MAIMULT A TINKTUUS010000511B2 U U UTORA (12 ) United States Patent ( 10 ) Patent No. : US 10 , 000 ,511 B2 Walensky et al. (45 ) Date of Patent: Jun . 19 , 2018

(54 ) SMALL MOLECULES FOR THE (2013 . 01 ) ; CO7D 249/ 12 (2013 . 01 ) ; CO7D MODULATION OF MCL - 1 AND METHODS 277/ 34 ( 2013 .01 ) ; C07D 279 /06 ( 2013 .01 ) ; OF MODULATING CELL DEATH , CELL C07D 307 /54 ( 2013 . 01 ) ; C07D 327/ 04 DIVISION , CELL DIFFERENTIATION AND (2013 .01 ) ; C07D 401/ 12 ( 2013 .01 ) ; C07D METHODS OF TREATING DISORDERS 403 /04 (2013 .01 ) ; C07D 403 / 06 (2013 . 01 ) ; CO7D 403 /08 ( 2013 .01 ) ; C07D 405/ 12 ( 71 ) Applicant: Dana - Farber Cancer Institute , Inc ., (2013 . 01 ) ; C07D 405 / 14 ( 2013 .01 ) ; C07D Boston , MA (US ) 409 / 06 ( 2013 .01 ) ; CO7D 409 / 12 ( 2013 .01 ) ; ( 72 ) Inventors : Loren D . Walensky , Newton Centre , C07D 409/ 14 ( 2013 .01 ) ; C07D 413 /06 MA (US ) ; Michelle L . Stewart , ( 2013 . 01 ) ; C07D 413 / 14 ( 2013 . 01 ) ; C07D 417 /04 ( 2013 .01 ) ; C07D 417 / 14 ( 2013 .01 ) ; Brookline , MA (US ) ; Nicole Cohen , C07C 2602/ 10 (2017 .05 ) Newton , MA (US ) (58 ) Field of Classification Search ( 73 ) Assignee : Dana -Farber Cancer Institute , Inc. , CPC .. CO7D 513 / 04 ; CO7D 249/ 12 ; C07D 401/ 12 ; Boston , MA (US ) CO7D 413 / 14 ( * ) Notice: Subject to any disclaimer , the term of this USPC ...... 514 /370 ; 544 / 54 patent is extended or adjusted under 35 See application file for complete search history . U .S .C . 154 (b ) by 0 days . days . (21 ) Appl. No. : 14 /705 , 764 (56 ) References Cited ( 22 ) Filed : May 6 , 2015 U . S . PATENT DOCUMENTS 2004 /0002117 AL 1 /2004 Hogan et al. (65 ) Prior Publication Data 2004 / 0157883 Al 8 / 2004 Chen et al. US 2015 /0315211 A1 Nov. 5 , 2015 2004 /0191328 Al 9 /2004 Warrell , Jr. et al. 2007 / 0203236 A1 8 / 2007 Smith et al . 2009 / 0054402 A1 2 / 2009 Wang et al. Related U . S. Application Data 2009/ 0069324 A1 3 / 2009 Reed et al . (62 ) Division of application No. 13 /576 , 116 , filed as 2009/ 0124616 A1 5 /2009 Song et al. application No. PCT/ US2011 /023220 on Jan . 31 , 2009 /0163545 A1 6 / 2009 Goldfarb 2010 /0004324 A11 /2010 Skaar et al . 2011, now abandoned . 2010 /0234456 AL 9 / 2010 Wendt et al. 2011/ 0077250 A1* 3 / 2011 Ryder ...... A61K 31/ 70 (60 ) Provisional application No . 61/ 299 , 803, filed on Jan . 514 /236 . 8 29, 2010 . 2012 /0142917 AL 6 /2012 Lawrence et al. (51 ) Int. Ci. FOREIGN PATENT DOCUMENTS C07D 513 /04 ( 2006 .01 ) C07D 249 / 12 ( 2006 .01 ) EP 1475372 11/ 2004 C07D 401/ 12 ( 2006 .01 ) 2004 /099518 A 4 /2004 2006516383 7 / 2006 C07D 413 / 14 ( 2006 . 01 ) JP 2007084494 4 / 2007 C07D 409 / 12 ( 2006 . 01 ) WO 2004 /069200 A2 8 / 2004 C07C 311 / 20 ( 2006 .01 ) (Continued ) C07D 409 / 14 ( 2006 . 01 ) CO7D 405 / 14 ( 2006 .01 ) C07D 417 / 14 ( 2006 . 01 ) OTHER PUBLICATIONS CO7D 307/ 54 ( 2006 . 01 ) ZINC 15913384 , PubChem SID 59797118 , PubChem CID : 6848561 CO7D 405 / 12 ( 2006 .01 ) (509102 -00 -5 ), Available Date: May 28 , 2009 . http :/ / pubchem .ncbi . C07D 409 / 06 ( 2006 . 01 ) nlm .nih .gov / substance /59797118 /version / 1. * C07D 413 /06 ( 2006 .01 ) (Continued ) CO7D 279 / 06 ( 2006 .01 ) C07D 327/ 04 ( 2006 .01 ) Primary Examiner — Yih -Horng Shiao CO7D 239 /60 ( 2006 .01 ) (74 ) Attorney , Agent , or Firm — Fish & Richardson P. C . C07D 403 / 06 ( 2006 . 01 ) CO7D 27734 (2006 .01 ) ( 57 ) ABSTRACT CO7D 417 /04 ( 2006 . 01 ) This invention relates to compounds which selectively bind C07C 337 / 08 ( 2006 . 01 ) to the survival protein MCL - 1 with high affinity and selec C07C 251/ 20 ( 2006 .01 ) tivity , pharmaceutical compositions containing such com C07C 251/ 84 ( 2006 . 01 ) pounds and the use of those compounds or compositions for C07C 335 / 16 (2006 .01 ) modulating MCL - 1 activity and for treating hyperprolifera C07D 403 / 04 ( 2006 .01 ) tive disorders , angiogenesis disorders , cell cycle regulation CO7D 403/ 08 (2006 . 01) disorders, autophagy regulation disorders , inflammatory dis (52 ) U . S . CI. orders, and / or infectious disorders and /or for enhancing CPC ...... C07D 513 / 04 ( 2013 .01 ) ; C07C 251/ 20 cellular engraftment and /or wound repair , as a sole agent or ( 2013 .01 ) ; C07C 251/ 84 ( 2013 .01 ) ; C07C in combination with other active ingredients . 311/ 20 ( 2013 .01 ); C07C 335 / 16 ( 2013 .01 ) ; C07C 337/ 08 (2013 . 01 ) ; C07D 239 /60 17 Claims, 19 Drawing Sheets US 10 ,000 , 511 B2 Page 2

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No . 13763682. 5 dated Mar. CAS Registry No . 478856 - 87 -0 ; STN Entry Date Jan . 13 , 2003 ; 14 , 2017 (7 pages ). 2 - ( cyclohexylimino ) - 4 -methyl - N ( phenylmethylene ) - 3 ( 2H ) JPO Official Action for JP App . No. 2016 - 097054 dated Apr. 13 , thiazolamine , 1 page . 2017 ( 10 pages) [With English Translation ]. CAS Registry No . 479363 -09 - 2 ; STN Entry Date Jan . 17 , 2003 ; U . S . Appl . No. 14 / 386 ,747 , filed Sep . 19 , 2014 , Walensky. 3 -[ [ [2 - ( cyclohexylimino ) -4 - (2 , 4 - dimethoxyphenyl) - 3 (2H ) thiazolyl] imino ]methyl ) - phenol, 1 page . * cited by examiner U . S . Patent Jun . 19 , 2018 Sheet 1 of 19 US 10 , 000, 511 B2

FIG . 1A W SAHB

MCL - 1 Small Molecules

Qyte Small Molecule Competition

FIG . 1B

Competitive MCL - 1 binding screen by FPA

Screen against FITC Screen against FITC SAHB selective for SAHB/ peptide MCL- 1 selective for BCL -XL WWW70 ,000 compounds 70 ,000wwwwwwww compounds MCL - 1 tightbinders BCL - X _ tight binders

Identify MCL - 1 Selective SmallMolecules 290 compounds (130 High Hits , 160 Medium Hits ) U . S . Patent Jun . 19, 2018 Sheet 2 of 19 US 10 ,000 ,511 B2

FIG . 2A

FIG . 2B

1 ) 1589P01 (Class G ) 2 ) 1597G03 (Class E ) 3 ) 1655B03 (Class E )

4 ) 1614B11 (Class E ) 5 ) 1654L06 (Unclass . ) 6 ) 1621122 (Class I) U . S . Patent Jun . 19, 2018 Sheet 3 of 19 US 10 ,000 ,511 B2 FIG . 2C

225 8 . V .. . OmO MCLWil -" 1? 2001 - D - MCL - 1 diluted - - 4 - x - - - A - MCL - 1 1570G15 . . D . MCL - 1 + 1570G15 diluted . WAKAMARI www Lounge condooooooooooodoodoodooooooooooo o dodhadhabaha Sedabadebodaodonccdcntotesde destes OL 10 102 106 No . 1 panna

225 - o - MCL - 1 200 - 0 - MCL - 1 diluted 175?? - - MCL -14 1725P16 du . . .. MCL - 1 1725P16 diluted Ver 100 Larsenal- 4 - & - * - * - * - A-- D v ????????????? Docomo papapapagos Podobdobodontotoooooooooooood 10 -9 10 % 10 % 10 (MCL - 1 ) , M

225 , o MCL - 1 200 wereQuersummer - 0 - MCL - 1 diluted - - MCL- ???1 i 1579E07 . 7V . - MCL - 1 * 1579E07 dilutedwa du wwwwwww were mage-- - - - D...... wanamume ------i------

WWWMWWW 01- 8 10 100 Fan VI ** 1 atent Jun . 19 , 2018 Sheet 4 of 19 US 10 ,000 ,511 B2

FIG . 2D

discrete binding site for MCL - 1 inhibition

:1:1 : 1 M canonical BH3 binding site engaged by MCL - 1 SAHB TINQESCIEPLAESITDVLVR al cys- fragment .MW Unmodified MCL- 1ANAC 2330 . 7 Da 1923A19 -modified MCL - 1ANAC 2777 . 4 Da U . S . Patent Jun . 19, 2018 Sheet 5 of 19 US 10 , 000 , 511 B2

FIG . 2E

------O MCL - 1 WT 100QQ - O - C286S begin -- - -- D %BIDBH3bound 6 ?6 ?? www bawawasawwwwwwwwwww dos (1929A19 ), M

100 % omeland ------MCL - 1 WT - O - C2865 o ?enasano maresmenhos %BIDBH3bound ha

ododonobooooooooooooo??oooooo???????????????????onsoo?????????????????? d OL. LOL GOL GOL )lOON9191 W atent Jun . 19 , 2018 Sheet 6 of 19 US 10 ,000 ,511 B2 FIG . 3A

Celastrol MCL - 1 I BCL - W ?????? 1 1 - BCL - XL %BidBH3bound memenetime etentretien start

2 4 Celastrol ( log nM ) U73122 100 - - MCLBCL - W 1 - * - BCL- XL %BidBH3bound

073122 (log nM ) Hexachlorophene 100 MCL - 1 - -+ - BCLBCL -- WXL %BIOBH3bound bodoxoxo2 4 dowosopposes5 Hexachlorophene ( log nM ) Gossypol - MCL- 1 100* * - # - BCL - W - # - BCL -XL %BidBH3bound

P r - - - 4 Gossypol (log nM ) atent Jun . 19 , 2018 Sheet 7 of 19 US 10 ,000 ,511 B2

FIG . 3A , cont.

Manoalide - MCL - 1 $ 100 * ** - - BCL - W wordtdoor BCL - XL de %BidBH3bound ????? sentiment

o w * * * * * * * * * * * * wwwwww 2 4 6 Manoalide ( log nM Woo) Bithionol

B MCL - 1 - + - BCL - W www BCL - XL %BidBH3bound KKKKKKKKKKKKKK B3

2 4 6 Bithionolw (log nM ) U , S , Patent Jul . 19, 2018 Sheet 8 0f19 Us 10, 000, 511 B2

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? ? \ \? :25>,UM?5< ???????????? ?????????? <5if'f ??????????? ??????????? ???????? ????????????????????????????????????????????? >25£IM ????????????????????? 792%{73 CompoundID '02/#3 [62}{€3 1923413 f920DF _5;$15

???????????????????????????????????????????????? Celestrol 7322 Gossypol Bithionol ????? FIG.38 Bioactive Hexachlorophene Manoalide U . S . Patent Jun . 19 , 2018 Sheet 9 of 19 US 10 , 000 , 511 B2

BCL-XL 1-MCL-BCL-XL .-MCL1 -ABCLW BCL-WA

smeta Compound[lognM) Compound?lognM) odbox 1 ithLITT Dodo Habits 104 hi LIL 1 *1007 W o Bound BH3 BID % Bound BH3 BID %

BCL-XL MCL-1 -BCLXL .-MCL1 -ABCLW -BCLW

Compound(lognM) ' Compound[lognM) 1 1673N01 1644L16 1994. ?55 11 ?? ? FIG.4A Bound BH3 BID % Bound BH3 BID % U . S . Patent Jun . 19 , 2018 Sheet 10 of 19 US 10 ,000 ,511 B2

-MCL1 ABCL-W GBCL-XL -MCL1 -BCLXL -BCLW

6 6

b haymom Compound[lognM) Compound(lognM)

WW 2345 - 2345 1756F05 1752L04 ? - ,IT - - io *HI s HAA Bound BH3 BID % Bound BH3 BID %

1-MCL- ABCL-W #BCL-XL .-MCL1 *BCL-XL-BCLW

6 6

dood la. 1 II 5 1 - Compound(lognM] - Compound[lognM) -- - -- 4 * - 1618K12 - ?345 1588L05 - IIL

*C FIG.4A,cont WA " 8 ? 2 Bound BH3 BID % Bound BH3 BID % U . S . Patent Jun . 19, 2018 Sheet 11 of 19 US 10 ,000 ,511 B2

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FIG . 4C

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FIG.5B U . S . Patent Jun . 19, 2018 Sheet 18 of 19 US 10 , 000 , 511 B2

FIG . 5C

Combination Index Values (CalcuSyn analysis ) Bioactive Compound ID EC50 EC75 ECgo SynergyTE VYCYEYOTFIELET Celastrol 1923A191 0. 366 1 0 .279 1 0 . 241 ? ???? ??????? ? Gossypol T1570G150 .507 1 0 .475 0 .561 WUXXXXXXXX MM Bithionol 1920103 0 .574 0 .574 1 0 . 661 ?????? ?? ?? Manoalide 1792M11 0 .635 0 .592 10. 576 ???? ?????? ???? U73122 1792NO341 + + + 12 + 0 .911 0 . 782 10. 686 THEbococoo ooooooooo FIG . 5D

Combination Index Values ( CalcuSyn analysis ) * * * * Compound* ** * * * * * * ** * * * ** * * * * * * * * * * * * * ID* * * * ** ** * * * * * * * * * * Class* * * MMXXXXXXXXXEC50 * * EC75* * * * * * * * * * * * * EC90* * ** * * * * * * * * * * * * * | * SynergyW W XX 1585105 A 0 . 850 0 .568 0 . 389 a moeten 1644L14WWWWWW WWWWWWW 1 . 110 10 .314 0WWWWWWWWWWWW .090 marathif new otoago to the 1134 1612H17 0 .687 0 .640 0 .682 ????? ????? 1789N21 0 .667 1 0 .574 0 .599 1784004www ???? 0 .341 0 .411 0 .504 wwwwwwwwwwwof hone ww ??????????? - SE 0 .332 1 0 .417 0 . 527 1586C04 KUXX 1754M20 J 0 . 395 1 0 .417 0 . 477 otho ba otet WWW WWW U . S . Patent Jun . 19, 2018 Sheet 19 of 19 US 10 , 000 , 511 B2

FIG . 6

mir PUZZZZZZZZARD Iz BAX + BID S(normalizedtoBID/BAX) o BAX + BID + MCL- 1 BAX + BID + MCL - 1 "{ e3000 optie 1923A19

20 40 60 80 Time (min )

* BAX BID (normalizedtoBID/BAX) O BAX BID + MOL - 1 %release BAX BID * MCL - 1 ANA Margarita + 1618N08 MARE verantw orth sens o ri* wwwwwwwwwwww WY 2020 40 60 80 Time (min ) US 10 , 000 ,511 B2 SMALL MOLECULES FOR THE anti -apoptotic pockets , others are more selective like the MODULATION OF MCL - 1 AND METHODS BAD BH3 that binds BCL - 2 , BCL - Xy , and BCL - w and the OF MODULATING CELL DEATH , CELL NOXA BH3 that targets MCL - 1 and BFL1 /A1 %. The differ DIVISION , CELL DIFFERENTIATION AND ential binding capacity of BH3 domains and their mimetics METHODS OF TREATING DISORDERS 5 is clinically relevant, as exemplified by the close relation ship between inhibitor binding spectrum and biological This application is a divisional and claims priority, of activity . For example , ABT- 737 , the prototype small mol co -pending U . S . application Ser. No. 13 /576 , 116 , filed Jul. ecule BH3 mimetic modeled after the BH3 domain of BAD , 30 , 2012 and having a 371 completion date of Oct . 22 , 2012 , was designed to specifically target BCL - 2 and BCL - XL " , which is a U . S . National Stage application , and claims 10 and induces apoptosis in select cancers that are driven by priority of International Application No. PCT /US2011 / these proteins14 - 16 . However , ABT- 737 fails to show efficacy 023220 , filed Jan . 31 , 2011 , which claims priority of U . S . Provisional Application Ser . No. 61/ 299 , 803 , filed Jan . 29 , against cancer cells that overexpress MCL - 1 , as this anti 2010 . The contents of all of the prior applications are apoptotic lies outside the molecule ' s binding spectrum 15- 17 . 15 In an effort to overcome the challenge of designing precision incorporated herein by reference in their entirety . small molecules to selectively target interaction surfaces that STATEMENT REGARDING FEDERALLY are comparatively large and more complex , we investigated whether Nature ' s selective BH3 domains could be used to SPONSORED RESEARCH OR DEVELOPMENT rapidly identify precise small molecule modulators . This invention was made with government supportort under 2020 The development of precise inhibitors for discrete anti grant number P01CA92625 awarded by The National Insti apoptotic BCL - 2 family proteins implicated in pathologic tutes of Health . The government has certain rights in the cell survival remains a formidable but pressing challenge . invention . Such compounds would provide finely - tuned molecular probes to study and treat human diseases driven by specific BACKGROUND OF THE INVENTION 25 anti - apoptotic blockades . For example , anti- apoptotic MCL - 1 has emerged as a major resistance factor in cancer. Beginning with the discovery ofof BCL - 2 atat the t14t14 : 1818 . MCL - 1 overexpression has been linked to the pathogen chromosomal breakpoint of follicular lymphomal- 3 , the esis of a variety of refractory cancers , including multiple anti - apoptotic members of the BCL - 2 family have emerged myeloma' , , acute myeloid leukemia ' ' , melanoma “ , and as key pathogenic proteins in human diseases characterized 30 poor prognosis breast cancer “ . MCL - 1 exerts its pro - sur by unchecked cellular survival, such as cancer and autoim - vival activity at the mitochondrial outermembrane where it munity . A series of anti -apoptotic proteins including BCL - 2 , neutralizes pro - apoptotic proteins such as NOXA , PUMA , BCL - X , , BCL - W , MCL - 1 , BFL1/ A1, and BCL - B promote BIM , and BAK . The critical role of MCL - 1 in selective cellular survival by trapping the critical apoptosis - inducing apoptotic resistance has been highlighted by the sensitizing BCL - 2 homology domain 3 (BH3 ) a -helix of pro - apoptotic 35 effects of small interfering RNAs that downregulate MCL - 1 BCL - 2 family members4 . Cancer cells exploit this physi - protein levels . ologic survival mechanism through anti- apoptotic protein Despite the formidable challenges associated with devel overexpression , establishing an apoptotic blockade that oping precise small molecule modulators of biomedically secures their immortality . To overcome this potentially fatal relevant protein targets , the identification of novel and resistance mechanism , a pharmacologic quest is underway 40 selective small molecules modulators of MCL - 1 is described to develop targeted therapies that bind and block BCL - 2 herein . family survival proteins . Anti- apoptotic proteins contain a hydrophobic binding BRIEF SUMMARY OF THE INVENTION pocket on their surface that engages BH3 a - helices4, 5 . Because Nature ' s solution to anti - apoptotic targeting 4545 One embodiment of this invention encompasses a com involves selective interactions between BH3 death domains pound of the formula : and anti -apoptotic pockets® , 7 , molecular mimicry of the BH3 a -helix has formed the basis for developing small molecule modulators of anti - apoptotic proteins8 -10 . Promising com pounds undergoing clinical evaluation , such as ABT- 26311 , 50 obatoclax ” , and AT- 10112 , each target three or more anti RAN apoptotic proteins. The development of more precise inhibi tors that target individual anti- apoptotic proteins remains a significant challenge due to the often subtle differences among BH3 -binding pockets . Reminiscent of the long - term 55 goals in kinase therapeutics , anti- apoptotic inhibitors with greater specificity would provide finely - tuned therapies to treat distinct diseases while potentially avoiding unwanted side -effects , such as those observed for ABT- 26311 and Wherein : AT- 10113 . In addition , such compounds would serve as 60 X is = O or - O - R ; invaluable research tools to dissect the differentialbiological R ! is substituted or unsubstituted aryl, substituted or functions of anti - apoptotic proteins . unsubstituted heteroaryl, substituted or unsubstituted The specificity of anti- apoptotic proteins for BH3 cycloalkyl, substituted or unsubstituted heterocy domains is conferred by the topography of the canonical cloalkyl, ?S - R24 , - S02 R2a ; binding groove and the distinctive amino acid composition 65 R2 is hydrogen , halogen , O - R24, NH - R2a , of the interacting BH3 helix . Whereas some BH3 domains , - S — CN , S R2a , - S CH _ R24 or - S02 – such as that of pro - apoptotic BIM , can tightly engage all R2a ; US 10 ,000 ,511 B2 R2a is C -C , alkyl, substituted or unsubstituted aryl, and R & a is substituted or unsubstituted aryl, substituted or substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl , substituted or unsubstituted unsubstituted cycloalkyl , or substituted or unsubsti tuted heterocycloalkyl; cycloalkyl , or substituted or unsubstituted heterocy and R ’ is hydrogen , C . -C5 alkyl substituted or unsubsti- 5 cloalkyl; or tuted aryl, substituted or unsubstituted heteroaryl, sub stituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; or

B) 10 R9 wherein Rº is hydrogen , C . - C . alkyl, C2- C6 alkeynyl, a C2- C6 alkynyl, - NH2, - NHR9a , 15 - NR94R ” , COH , COR ” , COOH , or OH?? N COOR , R - RS Rºa and Rºb are independently C . - C . alkyl , C2 -Co alkey RA nyl, C2- C6 alkynyl , substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or wherein : 20 unsubstituted cycloalkyl, or substituted or unsubsti R4 and R are independently hydrogen , substituted or tuted heterocycloalkyl unsubstituted aryl, substituted or unsubstituted het eroaryl, substituted or unsubstituted cycloalkyl, or sub W is stituted or unsubstituted heterocycloalkyl or are taken Rio together to form a 5 - 7 membered , optionally substi - 25 - COR10 tuted , heteroaryl or heterocycloalkyl, provided that no - CO - NH ( CH2) , R1 , more than one of R4 and R is hydrogen ; - CO - NH - CO - NH ( CH?) , R1 , And Rºis substituted or unsubstituted aryl, substituted or _ CO _ NH _ SO _ NH ( CH2 ), R1 , unsubstituted heteroaryl, substituted or unsubstituted SO (CH2 ) n R10 , cycloalkyl, or substituted or unsubstituted heterocy - 30 - SONH — (CH , ) - R10 , cloalkyl; or _ SO _ NH _ CO _ NH ( CH?) , R1 , SONH — SONH — (CH ), R10 , or the group C 35 NC = Hic N — R12 40 SR> 80 *

wherein * represents the point of attachment ; 45 R1° is Z or substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl , substituted or unsubsti Wherein R ’ is CH2- CO - R7a ; - CH2 - NH - 302 - R7a ; tuted cycloalkyl, or substituted or unsubstituted het and Ria is substituted or unsubstituted aryl, substituted or erocycloalkyl; unsubstituted heteroaryl , substituted or unsubstituted R12 is Z or hydrogen , C . - C . alkyl or — (CH2 ) n cycloalkyl , or substituted or unsubstituted heterocy - 30se R12a , cloalkyl ; or R124 is Z or substituted or unsubstituted aryl, substi tuted or unsubstituted heteroaryl, substituted or 1 unsubstituted cycloalkyl, or substituted or unsubsti tuted heterocycloalkyl; or 55

R8 NC 60 ?? R13 s s R 14 Wherein R8 is is COR8a ; - CO - NH - (CH ) n Rºa ; CÓ CH , Rºa , _ NH _ CO _ NH _ Rºa ; 65 wherein _ NH _ SO, NHR8a ; or – NH CO Rºa , R13 is C - C , alkyl, C2- Cg alkenyl, C2- C , alkynyl , or _ NH _ SO , Rºa C1 -C6 alkoxy, US 10 ,000 , 511 B2 and R14 is substituted or unsubstituted aryl, substituted or R20 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl , substituted or unsubstituted unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocy cycloalkyl, or substituted or unsubstituted heterocy cloalkyl, wherein R20 may be further substituted by Z ; cloalkyl wherein R14 may be further substituted by Z ; R21 represents = S , = NH , NHR21a , CHR21a , or = NR21a , or = CCO - R21a ; and G ) R21a is substituted or unsubstituted aryl, substituted or R 15 (2 unsubstituted heteroaryl, substituted or unsubstituted R150 cycloalkyl, or substituted or unsubstituted heterocy 10 cloalkyl, wherein R21a may be further substituted by Z ; or

S R16 R22 wherein R15 is hydrogen , hydroxyl, halogen , Co — ( CH2) n 15 R15a , CÓ NH ( CH?) , R154 , CO - NH CO _ NH ( CH? ), RS4, CÓ NH SO R24KANR24 NH ( CH3 ), R134 , NHCO ( CH2 ) , Red, NH CÓ NH ( CH ), R14, SO ( CH , ) , - 20 R15a , So NH ( CH?) , R15a, _ SO _ NH R22 is substituted or unsubstituted aryl, substituted or CÓ NH ( CH2 ), R15a, _ SO _ NH _ SO unsubstituted heteroaryl , substituted or unsubstituted NH — ( CH )n - Risa , or — NH — SO2 - R15a; alkyl cycloalkyl, or substituted or unsubstituted heterocy C1- C8 -R15a cloalkyl wherein R22 may be further substituted by Z ; R15a is substituted or unsubstituted aryl, substituted or 25 R23 is C , - C , alkyl, C2- C , alkenyl, C2 - C , alkynyl , or unsubstituted heteroaryl, substituted or unsubstituted C - C . alkoxy, substituted or unsubstituted aryl, substi cycloalkyl, or substituted or unsubstituted heterocy tuted or unsubstituted heteroaryl, substituted or unsub cloalkyl; stituted cycloalkyl, or substituted or unsubstituted het and R16 is hydrogen , halogen , hydroxyl or substituted or erocycloalkyl wherein R may be further substituted unsubstituted aryl , substituted or unsubstituted het- 30 by Z , and eroaryl, substituted or unsubstituted cycloalkyl, or sub R24 is C . - C , alkyl, C . - C , alkenyl, C . - C , alkynyl, or stituted or unsubstituted heterocycloalkyl; or C . - C . alkoxy, substituted or unsubstituted aryl, substi tuted or unsubstituted heteroaryl, substituted or unsub stituted cycloalkyl, or substituted or unsubstituted het 35 erocycloalkyl wherein R24 may be further substituted by Z ; or R17 VR18 x 1 (2 ) Z 40 wherein R25 X is O or S ; CNH R17 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocy cloalkyl, wherein R17 may be further substituted by Z ; wherein R18 is hydrogen , C . - C , alkyl , Cz- C , alkenyl , C2- Cz- 50so R25 is _ CO _ R25a , CÓ NH ( CH ) R25a , alkynyl, or C1- C6 alkoxy , wherein R1® may be further | CO CH , R25a , NH CO _ NH _ R25a . substituted by Z ; _ NH _ SO _ NH _ R25d, or – NH COR25a ; and Rl is hydrogen , C . -C , alkyl, C2- C , alkenyl, C2- C , - NH _ SO , R25a alkynyl, or C , - C alkoxy wherein Rl9 may be further R25a is substituted or unsubstituted aryl, substituted or substituted by Z ; or 55 unsubstituted heteroaryl , substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocy cloalkyl wherein R22 may be further substituted by Z , R20 or §

R21 wherein Y is N , NH , CH or CH2; N Y ? is O , S , or NH ; 65 CV NH R26 - - - - represents an optional double bond ; • Dina US 10 , 000 ,511 B2 wherein tables Table 1 - A through Table 1 - P ). These compoundsmay R26 is substituted or unsubstituted aryl, substituted or be useful in the methods and compositions described herein . unsubstituted heteroaryl, substituted or unsubstituted Another aspect of the invention encompasses a pharma cycloalkyl, or substituted or unsubstituted heterocy - ceutical composition comprising one or more compounds cloalkyl wherein R22 may be further substituted by Z ; 5 according to Formula A - M or physiologically acceptable or salts , solvates, hydrates , stereoisomers , or fusion com and wherein pounds thereof and a pharmaceutically acceptable diluent or in each of formulas A - L : carrier. In some embodiments , the pharmaceutical compo each occurrence of n is independently an integer from sition the invention encompasses a composition wherein the 0 - 4 ; 10 compound is present in a therapeutically effective amount . each occurrence of m is independently an integer from In still another embodiment, the pharmaceutical compo 0 - 3 ; sition the invention further encompasses at least one further each occurrence of p is independently an integer from active compound . In one embodiment, the further active 0 - 2 ; compound is a death receptor stimulant, an HDAC inhibitor, each occurrence of q is independently an integer from 15 a proteasome inhibitor, a BCL - 2 family inhibitor, a kinase 0 - 1 ; inhibitor, a mitotic inhibitor, a nucleoside analog , an anti each occurrence of Z , Z ' and Z " is independently cancer monoclonal antibody, a corticosteroid , a DNA -dam halogen , hydroxyl, C1- C , alkyl, C2- C , alkenyl, aging agent , an antimetabolite , or other cell death - activating C2- C , alkynyl, or C . - C alkoxy , substituted or stimulant. unsubstituted aryl, substituted or unsubstituted het - 20 Another aspect of the invention encompasses a packaged eroaryl, substituted or unsubstituted cycloalkyl , or pharmaceutical composition comprising a container, the substituted or unsubstituted heterocycloalkyl, pharmaceutical composition of the invention and instruc or M ) tions for using the pharmaceutical composition to treat a a compound having the formula disease or condition in a mammal . 25 Yet another aspect of the invention encompasses a method T ' + L — T . - T2 of modulating MCL - 1 in a cell comprising contacting a cell wherein : with one or more compounds of the invention . In one T represents a compound of the formula A - L ; embodiment , the activity modulated is apoptotic cell death . each T2 independently represents another compound In another embodiment, the activity modulated is autophagy . of the formula A - L which may be the same or 30 In another embodiment, the activity modulated is necrotic different from T ' ; cell death . In another embodiment, the activity modulated is each Q represents a direct bond , a C7 - C alkylene cell metabolism . In another embodiment, the activity modu linker, a C , - C , alkenylene linker, a C , - C , alky - lated is cell division . In another embodiment, the activity nylene linker , an amide linker , or a sulfonamide modulated is transcription in a cell . In another embodiment, linker formed by joining together a Z , Z ', or Z " 35 the activity modulated is RNA Processing in a cell . In moiety of Twith a Z , Z ' , or Z " moiety of each another embodiment, the activity modulated is cell differ subsequent T ? ; and entiation . In another embodiment, the activity modulated is u represents an integer from 0 - 5 transcription . In another embodiment, the activity modu or a pharmaceutically acceptable salt, solvate , lated is RNA processing . In another embodiment, the activ hydrate or stereoisomer thereof. 40 ity modulated is protein multimerization or dissociation . In some embodiments , the invention encompasses a com - Still another aspect of the invention encompasses a pound of having the formula M : method of treating a hyperproliferative disorder in a mam mal comprising administering to a mammal in need thereof, T ' + L — T? 70 - T2 a therapeutically effective amount of one or more com wherein : 45 pounds of the invention . In some embodiments , the hyper T represents a compound of the formula A - L ; proliferative disorder is cancer, including but not limited to , each T2 independently represents another compound of cancer of the breast , respiratory tract, brain , reproductive the formula A - L which may be the same or different organs , digestive tract , urinary tract, eye , liver, skin , head from T '; and neck , thyroid , parathyroid or a distant metastasis of a each Q represents a direct bond , a C -C , alkylene linker , 50 solid tumor, a lymphoma, sarcoma, melanoma or leukemia . a C2- C , alkenylene linker , or a C2- C , alkynylene linker Yet another aspect of the invention encompasses a method formed by joining together a Z , Z ', or Z " moiety of T of treating an angiogenesis disorder in a mammal compris with a Z , Z ', or Z " moiety of each subsequent T ? ; and ing administering to a mammal in need thereof, a therapeu u represents an integer from 0 -5 tically effective amount of one or more compounds of the or a pharmaceutically acceptable salt, solvate , hydrate or 55 invention . stereoisomer thereof. Still yet another aspect of the invention encompasses a In some embodiments , the invention encompasses a com method of treating an inflammatory disorder in a mammal pound having the formula M wherein u is O and Q is a direct comprising administering to a mammal in need thereof, a bond . therapeutically effective amount of one or more compounds In still other embodiments , the invention encompasses a 60 of the invention . compound having the formula M wherein u is O and Q is a Another aspect of the invention encompasses a method of C , - C , alkylene linker . treating an infectious disease in a mammal comprising In yet other embodiments , the invention encompasses a administering to a mammal in need thereof, a therapeutically compound having the formula M wherein u is 1 and each Q effective amount of one or more compounds of the inven is independently a direct bond or a C . - C , alkylene linker . 65 tion . Representative compounds and subclasses of compounds Still another aspect of the invention encompasses a of the invention are identified in Table 1 ( including Sub - method of treating a cell cycle regulation disorder in a US 10 ,000 ,511 B2 10 mammal comprising administering to a mammal in need FIG . 2C shows the results of a dilutional assay that thereof, a therapeutically effective amount of one or more revealed selective small molecule binders can be subclassi compounds of the invention . fied based on engaging MCL - 1 through noncovalent or Yet another aspect of the invention encompasses a method covalent interactions. FITC - BID BH3 binding to MCL - 1 is of treating an autophagy regulation disorder in a mammal 5 restored upon dilution in the top panel (molecule 1570G15 ) , comprising administering to a mammal in need thereof, a whereas competitive FITC - BID BH3 binding cannot be therapeutically effective amount of one or more compounds restored by dilution once MCL - 1 is exposed to small mol of the invention . Still yet another aspect of the invention encompasses a ecules 1725P16 and 1579E07 . These data suggest that method of treating an autoimmune disorder in a mammal 10 tion1570615 engages MCL - 1 through non -covalent interac comprising administering to a mammal in need thereof, a 10 tions , whereas 1725P16 and 1579E07 covalently modify therapeutically effective amount of one or more compounds MCL - 1, preventing re -engagement by FITC -BID BH3 even of the invention . after protein dilution . Another aspect of the invention encompasses a method of FIG . 2D shows that a subgroup of MCL - 1 inhibitor enhancing wound repair in a mammal comprising adminis - 15 molecules covalently reacted with C286 of MCL - 1 at a tering to a mammal in need thereof, a therapeutically effec novel interaction site . Proteomic analysis determined that a tive amount of one or more compounds of the invention . subset ofmolecules that covalently modify MCL - 1 react at Yet another aspect of the invention encompasses a method C286 of the identified tryptic fragment, revealing a discrete of enhancing cellular engraftment in a mammal comprising binding site for MCL - 1 that differs in location from the administering to a mammal in need thereof, a therapeutically 20 canonical BH3 binding site engaged by MC1- 1 SAHB . effective amount of one or more compounds of the inven - FIG . 2E demonstrates that C286S mutagenesis abrogates tion . binding of this subgroup of molecules , confirming engage Another aspect of the invention encompasses a method of ment of a novel interaction site for MCL - 1 inhibition . sensitizing a cell to treatment by a secondary active ingre - Molecules that covalently modify MCL - 1 at C286 dient comprising administering to a cell a sensitizing amount 25 ( 1929A19 ; 161N08 [Class A ]) effectively block FITC - BID of one or more compounds of the invention . In certain BH3 binding to MCL - 1. This small molecule activity is embodiments , the secondary active ingredient is a death specifically abrogated by C286S mutagenesis , implicating receptor stimulant, an HDAC inhibitor, a proteasome inhibi- interactions at a novel site in the modulation of binding tor, a BCL - 2 family inhibitor, a kinase inhibitor, a mitotic activity at the canonical BH3 binding pocket of MCL - 1 . inhibitor , a nucleoside analog , an anti -cancer monoclonal 30 FIG . 3A shows the degree of MCL -1 selectively as antibody , a corticosteroid , a DNA - damaging agent, an anti - determined by competitive fluorescence polarization bind metabolite , or other cell death -activating stimulant. ing assays (FPA ) in a secondary screen of small molecule It is noted that in this disclosure and particularly in the hits from a bioactive library against FITC -BID BH3/ anti claims and /or paragraphs, termssuch as “ comprises ” , “ com prised ” , “ comprising ” and the like can have the meaning 35 FIG . 3B demonstrates that small molecule bioactives attributed to it in U . S . Patent law ; e . g . , they can mean identified as MCL - 1 selective indeed exhibit preferential “ includes ” , “ included ” , “ including ” , and the like ; and that displacement of FITC - BID BH3 from MCL - 1ANAC com terms such as “ consisting essentially of” and “ consists pared to other anti -apoptotic proteins. The tabulated IC50 essentially of” have the meaning ascribed to them in U . S . values are shown. Patent law , e . g ., they allow for elements not explicitly 40 FIG . 4A shows the degree of MCL - 1 selectivity as deter recited , but exclude elements that are found in the prior art mined by competitive fluorescence polarization binding or that affect a basic or novel characteristic of the invention . assays ( FPA ) in a secondary screen of hits from small These and other embodiments are disclosed or are obvious molecule libraries against FITC -BID BH3 / anti -apoptotic from and encompassed by, the following Detailed Descrip protein interactions. Competitive FPA binding curves as tion . 45 shown . FIG . 4B shows tabulated IC50 values in uM , demonstrat BRIEF DESCRIPTION OF THE DRAWINGS ing that small molecules identified as MCL - 1 selective exhibit preferential displacement of FITC -BID BH3 from FIG . 1A shows the application of a selective stapled MCL - 1ANAC compared to other anti - apoptotic proteins. peptide in a competitive binding screen to identify selective 50 FIG . 4C shows structure -activity relationship (SAR ) small molecules against a protein target . analysis for select derivatives of Class A molecular inhibi FIG . 1B shows workflow for identifying small molecules tors of MCL - 1 , using the FITC -BID BH3/MCL - 1ANAC that selectively target MCL - 1 . competitive fluorescence polarization binding assay . FIG . 2A shows a surface view of the BH3 -binding pocket FIG . 4D shows structure - activity relationship (SAR ) of MCL - 1ANAC from the X -ray structure of MCL - 1 (PDB 55 analysis for select derivatives of Class J molecular inhibitors ID : 2PQK ), with discrete topographic regions of the binding of MCL - 1, using the FITC -BID BH3 /MCL - 1ANAC com surface labeled A - E . petitive fluorescence polarization binding assay . FIG . 2B shows examples of selective small molecule FIG . 5A shows that a SAHB peptide selective for MCL - 1 binders of MCL - 1ANAC engaging discrete topographic sensitizes TRAIL -induced and caspase - dependent cell death regions ( labeled A - E ) of the BH3 -binding pocket, as ren - 60 of Jurkat T -cell leukemia and OPM2 multiple myeloma dered by molecular docking . Since certain small molecules cells . MCL - 1 -targeting small molecule hits synergize with cover distinct regions of the pocket , synthetic combinations TRAIL to induce cell death of OPM2 cells. of molecules or fragments thereof can yield a larger mol FIG . 5B shows that small molecule bioactives that target ecule that selectively binds a greater surface area of the MCL - 1 also sensitize the capacity of TRAIL to inhibit BH3- binding pocket . The identified topographic coverage of 65 viability of OPM2 cells, as has been shown for selective select molecules include compounds 1 , ( A + B ) ; 2 , ( B + C ) ; 3 , targeting of MCL - 1 with MCL - 1 SAHB ( FIG . 5A ) and for ( C + D ) ; 4 , ( A + E ) ; 5 , (A + B + C ) ; 6 , (E + B + C ) . selective knock -down of MCL - 1 with siRNA . US 10 ,000 , 511 B2 12 FIG . 5C shows a combination treatment analysis using The term “ aryl” refers to aromatic radicals having in the CalcuSyn software revealing combination index values that range of 6 up to 14 carbon atoms such as phenyl, naphthyl , reflect synergy between treatment with TRAIL and the indanyl, and biphenyl. identified small molecule bioactive hits that target MCL - 1 . The term "heteroaryl ” refers to a stable 5 - to 13 -mem The tabulated combination index values document synergy 5 bered aromatic heterocycle having in the range of from 1 up of MCL - 1 -targeting molecules from bioactive library in to 4 heteroatoms from the group consisting of nitrogen , reducing OPM2 cell viability when combined with TRAIL . phosphorus, oxygen and sulfur, which ring or ring system FIG . 5D shows a combination treatment analysis using can be linked via a carbon atom or a nitrogen atom , if such CalcuSyn software revealing combination index values that an atom is present. For purposes of this invention , the reflect synergy between treatment with TRAIL and the 10 heteroaryl ring radical may be a monocyclic , bicyclic or identified small molecule hits that targetMCL - 1 . The tabu - tricyclic ring system . Examples of such heteroaryl radicals lated combination index values document synergy of MCL are : pyridyl, pyridyl N - oxide , pyrimidyl, pyridazinyl , 1 -targeting molecules from small molecule libraries in pyrazinyl, thienyl, furyl , pyrrolyl, pyrazolyl, imidazolyl , reducing OPM2 cell viability when combined with TRAIL . thiazolyl , oxazolyl or isoxazolyl, indolicenyl, indolyl, benzo (CI < 0 . 9 , synergy ; 0 . 9 < CI < 1 . 1 , additive effect; CI> 1. 1 , 15 [b ] thienyl, benzo [ b ] furyl, benzothiazolyl, benzothiadiaz antagonism ) . olyl , indazolyl, quinolyl, isoquinolyl, isoquinolyl, naphthy FIG . 6 shows that selective MCL - 1 inhibitor molecules ridinyl, quinazolinyl, oxadiazolyl, benzoxazolyl , tetrazoyl , block the anti -apoptotic activity of MCL - 1 in a BAX - triazolyl , thiadiazolyl, and benzimidazolyl. mediated liposomal release assay . MCL - 1 blockade of tBID - The term " heterocycloalkyl” refers to a stable 3 to 13 induced , BAX -mediated liposomal release is inhibited by 20 membered saturated or partially unsaturated heterocycle pretreatment of MCL - 1 with the selective small molecule having in the range from 1 up to 4 heteroatoms from the MCL - 1 inhibitors . group consisting of nitrogen , phosphorus , oxygen and sulfur, which ring or ring system can be linked via a carbon atom DETAILED DESCRIPTION OF THE or a nitrogen atom , if such an atom is present. For purposes INVENTION 25 of this invention , the heterocyclic ring radical may be a monocyclic , bicyclic or tricyclic ring system , which may Definitions include fused , bridged or spiro ring systems. Examples of such heterocyclyl radicals are : tetrahydropyranyl, aziridyl, In order that the invention may be more readily under - azepanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolinyl, piperidi stood , certain terms are first defined and collected here for 30 nyl, 1 , 2 dihydropyridinyl, 1 ,4 dihydropyridinyl, piperazinyl , convenience . Other definitions appear in context throughout morpholinyl, thiomorpholinyl, azepinyl, oxazolinyl, thiaz the application . olinyl and 1 , 4 diazepinyl. In this disclosure, " comprises, " " comprising, " " contain - The term “ alkylamino ” refers to an alkyl group as defined ing” and “ having ” and the like can have the meaning herein attached via amino linkage to the restof the molecule . ascribed to them in U . S . Patent law and can mean 35 The term alkylamino further includes dialkyl amino moieties " includes ,” “ including , ” and the like ; " consisting essentially in which two alkyl groups as define herein are attached via of" or " consists essentially ” likewise has the meaning amino linkage to the rest of the molecule . Representative ascribed in U . S . Patent law and the term is open - ended , examples of those groups are methylamino and dimethyl allowing for the presence of more than that which is recited amino . so long as basic or novel characteristics of that which is 40 Where the plural form of the word compounds , salts , recited is not changed by the presence of more than that polymorphs , hydrates , solvates and the like, is used herein , which is recited , but excludes prior art embodiments . this is taken to mean also a single compound , salt, poly The term “ halogen ” refers to radicals of fluorine , chlorine , morph , isomer , hydrate , solvate or the like . bromine and iodine . The compounds of this invention may contain one or The term “ alkyl” refers to a straight or branched hydro - 45 more asymmetric centers , depending upon the location and carbon chain radical, containing solely carbon and hydrogen nature of the various substituents desired . Asymmetric car atoms, having in the range from one up to eight carbon bon atoms may be present in the ( R ) or ( S ) configuration , atoms, and which is attached to the rest of the molecule by resulting in racemic mixtures in the case of a single asym a single bond , such as illustratively , methyl, ethyl, n -propyl metric center, and diastereomeric mixtures in the case of 1 -methylethyl (iso - propyl) , n -butyl , n - pentyl , and 1 , 1 - dim - 50 multiple asymmetric centers . In certain instances , asymme ethylethyl ( tert -butyl ) . try may also be present due to restricted rotation about a The term “ cycloalkyl” denotes a non - aromatic mono or g iven bond, for example , the central bond adjoining two multicyclic ring system having in the range of 3 up to 14 substituted aromatic rings of the specified compounds. Sub carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, stituents on a ring may also be present in either cis or trans and cyclohexyl. Examples of multicyclic cycloalkyl groups 55 form . It is intended that all such configurations ( including include decahydronapththyl. Examples of bridged enantiomers and diastereomers ) , are included within the cycloalkyl groups or sprirobicycloalkyl groups include ada scope of the present invention . Preferred compounds are mantyl norbornyl, and sprio [ 4 .4 ]nonyl groups . those which produce the more desirable biological activity . The term “ alkoxy ” denotes an alkyl group as defined Separated, pure or partially purified isomers and stereoiso herein attached via an oxygen linkage to the rest of the 60 mers or racemic or diastereomeric mixtures of the com molecule . Representative examples of those groups are pounds of this invention are also included within the scope methoxy , ethoxy , iso - propoxy, n -butoxy , and tert- butoxy . of the present invention . The purification and the separation The term “ cycloalkoxy ” denotes a cycloalkyl group as of such materials can be accomplished by standard tech defined herein attached via an oxygen linkage to the rest of niques known in the art . the molecule . Representative examples of those groups are 65 The optical isomers can be obtained by resolution of the cyclopropoxy, cyclobutoxy , cyclopentoxy, cyclohexoxy, and racemic mixtures according to conventional processes , for cycloheptoxy. example , by the formation of diastereoisomeric salts using US 10 ,000 , 511 B2 13 14 an optically active acid or base or formation of covalent methyl, ethyl , propyl, and butyl chlorides, bromides and diastereomers . Examples of appropriate acids are tartaric , iodides ; dialkyl sulfates like dimethyl, diethyl, and dibutyl diacetyltartaric , ditoluoyltartaric and camphorsulfonic acid sulfate ; and diamyl sulfates , long chain halides such as Mixtures of diastereoisomers can be separated into their decyl, lauryl, myristyl and stearyl chlorides, bromides and individual diastereomers on the basis of their physical and/ or 5 iodides , aralkyl halides like benzyl and phenethyl bromides chemical differences by methods known in the art, for and others . example , by chromatography or fractional crystallization . A solvate for the purpose of this invention is a complex of The optically active bases or acids are then liberated from a solvent and a compound of the invention in the solid state . the separated diastereomeric salts . A different process for Exemplary solvates would include , but are not limited to , separation of optical isomers involves the use of chiral 10 complexes of a compound of the invention with ethanol or chromatography ( e . g ., chiral HPLC columns ), with or with methanol. Hydrates are a specific form of solvate wherein out conventional derivitization , optimally chosen to maxi- the solvent is water. mize the separation of the enantiomers . Suitable chiral The term “ subject” refers to animals such as mammals , HPLC columns are manufactured by Diacel, e . g . , Chiracel including , but not limited to , primates ( e . g . , humans) , cows, OD and Chiracel OJ among many others , all routinely 15 sheep , goats , horses, dogs , cats , rabbits , rats, mice and the selectable . Enzymatic separations , with or without deriviti - like . In certain embodiments, the subject is a human . zation , are also useful. The optically active compounds of It is also to be understood that the terminology used herein this invention can likewise be obtained by chiral syntheses is for purposes of describing particular embodiments only , utilizing optically active starting materials . and is not intended to be limiting . As used in the specifica The present invention also relates to useful forms of the 20 tion and the appended claims, the singular forms “ a ” , “ an ” , compounds as disclosed herein , such as pharmaceutically and “ the ” include plural referents unless the context clearly acceptable salts , co - precipitates, metabolites, hydrates , sol- indicates otherwise . Thus, for example , reference to " a vates and prodrugs of all the compounds of examples . The peptide” includes multiple peptides, reference to “ a spacer " term “ pharmaceutically acceptable salt ” refers to a relatively includes two or more spacers . non - toxic , inorganic or organic acid addition salt of a 25 Unless otherwise defined , all technical and scientific compound of the present invention . For example , see S . M . terms used herein have the same meaning as commonly Berge , et al. “ Pharmaceutical Salts ,” J. Pharm . Sci. 1977 , 66 , understood by one of ordinary skill in the art to which this 1 - 19 . Pharmaceutically acceptable salts include those invention belongs. In case of conflict, the present applica obtained by reacting the main compound , functioning as a tion , including definitions will control. All publications , base , with an inorganic or organic acid to form a salt, for 30 patent applications, patents , and other references mentioned example , salts of hydrochloric acid , sulfuric acid , phos - herein are incorporated by reference . phoric acid , methane sulfonic acid , camphor sulfonic acid , Compounds oxalic acid , maleic acid , succinic acid and citric acid . As set forth above , the invention provides compounds Pharmaceutically acceptable salts also include those in which selectively bind to the survival protein MCL - 1 with which a compound of the invention functions as an acid and 35 high affinity and selectivity . is reacted with an appropriate base to form , e . g . , sodium , Table 1 ( including Subtables Table 1 - A through Table potassium , calcium , magnesium , ammonium , and chorine 1 - P ) lists of representative compounds and subclasses iden salts . Those skilled in the art will further recognize that acid tified as selective or preferential MCL - 1 binders based upon addition salts of the claimed compounds may be prepared by a small molecule competitive binding assay using FITC reaction of a compound of the invention the appropriate 40 MCL - 1 SAHB /MCL - 1ANAC and FITC -BAD BH3/ BCL inorganic or organic acid via any of a number of known X , AC interaction pairs . FIGS . 4C and 4D tabulates select methods. Alternatively , alkali and alkaline earth metal salts derivatives of Class A and Class J compounds that exhibit of acidic compounds of the invention are prepared by differential MCL - 1 targeting activity based on chemical reacting the compounds of the invention with the appropri - variation of discrete R groups. ate base via a variety of known methods. 45 Certain compounds of Table 1 describe certain known Representative salts of a compound of the invention bioactive compounds. These compounds are particularly include the conventional non - toxic salts and the quaternary useful in targeting MCL - 1 and may be useful in the treat ammonium salts which are formed , for example , from ment of diseases as described herein . Thus , in certain inorganic or organic acids or bases by means well known in aspects , the compounds of Formula A - L may exclude certain the art . For example , such acid addition salts include acetate , 50 compounds of Table 1 . adipate , alginate , ascorbate , aspartate , benzoate , benzene The compounds of the invention may contain one or more sulfonate , bisulfate , butyrate , citrate , camphorate , camphor - asymmetric centers and thus occur as racemates and racemic sulfonate , cinnamate , cyclopentanepropionate, digluconate , mixtures , single enantiomers, individual diastereomers and dodecylsulfate , ethanesulfonate, fumarate , glucoheptanoate , diastereomeric mixtures. All such isomeric forms of these glycerophosphate , hemisulfate , heptanoate , hexanoate , 55 compounds are expressly included in the invention . The hydrochloride , hydrobromide , hydroiodide , 2 -hydroxy compounds of this invention may also be represented in ethanesulfonate , itaconate , lactate , maleate , mandelate , multiple tautomeric forms, in such instances , the invention methanesulfonate , 2 -naphthalenesulfonate , nicotinate , expressly includes all tautomeric forms of the compounds nitrate , oxalate , pamoate , pectinate , persulfate , 3 - phenylpro - described herein . All such isomeric forms of such com pionate , picrate , pivalate , propionate , succinate , sulfonate , 60 pounds are expressly included in the invention . All crystal tartrate , thiocyanate , tosylate , and undecanoate . forms of the compounds described herein are expressly Base salts include alkali metal salts such as potassium and included in the invention . sodium salts , alkaline earth metal salts such as calcium and Naturally occurring or synthetic isomers can be separated magnesium salts , and ammonium salts with organic bases in several ways known in the art . Methods for separating a such as dicyclohexylamine and N -methyl - D - glucamine. 65 racemic mixture of two enantiomers include chromatogra Additionally, basic nitrogen containing groups may be quat - phy using a chiral stationary phase ( see , e . g ., " Chiral Liquid ernized with such agents as lower alkyl halides such as Chromatography, " W . J . Lough , Ed . Chapman and Hall , US 10 , 000 ,511 B2 15 16 New York ( 1989 )) . Enantiomers can also be separated by MCL - 1 not associated with the established BH3 binding site classical resolution techniques . For example , formation of ofMCL - 1 . In still other embodiments , the modulation may diastereomeric salts and fractional crystallization can be be the result of a covalent bond between the compound with used to separate enantiomers . For the separation of the C286 reside of MCL - 1 . In certain embodiments , the enantiomers of carboxylic acids , the diastereomeric salts can 5 modulation may be the result of a non - covalent bond be formed by addition of enantiomerically pure chiral bases between the compound in the region of the C286 residue of such as brucine , quinine, ephedrine , strychnine, and the like. MCL - 1. Alternatively , diastereomeric esters can be formed with In one aspect , the invention encompasses a method of enantiomerically pure chiral alcohols such as menthol, fol- treating a hyperproliferative disorder in a mammal compris lowed by separation of the diastereomeric esters and hydro - 10 ing administering to a mammal in need thereof, a therapeu lysis to yield the free , enantiomerically enriched carboxylic tically effective amount of one or more compounds of the acid . For separation of the optical isomers of amino com invention . pounds, addition of chiral carboxylic or sulfonic acids, such As used herein the term “ hyperproliferative disorder ” as camphorsulfonic acid , tartaric acid , mandelic acid , or refers to a disorder associated with an irregular or an lactic acid can result in formation of the diastereomeric salts . 15 abnormally high rate of cell division (which results in a rapid Methods of obtaining a compound of the invention proliferation of the cells ) or a blockade in the natural cell include purchasing , synthesizing or otherwise acquiring the death pathway resulting in the accumulation of cells , or a compound . Synthesizing a compound of the invention is combination thereof . Hyperproliferative disorders include within the means of chemists of ordinary skill in the art. but are not limited , e . g . , psoriasis , keloids, and other hyper Methods for optimizing reaction conditions, if necessary 20 plasias affecting the skin , benign prostate hyperplasia minimizing competing by - products , are known in the art . (BPH ) , solid and liquid tumors , such as cancers of the breast , The methods may also additionally include steps, either respiratory tract, brain , reproductive organs , digestive tract, before or after the steps described specifically herein , to add urinary tract, eye , liver, skin , head and neck , thyroid , para or remove suitable protecting groups in order to ultimately thyroid and their distantmetastases , lymphomas, sarcomas, allow synthesis of the compounds herein . In addition , vari- 25 and leukemias. ous synthetic steps may be performed in an alternate Still another aspect of the invention encompasses a sequence or order to give the desired compounds. Synthetic method of treating a cell cycle regulation disorder in a chemistry transformations and protecting group methodolo - mammal comprising administering to a mammal in need gies ( protection and deprotection ) useful in synthesizing the thereof , a therapeutically effective amount of one or more applicable compounds are known in the art and include , for 30 compounds of the invention . example , those described in R . Larock , Comprehensive As used herein , the term " cell cycle regulation disorder” Organic Transformations, VCH Publishers ( 1989) ; T . W . relates to a disorder of the cell cycle regulation mechanisms, Greene and P . G . M . Wuts , Protective Groups in Organic observed in many malignant tumors, are directly related to Synthesis , 3rd Ed ., John Wiley and Sons (1999 ) ; L . Fieser uncontrolled proliferation of cancer cells . Cell cycle regu and M . Fieser , Fieser and Fieser ' s Reagents for Organic 35 lation disorders can be the result of the degradation of the Synthesis , John Wiley and Sons ( 1994 ); and L . Paquette , ed. , regulation of one or more stages of cell growth , G1, S , G2, Encyclopedia of Reagents for Organic Synthesis , John Wiley and M phases of the growth cycle and may include the and Sons (1995 ) and subsequent editions thereof. degradation of one or more cell cycle regulators including , Methods of Treatment but not limited to , p27Kip1, cdk2 , cdk4 , cdk6 , chki, cdc25 , In one aspect, the invention encompasses a method of 40 cyclin B , cyclin H , cyclin D , and cyclin E . modulating MCL - 1 in a cell comprising contacting a cell Examples of breast cancer include , but are not limited to with one or more compounds of the invention . Such modu - invasive ductal carcinoma, invasive lobular carcinoma, duc lation is useful in the modulation of programmed cell death tal carcinoma in situ , and lobular carcinoma in situ . or apoptosis , autophagic cell death ; necrotic cell death ; cell Examples of cancers of the respiratory tract include , but metabolism ; cell division ; cell differentiation ; cell migra - 45 are not limited to small - cell and non - small - cell lung carci tion ; cell engraftment ; tissue repair ; RNA transcription and noma, as well as bronchial adenoma and pleuropulmonary processing ; or protein multimerization or dissociation . blastoma . As used herein , the term “ modulation ” refers to a change Examples of brain cancers include, but are not limited to in activity as a direct or indirect response to the presence of brain stem and hypophtalmic glioma, cerebellar and cerebral at least one compound of the invention described herein , 50 astrocytoma , medulloblastoma , ependymoma, as well as relative to the activity in the absence of the compound . The neuroectodermal and pineal tumor. change may be an increase in activity or a decrease in Tumors of the male reproductive organs include, but are activity , and may be due to the direct interaction of the not limited to prostate and testicular cancer. Tumors of the compound with MCL - 1 , or due to the interaction of the female reproductive organs include , but are not limited to compound with one ormore other factors that in turn interact 55 endometrial, cervical, ovarian , vaginal, and vulvar cancer , as with MCL - 1 or affect MCL - 1 activity . In certain embodi well as sarcoma of the uterus. ments , the modulation may be the result of a covalent bond Tumors of the digestive tract include , but are not limited between the compound with MCL - 1 . In certain embodi - to anal, colon , colorectal, esophageal, gallbladder , gastric , ments , the modulation may be the result of a non - covalent pancreatic , rectal , small - intestine, and salivary gland can bond between the compound with MCL - 1 . In certain 60 ?ers . embodiments , themodulation may be the result of a covalent Tumors of the urinary tract include , but are not limited to bond between the compound with the established BH3 bladder, penile , kidney , renal pelvis , ureter, urethral and binding site of MCL - 1 or with a residue of MCL - 1 not human papillary renal cancers. associated with the established BH3 binding site of MCL - 1. Eye cancers include , but are not limited to intraocular In certain embodiments, the modulation may be the result of 65 melanoma and retinoblastoma. a non - covalent bond between the compound with the estab - Examples of liver cancers include , but are not limited to lished BH3 binding site of MCL - 1 or with a residue of hepatocellular carcinoma ( liver cell carcinomas with or US 10 , 000 ,511 B2 17 18 without fibrolamellar variant) , cholangiocarcinoma ( intrahe pneumoniae Infection , Cholera , Chronic Fatigue Syndrome, patic bile duct carcinoma ), and mixed hepatocellular cho CID (Creutzfeldt - Jakob Disease ) , Clonorchiasis (Clonorchis langiocarcinoma. Infection ), CLM (Cutaneous Larva Migrans , Hookworm Skin cancers include, but are not limited to squamous cell Infection ) , Coccidioidomycosis , Conjunctivitis , Coxsacki carcinoma , Kaposi ' s sarcoma, malignant melanoma, Merkel 5 evirus A16 (Hand , Foot and Mouth Disease ), Cryptococco cell skin cancer, and non -melanoma skin cancer. sis , Cryptosporidium Infection (Cryptosporidiosis ) , Culex Head -and -neck cancers include, but are not limited to mosquito (Vector of West Nile Virus ) , Cutaneous Larva laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal Migrans ( CLM ) , Cyclosporiasis (Cyclospora Infection ) , cancer , lip and oral cavity cancer and squamous cell . Lym - Cysticercosis (Neurocysticercosis ), Cytomegalovirus Infec phomas include, but are not limited to AIDS -related lym - 10 tion , Dengue /Dengue Fever, Dipylidium Infection (Dog and phoma , non -Hodgkin 's lymphoma, cutaneous T -cell lym - Cat Flea Tapeworm ) , Ebola Virus Hemorrhagic Fever , Echi phoma, Burkitt lymphoma, Hodgkin ' s disease , and nococcosis ( Alveolar Hydatid Disease ) , Encephalitis , Enta lymphoma of the central nervous system . moeba coli Infection , Entamoeba dispar Infection , Entam Sarcomas include , but are not limited to sarcoma of the ceba hartmanni Infection , Entamoeba histolytica Infection soft tissue , osteosarcoma, malignant fibrous histiocytoma, 15 (Amebiasis ) , Entamoeba polecki Infection , Enterobiasis lymphosarcoma, and rhabdomyosarcoma . (Pinworm Infection ), Enterovirus Infection (Non - Polio ), Leukemias include, but are not limited to acute myeloid Epstein -Barr Virus Infection , Escherichia coli Infection , leukemia , acute lymphoblastic leukemia , chronic lympho Foodborne Infection , Foot and mouth Disease , Fungal Der cytic leukemia , chronic myelogenous leukemia , and hairy matitis, Gastroenteritis, Group A streptococcal Disease , cell leukemia . 20 Group B streptococcal Disease, Hansen ' s Disease (Lep In one aspect , the invention encompasses a method of rosy ), Hantavirus Pulmonary Syndrome, Head Lice Infes treating angiogenesis in a mammal comprising administer - tation (Pediculosis ), Heliobacter pylori Infection , Hemato ing to a mammal in need thereof, a therapeutically effective logic Disease, Hendra Virus Infection , Hepatitis (HCV , amount of one or more compounds of the invention . HBV ) , Herpes Zoster ( Shingles ) , HIV Infection , Human As used herein the term “ angiogenesis ” refers to a disor- 25 Ehrlichiosis , Human Parainfluenza Virus Infection , Influ der associated with excessive and / or abnormal angiogenesis . enza , Isosporiasis ( Isospora Infection ), Lassa Fever, Leish Inappropriate and ectopic expression of angiogenesis can maniasis , Kala -azar (Kala -azar , Leishmania Infection ), Lice be deleterious to an organism . A number of pathological (Body lice , Head lice , Pubic lice ), Lyme Disease, Malaria , conditions are associated with the growth of extraneous Marburg Hemorrhagic Fever , Measles , Meningitis , Mos blood vessels . These include , e . g . , diabetic retinopathy , 30 quito - borne Diseases , Mycobacterium avium Complex ischemic retinal vein occlusion , and retinopathy of prema MAC( ) Infection , Naegleria Infection , Nosocomial Infec turity ( Aiello et al . New Engl. J . Med . 1994 , 331, 1480 ; Peer tions, Nonpathogenic Intestinal Amebae Infection , et al. Lab . Invest. 1995 , 72 , 638 ) , age - related macular Onchocerciasis (River Blindness) , Opisthorciasis (Opisthor degeneration (AMD ; see , Lopez et al. Invest . Opththalmol. cis Infection ) , Parvovirus Infection , Plague, PCP (Pneumo Vis . Sci. 1996 , 37 , 855 ) , neovascular glaucoma, psoriasis , 35 cystis carinii Pneumonia ) , Polio , Q Fever, Rabies, Respira retrolental fibroplasias , angiofibroma, inflammation , rheu - tory Syncytial Virus (RSV ) Infection , Rheumatic Fever, Rift matoid arthritis (RA ), restenosis , in - stent restenosis , vascu - Valley Fever, River Blindness (Onchocerciasis ), Rotavirus lar graft restenosis , etc . In addition , the increased blood Infection , Roundworms Infection , Salmonellosis , Salmo supply associated with cancerous and neoplastic tissue , nella Enteritidis , Scabies , Shigellosis , Shingles, Sleeping encourages growth , leading to rapid tumor enlargement and 40 Sickness, Smallpox , Streptococcal Infection , Tapeworm metastasis . Moreover, the growth of new blood and lymph Infection ( Taenia Infection ) , Tetanus , Toxic Shock Syn vessels in a tumor provides an escape route for renegade drome, Tuberculosis , Ulcers (Peptic Ulcer Disease ) , Valley cells , encouragingmetastasis and the consequence spread of Fever , Vibrio parahaemolyticus Infection , Vibrio vulnificus the cancer. Thus , compounds of the present invention can be Infection , Viral Hemorrhagic Fever , Warts , Waterborne utilized to treat and /or prevent any of the aforementioned 45 infectious Diseases, West Nile Virus Infection (West Nile angiogenesis disorders , e .g . , by inhibiting and /or reducing Encephalitis ), Whooping Cough , Yellow Fever , tuberculo blood vessel formation ; by inhibiting, blocking , reducing , sis , leprosy , mycobacteria - induced meningitis , Chagas dis decreasing, etc . endothelial cell proliferation or other types ease , effects of Shiga - like toxin resulting from Staphylococ involved in angiogenesis , as well as causing cell death or c us infection , meningococcal infection , and infections from apoptosis of such cell types. 50 Borrelia burgdorferi or Treponema pallidum . Another aspect of the invention encompasses a method of Yet another aspect of the invention encompasses a method treating an infectious disease by blocking pathogen or of treating an autophagy regulation disorder in a mammal infected cell survival in a mammal comprising administering comprising administering to a mammal in need thereof, a to a mammal in need thereof, a therapeutically effective therapeutically effective amount of one or more compounds amount of one or more compounds of the invention . 55 of the invention . Examples of infectious diseases include , but are not As used herein , the term “ autophagy regulation disorder” limited to , AIDS , Alveolar Hydatid Disease ( AHD , Echi- relates to a dissorder of the autophagy system of a cell i . e . nococcosis ) , Amebiasis ( Entamoeba histolytica Infection ), the self -digestion by a cell through the action of enzymes Angiostrongylus Infection , Anisakiasis , Anthrax , Babesiosis originating within the same cell including , but not limited to , (Babesia Infection ), Balantidium Infection ( Balantidiasis ), 60 chaperone -mediated autophagy, macroautophagy, microau Baylisascaris Infection (Raccoon Roundworm ) , Bilharzia tophagy . Disorders of autophagy regulation include but are ( Schistosomiasis ) , Blastocystis hominis Infection ( Blasto - not limited to , Danon disease X - linked myopathy with mycosis ) , Boreliosis , Botulism , Brainerd Diarrhea, Brucel excessive autophagy, Infantile autophagic vacuolar myopa losis , BSE ( Bovine Spongiform Encephalopathy ), Candidi- thy, Childhood autophagic vacuolar myopathy, Adult -onset asis , Capillariasis (Capillaria Infection ) , CFS ( Chronic 65 autophagic vacuolar myopathy with multiorgan involve Fatigue Syndrome ) , Chagas Disease (American Trypanoso - ment, LGMD 1A , Toxic myopathy , Inflammatory myopa miasis ), Chickenpox (Varicella - Zoster virus) , Chlamydia thies, Hypokalemic myopathy , Acid maltase deficiency , US 10 ,000 , 511 B2 19 20 Inflammatory bowel disease associations, Autophagy langitis ; serum sickness ; Sezary syndrome; Sjogren ' s syn 16 - like 1 (ATG16L1 ), LRG47 , and autophagy -mediated drome; Stevens- Johnson syndrome; Still ' s disease ; subacute survival of cancer cells or cancer stem cells to escape sclerosing panencephalitis ; sympathetic ophthalmia ; sys chemotherapy or radiation treatments . temic lupus erythematosus ; transplant rejection ; ulcerative Still yet another aspect of the invention provides a method 5 colitis; undifferentiated connective tissue disease ; urticaria , of treating an autoimmune disorder in a mammal comprising chronic ; urticaria , cold ; uveitis ; vitiligo ; Weber- Christian administering to a mammal in need thereof, a therapeutically disease ; Wegener 's granulomatosis ; and Wiskott - Aldrich effective amount of one or more compounds of the inven syndrome. tion . In another aspect , the invention provides a method of Examples of autoimmune disorders include, but are not 10 enhancing cellular engraftment in a subject comprising limited to , allergic bronchopulmonary aspergillosis ; autoim - administering to a subject in need thereof of a therapeuti mune hemolytic anemia ; acanthosis nigricans ; allergic con - cally effective amount of one or more compounds of the tact dermatitis ; Addison ' s disease ; atopic dermatitis ; alope invention . cia greata ; alopecia universalis ; amyloidosis ; anaphylactoid As used herein the phrase " enhancing cellular engraft purpura ; anaphylactoid reaction ; aplastic anemia ; 15 ment ” refers to an improvement in efficiency , quality or angioedema, hereditary ; angioedema, idiopathic ; ankylosing rapidity of cell transplantation which may result from spondylitis ; arteritis , cranial; arteritis , giant cell ; arteritis , improved homing to the target tissue, improved adhesion , Takayasu ' s ; arteritis , temporal; asthma; a -telangiectasia ; reduced rejection and the like . Methods for assessing cell autoimmune oophoritis ; autoimmune orchitis ; autoimmune engraftinent potential include , for example , cell migration polyendocrine failure ; Behcet' s disease; Berger ' s disease; 20 and other in vitro techniques , and histological, immunologi Buerger ' s disease ; bullous pemphigus ; candidiasis , chronic cal and / or radiological assessment of tissues and organs mucocutaneous; Caplan ' s syndrome; post -myocardial from actual in - vivo transplantation . In certain aspects , the infarction syndrome; post - pericardiotomy syndrome; cardi- term " enhancing cellular engraftment " also includes , but is tis; celiac sprue ; Chagas 's disease ; Chediak -Higashi syn - not limited to , the treatment of a disorder in which the drome; Churg -Strauss disease ; Cogan ' s syndrome; cold 25 subject is treated with cells , such as in the context of bone agglutinin disease ; CREST syndrome; Crohn ' s disease ; marrow transplantation , stem cell replacement, and skin cryoglobulinemia ; cryptogenic fibrosing alveolitis ; dermati- engraftment. tis herpetifomis ; dermatomyositis ; diabetes mellitus ; Dia In another aspect, the invention provides a method of mond - Blackfan syndrome ; DiGeorge syndrome; discoid enhancing wound repair in a subject comprising adminis lupus erythematosus ; eosinophilic fascitis ; episcleritis ; dry - 30 tering to a subject in need thereof of a therapeutically thema elevatum diutinum ; erythema marginatum ; erythema effective amount of one or more compounds of the inven multiforme; erythema nodosum ; familial Mediterranean tion . fever; Felty ' s syndrome; fibrosis pulmonary ; glomerulone - As used herein the term , “ enhancing wound repair ” phritis , anaphylactoid ; glomerulonephritis, autoimmune; includes, but is not limited to , the formation of granulation glomerulonephritis , post- streptococcal; glomerulonephritis, 35 tissue; of wound contraction ; and / or the induction of epi post- transplantation ; glomerulopathy , membranous ; Good - thelialization ( i. e ., the generation of new cells in the epithe pasture ' s syndrome; graft - vs . - host disease ; granulocytope- lium ) . Wound repair is conveniently measured by decreasing nia , immune -mediated ; granuloma annulare ; granulomato - wound area . In certain aspects, the term " enhancing wound sis , allergic ; granulomatous myositis ; Grave ' s disease ; repair ” also includes, but is not limited to , the treatment a Hashimoto ' s thyroiditis ; hemolytic disease of the newborn ; 40 disorder in which the subject is being treated for tissue hemochromatosis , idiopathic ; Henoch - Schoenlein purpura ; damage, either internal or external, such that the goal is to hepatitis , chronic active and chronic progressive ; histiocy - restore organ or tissue integrity . tosis X ; hypereosinophilic syndrome; idiopathic thrombo - In another aspect , the invention provides a method of cytopenic purpura ; Job ' s syndrome; juvenile dermatomyo - treating an inflammatory condition in a subject comprising sitis ; juvenile rheumatoid arthritis juvenile chronic arthritis ) ; 45 administering to a subject in need thereof an effective Kawasaki' s disease ; keratitis ; keratoconjunctivitis sicca ; amount of a composition comprising a compound of the Landry -Guillain - Barre - Strohl syndrome; leprosy, leproma - invention or a pharmaceutically acceptable salt thereof. tous; Loeffler' s syndrome; Lyell ' s syndrome; Lyme disease ; As used herein , the term “ inflammatory condition ” refers lymphomatoid granulomatosis ; mastocytosis , systemic ; to a condition or disorder associated with one or more mixed connective tissue disease ; mononeuritis multiplex ; 50 aberrant physiological processes or other physiological Muckle - Wells syndrome; mucocutaneous lymph node syn - responses (such as responses to an injurious or noxious drome; mucocutaneous lymph node syndrome; multicentric stimulus ) which result in a pathophysiological state of reticulohistiocytosis ; multiple sclerosis ; myasthenia gravis ; inflammation . An inflammatory condition may be either an mycosis fungoides ; necrotizing vasculitis, systemic ; neph acute or chronic inflammatory condition , which can result rotic syndrome; overlap syndrome; panniculitis ; paroxysmal 55 from infections or non - infectious causes . Various infectious cold hemoglobinuria ; paroxysmal nocturnal hemoglobinu causes include meningitis , encephalitis , uveitis , colitis , ria ; pemphigoid ; pemphigus; pemphigus erythematosus ; tuberculosis , dermatitis , and adult respiratory distress syn pemphigus foliaceus ; pemphigus vulgaris ; pigeon breeder ' s drome. Non - infectious causes include trauma (burns , cuts , disease; pneumonitis , hypersensitivity; polyarteritis nodosa ; contusions, crush injuries ), autoimmune diseases, and organ polymyalgia rheumatica ; polymyositis ; polyneuritis , idio - 60 rejection episodes . Thus , in specific aspects , an inflamma pathic ; Portuguese familial polyneuropathies; pre - eclamp- tory condition results from a condition selected from the sia / eclampsia ; primary biliary cirrhosis ; progressive sys - group that includes : atherosclerosis ( arteriosclerosis ) ; auto temic sclerosis ( scleroderma) ; psoriasis ; psoriatic arthritis ; immune conditions, such as multiple sclerosis, systemic pulmonary alveolar proteinosis ; pulmonary fibrosis , Ray - lupus erythematosus, polymyalgia rheumatica (PMR ) , gouty naud ' s phenomenon / syndrome; Reidel ' s thyroiditis ; Reit - 65 arthritis , degenerative arthritis , tendonitis , bursitis , psoriasis , er ' s syndrome, relapsing polychrondritis ; rheumatic fever; fibrosis , arthrosteitis , rheumatoid arthritis and other forms of rheumatoid arthritis ; sarcoidosis ; scleritis ; sclerosing cho inflammatory arthritis , Sjogren ' s Syndrome, progressive US 10 ,000 , 511 B2 21 22 systemic sclerosis ( scleroderma) , ankylosing spondylitis , pound of the invention or a pharmaceutically acceptable salt , polymyositis , dermatomyositis , pemphigus , pemphigoid , solvate or hydrate thereof or an a compound of the invention Type I diabetes mellitus, myasthenia gravis , Hashimoto ' s pharmaceutical composition is suitable for topical , intrave thyroditis , Graves ' disease , Goodpasture ' s disease , mixed nous , parental, or oral administration . The methods of the connective tissue disease , sclerosing cholangitis , inflamma- 5 invention further include administering to a subject a thera tory bowel disease including Crohn ' s Disease ( regional peutically effective amount of a compound of the invention enteritis ) and ulcerative colitis , pernicious anemia , inflam or a pharmaceutically acceptable salt, solvate or hydrate matory dermatoses; usual interstitial pneumonitis (UIP ) , asbestosis , silicosis , bronchiectasis , berylliosis , talcosis , all thereof. formsof pneumoconiosis , sarcoidosis ( in the lung and in any 10 The phrase " pharmaceutically acceptable” refers to a other organ ) , desquamative interstitial pneumonia , lymphoid compound of the invention , compositions containing a com interstitial pneumonia , giant cell interstitial pneumonia , cel pound of the invention , and / or dosage forms which are , lular interstitial pneumonia , extrinsic allergic alveolitis , within the scope of sound medical judgment, suitable for use Wegener ’ s granulomatosis and related forms of angiitis in contact with the tissues of human beings and animals ( temporal arteritis and polyarteritis nodosa ); sepsis ; inflam - 15 without excessive toxicity , irritation , allergic response , or matory dermatoses not presumed to be autoimmune ; chronic other problem or complication , commensurate with a rea active hepatitis ; delayed - type hypersensitivity reactions sonable benefit /risk ratio . ( e. g ., poison ivy dermatitis ); pneumonia or other respiratory The phrase " pharmaceutically - acceptable carrier " tract inflammation due to any cause ; Adult Respiratory includes pharmaceutically -acceptable material, composition Distress Syndrome (ARDS ) from any etiology ; encephalitis 20 or vehicle , involved in carrying or transporting the subject with inflammatory edema; immediate hypersensitivity reac - chemical from one organ , or portion of the body, to another tions including, but not limited to , asthma , hayfever, cuta - organ , or portion of the body. Each carrier must be “ accept neous allergies , acute anaphylaxis ; diseases involving acute able ” in the sense of being compatible with the other deposition of immune complexes, including , but not limited ingredients of the formulation and not injurious to the to , rheumatic fever, acute and /or chronic glomerulonephritis 25 patient. due to any etiology , including specifically post - infectious Methods of preparing these compositions include the step ( e . g ., post - Streptococcal) glomerulonephritis , acute exacer - of bringing into association a compound of the invention or bations of Systemic Lupus Erythematosus ; pyelonephritis ; a pharmaceutically acceptable salt , solvate or hydrate cellulitis ; cystitis ; acute and /or chronic cholecystitis ; and thereof with the carrier and , optionally , one or more acces conditions producing transient ischemia anywhere along the 30 sory ingredients . These compositions may also contain adju gastrointestinal tract, bladder , heart, or other organ , espe vants such as preservatives , wetting agents , emulsifying cially those prone to rupture ; sequelae of organ transplan - agents and dispersing agents . tation or tissue allograft , including allograft rejection in the Regardless of the route of administration selected , a acute time period following allogeneic organ or tissue trans - compound of the invention , which may be used in a suitable plantation and chronic host - versus - graft rejection . The term 35 salt, solvate , or hydrate form , and / or the pharmaceutical " inflammatory condition ” also includes appendicitis , arteri compositions of a compound of the invention , are formu tis , blepharitis , bronchiolitis , bronchitis , cervicitis , cholan - lated into pharmaceutically acceptable dosage forms by gitis , chorioamnionitis , conjunctivitis , dacryoadenitis , der - conventional methods known to those of skill in the art. matomyositis , endocarditis , endometritis , enteritis , Formulations are provided to a subject in an effective enterocolitis, epicondylitis , epididymitis , fasciitis , fibrositis, 40 amount. The term " effective amount ” includes an amount gastritis , gastroenteritis , gingivitis , ileitis , iritis , laryngitis , effective, at dosages and for periods of time necessary, to myelitis , myocarditis , nephritis , omphalitis , oophoritis , achieve the desired result . An effective amount of a com orchitis , osteitis , otitis , pancreatitis , parotitis , pericarditis, pound of the invention may vary according to factors such pharyngitis, pleuritis , phlebitis , pneumonitis , proctitis , pros as the disease state , age, and weight of the subject, and the tatitis , rhinitis , salpingitis , sinusitis , stomatitis, synovitis , 45 ability of the compound to elicit a desired response in the tonsillitis , uveitis , vaginitis , vasculitis , vulvitis , and vul- subject. Dosage regimens may be adjusted to provide the vovaginitis, angitis , chronic bronchitis , osteomylitis , optic optimum therapeutic response. neuritis , temporal arteritis , transverse myelitis , necrotizing The effective amount is generally determined by the fascilitis , hepatitis , and necrotizing enterocolitis . physician on a case - by - case basis and is within the skill of In a specific aspect , the invention encompasses a method 50 one in the art. As a rule , the dosage for in vivo therapeutics of targeting MCL - 1 in a cell comprising contacting a cell or diagnostics will vary . Several factors are typically taken with one or more compounds selected from gossypol, cel into account when determining an appropriate dosage . These astrol, manoalide, u73122 , bithionol and hexachlorophene . factors include age , sex and weight of the patient, the Such targeting may useful in the modulation of programmed condition being treated , and the severity of the condition . In cell death or apoptosis , autophagic cell death ; necrotic cell 55 addition , an effective amount is preferably selected to mini death ; cell metabolism ; cell division ; cell differentiation ; mize adverse side effects . For example , an effective amount RNA transcription and processing ; or protein multimeriza - of a compound of the invention is preferably selected to treat tion or dissociation . or prevent a disorder as disclosed herein , while minimizing As used herein , the term “ targeting ” refers to the direct side effects such as hypotension . interaction of the compound with MCL - 1 , or to the inter - 60 Suitable dosages and formulations of a compound of the action of the compound with one or more other factors that invention or a pharmaceutically acceptable salt , solvate or in turn interact with MCL - 1 or affect MCL - 1 activity . hydrate thereof can be empirically determined by the admin Administration istering physician . Standard texts , such as Remington : The In certain embodiments , a compound of the invention or Science and Practice of Pharmacy , 17th edition , Mack a pharmaceutically acceptable salt, solvate or hydrate 65 Publishing Company, and the Physician ' s Desk Reference , thereof is administered to the subject in a pharmaceutically - each of which are incorporated herein by reference , can be acceptable formulation . In certain embodiments , a com consulted to prepare suitable compositions and doses for US 10 , 000 ,511 B2 23 24 administration . A determination of the appropriate dosage is the patient' s system and , thus , is subject to metabolism and within the skill of one in the art given the parameters for use other like processes , for example , subcutaneous administra described herein . tion . Standard texts , such as Remington : The Science and Such dosages may vary, for example , depending on Practice of Pharmacy, 17th edition , Mack Publishing Com - 5 whether multiple administrations are given , tissue type and pany, incorporated herein by reference , can be consulted to route of administration , the condition of the individual, the prepare suitable compositions and formulations for admin desired objective and other factors known to those of skill in istration , without undue experimentation . Suitable dosages the art . can also be based upon the text and documents cited herein . Available routes of administration include subcutaneous, A determination of the appropriate dosages is within the skill 10 intramuscular, intraperitoneal, intradermal , oral , intranasal , of one in the art given the parameters herein . intrapulmonary ( i . e . , by aerosol ) , intravenously , intramus In terms of treatment, an effective amount is an amount cularly , subcutaneously , intracavity, intrathecally or trans that is sufficient to palliate , ameliorate , stabilize , reverse or d ermally , alone or in combination with other pharmaceutical slow the progression of an inflammatory condition or other 16 agents . disorder as disclosed herein , or the symptoms thereof. A . A cell can be contacted with a composition including a therapeutically effective amount can be provided in one or a compound of the invention , in certain embodiments in vivo series of administrations . In terms of an adjuvant, an effec or, in certain embodiments , in vitro . Contacting a cell in vivo tive amount is one sufficient to enhance the anti - inflamma can include administration of the composition to a subject , tory or immune response to the anti - inflammatory or immu - 20 or to a tissue , such that a cell is contacted with a compound nogen . The effective amount is generally determined by the of the invention . Contacting a cell in vitro can include , e. g ., physician on a case -by - case basis and is within the skill of contacting the cell with a compound of the invention , or a one in the art. composition consisting essentially of a compound of the As a rule , the dosage for in vivo therapeutics or diagnos - invention , directly or by addition of irinadalone or the tics will vary . Several factors are typically taken into account 25 composition to a growth medium for the cell . when determining an appropriate dosage. These factors Additional/ Secondary Therapeutic Agents include age , sex and weight of the patient, the condition The compounds and compositions of the invention can be being treated , the severity of the condition and the method administered with one or more additional additional thera of administration . peutic agents either separately or in the same formulation as The dosage of a compound of the invention or a pharma - 30 a compound of the invention . ceutically acceptable salt, solvate or hydrate thereof can vary In certain embodiments the compounds and compositions from about 0 .01 mg to about 1 ,000 mg per day ; about 0 . 1 mg of the invention are useful in sensitizing a cell or a subject to about 250 mg per day ; about 0 . 5 mg to about 100 mg per to one or more secondary therapeutic agents. In such day ; or about 1 to about 5 mg per day . Ascertaining dosage embodiments , the invention provides a method for sensitiz ranges is well within the skill of one in the art. The dosage 35 ing a cell to a secondary therapeutic agent comprising of a compound of the invention or a pharmaceutically administering to said cell a sensitizing amount of one or acceptable salt , solvate or hydrate thereof can range from more compound of the invention . A sensitizing amount may about 0 .001 to 25 mg/ kg of body weight. Such dosages may be any amount capable of generating a greater response by vary , for example , depending on whether multiple adminis the cell to the secondary therapeutic agent as compared to trations are given , tissue type and route of administration , 40 the response by the cell without a compound or composition the condition of the individual, the desired objective and of the invention . In certain embodiments a sensitizing other factors known to those of skill in the art. Administra - amount is a therapeutically effective amount. In other tions can be conducted infrequently , or on a regular weekly embodiments , a sensitizing amount is less than a therapeu basis until a desired ,measurable parameter is detected , such tically effective amount. as diminution of disease symptoms. Administration can then 45 The compounds and compositions of the invention can be be diminished , such as to a biweekly or monthly basis , as administered with one or more additional therapeutic agents appropriate . either separately or in the same formulation as a compound A therapeutically effective amount can be administered in of the invention . one or more doses . The term “ administration ” or “ adminis - Additional Therapeutic agents include, but are not limited tering” includes routes of introducing the compound ( s ) to a 50 to antacids , antibiotics , antiemetic agents , antidepressants , subject to perform their intended function . Examples of and antifungal agents, anti - inflammatory agents , antiviral routes of administration which can be used include injection agents , anticancer agents , immunomodulatory agents , beta ( subcutaneous , intravenous , parenterally , intraperitoneally, interferons , hormones or cytokines . intrathecal) , oral, inhalation , rectal and transdermal. Additional Therapeutic agents further include, but are not The phrases " parenteral administration ” and “ adminis - 55 limited to death receptor stimulants ( eg . TRAIL ), HDAC tered parenterally ” as used herein means modes of admin - inhitor ( eg . SAHA ), proteasome inhibitor ( eg . Bortezomib ) , istration other than enteral and topical administration , usu BCL - 2 family inhibitor (eg . ABT- 737 , ABT - 263 , obatoclax ) , ally by injection , and includes, without limitation , kinase inhibitor ( e . g . Gleevac , Raf inhibitor ), mitotic inhibi intravenous, intramuscular, intraarterial, intrathecal , intraca - tors ( e . g . Taxol) , nucleoside analogs ( e . g . Gemcitabine ) , and psular, intraorbital, intracardiac , intradermal, intraperito - 60 anti - cancer monoclonal antibodies ( e . g . cetuximab ) , corti neal, transtracheal, subcutaneous, subcuticular, intraarticu - costeroids ( e . g . dexamethasone ) , DNA - damaging agents lar, subcapsular , subarachnoid , intraspinal and intrasternal ( e . g . cisplatin ), antimetabolites ( e . g . methotrexate ) . injection and infusion . The compounds of the invention can be formulated in The phrases “ systemic administration ,” “ administered combination with antacids. For example , they can be for systemically ” , “ peripheral administration ” and “ adminis - 65 mulated with aluminum carbonate , aluminum hydroxide , tered peripherally ” as used herein mean the administration bismuth subsalicylate , calcium carbonate , calcium hydrox of a compound (s ), drug or other material, such that it enters ide , calcium phosphate , dihydroxyaluminum sodium car US 10 ,000 , 511 B2 25 26 bonate , magnesium hydroxide, magnesium oxide, magne - pizotyline , propizepine, protriptyline, quinupramine , tianep sium trisilicate , sodium bicarbonate , simethicone, glycine , tine , trimipramine , adrafinil , benactyzine , bupropion , buta or combinations thereof. cetin , dioxadrol, duloxetine , etoperidone, febarbamate , fer The compounds of the invention can be formulated in noxetine , fenpentadiol, fluoxetine , fluvoxamine , combination with antibiotics . For example, they can be 5 hematoporphyrin , hypericin , levophacetoperane , medifox formulated with a macrolide ( e. g . , tobramycin ), a cepha amine, milnacipran , minaprine, moclobemide , nefazodone , losporin (e . g. , cephalexin , cephradine, cefuroxime, cefpro - oxaflozane, piberaline , prolintane , pyrisuccideanol, ritan zil, cefaclor, cefixime or cefadroxil ) , a clarithromycin ( e. g ., serin , roxindole , rubidium chloride, sulpiride , tandospirone , clarithromycin ), an erythromycin ( e . g . , erythromycin ) , a thozalinone , tofenacin , toloxatone , tranylcypromine, L - tryp penicillin ( e . g . , penicillin V or a quinolone ( e . g . , ofloxacin , 10 tophan , venlafaxine , viloxazine , and zimeldine . ciprofloxacin or norfloxacin ), aminoglycoside antibiotics The compounds of the invention can be formulated in ( e . g . , apramycin , arbekacin , bambermycins, butirosin , combination with an antifungal agent. Suitable antifungal dibekacin , neomycin , neomycin , undecylenate , netilmicin , agents include but are not limited to amphotericin B , itra paromomycin , ribostamycin , sisomicin , and spectinomy - conazole , ketoconazole , fluconazole , intrathecal, flucyto cin ) , amphenicol antibiotics ( e . g . , azidamfenicol, chloram - 15 sine, miconazole , butoconazole , clotrimazole , nystatin , ter phenicol, florfenicol , and thiamphenicol) , ansamycin anti - conazole , tioconazole , ciclopirox , econazole , haloprogrin , biotics ( e . g . , rifamide and rifampin ) , carbacephems ( e . g ., naftifine , terbinafine , undecylenate , and griseofuldin . loracarbef) , carbapenems ( e. g ., biapenem and imipenem ), The compounds of the invention can be formulated in cephalosporins ( e . g . , cefaclor, cefadroxil , cefamandole , combination with an anti - inflammatory agent. Useful anti cefatrizine , cefazedone, cefozopran , cefpimizole , cefpir- 20 inflammatory agents include , but are not limited to , non amide , and cefpirome) , cephamycins (e . g ., cefbuperazone, steroidal anti - inflammatory drugs such as salicylic acid , cefrnetazole , and cefminox ) ,monobactams ( e . g . , aztreonam , acetylsalicylic acid , methyl salicylate , diflunisal, salsalate , carumonam , and tigemonam ), oxacephems (e . g. , flomoxef, olsalazine, sulfasalazine , acetaminophen , indomethacin , and moxalactam ) , penicillins ( e . g . , amdinocillin , amdinocil sulindac , etodolac, mefenamic acid , meclofenamate sodium , lin pivoxil, amoxicillin , bacampicillin , benzylpenicillinic 25 tolmetin , ketorolac , dichlofenac , ibuprofen , naproxen , acid , benzylpenicillin sodium , epicillin , fenbenicillin , floxa - naproxen sodium , fenoprofen , ketoprofen , flurbinprofen , cillin , penamccillin , penethamate hydriodide, penicillin oxaprozin , piroxicam , meloxicam , ampiroxicam , droxicam , o -benethamine , penicillin 0 , penicillin V , penicillin V ben - pivoxicam , tenoxicam , nabumetome, phenylbutazone , oxy zathine , penicillin V hydrabamine , penimepicycline , and phenbutazone , antipyrine, aminopyrine , apazone and nime phencihicillin potassium ) , lincosamides (e . g ., clindamycin , 30 sulide ; leukotriene antagonists including, but not limited to , and lincomycin ), amphomycin , bacitracin , capreomycin , zileuton , aurothioglucose , gold sodium thiomalate and aura colistin , enduracidin , enviomycin , tetracyclines (e .g ., apicy - nofin ; steroids including , but not limited to , alclometasone cline, chlortetracycline , clomocycline , and demeclocycline ), diproprionate , amcinonide , beclomethasone dipropionate , 2 , 4 - diaminopyrimidines ( e . g . , brodimoprim ) , nitrofurans betametasone , betamethasone benzoate , betamethasone ( e . g . , furaltadone, and furazolium chloride ) , quinolones and 35 diproprionate , betamethasone sodium phosphate , betame analogs thereof ( e . g . , cinoxacin , clinafloxacin , flumequine , thasone valerate , clobetasol proprionate , clocortolone piva and grepagloxacin ), sulfonamides ( e . g ., acetyl sulfame - late , hydrocortisone , hydrocortisone derivatives, desonide , thoxypyrazine , benzylsulfamide , noprylsulfamide, phthalyl - desoximatasone , dexamethasone , flunisolide, flucoxinolide , sulfacetamide , sulfachrysoidine, and sulfacytine ), sulfones flurandrenolide , halcinocide, medrysone , methylpredniso ( e . g ., diathymosulfone , glucosulfone sodium , and solasul - 40 lone, methprednisolone acetate , methylprednisolone sodium fone ), cycloserine, mupirocin and tuberin . succinate , mometasone furoate , paramethasone acetate , The compounds of the invention can be formulated in prednisolone , prednisolone acetate, prednisolone sodium combination with an antiemetic agent . Suitable antiemetic phosphate , prednisolone tebuatate , prednisone , triamcino agents include, but are not limited to , metoclopromide, lone, triamcinolone acetonide , triamcinolone diacetate , and domperidone , prochlorperazine , promethazine , chlorprom - 45 triamcinolone hexacetonide ; and other anti- inflammatory azine , trimethobenzamide , ondansetron , granisetron , agents including, but not limited to , methotrexate , colchi hydroxyzine, acethylleucine monoethanolamine , alizapride , cine , allopurinol , probenecid , sulfinpyrazone and benzbro azasetron , benzquinamide , bietanautine , bromopride , bucl - marone . izine , clebopride , cyclizine , dimenhydrinate , diphenidol, The compounds of the invention can be formulated in dolasetron , meclizine, methallatal, metopimazine , nabilone, 50 combination with another antiviral agent. Useful antiviral oxypemdyl, pipamazine, scopolamine , sulpiride , tetrahydro - agents include , but are not limited to , protease inhibitors , cannabinols , thiethylperazine , thioproperazine, tropisetron , nucleoside reverse transcriptase inhibitors, non -nucleoside and mixtures thereof . reverse transcriptase inhibitors and nucleoside analogs . The The compounds of the invention can be formulated or antiviral agents include but are not limited to zidovudine , formulated in combination with an antidepressant. Suitable 55 acyclovir , gangcyclovir , vidarabine, idoxuridine , trifluri antidepressants include , but are not limited to , binedaline , dine, and ribavirin , as well as foscarnet, amantadine, rim caroxazone, citalopram , dimethazan , fencamine , indalpine , antadine , saquinavir , indinavir , amprenavir , lopinavir , rito indeloxazine hydrocholoride , nefopam , nomifensine , navir , alpha - interferons; adefovir , clevadine, entecavir , oxitriptan , oxypertine , paroxetine , sertraline, thiazesim , tra - pleconaril . zodone, benmoxine , iproclozide , iproniazid , isocarboxazid , 60 The compounds of the invention can be formulated in nialamide , octamoxin , phenelzine , cotinine , rolicyprine , combination with an immunomodulatory agent. Immuno rolipram , maprotiline , metralindole , mianserin , mirtazepine , modulatory agents include , but are not limited to , metho adinazolam , amitriptyline , amitriptylinoxide , amoxapine, threxate , leflunornide , cyclophosphamide , cyclosporine A , butriptyline , clomipramine , demexiptiline, desipramine, mycophenolate mofetil , rapamycin ( sirolimus) , mizoribine , dibenzepin , dimetacrine , dothiepin , doxepin , fluacizine, imi- 65 deoxyspergualin , brequinar, malononitriloamindes ( e . g . , pramine , imipramine N - oxide, iprindole , lofepramine , meli - leflunamide ), T cell receptor modulators , and cytokine tracen , metapramine, nortriptyline , noxiptilin , opipramol, receptor modulators, peptide mimetics, and antibodies ( e. g ., US 10 , 000 ,511 B2 27 human , humanized , chimeric , monoclonal, polyclonal, Fvs , is hereby incorporated by reference in its entirety. Other ScFvs, Fab or F (ab ) 2 fragments or epitope binding frag - known absorption enhancers can also be used . Thus, the ments ) , nucleic acid molecules ( e . g . , antisense nucleic acid invention also encompasses a pharmaceutical composition molecules and triple helices ) , small molecules , organic comprising one or more sulfated polysaccharides of the compounds , and inorganic compounds . Examples of T cell 5 invention and one or more absorption enhancers . receptor modulators include , but are not limited to , anti - T The additional therapeutic agent can act additively or, cell receptor antibodies ( e . g . , anti -CD4 antibodies ( e . g ., more preferably , synergistically . In a preferred embodiment, cM - T412 (Boeringer ), IDEC - CE9. 1® (IDEC and SKB ), a composition comprising a compound of the invention is MAB 4162694 , Orthoclone and OKTcdr4a (Janssen - administered concurrently with the administration of Cilag ) ), anti -CD3 antibodies ( e . g ., Nuvion ( Product Design 10 another therapeutic agent, which can be part of the same Labs ) , OKT3 (Johnson & Johnson ), or Rituxan ( IDEC ) ) , composition or in a different composition from that com anti - CD5 antibodies ( e . g . , an anti- CD5 ricin - linked immu- prising the compounds of the invention . In another embodi noconjugate ), anti -CD7 antibodies ( e . g ., CHH - 380 (Novar - ment , a compound of the invention is administered prior to tis ) ) , anti -CD8 antibodies , anti -CD40 ligand monoclonal or subsequent to administration of another therapeutic agent. antibodies ( e . g . , MEC - 131 (IDEC )) , anti -CD52 antibodies 15 In a separate embodiment, a compound of the invention is ( e . g ., CAMPATH 1H ( Ilex ) ) , anti -CD2 antibodies, anti - administered to a patient who has not previously undergone CD1la antibodies ( e. g ., Xanelim (Genentech )) , and anti- B7 or is not currently undergoing treatment with another thera antibodies ( e . g . , IDEC -114 (IDEC ) ) and CTLA4 - immuno - peutic agent , particularly an antiviral agent . globulin . Examples of cytokine receptormodulators include, Oral Dosage Forms but are not limited to , soluble cytokine receptors ( e . g . , the 20 A compound of the invention or a pharmaceutically extracellular domain of a TNF - alpha . receptor or a fragment acceptable salt , solvate or hydrate thereof and compositions thereof, the extracellular domain of an IL - 1 .beta . receptor or comprising a compound of the invention that are suitable for a fragment thereof, and the extracellular domain of an IL - 6 oral administration can be presented as discrete dosage receptor or a fragment thereof ), cytokines or fragments forms, such as , but are not limited to , tablets ( e . g ., chewable thereof ( e. g ., interleukin (IL )- 2 , IL -3 , IL - 4 , IL - 5 , IL -6 , IL - 7 , 25 tablets ), caplets , capsules, and liquids (e . g. , flavored syrups ). IL -8 , IL - 9 , IL - 10 , IL - 11 , IL - 12 , IL - 15 , TNF -alpha ., inter - Such dosage forms contain predetermined amounts of active feron ( IFN ) - alpha ., IFN -beta . , IFN - gamma, and GM - CSF ) , ingredients , and may be prepared by methods of pharmacy anti - cytokine receptor antibodies ( e . g ., anti - IFN receptor well known to those skilled in the art . See generally , antibodies , anti - IL - 2 receptor antibodies ( e . g ., Zenapax Remington 's Pharmaceutical Sciences, 18th ed ., Mack Pub ( Protein Design Labs )) , anti -IL - 4 receptor antibodies , anti - 30 lishing, Easton Pa . ( 1990 ) . IL -6 receptor antibodies, anti - IL - 10 receptor antibodies, and Typical oral dosage forms of the invention are prepared by anti - IL - 12 receptor antibodies ), anti -cytokine antibodies combining the active ingredient( s ) in an intimate admixture ( e . g . , anti- IFN antibodies, anti - TNF - alpha . antibodies, anti - with at least one excipient according to conventional phar IL - 1beta antibodies, anti - IL -6 antibodies , anti - IL - 8 antibod maceutical compounding techniques. Excipients can take a ies ( e . g ., ABX - IL - 8 (Abgenix ) ) , and anti - IL - 12 antibodies ). 35 wide variety of forms depending on the form of preparation The compounds of the invention can be formulated in desired for administration . For example , excipients suitable combination with cytokines . Examples of cytokines include, for use in oral liquid or aerosol dosage forms include , but are but are not limited to , interleukin - 2 ( IL - 2 ) , interleukin - 3 not limited to , water , glycols , oils , alcohols , flavoring agents , ( IL -3 ), interleukin - 4 ( IL -4 ), interleukin -5 ( IL - 5 ), interleu preservatives, and coloring agents . Examples of excipients kin - 6 ( IL - 6 ), interleukin - 7 ( IL - 7 ) , interleukin - 9 ( IL - 9 ) , inter - 40 suitable for use in solid oral dosage forms ( e . g . , powders , leukin - 10 ( IL - 10 ) , interleukin - 12 ( IL - 12 ), interleukin 15 tablets , capsules , and caplets ) include, but are not limited to , ( IL - 15 ) , interleukin 18 (IL - 18 ) , platelet derived growth starches, sugars , micro - crystalline cellulose , diluents , granu factor (PDGF ) , erythropoietin (Epo ) , epidermal growth fac - lating agents , lubricants , binders , and disintegrating agents . tor (EGF ) , fibroblast growth factor ( FGF ) , granulocyte mac Because of their ease of administration , tablets and cap rophage stimulating factor (GM -CSF ) , granulocyte colony 45 sules represent the most advantageous oral dosage unit stimulating factor (G -CSF ) , macrophage colony stimulating forms, in which case solid excipients are employed . If factor (M -CSF ), prolactin , and interferon ( IFN ) , e. g ., IFN desired , tablets can be coated by standard aqueous or non alpha , and IFN - gamma ) . aqueous techniques . Such dosage forms can be prepared by The compounds of the invention can be formulated in any of the methods of pharmacy . In general , pharmaceutical combination with hormones . Examples of hormones 50 compositions and dosage forms are prepared by uniformly include , but are not limited to , luteinizing hormone releasing and intimately admixing the active ingredients with liquid hormone ( LHRH ), growth hormone (GH ) , growth hormone carriers , finely divided solid carriers , or both , and then releasing hormone , ACTH , somatostatin , somatotropin , shaping the product into the desired presentation if neces somatomedin , parathyroid hormone , hypothalamic releasing sary. factors , insulin , glucagon , enkephalins , vasopressin , calci- 55 For example , a tablet can be prepared by compression or tonin , heparin , low molecular weight heparins , heparinoids , molding. Compressed tablets can be prepared by compress synthetic and natural opioids, insulin thyroid stimulating ing in a suitable machine the active ingredients in a free hormones, and endorphins . flowing form such as powder or granules , optionally mixed The compounds of the invention can be formulated in with an excipient. Molded tablets can be made by molding combination with beta - interferonswhich include, but are not 60 in a suitable machine a mixture of the powdered compound limited to , interferon beta - la and interferon beta - lb . moistened with an inert liquid diluent. The compounds of the invention can be formulated in Examples of excipients that can be used in oral dosage combination with an absorption enhancer , particularly those forms of the invention include , but are not limited to , which target the lymphatic system , including, but not limited binders , fillers , disintegrants , and lubricants . Binders suit to sodium glycocholate ; sodium caprate ; N - lauryl- D -malto - 65 able for use in pharmaceutical compositions and dosage pyranoside ; EDTA ; mixed micelle ; and those reported in forms include, but are not limited to , corn starch , potato Muranishi Crit . Rev . Ther . Drug Carrier Syst. , 7- 1 -33 , which starch , or other starches , gelatin , natural and synthetic gums US 10 , 000 ,511 B2 29 30 such as acacia , sodium alginate , alginic acid , other alginates , tion and constant infusion ) , intramuscular, and intraarterial. powdered tragacanth , guar gum , cellulose and its derivatives Because their administration typically bypasses patients ' ( e . g . , ethyl cellulose , cellulose acetate , carboxymethyl cel natural defenses against contaminants , parenteral and intra lulose calcium , sodium carboxymethyl cellulose ), polyvinyl vascular dosage forms are preferably sterile or capable of pyrrolidone , methyl cellulose , pre - gelatinized starch , 5 being sterilized prior to administration to a patient. hydroxypropyl methyl cellulose , ( e. g ., nos. 2208 , 2906 , Examples of parenteral dosage forms include , but are not 2910 ), microcrystalline cellulose , and mixtures thereof. Examples of fillers suitable for use in the pharmaceutical limited to , solutions ready for injection , dry products ( in compositions and dosage forms disclosed herein include , but cluding, but not limited to lyophilized powders , pellets , and are not limited to , talc , calcium carbonate (e .g ., granules or 10 tablets ) ready to be dissolved or suspended in a pharmaceu powder) , microcrystalline cellulose, powdered cellulose , tically acceptable vehicle for injection , suspensions ready dextrates , kaolin , mannitol, silicic acid , sorbitol, starch , for injection , and emulsions . pre - gelatinized starch , and mixtures thereof. The binder or Suitable vehicles that can be used to provide parenteral filler in pharmaceutical compositions of the invention is dosage forms of the invention are well known to those typically present in from about 50 to about 99 weight percent 15 skilled in the art . Examples include , but are not limited to : of the pharmaceutical composition or dosage form . Water for Injection USP ; aqueous vehicles such as , but not Suitable forms of microcrystalline cellulose include , but limited to , Sodium Chloride Injection , Ringer ' s Injection , are not limited to , the materials sold as AVICEL -PH - 101 , Dextrose Injection , Dextrose and Sodium Chloride Injec AVICEL - PH - 103 AVICEL RC -581 , AVICEL - PH - 105 tion , and Lactated Ringer 's Injection ; water -miscible (available from FMC Corporation , American Viscose Divi- 20 vehicles such as, but not limited to , ethyl alcohol, polyeth sion , Avicel Sales, Marcus Hook , Pa .) , and mixtures thereof. ylene glycol, and polypropylene glycol; and non -aqueous A specific binder is a mixture of microcrystalline cellulose vehicles such as, but not limited to , corn oil , cottonseed oil , and sodium carboxymethyl cellulose sold as AVICEL peanut oil, sesame oil , ethyl oleate , isopropyl myristate , and RC -581 . Suitable anhydrous or low moisture excipients or benzyl benzoate . additives include AVICEL - PH - 103TM and Starch 1500 LM . 25 Compounds that increase the solubility of one or more of Disintegrants are used in the compositions of the inven - the active ingredients disclosed herein can also be incorpo tion to provide tablets that disintegrate when exposed to an rated into the parenteral dosage forms of the invention . aqueous environment. Tablets that contain too much disin - For intravascular administration , for instance by direct tegrant may disintegrate in storage , while those that contain injection into the blood vessel, or surrounding area , it may too little may not disintegrate at a desired rate or under the 30 be desirable to administer the compositions locally to the desired conditions . Thus , a sufficient amount of disintegrant area in need of treatment . This can be achieved , for example , that is neither too much nor too little to detrimentally alter by local infusion during surgery , by injection , by means of the release of the active ingredients should be used to form a catheter, or by means of an implant, said implant being of solid oral dosage forms of the invention . The amount of a porous, non -porous , or gelatinous material, including disintegrant used varies based upon the type of formulation , 35 membranes , such as silastic membranes , or fibers . A suitable and is readily discernible to those of ordinary skill in the art. such membrane is Gliadel® provided by Guilford Pharma Typical pharmaceutical compositions comprise from about ceuticals Inc . 0 . 5 to about 15 weight percent of disintegrant, specifically Transdermal, Topical, and Mucosal Dosage Forms from about 1 to about 5 weight percent of disintegrant. Transdermal, topical, and mucosal dosage forms of the Disintegrants that can be used in pharmaceutical compo - 40 invention include , but are not limited to , ophthalmic solu sitions and dosage forms of the invention include , but are not tions , sprays , aerosols , creams, lotions, ointments , gels , limited to , agar -agar , alginic acid , calcium carbonate , micro - solutions, emulsions, suspensions , or other forms known to crystalline cellulose , croscarmellose sodium , crospovidone, one of skill in the art. See , e . g . , Remington ' s Pharmaceutical polacrilin potassium , sodium starch glycolate, potato or Sciences , 16th and 18th eds. , Mack Publishing , Easton Pa . tapioca starch , pre - gelatinized starch , other starches, clays , 45 ( 1980 & 1990 ) ; and Introduction to Pharmaceutical Dosage other algins, other celluloses , gums, and mixtures thereof. Forms, 4th ed ., Lea & Febiger , Philadelphia ( 1985 ) . Dosage Lubricants that can be used in pharmaceutical composi forms suitable for treating mucosal tissues within the oral tions and dosage forms of the invention include , but are not cavity can be formulated as mouthwashes or as oral gels . limited to , calcium stearate, magnesium stearate , mineral oil, Further, transdermal dosage forms include “ reservoir type ” light mineral oil , glycerin , sorbitol , mannitol, polyethylene 50 or " matrix type ” patches, which can be applied to the skin glycol, other glycols, stearic acid , sodium lauryl sulfate , talc , and worn for a specific period of time to permit the pen hydrogenated vegetable oil ( e . g . , peanut oil , cottonseed oil , etration of a desired amount of active ingredients . sunflower oil, sesame oil , olive oil, corn oil, and soybean Suitable excipients ( e . g . , carriers and diluents ) and other oil ) , zinc stearate , ethyl oleate , ethyl laureate , agar, and materials that can be used to provide transdermal , topical , mixtures thereof. Additional lubricants include , for example , 55 and mucosal dosage forms encompassed by this invention a syloid silica gel (AEROSIL 200 , manufactured by W . R . are well known to those skilled in the pharmaceutical arts , Grace Co . of Baltimore , Md. ) , a coagulated aerosol of and depend on the particular tissue to which a given phar synthetic silica (marketed by Degussa Co . of Plano , Tex . ) , maceutical composition or dosage form will be applied . CAB - O -SIL ( a pyrogenic silicon dioxide product sold by With that fact in mind, typical excipients include , but are not Cabot Co . of Boston , Mass . ), and mixtures thereof. If used 60 limited to , water , acetone , ethanol, ethylene glycol, propyl at all , lubricants are typically used in an amount of less than ene glycol, butane - 1 , 3 - diol , isopropyl myristate , isopropyl about 1 weight percent of the pharmaceutical compositions palmitate , mineral oil , and mixtures thereof to form lotions , or dosage forms into which they are incorporated . tinctures , creams, emulsions, gels or ointments , which are Parenteral and Intravascular Dosage Forms non - toxic and pharmaceutically acceptable . Moisturizers or Parenteral and intravascular dosage forms can be admin - 65 humectants can also be added to pharmaceutical composi istered to patients by various routes including , but not tions and dosage forms if desired . Examples of such addi limited to , subcutaneous, intravenous (including bolus injec - tional ingredients are well known in the art. See , e. g ., US 10 ,000 ,511 B2 31 32 Remington ' s Pharmaceutical Sciences, 16th and 18th eds. , cible vehicles such as, but not limited to , ethyl alcohol, Mack Publishing , Easton Pa . ( 1980 & 1990 ) . polyethylene glycol, and polypropylene glycol; and non Depending on the specific tissue to be treated , additional aqueous vehicles such as , but not limited to , corn oil , components may be used prior to , in conjunction with , or cottonseed oil , peanut oil , sesame oil, ethyl oleate , isopropyl subsequent to treatment with active ingredient of the inven - 5 myristate , and benzyl benzoate . tion . For example , penetration enhancers can be used to All documents mentioned herein are incorporated herein assist in delivering the active ingredients to the tissue . Suitable penetration enhancers include , but are not limited by reference in their entirety . to : acetone ; various alcohols such as ethanol, oleyl, and EXAMPLES tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide ; 10 dimethyl acetamide ; dimethyl formamide ; polyethylene gly col; pyrrolidones such as polyvinylpyrrolidone; Kollidon In order that the invention may be more fully understood , grades ( Povidone , Polyvidone ) ; urea ; and various water the following examples are provided . It should be under soluble or insoluble sugar esters such as Tween 80 (poly stood that these examples are for illustrative purposes only sorbate 80 ) and Span 60 ( sorbitan monostearate ) . and are not to be construed as limiting the invention in any The pH of a pharmaceutical composition or dosage form , way. or of the tissue to which the pharmaceutical composition or General Experimental Methodology dosage form is applied , may also be adjusted to improve 1 . To identify potent and selective small molecule modu delivery of one or more active ingredients . Similarly, the lators of MCL - 1 , a small molecule binding screen by polarity of a solvent carrier , its ionic strength , or tonicity can 20 competitive fluorescence polarization assay was con be adjusted to improve delivery . Compounds such as stear ducted . Small molecules were screened in parallel ates can also be added to pharmaceutical compositions or against the FITC -MCL - 1 SAHB /MCL - 1ANAC inter dosage forms to advantageously alter the hydrophilicity or action and the FITC -BAD BH3/ BCL - X , AC interac lipophilicity of one or more active ingredients so as to tion . Those compounds that preferentially disrupted the improve delivery . In this regard , stearates can serve as a lipid 25 FITC -MCL - 1 SAHB MCL/ - 1ANAC interaction were vehicle for the formulation , as an emulsifying agent or advanced to secondary screening analyses . surfactant, and as a delivery - enhancing or penetration - en 2 . A series of confirmatory binding assays were per hancing agent. Different salts , hydrates or solvates of the formed . First , the competitive binding assay using active ingredients can be used to further adjust the properties FITC -MCL - 1 SAHB /MCL - 1ANAC was repeated using of the resulting composition . 30 dose - response analysis to confirm dose - responsive Although methods and materials similar or equivalent to binding and determine small molecule K , values. Sub those described herein can be used in the practice of the sequently specificity analysis was performed by com present invention , preferred methods and materials are petitive fluorescence polarization binding assay of described below . The materials , methods, and examples are illustrative only and not intended to be limiting . Other 35 small molecules against the interactions of FITC - BID features and advantages of the invention will be apparent BH3 in combination with MCL - 1ANAC , BCL - 2AC , from the detailed description and from the claims. BCL - X AC , BCL -WAC , and BFL1/ A1AC . Small mol Kits ecules exhibiting high fidelity specificity for MCL This invention therefore encompasses kits which , when 1ANAC over the other anti -apoptotic proteins were used by the medical practitioner, can simplify the identifi- 40 advanced to SAR , binding site , and functional analysis . cation of subjects and the administration of appropriate Docking analysis was performed to confirm the com amounts of a compound of the invention or a pharmaceuti patibility of small molecule structure with an energeti cally acceptable salt, solvate or hydrate thereof to a patient. cally favorable docking site at the structurally defined A typical kit of the invention comprises one or more unit ( i. e . X - ray crystallography ) BH3- binding pocket of dosage forms of a compound of the invention or a pharma - 45 MCL - 1ANAC . Dilutional assays were performed to ceutically acceptable salt , solvate or hydrate thereof, and distinguish between non - covalent and covalent MCL - 1 instructions for identification of a subject. binders . These studies identified a series of covalent Kits of the invention can further comprise devices that are interactors ; for example , a novel site of interaction for used to administer a compound of the invention or a phar inhibiting MCL - 1 binding activity was identified based maceutically acceptable salt, solvate or hydrate thereof. 50 on covalent modification of MCL - 1 by the molecule at Examples of such devices include , but are not limited to , cysteine 286 . Finally , selective MCL - 1 inhibitors were intravenous cannulation devices, syringes, drip bags , patches , topical gels , pumps , containers that provide pro tested in a BAX -mediated liposomal release assay to tection from photodegradation , autoinjectors , and inhalers . evaluate their capacity to block the inhibitory /anti Kits of the invention can further comprise pharmaceuti - 55 apoptotic activity of MCL - 1. cally acceptable vehicles that can be used to administer one 3 . Small molecule modulators of MCL - 1 were then tested or more active ingredients. For example , if an active ingre in a cellular apoptosis sensitization assay using OPM2 dient is provided in a solid form that must be reconstituted cells exposed to subcytotoxic doses of TRAIL in the for parenteral administration , the kit can comprise a sealed presence or absence of serial dilutions of small mol container of a suitable vehicle in which the active ingredient 60 ecule . Sensitization activity was also confirmed by use can be dissolved to form a particulate - free sterile solution of CalcuSyn analysis of combination treatments. that is suitable for parenteral administration . Examples of Methods pharmaceutically acceptable vehicles include , but are not Sahb Synthesis . limited to : Water for Injection USP ; aqueous vehicles such Hydrocarbon -stapled peptides corresponding to BCL - 2 as, but not limited to , Sodium Chloride Injection , Ringer ' s 65 family BH3 domains and their FITC -BAla derivatives were Injection , Dextrose Injection , Dextrose and Sodium Chlo - synthesized , purified , and characterized using previously ride Injection , and Lactated Ringer 's Injection ; water -mis - described methodologies24 ,27 , 29 . US 10 ,000 ,511 B2 33 34 Anti -Apoptotic Protein Preparation . compound -modified MCL - 1 and unmodified MCL - 1, both Recombinant and tagless MCL - 1ANAC , BCL - 2 AC , before and after dilution , followed by FITC -BID BH3 ( 15 BCL -XLAC , BCL -WAC , and BFL1/ A1AC were produced nM ) to a 96 -well black flat bottom plate ( Costar) . FP using previously described methods27, 29 , 31 . measurements and analyses are then performed as described Small Molecule Screening . 5 above . Small molecule screens were performed at the Institute Identification of Covalent Modification Site by Mass for Chemistry and Cellular Biology at Harvard Medical Spectrometry . School and employed commercial libraries ( Asinex , Chem MCL - 1ANAC (90 ?M ) was incubated with a 1 : 1 . 25 ratio bridge , ChemDiv , Enamine , Life Chemicals, and May - of compound for 2 hours at room temperature in 150 mm bridge ) . A high - throughput competitive FP binding assay 10 NaC1, 50 mM Tris , pH 7 . 4 . Excess compound was removed was employed to screen for small molecules that disrupted by gel filtration and the protein was digested with trypsin the interaction between FITC -MCL - 1 SAHB ( 15 nM ) and overnight at 37° C . Peptides were analyzed by nano - LCI MCL - 1ANAC ( 45 nM ) . MCL - 1ANAC was expressed and ESI/ MS using a vented column assembly as described purified by FPLC and delivered by automated liquid handler (Ficarro et al. , 2009 ) 34. Briefly , peptides were injected using to 384 well plates , followed by addition of small molecule 15 an autosampler and HPLC (Waters NanoAcquity ) onto a libraries . After a 15 minute incubation at room temperature, self- packed precolumn ( 4 cm , 100 um I . D ., POROS10R2, FITC -MCL - 1 SAHB ( 15 nM ) was added to each well by Applied Biosystems) and gradient eluted ( - 30 % B in 20 liquid handler and FP read at equilibrium (e .g . 1 hr ). Small minutes, A = 0 . 2 M acetic acid in water, B = acetonitrile with molecule hits were re - examined in this assay using serial 0 . 2M acetic acid ) to the resolving column (self -packed 30 dilutions of the compounds to confirm dose -responsive 20 um I. D ., 12 cm of 5 um Monitor C18 , Column Engineering ) inhibition of FITC -MCL - 1 SAHB binding . and introduced to the mass spectrometer ( Thermo Fisher Confirmatory Binding Analyses . LTQ -Orbitrap XL ) via EST ( spray voltage = 2 . 2 kV) . The top The most potent hits were advanced to rigorous quanti - 8 most abundant precursors in each MS scan (image current fication of binding activity and specificity . First , serial detection , resolution = 30 ,000 ) were subjected to CAD ( elec dilutions of small molecule in triplicate were mixed with 25 tron multiplier detection , collision energy = 35 % ). A separate FITC -MCL -1 SAHB ( 15 nM ), followed by addition to targeted nano -LC /ESI / MS experiment was performed with 384 -well black Costar plates containing MCL - 1ANAC (45 compound -modified peptide (e . g. nM ) diluted in binding buffer (50 mM Tris , 100 mM NaCl, TINQES * CIEPLAESITDVLVR * C = modified cysteine ) ]) , 0 . 0625 % CHAPS , pH 8 . 0 ) . The plates were incubated in the which was subjected to HCD (higher energy collisionally dark at room temp until equilibrium was reached ( i. e . 30 activated dissociation ) . stabilization of binding isotherms) and FP (mP units ) deter - Liposomal Release Assay . mined using a microplate reader ( e . g . Spectramax ) . K ; Liposomes are prepared from a mixture of lipids that values were calculated by nonlinear regression analysis of reflect the composition of the outer mitochondrialmembrane dose - response curves using Prism software (Graphpad ). as previously described in detail (Pitter et al ., 2008 ) 31. Small molecule hits were then tested for their ability to 35 Aliquots ofmixed lipids ( 1 mg total ) are stored in glass at dissociate FITC - BAK BH3 from MCL - 1ANAC in a com - - 20° C . under nitrogen , and before use , resuspended in petitive FP assay, performed as described above except that liposome assay buffer ( 10 mM HEPES , 200 mM KC1, 1 mM small molecule dilutions were added to a solution of FITC - MgCl2 , pH 7 ) with 12 . 5 mM of the fluorescent dye ANTS BAK BH3 (25 nM ) and MCL - 1ANAC (250 nM ) to simulate ( 8 - aminonaphthalene - 1 , 3 , 6 - trisulfonic acid , disodium salt ) a physiologic displacement of pro - apoptotic BAK from the 40 and 45 mM of the quencher DPX ( p -xylene - bis - pyridinium inhibitory MCL - 1 protein . For rigorous specificity analysis , bromide ) . The resulting slurry is vortexed for 10 minutes the identical competitive FP - type experiment was performed and freeze - thawed five times alternating between liquid except that serial dilutions of small molecule hits were nitrogen and a 40° C . water bath . The solution is then passed mixed with the pan -anti -apoptotic binder FITC - BID BH3 through an Avanti Mini- Extruder Set (# 610000 ) equipped ( 15 nM ) , followed by addition to plates containing either 45 with a 100 - nm filter , followed by passage through a Sep MCL - 1ANAC , BCL - 2AC , BCL - X AC , BCL -WAC , or harose column (GE Healthcare ) to remove residual ANTS/ BFL1/ A1AC . We used PGEX vectors to express GST- MCL - DPX . The lipsomes are brought up to a volume of 3 mL to 1ANAC , BCL - 2AC , BCL -X , AC , BCL -WAC , and BFL1 / produce a final liposome stock . For the liposomal release A1AC , followed by thrombin cleavage and FPLC -based gel a ssay , a total volume of 30 uL is used in 384 well black flat filtration chromatography. Of note , the C -terminal alpha - 50 bottom plates ( Costar ) , and baseline fluorescence measure helix was deleted from each of these constructs to facilitate ments of 8 uL liposomes are made for 10 minutes using the protein expression and purification ; an additional N - terminal Tecan Infinite M1000 (excitation : 355 nm , emission : 520 deletion of MCL - 1 was performed to further enhance nm ) . Following the baseline read , recombinant MCL expression , purity , and stability . FP analysis was performed ANAC pre - incubated with a 1 : 1 ratio of compound is added as above and those molecules showing exclusive binding 55 to the liposomes . Next, 20 nM caspase -cleaved mouse BID activity for MCL - 1ANAC were advanced to structural dock - ( R & D systems) and 250 nM purified recombinant mono ing and functional testing . meric BAX is added , and fluorescence measurements ( F ) are Dilution Assay . recorded every minute from time zero ( FO ) until the release MCL - 1ANAC ( 2 uM , 500 mL) was incubated with com - readings plateau . The liposomes are then quenched with 1 % pound ( at a ratio well above each previously determined 60 Triton X - 100 ( 100 % release ; F100 ), and percent ANTS /DPX EC50 value ) for 1 hour at room temperature in FP buffer ( 100 release is calculated as ( ( F - FO ) / (F100 - F0 ) ) x100 . mM NaC1, 50 nM Tris , pH 8 ) . A small sample was taken as Cytochrome c Release Assays . a pre - dilution control. Compound - treated protein and Mouse liver mitochondria ( 0 . 5 mg/ mL ) are isolated and unmodified protein were then diluted to a volume of 20 mL , release assays performed as described31. Mitochondria are and subsequently re - concentrated to 500 mL using 10K 65 incubated with a serial dilution of MCL - 1 targeting small centrifugal filter units ( Amicon ) . FP binding analysis was molecule , singly or in combination with BID BH3 , and after performed as described above by adding serial dilutions of 40 minutes, the pellet and supernatant fractions are isolated US 10 , 000 ,511 B2 35 36 and cytochrome c quantitated using a colorimetric ELISA small molecule activity is also confirmed by monitoring for assay ( R & D Systems) . Percent cytochrome c released into blockade of activity upon co - treatment with the pan -capase the supernatant ( % cytoczup ) from releasable mitochondrial inhibitor Z - VAD . pools was calculated according to the following equation : % Pharmacokinetic Analyses. cytoc = [ (cytoc - cytoc )/ (cytoc . . ; - cytoc ) * 100 . 5 Lead small molecules undergo pharmacokinetic (PK ) where background release represents cytochrome c detected analysis in mice , performed in conjunction with the DF /HCC Clinical Pharmacology Core . LC /MS - based ana in the supernatant of vehicle - treated ( 1 % DMSO ) samples lytical assays are developed in order to detect and quantify and total release represents cytochrome c measured in 1 % compound levels in plasma. For PK analysis , small mol Triton - X 100 treated samples. All experimental conditions 10 ecules (e . g . 10 , 50 , 100 mg/ kg ) are injected by tail vein or are also tested on Bak - ' - mitochondria to ensure that the 10 intraperitoneally into male C57 /BL6 mice . Blood samples observed cytochrome c release from wild - typemitochondria are withdrawn by retro - orbital bleed at various time points derives from BAK activation . and plasma isolated for compound quantification , followed Immunoprecipitation Assay . by calculation of plasma half - life, peak plasma levels , total MCL - 1 - expressing cancer cells ( 10x10 ' ) are incubated 16 plasma clearance , and apparent volume of distribution . with the MCL - 1 targeting small molecule or vehicle in Small molecules that exhibit selective MCL - 1 targeting in serum - free media at 37° C . for 4 hours , followed by serum cells and exhibit a favorable pharmacokinetic profile re replacement for an additional 6 hours . After cellular lysis in advanced to in vivo testing. 50 mM Tris (pH 7 . 4 ) , 150 mM NaCl, 1 mM EDTA , 1 mM In Vivo Efficacy Studies . DTT , 0 . 5 % NP40 and complete protease inhibitor pellet , 20 Small molecule - sensitive cancer cell lines are retrovirally cellular debris is pelleted at 14 ,000 g for 10 minutes at 4° C . 20 transduced to achieve stable luciferase expression (PMMP The supernatant is exposed to pre - equilibrated protein A / G LucNeo ) and transplanted into SCID beige mice as previ ously described27 ,32 . Initial xenograft studies examine 5 sepharose beads and the pre -cleared supernatant subse mouse cohorts ( n = 10 ) , treated with either vehicle alone , low quently incubated with anti -MCL - 1 antibody for 1 . 5 hours at or high dose small molecule alone , or low /high dose small 4° C ., followed by the addition of protein A /G sepharose 25 molecule in combination with subtherapeutic dosing of beads for 1 hour . The beads are pelleted and washed with pro -apoptotic stimuli ( e . g . TRAIL , ABT- 737 , doxorubicin , lysis buffer for 10 minutes at 4° C . The washed bead are then etoposide , dexamethasone ) . Starting on experimental day 1 , pelleted , heated to 90° C . for 10 minutes in SDS loading mice receive a once daily tail injection of small molecule buffer , analyzed by SDS /PAGE , and then immunoblotted for ( e . g . 25 or 100 mg /kg , with or without combination treat MCL - 1 and BAK . 30 ment ) . For alternate day in vivo tumor imaging , mice are Crystallography . anesthetized with inhaled isoflurane and treated concomi Crystallization conditions for lead small molecule -MCL - tantly with intraperitoneal injection of D -luciferin . Photonic 1ANAC complexes are screened using 96 -well sitting drop emission is imaged ( 2 min exposure) using a Xenogen In plates set up using a Phoenix crystallization robot. Initial Vivo Imaging System and totalbody bioluminescence quan conditions include HT Index Screen , JSCG + Suite , and 25 tified by the integration of photonic flux (photons / sec) using Pro - Complex Suite . Screening around the best hit , including Xenogen ' s Living Image Software . The survival distribu varying pH , salt , and detergent concentrations , are per tions of experimentalmice are determined using the Kaplan formed to identify the best condition for crystal growth . Meier method and compared using the log - rank test. The Once generated , the crystals are removed , washed in the Fisher ' s exact test is used to compare the proportion ofmice crystallization buffer , and subjected to mass spectroscopy to who fail treatment, where treatment failure is defined as verify the presence of compound and protein within the 40 progression or death , and success as stable disease or crystal. The crystal is then soaked in cyroprotectant, flash regression . If a treatment response is observed with a frozen , and stored in liquid nitrogen . Suitable crystals are particular small molecule , three additional cohorts , treated examined at the Argonne National Laboratory synchrotron with either vehicle, small molecule , or small molecule facility . Phases are obtained by molecular replacement fol combination will be used for pharmacodynamic studies in lowed by data analysis and refinement ( Phaser, Phenix , and 45 which pro -apoptotic activity is evaluated in tissues by Coots software ) . TUNEL and activated caspase - 3 immunohistochemical Cellular Apoptosis Induction Assays . staining Small molecule inhibitors of MCL - 1 were screened in MCL - 1 - expressing cancer cell lines, such as OPM - 2 (mul Examples tiple myeloma ) cells . The cells are treated with the small 50 molecule MCL - 1 inhibitor, alone or in combination with Identification of MCL - 1 Selective Small Molecules by a subtherapeutic pro -apoptotic stimuli (e . g. TRAIL , Fas Competitive Fluorescence Polarization Binding Assay ligand , ABT- 737 ) , and then cell viability is measured at 48 Using FITC -MCL - 1 SAHB and MCL - 1ANAC . hours by MTT assay performed according to the manufac A high - throughput competitive FP binding assay was turer ' s protocol (Roche ) and quantitated by an ELISA employed to screen for small molecules that disrupted the microplate reader (Biorad ) . IC50 values were determined by 3 interactions between FITC -MCL -1 SAHB/ MCL - 1ANAC nonlinear regression analysis using Prism software (Graph and FITC - BAD BH3/ BCL - X , AC ( FIG . 1 ) . Compounds pad ) . Small molecules that decrease cell viability are then were ranked based upon their selectivity for MCL - 1ANAC screened for cellular apoptosis induction by annexin V over BCL - X , AC targeting , and tabulated ( see Table 1 ( in binding and FACS analysis , and by cell fractionation -based cluding Subtables Table 1 - A through Table 1 - P ) ) . mitochondrial cytochrome c release , as described28 . Apop - 60 The Structures of Small Molecule/ MCL - 1 Complexes tosis is also correlated with in situ dissociation of the Provide a Template for Refining MCL - 1 Binding Activity MCL - 1 /BAK complex ( immunoprecipitation ) performed as and Specificity . described above . As an additional measure of small mol - Using the crystal structure ofMCL - 1ANAC , the identified ecule MCL - 1 specificity , the identical experiments are con - small molecules were docked onto the BH3 -binding pocket ducted on wild - type vs . Mcl - 1 - - MEFs, to explicitly link the 65 ofMCL - 1ANAC to analyze the location and energetic favor selective MCL - 1 targeting capacity of the small molecules ability of interaction . The compounds covered discrete sub to sensitization of apoptosis . The caspase - dependency of regions of BH3- binding pocket topography ( FIG . 2 ) , pro US 10 ,000 , 511 B2 37 38 viding a blueprint for engineering selective , larger MCL -1ANAC C , BCL -2AC , BCL - XLAC , BCL -WAC , and molecules that incorporate combinations of molecules or BFL1/ A1AC . As exemplified in FIGS. 3 and 4 , small mol their subfragments to engage the complex and extended ecules identified as MCL - 1ANAC selective indeed exhibited BH3- binding surface of MCL - 1 . preferential displacement of FITC - BID BH3 from MCL Subclassification of Selective MCL -1 Inhibitors Based on 5 1ANAC compared to the other anti -apoptotic proteins tested . Non - Covalent Vs. Covalent Interactions. Structure - Activity Relationship Analysis of Selective To examine the mechanism of small molecule binding to MCL - 1 , the compounds were subjected to a rapid dilution MCL - 1 Inhibitors. assay ( FIG . 2C ) . Briefly , MCL - 1ANAC was pre - incubated Chemical derivatives of class A (FIG . 4C ) and J ( FIG . 4D ) with the small molecule inhibitors prior to dilution with 10 molecules demonstrated significant differences in binding buffer. The solution was concentrated to its original volume activity . Importantly , the five -member thiotriazole ring at and fluorescence polarization was performed . Compounds position R1 of class A increased binding affinity for MCL that bind covalently will remain bound to the protein , 1ANAC compared to smaller, non - aromatic substituents . At whereas compounds that bind reversibly will be exchanged the R2 position , aromatic residues were favored with small upon dilution . As a positive control, the rapid dilution assay 15 hydrophobic side chains or hydrogen bond acceptors at the confirmed that gossypol (1570G15 ) bound reversibly to MCL - 1ANAC ( FIG . 2C , top panel ) . In contrast, 1725P16 para -position of the phenyl ring. For class J, small aliphatic and 1597E07 failed to exchange upon dilution , indicating groups were well tolerated at the R1 position , and the that the compounds bind and covalently modified MCL trihydroxyphenyl ring at the R2 position demonstrated supe 1ANAC (FIG . 2C , middle , bottom panels ) . 20 rior binding affinity for MCL - 1ANAC . Finally , five - or Identification of a Novel Interaction Site for MCL - 1 six -member aromatic rings were favored at the R3 position . Inhibition Based on C286 Engagement. Specifically , analogs that contained methyl substituents at To localize sites of covalent modification . small mol- R3 displayed no activity unless paired with a large aliphatic ecule -treated MCL -1ANAC ( e . g . 1929A19 ) was subjected to group at the Rl position . mass spectrometry . An additional MALDI peak was 25 Small Molecule MCL - 1 Binders Sensitize Cancer Cells to observed for small molecule - treated protein , with the Pro -Apoptotic Stimuli . increase in mass corresponding to the molecular weight of Cancer cells that depend on MCL - 1 for survival were the covalently tethered compound . The MCL - 1ANAC -ad - treated with selective small molecule MCL - 1ANAC binders duct was further subjected to tandem MS /MS , which 20 in combination with other pro -apoptotic agents to assess for revealed cysteine 286 as the site of modification for synergistic anti - tumor activity . As exemplified in FIG . 5 , 1929 A19 (FIG . 2D ). Importantly , cysteine 286 is not located small molecule bioactives synergized with TRAIL to kill at the canonical BH3 binding pocket (FIG . 2D ), indicating OPM2multiple myeloma cells , as assessed by MTT viabil that engagement of this novel binding site can allosterically ity assay and by combination treatment analysis using regulate MCL - 1 ' s anti - apoptotic /BH3 -binding activity . To 35 CalcuSyn software . confirm the functional importance of C286 engagement, a Selective MCL - 1 Inhibitors Block the Anti -Apoptotic C286S construct of MCL -1ANAC was generated by site Function of MCL - 1 in a BAX -Mediated Liposomal Release directmutagenesis and small molecule binding activity was Assay . compared in competitive FP assays using FITC - BID BH3 4 The liposomal release assay is designed to mimic the and the wild - type and mutant MCL - 1ANAC proteins. functional release of mitochondrial cytochrome c by forma Whereas 1929A19 and 1616N08 competed with FITC -BID tion BAX - containing pores within the outer mitochondrial BH3 for MCL - 1ANAC binding, the molecules failed to membrane . Here , recombinant full length BAX was incu engage the C286S construct of MCL - 1ANAC , as demon bated with recombinant tBID in the presence or absence of strated by the capacity of FITC -BID BH3 to maintain 45 MCL - 1ANAC . As demonstrated in FIG . 6 , tBID triggered binding activity toward MCL - 1ANAC C286S in the presence BAX -mediated release of the fluorophore from the liposome of the compounds . in a time- dependent manner. MCL - 1ANAC blocked BAX Confirmation of Small Molecule Binding Activity and activation , inhibiting fluorophore release . By inhibiting Selectivity by Use of Competitive Binding Assays. MCL - 1ANAC , 1929A19 and 1616N08 dose - responsively To verify the capacity of the identified small molecules to 30 re - instated tBID - induced , BAX -mediated liposomal release selectively target MCL - 1ANAC , small molecule hits were of the fluorophore , highlighting the capacity of the small tested for their differential ability to dissociate FITC -BID molecule MCL - 1 inhibitors to block the anti - apoptotic activ BH3 from a panel of anti -apoptotic proteins, including ity of MCL - 1.

TABLE 1 - A Structure MCL- 1 % Bound BCL - XL % Bound Spec Values HO 34. 13126397 102. 3629331 68. 231669 ??

- OH

6 OH?? ?? US 10, 000 ,511 B2 39 40 39 US 10- continued. 0 . 31 B2 1.40 HO 16 . 68871081 105 . 341 6001 88 .652889

OH

?? OH OH

34 . 5462316354623163 100 . 760843760843 66. . 214611

NH2

35 .01128141 106 . 8345939 71 . 823312

odOH OH

56 . 60157162 106 . 2747212 49 .67315

CH, HN

Love 10 . 32290702 104 . 1661092 93 .843202

- OH

OUROH 1 .259544221 90 . 57802572 89 . 318482 HO .

TO Y * ??OH OH US 10, 000 ,511 B2 -continued 27 . 20036677 100 .4228001 73 . 222433

Cl Na+ Nat 3 . 955080904 101. 0243434 97 .069262 ?? HO

PL - 157DG15

HO

OH

Y OH

OH

41. 01271009 100 . 3606236 59 . 347914 OH HO . ol

32 . 90443509 97 . 09434568 64 . 189911 10(

HO 1111111 int - OH

111111** 1111

15 .93536133 98 . 2573418 82 . 32198 HO OH HO OHO - OH HO?? ?? ?? HOK ??

?? OH HO OH HO OH HO OH = o HOW > HOOH US 10 ,000 ,511 B2 44 -continued TABLE 1 - B

O= s= 0 Z

R1 = heterocycle or aromatic R2 = H or S -heterocycle or aromatic

Structure MCL - 1 % Bound BCL - XL % Bound Spec Values

2 . 332623997 103 . 4341651 101. 1015411 OST

OSNO 4 . 951710591 96 . 11650485 91 . 16479426

19 .67020024 96 . 38730675 76 . 71710652 HO

IZ ogsHN 19 .74954984 93 . 16376474 73 .4142149 US 10, 000 ,511 B2 45 -continued 35 . 6 98 . 3 62 . 7

S= 0

ZE 36 . 15465782 91. 34577213 55 . 19111431

=

O=

TABLE 1 - C

en OH R1 R2 where R3 = heterocycle or aromatic

Structure MCL- 1 % Bound BCL -XL % Bound Spec Values

9 .463295931 115 . 1848233 105 .7215274

OH N

?? - 0 . 3 108 . 4 108 . 7

00HN US 10, 000 ,511 B2 48 - continued

42 .48636709 113. 7228826 71 . 23651547

OH HN

??

27 . 16644285 93. 0177803 65 . 85133746

HO

HN

N :

43 .7012494 106 .4084449 62. 70719545

SOH OH N .

58 . 33277892 103. 7511722 45 . 41839332

OH HN US 10, 000 , 511 B2 50 -continued TABLE 1 - D

? -

Structure MCIL - 1 % Bound BCI - XI % Bound Spec Values

14. 6 101 . 3 86 . 7 N ??? - 2 . 04714193 75 . 96629122 78 . 01343315

NH -

-- 8 . 33210844 81 . 17127433 72 83916559

HN

?? 43 . 2 109 . 5 66 . 3 43 104 63 )

NH - - N - -- US 10, 000 ,511 B2 52 -continued 35 .72624681 99 . 74101397 64 .01476715

HN

37 . 6 98 . 3 60. 7

VN Hotthongolden 45 . 37762426 98 .52069172 53 . 14306746 DyTABLE 1 -- E where R1 = amide or ester R2 = heterocycle or aromatic

Structure MCL - 1 % Bound BCL -XL % Bound Spec Values

- 0 .733372059 80 . 39676939 81. 13014145 US 10 , 000 , 511 B2 53 54 -continued 11 . 728005051 79 . 877899881 68 . 14989482 :?? . NH

31 . 16639478 95. 42028511 64 . 25389033

OH

??? 38 .47471452 101. 451065 65. 97539199 18 . 740375731 77. 59057823 58 . 85020251

BT

???? 47. 00574838 99 .25113873 52 . 245390351

F F F \ / NH H US 10 ,000 ,511 B2 55 56 -continued H 53 . 4905597 105 . 62956951 52 . 139009761

47 .953523241 9690361633 48 . 95009309

Br NH ?????? 56 . 33127545 104 . 43159921 48 . 100323781

HN

?? 55 . 14812663 103 . 2306124 48. 08248577 US 10, 000 ,511 B2 57 58 - continued 64 .63766028 112 . 7047864 48 . 0671261 ??

62 . 71844661 110 .0825659 47 . 3641Hm :

40 . 50995892 86 . 74650699 46 . 23654860

66 .09053804 111 . 9999168 45 .90937874 obcoNH oslo -56 .10428305 101 . 763264 45 .65898096 US 10, 000 ,511 B2 59 -continued 69. 53461354 115 . 1985743 45 .66396079

= O= 68. 79611916 114 . 1804937 45. 38437457

TABLE 1 - F

HNE

Structure MCL - 1 % Bound BCL - XL % Bound Spec Values 14 . 30073607 84 .68236855 70 .38163248

HN US 10 , 000 ,511 B2 -continued 25 . 28012412 92 . 88964225 67. 60951814

NH

19 . 17808219 73 . 82692962 54 . 64884742

HNE

33 . 27874504 82 .61218664 49 . 33344159 s= 0 33. 27874804 O=

HN

TABLE 1 - 6

NC y NH Wss where R1 = heterocycle or aromatic

Structure MCL - 1 % Bound BCL - XL % Bound Spec Values

24 . 59486004 99 . 32600497 74 .73114493

NH

' s US 10, 000 ,511 B2 63 64 -continued 18 .96382387 92 .90700473 73. 94318087

s s

15 . 16614831 84 . 81906443 69 . 65291612

NH

OH

30 .48780488 79 .68386424 49 . 19605936

NH

TABLE 1 - H

NC

where R1 = aromatic or heterocycle R2 = CH2

Structure MCL - 1 % Bound BCL -XL % Bound Spec Values

- 10 . 5954323 87 . 16205145 97 .75748375 SM US 10 , 000 ,511 B2 65 - continued o us and 7 .037265168 we 97 . 35012677 90 . 3128616 HN o=n0 09

3 . 36867863 86 .24446993 82 . 8757913

- 7 . 07177814 75 . 49565787 82 . 56743601

N

BarHO 20 . 58823529 102. 1918705 81 .60363516

Br

30 . 19710502 107 .6879038 77 . 4907988

road?? US 10, 000 ,511 B2 -continued NH 21 . 8 98 . 2 76 . 4 O = S = O

HO .

15 . 04465661 89 . 36779259 74 . 32313598

oardsHO

31. 4290114 89 .23693465 57 . 80792326

?? pate Dat 30 . 81305821 84 . 78776478 53 . 97470658 US 10, 000 ,511 B2 69 70 -continued OH 3636. . 80576864 8989 . 70132202 70132202 5252. . 89555338 8955533 EZ HO .

38 . 82044965 91. . 5923775359237783 52 . 77192788

229. . 334770563471056 7878 . 7829676829966 49 .43352911

TABLE 11 - - 1 Ris

where R1 = H or sulfonamide -, ester - , or amide linked heterocycle or aromatic R2 = H or aromatic Structure MCL - 1 % Bound BCL -XL % Bound Spec Values CI e - 5 .219264716 el104 sector .6841563 109 . 903421

11. 19902121 76 . 15107324 64 .95205204

HO US 10, 000 ,511 B2 72 - continued 25 . 38058602 86 .7447645 61 . 36417848

grica 44 . 3557943 92 . 23836973 47 . 88257544 TABLE 1 - 1

N - R

N R2 where X = S or o R3 = aromatic or heterocycle

Structure MCL - 1 % Bound BCL - XL % Bound Spec Values

19 .08127208 95 . 08543531 76 . 00416323 - NH S

43. 51306119 106 . 9095962 63 .39656504 ;

N 08 asto 39 . 32183657 100 . 3628503 61. 041013731 HODO 0 N ' S US 10, 000, 511B2 73 74 -continued 4 41 . 816411 102 . 1683264 60. 35158527

HN

38 . 17542436 95 : 91685226 5 . 4142791 -

Br

45 .19897498 102. 0166733 56. 81769831

H

4198533 98 .204 ' 09 ' 2 56 . 2189f6f2 ?

HO

52 , 01193335 105643612 53, 6624278 US 10 , 000 , 511 B2 75 76 -continued 46. 42363385 99. 004091721 52 .58045787

HN

39 .96352925 92 . 21643287 52 .25290362 HN NH

30 .59011861 81. 340) 23006 50 .75011146 HN

HO DH

33 .00999412 82. 65729799 49 .64730387

N

HN . o ?? ?? 51 .18862475 99 . 22340759 48 . 03478284 US 10, 000 ,511 B2 77 78 -continued 55 . 45434348 100 .8550361 45 . 4006926

TABLE 1 - K

RY X - R3

where R1 = phenyl or furan X = N or C

Structure MCL - 1 % Bound BCL - XL % Bound Spec Values

2 .606828112 96 . 58770003 93 . 98087192

HO OH

15 . 57226143 89 . 44018281 73 . 86792137 attyNH

48 .21516581 111 .214653 62 . 99948715

NH US 10 , 000 , 511 B2 | 1979 | 80 -continued 43. 16379248 101. 2275149 58 .06372239

NH

13 . 28219273 66 .62503346 53 .34284073

HN

HN

48 . 95344507 97 .58836088 48 . 63491581

= N

215 .6015433 263 . 737406 48 . 13586273

H (

40 . 42192793 87 . 79749734 47 . 37556941

???HN ? US 10 , 000 , 511 B2 81 -continued

TABLE 1 - L

????? N R2 where X1 = 0 orS X2 = S or N

Structure MCL - 1 % Bound BCL - XL % Bound Spec Values

- 9 .610778443 87. 82745228 / 97. 43823072 !

( H

??? 19 . 68883273 12. 4094424 82 . 72060968 | | HN ?? - NH

33 . 27029276 112 .3457833 79 .07549058 ; | Br

???S HO 26. 07784431 102 . 7683663 ; 76 . 69052194

?????? 19 . 8 95 . 8 76 . 1 NH

HO US 10, 000 ,511 B2 83 84 * US 10-continued . 11 .02 38 .57217917 107 .8998073 69 .32762815

Doch NH 2121. . 28742515 28742515 9090 .. 2074208720742087 6868. . 91999572 91999572

0 OH

23 . 0 89 . 9 66 . 9

-z voo=no0

29 . 45492662 92 . 21556886 62 . 76064224

NH

27. 92957859 85 . 64814815 57 .71856956

HN

= to 38 .04746419 93 . 34466638 55 . 29720219

NH US 10, 000 ,511 B2 8585 86 -continued 21 . 54074074 76 .08572124 54 . 54498051

OH

OH Ö

42 .5929832 94 . 16497422 51. 57199103

N F

39 . 83535868 91 . 73339804 51 . 89803936

48 . 7076967' 98 . 89474361 50 . 1870469

NH

OH

58 . 50862999 105 . 8574397 47 . 34880968 NH , S ret US 10 ,000 ,511 B2 87 88 -continued TABLE 1 - M Ri,

R2 N where R1 = aromatic or heterocycle

Structure MCL - 1 % Bound BCL -XL % Bound Spec Values

- 23 . 10049446 101. 7468716 124 . 8473661

HO

- HO OHOH

- 16 . 27483907 89 . 94614004 106 . 2209791

HOMHO . - HOOH

TABLE 1 - N

Structure MCL - 1 % Bound BCL -XL % Bound Spec Values

HS - 65. 83063646 102 .6892674 168. 5199038 IN

N OH

- 6 . 59173964 129 . 3090909 135 . 9008305

HN NH ) US 10, 000 ,511 B2 8989 90 -continued - 23 . 10049446 101 . 7468716 124 . 8473661

?? . N - N HOHO OH

- 30 . 96202705 88 .78573776 119 . 7477648

- 5 . 219264716 104 .6841563 109. 903421 HO 1

- 0 . 3 108 . 4 108 . 7

OH oseHN - 1 .785938876 1106 .6729323 108. 4588712

- 11 . 55743971 96 . 56812237 108 . 1255621 OH HODot . OH US 10 , 000 ,511 B2 91 92 US 10-, continued 50 ,511 82 92 - 16 . 27483907 89 . 94614004 106 .2209791

HO?? N - N ?? ??

9 . 463295931 115 . 1848233 105 . 7215274

OH?? N

- 7 .035327937 98 . 55501099 105 .5903389 EZ

10 . 7155014 114 .7223418 104 . 0068404

NH2

NH2

2 . 332623997' 103 . 4341651 101 . 1015411

14 . 04124001 112 . 8657315 98 . 82449146 NH

HN NH US 10, 000 ,511 B2 93 94 -continued - 10 . 8685997 87 .36357022 98 .23216992

HN HN1

HO

HO OH

- 14 . 50299142 83 .61559031 98 . 11858173

NH?? VN 0

Orlaio - 10 . 5954323 87 . 16205145 97 . 75748375 ZI . S

- 9 .610778443 87 . 82745228 97 .43823072

- OH

OH 3 . 955080904 101. 0243434 97 . 0692625 ??HO HO OH

?? US 10, 000 ,511 B2 95 - continued - 18 . 5 78 . 1 ! 96 . 6

2 .606828112 96 / 58770003 93 .98087192 Br

HO OH

100. 32290702. 32290702 104 . 1661092 93 .84320213 - OH or OH 6 . 6 99 . 9 93 . 2

- NH2

NH OokO

4 .951710591 96 . 11650485 91 . 16479426 US 10, 000, 511 B2 97 ) $10, 00 , 98 -continued 7 . 0372651 68 97 . 35012677 90. 3128616

= HONS - -= -

1 . 259544221 90. 57802572 89. 3184815 H .

GS OH OH

NH 14. 82516366 103 . 9217319 89. 09656823 R

HO 16. 68871081 105 .341 6001 88 . 65288929

- OH OH OH- 14. 6 101 . 3 86. 7

?? - 13 . 00667806 71 .10363607 84. 11031413

OH ??? US 10, 000 ,511 B2 9999 100 -continued N 30 . 17353579 114 . 1379598 83 . 964424

16 . 19966207 199 . 90532748 83. 70566541

NH

9 . 240186268 92 . 49338093 83 .25319466 HO

HO auto9??

be 3 . 36867863 86 . 24446993 82 . 8757913

0 = 19 .68883273 102 . 4094424 82 .72060968

HN

NH US 10 , 000 ,511 B2 101 102 - continued 20 .72333282 103 .4206261 82 .69729329

6 . 516370589 89 .09642401 82 . 58005342

- 7 .07177814 75 .49565787 82 . 56743601

N

HO??

HO OH 15 . 93536133 98 . 2573418 82 . 32198048

HO . HO © OH Ho - O HO OH OH

??

?? OH

HO

HO OH OH O HÓHO OH

HoHO - O HO OH US 10 , 000 ,511 B2 103 104 10 us to- continued 20 . 58823529 102 . 1918705 81 .60363516 Br boute ogno - 0 . 733372059* 80 . 39676939 81. 13014145

2222. .9770388 104. . 006981006981 8181.. 02905928 02905928 olabo 1114. 4 9222 . 2 80880 . 8 - 3 .913592369 75 . 92785571 79 . 841448081

OH US 10, 000 ,511 B2 105 106 - continued 21. 93834459 101 . 5900478 79 .65170319

33 . 27029276 112 . 3457833 79 .07549058

Br

- 2 . 04714193 75 . 96629122 78 .01343315

NH

30 . 19710502 107 .6879038 77 . 4907988 oboroO soodsOH 19 .67020024 96 . 38730675 76 . 71710652

N

13 . 3744856 90 . 06707122 76 .69258563 OH

2004SHO 26 .07784431 102 . 7683663 76 .69052194 US 10 , 000 , 511 B2 107 108 -continued | NH2 21 . 8 98 . 2 76 . 4 0 = S = 0

HO

17 . 4 93. 7 76 . 4

HO

1919. . 8 95.. 8 8 76176 . 1 ??NH HO

19 .08127208 95 .08543531 76 .00416323 - NH S US 10, 000 ,511 B2 109 110 - continued 21 .79034158 97 . 16842962 75 . 37808804

21. 55274889 96 . 34785598 74 .79510709

24 . 59486004 99 . 32600497 74 . 73114493

NH

' s

15 .04465661 89 . 36779259 74 . 32313598

HO

18 . 96382387 92 . 90700473 73 . 94318087

??

15 . 57226143 89 . 4401828 73 . 86792137 N

NH CU US 10 , 000 , 511 B2 111 ? 112 -continued HN \ 19 . 74954984 93 . 6376474 73 .4142149 N /

= =0 : (

27 .20036677 100 .4228001 73 . 22243335

??Na + NatNa* 37, 92480367 111 . 0248078 73 . 1 ( ( ( ( ] 413 HO ??? ?? 30 ,22510381 103. 1419548 72 .916851 8 .322108744 81 . 17127433 72 .83916559

HN

( H 12 . 58907363 84 . 89878885 72 . 30971522

OH ??SO US 10, 000 , 511 B2 ii3 114 -continued p 1 on35 . 1128141 106. 8345939?? 71. 8233125

OH

OH

42 . 48636709 113. 7228826 71 . 236515471

OH HN <> HO - 5 . 849688118 65 . 32554791 71. 17523603 ??c .OH IINH , N 14 . 29140445 85. 26500639 70. 9736194 F F

???? ???? 58. 7 129 . 5 70. 8 11 . 97648788 82. 61082938 70. 6343415, P???

F US 10, 000 ,511 B2 115 116 -continued 14 . 30073607 84 .68236855 70 . 381632481

' N N

HN

- 7 . 305502846 62. 96737927 70 . 27288212

ZEZ

15 . 16614831 84 . 8190644 69. 65291612

NH

s

TOH sta 38 .57217917 107 . 8998073 69 .32762815 O=

NH otvaraNN 38 .64682418 107 . 8504978 69. 20367362 US 10 , 000 ,511 B2 117 118 - continued 21 .28742515 90 . 20742087 68 .91999572

??

31 .28187691 100 . 1843318 68 . 90245488

NH NE

19 . 37221111 87 .66541823 68 .29320712

HO 20 14 . 5 82 . 8 68 . 3

NH

HO 34 . 13126397 102 . 3629331 68 . 23166909 OH

?? HO ? ?? ?? US 10 , 000 , 511 B2 19 120 -continued 11. 72800505 79 .87789988 68 .14989482

??. NH

z | 39. .1 1 10710 .. 0 ( 6967. 9

SH

NH

25 . 28012412 9288964225 67. 60951814

NH

34 .53258845 102 .0162884 67 . 48369999

HN

23. 0 899 66 . 9

0- HN - s =

0 US 10 , 000 , 511 B2 121 | 122 | 121 -continued

3792480367 104 , 5573433 66 .6325396

HO

19243 . 2 19931095 66?? . 3

NH

SN

?? 30 . 47645315 96 . 72727273 66 . 25081958 ??? 3454623163 100 . 760843 66 . 2146114 HO .

NH , HO??? 23. 45468053 89 . 61072441 66 . 1564388

24 . 12504492 90 .1231625 6599811758

LZ US 10, 000 ,511 B2 123123 124 - continued 27 . 16644285 93 .0177803 65 . 85133746

HO

HN

. OH

- 8 .097006083 57 .6845011 65. 78150718

HN

??

66 '13604597 131 .2029473 65 . 06690133

HO NH

1119902121 76 . 15107324 6495205204

HO

4 . 577793748 69 . 52721494 64 . 94942119

IZ ast?? 18 . 95094399 83 .66485345 64 .71390946 US 10, 000 ,511 B2 125 126 - continued 26 . 7 91 . 4 64 . 7

34 . 3965106 99 .00834642 64 .61183582

IZ

31. 16639478 95 . 42028511 64 . 25389033

“ ??

32 .90443509 97. 09434568 64 . 18991059 .inter HO 1111 *01111 OH

ttitill*

36 .11902501 100 . 2785294 64 . 15950436

HN

HO o US 10, 000 ,511 B2 127 128 -continued 35 . 72624681 99 .74101397 64 .01476715

N

HN

21 .78361457 85 . 37461774 63 .59100317

39 .7488359 103 .3154122 63 . 56657629 0 . NH

41 . 28528975 104 . 7348973 63 .44960756 F F IN HN O S= toshxO= 43 . 51306119 106 . 9095962 63 .39653504 asteroO = NH US 10 , 000 , 511 B2 129 130 - continued Br 18 .57176712 81 , 71632703 63 .14455991

N - NH

29 . 27084059 92. 34063996 63 .06979937

HN ??S -

31 .39551493 94 .41842001 63. 02290508

??NH

??N

0H ??? 48 . 21516581 111 . 214653 62 .99948715 NH

?? OH 27 . 71898883 90 . 70584478 62 . 98685595

???? 38 . 47471452 101 . 4501065 6297539199 ( ] ?? | US 10 , 000 , 511 B2 131131 132 -continued - 19 .61126884 43. 27239348 62. 88366232

6 . 012957705 68 . 85183202 62 . 83887431

N " NH

??? 29 .45492662 92 .21556886 62 .76064224

NH ?????? 35 . 6 98 . 3 62 . 7 0 0

43 . 7012494 106 . 4084449 62 . 70719545

OH ) OH ?

40 .93873164 103 .0201067 62 . 08137505 |

31 . 21264846 93 .01166131 61 . 79901285 US 10 ,000 , 511 B2 | 133133 134 - continued 39. 52157023 101. 2560945 61 . 73452431

N

9 . 41744 120 . 9256794 61. 48393538

OH

25 . 38058602 86.747645 61 . 36417848

GT 39. 32183657 100 . 3628503 61 . 04101373

HO ' \ n Ss

18 . 43595701 79 . 39308398 60 . 95712697

- NH )

SearHO - 29642702780278 3129614531 . 23996145 4608264360 , 88266423 Brorg Jo 27. 27621091 88 . 11755764 60 . 84134673 US 10 , 000 , 511 B2 135135 136 us1960s -continued 1 8 37 . 6 98 .16 3 60 . 7

4.4 .6132808756673280876 65656028457. 02894576 60 . 35566489

0H ???

41 .8167411 102 . 1683264 60 . 35158527

HN : ???

39 . 93161377 99 .39343581 59. 46182205 ????????=? 41 .01271009 100 . 360 6236 59 . 34791354 OH HO ??? 47 . 18965126 106 . 5320264 59 . 34237511 US 10, 000 ,511 B2 137 138 -continued 36 .55154869 95 . 64034877 59 .08880009 Bgrey 41 .78912826 100 . 8274721 59 .03834381

OH

Br 34 .66750489 93 .68924174 59 .02173685

N , NHNH

18 . 74037573 77 . 59057823 58 . 85020251

IZ.

OH 22 .81374655 81 . 47543659 58 .66169004

HN CNH US 10, 000 ,511 B2 139 140 - continued 39 .52194519 98 .09873249 58 . 5767873

F NH OS

?? OS

Z

Z-

OH 39 . 11594696 97 .6372151 58 . 52125455

30 . 40891164 88 . 85677858 58 .44786694

25 . 2061285 83 . 4987795 58 .29265464 d o OH tenen US 10 , 000 , 511 B2 | 141 | 142 -continued 43 .16379248 101 . 2275149 58 .06372239

NH

31 . 4290114 89 . 23693465 57 , 80792326

H (

7 . 609675481 65 .35257732 57 . 742900184

HN

?? 3838. . 17542436 17542436 959591685226. 91685226 5757. . 74142791 74142791 ON- !

Br

2792957859 85 . 64814815 57. 71856956

F

48 ,94267421 106 .2615101 57 ,31883592 HN US 10, 000 ,511 B2 143 144 - continued 32 . 32116549 89 .4407446 57 . 11957911

38 . 73289121 95 . 62561095 56 . 89271974

Choo 45 . 19897498 102 .0166733 56 .81769831

NH

IZIZ box 35 . 39582849 92 . 03372469 56 .6378962

34 . 63356974 90 . 94954311 56 . 31597337

??

44 . 3733406 100 .6715454 56 .29820482

NH DioumitryO=n0 41. 985733 98 . 20470972 56 . 21897672

HO?? TO US 10 ,000 , 511 B2 145 146 145 -continued F 45 . 61615856 101 . 6646949 56 .04853639 F

F

F *

41 . 13775626 97 .06777083 55 . 93001457 ?

7 . 821811681 63 . 5443038 55 . 72249212 ? - NH2

45. 56074766 101 . 2544661 55 . 69371839 ? ??

43. 76933274 99 .37348265 55 . 60414991

43. 36958596 98 .96808255 55 . 5984966

HN US 10 , 000 ,511 B2 147147 148 -continued Nw 35 . 11114267 90 .69859962 55 .58745694

HNY 29 .48940031 85 .03813193 55 . 54873162

| H2N NN 35 . 03258269 !90 . 44736221 55 .41477952

55 .47976701 110 . 818343 55 . 3385760

38 .04746419 93 . 34466638 55 .29720219

NH

40 . 13238539 95 . 37388609 55 . 2415007 ta Osta 44 . 19099554 : 99 . 42563929 55 . 23464375 US 10 , 000 ,511 B2 149149 150 - continued LOH 33 . 24656324 87 . 76949553 54 . 52293229

??

ZE 36 . 15465782 91 . 34577213 55 . 19111431

2721. . 275491 2754491 8282274192 . 27419222 54. . 99874311 HN

36 .59699818 91 . 44689379 54 . 84989561 Ô 37 . 48100881 !92 . 20830691 54 . 7272981 HN

19 . 17808219 73. 82692962 54 . 64884742

HN ?