| Hai Lala Mo at Alt a La La La Manual

|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 . 3 100 .0 , 10. 08 Plasmaconcentration(ug/ml) + Anti- Her2 - LC - S159C - 38 1. 0 - Anti- Her2 -LC - S159C - 2 + Anti - Her2- LC - S159C - 5 • Anti- Her2 - LC - S159C - 32 ? O - Anti- Her2 - LC - S159C - 6 Anti- Her2 -LC - S159C - 18 + Anti- Her2 - LC -S159C - 4 0 Anti- Her2 100 200 300 400 500 600 Time (hours ) U . S . Patent Apr . 10 , 2018 Sheet 7 of 7 US 9 , 938 ,323 B2

TumorVolume(mm)Mean+SEM . TH. .

8 12 16 20 24 28 32 36 40 44 Days Post Implantation US 9 ,938 ,323 B2 AMATOXIN DERIVATIVES AND in humans in the U .S . was launched in 2000 (and later CONJUGATES THEREOF AS INHIBITORS withdrawn from the market ), a decade later only a few OF RNA POLYMERASE chemical classes of drug compounds (maytansinoids , auristatins , calicheamycins and duocarmycins) had reached 5 clinical trials as payloads for ADCs. Antibody - Drug Conju CROSS -REFERENCE TO RELATED gates : the Next Generation of Moving Parts , A . Lash , APPLICATIONS Start -Up , December 2011 , 1 - 6 . The use of amatoxins as cytotoxic moieties in ADC ' s for This application is a 371 U . S . national phase application tumour therapy was explored in 1981 (Davis & Preston , of international application number PCT /IB2015 /058537 Science 1981 , 213 , 1385 - 1388 ) by coupling an anti - Thy 1 . 2 filed 5 Nov. 2015 , which claims the benefit of U . S . Provi - 10 antibody to alpha -amanitin using a linker attached to the 7 ' sional Application No. 62 /076 ,023 , filed 6 Nov . 2014 , which position of the indole ring via diazotation . Morris & Venton is incorporated by reference herein in its entirety . (Morris & Venton , Int. J . Peptide Protein Res. 1983 , 21 419 -430 ) also demonstrated that substitution at the 7 ' posi FIELD OF THE INVENTION tion resulted in a derivative which maintained cytotoxic 15 activity . The invention relates to amatoxins and conjugates of Patent application EP 1 859 811 A 1 (published Nov . 28 , amatoxins to a target - binding moiety , e . g . an antibody, and 2007 ) described the direct conjugation ( i. e . without a linker the use of such conjugates to treat cancer . structure ) of albumin or a monoclonal antibody (HEA125 , OKT3 , or PA - 1 ) to the gamma C -atom of amatoxin amino BACKGROUND 20 acid 1 of beta -amanitin . The inhibitory effect of these conjugates on the proliferation of breast cancer cells (MCF Amatoxins are cyclic peptides comprised of eight amino 7 ), Burkitt' s lymphoma cells (Raji ) , and Tlymphoma cells acid units which can be prepared synthetically , or can be ( Jurkat) was shown . The use of linkers was suggested , isolated from a variety of mushroom species, such as however no such constructs were exemplified and no details Amanita phalloides ( green death cap mushroom ). Amanita 25 regarding linker attachment sites on Amatoxins were pro vided . bisporigera (destroying angel ), Amanita ocreata (destroying Patent applications WO 2010 / 115629 and WO 2010 / angel ) , Amanita virosa ( destroying angel) , Amanita bisp 115630 (both published Oct. 14 , 2010 ) describe conjugates , origera ( fool' s mushroom ), Lepiota brunneo - incamata where antibodies , such as antiEpCAM antibodies such as (deadly dapperling) , Conocybe filaris and Galerina margi humanized antibody huHEA125 , are coupled to amatoxins nata . via ( i ) the gamma C - atom of amatoxin amino acid 1 , ( ii ) the There are currently ten known members of the Amatoxin 6 ' C - atom of amatoxin amino acid 4 , or ( iii) via the delta Family : alpha - Amanitin , beta -Amanitin , gamma- Amanitin , C - atom of amatoxin amino acid 3 , in each case either epsilon -Amanitin , Amanullin , Amanullinic acid , Amanina directly or via a linker between the antibody and the ama mide , Amanin and Proamanullin . Different mushroom spe toxins. The inhibitory effects of these conjugates on the cies contain varying amounts of different Amatoxin family 35 proliferation of breast cancer cells ( cell line MCF - 7 ) , pan members . creatic carcinoma ( cell line Capan - 1 ) , colon cancer ( cell line Amatoxins are potent and selective inhibitors of RNA Colo205 ) and cholangiocarcinoma (cell line OZ ) were polymerase II , a vital enzyme in the synthesis ofmessenger shown . RNA (mRNA ) , microRNA , and small nuclear RNA (sn - Patent applications WO 2012 / 119787 ( published Sep . 13 , RNA ). By inhibiting the synthesis of mRNA , Amatoxins 40 2012 ) describes conjugating a target- binding moiety via a thereby stop cell metabolism by inhibiting transcription and linker attached to the amatoxin indole nitrogen . The cyto protein biosynthesis , which results in cellular apoptosis . toxic activity of such conjugates on a HER2- positive tumor Consequently Amatoxins stop cell growth and proliferation . cell line in vitro was disclosed . Alpha -amanitin , is known to be an extremely potent Patent applications WO 2014 /043403 ( published Mar. 20 , inhibitor of eukaryotic RNA polymerase II (EC2 . 7 . 7 .6 ) and 45 2014 ) describes conjugating a target -binding moiety via a to a lesser degree , RNA polymerase III, thereby inhibiting linker attached to the 7 ' position of the amatoxin indole . The transcription and protein biosynthesis . Wieland ( 1983 ) Int. J . cytotoxic activity of such conjugates on Herceptin and IgG1 Pept. Protein Res . 22 (3 ): 257 -276 . Alpha -amanitin binds in MDA -MB -468 cells was disclosed . Also , the cytotoxic non - covalently to RNA polymerase II and dissociates activity of such conjugates on Herceptin in PC3, HCC - 1954 slowly, making enzyme recovery unlikely. 50 and MDA -MB - 46 cells was disclosed . The use of antibody - drug conjugates (ADCs ) for the In view of the toxicity of amatoxins , particularly for liver targeted delivery of cell proliferation inhibitors and/ or cyto - cells , it is important that ADC' s comprising a linked ama toxic agents to specific cells has been the focus of significant toxin remain highly stable in plasma prior to the release of research . Antibody -Drug Conjugate , Methods in Molecular the amatoxin after internalization into the target cells . In this Biology , Vol. 1045 , Editor L . Ducry, Humana Press (2013 ) . 55 regard , improvements of the conjugate stability may have ADCs include an antibody selected for its ability to bind to drastic consequences for the therapeutic window and the a cell targeted for therapeutic intervention , linked to a drug safety of the amatoxin conjugates for therapeutic selected for its cytostatic or cytotoxic activity . Binding of the approaches . Thus , given the widely acknowledged value of antibody to the targeted cell thereby delivers the drug to the ADCs as therapeutics for treating cancer there remains a site where its therapeutic effect is needed . Many antibodies 60 need for the stable delivery of potent RNA polymerase that recognize and selectively bind to targeted cells, like inhibitors to the target cells prior to internalization of the cancer cells , have been disclosed for use in ADCs, and many RNA polymerase inhibitors . methods for attaching payload ( drug ) compounds such as cytotoxins to antibodies have also been described . In spite of SUMMARY OF THE INVENTION the extensive work on ADCs, though , only a few classes of 65 cell proliferation inhibitors have been used extensively as The invention provided herein includes cytotoxic cyclic ADC payloads . Even though the first ADC approved for use peptides of formula (I ) , which are analogs of alpha -amanitin US 9 ,938 ,323 B2 and beta -amanitin , and methods of using such cytotoxic (CH2 ) mX3 ( CH2 ) m , - (CH2 ) mNRS — , cyclic peptides as the drug component of an antibody - drug conjugate ( ADC ) . The present invention includes novel cytotoxic cyclic peptides and the use of such novel cytotoxic cyclic peptides as payloads for ADCs. The invention further 5 - wim includes methods and intermediates useful for incorporating mm such novel cytotoxic cyclic peptides into ADCs. S10 The invention further includes immunoconjugates com prising such cyclic peptides (i . e Antibody Drug Conjugates ) , 10 mm pharmaceutical compositions comprising such cyclic pep mm mm 01 tides immunoconjugates and compositions comprising such tot cyclic peptides immunoconjugates with a therapeutic co - L , is (CH ) . NRS( CH , , , — , - ( CH , ) , NRC( O ) agent. (CH2 ) m , X4 , (CH2 ) - NRŠCEO )X4 , + (CH2 ) m The invention further includes methods of inhibiting 15 NR CEO) - , - (( CH2 ) mO ) n (CH2 ) m - , - ( ( CH2) mO )n RNA polymerase using such immunoconjugates comprising (CH2 ) mX3 ( CH2 ) m , NR ( (CH2 ) mO ), X3( CH2 ) m - , such cyclic peptides and methods of treating cell prolifera - NR $ ( (CH2 ) mO ), (CH2 ) mX3 ( CH2 ) m - , - X , X , C ( = O ) tion diseases using such immunoconjugates comprising such (CH2 ) m , (CH2 ) m ( O (CH2 ) m ) n , ( CH2) mNR cyclic peptides. (CH2 )m ( CH2) mNR $ C ( O )( CH2 )mX2 (CH2 )m - , 20 — (CH2 ) mC (= O )NR ( CH2) mNR CEO ) (CH2 ) m , In one aspect the cytotoxic cyclic peptides of the inven - ( CH2) mC ( = O ) , ( CH2)mNR (CH2 ) mCEO ) tion , or stereoisomer and pharmaceutically acceptable salts X X , C (= O ) , (CH2 )mX3 (CH2 )mC (= O ) XXX , C thereof, have the structure of Formula ( A ) ( O ) - , - (CH2 )mC ( = O ) NR $( CH2)m , (CH2 ) mC ( O )NR® (CH2 ) mX3 ( CH2) m , – (CH2 ) mX3 (CH2 ) m 25 NR CEO) ( CH2 ) m — — (CH2 ) mX (CH2 ) mC O )NRS Formula ( A ) ( CH ) m > , < ( CH2) 2) ,( CH2) NR°C ( ~ O )( CH2) n … , HO - (CH2 ) mC ( = O )NR ( CH2) m ( O (CH2 ) m ) n — , - (CH2 ) m (O ( CH2) m ) C (= O ) , (CH2 ) „ NR (CH2 ) mC = - , (CH2 ) , C = O )NR $ (CH2 ) „ NRC = 0 ) (CH2 ) m HOS 30 (O ( CH2) m ), X3( CH2 ) m , ( CH2) mX3 ( CH2) mO ). (CH2 ) (CH2 ) . X2( CH2 ) mC (= O ) - , - (CH2 ) C (= ONR $ (CH2 )mO ) ,( CH2 ) mX , (CH2 ) m , (CH2 ) mX ; (CH2 )m ) (O ( CH2) m ), NR $ C = O ) (CH2 ) m , (CH2 ) „ X ; 35 ( CH2) m (O (CH2 )m ) „ CeO ) , ( CH2)mX2 ( CH2) m (0 HN (CH2 ) m )n (CH2 ) mCO )NR (CH2 ) mCEO ) , - (CH2 ) mC ( O ) NR (CH2 ) m (O (CH2 ) m )^ 0 ) — , HO - ( (CH2 ) mO ) , (CH2 ) „ NRSC ( = O )( CH2 ) m , (CH2 ) mC ( O ) NR ( CH ,) , C ( O ) NR ( CH, ) , ( CH2) , NRC 40 ( O ) (CH2 )mNR C (= O ) (CH2 ) m , (CH2 )mX3 (CH2 )m o ? CEO ) NRS - , ( CH2) mCEO ) NR — , ( CH2) mXz - , R C (R $ ) 2 (CH2 ) m , (CH2 ) mC ( R $) 2NR54 , ( CH2)mC ( O )NR (CH2 )mNRS , ( CH2) CEO )NR (CH2 ) m 45 NRSC (= O )NRS , ( CH2) mC ( = O ) X ,X , C = O ) , NH - C( R $) 2 ( CH2 ) mNRC = O )( CH2 ) , (CH2 ) mC ( = O )NR (CH2 ) mC (R5 ) NRS , C (R5 ) , ( CH2) mX ; ( CH2) m - , - (CH2 )mX3 ( CH2 )mC ( R ) 2NR — , - C ( R $ ) 2 wherein : (CH2 ) mOCEO ) NR (CH2 ) m - , - (CH2 ) mNR CEO) O 50 (CH2 ) mC ( R ) 2NRS , (CH2 ) mX2 ( CH2 ) mNRS , X is S (= O ), S ( = O )2 or S ; 50 (CH2 )mX3 ( CH2 ) m ( O (CH2 ) m )„ NR5 – (CH2 ) m R ! is H or CH3; R2 is - L ; L2R +, -LzR or - (CH2 )mXz NR - ( CH2) , C, ZO) NR ( CH2) , ( O( CH2) , ), NR , (CH2 ) mR4; R3 is — NH , or — OH ; L , is (CH2 ) NRC - (CH2 ) m ( O ( CH2) m ) , NR , (CH2CH2O ), (CH2 ) m - , FO ) - (CH2 ) m (OCH2CH2 ) n ) — (CH2 ) mO (CH2 ) m , (CH2 ) m 55 SE02 , (CH2 )mCEO ) NR® (CH2 ) mS ( 0 ) 2 - , - (CH2 ) mX3 ( CH2 ) S (= O )2 - , (CH2 ) mX , X C my GO ) , ( CH2) m ( O ( CH2) m )C, O ) X X CEO) - , (CH2 ) m ( O (CH2 ) m ) X2X C, O ) , ( CH2) mXz mm , mo (CH2 ) mX X CEO ) , (CH2 ) mX3 ( CH2) m ( O ( CH2) m ) n mm 60 X X , C ( O ) - , - ( CH2) mX3 (CH2 ) mCEO ) NR (CH2 ) m NR C ( O ) - , - ( CH2) mX ( CH2) mC ( O )NR (CH2 ) mC WA ( O ) , ( CH2) mX3 (CH2 ) mC (= O )NR (CH2 ) m (O (CH2 ) m )C , 0 — , (CH2 ) mC ( = O ) X X , C ( = O ) NR ( CH2) m , ( CH2)mX3 ( O ( CH2) m ), C ) , (CH2 ) m mw 65 NR C (= O ) (( CH2 ) mO ), (CH2 ) m - , - (CH2 ) m ( O (CH2 ) m )n CEO) NR (CH2 ) m — (CH2 ) mNR CEO) NR (CH2 ) m - or - (CH2 )mX3 (CH2 )mNRCO ) , US 9 ,938 ,323 B2 wherein : - continued Ph N NH mi wi my or *Xi is Xosso min NH2 mm N mi in or mm m X31 nim and OH s tisu mogliere

HO ?? X4 is win 30 mm ??

Lz is (CH2 )mNRC = 0 ) ; mun nyt site35 R4 is sunt com

?? P 40

OH wion

?? 45 min NH ?? 50 man NH

X2 is NH . 55 JNRgCK _ OCH = CH2, — NG, HNH2N ZO

w HN . - C = CH , 60

SH , - S ( = O ) (CH = CH2) , (CH2 ) 2S ( O ) 2 mi ( CH CH2) , LNR°S( ~ O ) ( CH = CH2) , LNR°C ~ O ) NH og CHAR ”, JNRC( LO?CH, Br , JNR°C ( LO?CH , I, _ NHC _ OCHBr, _ NHCUZO) CHAI, LONH , - CEO) NHNH2 , 7 US 9 ,938 ,323 B2

R10 IO N (R !! ) 1- 2 , min mo R10 4x deRR 10 - L (R19 1- 2 ( R !! ) 1 - 3 man min — CO2H , — NH2, RIO — NC ( = O ) , — NC ( = S ) , - HO O=AFO

?? NH2, HO OH - P = O KnyustgartHO

) HO O=A

??HO HO - NH2, HO ?? P = tegusitzenHO O 0. - OH HO OH

NH NH2, there?? is the

HO HOS OH

2 NH2NH , terkesterHOO .HO US 9 ,938 , 323 B2 - continued HO O=- o=— min OH OH NH ) , tuottaaHOP OH OH OTO tyy OH OH HO ??HO N ' to

OH O = A SN OH ?? NH , HO OH N . XyryttigasteHO HOPE

n mi min Hus Huse HNH2Nting min this HN 10 H2NXXXX mm mm each R is independently selected fromLa H and C . - Cgalkyl; - continued Rºis

w m

! II

NO2, or w F : US 9 ,938 ,323 B2 R ? is S (CH2 ) , CHRÅNHC ( O ) Rs or 12

OH N ww OH OH NH2; August HO OH N HO o

R® is Rs or CTO) ORS; -continued each Rºis independently selected from H , C7 -Cgalkyl , F , C1, 15 and OH ; each R1° is independently selected from H , C , -Coalkyl , F , C1, - NH2, OCH3, OCH2CH3, — N (CH3 ) 2 , CN , w mi - NO , and OH ; each Rll is independently selected from H , C1- alkyl, fluoro , 20 benzyloxy substituted with C O OH , benzylShu substi - ( CH2 )mX3 ( tasCH2 ) - , - (CH2 ) mNR — tuted with CCOOH , C alkoxy substituted with CEO) OH and C -4alkyl substituted with CEO ) OH ; each m is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 25 – ZI 9 and 10 , and w mm each n is independently selectedautom from 1 1., 22 , . 3 4, 4. ,5 5. ,0 6. , 71 7 , 8 , tot tot 9 , 10 , 11 , 12 , 13 and 14 . In one aspect the cytotoxic cyclic peptides of the inven mm tion , or stereoisomer and pharmaceutically acceptable salts 30 min or thereof, have the structure of Formula (en I ) el sentit a totmi L , is (CH2 ) m , - NR (CH2 ) m - , - (CH2 ) mNR CEO) Formula (I ) (CH2 ) m , X4, (CH2 ) mNRC ( = O ) X4, ( CH2) m HO 35 NRSCO ) , ( CH2) mO ), (CH2 ) - , - ( CH2) mO )n ( CH2)mX3 (CH2 ) m , NR ( (CH2 )mO ), X3( CH2) m , - NR $ (( CH2 ) mO ), (CH2 ) mX3 ( CH2 ) m , X , X CEO) (CH2 ) = (CH2 ) m (O ( CH2) mn , (CH2 )mNR $ (CH2 ) m , (CH2 )mNR CEO) (CH2 ) mX2 ( CH2 ) m , 40 - (CH2 ) mC O ) NR (CH2 ) mNRC = O )( CH2 ) m HN (CH2 )mC = O ) - , - ( CH2) mNR (CH2 ) 771 CEO ) X X , C ( 0 ) — - ( CH2) mX3 (CH2 ) mCe= O )X2X , C NH FO , (CH2 )mC (= O )NR (CH2 ) m , (CH2 )mC HN FO ) NR (CH2 ) mX3 (CH2 ) (CH2 ) mX3 (CH2 ) m 45 NR CEO) (CH2 ) m - (CH2 ) mX3 (CH2 )mC ( = O ) NR5 ( CH2) m , ( CH2) mO ), (CH2 ) mNR CEO) (CH2 ) m - , HO (CH2 ) mC ( O )NR $ (CH2 ) m ( O (CH2 ) m )n — , (CH2 ) m Z ( O (CH2 ) m _ C = 0 ) , (CH2 ) mNR (CH2 ) mC = O ) , - (CH2 ) mCEO) NR (CH2 ) mNR C ( O ) - , - ( CH2) m s - R2 50 ( O ( CH2 ) n ) , X ( CH2 ) n ” , ( CH2) Xg( ( CH2 ) , 0 ) , ( CH2= O )) NR m $, (CH2 ()CH2 mO ),) mX3 (CH2 ( CH2) mX2 ) mC ( CH2 ( ) m0 ) , , (CH2 (CH2 )mXg ) mC N IZ (CH2 ) m ( O (CH2 ) m )„ NRCEO) (CH2 ) (CH2 ) mX ; (CH2 ) m ( O ( CH2)m ), 0 , (CH2 ) mX3( CH2 ) m (0 55 (CH2 ) m )n ( CH2) mCEO ) NR (CH2 ) mCO ) , — (CH2 )mC ( = O ) NR ( CH2)m (O (CH2 ) m ), Ce= ) ; wherein : - ( (CH2 )mO ), (CH2 ) mNR $ C = O ) (CH2 ) m , ( CH2) mC R ! is H or CH2; R2 is - L , L2R + , -L3R or — (CH2 ) mXz ( = O )NR $ (CH2 ) . CEO) NR (CH2 ) m , (CH2 ) mNRC (CH2 ) mR4 ; R is — NH , or — OH ; ( O )( CH2 )mNR CEO) ( CH2) m - , (CH2 )mX3 (CH2 ) m Lj is (CH2 ) mNR C$ O ) , 60 C ( O )NR — , (CH2 )me = O ) NR - , (CH2 ) mXz - , - C ( R ) 2 (CH2 ) m - , ( CH2) , C ( R ) NR , - (CH ) . C FO( ) NR $ (CH2 ) mNR , ( CH ) C (= O )NR $ (CH2 ) m mu NR $CEO ) NRS , ( CH2) mCEO ) X , X ,CO ) , - C (R $) , ( CH2 )mNR CEO) (CH2 ) m - (CH2 )mC 65 ( O )NR $ (CH2 ) . C ( R ) 2NRS , C ( R $ ) 2 (CH2 ) mXz mu mu (CH2 ) m (CH2 ) mX ; (CH2 )mC (RS ) NRS , C ( R ' ) 2 (CH2 ) mOCO )NR® ( CH2) m , (CH2 ) mNR $ C ( = O ) 0 US 9 ,938 ,323 B2 13 14 (CH2 ) „ C (R $) NRS , (CH2 )mXz ( CH2 ) „ NRS , - continued (CH2 ) mX2 (CH2 ) m ( O (CH2 ) m ) „ NRG ( CH2) m NRG (CH2 ) mC ( O )NR (CH2 ) m (O (CH2 ) m )n NR ” , ( CH2) ( O ( CH2) n ) , NR , < ( CH3CH2O) , (CH2 ) m — (CH2 ) m (OCH2CH2 ) n = (CH2 ) mO 5 mm ( CH2) m , (CH2 ) mS ( 0 )2 , (CH2 )mC ( = O )NR NH (CH2 ) , SEO) 2 - , (CH2 ) mX3 ( CH2 ) mS ( O ) 2 - , (CH2 ) mX , X , CC= 0 ) , 7 (CH2 ) m (O ( CH2) m _„ Ce= 0 ) XXXCO ) , ( CH2) m ( O (CH2 ) m ) „ X , X , C ( = O ) , - (CH2 ) mX3 (CH2 ) mX X , C ( O ) - , - (CH2 )mX3 ( CH2) m NH ( O (CH2 ) m ), X , X ,CE ) , (CH2 ) X2( CH ) CEO ) NR® (CH2 )mNRCO ) , (CH2 )mX3 (CH2 ) mCEO ) X2 is 0 = NH2, NR * (CH2 ) mC ( O ) , (CH2 ) mX3 ( CH2)mC ( O ) NR (CH2 ) m ( O ( CH2) m ), C ( = O ) , ( CH2) mC = 0 )X , X , C 15 H2N20 FO) NR *( CH2)m , (CH2 )mX3 ( O (CH2 ) m ),CO ) , (CH2 ) mNRC ( = O )( ( CH2 ) mO ), (CH2 )m , (CH2 ) m ( O (CH2 ) m ),C O )NR $ (CH2 ) m , (CH2 ) NR CEO) NR (CH2 ) m - or - (CH2 ) mX3 ( CH2 )mNR°C ( = O ) — , wherein : 20 2min EZ

25 ? nin . X is N or + X 30 mi mo tox 35 NH2 mm NH mm XOX 40 or

w v

w 45 LOH X3 is a or and HO . Bu mafua mi Y OH 2 50 OH man X4 is mia w mi Lz is (CH2 ) mNRC = 0 ) ; tyrt R4 is 60 en ensamento OH

OH 65 M OH : + + US 9 ,938 ,323 B2 15 16 - continued -continued - R10 shine NH – NRC = OCH = CH2, — NG, — (R !! ) 1- 2 wim 10 mi · RIO Rio CECH

winan 15 (R101 -2 SH , - S ( = O ) (CH = CH2 ), (CH2 ) 2S ( = O ) 2 ( CH = CH2) , NRS( 20) ( CH = CH2) , NRC = O ) CHAR ” , _ NR°C( LO?CH , Br, LNR°C( LO?CH , I, _ NHC ( ~ O ) CH2Br , — NHC( LO?CH , I, LONH2, 20 CEO) NHNH , tot R9 2 25 min

w 4x0s .– CO. H , NH?NCat( 50) , – NC( = S ) , OH

N A TOTO OH OH NH2 HO . OH N KryyttingsHOHO

F He then mmthe 1 thesemi HN min H?N H2N w

*ox yox or yox US 9 ,938 ,323 B2 17 18 each R is independently selected from H and C -Cgalkyl ; enRis el tracta de tres mese, Cast Formula ( I ) HO .

HO mmaa HN NO2, or 1010 mi HN 15 HO — OSS F N - R2

20 IZ Pue R ? is S ( CH2) „ CHR®NHC ( = O )Rs or

o OH O= HN OH NH2; XwgrybyHO . OH N HOTHO ) DH

R $ is Rs or CEO )ORS ; wherein : R ' is H or CH3; R² is - L ,L , R4, -LzR6 or — (CH2 ) mX (CH2 ) mR4 ; R * is — NH , or — OH ; each Rº is independently selected from H , C ,- Cgalkyl, F, C1, 40 Li is (CH2 ) mNRC O ) , and — OH ; each Rl0 is independently selected from H , C , -Calkyl , F , C1, - NH2, OCH3, OCH CH3, N (CH3 ) 2 , CN , 45 min mm — NO2 and enOH ; conta come to ni takmi each R is independently selected from H , C1- alkyl, fluoro , benzyloxy substituted with C ( O )OH , benzyl substi- > tuted with CEO )OH , C1- alkoxy substituted with ni - Ce= O )OH and C1- alkyl substituted with CEO) tos - (CH2 ) mX2 ( CH2 ) m - , - (CH2 )mNR — , OH ; 5555 each m is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , me ang9 andmga 10 , andtaganteny , obed mom1 . 2. 3. 4. 5. 6 . 7 . 4 . tou mnim ; § t each n is independently selected from 1 , 2, 3 , 4 , 5 , 6 , 17,, 88 , 60 9 , 10 , 11 , 12 , 13 and 14 . 1234567. 4 " X or tot In certain embodiments the cytotoxic cyclic peptides of 65 L , is -- ( CH .) . — . - NR5( CH ). — . - ( CH . ) . NRSC ( = O ) the invention , or stereoisomer and pharmaceutically accept (CH2 ) m , - X4, (CH2 ) mNRCO ) X4, (CH2 ) m able salts thereof, have the structure of Formula (I ) NR°C( 20) – , ( ( CH2) nO) , ( CH2) 2 , ( CH2) 2 ) , US 9 ,938 ,323 B2 19 20 ( CH2) mX3 (CH2 ) m - , - NR ( (CH2 ) mO ) X3( CH2) m - , -continued - X , X CEO) ( CH2) m , ( CH2) m ( O ( CH2)mn - , ( CH2) mNR (CH2 ) m , (CH2 ) mNRC = O ) (CH2 ) m X (CH2 ) m , (CH ) CEO )NR $ (CH2 ) NR $ CEO) or (CH2 ) m (CH2 )mC = O ) , ( CH2) mNR * (CH2 ) mC5 mm ( O ) XXX , CEO) - (CH2 )mX3 ( CH2)mC ( = O ) mu XX, C = 0 ) — , - (CH2 )mCEO ) NR (CH2 ) m - , (CH2 ) mCEO772 ) NR *( CH2 )mX2 ( CH2)m , (CH2 ) mXz (CH2 ) mNRC = O ) (CH2 ) m - , (CH2 ) mX3 ( CH2) mC FO )NR ( CH2) m , ( CH2) mO ), (CH2 ) mNR CEO) ( CH? ) , ( CH2) C ( CO )NR° ( CH2) , ( O( CH2 ) n ), , HO . (CH2 ) m ( O (CH2 )m ) C = ) - , - (CH2 ) mNR (CH ) , C OH - 0 , (CH2 ) C ( = O )NR $ (CH2 ) mNRCO ) , OH ( CH2) m ( O (CH2 ) m ) , X , (CH2 ) m - , (CH2 ) mX2( CH2) m w O ) n (CH2 ) m , ( CH2)mX3 ( CH2) mCEO ) - , - ( CH2) m w CEO) NR $( CH2 ) mO (CH2 ) mX3 ( CH2 ) m , (CH2 ) m 15. X3( CH2) m (O (CH2 ) m )„ NR CEO) ( CH2 ) m , (CH2 ) m X ( CH ) ( O( CH ) C( O) - ( CH ) , X, ( CH2) , ( O (CH2 ) m )n ( CH2) mCEO )NR ( CH2)mC40 ) — , ( CH2) mC ( O )NR ( CH2) m ( O ( CH2) m )C, O ) , ( (CH2 ) mO ), (CH2 ) mNR CEO ) (CH2 ) m - , - ( CH2)mC 20 ?? EO )NR (CH2 ) mCO )NR $ (CH2 ) m - , (CH2 )mNRC FO )( CH )- NRC = O ) (CH2 ) m " = (CH2 ) X , ( CH2) m HO . C (= O )NR , (CH2 ) mCEO) NR — , - (CH2 ) m OH X3 — , - C ( R ) 2 ( CH2) m , ( CH2) mC ( R ) 2NR - , (CH2 ) mCIO ) NR (CH2 ) mNRS , (CH2 ) mCEO ) OH NR ( CH2) mNR CEO NRS , (CH2 ) mC = OX X ? FO- (CH2 ) - ) , - C ( O )NR - C (( RCH2 ) 2) ( CH2 , C (R ) . NRCE) NRS = , O )( CH2C )( mR —) 2 , ( CH2CR) „ )X3 2( (CH2 CH2) mOC) m , ( = O- ()CH2 NR *) (mX3 CH2 ( ) CH2 m 5) . C (RS ) NRS(CH2 ) , mm NH NR°C( CO) ( CH, ) , C( R ), NR , ( CH2) , X , ( CH ) NRS- ( CH2 ,) . NR ,( CH2) mX2 C ( CH2 (CH2 ) )C m (- O ONRCH(CH2 ) m ) » NRS ) ,( O, (CH2 ) m ). NRS , (CH2 ) m ( O (CH2 ) m ) , NR — , NH ( CH2CH2O )n ( CH2) m — — (CH2 ) m ( OCH2CH2) n , (CH2 ) mO (CH2 ) m , ( CH2) mS = 0 , - , - (CH2 ) mC 35 FO )NR ( CH2)mS = 0 ) 2 - , - ( CH2)mX3 (CH2 ) mS X2 is NH2 ( 0 ) 2 , (CH2 ) mX2X C, O ) - , ( CH2) m ( O ( CH2) m ) , CEO ) X , X , C40 ) — , (CH2 ) m ( O (CH2 ) m ) n H2N 20 XXX C, O ) - , - ( CH2) mX3 (CH2 ) mX X , C40) , (CH2 ) mX2 ( CH2 ) m ( O (CH2 ) m ), X X C, ) , 40 HN (CH2 CH )) mX2 » X ( CH2 CH2)) m C == OJNRO )NR ( CH2 CH )) mC NR = $ C0 )= 0, ) , (CH2 ) mX , (CH2 ) CEO )NR (CH2 ) m (O (CH2 ) m ), C FO , - (CH2 ) m?EO ) XX CO, )NR (CH2 ) m — , (CH2 ) mX3 ( O (CH2 ) m ) C ( = O ) — , (CH2 ) mNR°C 45 mm FOO )NR (( CH2 (CH2 ) mO ) ) m , (CH2 ) m , (CH2 (CH2 ) mNR ) m ( O CEO (CH2 ) m)m NRS ), , c C EZ ( CH2) m - or - (CH2 ) mX3 ( CH2) mNR C O ) , wherein : N mm or Xi is mi NH min } N US 9 ,938 ,323 B2 21 22 - continued CHAR ”, JNRCC _ O ) CHBr , NRC = O?CH, I , – NHC ( OCHBr, … NHCUO?CH , I , ANH , in - C ( O )NHNH2 , m

X3 is met or , and * www N m w mi - X4 is u maxw R 10 Lz is (CH2 ) mNRCE = 0 ) ; R4 is - (R !! ) 1 - 2

P > mai mi RIO par minn Rio (R19 ) 1- 2

ZNH w mm - NR, C ( - O?CH = CH - N ,

CECH ,

to 40 at SH , - S ( O ) 2 (CH = CH2) , ( CH2) 2S ( O ) 2 * ( CH = CH2 ), NR ' S( 20) ( CH = CH2) , NR°C( < 0) CO, H, NH , NC( ~ O) , – NC( = S ),

OH 0 0 ma TOTO ?? ?? NH2, HO , À OH N Xwymytty-P = HO O

H2N miHuse Y F thismoto box US 9 ,938 , 323 B2 23 24 -continued HN .

HON

pox or yax each R is independently selected from H and C , -C alkyl; R8 is R or - C ( O )OR " ; Ris 15 each Rº is independently selected from H , C - C alkyl, F , CI , and OH ; 20

each Rlº is independently selected from H , C1- Cgalkyl, F , + to CI , NH ,, OCH3, OCH CH3, N (CH3 ) 2 , CN , 25 - NO , and OH ;

30 each Rll is independently selected from H , C1- alkyl, fluoro , benzyloxy substituted with C (= O )OH , benzyl substi NO2, or tuted with C ( O )OH , C1. 4alkoxy substituted with -C O OH and C -4alkyl substituted with C ( O ) ton 35 OH ; 40 each m is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 and 10 , and

45 each n is independently selected from 1, 2 , 3, 4, 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 and 14 .

50 In certain embodiments of this aspect of the cytotoxic F cyclic peptides having the structure of Formula ( I) , are cytotoxic cyclic peptides having the structure of Formula R ’ is S ( CH2) , CHR®NHC( = O ) R $ or (la ) :

OH O= o ?? ?? - NH2; HO OH N XuyustimP= HO O US 9 , 938 , 323 B2 25 2626 X , is Formula ( la ) X, is HO H2N 20

HN ** * 11111

HN 10 HN HN NH w HOMME $ EZ . 15 X2 is Z

N 20 in

R3 n In certain embodiments of such cytotoxic cyclic peptides as min of Formula (I ) and Formula ( la ): R ' is H or CH3; R2 is - L ,L2R “, - L3R6 or (CH2 ) mXz (CH2 ) mR4 ; R * is - NH , or OH ; and X4 is Lj is – (CH2 ) mNRSCO ) , 30 mi mi mun mo mi 35 tof Xox Lz is (CH2 ) mNRCE tox= ) ; R is 40 tosmi :45 + or - (CH2 ) mX3 (CH2 ) m = ; R6 is L2 is (CH2 ) m , - NR $( CH2 ) m (CH2 ) . NR $ C = O ) 30 ( CH2) m - , - X4, (CH2 ) mNR CEO ) X4, (CH2 ) m NR°C( 0 ) , ( CH2) , 0 ), ( CH2 ) n , ( CH2 ) , 0 ) , (CH2 )mX , (CH2 ) m , NR $ (( CH ) 0 ), X3 (CH2 )m - or - X1X2C = O ) (CH2 ) m - , wherein : 55 bo X , is each R is independently selected from H and C . -Coalkyl ; each m is independently selected from 1, 2 , 3 , 4 , 5 , 6 , 7, 8 , 9 and 10 , and m each n is independently selected from 1, 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 and 14 . In certain embodiments of such cytotoxic cyclic peptides mi 65 of Formula (I ) and Formula ( la ): R ' is H or CH3; R2 is -L , L2R4, -LzR6 or (CH2 ) mXz ( CH2) mR4 ; R * is -- NH , or OH ; US 9 ,938 ,323 B2 27 28 Lj is (CH2 ) mNR CEO) , andand X X4, is is 28 way 5 min mvh mi 10 Lz is – (CH2 ) mNRCO ) - or ( CH2) mNRC ( = O ) ( CH2) mCEO ) ; - ww min ° R4C is , H, NRG - )- (CH NRC 0)

or - (CH2 ) mX3 ( CH2 ) m ; L2 is – (CH2 ) m - , - NR $ (CH2 ) m , ( CH2) . NR $ CEO) ONH , (CH2 ) m - , - X4, (CH2 )mNR CEO )X4 , (CH2 ) m win NR°C( = ) - (( CH2 ) O) , ( CH2) n -, - ( CH2) O ), mu (CH2 ) mX2 ( CH2 ) m , NR $ (( CH )mO ), X2( CH2 ) m -, 20 - NR > ( (CH2 ) mO ) , (CH2 )mXz ( CH2) m - or - X X _ C (= O ) (CH2 ) m , wherein : or X is 25 mi min thos : Home- F Ris X2 is

H2N20

HN mm 45 each RS is independently selected from H and C . - C alkyl; each m is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 and 10 , and N each n is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 50 9 , 10 , 11 , 12 , 13 and 14 . N mm In certain embodiments of such cytotoxic cyclic peptides . V of Formula (I ) and Formula ( la ): + Rl is H or CH3 ; R2 is - LiL2R + , - L3Rº or ( CH2) mXz (CH2 ) mR4 ; R * is — NH , or — OH ; XoXzis is ss Lj is – (CH2 ) mNRC = 0 ) ,

min 60 mw mi ma w 65 mm mi US 9 ,938 , 323 B2 29 30 or (CH2 )mX3 ( CH2 ) m and X4 is L2 is – (CH2 ) m - , - NR (CH2 ) m - , - (CH2 ) mNR CEO) ( CH2) m - , - X4, (CH2 ) mNR CEO ) X4 , — ( CH2) m NRCE= 0 ) , ( (CH2 ) mO ), (CH2 ) , ( (CH2 ) mOn (CH2 ) mX3 (CH2 ) m - , - NR (( CH2 )mO ), X3 (CH2 ) m , 5 mu in NR $ (( CH2 ) mO (CH2 ) X3( CH2 ) m - or - X , X , C m GO )( CH2) m - , wherein : X? is 10 Lz is ( CH2)mNR C ( O ) - or ( CH2)mNR CEO ) (CH2 )mCE = 0 ) ; IZ R4 is

mim or - ONH2; X , isthors wana

H2N

HN in

R6 is Xz is mw O w each R is independently selected from H and C , -Coalkyl ; each m is independently selected from 1, 2 , 3, 4 , 5 , 6 , 7 , 8 , 40 9 and 10 , and each n is independently selected from 1 , 2 , 3 , 4, 5, 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 and 14 . mun In preferred embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ) : R ? is

mi wM NH ????NH ???? NH NH nun ww NH X O= ing star US 9 , 938 , 323 B2 US 9. 33 02 32 - continued

mm NH mm NH O=

w tinisNH mintrims SingingHN tingos. HN temwegemu . HN

. N - N N

. HN N

NH ,

IZ mia . HN

min NH US 9 ,938 , 323 B2 34 1598-continued . 23 8 . NH mm NH misO tahan mi . HN NH xusO= xen . HN HN . HN man

mm NH

oyun HN

mayanmm NH

mm . HN

mm tugasNN up to US 9, 938 ,323 B2 35 36 35 - continued

mi mi

wania yohuyors

min mi

05 NH2

mm muno se mmtope US 9 ,938 , 323 B2 3731 US 9, 938 , 323 B2 38 - continued ???liliti111? 2 IIIIIIIIIIIIIII??? .or In certain embodiments of any of the aforementioned 15 cytotoxic cyclic peptides of Formula (I ) and Formula ( la ): R2 is

????? NH ?????NH NH 'NH * * ?????NH * NH ??? ????? * NH NH 0 * * * NH ???? NH NH NH H , 0 NH2

????? NH 0 ???? " NH

*????? NH *??? ????NH US 9 , 938 , 323 B2 40 - continued

NH NH mi NH

HN mi HN the thingsIZ

NH HN XemHN tinguts * . HN 23 * NH wem NH

N mm NH

N US 9 , 938 , 323 B2 41 -continued or 0=

NH2

mi NH

In certain embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula (I ) and Formula (la ) : 25 R is

?? * NH * ?? NH NH tugasNH min dan

NH i NH XanXamn NH2, tenisNH man angNH US 9 , 938 , 323 B2 43 US 98. 33 B2 44 - continued

. HN

KanyondNH

mm NH

NH2 N

11 IZ

mon HN US 9 ,938 ,323 B2 45 46 In certain embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula ( I ) and Formula (la ) : theR² is imagem communes

. HN xringmi thingNH

HN kurang tuanNH

HN kurangHN tuan

O HN NH xwe? Xu NH2 NH mm NH NH2 . HN

mi . HN NH tranaONH , traw NH2, xiisHN 0 kuiNH

mm NH ??????NH US 9 ,938 , 323 B2 47 -continued . NH mino NH XingOs HN more ?

NH NH min w kung tunayIZ mwingingHN Xwengososmm HN kuinummm HN

mm , HN

ia HN 0 N NN US 9 , 938 , 323 B2 49 50 • skolen- continued

NH ,

OS **SILA win Nooror ulll NN

min * ??

In certain embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ): R² is

NH " NH

kwan?O na NH mi NH twintunt? 0 =

NH tranmm NH tugan " NH2, Xunque mu NH

XumsNH tungongsmm US 9 ,938 , 323 B2 US9 , 938 , 323 B2 52 -continued 52

NH tuimmmm NH 's

mm N

mi ??

In certain embodiments of any of the aforementioned 50 cytotoxic cyclic peptides of Formula ( I) and Formula ( la ) : R2 is

ww mm

O= US 9 ,938 , 323 B2 ? US9 ,938 ,323 B2 54 - continued

???????? ???????? ????????

???? ?? ???????????

???????? ??????? ?? ??? * N ??? ?

???? ?? ??????? ??? US 9 , 938, 323 B2 | 5555 56 - continued www

www

N NN

www

NH NN

0 : NH 11111 NH www US 9, 938 , 323 B2 57 S8 In certain embodiments of any of the aforementioned -continued cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ): Re is www

or ? 10

In certain embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula (I ) and Formula ( la) : Re is

None nnnnnn

0 =

PAN ???mons ?

VMAun

nnnn ?? ANNA

O ???? NH ? NH US 9, 938 ,323 B2 -continued

i mm mm

NH . NH2 OS NH2 O NH2, 07

nim mm

O H2

wins mi

man 5oomin mums wi US 9, 938 ,323 B2 6161 622 - continued

inn doline

In certain embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula ( I) and Formula ( la ): * - continued R2 is www

toyO=

toy NH2, US 9 ,938 ,323 B2 63 64 - continued -continued

min

10 mu

mm 15 vºn

20 In certain embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula ( I) and Formula ( la ): Lugones R2muy is Luis miEmpire mi min wim mi

mm min

N 20 IZ HN US 9 ,938 , 323 B2 65 - continued minn mo

O HN 0 = IZ XosXomm mi

O ZI Ennyt mo

O N vorovo O, US 9 , 938 , 323 B2 67 68 67 -continued or

NH2

IZ Stiil

min 25 In certain embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula ( I) and Formula ( la ) : - continued R2 is ni

30 min

O = or 35

In certain embodiments of any of the aforementioned 40 cytotoxic cyclic peptides of Formula ( I) and Formula ( la ): R ? is mm win (R )

w mm ( R ) US 9, 938 , 323 B2 69 - continued MAN new ( R) ( R )

NH *ANN NNNN (R ) Mine

HN HN *ANAN

*( R )

Now * US 9 , 938 , 323 B2 72 71 - continued nnnnnn

0 NN or

NH2 NH . 11 YTI NH

35 In certain embodiments of any of the aforementioned -continued cytotoxic cyclic peptides of Formula ( I) and Formula ( la ) : R2 is

OS or #

In certain embodiments of any of the aforementioned 50 cytotoxic cyclic peptides of Formula (I ) and Formula (la ): R² is

**1111kum 111111111110 *1111000 . US 9 ,938 , 323 B2 73 ? 74 panulqu00 *IIIIIIIIIIIIII IIIIIIIIIIIIII 11/1IIIIIIIIIIII

*iiiliilililil 1IIIIIIIIIIIIIII

???

?? *1IIIIIIIIIIIII?? IIIIIIIIIIII10 ?=11IIIIIIIIIIII

Z LZ *:IIIIIIIIIIII IIIIIIIIIIIIIII??

ZL *IIIIIIIIIIII

0 *IIIIIIIIIIIIII ???

NN US 9 , 938 , 323 B2 75 76 - continued 16 *111111111111

N SAU NH

N IIIIIIIIIIII

40 tourIn certain embodiments of any of the aforementioned -continued cytotoxic cyclic peptides of Formula (I ) and Formula ( la ) : R2 is 45 1102

50 or

55 In certain embodiments of any of the aforementioned cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ) : R ? is

F F

m F NH mi NH 0

F . XingF F US 9, 938 ,323 B2 77 - continued

mu NH HN

tipsF XeniaF F NH NH O XogF tweetedF min . HN F F NH

O =

winan

O =

F F

Hantew tutte le

F -T) F

HN , HN 0 YE LuisF xxxvideo

* ?? tipoF xaneta US 9 ,938 , 323 B2 64 p?nujuoy as

min HN ??HN 0 pory gry-IL ann NH m scenes AmexHN ma L thay thing- they men

mostrangexHN munmenging

mm Z N - US 9, 938 ,323 B2 82 - continued

w XimenemZ mi toxto HN

min

F tak F F

F Xushningmi N win

F US 9 , 938 , 323 B2 83 84 -continued F F mw N XylenmeyeF ???

Yunmi HN F F toyto F

me - TT

- mi

X NH

NH XylemnuniyF US 9, 938 ,323 B2 85 86 -continued W N

Or.

In certain embodiments of any of the aforementioned 6 'O -methyl -7 ' C -( (2 - (3 - ( 23 - ( (4 -maleimido )methyl - 1H -1 ,2 , 3 cytotoxic cyclic peptides of Formula (I ) and Formula ( la ) R triazol- 1 - yl ) - 3, 6 , 9 , 12 ,15 , 18 , 21- heptaoxatricosyl ) ureido ) is — CH3, while in certain other embodiments of any of the ethylthio )methyl ) - a - Amanitin ; aforementioned cytotoxic cyclic peptides of Formula ( I ) and 30 6 'O -methyl - 7 'C - ( ( 2 - ( 4 - (maleimido ) ethanecarboxamido ) bu Formula ( la ) R ' is H . tylthio )methyl ) -a - Amanitin ; In certain embodiments of any of the aforementioned 6 'O -methyl - 7 ' C - ([ 4 -( 5 -( maleimido )pentanecarboxamido ) cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ) R3 butylthio )methyl ) - a - Amanitin ; is — NH ,, while in certain other embodiments of any of the 6 ' O -methyl - 7 'C -( 4 -( (2 , 5 - dioxopyrrolidin -1 -yl ) oxycarbo aforementioned cytotoxic cyclic peptides of Formula (I ) and 35 nylamino )butylthiomethyl ) -a -Amanitin ; Formula ( la ) R3 is — OH . 6 ' O - methyl- 7 ' C - ( ( 4 - ( 4 - ( (maleimido )methyl ) cyclohexan In certain embodiments of any of the aforementioned ecarboxamido )butylthio methyl) - a - Amanitin ; cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ), 6 ' O -methyl - 7 ' C - ( ( 4 - ( 3 - ( 6 - (maleimido )hexyl ) ureido )butyl the cytotoxic cyclic peptides ofFormula ( I ) and Formula ( la ) thio )methyl ) - a - Amanitin ; is selected from : 6 ' O -methyl - 7' C -( ( 4 -( 5 - (5 - (maleimido )pentanecarboxamido ) 6 'O -methyl -7 ' C -( ( 2 - (2 -( maleimido Jethanecarboxamido leth pentanecarboxamido )butylthio )methyl ) - a -Amanitin ; ylthio )methyl ) - a -Amanitin ; 6 ' O -methyl - 7 ' C - ( ( 4 - ( 5 - ( 4 - ( (maleimido )methyl ) cyclohexan 6 ' O -methyl - 7 ' C - ( ( 2 - ( 3 - (maleimido )propanecarboxamido ) ecarboxamido )pentanecarboxamido )butylthio )methyl ) ethylthio )methyl ) - a - Amanitin ; 45 C -Amanitin ; 6 ' O -methyl - 7 ' C - ( ( 2 - ( 4 -( maleimido )butanecarboxamido eth - 6 'O -methyl - 7 'C - ( [ 4 - ( 14 - (maleimido ) - 3 ,6 , 9 , 12 - tetraoxatetra ylthio )methyl ) - a - Amanitin ; decancarboxamido )butylthio )methyl ) - a - Amanitin ; 6 'O -methyl - 7 ' C -( (2 - ( 5 - (maleimido )pentanecarboxamido ) 6 'O -methyl - 7' C -( [4 - ( 14 -( (4 -maleimido )methyl - 1H - 1 ,2 , 3 ethylthio )methyl ) - a - Amanitin ; triazol- 1 - yl) - 3 , 6 , 9 , 12 - tetraoxatetradecancarboxamido ) bu 6 'O -methyl - 7' C -( 2 -( ( 4 - (( maleimido )methyl ) cyclohexan - 50 tylthio )methyl ) -a - Amanitin ; ecarboxamido ) ethylthio )methyl ) - a - Amanitin ; 6 '0 -methyl - 7 ' C - ( [ 4 - ( 3 - (23 - ( ( 4 -maleimido )methyl - 1H - 1 , 2 , 3 6 'O -methyl -7 ' C -( ( 2 - (5 - (5 - (maleimido )pentanecarboxamido ) triazol- 1 - yl) - 3 ,6 , 9 , 12 ,15 , 18, 21- heptaoxatricosyl ) ureido ) pentanecarboxamido )ethylthio )methyl ) -a -Amanitin ; butylthio )methyl ) - a - Amanitin ; 6 'O -methyl - 7' C -( (2 - (6 - (4 -( ( maleimido )methyl ) cyclohexan - 7 'C - (( 2 -( 5 -( maleimido )pentanecarboxamido ) ethylthio ) ecarboxamido )hexanecarboxamido ) ethylthio )methyl ) - a - 55 methyl ) - a - Amanitin ; Amanitin ; 6 ' O -methyl - 7 ' C - (( 1 -( 3 - (maleimido )propanoyl ) - azetidin - 3 6 ' O -methyl - 7 ' C - ( ( 2 - ( 14 - (maleimido ) - 3 , 6 , 9 ,12 - tetraoxatetra - thio )methyl ) - a - Amanitin ; decancarboxamido ) ethylthio )methyl ) - a - Amanitin ; 6 '0 -methyl -7 ' C - ( ( 1 - ( 6 - (maleimido )hexanoyl ) - azetidin - 3 6 ' O -methyl - 7 ' C - ( ( 2 - ( 14 - ( ( 4 - maleimido )methyl - 1H - 1 , 2 , 3 thio )methyl ) - a - Amanitin ; triazol - 1 - yl) - 3 ,6 , 9 , 12 - tetraoxatetradecancarboxamido ) 60 6 ' O -methyl - 7 ' C - ( ( 1 - ( 3 - (maleimido )propanoyl ) - piperidine ethylthio )methyl ) - a - Amanitin ; 4 -thio )methyl )- a - Amanitin ; 6 ' O -methyl - 7 ' C - ( 2 - ( ( 2 , 5 - dioxopyrrolidin - 1 -yl ) oxycarbo 6 '0 -methyl - 7 'C - ( ( 1 - (6 - (maleimido )hexanoyl ) -piperidine - 4 nylamino )ethylthiomethyl ) - a - Amanitin ; thio methyl) - a - Amanitin ; 6 -O -methyl - 7 '- C -( ( 2 -( 3 - (6 -( maleimido )hexyl ) ureido )ethyl - 6 ' O -methyl - 7' C — (( N -( 4 -( (maleimido )methyl )cyclohexan thio )methyl ) - a - Amanitin ; 65 ecarbonyl )piperidine - 4 - thio )methyl ) - a - Amanitin ; 6 '- O -methyl - 7 'C - (MC - Val- Cit - PABC - ( 2 - aminoethyl) thiom - 6 ' 0 -methyl - 7 ' C - ( ( 1 - ( 6 - ( 6 -( maleimido )hexanamido ) ethyl- ) - a - Amanitin ; hexanoyl) - piperidine - 4 - thio )methyl ) - a - Amanitin ; US 9 ,938 ,323 B2 87 88 6 'O -methyl - 7' C -( (1 - (6 - (4 -( ( maleimido )methyl ) cyclohexan In other embodiments of any of the aforementioned ecarboxamido ) hexanoyl) -piperidine - 4 - thio )methyl ) - a cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ), Amanitin ; the cytotoxic cyclic peptides ofFormula (I ) and Formula ( la ) 6 'O -methyl - 7 ' C - ( ( 1 - ( 15 - (maleimido ) - 4 , 7 , 10 , 13 - tetraoxa is selected from : pentadecanoyl) - piperidine - 4 - thio )methyl ) - a - Amanitin ; 5 60 -methyl - 7°C - ( ( 2 - ( 3 - ( 2 6 ' O -methyl - 7 'C — (R ) - ( N - ( 3 - (maleimido )propanoyl ) pyrro 6 ' O - methyl - 7 ' C - ( ( 2 - ( 3 - ( 23 - ( ( 4 -maleimido )methyl - 1H - 1 , 2 , 3 lidine - 3 - thio )methyl - a - Amanitin ; triazol- 1 - yl) - 3 ,6 , 9, 12 , 15 , 18 ,21 - heptaoxatricosyl) ureido ) 6 ' O -methyl - 7 ' C — ( R ) ( N - ( 6 - (maleimido )hexanoyl ) pyrro ethylthio )methyl ) - a - Amanitin ; lidine - 3 - thio )methyl - a - Amanitin ; 6 'O -methyl - 7' C -( [4 - ( 3 -( 23 -( (4 -maleimido )methyl -1H -1 ,2 , 3 6 'O -methyl - 7' C — (S ) (N -( 3 -( maleimido )propanoyl ) pyrro - 1010 triazol- 1 -yl ) - 3 ,6 , 9, 12 ,15 ,18 ,21 - heptaoxatricosyl ) ureido ) lidine- 3 -thio )methyl - a - Amanitin ; butylthio )methyl ) - a -Amanitin , and 6 ' O -methyl -7 ' C — ( S ) - (N -( 6 -( maleimido )hexanoyl ) pyrroli 6 'O -methyl - 7 'C - ( [4 -( 14 - (maleimido )- 3, 6 , 9, 12 - tetraoxatetra dine - 3 - thio )methyl -a -Amanitin ; decancarboxamido )butylthio )methyl ) - a - Amanitin . 6 ' O -methyl - 7 ' C — ( N -( 6 - (maleimido ) hexanoyl) pyrrolidine - 15 The present invention provides immunoconjugates , also 3 - thio )methyl - Q - Amanitin ; referred to herein as ADCs, containing cytotoxic cyclic 6 ' O -methyl - 7 ' C - ( (( 4 - ( 4 -maleimido )methyl - 1H - 1 , 2 , 3 - tri peptides linked to an antigen binding moiety , such as an azol- 1 - yl) butylthio )methyl ) - a - Amanitin ; antibody or antibody fragment. These conjugates compris 6 'O -methyl - 7' C -( 6 - ( aminoxy )hexaneamido ) - ethylthiom ing cytotoxic cyclic peptides are useful to treat cell prolif ethyl) - O - Amanitin ; 20 eration disorders , particularly when the cytotoxic cyclic 6 ' O -methyl - 7 ' C - ( ( 1 - ( 6 - ( aminooxy ) hexanoyl ) -piperidine - 4 peptides are linked to an antibody that recognizes cancer thio )methyl ) - a - Amanitin ; cells and thus promotes delivery of the cytotoxic cyclic 6 ' O -methyl - 7 ' C - ( (6 -( aminoxy ) hexaneamido ) - butylthiom peptides to a cell targeted for attack . The immunoconjugates ethyl) - O - Amanitin ; are especially useful for treating certain cancers as further NHS ester of 6 'O -methyl - 7 'C - ( ( 2 - ( 3 - ( carboxy ) propanecar - 25 detailed herein . Data provided herein demonstrate that these boxamido ) ethylthio )methyl ) - a - Amanitin ; immunoconjugates are effective inhibitors of cell prolifera NHS ester of 6 ' O -methyl - 7' C - ([ 4 - (3 - ( carboxy )propanecar tion . boxamido )butylthio )methyl ) - a - Amanitin ; In one aspect of the immunoconjugates of the invention NHS ester of 6 ' O -methyl - 7 ' C - ( ( 1 - ( 4 - ( carboxy )butanoyl ) - pi include immunoconjugates of Formula ( B ) : peridine - 4 - thio )methyl ) - a - Amanitin , and 30 6 ' O -methyl - 7 ' C - ( ( 1 - ( 14 - ( ( 4 - maleimido )methyl - 1H - 1 , 2 , 3 triazol- 1 - yl ) - 3 ,6 , 9 , 12 - tetraoxatetradecancarbonyl) - piperi Formula ( B ) dine - 4 - thio )methyl ) - a - Amanitin . HO In other embodiments of any of the aforementioned 35 cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ), HO the cytotoxic cyclic peptides of Formula ( I ) and Formula ( la ) is selected from : Tetrafluorophenyl ester of 6 '0 -methyl - 7' C -( (4 - (3 - ( carboxy ) HN propanecarboxamido )butylthio )methyl ) - a - Amanitin ; 40 7 ' C - ( [ 4 - ( 3 - (23 - ( ( 4 -maleimido )methyl - 1H - 1 , 2 , 3 - triazol- 1 HN yl) - 3, 6 , 9 , 12 , 15 ,18 ,21 -heptaoxatricosyl ) ureido )butylthio ) methyl) - a - Amanitin ; HO 7 'C -( ( 4 - 14 -( maleimido )- 3 ,6 ,9 ,12 - tetraoxatetradecancar 45 boxamido )butylthio )methyl ) - a - Amanitin ; - Ab 6 ' O -methyl - 7 ' C - ( [ 4 - ( 26 - (maleimido ) - 3 , 6 , 9 , 12, 15 , 18 ,21 , 24 S - R20 octaoxahexacosancarboxamido )butylthio )methyl ) - a Amanitin ; NH 6 ' O -methyl - 7 ' C - ( ( 4 - ( 3 - ( 2 - ( 2 - ( 2 - ( 2 - ( ( 4 -maleimido )methyl 1H - 1, 2, 3 -triazol - 1 -yl ) - ethoxy ) ethoxy) ethoxy ) ethyl) 50 ureido )butylthio )methyl ) -a - Amanitin ; 6 ' O -methyl - 7 ' C - ( ( 1 - ( 26 - (maleimido ) - 3 , 6 , 9 , 12, 15 , 18 ,21 , 24 wherein : octaoxahexacosancarboxamido ) -piperidine - 4 - thio ) X is S (= O ), S (= O )2 or S ; methyl) - a - Amanitin ; Ab represents an antigen binding moiety ; Perfluorophenyl ester of 6 ' O -methyl - 7 ' C - ( ( 4 - ( ( 23 -carboxy ) y is an integer from 1 to 16 ; 3 , 6 , 9 , 12, 15 , 18 ,21 - heptaoxatricosancarboxamido )butyl thio )methyl ) - a -Amanitin ; R ' is Hor - CH3; R * is - NH , or — OH ;R20 is - L L , R40 , Tetrafluorophenyl ester of 6 'O -methyl - 7' C -( ( 4 - ( 14 -carboxy -L3Rº or (CH2 ) mX3( CH2)mR40 ; 3 ,6 , 9 , 12 -tetraoxatetradecancarboxamido )butylthio ) 60 Lj is ( CH2) mNRCO ) , methyl ) - a - Amanitin ; 6 'O -methyl - 7 ' C -( [4 - ( 26 - ( 3 -( maleimido )propanamido )- 3 ,6 , 9 , 12, 15 ,18 ,21 , 24 -octaoxahexacosancarboxamido )butyl thio )methyl ) - a - Amanitin ; and Perfluorophenyl ester of 6 ' O -methyl - 7 ' C - ( 1 - (23 -carboxy - 3 , 65 wir 6 , 9 , 12 , 15 ,18 ,21 -heptaoxatricosancarbonyl ) -piperidine - 4 thio )methyl ) -a -Amanitin . mm US 9 ,938 ,323 B2 89 90 - continued (CH2 )m (O ( CH2)mn m )„ X X C ( 0 ) (CH2 )mXz (CH2 ) mC ( O )NR $ ( CH2)mNR CEO) - , - (CH2 ) m X ;( CH2 ) mC = O )NR $ (CH2 ) mC = 0 ) , (CH2 ) mXz (CH2 )mC ( O )NR (CH2 ) m (O (CH2 ) m ) CEO ) , min . ( CH2) mC ( O ) X X , C ( O )NR (CH2 ) m , (CH2 ) mXz ( O (CH2 ) m )C , O) , (CH2 ) mNRC GO ( CH2) mO ) , ( CH2) m , (CH2 ) m ( O (CH2 ) m ), C (CH2 ) mX , (CH2 ) m - , (CH2 ) . NRS — , ( O )NR (CH2 ) m — (CH2 ) mNR m $ C ( O )NRS 10 (CH2 )m , or (CH2 ) mX2 ( CH2 )mNR $ C = 0 ) wherein : X , is

Rutatm 15 tot or totm mu L2 is (CH2 ) m , - NR $ (CH2 ) , (CH2 ) mNRC 20 mat N ( O )( CH2) m , X4, (CH2 )mNR $CEO )X4 , (CH2 ) mNR C ) , ( (CH2 ) mO ), (CH2 ) m - , = ( CH2 ) , 0 ), ( CH ) , X ,( CH2) 2 , NR ( CH2) , 0 ) , X3( CH2) m — — NR * (( CH2 ) mO ) n (CH2 )mX3 ( CH2 ) m - , - X , X CEO) (CH2 ) m , ( CH2) m ( O ( CH2) m ) , 25 - ( CH2) mNR (CH2 ) m — — ( CH2) mNR C ( O ) ( CH2) m X3( CH2 ) m , ( CH2) mCEO )NR (CH2 )m R?C ( = O ) ( CH2) m - , - (CH2 ) mCEO) — , - (CH2 ) m tox NR (CH2 ) mCO ) X X , C ( O ) - , - ( CH2) mXz ( CH2 ) C( LOX, XC ( CO) , ( CH ), C( - O ) NR° 30 (CH2 ) m - , (CH2 )mCEO ) NR (CH2 ) mX3( CH2) m , - (CH2 )mX2 ( CH2 )mNR $ C = O )( CH2 )m , (CH2 ) m X2( CH2 ) CEO) NR $ (CH2 ) m , (CH2 ) mO ), (CH2 ) m mw NRC ( O ) (CH2 ) m , (CH2 ) mCEO ) NR (CH2 ) m min ( O (CH2 ) m )n — — (CH2 ) m ( O (CH2 ) m ) , C ( 0 ) — , 35 (CH2 ) mNR $ (CH2 ) mC = O ) , (CH2 ) C = O ) NR >( CH2 ) mNRCO ) , (CH2 ) m ( O ( CH2) m ), Xz (CH2 ) m , ( CH2) mX3 ( (CH2 ) mO ) , (CH2 ) m - - ( CH2)mX3 ( CH2) mCEO ) , (CH2 ) mC ( O )NR (CH2 ) m ), (CH2 ) X , (CH2 ) m , (CH2 ) mX , (CH2 ) m 40 (O ( CH2) m ), NRCO )( CH2)m , ( CH2)mX3 ( CH2) , ( O ( CH ) ) , C ZO ) , ( CH ) X ( CH2 ) , ( O wim (CH2 )m ) - , ( CH2) mCEO )NR (CH2 )mCO ) , - ( CH2)mCEO )NR (CH2 ) m ( O (CH2 ) m ) CEO ) , ( (CH2 ) mO ) , ( CH2) mNRC ( O ) (CH2 ) m , 45 OH or (CH2 ) m CEO) NR (CH2 ) . C ( = O )NR > (CH2 ) m — , (CH2 ) mm NRC = O )( CH2 ) mNR $CÉ = O )( CH2 ) HO . (CH2 )mX , (CH2 )mCEO ) NR — , — ( CH2) mCEO ) Y OHOH NR — , (CH2 )mX ; C (R ) 2 (CH2 ) m , - ( CH2) m C ( R ' ) 2NR — (CH2 ) mC ( = O ) NRS 50 OH (CH2 ) m NRS - , (CH2 ) mC ( = O )NR (CH2 ) „ NRC FO )NR 4 , ( CH2) mC = O )X , X , CEO ) , C (R $ )2 (CH2 ) mNRC = O ) (CH2 ) m = , (CH2 ) mC CONR (CH2 ) . C (RS ) , NRS , C (R ) ,( CH ) „ X m (CH2 ) m — , - (CH2 ) mX , (CH2 ) mC ( R $) 2NRS - , 55 C (R ), (CH2 ) mOC ( = O )NR (CH2 ) m — , - (CH2 )m um NRÁCEO ) O (CH2 ) mC ( R ) NR - - ( CH2) mX2 min (CH2 ) mNRS , (CH2 ) mX2 (CH2 ) m ( O (CH2 )m )n NR — , - (CH2 ) mNRS , (CH2 ) mC (= O )NR (CH2 ) m ( O ( CH2) m ) „ NR — (CH2 ) m ( O ( CH2) m ) n 60 OH NR — (CH2CH2O ) , ( CH2) m , (CH2 ) m (OCH2CH2 ) n (CH2 ) mO ( CH2) m , (CH2 )mS HO . F02 , (CH2 ) mCO )NRS (CH2 ) „ S = 0 ) 2 - , ?? (CH2 ) mX2 ( CH2 ) SE02 (CH2 ) mX , X , C ( 0 ) , ( CH2) m ( O ( CH2) m ) CEO) X , X , C 65 © OH ( 0 ) — , (CH2 ) m (O ( CH2) m ) , X2X , C ( = O ) — (CH2 ) mX3 (CH2 ) mX2X CO, (CH2 ) mX3 US 9, 938 , 323 B2 911 X,X , is R10 is

|. ?

?NH ??? ww UN NH SS nudenne ? A NH NAN NH) , NH HEN ? 20 NV Som HN ?

? fe, ? HN now NNN - NH mufa un NA x ? - NR°C( O) CH , , – NHC( 20) CH , … , S ( 80) CH?CH, , -( CH , SK2O ) CHÚCH , … , Ph - NR ' S ( = O ), CHACH , NR- C ( = O )CHACH , 30 _ NH?, C( O ) , _ NHC( < 0 ) , - CHANHCHACH , , - NHCHACH , , - S ,

NH or

unununw NH) warna ????? NNMN NAM ?NH New ??? AMAN RO NNNNN or X , is - NH NNNA W??? wwww Mun

and X , is N?N UNA ANN NNNNN NANA |L , is (CH ) +NR - C ( = O) - : + S US 9 ,938 ,323 B2 93 94 - continued -continued R 10 NN man nh (R1991 - 2

w = 10 ni RIO VEN nim mm w EZ 20 RIO man 25 inn R12 H - R10 m V = N 30 R www - m

mi R12

35 H2N maxRG mi

40 mi R12

45 Box RS On R12 ZZ 50 RIO man

55 OS R12 . HN min (R101 -2 RY

wa R12 US 9 ,938 ,323 B2 95 96 -continued Lj is (CH2 ) mNRCO ) , O

= w S w

mu , each RS is independently selected from H and C . - Cgalkyl; R60 is — NH — ; mm R70 is 10 min mi OHOH 0 Xnly O w 15 (CH2 83)). mX3 x . ( chCH2 )) m - , -- ch( CH2) ) mNR ok — , R $ is H , CZ -Cgalkyl or C ( O )ORS ; koeach Rº is independentlyposla, selected opreme from H. , CC. . -Cgalkyl COM, F , 20 in Cl, and OH ; tou t each R10 is independently selected from H , C . -Cgalkyl , F , Cl, - NH2, - OCH3, - OCH2CH3, — N ( CH3 ) 2 , — CN , - NO2 and — OH ; 25 Xy totmm each Rll is independently selected from H , C1- alkyl, man fluoro , benzyloxy substituted with C ( = O )OH , ben zyl substituted with CCOOH , C , alkoxy substi L2 is – (CH2 ) m - , - NR (CH2 ) m , (CH2 ) mNRC tuted with CEO) OH and C1- alkyl substituted with FO ( CH2) m , - X4, (CH2 ) mNR CEO ) X4 , 30 - ( CH2)mNR°C ) — , - ( CH2) mO ) , (CH2 ) m - , C ( O )OH ; 30 (( CH2) mO ), ( CH2) mX3 ( CH2) m , - NR *( ( CH2) mO )n each R12 is independently selected from H , CH3 and X3( CH2) m ; - X X , C ( = O )( CH2) m , (CH2 ) m (O phenyl; ( CH2) m )n - , ( CH2) mNR ( CH2) m , ( CH2) mNRC each m is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , FO) (CH2 ) mX2( CH2) m - , - ( CH2) mC ( O ) NR 8 , 9 and 10 , and 35 (CH2 )mNRC ( = O ) (CH2 ) m , - (CH2 ) mCEO ) , each n is independently selected from 1 , 2, 3, 4 , 5 , 6 , 7 , ( CH2) mNR (CH2 ) mCEO ) X X X ( 0 ) , 8 , 9 , 10 , 11, 12 , 13 and 14 . (CH2 ) mX2 (CH2 ) mC = O )X , X C, O ) , (CH2 ) m In one aspect of the immunoconjugates of the invention CEO) NR (CH2 ) m , (CH2 ) mCEO) NR (CH2 ) m include immunoconjugates of Formula ( II ) : X3 (CH2 ) m - , ( CH2) mXz (CH2 )mNR CEO) 40 (CH2 ) m , – (CH2 ) 777 , X , (CH ) CEO) NRS (CH2 ) m - - (CH2 ) mO777 (CH2 ) mNRC = O )( CH2 )m - , (CH2 ) m Formula ( II) C O ) NR° ( CH2) , ( O ( CH2) , ) , … , - CO(CH2 ) m (. O (CH2 ) m )» Ce0 - , (CH2 ) mNR (CH2 ) m ( 0 ) , HO - ( CH2) mC4O )NR (CH2 )mNR VRNRECH %C40 ) , 45 (CH2 ) m (O (CH2 ) m ) , X2( CH2 ) m , (CH2 ) mXz HOM ( CH2) , O ), (CHCI CH ) - ( CH2) X, ( CH2 ) C O ) (CH ) CEO )NR $( CH 2mn) O ), (CH2 ) , X , (CH2 ) HN (CH2 ) mX3( CH2 ) m (O ( CH2) m ), NR $ C (= O )( CH2) m , - ( CH2) mX (CH2 ) m (O (CH2 ) m ) , CeO ) - , - (CH2 ) m 50 X3( CH2 )m (O (CH2 )mn , — (CH2 ) mCEO ) NR HN (CH2 ) m Ce= 0 ) , – (CH2 ) mCEO ) NR (CH2 )m (0 (CH2 ) m )„ CEO) — , (( CH2 ) mO ) , (CH2 ) mNR CEO ) HO (CH2 ) m , (CH2 ) mCEO )NR (CH2 ) mCEO )NRS 04 (CH2 ) m , ( CH2)mNR $ CEO) (CH2 ) mNRÁCEO ) 55 (CH2 ) m , (CH2 ) mX3 ( CH2 ) mCEO ) NRS , N An - ( CH2)mCEO )NR — , - (CH2 ) mX - C ( R ' ) 2 ? (CH2 ) m - , (CH2 ) mC ( R ' ) 2NR — (CH2 ) mCEO ) N NR (CH2 )mNRS ( CH2) mceO )NR $ (CH2 )m IR NR CEO )NR5 — , - ( CH2) mCEO )X , X , CEO ) , 60 C (R $) 2( CH2)mNRC ( = O ) (CH2 ) m , (CH2 )mC O )NR $ (CH2 ) C ( R $ ) ,NRS C (RS ) , (CH ) , Xz wherein : (CH2 ) m , (CH2 ) mX2 ( CH2 ) mC ( R $ )2NRS - , Ab represents an antigen binding moiety ; C (R ') 2 (CH2 ) mOCONR ( CH2) m , - (CH2 ) m NRC( O ) O (CH2 ) mC (R™ ) 2NR ( CH2)mX3 y is an integer from 1 to 16 ; (CH2 ) mNR = — (CH2 ) mX3 ( CH2 ) m (O (CH2 ) m )n Rl is H or — CHz; R3 is — NH , or — OH ;R20 is - L , L2R40PR40 , 05 NRS , (CH2 ) mNRS — (CH2 ) mC = O )NR - L3Rºor (CH2 ) mX (CH2 )mR40 ; (CH2 ) m ( O (CH2 ) m ] „ NRS , (CH2 ) m ( O (CH2 ) m )n US 9 ,938 , 323 B2 97 98 NRG (CH2CH20 ) , (CH2 ) m , (CH2 ) m X2 is 98 (OCH CH2) = (CH2 ) mO (CH2 ) m — (CH2 ) m SE02 (CH2 ) mCEO )NR (CH2 ) mS ( 0 ) 2 - , (CH2 ) mX3 ( CH2 ) mSe = 0 ) 2 - , - (CH2 ) mX , X C FO) , (CH2 )m (O (CH2 ) m ), CEO) X X , C _ 5 m FO , (CH2 )m (O ( CH2) m )„ X2X , CEO ) , - (CH2 ) mX3 ( CH2 ) mX2X , C ( = O ) , (CH2 ) mXz (CH2 ) m (O (CH2 ) m ), X _ X , C ( O ) - , (CH2 ) . X2 ( CH2) mC ( O )NR *( CH2 ) mNR %CEO ) , (CH2 ) m X3( CH2 ) „ CEO )NR (CH2 ) mC = O (CH2 ) , X , 10 (CH2 ) mC ( = O ) NR (CH2 ) m ( O (CH2 ) m )»C O ) , (CH2 ) mC ( = O ) X , X , C ( O )NR $ ( CH2) m ?? ( CH2) mX3 ( O (CH2 ) m ) „ CEO) — , ( CH2)mNRC FO ( CH2) mO ) , (CH2 ) m , (CH2 ) m ( O (CH2 ) m ), C NH2, FO) NR (CH2 ) m - ( CH2)mNR°C ( O ) NR HNH2NO . (CH2 ) m , or — (CH2 ) mX3 ( CH2 ) mNRCO ) , wherein : HN X , is mm mo NH

N toxmi OT min NH NH2 min mi

?? or 45 mi N

HO . ?? w mi OH Xz is N Or mu Wim

wim mm and X4 is

OH min HO . OH WM OH Lz is (CH2 ) mNR $ C = 0 ) ; US 9, 938 , 323 B2 66 100 R40 is -continued R10 unny ????? AMAN ? 10 Now R10

5 R10 + mino NHP . - NRCC _ ONCH , , NHC ( CO ) CH2 , 20 - SC - 0 ) , CHACH, , - ( CH ) SC - 0 ), CHACH) , - NR ' S( = O ), CHACH , ANREC ( = O )CH , CH , CH, NICHACH , , - NICH CH 25

Nuno www ?S? ( hon RIO Mons ???

NH www ANNAN ??? A N? ????~ AN winan www ??N R10 N?N NANNNNN 1 - 2 NNNN

? NNN ) ! R10 US 9, 938 ,323 B2 101 102 - continued each R1° is independently selected from H , C / -Cgalkyl , F , Cl, - NH2, OCHz, OCH CH3, N (CH3 ) 2 , CN , - NO2 and OH ; min each Rll is independently selected from H , C1- alkyl, 5 fluoro , benzyloxy substituted with CCOOH , ben zyl substituted with C ( O )OH , C1- 4alkoxy substi R12 tuted with CEO )OH and C1- alkyl substituted with nina C ( O )OH ; each R12 is independently selected from H , CH2 and 10 phenyl; each m is independently selected from 1, 2 , 3 , 4, 5 , 6 , 7 , 8 , 9 and 10 , and each n is independently selected from 1, 2 , 3 , 4 , 5 , 6 , 7 , R12 15 8 , 9 , 10 , 11 , 12 , 13 and 14 . min X In certain embodiments of this aspect of immunoconju gates having the structure of Formula ( II ) , are immunocon HON jugates having the structure of Formula ( IIa ) :

20 Formula ( lla ) HO noun R12

25 ?? . Box ** * 1111 HN man IZ 30

HOM /

' s - R20 Ab .

OS R12 ni pot 40 H2N In certain embodiments of such immunoconjugates of Formula ( II ) and Formula ( Ila ) : or Ab represents an antigen binding moiety ; y is an integer from 1 to 16 ; 45 RI2 R ' is Hor — CHz; R3 is - NH , or - OH ; R20 is -L , L2R40 , mi yet. -LzR6 or ( CH2) mXz ( CH2) mR40 ; L , is - (CH3 ) NRC ( = O ) — , OH O = XmynstA 50 minn each R? is independently selected from H and C -Coalkyl 11syl ; 55 mm min mm R60 is — NH — ;

R70 is w

OH O mi = 60 A or - (CH2 )mX3 ( CH2 ) m = ; L , is – (CH2 ) - , - NR $ (CH2 ) - , - (CH2 ) mNRC FO( ) (CH2 ) m , - X4, (CH2 ) mNR $CEO ) X4, R $ is H , C . - C alkyl orCEO) ORS ; 65 - ( CH2)mNR°C ) — , ( CH2) mO ) , (CH2 )m - , each R is independently selected from H , C , -Calkyl , F , (( CH2 ) mO ), (CH2 ) mX3 ( CH2 ) m , - NR * ( (CH2 ) mO )n Cl, and OH ; X3 (CH2 ) m - , or — X , X , C ( O )( CH2) m - , US 9 ,938 ,323 B2 103 104 wherein : 103 In certain embodiments of such immunoconjugates of Formula ( II ) and Formula ( lla ) : WhereinX is : Ab represents an antigen binding moiety ; y is an integer from 1 to 16 ; Rl is Hor - CHz; R3 is - NH , or - OH ; R20 is - L , L , R40 , -L3R® or (CH2 ) mX3( CH2 ) mR40 ; Lj is (CH2 ) mNRCO ) — , twork mm X , is win moto mun H2N20 ti HN . mm or - (CH2 ) mX3 ( CH2 ) m ; L2 is (CH2 ) - , - NR $ (CH2 ) m , (CH2 ) mNRC ma N FO ) ( CH2) m , — X4, (CH2 ) mNR CEO) X4 , NH - ( CH2)mNRC ( O ) - , (( CH2)mO ) , (CH2 ) m - , 25 ( CH ,) O) , ( CH2) X ( CH ) , , NR (( CH ,) , O) , X3( CH2 )m , NR (( CH2)mO ), (CH2 )mX3 ( CH2)m - , or — X X C, O ) (CH2 ) m - , X2 is wherein : 30 X is

min mm HA X2 is and X4 is H2NO tax HN . Lz is – (CH2 ) mNRC ( 0 ) R40 is mi min

Z X2 is

9 mm R is N1 each R is independently selected from H and C -Cgalkyl ; each m is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , mm 8 , 9 and 10 , and 65 each n is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9, 10 , 11 , 12 , 13 and 14 . US 9 ,938 ,323 B2 105 106 and X X4, is is 105 NH , CEO) - , - NHCO ) or

n tax s mmenfant Lz is (CH2 ) mNRC ( = 0 ) ; 10 R40 is much 15 HO R60 is NH ; each R is independently selected from H and C -Cgalkyl ; each m is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , Tetots20 8 , 9 and 10 , and mi each n is independently selected from 1 , 2 , 3 , 4 , 5 , 6 , 7 , NH 8 , 9 , 10 , 11 , 12 , 13 and 14 . min 25 In preferred embodiments of any of the aforementioned immunoconjugates of Formula ( II ) and Formula (IIa ): R20 is

m in ? min NH NH tingnastNH mm NH ????? nu NH mi NH IZ tin US 9 , 938 , 323 B2 107 108 - continued

mm NH mi HN XumatM Kant NH NH mi ??? mo mus win man mo O wi

im mm

m in ' mm min NH mi HN mina im mo . HN estmin timp

NH ma Myst time. HN US 9 ,938 ,323 B2 109 1100 - continued

UH ?????? NH ????????

nn? ????

??????? *???????? ???????? ???????? ??????? ??????? ???????? ????????

? ?

? ???????? Q ????? ??tifllii

nnnnnhw or

??? ??????? ???????? ???

In certain embodiments of any of the aforementioned as immunoconjugates of Formula ( II ) and Formula ( la ) : R20is

NH ?????? ?nnon NH ? 0 ? ???????? 8 US 9 ,938 , 323 B2 111 III -continued min . HN mi mo NH

XenotNH the sinn min NH mi min HN NH nan mu HN ma Xxxbutmi , HN mount NH mm mm mi . HN NH terestmi Karst mim *

threat the?? mm HN minn mo min ?? mm . HN men Xindy tematminn

HN NH

Xinyt tinyim US 9 ,938 ,323 B2 113 114114 mm - continued HN man NH mm wi mu w HN . HN mi min N

HN ?? min ZI OS mi

mu ? NH tamaxHN mu 1 IZ +wa test stramtmm . TIIVHN HN mu AH XIXmi ? ist min NH MamyO= min VH tingnyttN

NH we N

NH wa US 9 , 938 , 323 B2 115 116 115 - continued 116 min ?? www

> Or mm NH min

Z mm

NH2

In certain embodiments of any of the aforementioned immunoconjugates of Formula ( II ) and Formula ( Ila ) : R20 is

mm VH khiNH mi ?i mi mm NH XatNH mim NH

NH NH mm US 9 ,938 , 323 B2 117 118 117 -continued 118

mnm NH mi HN ??- N , HN . HN mi w min NH ??????mi ? NH XemO= AH mm NH minn an ?????NH minn min NH timmtw man NH mi

yor who HN

- N US 9 ,938 , 323 B2 119 120 - continued

NH2

In certain embodiments of any of the aforementioned R20 is immunoconjugates of Formula ( II ) and Formula ( IIa ):

mm . HN

w NH twittNH min mu ?

HN . HN min xung istni

NH maymin tumatN min

NH * ?? minn Xuand windmin US 9 , 938 , 323 B2 121121 122122 . - continued NH men NH w min

mm . HN ma win tant thingine mm HN mu mu mm man

Xem . HN in

mm NH n

tusentNH tenet NH kwenymi , mennyt minn mm NH Xumar mm NH mikuin muutma NH NHO . HN XuntHN muzyx US 9, 938 ,323 B2 123 124 -continued 124 mi mu HN HN mu IZ

NH xwestm mim NH NH trynymo they IZ yungmm HN O= xungettmm . HN mungest. HN ma HN

HN kuimmitm , ????? mi min NH min NN US 9 ,938 , 323 B2 125 126 -continued mo min or N XuyurtN = N .

NH2 mm NH

NH w FO

In certain embodiments of any of the aforementioned 30 R20 is aimmunoconjugates la croin co nditionsof Formula are ( II ) and Formulathe legendamined ( IIa ) : so

min HN mm NH kwambtFO navia wat mm , HN mon NH muzaftw knetNH mm . HN mm min

HN ON mo US 9 ,938 , 323 B2 127 128 -continued um 92 m Xumatmm . HN wi , XurstNH NH my NH

mom . HN

- > tingnyttOS minoxwengordt NH kummight. HN mm or HN

mon wa NN US 9 ,938 ,323 B2 129 130 In certain embodiments of any of the aforementionedU1598632 22 immunoconjugates of Formula ( II ) and Formula ( IIa ) : BeR20 isste boererater

w Sasakini telahmann mann mi EM mm wi

mum mi ist min

niin

NH wimu mu

mi ErstN wa timeNH US 9, 938 ,323 B2 131131 132 - continued

non

NH O NuAnne

NANA ~ ~ ~ ~

nnnnne ???

nnnnnnn

Mine US 9, 938 ,323 B2 133 134 - continued or

NH2

OE N

min

In certain embodiments of any of the aforementioned immunoconjugates of Formula ( II ) and Formula ( Ila ): 25 - continued R20 is wwwen forma conimel

30 win O = N 35 In certain embodiments of any of the aforementioned or immunoconjugates of Formula ( II ) and Formula ( Ila ) : imat R20 is

nim mu Engst metmin mm man ni mi ) ni wa US 9 , 938 , 323 B2 135 136 -continued

inn min

nnnnnn mu

win

min NH tubig

w Exosttw . mi US 9 , 938 , 323 B2 137 138 -continued

wa mine

mani im O= menna

mu mi mi OS nh min wi , fitostextes inn mm

mm m

O = Lucy Lut mann

un ni

o nin mi my mimo ww V = US 9 ,938 , 323 B2 139 140 139 - continued

m in ta mm ni Sunt LextotN ni Lyselmm

mu tami hetNH ni minst

Z- US 9 , 938 , 323 B2 141 142 -continued or mm

NH2

NH 111111

mo In certain embodiments of any of the aforementioned immunoconjugates of Formula ( II ) and Formula ( Ila ) : tacostR20 is ni maria -:+OM minn

mi wan

man

IZ ni US 9 ,938 ,323 B2 143 144 143 -continuedand Lugnytt min Lugosstni .or nenLumnytt In certain embodiments of any of the aforementioned immunoconjugates of Formula (II ) and Formula ( IIa ): R20 is mi ni minn

mu mi min wan min US 9, 938 ,323 B2 145 146 - continued NA WW NAN

0= VAN NH NNN

NH ?* Andonne AN Aw nudion 0 =

vvvvv

* HN +SSSSS NNNN SSSSS US 9 , 938 , 323 B2 147 148 - continued mi

min mm

or mu

IVNI

50 In certain embodiments of any of the aforementioned -continued immunoconjugates of Formula ( II ) and Formula ( Ila ) : R20 is Xocolata55 min min nim

O US 9 ,938 ,323 B2 149 150 In certain embodiments of any of the aforementioned immunoconjugates of Formula ( II ) and Formula ( IIa ) : R20 is

mun mm min minn 1xxma mm wan mas winan wa mm mm man mi NH

mm xmm( R ) min min O = IZ

m man ann min ??? HN US 9 , 938 , 323 B2 151 152 - continued mm Lunstmui mm

m FO tecom mm

towningmi

X? Mim

in W

Z - 0 -

Z US 9 , 938 , 323 B2 153 154 153 -continued or min

IZ 1111 NH

In certain embodiments of any of the aforementioned -continued immunoconjugates of Formula ( II ) and Formula ( Ila ) : R20 is 30 mm

mi 35 mm 40# In certain embodiments of any of the aforementioned Or immunoconjugates of Formula ( II) and Formula ( IIa ): R20 is

U11 !111111 mm

O =

m mm my100lt 1111111 mm mi mo US 9 ,938 , 323 B2 155 156 155 p?nuljuoy 1111111

ni ms 2017121111111

m ZI WM ZI

M 2103 mm yobxarxaxalxo? 0 HN wan Millis

II ??? Emmast US 9 , 938 , 323 B2 157 158 - continued in

**1 m mm

tu mi

11111 NH YocongtNH bo

JIMI ?

50 In certain embodiments of any of the aforementioned - continued immunoconjugates of Formula (II ) and Formula ( Ila ): R20 is Pinilil 55 i 1111111 min

65 or In certain embodiments of any of the aforementioned FO immunoconjugates of Formula ( II ) and Formula ( IIa ): R20 is US 9 ,938 ,323 B2 159 160

?? NH HNOHN mu HN * i HN x * HN x we NH mo HN teritmin tim IZ mm NH mi HN xatmi tyyma mm

. HN ma ZI Xinst tax IZ

m O HN Devedesetilet mm ma HN HN NS mm Ximentm ngumumanmi NEN US 9 , 938 , 323 B2 161 162 161 - continued ta Xylomenimistwin

mwin IZ

nim

14 mm mm

IZ mm ZI

ZI mi mm min in HN IZ m

w Rxxxx IZ US 9 ,938 , 323 B2 163 164 -continued Xylowanemagtigt or

In certain embodiments of any of the aforementioned - NH2, while in certain other embodiments of any of the immunoconjugates of Formula (II ) and Formula (Ila ), R ' is 20 aforementioned immunoconjugates of Formula ( II ) and For - CH3, while in certain other embodiments of any of the mula ( IIa ) R3 is OH . aforementioned immunoconjugates of Formula ( II) and For mula ( Ila ) Rl is H . In certain embodiments of any of the aforementioned In certain embodiments the immunoconjugates of the immunoconjugates of Formula ( II) and Formula ( IIa ) R * is invention are selected from :

HN 4111111

= IZ fo OH : NH2 Ab NH N

IZ o= =0 OH : IZ FO 0 NH2 E NH US 9 , 938 , 323 B2 165165 166 - continued HO HO

O=ts N HO |:

NH ,

O OVE HN = IZ

HO ONE E

NH HI i HO : N O= to HN

= HN

NH - H

o=ih IZ FO , ?? :HO NH IHN NH =O NH US 9 , 938 , 323 B2 167 168 -continued OHI OH FO HN

= OH ;

HO HO Os HN OSW null? HO O= ! NH2 AL =O NH

OH ??

, HO : 0 NH2NH , NH Ab o= US 9 , 938 ,323 B2 169 170170 - continued

H HN

O IZ

HN - NH2 O= NH Ab HO HNH

ul.

= HO 0 NH2 NH TZ Ab

MO HO 03 HN IZ

11111 V = HO |: O OH IZFO NH2 HN NH US 9 , 938, 323 B2 171171 172 - continued HO HO j HN HNH

OH IZ O= © NH2

=O NH ?? NH Ab HNH

HO OH

HN H

= HO C NH NH

HO HO ?HNH HN

11111.

HO V HO HN IZ 0 NH2

=O NH =

IZ US 9 , 938 , 323 B2 173 174174 - continued OH O E =O HN Ž H

= OH : IZ

O Ah NH

OH HO

HN H

= O H OH CO OE NH2

0= HN - H NH

HN . HII U

= À Ö NH2 - OH HN HN . HNH NH US 9 , 938 , 323 B2 175 176 - continued HO HO

HN H 111111 = IZ HO NH2

HN . HO NH NH

HN @ 111111 H = =0 ?? (R ) OH 1. OH TNH HN

HN . HN NH US 9 , 938 , 323 B2 177 178 - continued HO

HN ÖSEZE

S ) s( LO IZ ( R ) OH o= NH2

HN . HNH NH

HO H HN N

O=? NH O=

NH OF HNH NH

on HO

=ŽE HN

HO = H HN O FO | 20 HN OF HN HNH HN US 9 , 938 , 323 B2 179179 180 - continued OH HO

OS ZE HN

HON HO VEO HN NH2

HNH NHNH

OH OH

0 HN HNH

Z 0

O= Z NH2

A HN NH HNH NH US 9 , 938 , 323 B2 181181 US98 ,323 B2 182 181 - continued OH

0 HN ?

- OH Z HN HN

=O a -Z NH2

NH HN

HO HO

HN HNH

- O=1 N

HO Z OF HNH NH