US 20190202839A1 ( 19) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2019 /0202839 A1 JAIN et al. ( 43 ) Pub . Date: Jul. 4 , 2019 (54 ) NOVEL CYTOTOXIC AGENTS AND Publication Classification CONJUGATES THEREOF (51 ) Int . CI. (71 ) Applicant : POLYTHERICS LIMITED , C07D 498 / 18 (2006 .01 ) Cambridge (GB ) A61K 31 /537 ( 2006 . 01) A61K 47/ 68 (2006 . 01 ) ( 72 ) Inventors: Nareshkumar JAIN , Ringoes, NJ A61P 35 /00 ( 2006 .01 ) (US ) ; Sanjeevani GHONE , Plainsboro , 2 ) U .S . CI. NJ (US ) ; Sean SMITH , Hamilton , NJ PO . C07D 498 / 18 (2013 . 01 ) ; A61K 31/ 537 (US ) ; Ian GLASSFORD , King of ( 2013 .01 ) ; A61P 35 / 00 ( 2018 .01 ) ; A61K Prussia , PA (US ) ; Sylvia J . 47 /6803 ( 2017 .08 ) ; A61K 47/ 6851 (2017 .08 ) DEGRADO , Newtown , PA (US ) ; Fu -an KANG , Collegeville , PA (US ) ; Senzhi ZHAO , Bensalem , PA (US ) (57 ) ABSTRACT (21 ) Appl. No. : 16 /332 , 797 Provided herein are novel maytansinoid compounds of gen (22 ) PCT Filed : Sep . 15 , 2017 eral formula I . Also provided herein are conjugates com prising the compounds linked to a binding protein via a ( 86 ) PCT No. : PCT/GB2017 / 052731 linker , and conjugating reagents comprising the compounds attached via a linker to at least one functional group capable $ 371 (c ) ( 1 ) , of reacting with a binding protein . Also provided herein are ( 2 ) Date : Mar. 13 , 2019 pharmaceutical compositions comprising the compounds ( 30 ) Foreign Application Priority Data and conjugates, therapeutic methods and uses involving the compounds and conjugates , for example in cancer therapy, Sep . 15 , 2016 (GB ) . .. .. 1615725. 7 and novel synthetic processes . Patent Application Publication Jul . 4 , 2019 Sheet 1 of 3 US 2019 /0202839 A1 Maytansinoid derivatives SK -BR - 3 cell line • 3 v DM1 Cellviability(%) III . Compound Concentration (nM ) Figure 1 Maytansinoid Derivatives Karpas - 299 cell line # 3 Cellviability(%) V DM1 0.0001 100 Concentration ( nM ) Figure 2 -Patent Application- Publication - .Jul. 4 , 2019 . Sheet. 2 of . 3 .US. 2019 / 0202839 A1. W Vehicle W 29 ( 30 mg/ kg ) Kadcyla® ( 30 mg/ kg ) IIIIIITITITITI IIIIII TI IIIIITITUTT D IIIIIIIIIIIIIIIIIII IIIIII DIIII IIIIII LLLLLLL IIIIIIIII DIT IIIIIIIIIII Meantumourvolume(r III LIIT LLLLLLL LLLLLLLL IIIIIIIII IIIIII JULI IIIIIIIIIII LLLLLLLLL IIIIII LLLLLLLL III LIITLI IIIIIII III HILLIP IT LIIIII IIIIII ITIT IIIIII TTTTTT TITITITI LLLLLILL IIIIII LIITUN IIIII TILL LLLLLL UUUUUUITIL • 14 14 © 14 Days post treatment Figure 3 Patent Application Publication Jul. 4 , 2019 Sheet 3 of 3 US 2019 / 0202839 A1 Degradation product A Degradation Product B 0 78 %totaldrugrelatedionsobserved ° 6 4 7 6 4 I Days incubation in serum Figure 4A Degradation Product C - Degradation Product D VIZ W 26 %totaldrugrelatedionsobserved ó À í ó À í ? Days incubation in serum Figure 4B US 2019 /0202839 A1 Jul . 4 , 2019 NOVEL CYTOTOXIC AGENTS AND -continued CONJUGATES THEREOF OH FIELD OF INVENTION OMe [0001 ] This invention relates to novel cytotoxic agents , novel conjugates , particularly antibody - drug conjugates , and intermediates for making the conjugates , including novel conjugating reagents . It also relates to a novel process for making cytotoxic agents . BACKGROUND TO THE INVENTION [0002 ] Much research has been devoted in recent years to ?? ? . the conjugation of therapeutic agents as payloads to peptides and proteins for a wide range of applications. The protein or Maytansinol peptide itself may have therapeutic properties , and / or it may be a binding protein . In addition , conjugation to polymers [0005 ] U . S . Pat. No . 4 , 190 ,580 described a method of has been used to improve the properties of certain therapeu synthesis of maytansinoids , while U . S . Pat . No . 4 , 260 , 608 tic agents . For example , water soluble , synthetic polymers , identified a number of maytansine derivative compounds particularly polyalkylene glycols, are widely used to conju with potent anti- microbial and anti tumour activities as a gate therapeutically active peptides or proteins . These thera result of their anti -mitotic effects upon cells . U . S . Pat. No. peutic conjugates have been shown to alter pharmacokinet 4 , 260 ,608 disclosed maytansine derivatives with an option ics favourably by prolonging circulation time and decreasing ally substituted alkyl group at the R3 position . Since then clearance rates , decreasing systemic toxicity , and in several numerous structure - activity relationship studies have been cases , displaying increased clinical efficacy . The process of performed to determine other potent structures of maytansi covalently conjugating polyethylene glycol, PEG , to pro noids. Kawai et al . , Chem . Pharm . Bull. 32 ( 9 ) , 3441 -3451 teins and other payloads is commonly known as “ PEGY ( 1984 ) published a number of structures that were modified lation ” . at the C3 position of Maytansinol, which displayed potent [ 0003] Binding proteins ( i. e . proteins or peptides capable anti - tumour effects in vivo . EP O 004 466 , U . S . Pat. No . of binding to a binding partner on a target ) , particularly 4 , 137 ,230 and WO 2012 / 061590 all describe novel may antibodies or antibody fragments , are frequently used in tansinoids. Various modifications of maytansinoids are conjugates . For example , they may be conjugated to cyto described in US 2009 / 258870 , Taft et al , Chem . Eur. J . 2012 , toxic agents and chemotherapy drugs to produce antibody 18 , 880 - 886 , and Harmrolfs et al , Beilstein J . Org . Chem . drug conjugates (ADCs ) , allowing targeted delivery of such 2014 , 10 , 535 - 543 . agents to specific tissues or structures , for example particular 10006 ]. Well -known maytansinoids include those known as cell types or growth factors , minimising the impact on DM1 and DM4 as described within U . S . Pat . No . 5 ,208 , 020 normal, healthy tissue and significantly reducing the side and WO2004 /103272 . These have the structures : effects associated with chemotherapy treatments . Such con jugates have extensive potential therapeutic applications in several disease areas , particularly in cancer . HS . 00041 Kupchan et al. , J . Am . Chem . Soc . , 94 , 1354 ( 1972 ) first isolated Maytansine from the bark of the African shrub Maytenus ovatus, where it was noted for its anti- leukemic JOMe properties . Maytansinoids, such as maytansinol and C - 3 esters of maytansinol were found to be made by microbes ( U . S . Pat. No. 4 , 151 ,042 ) , mosses (Sakai et al , J . Nat . Prod ., 51 ( 5 ) , 845 - 850 , 1988 ) , and could also be generated by synthetic routes (Kupchan et al . , J . Med . Chem ., 21, 31 - 37 , VU 1978 , Higashide et al. , Nature 270 , 721 - 722 , 1977 ) . May tansine and maytansinol have the structures : DM1 orMertansine DM4 uillllll HS 111 OMe OMe N LO O H HO O Maytansine US 2019 /0202839 A1 Jul . 4 , 2019 [0007 ] Ansamitocins are a sub -group of maytansinoids, payloads need to be conjugated efficiently with an antibody the synthesis of which is described by Taft et al, ChemBio using a conjugation technology which allows more stable Chem 2008 , 9 , 1057 - 1060 . A well - known ansamitocin is ADCs to be generated . There is also a need for ADCs which AP - 3 , which has the structure : show greater efficacy or potency in relevant cell or animal models of cancers , or alternatively , show a similar level of efficacy /potency but a reduced amount of non - specific tox icity within the cell or test subject . [0011 ] We have now found that certain novel maytansi noid compounds possess improved cytotoxic activity , and are particularly suited for inclusion in conjugates with binding proteins . The maytansinoids further have improved stability compared with comparator compounds. We have also found a novel method of synthesis which enables efficient preparation of these novel compounds as well as a novel and improved method of synthesis of other maytansi .) noids . OHON SUMMARY OF THE INVENTION [ 0012 ] The invention provides a compound of the general formula ( I ) or a salt thereof: [0008 ] Maytansinoids and maytansinoid esters were sub sequently used as a cytotoxic payload within an ADC context within EP 0 425 235 and have been used extensively since, see for example Liu et al , Proc . Natl. Acad . Sci. USA , 93 , 8618 - 8623 ( 1996 ) ; and Kieda et al, Clin . Cancer Res . , 15 ( 12 ) , 2009 . Widdison et al, J . Med . Chem . 49 , 4392 - 4408 (Rein (2006 ) described the thiol- linked DM1 and DM4 maytansi 0 0 ' Rg noid payloads. These payloads were conjugated via a two step process in which first a heterobifunctional, thiol- con taining linker was reacted with lysine residues within the Rd antibody , followed by conjugation of the thiol- containing payloads to the linker by disulfide exchange . An alternative means of producing ADCsby this two - step process is by use of a succinimidyl trans - 4 - maleimidylmethyl) cyclohexane RA - 1 - carboxylate ( SMCC ) heterobifunctional linker , which has a succinimide group at one end to react with lysine residues within the antibody and a maleimide group at the other end Rb Rovó to react with the thiol group of the maytansinoid . The commercially -available pharmaceutical Kadcyla® is an in which R represents a group — Y - OH , — Y — 0 ~ R * , example of an ADC produced using an SMCC linker with a - Y - SH , - Y - S R * , - Y - S ( O ) NH - R " , - Y - NHS DM1 maytansinoid payload . Kadcyla® is currently one of ( 0 ) 2 - R * , - Y - C ( O ) H , — Y - CO , H , - Y - C ( O ) - R * , the leading ADCs in the clinic and is used for