK293 reporter cells expressing human TLR7 or human TLR8 were purchased from Invivogen and vendor protocols were followed for cellular propagation and experimentation. Briefly, cells were grown to 80-85% confluence at 5% CO 2 in DMEM supplemented with 10% FBS, Zeocin, and Blasticidin. Cells were then seeded in 96-well flat plates at 4x10 4 cells/well with substrate containing HEK detection medium and immunostimulatory molecules. Activity was measured using a plate reader at 620-655 nm wavelength. Example 203 Assessment of Immunoconjugate Activity In Vitro This example shows that Immunoconjugates of the invention are effective at eliciting myeloid activation, and therefore are useful for the treatment of cancer. Isolation of Human Antigen Presenting Cells: Human myeloid antigen presenting cells (APCs) were negatively selected from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation using a ROSETTESEP TM Human Monocyte Enrichment Cocktail (Stem Cell Technologies, Vancouver, Canada) containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123, and HLA-DR. Immature APCs were subsequently purified to >90% purity via negative selection using an EASYSEP TM Human Monocyte Enrichment Kit (Stem Cell Technologies) without CD16 depletion containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123, and HLA-DR. Myeloid APC Activation Assay: 2 x 10 5 APCs were incubated in 96-well plates (Corning, Corning, NY) containing iscove’s modified dulbecco’s medium, IMDM (Lonza) supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL (micrograms per milliliter) streptomycin, 2 mM L-glutamine, sodium pyruvate, non-essential amino acids, and where indicated, various concentrations of unconjugated (naked) PD-L1 or HER2 antibodies and immunoconjugates of the invention (as prepared according to the Example above). Trastuzumab and avelumab were used as the antibody constructs. Cell-free supernatants were analyzed after 18 hours via ELISA to measure TNF ^ secretion as a readout of a proinflammatory response. Activation of myeloid cell types can be measured using various screen assays in which different myeloid populations are utilized. These may include the following: monocytes isolated from healthy donor blood, M-CSF differentiated Macrophages, GM-CSF differentiated Macrophages, GM-CSF+IL-4 monocyte-derived Dendritic Cells, classical Dendritic Cells isolated from healthy donor blood, and myeloid cells polarized to an immunosuppressive state (also referred to as myeloid derived suppressor cells or MDSCs). Examples of MDSC polarized cells include monocytes differentiated toward immunosuppressive state such as M2a MΦ (IL4/IL13), M2c MΦ (IL10/TGFb), GM-CSF/IL6 MDSCs and tumor-educated monocytes (TEM). TEM differentiation can be performed using tumor-conditioned media (e.g.786.O, MDA-MB-231, HCC1954). Primary tumor-associated myeloid cells may also include primary cells present in dissociated tumor cell suspensions (Discovery Life Sciences). Assessment of activation of the described populations of myeloid cells may be performed as a mono-culture or as a co-culture with cells expressing the antigen of interest which the ISAC may bind to via the CDR region of the antibody. Following incubation for 18- 48 hours, activation may be assessed by upregulation of cell surface co-stimulatory molecules using flow cytometry or by measurement of secreted proinflammatory cytokines. For cytokine measurement, cell-free supernatant is harvested and analyzed by cytokine bead array (e.g. LegendPlex from Biolegend) using flow cytometry. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
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