US010548864B2

(12 ) United States Patent ( 10 ) Patent No.: US 10,548,864 B2 Lu et al. (45 ) Date of Patent : Feb. 4 , 2020

(54 ) ENHANCED ATRA - RELATED COMPOUNDS (58 ) Field of Classification Search FOR THE TREATMENT OF None PROLIFERATIVE DISEASES , AUTOIMMUNE See application file for complete search history . DISEASES , AND ADDICTION CONDITIONS (56 ) References Cited (71 ) Applicants :Beth Israel Deaconess Medical U.S. PATENT DOCUMENTS Center, Inc., Boston , MA (US ) ; 4,683,195 A 7/1987 Mullis et al . Pinteon Therapeutics , Inc. , Concord , 4,683,202 A 7/1987 Mullis MA (US ) 5,223,409 A 6/1993 Ladner et al . 5,283,317 A 2/1994 Saifer et al. (72 ) Inventors : Kun Ping Lu , Newton , MA (US ) ; Xiao 5,459,039 A 10/1995 Modrich et al . 5,498,531 A 3/1996 Jarrell Zhen Zhou , Newton , MA (US ) ; Shuo 5,952,467 A 9/1999 Hunter et al. Wei , Chestnut Hill , MA (US ) ; Lijun 5,972,697 A 10/1999 Hunter et al. Sun , Harvard , MA (US ) ; Michelle 6,462,173 B1 10/2002 Lu et al. Lynn Hall , Somerville , MA (US ) 6,495,376 B1 12/2002 Lu et al . 6,596,848 B1 7/2003 Hunter et al. (73 ) Assignee : Beth Israel Deaconess Medical 6,649,611 B2 11/2003 Blumberg et al. 6,764,698 B1 7/2004 Byun et al. Center, Inc. , Boston , MA (US ) 7,125,677 B2 10/2006 Hunter et al. 7,125,955 B2 10/2006 Hunter et al. ( * ) Notice : Subject to any disclaimer, the term of this 7,148,003 B2 12/2006 Hunter et al. patent is extended or adjusted under 35 7,161,060 B1 1/2007 Duff et al. U.S.C. 154 ( b ) by 0 days. 7,164,012 B2 1/2007 Hunter et al. (Continued ) ( 21) Appl. No.: 15 /557,731 FOREIGN PATENT DOCUMENTS ( 22 ) PCT Filed : Mar. 10 , 2016 JP 2004-532390 A 10/2004 (86 ) PCT No.: PCT /US2016 / 021759 WO WO - 94 / 10300 A1 5/1994 $ 371 (C )( 1 ), (Continued ) (2 ) Date : Sep. 12 , 2017 OTHER PUBLICATIONS (87 ) PCT Pub . No .: WO2016 / 145186 Jaeger et al. (European Journal of Medicinal , 28 ( 4 ), 275-290 , 1993 ). * PCT Pub . Date : Sep. 15 , 2016 U.S. Appl . No. 61/ 490,338 , Lu et al . U.S. Appl. No. 61/ 968,862, Lu et al. (65 ) Prior Publication Data Curtis et al. , “ The genomic and transcriptomic architecture of 2,000 US 2018/0064666 A1 Mar. 8 , 2018 breast tumours reveals novel subgroups, " available in PMC Dec. 21, 2012 , published in final edited form as : Nature 486 (7403 ) : 346-52 ( 2012 ) ( 15 pages ). Al- Hajj et al. , “ Prospective identification of tumorigenic breast Related U.S. Application Data cancer cells, ” Proc Natl Acad Sci U.S.A. 100 ( 7) :3983-8 (2003 ). Bild et al. , “ Oncogenic pathway signatures in human cancers as a (60 ) Provisional application No. 62 / 177,395 , filed on Mar. guide to targeted therapies ,” Nature . 439 ( 7074 ) :353-7 (2006 ) . 12 , 2015 . Carell et al ., “ A novel procedure for the synthesis of libraries containing small organic molecules ,” Angew Chem Int Ed Engl . (51 ) Int. Ci. 33 (20 ) :2059-2061 ( 1994 ) . A61K 31/196 ( 2006.01 ) (Continued ) A61K 45/06 ( 2006.01 ) A61K 31/122 (2006.01 ) Primary Examiner Bong- Sook Baek A61K 31/4152 ( 2006.01) (74 ) Attorney, Agent, or Firm — Clark & Elbing LLP A61K 31/4166 ( 2006.01 ) ( 57 ) ABSTRACT A61K 31/4192 ( 2006.01 ) The invention features all -trans retinoic acid (ATRA ) -re A61K 31/4245 ( 2006.01) lated compounds capable of associating with Pinl and A61K 31/452 ( 2006.01) methods of treating a proliferative disorder characterized by A61K 31/5375 ( 2006.01 ) elevated Pinl marker levels, Pin1 degradation , and / or A61K 31/203 (2006.01 ) reduced Pin1 Ser71 phosphorylation in a subject by admin A61K 31/4196 ( 2006.01) istering an ATRA -related compound . The invention also (52 ) U.S. Ci. features methods of treating proliferative disorders , autoim ??? A61K 31/196 ( 2013.01) ; A61K 31/122 mune diseases , and addiction conditions (e.g. , diseases, (2013.01 ) ; A61K 31/203 (2013.01 ) ; A61K disorders, and conditions characterized by elevated Pin1 31/4152 ( 2013.01) ; A61K 31/4166 (2013.01 ) ; marker levels )by administering an ATRA - related compound A61K 31/4192 (2013.01 ) ; A61K 31/4196 in combination with another therapeutic compound . ( 2013.01 ) ; A61K 31/4245 (2013.01 ) ; A61K 31/452 (2013.01 ) ; A61K 31/5375 (2013.01 ) ; 17 Claims, 83 Drawing Sheets A61K 45/06 (2013.01 ) Specification includes a Sequence Listing . US 10,548,864 B2 Page 2

( 56 ) References Cited Davis et al. , “ RAC1P29S is a spontaneously activating cancer associated GTPase .” Proc Natl Acad Sci U.S.A. 110 ( 3 ) :912-7 U.S. PATENT DOCUMENTS ( 2013 ) . Dontu et al. , “ In vitro propagation and transcriptional profiling of 7,175,830 B2 2/2007 Collins et al. 7,592,145 B2 9/2009 Bao et al. human mammary stem /progenitor cells ,” Genes Dev. 17 ( 10 ): 1253 8,129,131 B2 3/2012 Lu et al. 70 ( 2003 ). 8,258,099 B2 9/2012 Lu et al. Elenbaas et al. , “ Human breast cancer cells generated by oncogenic 8,771,693 B2 7/2014 Lu et al. transformation of primary mammary epithelial cells ,” Genes Dev . 2002/0002552 Al 1/2002 Schultz et al . 15 ( 1 ): 50-65 ( 2001) . 2002/0025521 Al 2/2002 Lu et al . Erb et al. , “ Recursive deconvolution of combinatorial chemical 2002/0106348 A1 8/2002 Huang et al . libraries, ” Proc Natl Acad Sci US A. 91 (24 ) : 11422-11426 ( 1994 ). 2004/0176912 A1 9/2004 Sowadski et al . 2005/0159485 A1 7/2005 Jost - Price et al. Esnault et al ., “ Pin1 modulates the type 1 immune response, ” PLoS 2005/0239095 Al 10/2005 Lu et al. One. 2 ( 2 ) :e226 ( 2007 ) ( 9 pages ) . 2005/0250742 A1 11/2005 Dagostino et al . Extended European Search Report for European Patent Application 2006/0018899 Al 1/2006 Kao et al . No. 13800857.8 , dated Dec. 1 , 2015 (7 pages ) . 2006/0074222 A1 4/2006 Lu et al. Forbes et al. , “ COSMIC : mining complete cancer genomes in the 2007/0072875 A1 3/2007 McMaster Catalogue of Somatic Mutations in Cancer, ” Nucleic Acids Res. 2007/0203236 A1 8/2007 Smith et al. 2008/0118505 Al 5/2008 Tedder 39: D945-50 (2011 ). 2008/0214470 A1 9/2008 Lu et al. Gianni et al. , “ Inhibition of the peptidyl -prolyl - isomerase Pinl 2008/0248043 A1 10/2008 Babcook et al. enhances the responses of acute myeloid leukemia cells to retinoic 2009/0053209 A1 2/2009 Malter et al . acid via stabilization of RARalpha and PML -RARalpha , ” Cancer 2009/0105249 Al 4/2009 Benjamin et al . Res. 69( 3 ) : 1016-26 ( 2009 ). 2009/0258352 A1 10/2009 Lu et al. Ginestier et al. , “ Distinct and complementary information provided 2010/0010084 Al 1/2010 Yu by use of tissue and DNA microarrays in the study of breast tumor 2010/0278832 A1 11/2010 Kamogawa et al . markers, ” Am J Pathol. 161( 4 ) : 1223-33 (2002 ). 2011/0034554 A1 2/2011 Washington International Preliminary Report on Patentability for International 2011/0065704 Al 3/2011 Ryder 2011/0077250 A1 3/2011 Ryder Application No. PCT /US2015 / 040771 , dated Jan. 17 , 2017 ( 9 2011/0077298 A1 * 3/2011 Chen C07C 69/734 pages) . 514/529 International Preliminary Report on Patentability for International 2011/0104756 A1 5/2011 Rodriguez et al . Patent Application No. PCT/ US2015 /021522 , dated Sep. 21, 2016 2012/0183560 A1 7/2012 Akassoglou (8 pages) . 2013/0028900 A1 1/2013 Lu et al. International Search Report and Written Opinion for International 2014/0086909 A1 3/2014 Lu et al . Application No. PCT/ US13 / 44747, dated Nov. 12, 2013 ( 18 pages) . 2014/0219957 A1 8/2014 Lu et al. International Search Report and Written Opinion for International 2014/0242100 Al 8/2014 Lu et al. Application No. PCT/ US14 / 27017 , dated Oct. 28 , 2014 ( 19 pages ). 2015/0044278 A1 2/2015 Lu et al . International Search Report and Written Opinion for International 2015/0133442 Al 5/2015 Lu et al . Application No. PCT /US15 /21522 , dated Aug. 10 , 2015 ( 19 pages) . International Search Report and Written Opinion for International FOREIGN PATENT DOCUMENTS Application No. PCT/ US15 /40771 , dated Jun . 30 , 2016 ( 13 pages) . International Search Report and Written Opinion for International WO WO -94 / 16101 A2 7/1994 Application No. PCT/ US16 /21759 , dated Aug. 12 , 2016 ( 18 pages) . WO WO - 97 / 17986 A1 5/1997 International Search Report and Written Opinion for International WO WO - 99 / 09969 A1 3/1999 Application No. PCT/ US2012 /029077 , dated Jul. 18 , 2012 (8 pages) . WO WO -02 /064015 A2 8/2002 WO WO -02 /065091 A2 8/2002 International Search Report for International Application No. PCT/ WO WO - 02 /092765 A2 11/2002 US2012 /039850 , dated Oct. 3 , 2012 ( 3 pages ). WO WO - 03 /073999 A2 9/2003 Jeong et al. , “ Novel role of Pinl induction in type II collagen WO WO - 2004 /016751 A2 2/2004 mediated rheumatoid arthritis ,” J Immunol . 183 ( 10 ): 6689-97 (2009 ). WO WO - 2004 / 101745 A2 11/2004 Kao et al. , “ Correlation ofmicroarray -based breast cancer molecu WO WO - 2005 / 027727 A2 3/2005 lar subtypes and clinical outcomes: implications for treatment WO WO - 2006 / 002097 A2 1/2006 optimization ,” BMC Cancer . 11: 143 ( 2011) ( 15 pages ). WO WO - 2006 / 028576 A2 3/2006 Keller et al. , “ Defining the cellular precursors to human breast WO WO - 2007 / 133702 A2 11/2007 cancer, ” Proc Natl Acad Sci U.S.A. 109 ( 8 ): 2772-7 (2012 ) . WO WO - 2008 / 137488 A1 11/2008 Kunju et al. , “ EZH2 and ALDH - 1 mark breast epithelium at risk for WO WO - 2009 / 146218 A2 12/2009 breast cancer development, ” Mod Pathol. 24 (6 ): 786-93 (2011 ) . WO WO - 2010 / 081488 A1 7/2010 Lam et al. , “ Prolyl isomerase Pinl is highly expressed in Her2 WO WO - 2010 / 141738 A2 12/2010 WO WO - 2011 /056561 Al 5/2011 positive breast cancer and regulates erbB2 protein stability ,” Mol WO WO - 2011 / 104671 Al 9/2011 Cancer 7 (91 ) : 1-12 (2008 ) . WO WO - 2012 / 125724 A1 9/2012 Lee et al. , “ Death -associated protein kinase 1 phosphorylates Pin1 WO WO - 2012 / 149334 A2 11/2012 and inhibits its prolyl isomerase activity and cellular function ,” Mol WO WO - 2012 / 162698 Al 11/2012 Cell. 42 ( 2 ): 147-59 (2011 ). WO WO - 2013 / 185055 A1 12/2013 Liou et al. , “ Loss of Pinl function in the mouse causes phenotypes WO WO - 2014 / 152157 A2 9/2014 resembling cyclin D1- null phenotypes ,” Proc Natl Acad Sci U.S.A. WO WO - 2015 / 143190 A1 9/2015 99 ( 3 ) : 1335-40 ( 2002 ) . WO WO - 2016 /011265 A2 1/2016 Luo et al. , “ Prolyl isomerase Pinl acts downstream of miR200c to WO WO - 2016 / 145186 A1 9/2016 promote cancer stem - like cell traits in breast cancer, ” Cancer Res. 74 ( 13 ) : 3603-16 (2014 ). OTHER PUBLICATIONS Ma et al. , “ A functional polymorphism in PIN1 that prevents its Cerami et al. , “ The cBio cancer genomics portal: an open platform suppression by AP4 is associated with delayed onset of Alzheimer's for exploring multidimensional cancer genomics data ,” Cancer disease, " available in PMC Apr. 1 , 2013 , published in final edited Discov. 2 ( 5 ) :401-4 (2012 ) . form as : Neurobiol Aging. 33 ( 4 ) : 804-13 (2012 ) ( 18 pages ) . Cho et al. , “ An unnatural biopolymer, ” Science. 261( 5126 ): 1303 Mani et al. , “ The epithelial- mesenchymal transition generates cells 1305 ( 1993 ). with properties of stem cells ,” Cell 133 : 704-715 (2008 ). US 10,548,864 B2 Page 3

( 56 ) References Cited Schmidt et al. , “ The humoral immune system has a key prognostic impact in node - negative breast cancer, ” Cancer Res. 68 ( 13 ): 5405 OTHER PUBLICATIONS 13 ( 2008) . Tun -Kyi et al. , “ Essential role for the prolyl isomerase Pin1 in Mori et al. , “ A dual inhibitor against prolyl isomerase Pinl and Toll - like receptor signaling and type I interferon -mediated immu nity ,” Nat Immunol. 12 ( 8 ): 733-41 (2011 ) (27 pages ) . cyclophilin discovered by a novel real - time fluorescence detection Written Opinion of the International Searching Authority for Inter method ,” Biochem Biophys Res Commun . 406 (3 ) :439-43 ( 2011 ). national Application No. PCT/ US2012 /039850 , dated Oct. 3 , 2012 Nagaoka et al. , " Possible involvement of peptidylprolyl isomerase (5 pages) . Pin1 in rheumatoid arthritis ,” Pathol Int . 61( 2 ) : 59-66 (2011 ). Wulf et al. , " Pin1 is overexpressed in breast cancer and cooperates Office Action for U.S. Appl. No. 14 / 334,052 , dated Nov. 20 , 2014 with Ras signaling in increasing the transcriptional activity of c - Jun (21 pages) . towards cyclin D1, ” EMBO J. 20 ( 13 ) : 3459-72 ( 2001) . Parker et al. , “ Supervised risk predictor of breast cancer based on International Preliminary Report on Patentability for International intrinsic subtypes, ” J. Clin . Oncol. 27 ( 8 ): 1160-7 (2009 ). Patent Application No. PCT/ US2013 / 044747 , dated Dec. 9 , 2014 ( 8 Ranganathan et al. , “ Structural and functional analysis of the mitotic pages ). rotamase Pinl suggests substrate recognition is phosphorylation Notice of Reasons for Rejection and English Translation for Japa dependent ,” Cell. 89 (6 ): 875-86 ( 1997 ). nese Patent Application No. 2015-516246 , dated Mar. 28 , 2017 ( 12 Ryo et al. , “ Pinl regulates turnover and subcellular localization of pages) . beta - catenin by inhibiting its interaction with APC ,” Nat Cell Biol. 3 ( 9 ) :793-801 ( 2001) . * cited by examiner U.S. Patent Feb. 4 , 2020 Sheet 1 of 83 US 10,548,864 B2

HIF-1* Rab2A S6K Oct4* RBBP8# ]??????GRK2* PTP-PESTNanog* 11)Smad# RUNX3#

Survivin 1 SF-1 #KLF10 PTP FBW7# ??? Tax* AMPK Rb PKM2V-Rel HBX* #:preventproteindegradation *:preventproteindegradation Cdk10 21 FAK GRK2 p53M Btk ER* 19Tumorsuppressors/growth 32Oncogenes/ Smad# inhibitoryregulators Stat3* ALLIIRARa# growth-promotingregulators Raf-1 Activate Inactivate Fox04# NF-kB* AIB1 Bax PLK TRF1#

C-Myb SUV39H1# D1*Cyclin Daxx# Fos*C- Pin1 SMRT# AKT* les PML# C-Jun* inhibitors

Notchß-catenin* Mcl-1* F Neu DrugTargets DDream targets HotTargets but,non-druggable Figure1 U.S. Patent Feb. 4 , 2020 Sheet 2 of 83 US 10,548,864 B2

. K63 ????

WW

Q13 ONF134 2B M130 R69

2A Figure2 U.S. Patent Feb. 4 , 2020 Sheet 3 of 83 US 10,548,864 B2

OH

vice,Fenretinide Boxarotene numberCompound you Compound(uMI *Retinol Retinal* ATRA13CRA+ 150 2J CompoundIuM 2.37 Basededateandtheha WW 2D 20 7A ??????? NA 2F Photoaffinity 13-ctsgetinoicacid13CRA()AlltransretinoicacidATRA 212 a ** Conc.M 0.02.55.07.510.0 THATRAIUMI Yata 13CRA 30 2E 80 1802C 2G 2H Figure2 U.S. Patent Feb. 4 , 2020 Sheet 4 of 83 US 10,548,864 B2

*** 698 K63

Q131 F134 M130 1122 2L

2K Figure2 U.S. Patent Feb. 4. 2020 Sheet 5 of 83 US 10,548,864 B2

R69 Residuesidechaindistribution 0131 H59

Q131 $154 H157 3C Residueswithin4AngstromsofATRA S72 D112 L122 M130 H59 K63 R68 R69 S71 Interfacesurface

?? 3B Figure3 U.S. Patent Feb. 4. 2020 Sheet 6 of 83 US 10,548,864 B2

Residuesidechaindistribution R69 C113 D153 $154 G155 1156 1159 BE P133 F134 E135 S138 V150 T152 Q129 125 Q131 K132 4C Residueswithin8AngstromsofATRA D112 C113 S114 $115 1122 0129 M130 S71 572 W73 R74 Q75 E76 178 5111 H59 L60 L61 K63 S67 R68 R69 Interfacesurface

4A 4B Figure4 U.S. Patent Feb. 4 , 2020 Sheet 7 of 83 US 10,548,864 B2

1156 H157 1159 F134 E135 S138 V150 T152 D153 $154 G155 RSR69 115$ L122 F125 Q129 M130 Q131 K132 P133 F134 44122 L132 138$ H59 L60 L61 K63 R68 R69 D112 C113 Residuesclosetocyclohexenylmoiety within8Angstromswithin4Angstroms5B

R68 $154 HX57 H59 R68 L122 M130 0131 F134 S154 H157

Figure5 U.S. Patent Feb. 4 , 2020 Sheet 8 of 83 US 10,548,864 B2

T152 D153 S154 G155 H157 1113 within8Angstroms R68Q131 S72$114 115W73$ R74L122 F125075 Q129178 S111M130 D112Q131 C113F134 M230 122 Q129 H59 L61 K63 67$ R68 R69 P70 $71 todoublebondsResiduesclose

11

within4Angstroms K63 R68 R69 S71 72$ D112 S154

6A 6Figure U.S. Patent Feb. 4 , 2020 Sheet 9 of 83 US 10,548,864 B2

E7%

572 K63 D211$ H157L122E135 R69 H59S71D112Q131D153 L60S72C113K132S154 L61W73S114P133G155 K63R74$115F1341156 R68E76F125S1381159 within8Angstroms V150R69Q129178 P70S111M130T152 L122Q75$67 Residuesclosetocarboxylicgroup 7B

within4Angstroms K63 R69 71

7? Figure7 U.S. Patent Feb. 4. 2020 Sheet 10 of 83 US 10,548,864 B2

P5pocket P4pocket P6pocket

PotentialpocketsforenhancedATRA-relatedcompounds Interfacesurface

P1pocket Potential P3pocket P2pocket Figure8 U.S. Patent Feb. 4. 2020 Sheet 11 of 83 US 10,548,864 B2

119 Residuesidechaindistribution G120

PotentialpocketP1-within4Angstroms 9C

extendedfrom1122)( residues:Important K117C113D121 118L122114$ R119115$ G120A116 Interfacesurface

9A 9B Figure9 U.S. Patent Feb. 4. 2020 Sheet 12 of 83 US 10,548,864 B2

Residuesidechaindistribution (113, 1 Mi30 BIZI PotentialpocketP1-within8Angstroms 10C

extendedfrom1122)( residues:Important R119A124S114C57 F125G120$115659 Q129D121A116161 M130K117L122D112 F134A118G123C113 Interfacesurface

10A 10B 10Figure U.S. Patent Feb. 4. 2020 Sheet 13 of 83 US 10,548,864 B2

R68

Residuesidechaindistribution +134

PotentialpocketP2within-Angstroms4 11C (extendedfromhexa-carbonmoietyofATRA) residues:Important Q131H59 F134R68 $154L122 H157M130 Interfacesurface

11A 11B Figure11 U.S. Patent Feb. 4. 2020 Sheet 14 of 83 US 10,548,864 B2

$1 0123 Residuesidechaindistribution Q131 130

PotentialpocketP2within-Angstroms8 12C $138S1541159D112F125K132V62 (extendedfromhexa-carbonmoietyofATRA) P133V150G155K63C113H59Q129 R68L60T1521156M130F134$115 H157D153E135L122Q131L61R69 residues:Important Interfacesurface

12A 12B 12Figure U.S. Patent Feb. 4. 2020 Sheet 15 of 83 US 10,548,864 B2

R68 Residuesidechaindistribution Q131 K132 Q129 PotentialpocketP3-within4Angstroms 13C Q131)fromR68,M130and(extended residues:Important Q131R68 K132Q129 D153M130 Interfacesurface

13A 13B 13Figure U.S. Patent Feb. 4. 2020 Sheet 16 of 83 US 10,548,864 B2

sidechaindistributionResidue HX57 K132 PotentialpocketP3-within8Angstroms 14C (extendedfromR68,M130andQ131) S154E135Q131 residues:Important D153F134M130R68 G155F151K132G128 H157T152P133Q129 R69 Interfacesurface

14A 14B 14Figure U.S. Patent Feb. 4. 2020 Sheet 17 of 83 US 10,548,864 B2

distributionsidechainResidue

PotentialpocketP4-within4Angstroms 15C (extendedfromK63,R68R69andS154) residues:Important R69 S154 K63 S67 R68 surfaceInterface

15A 15B 15Figure U.S. Patent Feb. 4. 2020 Sheet 18 of 83 US 10,548,864 B2

567 Residuesidechaindistribution 1152

PotentialpocketP4-within8Angstroms 16C (extendedfromK63,R68R69andS154) R69D153H157178 residues:Important Q66P70S154D112L61 G155S67Q131V62S71 1156R68T152K63972 Interfacesurface

16A 16B 16Figure U.S. Patent Feb. 4. 2020 Sheet 19 of 83 US 10,548,864 B2

31 Residuesidechaindistribution

PotentialpocketP5-within4Angstroms 17C from71)extended$( residues:Important Q75571 E76S72 077W73 Interfacesurface

17A 17B 17Figure U.S. Patent Feb. 4. 2020 Sheet 20 of 83 US 10,548,864 B2

E76 Q77 075 $71 R74 72$0112 W73 Residuesidechaindistribution R69

PotentialpocketP5-within8Angstroms 18C

R69W73Q77$114 extended71)from$( 972E76D112K63 residues:Important 075T79S71 P70R74178 Interfacesurface

18A 18B 18Figure U.S. Patent Feb. 4. 2020 Sheet 21 of 83 US 10,548,864 B2

S72 Residuesidechaindistribution 0113

PotentialpocketP6within-4Angstroms 19C extended$71)from( residues:Important D112 C113 S114 S71 S72 W73 Interfacesurface

19A 19B 19Figure U.S. Patent Feb. 4. 2020 Sheet 22 of 83 US 10,548,864 B2

6 Residuesidechaindistribution C113

20C PotentialpocketP6-within8Angstroms S108D112A116G120 S115S111R119 C113K117S72$105Q109 W73L106F110S114A118 extendedfrom$71)( residues:Important S71 Interfacesurface

20A 20B Figure20 U.S. Patent Feb. 4 , 2020 Sheet 23 of 83 US 10,548,864 B2

1 Conc.(um) 1000 1804GST GST.Pin1 **150 120 0 tapiATRA Conc.UM) 210 ,0 0.01 101001000 KE90 0 probeHiLyteinM)488 0.0001 21E

1

0 Conc.IuM) 21B LL "Milyte" probe

1 GST-Pin1Conc.LuM) 21D 180 120120 21A 21Figure U.S. Patent Feb. 4 , 2020 Sheet 24 of 83 US 10,548,864 B2

22A 22B 150 90 60 Aspirin

22C 22D

3951Pintcatalyticactivity(OD395)Cyclophilin FKBP12activity(OD395)Pintcatalyticactivity(OD395) Time ( sec) 22E 22F

Time (sec ) Figure 22 U.S. Patent Feb. 4. 2020 Sheet 25 of 83 US 10,548,864 B2

23A Carboxylic % to Retinoid group ATRA a 13CRA ATRA Salicylic13CRA acid Retinol No Retinyl No acetate No Retinal B - carotene No Next Carboxylic % to generation group ATRA ATRA Acitretin Fenretinide No Bexarotene Yes lessur Tamibarotene Yes 63 ATRA Carboxylic % to analog group ATRA Indo - 3 acetic acid 0 Pravastatin Yes

Figure 23 U.S. Patent Feb. 4. 2020 Sheet 26 of 83 US 10,548,864 B2

23B

PPlase domain

Figure 23 U.S. Patent Feb. 4 , 2020 Sheet 27 of 83 US 10,548,864 B2

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0.0.01 2 0 20 24A 1 * 10 *( number Cell 24E www 24Figure U.S. Patent Feb. 4 , 2020 Sheet 28 of 83 US 10,548,864 B2

MISKAMMAR haraan .ul 24J 600 24L

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80li.li. 20 1 24KVec 241

24H Ctr ATRA 25UM ATRA SOUM 24Figure U.S. Patent Feb. 4 , 2020 Sheet 29 of 83 US 10,548,864 B2

AC-93253(MM) TO0 25 25C meactin8- 25F ATRA+RO(UM) 0151001510 Flag

25B

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] x109[ number Cell 25A 25D 25Figure U.S. Patent Feb. 4 , 2020 Sheet 30 of 83 US 10,548,864 B2 institutio

25K 88

251 U 253 W34/ K63A

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2013 00 25P C

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26A OŠO 26Figure U.S. Patent Feb. 4 , 2020 Sheet 33 of 83 US 10,548,864 B2

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RAROPML-27B 27C

27A 27Figure U.S. Patent Feb. 4 , 2020 Sheet 34 of 83 US 10,548,864 B2

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.VGGGG 100 28DVec VO 28E

RARPML-Flag Time[hour] stoneshPin1 100 20 ] %[ intensity RARa- PML 28B 28C RARA- PLZF RARA- PML son Flag Flag-tagged RARafusion 28A 28Figure U.S. Patent Feb. 4 , 2020 Sheet 35 of 83 US 10,548,864 B2

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W WMN dys xoa W US WWW

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osobnosti Fuglona + RemissiOH ATRA AIRASYS10 ATRA 30E 30H Worma wwwing

1 ROMission PlaceboATRAEGCGJuglone wasilkan daysJOATRAATRA 30G Motihar 30D RARO Remissiondays10 days

$ Normal 30C 30F Figure30 U.S. Patent Feb. 4 , 2020 Sheet 38 of 83 US 10,548,864 B2 R69

K63 31C 2319+WBSKBR3 -MDA HO01937 BT474 1,2033%g?????????? 10AAU585#MC 31BImmor

Onnumuruspore180ccorgen10-910-810-710-610-5 Conc,IM 40 31A ] % [ Proliferation Cell of Inhibition 31Figure U.S. Patent Feb. 4 , 2020 Sheet 39 of 83 US 10,548,864 B2

TEL HER2 PK12 31F

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33Figure U.S. Patent Feb. 4 , 2020 Sheet 44 of 83 US 10,548,864 B2

tin ,0+ 0.05 34C P 0.1+0.01 34E wa

33.390.03333%!2.12.36.03.3 ATRA/ROA15253Conc.IAM) ???????????" IRA

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100% 3100x KIRA 56A 56C 56Figure US 10,548,864 B2 1 2 ENHANCED ATRA - RELATED COMPOUNDS studies using mouse models , where the IRAK1 gene is FOR THE TREATMENT OF removed , have demonstrated a key role for this kinase in the PROLIFERATIVE DISEASES , AUTOIMMUNE TLR7 / 9 / IRF pathway that produces large quantities of IFNa DISEASES , AND ADDICTION CONDITIONS in response to viral infection . IRAK1 gene deletion prevents 5 TLR dependent activation of IRF5 / 7 in PDCs, the immune STATEMENT AS TO FEDERALLY SPONSORED cells responsible for IFNa production . Significantly , autoan RESEARCH tibody complexes obtained from SLE patients contain DNA and RNA and are taken up by pDCs to activate TLR7 and This invention was made with government support under TLR9 leading to secretion of cytokines and IFNa . More Grant Nos . CA122434 , CA167677 , AG039405 , DA031663 , 10 over , TLR activation is known to inhibit activity of gluco and HL111430 awarded by the National Institutes of Health . corticoids, a frontline drug class used to treat SLE . Although The government has certain rights in the invention . IRAK1 activity is regulated by phosphorylation upon TLR STATEMENT AS TO JOINT RESEARCH activation , little is known about whether it is subject to 15 further control after phosphorylation and whether such regu AGREEMENT lation has any role in SLE . This invention was made by or on behalf of the below The prevalence of asthma is increasing in the developed listed parties to a joint research agreement. The joint world , but the underlying mechanisms are not fully under research agreement was in effect on or before the date the stood , and therapeutic modalities remain limited . Asthma is claimed invention was made and the claimed invention was 20 a chronic inflammatory disease of the airways that is made as result of activities undertaken within the scope of induced by overexpression of multiple proinflammatory the joint research agreement. The parties to the joint research genes regulated by various signal pathways in response to agreement are BETH ISRAEL DEACONESS MEDICAL exposure to any of numerous allergens, including Toll- like CENTER and PINTEON THERAPEUTICS , INC . receptor /interleukin - 1 receptor ( TLR / IL - 1R ) signaling acti 25 vated by house dust mite (HDM ) allergens and IL - 33 , FIELD OF THE INVENTION respectively. A major regulatory mechanism in these signal pathways and gene activation is Pro - directed phosphory In general , this invention relates to all - trans retinoic acid lation (pSer / Thr- Pro ), but until recently little was known ( ATRA ) -related compounds for modulation of Pinl. The about whether and how they are regulated following phos invention also relates to the treatment of proliferative dis- 30 phorylation . orders, autoimmune disorders , and addiction ( e.g., disorders , Current treatment regimens for immune disorders typi diseases, and conditions characterized by elevated Pin1 cally rely on immunosuppressive agents . However, the marker levels ) with retinoic acid compounds . effectiveness of these agents can vary and their use is often accompanied by adverse side effects . Thus, improved thera BACKGROUND OF THE INVENTION 35 peutic agents and methods for the treatment of autoimmune disorders are needed . Immune disorders are characterized by the inappropriate In addition , drug addiction affects millions of individuals activation of the body's immune defenses . Rather than worldwide . The prevalence of cocaine addiction , for targeting infectious invaders , the immune response targets example , is estimated at over one million persons in the and damages the body's own tissues or transplanted tissues . 40 United States alone . Dopamine receptor signaling is under The tissue targeted by the immune system varies with the stood to play a major role in addiction to drugs such as disorder. For example , in multiple sclerosis , the immune cocaine known to elicit dopamine responses . Dopamine response is directed against the neuronal tissue, while in induction is coupled to the phosphorylation of glutamate Crohn's disease the digestive tract is targeted . receptor protein mGluR5 , which in turn potentiates NMDA Immune disorders affect millions of individuals and 45 receptor- mediated synaptic plasticity and thus cocaine - in include conditions such as asthma, allergic intraocular duced sensation . MAP Kinase phosphorylates mGluR5 inflammatory diseases, arthritis , atopic dermatitis , atopic where it binds the adaptor protein Homer and in so doing is eczema, diabetes , hemolytic anaemia , inflammatory derma thought to create a binding site for proteins that catalyze toses , inflammatory bowel or gastrointestinal disorders ( e.g. , cis - trans isomerization of a phosphorylated serine -proline Crohn's disease and ulcerative colitis ), multiple sclerosis , 50 bond (pSer / Pro ). Despite this recognition , there are presently myasthenia gravis , pruritis / inflammation , psoriasis , rheuma no FDA -approved medications to treat cocaine addiction . toid arthritis , cirrhosis , and systemic lupus erythematosus . Accordingly , there is a need to identify and develop thera A major cellular pathway in the pathogenesis of autoim peutic agents for the treatment of cocaine addiction . munity is the TLR / IRAK1/ IRF / IFN pathway. For example , The increased number of cancer cases reported in the levels of IFNa ( type I interferon ) are elevated in patients 55 United States, and , indeed , around the world , is also a with autoimmune diseases, including systemic lupus erythe significant concern . There are currently only a handful of matosus (SLE ) , and are central to disease pathogenesis , detection and treatment methods available for some specific correlating with autoantibodies and disease development. types of cancer , and these provide no absolute guarantee of Recent genetic studies in SLE patients and lupus -prone mice success. In order to be most effective , these treatments have identified variants in the genes critical for the TLR / 60 require not only an early detection of the malignancy , but a IRAK1/ IRF / IFN pathways , including TLR7, IRAK1 and reliable assessment of the severity of the malignancy. IRF5 . In addition , several TLR inhibitors are in development It is apparent that the complex process of tumor devel for treatment of SLE . Notably, IRAK1 genetic variants have opment and growth must involve multiple gene products . It recently been identified in human SLE . IRAK1, a well is therefore important to define the role of specific genes established pivotal player in TLRs and inflammation , is 65 involved in tumor development and growth and identify located on the X chromosome, which may help account for those genes and gene products that can serve as targets for the fact that SLE is more common in women . Importantly , the diagnosis , prevention , and treatment of cancers. US 10,548,864 B2 3 4 In the realm of cancer therapy, it often happens that a are yet to be elucidated . For example , Rab2A , a small therapeutic agent that is initially effective for a given patient GTPase mainly localized to the ER -Golgi intermediate becomes, over time, ineffective or less effective for that compartment (ERGIC ) , is essential for membrane trafficking patient. The very same therapeutic agentmay continue to be between the ER and Golgi apparatus but has no known effective over a long period of time for a different patient. 5 function in cancer or CSCs. As disclosed herein , we have Further, a therapeutic agent that is effective , at least initially , unexpectedly found that Rab2A is a Pinl transcriptional for some patients can be completely ineffective from the target that is activated via its gene amplification or mutation outset or even harmful for other patients. Accordingly , it or Pinl overexpression in breast cancer and promotes BCSC would be useful to identify genes and / or gene products that expansion in vitro and in vivo as well as in human primary represent prognostic genes with respect to a given therapeu- 10 normal and cancerous breast tissues . Mechanistically , tic agent or class of therapeutic agents . It then may be Rab2A directly binds to Erk1/ 2 via a docking motif that is possible to determine which patients will benefit from a also used by an Erk1 / 2 phosphatase, MKP3 (MAP kinase particular therapeutic regimen and , importantly , determine phosphatase 3 ) to prevent Erk1/ 2 from being dephosphory when , if ever , the therapeutic regime begins to lose its lated / inactivated , leading to activation of the known BCSC effectiveness for a given patient. The ability to make such 15 regulators Zeb1 and B - catenin . We further describe a tight reasoned predictions would make it possible to discontinue association of Rab2A overexpression with ß -catenin or Zeb1 a therapeutic regime that was losing its effectiveness well downstream target expression in human breast cancer tissues before its loss of effectiveness becomes apparent by con as well as with poor outcome of breast cancer patients , ventional measures . especially in the most common subtypes , as defined by Recent advances in the understanding of molecular 20 HER2 -negative or non -triple -negative breast cancer. Thus , mechanisms of oncogenesis have led to exciting new drugs the Pin1/ Rab2A / Erk axis drives BCSC expansion and tum that target specific molecular pathways. These drugs have origenicity , contributing to high mortality in patients . Simi transformed cancer treatments , especially for those caused larly , Pinl has also been identified as a critical regulator by some specific oncogenic events , such as Herceptin for acting downstream of miR200c . breast cancer , caused by HER2/ Neu , and Gleevec® for 25 These and other results disclosed herein suggest that Pinl chronic myelogenous leukemia caused by Ber- Abl. How inhibitors may have a major impact on treating cancers , ever , it has been increasingly evident that, in many indi especially aggressive and /or drug - resistant cancers. A com vidual tumors, there are a large number of mutated genes mon and central signaling mechanism in many oncogenic that disrupt multiple interactive and / or redundant pathways. pathways is proline (Pro ) -directed phosphorylation (pSer / Thus, intervening in a single pathway may not be effective . 30 Thr- Pro ) . Proline adopts cis and trans conformations , the Furthermore , cancer resistance to molecularly targeted drugs isomerization of which is catalyzed by prolyl isomerases can develop through secondary target mutation or compen (PPlases ) including Pin1 . Phosphorylation on serine / threo satory activation of alternative pathways, so -called “ onco nine -proline motifs restrains cis /trans prolyl isomerization , genic switching. ” Thus, a major challenge remains how to and also creates a binding site for the essential protein Pinl. simultaneously inhibit multiple oncogenic pathways either 35 Pinl binds and regulates the activity of a defined subset of using a combination ofmultiple drugs , with each acting on phosphoproteins, as well as participating in the timing of a specific pathway , or using a single drug that concurrently mitotic progression . Both structural and functional analyses blocks multiple pathways . have indicated that Pinl contains a phosphoserine /threo Cancer stem - like cells (CSCs ) or tumor- initiating cells nine -binding module that binds phosphoproteins , and a ( TICs) have been hypothesized to retain the capacity of 40 catalytic activity that specifically isomerizes the phospho self - renewal and regeneration of the bulk of a heterogeneous rylated phosphoserinelthreonine -proline . Both of these Pin1 tumor comprised of CSCs and non - stem cells . CSCs have activities are essential for Pinl to carry out its function in important implications for understanding the molecular vivo . mechanisms of cancer progression and developing novel Pinl has been implicated in autoimmune diseases and targets for cancer therapeutics because they are thought to be 45 conditions such as SLE and asthma and in drug addiction responsible for tumor initiation , progression , metastasis , pathways . Further, we and others have shown that Pinl is relapse and drug resistance . A variety of regulators of breast prevalently overexpressed in human cancers and that high cancer stem -like cells ( BCSCs) , notably transcription factors Pinl marker levels correlate with poor clinical outcome in including Zeb1 and ß -catenin , and miRNAs, have recently many cancers . In contrast , the Pinl polymorphism that been identified . These modulators of transcription and /or 50 reduces Pinl expression is associated with reduced cancer translation are further regulated by upstream signaling path risk in humans. Significantly , Pinl activates at least 32 ways. For example , Erk signaling has been shown to regu oncogenes/ growth enhancers , including B -catenin , cyclin late BCSCs by increasing transcription of Zeb1 and nuclear D1, NF -KB , C - Jun , c - fos, AKT, A1B1 , HER2/ Neu , MC1-1 , accumulation of unphosphorylated (active ) ß - catenin . How Notch , Raf- 1, Stat3 , c- Myb , Hbx, Tax , and v - rel , and also ever , regulatory pathways upstream of Erk signaling that 55 inactivates at least 19 tumor suppressors/ growth inhibitors , regulates BCSCs are still not fully elucidated . including PML , SMRT, FOXOs, RARA , and Smad (FIG . 1 ) . Among the small GTPase superfamily , Ras has been Whereas Pinl overexpression causes cell transformation and shown to induce epithelial mesenchymal transition (EMT ) tumorigenesis , Pinl knockdown inhibits cancer cell growth and confer CSC traits to breast cells in vitro and in vivo , in cell cultures and mice . Pinl- null mice are highly resistant while the Rho family GTPase Rac1 is involved in the 60 to tumorigenesis induced either by oncogenes such as acti maintenance and tumorigenicity of CSCs in non - small cell vated Ras or HER2 /Neu , or tumor suppressors such as p53 . lung adenocarcinoma and glioma and is also required for Thus , Pinl inhibitors may have the desirable property to intestinal progenitor cell proliferation and LGR5 + intestinal suppress numerous oncogenic pathways simultaneously for stem cell expansion . Deletion of Racl in adult mouse treating cancers , especially those aggressive and /or drug epidermis stimulated stem cells to divide and undergo ter- 65 resistant cancers . Potent and selective Pinl inhibitors having minal differentiation . However, the roles of other GTPase low toxicity , high cell permeability , and long half- lives in the family members in CSCs in solid tumors or adult stem cells body are particularly desirable . US 10,548,864 B2 5 6 Pinl is highly conserved and contains active sites includ samples , blood , urine , biopsies , lymph , saliva, phlegm , and ing a protein -interacting module , called the WW domain , pus ) from the subject and administering an ATRA - related and a catalytically active peptidyl- prolyl isomerase (PPlase ) compound of the invention to the subject if the sample is portion , each of which include at least one binding pocket . determined to have elevated Pinl marker levels . Pinl is structurally and functionally distinct from members 5 In a further aspect, the invention provides a method of of two other well - characterized families of PPlases, the treating a proliferative disease , an autoimmune disease , or cyclophilins and the FKBPs. PPlases are ubiquitous an addiction condition in a subject previously treated with an enzymes that catalyze the typically slow prolyl isomeriza ATRA - related compound and shown to have Pinl degrada tion of proteins, allowing relaxation of local energetically tion ( e.g. , by comparing a Pinl marker level in a sample unfavorable conformational states . Phosphorylation on Ser / 10 obtained from a subject before administration of the ATRA Thr residues immediately preceding Pro not only alters the related compound with a Pinl marker level in a sample prolyl isomerization rate , but also creates a binding site for obtained from a subject after administration of the ATRA the WW domain of Pinl. The WW domain acts as a novel related compound ), the method comprising administering an phosphoserine- binding module targeting Pinl to a highly ATRA - related compound of the invention to the subject in conserved subset of phosphoproteins . Furthermore , Pin1 15 an amount sufficient to treat the subject. displays a unique phosphorylation - dependent PPlase that In a related aspect, the invention provides a method of specifically isomerizes phosphorylated Ser / Thr - Pro bonds identifying a candidate for treatment with an ATRA -related and regulates the function of phosphoproteins. The cis - trans compound , in which the candidate has a proliferative dis isomerization of certain pSer / Thr- Pro motifs can be detected ease, an autoimmune disease, or an addiction condition and by cis- and trans- specific antibodies . 20 has previously been treated with (e.g. , administered ) an Taken together , these results indicate that the Pinl sub ATRA -related compound ; the method comprising determin family of enzymes is a diagnostic and therapeutic target for ing whether the subject has Pinl degradation ( e.g. , Pinl diseases associated with signal pathways involving Pro degradation resulting from the prior administration of an directed phosphorylation and characterized by uncontrolled ATRA - related compound ), where a candidate for treatment cell proliferation , primarily malignancies . 25 with an ATRA - related compound has Pinl degradation . We have surprisingly found that an approved anticancer In the context of the present invention , an " all -trans reagentwith an unknown mechanism , all- trans retinoic acid retinoic acid (ATRA ) -related compound ” refers to a com ( ATRA ) , potently and reversibly binds and inhibits and pound that is structurally related to or an analog of ATRA . ultimately induces degradation of active Pinl. The use of For example , a compound that is structurally related to or an all- trans retinoic acid (ATRA ) to treat acute promyelocytic 30 analog of ATRA may have one or more components ( e.g., leukemia (APL ) is described as the first example of targeted one or more functional groups or structural motifs ) in therapy in human cancer. ATRA induces leukemia cell common with ATRA and / or may have one or more substi differentiation by activating RARa or the oncogene PML / tutions, elongations, eliminations , add or other differ RARa -dependent transcription and induces degradation of ences relative to ATRA , e.g., as described herein . For PML /RARQ . However, the mechanism by which ATRA 35 example , one or more components, functional groups, or mediates these anticancer effects is unknown. Though elements of ATRA may be modified , replaced , or eliminated , RARa and PML have been described as Pinl substrates , the e.g., by adding , changing , or eliminating one or more link between ATRA and Pinl is poorly understood . The substitutions, replacing one or more groups (e.g. , replacing establishment of the mechanism of interaction between a carboxyl group with an group ) , and / or increasing or ATRA and Pinl could facilitate the development and iden- 40 decreasing the size or length of a component of ATRA ( e.g., tification of selective Pinl inhibitors with low toxicity , high replacing a six -membered ring with a seven -membered cell permeability , and long half - lives for use in the treatment ring) . An ATRA - related compound may differ from ATRA of proliferative and other disorders. Accordingly , there is a by as few as one group , element, or feature ( e.g. , a single need for an improved understanding of the binding interac isotopic substitution , a single or absence tion between ATRA and Pinl . 45 thereof, etc. ). ATRA - related compoundsmay include isoto pically substituted species ( e.g. , ATRA including one or SUMMARY OF THE INVENTION more isotopic substitutions such as deuterium , tritium , 170 , 180 , 13C , 32p, 15N , and 18F ), functionally substituted species The present invention relates to all - trans retinoic acid ( e.g., ATRA with one or more methyl groups eliminated or ( ATRA ) -related compounds that act as Pinl substrates and 50 replaced by one or more other functional groups such as methods of treating a proliferative disorder, an autoimmune longer chain groups, hydroxyl groups, cycloalkyl disorder, or an addiction condition with the retinoic acid groups , and other groups ), and stereoisomers ( e.g. , ATRA compounds ( e.g., ATRA -related compounds ) of the inven including one or more cis groups along its backbone ) . tion . An ATRA -related compound of the invention may include In one aspect, the invention provides a method of treating 55 one or more unsaturations or substitutions ( e.g., 1, 2 , 3 , 4 , 5 , a proliferative disease , an autoimmune disease , or an addic 6 , or more unsaturations or substitutions ). An unsaturation tion condition in a subject comprising administering an may be a multiple bond such as a (alkene ) or ATRA - related compound of the invention to the subject in triple bond ( ) or a ring structure. A substitution may an amount sufficient to treat the subject, wherein the subject be selected from the group consisting of, but not limited to , is determined to have elevated levels of a Pinl marker ( e.g. , 60 a halogen atom , a , an ( e.g., a Ser71 phosphorylation or PML -RARa ) prior to the admin hydroxyl) , an ester , an , a carbonyl , an acyl halide, istration . a carbonate , an , a phosphate , a , a , a In another aspect, the invention features a method of sulfinic acid , a , a thial, a sulfate, a sulfonyl, an treating a proliferative disease , an autoimmune disease , or , an azido, a nitro , a cyano , isocyano , acyloxy, an an addiction condition in a subject comprising determining 65 amino, a carbamoyl, a sulfonamide, or another functional Pinl marker levels ( e.g., reduced Ser71 phosphorylation or group , or an optionally substituted alkyl ( e.g., C1-10 alkyl) , overexpression of PML -RAR? ) in a sample ( e.g., tumor alkenyl ( e.g., C2-10 alkenyl) , alkynyl ( e.g. , C2-10 alkynyl ) , US 10,548,864 B2 7 8 alkoxy ( e.g. , C1-10 alkoxy ) , aryloxy ( e.g. , C6-10 aryloxy ) , instance, the backbone may be 2,6 -dimethyl - octa - 1,3,5,7 cycloalkyl ( e.g., C3-5 cycloalkyl) , cycloalkoxy (e.g. , C3-8 tetraene . As described above , all cis and trans isomers are cycloalkoxy ), aryl ( e.g., C.- 10 aryl) , aryl- alkoxy ( e.g., C6-10 contemplated . aryl- C1-10 alkoxy ), heterocyclyl or heterocycloalkyl ( e.g. , In some embodiments , an ATRA -related compound C3-8 heterocycloalkyl ) , heterocycloalkenyl, ( e.g., C4-8 het- 5 includes a carboxylic acid group . In some embodiments , in erocycloalkenyl) , or heteroaryl (e.g. , C6-10 heteroaryl) . In place a carboxylic acid group , an ATRA -related compound some embodiments , the substituent groups themselves may includes one or more atoms and is a group selected be further substituted with , for example , 1 , 2 , 3 , 4 , 5 , or 6 from a hydroxyl , an ester , an aldehyde , a carbonyl , an acyl substituents as defined herein . For example , a C1-6 alkyl, halide , a carbonate , an acetal, a phosphate , a sulfoxide , a aryl, or heteroaryl group may be further substituted with 1, 10 , a sulfinic acid , a sulfonic acid , a sulfate , a sulfonyl, 2 , 3 , 4 , 5 , or 6 substituents as described herein . A heteroatom and an amide . In certain embodiments, an ATRA -related of a heteroaryl, heterocycloalkyl, heterocycloalkenyl, het compound includes in place of the carboxylic group of erocycloalkynyl, or other group may be, for example , a ATRA a group selected from a hydroxyl , an ester , an atom , oxygen atom , or nitrogen atom . A functional aldehyde, a carbonyl , an acyl halide , a carbonate , and an group such as a heteroaryl, heterocycloalkyl, heterocy 15 amide . In some embodiments , in place of the carboxylic cloalkenyl, heterocycloalkynyl, or other group may include group of ATRA , an ATRA - related compound includes one or more than one heteroatoms ( e.g., one sulfur atom and one more optionally substituted aryl, heteroaryl, cycloalkyl, nitrogen atom , two nitrogen atoms, three nitrogen atoms, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl four nitrogen atoms, one sulfur atom and one oxygen atom , 20 groups . one nitrogen atom and one oxygen atom , or any other In some embodiments , an ATRA - related compound is a combination ) compound according to Formula II In some embodiments , an ATRA -related compound may include one ormore rigid or sterically bulky groups such as one or more aryl, heteroaryl, cycloalkyl, cycloalkenyl, het- 25 ( Formula II ) erocycloakyl, or heterocycloalkenyl rings or a fusion R2 R3 thereof. For example , an ATRA -related compound may W include, in place of the cyclohexenyl group of ATRA , a naphthyl or hydronaphthyl ( e.g. , di-, tri-, tetra- , penta-, 30 hadag hexa- , hepta- , octa-, nona- , or deca- hydronaphthyl ) group . In Rí some embodiments , an ATRA - related compound may include, in place of the cyclohexenyl group of ATRA , a single carbon ring including a single double bond ( e.g. , a wherein n and m are independently selected from 0 , 1 , and cycloalkyl or cycloalkenyl group other than cyclohexene) . 35 2from , and the wherein group W consisting,R1 , R2, and of, R3 but are not independently limited to , a selectedhalogen In certain embodiments , an ATRA -related compound may atom , a carboxylic acid , an alcohol ( e.g., a hydroxyl ) , an include , in place of the cyclohexenyl group of ATRA , an ester , an aldehyde, a carbonyl, an acyl halide , a carbonate , an optionally substituted cylcohexene group ( e.g., a cyclohex acetal, a phosphate , a thiol, a sulfoxide, a sulfinic acid , a ene group having one ormore additional , fewer, or different sulfonic acid , a thial, a sulfate , a sulfonyl, an amide, an substitutions than the cyclohexenyl group of ATRA ). In 40 azido , a nitro , a cyano , isocyano , acyloxy, an amino , a some preferred embodiments , substitutions on a ring ( e.g. , a carbamoyl, a sulfonamide, or another , or an ring such as those described herein in place of the cyclo optionally substituted alkyl ( e.g. , C1-10 alkyl) , alkenyl ( e.g., hexenyl group of ATRA ) are not sterically bulky . For C2-10 alkenyl) , alkynyl ( e.g., C2-10 alkynyl) , alkoxy ( e.g., example , a ring preferably includes one or more short -chain C1-10 alkoxy ), aryloxy ( e.g. , C6-10 aryloxy ), cycloalkyl ( e.g., alkyl ( e.g., C -s alkyl ) substituents . In an embodiment, an 45 C3-8 cycloalkyl) , cycloalkoxy ( e.g. , C3-8 cycloalkoxy ), aryl ATRA - related compound includes a trimethylcyclohexene ( e.g., C6-10 aryl) , aryl - alkoxy ( e.g , C6-10 aryl- C1-10 alkoxy ) , such as 1,3,3 - trimethylcyclohexene . heterocyclyl or heterocycloalkyl ( e.g., C3-8 heterocy In some embodiments , an ATRA - related compound cloalkyl) , heterocycloalkenyl, ( e.g. , C4-8 heterocycloalk includes a " backbone " carbon chain such as the backbone enyl) , or heteroaryl ( e.g., C6-10 heteroaryl) . In some embodi carbon chain of ATRA ( e.g., the diterpene moiety ). In certain 50 ments , W is a bioisostere of a carboxyl group . For example , embodiments , an ATRA - related compound includes , in W can be selected from the group consisting of a phenolic place of the “ backbone” moiety of ATRA , a carbon chain group , a halophenolic group ( e.g. , 3 -chloro - 4 -hydroxyben ( e.g. , an alkyl chain ) including one or more rings . For zyl) , a heteroaryl group , a heterocycloalkyl group, a hetero example , an ATRA - related compound may include, in place cycloalkenyl group , a sulfonamide, and a sulfonic acid . Any of the “ backbone ” moiety of ATRA , an alkyl chain fused to 55 group may include one or more substitutions including an optionally substituted aryl, heteroaryl , cycloalkyl , halogen , hydroxyl, azido , and other substitutions described cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl herein , or any additional substitution . group . In some embodiments , an ATRA -related compound In certain embodiments , W is a carboxyl group . In some includes a “ backbone” moiety including one or more double embodiments , R1, R2, and Rz are independently selected bonds. In particular embodiments , the “ backbone” moiety 60 from an optionally substituted alkyl ( e.g. , C1-10 alkyl) group includes conjugation ( e.g., alternating single and double and an optionally substituted alkenyl (e.g. , C2-10 alkenyl) bonds) . For example , the “ backbone” moiety may be 4-10 group . carbon chain 2-5 double bonds, such as octa - 1,3,5,7 - tetra In certain embodiments , R1, R2, and Rz are independently ene. In certain embodiments , the “ backbone ” moiety may selected from optionally substituted ( e.g. , with amino , include one or more isoprene units and be , e.g. , a diterpene . 65 alkoxy, carboxyl, or sulfonyl groups or other hydrophilic In some embodiments , the " backbone ” moiety includes one moieties ) alkyl (e.g. , C1-10 alkyl) groups. In particular or more short - chain alkyl ( e.g. , C -5 alkyl) substituents . For embodiments , R1, R2, and Rz are methyl groups. US 10,548,864 B2 9 10 In some embodiments , n and m are independently ( e.g., C1-10 alkyl) . Examples include methoxy, ethoxy, selected from 0 and 1. In other embodiments , n and m are propoxy ( e.g., n -propoxy and isoproxy ) groups . The alkyl independently selected from 1 and 2. In certain embodi portion of an may include any additional ments , n and m are both 1 . substitution as defined herein . In some embodiments , an ATRA -related compound is a 5 As used herein , the term “ alkyl” includes straight chain compound according to Formula I and branched chain saturated groups including between 1 and 20 carbon atoms, unless otherwise specified . Examples include methyl , ethyl, n -propyl , and isopropyl. An alkyl ( Formula I ) 10 group may be optionally substituted with one or more R2 R3 substituents as defined herein . As used herein , the term “ alkenyl” represents an alkyl group including one or more double bonds. An alkene or alkenyl group may be a straight or branched alkyl chain with wadaysRí 15 two or more hydrogen atoms removed . Examples include methylene, , and isopropylene. An alkenyl group wherein W , R1, R2, and Rz are independently selected from may include between 2 and 20 carbon atoms, unless other the group consisting of, but not limited to , a halogen atom , wise specified , and may be optionally substituted as defined a carboxylic acid , an alcohol ( e.g. , a hydroxyl) , an ester, an 20 herein . Alkenyls include both cis and trans isomers . For aldehyde , a carbonyl, an acyl halide , a carbonate , an acetal, example , 2 -butene includes cis -but - 2 -ene [ ( Z ) -but - 2 - ene ] a phosphate , a thiol, a sulfoxide, a sulfinic acid , a sulfonic and trans -but - 2 - ene [ ( E ) -but- 2 -ene ] . acid , a thial , a sulfate , a sulfonyl , an amide, an azido , a nitro , As used herein , the term “ alkynyl” represents an alkyl a cyano , isocyano , acyloxy , an amino , a carbamoyl, a group including one or more triple bonds . An alkyne or sulfonamide , or another functional group, or an optionally 25 alkynyl group may be a straight or branched alkyl chain with substituted alkyl (e.g. C1-10 alkyl) , alkenyl ( e.g., C2-10 alkenyl ) , alkynyl ( e.g. , C2-10 alkynyl) , alkoxy ( e.g., C1-10 four or more hydrogen atoms removed . Examples include alkoxy) , aryloxy ( e.g., C6-10 aryloxy ), cycloalkyl ( e.g., C3-8 acetylene (ethyne ) , propyne, and butyne. An alkynyl group cycloalkyl) , cycloalkoxy ( e.g., C3-8 cycloalkoxy) , aryl ( e.g. , may include between 2 and 20 carbon atoms, unless other C6-10 aryl) , aryl -alkoxy ( e.g , C6-10 aryl- C1-10 alkoxy ) , het- 30 wise specified , and may be optionally substituted as defined erocyclyl or heterocycloalkyl ( e.g., C3-8 heterocycloalkyl ), herein . heterocycloalkenyl, (e.g. , C4-8 heterocycloalkenyl ), or het As used herein , the term “ cycloalkyl” represents a satu eroaryl ( e.g. , C6-10 heteroaryl) . rated or unsaturated non -aromatic cyclic hydrocarbon group In certain embodiments , one or more ofR1 , R2, and Rz are including 3 , 4 , 5 , 6 , 7, 8 , or more carbon atoms, unless methyl groups . In some embodiments , R , is a methyl group . 35 otherwise specified . A cycloalkyl group may optionally In some embodiments , R2 is a methyl group . In some include one or more substitutions, as defined herein . embodiments , Rz is a methyl group . In some embodiments , Examples include cyclopropyl, cyclobutyl, cyclopentyl , R , and Ry are methyl groups. In some embodiments , R2and cyclohexyl, cycloheptyl, and cyclooctyl groups. In some Rz are methyl groups . embodiments , the cycloalkyl is a polycyclic ( e.g., adaman In some embodiments , W is an group including one or 40 tyl ). A cycloalkyl group including one or more double bonds more electronegative atoms (e.g. , a carboxylic acid , alcohol, is referred to as a " cycloalkenyl” group . Examples of ester, aldehyde , carbonyl, acyl halide, carbonate , acetal , cycloalkenyl groups include cyclopentenyl, cyclohexenyl, phosphate , thiol, sulfoxide, sulfinic acid , sulfonic acid , thial , cycloheptenyl, and cyclooctenyl groups . sulfate , sulfonyl, , thioaldehyde , or amide ). In 45 As used herein , the term “ cycloalkoxy ” represents a particular embodiments , W is a carboxylic acid group . substituent of the form -OR , where R is a cycloalkyl grup , As used herein , the term “ acyl” represents an alkyl group as defined herein . or hydrogen that is attached to a parent molecular group As used herein , the term “ aryl ” represents a mono-, bi- , or through a . Examples include formyl, acetyl , multi -cyclic carbocyclic ring system having one or more and propionyl groups . 50 aromatic rings . For example , an aryl group may be a mono As used herein , the term “ acyloxy ” represents a group of or bicyclic Co -C14 group with [ 4n + 2 ] n electrons in conju the form - OC ( O ) R , in which R is a carbon - containing gation and where n is 1 , 2 , or 3. Phenyl is an aryl group group such as an alkyl group , as defined herein . where n is 1. Aryl groups also include ring systems where As used herein , the term “ acetal” represents a group of the 55 the ring system having [4n + 2 ], electrons is fused to a form -C (OR ') , R " , in which each OR ' are alkoxy groups, as non - aromatic cycloalkyl or a non - aromatic heterocyclyl. defined herein , and R " is a carbon -containing group such as Examples include phenyl, naphthyl, 1,2 -dihydronaphthyl , an alkyl group , as defined herein . The alkoxy groups of an 1,2,3,4 - tetrahydronaphthyl, anthracenyl, and indenyl. An acetal group may be the same ( e.g., a symmetric acetal) or aryl group may optionally include one or more substitutions, different (e.g. , a mixed acetal) . 60 as defined herein . As used herein , the term “ aldehyde” represents an acyl As used herein , the term “ heterocycloalkyl” or “ hetero group having the structure CHO . cyclyl” represents a cycloalkyl ( e.g. , a non - aromatic ring ) As used herein , the term “ carbonyl” represents a C ( O ) group including one or more heteroatoms independently group , alternatively represented by C = O . 65 selected from the group consisting of nitrogen , oxygen , and As used herein , the term " alkoxy ” represents a group of sulfur. A heterocycloalkyl group including one or more the formula OR , where R is an alkyl group of any length double bonds is referred to as a “ heterocycloalkeny? ” group . US 10,548,864 B2 11 12 A heterocyclyl group may be a multicyclic structure ( e.g. , a As used herein , the term " sulfinic acid represents an bicyclic structure or a bridged multicyclic structure ) . S (O )OH group . Examples of heterocycles include piperidinyl, pyrrolidinyl, and tetrahydrofuryl groups . Heterocyclyl groups may be As used herein , the term " sulfonic acid ” represents an unsubstituted or substituted with , e.g., 1 , 2 , 3 , or 4 substitu 5 S (O ) OH group . ent groups as defined herein . As used herein , the term “ sulfate ” represents an S ( O ) 422 As used herein , the term “ heteroaryl ” represents an aryl group . ( e.g. , aromatic ) group including one or more heteroatoms As used herein , the term “ sulfonamide ” represents a independently selected from the group consisting of nitro- 10 group of the form -S( O )2NR2 or —N ( R ) S ( O )2R , wherein gen , oxygen , and sulfur. Heteroaryls may be monocycles , each R is independently optionally substituted alkyl, aryl, bicycles , tricycles , or tetracycles in which any aromatic ring cycloalkyl , cycloaryl, or another group . is fused to one , two , or three heterocyclic or carbocyclic rings ( e.g. , an aryl ring ). Examples of heterocyclic aromatic As used herein , the term “ amide ” represents a group of the molecules include furan , thiophene, pyrrole, thiadiazole 15 form_CO )NR2 , or -N ( R ) C ( O ) R , wherein each R is ( e.g., 1,2,3 -thiadiazole or 1,2,4 -thiadiazole ), oxadiazole independently optionally substituted alkyl, aryl, cycloalkyl, (e.g. , 1,2,3 -oxadiazole or 1,2,5 - oxadiazole ) , oxazole , isox cycloaryl , or another group . azole , isothiazole , pyrazole , thiazole , triazole ( e.g. , 1,2,4 As used herein , the term " amino " represents an —NR2 triazole or 1,2,3 -triazole ) , , pyrimidine, pyrazine, 20 group, wherein each R is independently optionally substi pyrazine , triazine ( e.g , 1,2,3 - triazine 1,2,4 - triazine , or 1,3 , tuted alkyl, aryl, cycloalkyl, cycloaryl, or another group . 5 - triazine) , 1,2,4,5 -tetrazine , indolyl , quinolinyl, isoquino linyl, benzimidazolyl, benzothiazolyl, and benzoxazolyl. As used herein , the term “ azido ” represents an NE Heteroaryls may be unsubstituted or substituted with , e.g. , 1 , group . 2 , 3, or 4 substituents groups as defined herein . 25 As used herein , the term “ nitro ” represents an —NO2 As used herein , the term “ fused ” refers to one or more group . chemical elements that are connected to one another by one As used herein , the term “ cyano ” represents a CN or more chemical bonds. In particular , two rings ( e.g , group , while the term “ isocyano ” represents an -NC group . cycloalkyl or aryl groups) may be fused to one another , as 30 As used herein , the term “ carbamoyl” represents a group described above. Examples include indolyl, quinolyl , and of the form OC ( O )NR , or - N (R )C ( O )OR , wherein each isoquinolyl groups. R is independently optionally substituted alkyl, aryl , As used herein , the term “ alkaryl” represents an aryl cycloalkyl , cycloaryl, or another group . group , as defined herein , attached to a parent molecular group through an alkyl group , as defined herein . 35 In some embodiments , an ATRA - related compound of the As used herein , the term “ aryl- alkoxy” represents an invention may include one or more isotopic substitutions , alkaryl group , as defined herein , attached to a parent including deuterium , tritium , 170 , 180 , 13C , 32p, 15N , and molecular group through an oxygen atom . 18F . An ATRA -related compound may have any stereochem As used herein , the term “ aryloxy ” represents a group of istry. All possible isomeric and conformational forms of the form OR , where R is an aryl group , as defined herein . 40 ATRA - related compounds ( e.g., those disclosed herein ) are As used herein , the term “ halo ” represents a halogen contemplated , including diastereomers , enantiomers , and / or selected from the group consisting of bromine, chlorine , conformers of a given structure. Different tautomeric forms iodine , and fluorine . are also contemplated . The invention includes protonated , As used herein , the term “ carboxylic acid ” or “ carboxy ” 45 deprotonated , and solvated species , as well as salts of the represents a group of the form C (O )OH , also represented compounds of the invention . n some embodiments , an as — CO2H . ATRA -related compound is a compound according to For As used herein , the term “ ester ” represents a group of the mula la form C ( 0 )0— As used herein , the term “ acyl halide” represents a group 50 of the form C ( O ) X , in which X is a halide selected from (Formula Ia ) bromide , fluoride , chloride , and iodide. As used herein , the term “ carbonate ” represents a group of the form OC ( O ) O 55 As used herein , the term “ alcohol ” or “ hydroxyl” repre any sents a group of the form – OH . As used herein , the term " phosphate ” represents an P ( O )4- group . As used herein , the term “ thiol” represents an SH 60 wherein W comprises one or more optionally substituted group . ( e.g. , with one or more halogen , carbonyl, hydroxyl, alkoxy, As used herein , the term “ thial ” represents an —C ( S ) H amino , amide , sulfonyl, or other groups, such as those group . described herein ) aryl or heteroaryl groups or one or more As used herein , the term “ sulfoxide ” represents an optionally substituted cycloalkyl , heterocycloalkyl , S ( 0 ) group . 65 cycloalkenyl, or heterocycloalkenyl groups . For example , an As used herein , the term " sulfonyl” represents an ATRA - related compound may be selected from compounds -S( O )2 group . 1-101 of Table 1 . US 10,548,864 B2 13 14 TABLE 1

ATRA - related compounds of the invention including substitutions at W.

Compound MMGBSA Number Compound dG Bind 1 Sony -42.523

2 -41.676

HN 3 Saaday -40.719 4 pory -40.448 5 wony -40.365 6 -40.345

HNSony

7 -40.249

HN -??

8 -39.417

HN S US 10,548,864 B2 15 16 TABLE 1 - continued ATRA - related compounds of the invention including substitutions at W. Compound MMGBSA Number Compound dG Bind -39.232

0

10 -39.050

ci

HO worry 11 Domy -38.984 12 wory -38.958 13 -38.818

14 in -38.817

HN

15 -38.742

-??

16 -38.627

N 17 org -38.309 US 10,548,864 B2 17 18 TABLE 1 - continued ATRA - related compounds of the invention including substitutions at W. Compound MMGBSA Number Compound dG Bind 18 -38.247

HO NH

19 -38.124

HO 20 Xory -37.847 21 -37.846

HN 22 -37.804

NSorry 23 brong -37.628

24 -37.601 woryS 25 -37.585

HN 26 my -37.568 US 10,548,864 B2 19 20 TABLE 1 - continued ATRA - related compounds of the invention including substitutions at W. Compound MMGBSA Number Compound dG Bind 27 -37.558

-S

-37.542

..... S

29 -37.485

N

30 -37.460

N

31 from -37.390 HNSony 32 -37.361

HNSony 33 -37.135 HNfy 34 -36.909

HN 35 wory -36.848 US 10,548,864 B2 21 22 TABLE 1 - continued ATRA - related compounds of the invention including substitutions at W. Compound MMGBSA Number Compound dG Bind 36 -36.903

N - NH

37 -36.761

CI

0

38 - 36.718 HN HN ng 39 Wory -36.588 40 Sony -36.555 41 -36.527 H rong 42 prony -36.516 43 cony - 36.418 44 ong -36.392 US 10,548,864 B2 23 24 TABLE 1 - continued ATRA - related compounds of the invention including substitutions at W.

Compound MMGBSA Number Compound dG Bind

45 -36.388 HN org 46 -36.384 HNSony

47 -36.256 +HN Sony 48 -36.247 "fon S 49 trony -36.061 50 wony -35.965 51 wory -35.875 52 Goyang -35.849 US 10,548,864 B2 25 26 TABLE 1 - continued ATRA - related compounds of the invention including substitutions at W.

Compound MMGBSA Number Compound dG Bind

53 -35.784

54 -35.682

HN

55 -35.677

NH

56 -35.622

-?? w 57 -35.513

58 -35.493

wory0 59 wory -35.321 60 -35.277

myyN - NH US 10,548,864 B2 27 28 TABLE 1 - continued ATRA -related compounds of the invention including substitutions at W. Compound MMGBSA Number Compound dG Bind 61 -35.303

N

HN 62 tyyny -35.186 63 -35.164

N HNBryny 64 -35.152 F

N -NH ory 65 -35.142

66 from -34.986

67 -34.949

N HN 68 from -34.940

69 from -34.843 Oy