|HAI LALA MO ATUS009938323B2 ALT A LA LA LA MANUAL (12 ) United States Patent ( 10 ) Patent No. : US 9 ,938 , 323 B2 Grunewald et al. (45 ) Date of Patent : Apr . 10 , 2018 ( 54 ) AMATOXIN DERIVATIVES AND WO 2014135282 AL 9 / 2014 CONJUGATES THEREOF AS INHIBITORS WO 2014141094 Al 9 / 2014 OF RNA POLYMERASE WO 2016142049 AL 9 / 2016 (71 ) Applicant: NOVARTIS AG , Basel (CH ) OTHER PUBLICATIONS ( 72 ) Inventors : Jan Grunewald , San Diego , CA (US ) ; Yunho Jin , San Diego , CA (US ) ; Zhao , et al ., “ Synthesis of a Cytotoxic Amanitin for Biorthogonal Weijia Ou , San Diego , CA (US ) ; Conjugation ” , ChemBioChem , 2015 , pp . 1420 - 1425, vol . 16 , Tetsuo Uno , San Diego , CA (US ) Wiley - VCH Verlag GmbH & Co . Adair, et al ., “ Antibody -drug conjugates a perfect synergy ” , ( 73 ) Assignee : Novartis AG , Basel (CH ) Expert Opin . Biol. Ther. , 2012 , pp . 1191 - 1206 , vol. 12 , No . 9 , Informa UK , Ltd . ( * ) Notice : Subject to any disclaimer , the term of this Flygare , et al ., “ Antibody - Drug Conjugates for the Treatment of patent is extended or adjusted under 35 Cancer ” , Chem . Biol . Drug . Des. , 2013 , pp . 113 - 121 , vol. 81 , John Wiley & Sons A / S . U . S . C . 154 ( b ) by 0 days . Jackson , et al ., “ Using the Lessons Learned from the Clinic to Improve the Preclinical Development of Antibody Drug Conju (21 ) Appl. No. : 15 /524 , 197 gates ” , Pharm . Res . , 2014 , pp . 1 - 12 . (22 ) PCT Filed : Nov. 5 , 2015 (Continued ) ( 86 ) PCT No. : PCT/ IB2015 /058537 Primary Examiner — Jeanette Lieb $ 371 ( c ) ( 1 ) , ( 74 ) Attorney , Agent, or Firm — Daniel E . Raymond ; ( 2 ) Date : May 3, 2017 Genomics Institute of the Novartis Research Foundation ( 87) PCT Pub . No. : WO2016 /071856 (57 ) ABSTRACT PCT Pub . Date : May 12 , 2016 The invention disclosed herein relates to cytotoxic cyclic peptides of Formula (I ), methods of inhibiting RNA poly (65 ) Prior Publication Data merase with such cyclic peptides , immunoconjugates com US 2017/ 0355731 A1 Dec. 14, 2017 prising such cyclic peptides ( i. e Antibody Drug Conjugates) , pharmaceutical compositions comprising such cyclic pep tides immunoconjugates , compositions comprising such Related U . S . Application Data cyclic peptides immunoconjugates with a therapeutic co (60 ) Provisional application No . 62 /076 ,023 , filed on Nov. agent and methods of treatment using such cyclic peptides 6 , 2014 immunoconjugates: (51 ) Int. Ci. A61K 38 /00 ( 2006 .01 ) Formula ( I ) CO7K 7 /64 ( 2006 . 01 ) COOK 16 / 22 ( 2006 .01 ) HO COOK 16 / 30 ( 2006 .01 ) (52 ) U . S . CI. CO7K 7764 ( 2013 .01 ) ; CO7K 16 / 22 HO (2013 .01 ) ; CO7K 16 / 30 ( 2013 .01 ) ; CO7K 2319 / 30 (2013 .01 ) HN (58 ) Field of Classification Search None See application file for complete search history. 12Omn ( 56 ) References Cited HO FOREIGN PATENT DOCUMENTS EP 2774624 Al 10 / 2014 ' S - R2 EP 2683409 B1 12 / 2014 wo 2009114950 A1 9 / 2009 WO 2010115629 A2 10 / 2010 O WO 2011014973 A2 2 / 2011 WO 2012016186 A2 2 / 2012 WO 2012041504 A1 4 / 2012 WO 2012119787 A1 9 /2012 WO 2013104613 A1 7 / 2013 WO 2014009025 A1 1 / 2014 wo 2014043403 A13 / 2014 23 Claims, 7 Drawing Sheets US 9 ,938 ,323 B2 Page 2 ( 56 ) References Cited OTHER PUBLICATIONS Konig , et al. , “ Chemistry of Peptides and Proteins” , Proceeding sof the Sixth USSR -FRG Symposium on Chemistry of Peptides and PRoteins, Sep . 1 - 5 , 1987 . Moldenhauer , et al. , “ Therapeutic Potential of Amanitin -Conjugated Anti - Epithelial Cell Adhesion Molecule Monoclonal Antibody Against Pancreatic Cacinoma” , JNCI, Apr . 18 , 2012 , pp . 622 -634 , vol. 104 , Issue 8 , Oxford University Press . Senter , et al ., “ The discovery and development of brentuximab vedotin for use in relapsed Hogkin lymphoma and systemic anaplastic large cell lymphoma” , Nature Biotechnology , Jul. 6372012 , pp . 631 -637 , vol . 30 , Nature America , Inc. U. S . Patentatent Apr . 10 , 2018 Sheet 1 of 7 US 9 , 938 ,323 B2 FIG . 1A MDA -MB - 231 clone 40 - - - anti -Her2 - LC -S159C -21 5 o anti -Her2 -LC - S159C - 45 th + anti -Her2 - LC - S159C - 11 ! A anti- Her2 - LC -S159C - 18 • anti - Her2 - LC - S159C - 17 o anti- Her2 - LC - S159C - 22 - o - anti- Her2 - LC -S159C - 4 Normalizedviability 0 - anti- Her2 - LC -S159C - 32 * - anti -Her2 - LC - S159C - 23 # anti -Her2 - LC - S159C - 13 - - anti -Her2 - 16 1. E -07 1 . E - 06 1 . E -05 1 . E - 04 1 . E -03 1 . E -02 1 . E - 01 ADC concentration (UM ) FIG . 1B MDA - MB -231 clone 16 • anti -Her2 - LC - S159C - 21 o anti -Her2 -LC -S159C - 45 + anti- Her2 -LC -S159C - 11 A anti -Her2 - LC - S159C - 18 + anti -Her2 - LC - S159C - 17 Normalizedviability o anti- Her2 - LC - S159C -22 - o - anti -Her2 - LC - S159C - 4 anti - Her2 - LC - S159C - 32 Hoo- oo * anti- Her2 -LC -S159C -23 LETU + anti- Her2- LC - S159C - 13 * B S anti- Her2 -16 1. E - 07 1 . 6 - 06 1. 6 -05 1 . 5 -04 1. E -03 1. E -02 1 . 5 -01 ADC concentration (uM ) U . S . Patent Apr . 10 , 2018 Sheet 2 of 7 US 9 , 938 ,323 B2 FIG . 1C HCC1954 anti -Her2 -LC - S159C - 21 o anti - Her2 -LC - S159C - 45 Tab*XD + anti -Her2 -LC - S159C - 11 A anti -Her2 -LC - S159C - 18 anti- Her2 - LC - S159C - 17 o anti- Her2 - LC - S159C - 4 o anti -Her2 -LC - S159C - 22 Normalizedviability 0 - anti- Her2 - LC - S159C -32 * anti - Her2 - LC - S159C - 23 + anti -Her2 -LC - S159C - 13 anti - Her2 - 16 1 . E - 07 1 . E -06 1 . E - 05 1. E - 04 1 . E - 03 1 . E -02 1 . E -01 ADC concentration (uM ) FIG. Jim T - 1 - * * * anti- Her2 - LC - S159C - 21 anti- Her2 - LC - S159C - 45 + anti -Her2 -LC - S159C - 11 De A anti- Her2 - LC -S159C - 18 Home anti- Her2 - LC - S159C - 17 - - - anti- Her2 - LC - S159C - 4 o anti- Her2 - LC - S159C - 22 Normalizedviability 0 - anti- Her2 -LC - S159C - 32 * anti- Her2 - LC -S159C - 23 + anti- Her2 - LC - S159C - 13 - - - anti- Her2 - 16 1 . E -07 1 . E - 06 1 . 6 -05 1 . E -04 1 . E -03 1 . E -02 1 . E -01 ADC concentration (UM ) atent Apr . 10 , 2018 Sheet 3 of 7 US 9 , 938 ,323 B2 FIG . 2A Jim T - 1 1 . 2 anti- HER2 -HC -E388 -DS -ppan - CoA - 1 -40 -LEFIASKLA -N389 2 _ anti- HER2 - HC - E388 -DS -ppan - COA - 1 -39 - LEFIASKLA -N389 Normalizedviability J A anti -HER2 -HC -E388 - DS - ppan -CoA - 1 -41 -LEFIASKLA - N389 anti- HER2 -HC - E388 - DS -ppan - CoA - 1- 9 -40 -LEFIASKLA -N389 0 . 2 0 - anti- HER2 -HC -E388 -DS -ppan -CoA -1 - 9 -39 - LEFIASKLA -N389 A anti- HER2 -HC - E388 -DS -ppan - CoA - 1- 9 -41 -LEFIASKLA -N389 0 . 0 + 1. E -07 1. E - 06 1. E - 05 1 . 5 -04 1 . E -03 1 . E - 02 1 . E -01 ADC concentration (UM ) FIG . 2B A375 anti- HER2 - HC -E388 - DS -ppan -CoA - 1 -40 - LEFIASKLA -N389 + anti- HER2 - HC - E388 -DS -ppan - CoA - 1 - 39- LEFIASKLA -N389 Normalizedviability + anti- HER2 -HC - E388 -DS -ppan - CoA - 1 -41 -LEFIASKLA -N389 anti- HER2 - HC - E388 -DS -ppan - CoA - i- 9 - 40 -LEFIASKLA -N389 0 - anti- HER2 - HC - E388 -DS -ppan - CoA -1 - 9 -39 -LEFIASKLA -N389 A - anti- HER2 - HC -E388 -DS - ppan -CoA -i - 9 -41 - LEFIASKLA -N389 0 . 0 + 1 . E -07 1 . E - 06 1 . E - 05 1 . E - 04 1 . E -03 1 . E -02 1 . E -01 ADC concentration (UM ) atent Apr . 10 , 2018 Sheet 4 of 7 US 9 , 938 ,323 B2 FIG . 2C NCI- H526 anti -HER2 - HC - E388 -DS - ppan - COA - 1 -40 -LEFIASKLA -N389 + - anti- HER2- HC - E388 -DS -ppan - CoA - 1 - 39 -LEFIASKLA - N389 + anti -HER2 -HC - E388 -DS -ppan - CoA - 1 -41 -LEFIASKLA -N389 anti- HER2 -HC - E388 - DS - ppan -CoA - i- 9 - 40 -LEFIASKLA -N389 ñ anti- HER2 -HC -E388 - DS -ppan - CoA -i - 9 - 39 - LEFIASKLA -N389 ñ A - anti- HER2 -HC -E388 - DS -ppan -CoA - 1- 9 - 41 -LEFIASKLA -N389 ü Normalizedviability ö å 0 . 0 1 . E - 07 1. E - 06 1. E -05 1 . 5 - 04 1. E -03 1 . E - 02 1. 5 -01 ADC concentration (UM ) FIG . 2D HCC1954 anti- HER2 -HC - E388 -DS -ppan -CoA - 1 - 40 LEFIASKLA -N389 Ötodo - anti -HER2 -HC - E388 -DS - ppan - CoA - 1 - 39 LEFIASKLA -N389 - anti-HER2 - HC -E388 -DS - ppan - CoA - 1- 41 LEFIASKLA -N389 - anti- HER2 -HC - E388 - DS -ppan - CoA - 1 - 9 - 39 Normalizedviability LEFIASKLA -N389 SA anti- HER2 - HC -E388 - DS - ppan -CoA -i - 9 -41 TTTTTT LEFIASKLA -N389 0 . 0 + 1 . E -07 1. 5 -06 1 . E -05 1 .E - 04 1 . 5 -03 1. E -02 1 .E -01 ADC concentration (UM ) U .S . Pateatent Apr . 10 , 2018 Sheet 5 of 7 US 9 , 938 ,323 B2 FIG . 2E NCI- N87 anti- HER2 -HC -E388 -DS -ppan -CoA - 1 -40 LEFIASKLA -N389 anti- HER2 -HC -E388 - DS -ppan - CoA - 1 - 39 LEFIASKLA -N389 anti -HER2 - HC - E388 -DS -ppan -CoA - 1 -41 LEFIASKLA -N389 o anti- HER2 -HC - E388 - DS -ppan - CoA -i - 9 - 39 Normalizedviability LEFIASKLA -N389 1. E -07 1. 6 - 06 1. 6 - 05 1. 5 - 04 1. E -03 1. E -02 1. - 01 ADC concentration (uM ) FIG . 2F SK -BR - 3 - anti- HER2 -HC - E388 -DS -ppan -CoA - 1 - 40 LEFIASKLA -N389 anti- HER2 -HC - E388 -DS - ppan -CoA - 1 - 39 LEFIASKLA -N389 + anti- HER2 - HC - E388 -DS -ppan - COA - 1 - 41 LEFIASKLA -N389 anti- HER2 -HC -E388 -DS -ppan - CoA - i- 9 - 39 Normalizedviability II LEFIASKLA -N389 ASES 1. E - 07 1. 5 - 06 1 . 6 -05 1. -04 1. 5 -03 1 . -02 1. -01 ADC concentration (MM ) atent Apr . 10 , 2018 Sheet 6 of 7 US 9 , 938 ,323 B2 FIG .