(12) United States Patent (10) Patent No.: US 9,303,024 B2 Walensky Et Al

USOO93O3024B2

(12) United States Patent (10) Patent No.: US 9,303,024 B2 Walensky et al. (45) Date of Patent: Apr. 5, 2016

(54) PYRAZOL-3-ONES THAT ACTIVATE (56) References Cited PRO-APOPTOTC BAX U.S. PATENT DOCUMENTS (71) Applicant: Dana Farber Cancer Institute, Inc., 7, 160,870 B2 1/2007 Duffy et al. Boston, MA (US) 2009 OO12148 A1 1/2009 Maxfield et al. 2009,0163545 A1 6, 2009 Goldfarb 2011/0003851 A1 1/2011 Zhi et al. (72) Inventors: Loren D. Walensky, Newton, MA (US); Evripidis Gavathiotis, New York, NY FOREIGN PATENT DOCUMENTS (US) AR O49472 A1 8, 2006 CN 101343268 A 1, 2009 (73) Assignee: Dana-Farber Cancer Institute, Inc., CN 1017666O2 A T 2010 Boston, MA (US) DE 3728278 A1 6, 1988 s EP 2305250 A1 4/2011 WO O1-74769 A1 10, 2001 *) Notice: Subject to any disclaimer, the term of this WO 03-011855 A1 2, 2003 y WO WO2004O14902 A2 2, 2004 patent is extended or adjusted under 35 WO 2005-077939 A1 8, 2005 U.S.C. 154(b) by 0 days. WO WO2005077345 A1 8/2005 WO WO2005077368 A2 8, 2005 WO WO2007053.847 A2 5/2007 (21) Appl. No.: 14/350,847 WO 2009-071701 A1 6, 2009 WO WO2O11029046 A1 9, 2009 WO WO2O09130242 A1 10/2009 (22) PCT Filed: Oct. 11, 2012 WO WO2O10,042225 A2 4, 2010 OTHER PUBLICATIONS (86). PCT No.: PCT/US2012/059799 Chemical Abstracts Registry No. 7104-71-4, indexed in the Registry S371 (c)(1), file on STN CAS Online Nov. 16, 1984.* (2) Date: Apr. 10, 2014 Chemical Abstracts Registry No. 314292-25-6, indexed in the Reg istry file on STN CAS Online Jan. 17, 2001.* Chemical Abstracts Registry No. 314293-69-1, indexed in the Reg (87) PCT Pub. No.: WO2013/055949 istry file on STN CAS Online Jan. 17, 2001.* Chemical Abstracts Registry No. 314293-18-0, indexed in the Reg PCT Pub. Date: Apr. 18, 2013 istry file on STN CAS Online Jan. 17, 2001.* Gavathiotis et al., “Direct and selective small-molecule activation of O O proapoptotic BAX”. Nature Chemical Biology, 8(7):639-645 (May (65) Prior Publication Data 2012). Ren et al., “BID, BIM, and PUMA are Essential for Activation of the US 2014/O357687 A1 Dec. 4, 2014 BAX- and BAK-Dependent Cell Death Program'. Science, 330(6009): 1390-1393 (Dec. 2010). EP Patent Office, Partial Supplementary European Search Report, for Related U.S. Application Data EP Application No. 1284O7319-1453.2766355 PCT/ US2012059799, dated Jan. 18, 2015. (60) Provisional application No. 61/546,022, filed on Oct. International Search Report for PCT/US2012/059799 mailed Mar. 11, 2011. 13, 2013, 4 pages. Adam, et al., Synthesis and studies of Th(IV) and Ce(Ill) complexes with some azopyrazolone compounds, Delta Journal of Science, (51) Int. Cl. 11(3): 1089-103 (1987). CO7D 403/04 (2006.01) Amir, et al., Synthesis and antimicrobial activity of pyrazolinones CO7D 403/4 (2006.01) and pyrazoles having benzothiazole moiety, Med Chem Res, CO7D 417/04 (2006.01) 21:1261-1270 (2012). CO7D 417/4 (2006.01) Atta, Aly H., Reactions of 1-(2-benzothiazolyl)-4- A6 IK3I/4I55 2OO 6. O1 (dicyanomethylene)-3-methyl-2-pyrazolin-5-one towards amines, A6 IK3I/478 3:08: Heterocyclic Communications, 5(3):243-247 (1999). A6 IK3I/427 (2006.01) (Continued) gll, 17, 3.08: Primary Examiner — Laura L. Stockton (52) U.S. Cl (74) Attorney, Agent, or Firm — Fish & Richardson P.C. CPC ...... C07D 417/04 (2013.01); A61K 31/4155 (57) ABSTRACT (2013.01); A61K 31/4178 (2013.01); A61 K This application features pyrazol-3-one compounds that acti 31/427 (2013.01); A61 K3I/497 (2013.01); vate pro-apoptotic BAX. Also featured are methods of using C07D 403/04 (2013.01); C07D 403/14 Such compounds, e.g., for the treatment or prevention of (2013.01); C07D 413/04 (2013.01); C07D diseases, disorders, and conditions associated with deregu 417/14 (2013.01) lated apoptosis of cells (e.g., insufficient apoptosis of dis (58) Field of Classification Search eased or damaged cells or essentially the absence of apoptosis USPC 548/190, 312.4 of diseased or damaged cells). See application file for complete search history. 21 Claims, 65 Drawing Sheets US 9,303,024 B2 Page 2

(56) References Cited Mahesh, et al., Separation of some closely related 1-(2- benzothiazolyl)-3-methyl-4-arylhydrazono-pyrazoline-5-One OTHER PUBLICATIONS derivatives by thin layer chromatography, Fresenius' Zeitschrift fuer Baell, et al., New Substructure Filters for Removal of Pan Assay Analytische Chemie, 309(5):404 (1981). Interference Compounds (PAINS) from Screening Libraries and for Mamedov, et al., Reactions of Isomeric Arylchloropyruvates and Their Exclusion in Bioassays, J MedChem, 53(7): 2719-2740 (2010). Glycidates with Hydrazines, Russian Journal of Organic Chemistry, Bondock, et al., Ecofriendly solvent-free synthesis of thiazolylpyrazole derivatives, Monatshefte fuer Chemie 41(5):694-702 (2005). 139(11): 1329-1335 (2008). Naylor, et al., Identification of a chemical probe for NAACP by virtual Efros, et al., Benzothiazole derivatives. Preparation of screening, Nature Chemical Biology 5(4):220-226 (2009). I-benzothiazolyl-3-methyl-5(4H)-pyrazolone. Zhurnal Obshchei Parija, et al., Preparation of azamerocyanines and evaluation of the Khimi, 21:2046-50 (1951). effect of introduction of nitrogen atom in the chromophoric chain, El-Haty, A Co-Ordination and stability study. On some heterocyclic Journal of the Indian Chemical Society, 47(12): 1129-34 (1970). azopyrazolin-5-ones with Y(III), La(III), Ce(III) and UO, "ions, Jour Patel, et al., Synthesis and biological evaluation of some new nal of the Electrochemical Society of India, 40(3): 113-18 (1991). pyrazolones and imidazolinones, Oriental Journal of Chemistry Emandi, et al., 1-(2-Benzothiazolyl)-3-methyl-5-pyrazolone-based 19(2):435-440 (2003), Abstract Only). dyes, Revistade Chimie (Bucharest, Romania) 45(3): 179-82 (1994) Rosenbaum et al., Chemical screen to reducesterol accumulation in with English Abstract. Niemann–Pick Caisease cells identifies novel lysosomal acid lipase Erickson-Miller et al., Preclinical Activity of Eltromboapag (SB inhibitors, Biochimica et Biophysica Acta, Molecular and Cell Biol 4971 I5), an Oral, Nonpeptide Thrombopoietin Receptor Agonist, ogy of Lipids, 1791 (12), 1155-1165 (2009). Stem Cells, 27:424-430 (2009). Rout, M.K., Influence of structural changes on absorption. Ibrahim, et al., Synthesis of pyrazoles and fused pyrazoles. Novel Merocyanine dyes and aza-analogues of merocyanines, unsymmetri synthesis of pyrano 2,3-cpyrazole, thieno 2,3-cpyrazole and cal cyanines, and p-dialkylamino Styryl dives, Jour. & Proc. Inst. pyrazolo 3,4-bpyridine derivatives, Journal of the Indian Chemical Chem. (India), 35(3): 117-130 (1963). Society, 74(3):206-208 (1997). Chemical Structure Search Report by Science IP, dated Oct. 9, 2012 Kalluraya, et al., Synthesis of some triheterocyclic thiazole deriva (49 pages). tives of biological interest, Indian Journal of Heterocyclic Chemistry, 8(3):241-242 (1999). * cited by examiner U.S. Patent Apr. 5, 2016 Sheet 1 of 65 US 9,303,024 B2

Canonical BH3 Binding Site U.S. Patent Apr. 5, 2016 Sheet 2 of 65 US 9,303,024 B2

(S Hydrophobic O Hydrophilic Positive Charge & Negative Charge U.S. Patent Sheet 3 of 65 US 9,303,024 B2

21Y

2-42 O I I-O - Z| t

) LO

S21 XY2 4. O U.S. Patent Apr. 5, 2016 Sheet 4 of 65 US 9,303,024 B2

O ‘ºaaOO.,08'Hoov/ICTH

S Z] |-

U.S. Patent Apr. 5, 2016 Sheet 6 of 65 US 9,303,024 B2

‘TÁTOZeTUIT=TV7

U.S. Patent Apr. 5, 2016 US 9,303,024 B2

Z| U.S. Patent US 9,303,024 B2

OQ U.S. Patent US 9,303,024 B2

nHssºs'eseqs,6TU U.S. Patent Apr. 5, 2016 Sheet 11 of 65 US 9,303,024 B2

CN 4. Z

-(2 -

1. 4 T

CN 24 A dnoI5)ozei?GI

- ) t OOeuenTOLOp??uueleDV7–d 9Z//JejSUeII,OZTCI O- (y) |

S z=(Y ls U.S. Patent Apr. 5, 2016 Sheet 12 of 65 US 9,303,024 B2

750,000 in Silico Small Molecules

Glide/SPVS Docking

20,000 Top in Silico Hits

Glide/XPVS Docking

1,000 Top in Silico Hits

Interaction Analysis and Molecular Property-based Selection

100 Hits Tested Experimentally

FIG 3A U.S. Patent Apr. 5, 2016 Sheet 13 of 65 US 9,303,024 B2

U.S. Patent Apr. 5, 2016 Sheet 14 of 65 US 9,303,024 B2

EC50 FITC-BIMSAHB, BAX WT: 283 nM; 95% CI: 225-355 200 C ?h 5 18O c ge s 160 3 140 N 120 C ? 100 10-9 10-8 10-7 10-6 10-5 Protein), M FG. 4A

IC50 AC-BIMSAHB, BAX WT: 314 nM; 95% CI: 270-366 1 O O

8 O

6 O

4 O

2 O

1 O 9 10-8 10-7 10-6 10-5 Compound), M FIG. 4B U.S. Patent Apr. 5, 2016 Sheet 15 of 65 US 9,303,024 B2

SNNSNSNS

NSN SYYYYYYYYYYYYYY.

YYYYYYYYYYY. 6|&

(%)XYSO) pun09 SHVSWIGOL U.S. Patent Apr. 5, 2016 Sheet 16 of 65 US 9,303,024 B2

IC50 BAM7, BAX: 3.31 uM; 95% CI: 2.66-4.12

BAM7(resynthesis), BAX: 4.41 uM; 95% CI: 3.60-5.55 BAM8 BAM9 BAM10 BAM11 BAM7(resynthesis) 2 100 5 O - 80 n & 3X 60 225 40 an 9 2 2O O 10-7 10-6 10-5 10-4 Compound), M FIG. 4D

1 O

irSN' S FIG. 4E U.S. Patent Apr. 5, 2016 Sheet 17 Of 65 US 9,303,024 B2

10 9 8 7 6 5 4 3 2 1 -0 - 1 U.S. Patent Apr. 5, 2016 Sheet 18 of 65 US 9,303,024 B2

EC50 o FITC-BIMSAHB, BCL-X: 13.6 nM; 95%CI: 10.6-17.5 A FITC-BIMSAHB, MCL-1: 19.8nM; 95% CI: 17.3-22.7 D FITC-BIMSAHB, BFL1/A1: 17.9nM; 95% CI: 16.0-20.1 240 220 2 O O 180 160 140 120 100 10-9 10-8 10-7 106 10-5 Anti-Apoptotic Protein), M FIG. 5A IC50 o AC-BIMSAHB, BCL-X: 572nM; 95%CI:534-614 o BAM7, BCL-X. >50uM

100

10-9 10-8 10-7 10-6 10-5 10-4 Compound), M FIG. 5B U.S. Patent Apr. 5, 2016 Sheet 19 Of 65 US 9,303,024 B2

IC50 AAC-BIMSAHB, MCL-1: 136 nM; 95% Cl:117-158 A BAM7 MCL-1: >50M

100 2 - 80 an as 35 S.E. 60 is - in E 40 c - 20

O 10-9 10-8 10-7 10-6 10-5 10-4 Compound), M FIG. 5C IC50 AC-BIMSAHB, BFL-1: 603 nM; 95% Cl:559-650 BAM7 BFL-1: >50M 9 100 I D DoD no 80 On 3 35 3.G 60 > < on E 40 c5 2. 20

O 10-9 10-8 10-7 10-6 10-5 10-4 Compound), M FIG. 5D U.S. Patent Apr. 5, 2016 Sheet 20 of 65 US 9,303,024 B2

o2 o5 o6 of o8 o9

20 40 60 80 100 12O 140 16O 18O BAX Residue Number

FIG. 6A U.S. Patent Apr. 5, 2016 Sheet 21 of 65 US 9,303,024 B2

U.S. Patent Apr. 5, 2016 Sheet 22 of 65 US 9,303,024 B2

O 1 O6 O.7 O.8 O.9

40 60 80 100 120 140 160 180 BAX Residue Number

FIG. 7 U.S. Patent Apr. 5, 2016 Sheet 23 of 65 US 9,303,024 B2

3.75 7.5 10 15 FIG. 8C BAM7 uM) U.S. Patent Apr. 5, 2016 Sheet 24 of 65 US 9,303,024 B2

100

N Veh icle 2 N 15uMBAM7 Ø N Ø N^ N Ø N 2 30 uMBAM7 EGFP BAX EGFP-BAX POS ive Cells Translocation

U.S. Patent Apr. 5, 2016 Sheet 26 of 65 US 9,303,024 B2

BAKKO ZBAX KO DKO

100 OOKO<+CN Ø Ø OOOOO Ø N^10 Ø Ø NH-lo Ø Ø 2.5N NH-& 153-84 (uM) Ø r

FIG 9A U.S. Patent Apr. 5, 2016 Sheet 27 Of 65 US 9,303,024 B2

BAKKO BAX KO DKO

100

OOKO<+CN Ø Ø OOOOO N^ Ø 2.5N Ø Ø 1.25N Ø ØO.625 N Ø ØO.3125 N Ø 161-21 (uM) r O

FIG. 9B U.S. Patent Apr. 5, 2016 Sheet 28 of 65 US 9,303,024 B2

NBAKKO WBAXKO NDKO

100

COKO<+CN Ø Ø Ø Ø OOCDOCD N Ø 2.5N Ø 1.25N Ø 0.625N Ø Ø0.3125 N

FIG. 9C U.S. Patent Apr. 5, 2016 Sheet 29 Of 65 US 9,303,024 B2

BAKKO BAX KO DKO

100

OOCO<+CN Ø Ø OCDOCDCD N Ø 2.5N Ø Ø 1.25 Ø ØO.625 N Ø ØO.3125 Ø 161-79 (uM) N

FIG. 9D

U.S. Patent Apr. 5, 2016 Sheet 31 of 65 US 9,303,024 B2

code ||MWW IC50 (LM)(uM) || "|| r|| || | 4 O || 9 3

FIG 11 U.S. Patent Apr. 5, 2016 Sheet 32 of 65 US 9,303,024 B2

t 44

5 2

N O

FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 33 of 65 US 9,303,024 B2

Q - i. O 153-86 - 3 343. 4 1 OO N/ r:-N 153-92 4 12.5

153-95 4 O5 .. 5

153-96 4 19 .. 5

153-97 357. 4 NO binding

FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 34 of 65 US 9,303,024 B2 r s 5 t 5 4 3

7 6

1. 5 t 9 FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 35 of 65 US 9,303,024 B2

s 5 r 5 s s 1. s

re 1. FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 36 of 65 US 9,303,024 B2

1 6 1 - 67 481 .. 6 NO binding

1 6 1 - 79 419.5

1 61-91 4 O 5 .. 5

1 6 1 - 86 4 19 .. 5

161-87 42 6.5 ... 4

1.65 NO binding

FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 37 Of 65 US 9,303,024 B2

161-56 433.5 9

165 - 28 57 O. 6

165-42 4 67 .. 5

165 - 45 396 ... O r7. 165-53 481. O

165 - 6 O 4 67.1

165 - 62 497 - 2 K7 O

Eltrombopag 442.5

FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 38 of 65 US 9,303,024 B2

165 - 8 6-1

165 - 8 6-2

FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 39 Of 65 US 9,303,024 B2

1 7

FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 40 of 65 US 9,303,024 B2

Cr in 172–8 are 17 2-28 49 O. 62

17 2-29 47 6.5

FIG. 11 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 41 of 65 US 9,303,024 B2

5258 OT 9

518 OO 73 AN

5256949 ANA-BAM

5257582 ANA-BAM

525 99 41 ANA-BAM

526 O5 OO ANA-BAM

52 61856 U.S. Patent Apr. 5, 2016 Sheet 42 of 65 US 9,303,024 B2

52 62 O 94

52 67.338 ANA-BAM 8

52 691. 67 ANA-BAM 9

527 O356 ANA-BAM

527 O 896 ANA-BAM

527 1978 ANA-BAM

5 647 645 ANA-BAM

FIG. 12 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 43 of 65 US 9,303,024 B2

527 8 O 89 ANA-BAM 14

528 4438 ANA-BAM 15

5 646979

5 647 O19 ANA-BAM 17

5 647 O 28 ANA-BAM 18

5 647 O29

FIG. 12 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 44 of 65 US 9,303,024 B2

5 647 182

5 647.191

5 647 6O1

5 647 618 ANA-BAM

5 647 643

52.7474.2 ANA-BAM

FIG. 12 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 45 of 65 US 9,303,024 B2

5.647 655

5 64.77 65

57 O5788

57 O 58 O 6

57 O 58 O8 ANA-BAM

57 O 5811 ANA-BAM

FIG. 12 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 46 of 65 US 9,303,024 B2

57 O 5812

57 O 5813

57 O5814

57 O5815 ANA-BAM

57O 5816

57 O 5817. ANA-BAM

FIG. 12 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 47 of 65 US 9,303,024 B2

57 O 5824 ANA-BAM. 38

57 O 584 4 ANA-BAM

57 O 584 9 ANA-BAM

57 O 585 O ANA-BAM

57 O 5851. ANA-BAM

57 O 5852 ANA-BAM

FIG. 12 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 48 of 65 US 9,303,024 B2

57 O5853 ANA-BAM

57 O 84 60 ANA-BAM

57 O9214 ANA-BAM

5713 675 ANA-BAM

571 45 4 3 ANA-BAM

57 19952 ANA-BAM

FIG. 12 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 49 of 65 US 9,303,024 B2

572 O534 ANA-BAM

572 4 596 ANA-BAM

57 255 4 1 ANA-BAM

57 25 688 IANA-BAM

5844. 931. ANA-BAM

76 O 9381ANA-BAM

FIG. 12 (Cont.)

U.S. Patent Apr. 5, 2016 Sheet 51 of 65 US 9,303,024 B2

COImp Ound

FIG. 14 U.S. Patent Apr. 5, 2016 Sheet 52 of 65 US 9,303,024 B2

FIG. 14 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 53 Of 65 US 9,303,024 B2

FIG. 14 (Cont.) U.S. Patent Apr. 5, 2016 Sheet 54 of 65 US 9,303,024 B2

REH: Pre-BALL 125

O 100 O d O 75 CD O g 50 SS 25

O 10-7 10-6 10-5 10-4 103 Compound (M) F.G. 15A

DND41: T-ALL p53*, Ras

125

100 2 3 75 CD 50 D & 25

10-7 10-6 10-5 10-4 10-3 Compound M FIG. 15B U.S. Patent Apr. 5, 2016 Sheet 55 Of 65 US 9,303,024 B2

KOPT-K: T-ALL t|4:11) 125 100 d O 75 CD O S 50 & 25

O 10-7 10-6 10-5 10-4 10-3 Compound (M) FIG. 15C

MV(4:11): T-ALL t(4:11) 125

100 O g 75 CD 50 D & 25

10-7 10-6 10-5 10-4 10-3 Compound (M) FIG. 15D U.S. Patent Apr. 5, 2016 Sheet 56 of 65 US 9,303,024 B2

697: Pre-B-ALL EBV, t(7:19) 125

100

10-7 10-6 10-5 10-4 10-3 Compound M FIG 15E

RCH-ACV: Pre-BALL t(1:19) 125

100

10-7 10-6 10-5 10-4 10–3 Compound (M) F.G. 15F U.S. Patent Apr. 5, 2016 Sheet 57 Of 65 US 9,303,024 B2

SEMK2: Pre-BALL t(4:11) 125

100

75 O

O 10-7 10-6 10-5 10-4 10-3 Compound (M) FIG. 15G

Bin-1. Pre-BALL

125

100

O 10-7 10-6 10-5 10-4 10-3 Compound (M) FIG. 15H U.S. Patent Apr. 5, 2016 Sheet 58 Of 65 US 9,303,024 B2

125

100

10-7 10-6 10-5 10-4 1O-3 Compound M FIG. 16

BAM7 172-90

125 o KOPTK1 100 O MV(4:11) 2 A OC-AML3 3 75 p185'Arfl-McI-1del + BCL-X A p185*Arfl-McI-1del + MCL-1 s 50 & 25

Compound M FIG. 17

U.S. Patent US 9,303,024 B2 U.S. Patent Apr. 5, 2016 Sheet 61 of 65 US 9,303,024 B2

O JI™EI o,Og'eseqsõu?unHºHNEIZ TOUeUÏT O,O’TIOHINZ'ºoneN OVOEN TOUBUIT?EI O 9

S OO?IH N | O ÈN

YOeuexOTOTUTTOHINË "No. ºOOHeN-nes oogiliniº~** ?ON DO U.S. Patent US 9,303,024 B2

U.S. Patent Apr. 5, 2016 Sheet 64 of 65 US 9,303,024 B2

'OTO2OT130-L| O

9 NË}(ºeUexOICIU?TOH

S N () ºº!!!!!!!!!!!!!! O `r ()

II–ZLIQUIe6TJIOJ?UJèU?OS 24 TOH~º

US 9,303,024 B2 1. 2 PYRAZOL-3-ONES THAT ACTIVATE intercepted and sequestered by structurally-defined Surface PRO-APOPTOTC BAX grooves within the anti-apoptotic members (see, e.g., Sattler, M. et al. Structure of Bcl-XL-Bak peptide complex: recogni CROSS REFERENCE TO RELATED tion between regulators of apoptosis. Science 275, 983-6 APPLICATIONS (1997)). The relative levels of death-activating (pro-apop totic) BH3 domains and anti-apoptotic BH3-binding pockets This is the U.S. National Phase Application under 35 dictate the cellular response to stress. Cancer cells hijack the U.S.C. S371 of International Patent Application No. PCT/ survival circuitry of the BCL-2 family pathway, exploiting US2012/059799 filed Oct. 11, 2012, which claims the benefit pathologic overexpression of anti-apoptotic proteins to Sty of U.S. Provisional Application No. 61/546,022, filed on Oct. 10 mie physiologic and pharmacologic pro-apoptotic stimuli. 11, 2011, both of them are incorporated by reference herein. By overexpressing these anti-apoptotic proteins, cancer cells maintain a Survival advantage in the face of pro-apoptotic TECHNICAL FIELD stimuli. Thus, the over-expression of anti-apoptotic members This application features pyrazol-3-one compounds that 15 is believed to contribute to cancer pathogenesis. activate a pro-apoptotic function of BAX. Also featured are Whereas the mainstay of developmental BCL-2 family methods of using Such compounds, e.g., for the treatment or therapeutics has focused on the loss-of-function strategy of prevention of diseases, disorders, and conditions associated inhibiting anti-apoptotic proteins, direct activation of BAX with deregulated apoptosis of cells (e.g., diseased or damaged by select pro-apoptotic BCL-2 members that only contain a cells; e.g., insufficient apoptosis of diseased or damaged cells conserved BH3 domain (“BH3-only” proteins) has also or reduced apoptosis of diseased or damaged cells). Examples emerged as a physiologically relevant mechanism for induc of Such diseases, disorders, and conditions include, but are ing mitochondrial apoptosis during development and homeo not limited to, those associated with blockade(s) of cell death stasis (see Ren, D. et al. BID, BIM, and PUMA are essential pathways (e.g., over-expression of anti-apoptotic BCL-2 pro for activation of the BAX- and BAK-dependent cell death teins), e.g., hyperproliferative diseases, such as cancer. 25 program. Science 330, 1390-3 (2010)). BACKGROUND SUMMARY

BCL-2 family proteins are key regulators of the mitochon I drial apoptotic pathway in health and disease. The BCL-2 30 family includes both pro-apoptotic (e.g., BAX) and anti-apo This application features pyrazol-3-one compounds that ptotic proteins that form a complex protein interaction net activate a pro-apoptotic function of BAX, making them thera work of checks and balances that dictate cell fate (see, e.g., peutically useful for treating (e.g., controlling, relieving, Danial, N. N. & Korsmeyer, S. J. Cell death: critical control ameliorating, alleviating, or slowing the progression of) or points. Cell 116, 205-19 (2004)). 35 preventing (e.g., delaying the onset of or reducing the risk of The O.-helical BCL-2 homology 3 (BH3) domains of pro developing) diseases, disorders, and conditions associated apoptotic members (e.g., BAX) function as death ligands. with deregulated apoptosis of cells (e.g., diseased or damaged Pro-apoptotic member BAX is an executioner protein of the cells; e.g., insufficient apoptosis of diseased or damaged BCL-2 family that, when activated, undergoes a structural cells; or lack of apoptosis of diseased or damaged cells). transformation, which converts it from an inactive cytosolic 40 Examples of Such diseases, disorders, and conditions include monomer into a lethal mitochondrial pore (see Gavathiotis, (but are not limited to) those associated with blockade(s) of E., Reyna, D. E., Davis, M.L., Bird, G. H. & Walensky, L. D. cell death pathways (e.g., over-expression of anti-apoptotic BH3-triggered structural reorganization drives the activation BCL-2 proteins), e.g., hyperproliferative diseases, such as of proapoptotic BAX. Mol Cell 40, 481-92 (2010)). cancer (e.g., leukemia, e.g., acute lymphoblastic leukemia Oligomerization of BAX (and its close homologue BAK) 45 (ALL) or acute myelogenous leukemia (AML'); e.g., within the mitochondrial outer membrane enables the release chronic lymphoblastic leukemia (“CLL) or chronic myelog of apoptogenic factors such as cytochrome c and Smac? diablo enous leukemia (“CML)). While not wishing to be bound by that turn on caspases, the enzymatic effectors of apoptosis theory, it is believed that the compounds described herein (see Liu, X. Kim, C. N.Yang, J., Jemmerson, R. & Wang, X. induce and increase apoptosis in target cells (e.g., pathogenic Induction of apoptotic program in cell-free extracts: require 50 cells including, but not limited to, cancer cells), thereby Sup ment for dATP and cytochrome c. Cell 86, 147-57 (1996); Li, pressing tumor growth and/or proliferation. It is further P. et al. Cytochrome c and dATP-dependent formation of believed that increasing apoptosis in Such target cells reestab Apaf-1/caspase-9 complex initiates an apoptotic protease lishes the normal apoptotic control that, during homeostasis, cascade. Cell 91,479-89 (1997); Du, C., Fang, M., Li, Y., Li, is associated with a regulated balance between pro- and anti L. & Wang, X. Smac, a mitochondrial protein that promotes 55 apoptotic protein functions. cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33-42 (2000); Wei, M. C. et al. II Proapoptotic BAX and BAK: a requisite gateway to mito chondrial dysfunction and death. Science 292, 727-30 A. In some embodiments, the compounds described (2001)). The explicit mechanism by which BAX is triggered 60 herein directly activate BAX by direct binding to BAX. and how select pro-apoptotic BCL-2 proteins directly engage In some embodiments, the compounds described herein and activate BAX have been key questions in the apoptosis selectively bind to and activate BAX. For example, the com field (see, e.g., Youle, R. J. & Strasser, A. The BCL-2 protein pounds described herein selectively bind to and activate BAX family: opposing activities that mediate cell death. Nat Rev in the presence of one (or more) different BCL-2 proteins, Mol Cell Biol 9, 47-59 (2008)). 65 e.g., in the presence of one (or more) other pro-apoptotic The C-helical BCL-2 homology 3 (BH3) domains of acti BCL-2 proteins (e.g., BAK) and/or in the presence of one (or vated pro-apoptotic members (e.g., BAX) can, however, be more) anti-apoptotic BCL-2 proteins. US 9,303,024 B2 3 4 In some embodiments, the compounds described herein directly activate BAX by direct binding to BAX; and selec (I) tively bind to and activate BAX, e.g. selectively bind to and R O activate BAX in the presence of one (or more) different HN-A BCL-2 proteins, e.g., in the presence of one (or more) other n pro-apoptotic BCL-2 proteins (e.g., BAK) and/or in the pres N-X, ence of one (or more) anti-apoptotic BCL-2 proteins. R2 s Y BIt has been discovered that BAX contains a geographi cally distinct BH3 binding groove, which has been shown to mediate the direct activation of BAX. 10 wherein: Specifically, structural analysis of a BIM BH3 death A is N or CH: domain in complex with pro-apoptotic BAX uncovered a X is heteroaryl, which contains 5 ring atoms, wherein from BH3 interaction site that, when engaged, results in the direct 1-2 of the ring atoms is/are independently selected from N. activation of BAX (see Gavathiotis, E. etal. BAX activation is NH, N(C-C alkyl), O, and S.; wherein: initiated at a novel interaction site. Nature 455, 1076-81 15 X is connected to the pyrazolone nitrogen via a ring carbon (2008)). A BIM BH3 C-helix, structurally reinforced by atom in X; and hydrocarbon stapling, engages BAX at the opposite side of X is optionally further substituted with 1 R: the protein from the canonical BH3-binding groove of anti O apoptotic proteins (see Gavathiotis, E. etal. BAX activation is X is phenyl optionally substituted with from 1-5 R': initiated at a novel interaction site. Nature 455, 1076-81 O (2008)). See FIG. 1A. This BH3 trigger site on BAX is X is heteroaryl, which contains from 8-10 ring atoms, formed by the confluence of C-helices 1 and 6, and is struc turally defined by a hydrophobic groove comprised of amino wherein from 1-4 of the ring atoms is/are independently acids M20, A24, L27, I31, I133, M137, and L141, and a selected from N, NH, N(C-C alkyl), O, and S.; wherein: perimeter of charged and hydrophilic residues, including 25 X is connected to the pyrazolone nitrogen via a ring carbon K21, Q28, Q32, E131, and R134. See FIG. 1B. The flexible atom in X; and loop between C-helices 1 and 2 partially overlies the binding X is optionally further substituted with 1 R: site and its displacement by BIMBH3 has been implicated as Y is: the first ligand-induced conformational change of the BAX (i) C-C aryl, which is optionally substituted with from activation mechanism (see Gavathiotis, E., Reyna, D. E., 30 1-5 independently selected R'; or Davis, M. L., Bird, G. H. & Walensky, L. D. BH3-triggered (ii) heteroaryl, which contains from 5-10 ring atoms, structural reorganization drives the activation of proapoptotic wherein from 1-4 of the ring atoms is independently selected BAX. Mol Cell 40, 481-92 (2010)). For ease of exposition, from N. NH, N(C-C alkyl). O, and S; and wherein said this activating binding groove that is discussed at the start of heteroaryl ring is optionally substituted with from 1-3 inde section III is sometimes referred to herein as the “BAX 35 pendently selected R'; or trigger site' (iii) C-C alkyl, C-C haloalkyl, C-C alkoxy, C-C, In some embodiments, the compounds described herein thioalkoxy, C-Chaloalkoxy, or C-Chalothioalkoxy, each directly activate BAX by binding to BAX at the BAX trigger of which is optionally substituted with —OH, -NH2 or site. - SH: In some embodiments, the compounds described herein 40 R is: selectively activate BAX by binding to BAX at the BAX (i) C-C aryl, which is optionally substituted with from trigger site; e.g., selectively activate BAX in the presence of 1-5 independently selected R; or one (or more) different BCL-2 proteins, e.g., in the presence (ii) heteroaryl, which contains from 5-10 ring atoms, of one (or more) other pro-apoptotic BCL-2 proteins (e.g., wherein from 1-4 of the ring atoms is independently selected BAK) and/or in the presence of one (or more) anti-apoptotic 45 from N. NH, N(C-C alkyl). O, and S; and wherein said BCL-2 proteins. heteroaryl ring is optionally substituted with from 1-3 inde C. In some embodiments, the compounds described pendently selected R; or herein induce or activate BAX-dependent or mediated apop (iii) —C(O)—(Co-Co aryl or heteroaryl, which contains tosis (cell death). from 5-10 ring atoms as defined in R' definition (i) and (ii), D. In some embodiments, the methods described herein 50 respectively, above); or can include in vitro methods, e.g., contacting a sample con (iv) hydrogen; taining BAX (e.g., a cell or tissue containing BAX) with a each of RandR is, independently, selected from any one compound of formula (I) or a pharmaceutically acceptable of the substituents delineated collectively in (a), (b), (c), (d), salt thereof (e.g., including any Subgenera or specific com and (e) below: pound thereof of formula (I), e.g., formula (I-A)) 55 (a) C-C alkyl or C-Chaloalkyl, each of which is option In some embodiments, the methods described herein can ally substituted with from 1-2 R". include administering a compound of formula (I) or a phar (b) phenyl that is optionally substituted with from 1-4 R: maceutically acceptable salt thereof (e.g., including any Sub (c) heteroaryl containing from 5-6 ring atoms, wherein genera or specific compound thereof of formula (I), e.g., from 1-4 of the ring atoms is independently selected from N. formula (I-A)) to a subject (e.g., a Subject in need thereof, e.g., 60 NH, N(C-C alkyl), NC(O)(C-C alkyl). O, and S; and a mammal. Such as a human). wherein said heteroaryl is optionally substituted with from E. Accordingly, in one aspect, methods for activating 1-3 R: (e.g., directly, selectively, directly and selectively as defined (d) C-C cycloalkyl or C-C cycloalkenyl, each of which anywhere herein) BAX and/or inducing or activating BAX is optionally substituted with from 1-4 independently dependent apoptosis are featured, which include contacting 65 selected C-C alkyl groups; or BAX with a compound of formula (I) or a pharmaceutically (e) —NHC(O)(C-C alkyl), —C(O)(C-C alkyl); or acceptable salt thereof: —C(O)C(C-C alkyl); US 9,303,024 B2 5 6 R, at each occurrence, is independently selected from any (C-C alkyl); C(O)N(C-C alkyl); SO(C-C alkyl); one the substituents delineated collectively in (aa), (bb) and —SONH; SONHCC-C alkyl); and —SON(C-C, (cc) below: alkyl). (aa) C-C alkoxy; C-C haloalkoxy; C-C thioalkoxy; One or more of the following can apply. C-C thiohaloalkoxy; C-C alkyl, C-C haloalkyl, - NH 5 In some embodiments, X is not phenyl optionally Substi (C-C alkyl), N(C-C alkyl), or—NHC(O)(C-C alkyl). tuted with from 1-5 R. each of which is optionally substituted with —OH, -NH2, In some embodiments, R' is not substituted directly or azido ( N), or - SH: indirectly with one or more hydroxyl (—OH) groups. (bb) halo: —OH: —CN; nitro; —NH; azido: C-C alk In some embodiments, X is not phenyl optionally Substi enyl: C-C alkynyl; —C(O)H; —C(O)(C-C alkyl); C(O) 10 tuted with from 1-5 R"; and R' is not substituted directly or OH: —C(O)O(C-C alkyl); —OC(O)(C-C alkyl); —SO, indirectly with one or more hydroxyl (—OH) groups. (C-C alkyl); —SO(C-C haloalkyl); —C(O)NH2: In some embodiments, when R' is 2-methoxyphenyl, and —C(O)NH(C-C alkyl); C(O)N(C-C alkyl); —SO(C- R’ is C-Cs alkyl (e.g., CH4), then Y cannot be substituted Calkyl); SONH; SONHCC-C alkyl); SON(C- 15 phenyl, e.g., monosubstituted phenyl, e.g., phenyl monoSub C alkyl); —NHCO(C-C alkyl), or —NHSO(C-C, stituted at the para position, e.g., 4-chlorophenyl). alkyl); and In some embodiments, when R' is 2-carboxyphenyl, and (cc) C-C cycloalkyl, C-C cycloalkoxy, or heterocyclyl R’ is C-C alkyl (e.g., CH), then Y cannot be substituted containing from 5-6 ring atoms, wherein from 1-2 of the ring phenyl, e.g., monosubstituted phenyl, e.g., phenyl monoSub atoms of the heterocyclyl is independently selected from N. 20 stituted at the para position, e.g., 4-methoxyphenyl). NH, N(C-C alkyl), NC(O)(C-C alkyl), O, and S; and each In some embodiments, R' is other than 3-nitro-4-chlo of said ring systems is optionally substituted with from 1-3 rophenyl. In certain embodiments, R' is other than 3-nitro-4- independently selected C-C alkyl groups: chlorophenyl when R and Y are both unsubstituted phenyl. each occurrence of R and R is, independently, selected In some embodiments, the compound is other than the from any one the Substituents delineated collectively in (aaa), 25 compound sometimes referred to herein as "BAM7.” (bbb), (ccc), and (ddd) below: In another aspect, compounds having formula (I-A), or a (aaa) C-C alkoxy; C-C haloalkoxy; C-C thioalkoxy; pharmaceutically acceptable salt thereof, are featured: C-C thiohaloalkoxy; C-C alkyl, C-C haloalkyl, -NH (C-C alkyl), N(C-C alkyl), or—NHC(O)(C-C alkyl). each of which is optionally substituted with —OH, -NH, or 30 (I-A) —SH: (and optionally benzyloxy); (bbb) halo: OH: CN: nitro; NH; azido: C-C alk enyl: C-C alkynyl; —C(O)H; —C(O)(C-C alkyl); C(O) OH: —C(O)O(C-C alkyl); —OC(O)(C-C alkyl); —SO (C-C alkyl); SO(C-C haloalkyl); —C(O)NH; 35 O —C(O)NH(C-C alkyl); C(O)N(C-C alkyl); —SO(C- N ill C alkyl); —SONH; —SONHCC-C alkyl); —SON(C- N\, N X. C alkyl); —NHCO(C-C alkyl). —NHSO(C-C alkyl); N /N-( i —C(O)O-(CH2), is C(O)-(phenyl optionally sub R2 N X X" stituted as defined in (ddd) below (e.g., —C(O)O CH, C 40 (O)-(phenyl); (ccc) L-C-C cycloalkyl, C-C cycloalkoxy, or L-hetero In some embodiments of formula (I-A): cyclyl containing from 5-7 ring atoms, wherein from 1-2 of X is S; the ring atoms of the heterocyclyl is independently selected X" is unsubstituted phenyl, from N, NH, N(C-C alkyl), NC(O)(C-C alkyl), NC(O)O 45 X" is H or C-C alkyl: (C-C alkyl). O, and S; and each of said ring systems is R is: optionally substituted with from 1-3 independently selected C-C alkyl; or C-C alkyl groups; and wherein L is a bond or C-C alky phenyl that is optionally substituted with from 1-4 R; or lene; and heteroaryl containing from 5-6 ring atoms, wherein from (ddd) phenyl, -O-(phenyl), or heteroaryl containing from 50 1-4 of the ring atoms is independently selected from N. 5-6 ring atoms, wherein from 1-2 of the ring atoms of the NH, N(C-C alkyl), NC(O)(C-C alkyl). O, and S; and heteroaryl is independently selected from N, NH, N(C-C, wherein said heteroaryl is optionally substituted with alkyl), O, and S.; wherein each of said phenyl and heteroaryl from 1-3 R: is optionally substituted with from 1-3 substituents indepen R2 is: dently selected from halo: hydroxyl; cyano; —C(O)(C-C 55 C(O)OH: alkyl); C(O)CH; —C(O)O(C-C alkyl); nitro; NH; C-C alkoxy that is optionally substituted with NH, or —NH(C-C alkyl), N(C-C alkyl). —NHC(O)(C-C, heterocyclyl containing from 5-7 ring atoms, wherein from alkyl), C-C alkoxy; C-C haloalkoxy; C-C thioalkoxy; 1-2 of the ring atoms of the heterocyclyl is indepen C-C thiohaloalkoxy; C-C alkyl, and C-C haloalkyl, dently selected from N, NH, N(C-C alkyl), NC(O)(C- wherein said alkyl or alkyl portion is optionally substituted 60 C alkyl). NC(O)O(C-C alkyl), O, and S; and each of with —OH, -NH, or —SH; and which is optionally substituted with from 1-3 indepen R at each occurrence is, independently, selected from dently selected C-C alkyl groups; hydroxyl, C-C alkoxy; C-C thioalkoxy; C-C, each of RandR'' is H; and haloalkoxy; C-C thiohaloalkoxy; —NH; —NH(C-C, each occurrence of R is, independently, halo; cyano; alkyl); N(C-C alkyl); —NHC(O)(C-C alkyl): cyano: 65 —C(O)(C-C alkyl); C(O)OH: —C(O)O(C-C alkyl); —C(O)H; —C(O)(C-C alkyl); —C(O)(C-C haloalkyl); nitro; NH; —NH(C-C alkyl), N(C-C alkyl). —NHC C(O)OH: C(O)O(C-C alkyl); –C(O)NH; C(O)NH (O) (C-C alkyl), C-C alkoxy; C-C haloalkoxy; C-C, US 9,303,024 B2 7 8 thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, C-C, compound of formula (I), or a pharmaceutically acceptable cycloalkyl; and C-C haloalkyl. salt thereof (e.g., including any Subgenera or specific com One or more of the following can apply. pound thereof of formula (I), e.g., formula (I-A)). In certain embodiments, it is provided that R' cannot be In an aspect, methods for treating (e.g., controlling, reliev —C(O)CH when R is unsubstituted phenyl. ing, ameliorating, alleviating, or slowing the progression of) In certain embodiments, it is provided that R' cannot be a hyperproliferative disease in a subject in need thereofare —OCHCH when R is unsubstituted phenyl. featured. The methods include administering to the subject In certain embodiments, it is provided that R' cannot be (e.g., an effective amount of) a compound of formula (I), or a –OCHCH when R is CH. pharmaceutically acceptable salt thereof (e.g., including any In other embodiments of formula (I-A): 10 Subgenera or specific compound thereof of formula (I), e.g., X is NH; formula (I-A)). X" is unsubstituted phenyl, II. In still another aspect, methods for treating (e.g., con X" is H or C-C alkyl: trolling, relieving, ameliorating, alleviating, or slowing the R’ is: progression of) or preventing (e.g., delaying the onset of or C-C alkyl, or 15 reducing the risk of developing) cancer (e.g., leukemia, e.g., phenyl that is optionally substituted with from 1-4 R; or ALL or AML: e.g., CLL or CML) in a subject in need thereof heteroaryl containing from 5-6 ring atoms, wherein from are featured. The methods include administering to the sub 1-4 of the ring atoms is independently selected from N. ject (e.g., an effective amount of) a compound of formula (I), NH, N(C-C alkyl), NC(O)(C-C alkyl), O, and S; and or a pharmaceutically acceptable salt thereof (e.g., including wherein said heteroaryl is optionally substituted with any subgenera or specific compound thereof of formula (I), from 1-3 R: e.g., formula (I-A)). R’ is: In an aspect, methods for treating (e.g., controlling, reliev C(O)OH: ing, ameliorating, alleviating, or slowing the progression of) C-C alkoxy that is optionally substituted with —NH; or cancer (e.g., leukemia, e.g., ALL or AML) in a subject in need heterocyclyl containing from 5-7 ring atoms, wherein from 25 thereof are featured. The methods include administering to 1-2 of the ring atoms of the heterocyclyl is indepen the Subject (e.g., an effective amount of) a compound of dently selected from N, NH, N(C-C alkyl), NC(O)(C- formula (I), or a pharmaceutically acceptable salt thereof C alkyl), NC(O)O(C-C alkyl), O, and S; and each of (e.g., including any Subgenera or specific compound thereof which is optionally substituted with from 1-3 indepen of formula (I), e.g., formula (I-A)). dently selected C-C alkyl groups: 30 J. In yet another aspect, methods of modulating (e.g., each of R' and R'' is H; and increasing) apoptosis in vitro or in vivo are featured. Also each occurrence of R is, independently, halo: cyano; featured are methods of modulating (e.g., decreasing) cell —C(O)(C-C alkyl); C(O)OH: —C(O)C(C-C alkyl); division in vitro or in vivo are featured. The methods can nitro; NH; —NH(C-C alkyl), N(C-C alkyl). —NHC include contacting a sample containing BAX (e.g., a cell or (O)(C-C alkyl), C-C alkoxy: C-C haloalkoxy; C-C, 35 tissue containing BAX) with a compound of formula (I) or a thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, C-C, pharmaceutically acceptable salt thereof (e.g., including any cycloalkyl, and C-C haloalkyl. Subgenera or specific compound thereof of formula (I)); or F. In one aspect, methods for treating (e.g., controlling, administering a compound of formula (I) or a pharmaceuti relieving, ameliorating, alleviating, or slowing the progres cally acceptable salt thereof (e.g., including any Subgenera or sion of) or preventing (e.g., delaying the onset of or reducing 40 specific compound thereof of formula (I), e.g., formula (I-A)) the risk of developing) diseases, disorders, and conditions to a subject (e.g., a subject in need thereof, e.g., a mammal, associated with deregulated apoptosis of cells (e.g., diseased Such as a human). or damaged cells; e.g., insufficient apoptosis of diseased or K. In some embodiments, the methods described above damaged cells; or the lack of apoptosis of diseased or dam and throughout this disclosure can include one or more of the aged cells) in a subject in need thereof are featured. The 45 following features. methods include administering to the Subject (e.g., an effec The cancer can include carcinomas (originating in the tive amount of) a compound of formula (I), or a pharmaceu outer layer of cells of the skin and internal membranes, e.g., tically acceptable salt thereof (e.g., including any Subgenera breasts, lungs, intestines, skin, prostate, etc.); sarcomas (aris or specific compound thereof of formula (I), e.g., formula ing from connective tissue such as bone, muscle, cartilage and (I-A)). 50 blood vessels), and hematologic malignancies (e.g., lympho G In another aspect, methods for treating (e.g., control mas and leukemias, whicharise in the blood or blood-forming ling, relieving, ameliorating, alleviating, or slowing the pro organs Such as the spleen, lymph nodes and bone marrow, gression of) or preventing (e.g., delaying the onset of or e.g., leukemias, e.g., ALL or AML, e.g., CLL or CML). reducing the risk of developing) diseases, disorders, and con Cancer cells can include, for example, tumor cells, neoplastic ditions associated with blockade(s) of cell death pathways 55 cells, malignant cells, metastatic cells, and hyperplastic cells. (e.g., over-expression of anti-apoptotic proteins BCL-2 pro Non-limiting examples of cancers include breast cancer, teins) in a subject in need thereof are featured. The methods prostate cancer, lymphoma, skin cancer, pancreatic cancer, include administering to the Subject (e.g., an effective amount colon cancer, melanoma, malignant melanoma, ovarian can of) a compound of formula (I), or a pharmaceutically accept cer, brain cancer, primary brain carcinoma, head-neck cancer, able salt thereof (e.g., including any Subgenera or specific 60 glioma, glioblastoma, liver cancer, bladder cancer, non-Small compound thereof of formula (I), e.g., formula (I-A)). cell lung cancer, head or neck carcinoma, breast carcinoma, H In a further aspect, methods for treating (e.g., control ovarian carcinoma, lung carcinoma, Small-cell lung carci ling, relieving, ameliorating, alleviating, or slowing the pro noma, Wilms tumor, cervical carcinoma, testicular carci gression of) or preventing (e.g., delaying the onset of or noma, bladder carcinoma, pancreatic carcinoma, stomach reducing the risk of developing) a hyperproliferative disease 65 carcinoma, colon carcinoma, prostatic carcinoma, genitouri in a subject in need thereofare featured. The methods include nary carcinoma, thyroid carcinoma, esophageal carcinoma, administering to the Subject (e.g., an effective amount of) a myeloma, multiple myeloma, adrenal carcinoma, renal cell US 9,303,024 B2 10 carcinoma, endometrial carcinoma, adrenal cortex carci drugs (NSAIDs)); anti-inflammatory drugs (e.g., butazolidin, noma, malignant pancreatic insulinoma, malignant carcinoid DECADRON, DELTASONE, dexamethasone, dexametha carcinoma, choriocarcinoma, mycosis fungoides, malignant sone intensol, DEXONE, HEXADROL, hydroxychloro hypercalcemia, cervical hyperplasia, leukemia, acute lym quine, METICORTEN, ORADEXON, ORASONE, phocytic leukemia, chronic lymphocytic leukemia, chronic oxyphenbutaZone, PEDIAPRED, phenylbutaZone, granulocytic leukemia, acute granulocytic leukemia, acute PLAQUENIL, prednisolone, prednisone, PRELONE, and myelogenous leukemia, chronic myelogenous leukemia, TANDEARIL); and cancer chemotherapeutic drugs (e.g., hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLU Kaposi's sarcoma, polycythemia Vera, essential thrombocy DARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, tosis, Hodgkin’s disease, non-Hodgkin’s lymphoma, Soft 10 MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, tissue sarcoma, osteogenic sarcoma, primary macroglobu 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, beva linemia, and retinoblastoma. cizumab, TAXOTERE or TAXOL); cellular signaling mol In some embodiments, the patient has not been treated with ecules; ceramides and cytokines; and staurosporine, and the an agent that causes a thrombocytopenia-associated condi like. tion. In some embodiments the patient is not suffering from 15 In some embodiments, the Subject can be a Subject in need and/or is not a risk from developing a thrombocytopenia thereof (e.g., a Subject identified as being in need of Such associated condition. treatment, such as a Subject having, or at risk of having, one or In some embodiments, the methods further include admin more of the diseases or conditions described herein). Identi istering one or more additional therapeutic agents (e.g., anti fying a Subject in need of Such treatment can be in the judg cancer/chemotherapeutic agents) and/or techniques (e.g., ment of a Subject or a health care professional and can be radiation therapies, Surgical interventions, and the like) to a Subjective (e.g. opinion) or objective (e.g. measurable by a Subject or in vitro cells, tissues, and organs. test or diagnostic method). In some embodiments, the Subject In certain embodiments, the methods further include can be a mammal. In certain embodiments, the Subject can be administering one or more additional therapeutic agents such a human. as: agents that induce apoptosis; polynucleotides (e.g., anti 25 In certain embodiments, the Subject has not previously sense, ribozymes, siRNA); polypeptides (e.g., enzymes and undergone chemotherapy. In certain embodiments, the Sub antibodies); biological mimetics (BH3 mimetics); agents that ject is not suffering from, or at risk of thrombocytopenia, bind to and inhibit anti-apoptotic proteins (e.g., agents that Such as thrombocytopenia resulting from chemotherapy, inhibit anti-apoptotic BCL-2 proteins); alkaloids; alkylating radiation therapy, or bone marrow transplantation as treat agents; antitumor antibiotics; antimetabolites; hormones; 30 ment for cancer or lymphoma. platinum compounds; monoclonal or polyclonal antibodies LI In another aspect, methods of screening for (thereby (e.g., antibodies conjugated with anticancer drugs, toxins, identifying) compounds that activate BAX are featured. Said defensins, etc.), toxins, radionuclides; biological response methods generally include screening a compound of formula modifiers (e.g., interferons (e.g., IFN-alpha., etc.) and inter (I) or a pharmaceutically acceptable salt thereof (e.g., includ leukins (e.g., IL-2, etc.), etc.); adoptive immunotherapy 35 ing any Subgenera or specific compound thereof of formula agents; hematopoietic growth factors; agents that induce (I), e.g., formula (I-A)) and a test compound. The methods tumor cell differentiation (e.g., all-trans-retinoic acid, etc.); include providing: a compound of formula (I) or a pharma gene therapy reagents (e.g., antisense therapy reagents and ceutically acceptable salt thereof (e.g., including any Subgen nucleotides); tumor vaccines; angiogenesis inhibitors; pro era or specific compound thereof of formula (I)); a test com teosome inhibitors: NF kappa.beta. modulators; anti-CDK 40 pound; a first group of cells; and contacting the first group of compounds; HDAC inhibitors; and the like. cells with the formula (I) compound and the test compound; In certain embodiments, the methods further include and observing the effects of contacting the first group of cells administering one or more additional therapeutic agents that with the formula (I) compound and the test compound. In bind to and inhibit anti-apoptotic proteins (e.g., agents that some of these embodiments, the methods further provide the inhibit anti-apoptotic BCL-2 proteins), such as ABT-263, 45 additional step of comparing the effects observed in the first obatoclax, gossypol derivatives, IAP inhibitors, and Stapled cells against a second group of the cells contacted with the peptides that target anti-apoptotic proteins (MCL-1 SAHB formula (I) compound alone, or with the test compound (see, Stewart et al, Nature Chem Biol, 2010), BID SAHB alone. Effects that may be observed include, but are not lim (Walensky et al Science 2004), BAD SAHB (Danial et al ited to, those described in the Examples section. Nature Medicine 2008), BIMSAHB (Gavathiotisetal Nature 50 M In one aspect, pharmaceutical compositions are fea 2008), etc.). tured, which include a compound of formula (I), or a phar In certain embodiments, the methods further include maceutically acceptable salt thereof (e.g., including any Sub administering one or more additional therapeutic agents that genera or specific compound thereof of formula (I), e.g., induce or stimulate apoptosis. Agents that induce apoptosis formula (I-A)) and a pharmaceutically acceptable carrier. In include, but are not limited to, radiation (e.g., X-rays, gamma 55 Some embodiments, the compositions can include one or rays, UV); kinase inhibitors (e.g., Epidermal Growth Factor more additional therapeutic agents (e.g., anticancer/chemo Receptor (EGFR) kinase inhibitor, Vascular Growth Factor therapeutic agents) as defined anywhere herein. Receptor (VGFR) kinase inhibitor, Fibroblast Growth Factor In another aspect, methods of making the pharmaceutical Receptor (FGFR) kinase inhibitor, Platelet-derived Growth compositions described herein are featured. In some embodi Factor Receptor (PDGFR) kinase inhibitor, and Bcr-Abl 60 ments, the methods include taking any one or more of the kinase inhibitors such as GLEEVEC); antisense molecules: compounds of formula (I) (e.g., including any Subgenera or antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and specific compound thereof of formula (I), e.g., formula (I-A)) AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen); or a salt (e.g., a pharmaceutically acceptable salt) thereof as anti-androgens (e.g., flutamide, bicalutamide, finasteride, defined anywhere herein, and mixing said compound(s) with aminoglutethamide, ketoconazole, and corticosteroids); 65 one or more pharmaceutically acceptable carriers. cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, N In one aspect, methods of making the compounds meloxicam, NS-398, and non-steroidal anti-inflammatory described herein are featured. In some embodiments, the US 9,303,024 B2 11 12 methods include taking any one of the intermediate com thereof of formula (I)) or a salt (e.g., a pharmaceutically pounds described herein and reacting it with one or more acceptable salt) thereofas defined anywhere herein are fea chemical reagents in one or more steps to produce a com tured. In another aspect, any of the formula (I) compounds pound of formula (I) (and/or a compound of any of the other specifically described herein are featured. formulae described herein) or a salt (e.g., a pharmaceutically acceptable salt) thereofas defined anywhere herein. In some embodiments, the compounds of formula (I) are O In one aspect, the compounds of formula (I) them other than those described in the following printed publica selves (e.g., including any Subgenera or specific compound tions:

US 2011 OOO3851 U.S. Pat. No. 7,160,870 Stem Cells 2009, 27,424 Amir, Mohd.: Javed, Sadique A.: Hassan, Mohd. Zaheen. Synthesis and antimicrobial activity of pyrazolinones and pyrazoles having benzothiazole moiety. Medicinal Chemistry Research No. pp. yet given. CODEN: MCREEB ISSN: 1054-2523. AN 2011: 444952 CAPLUS Wang, Renxiao; Ma, Dawei, Li, Xun; Sun, Wei; Zhou, Bingcheng; Shi, Zhimin: Zhang, Xinglong; Zhu, Cuixia; Li, Wenwen. Preparation of thiazolylpyrazolone derivatives as Bcl-2 family proteins antagonists. Faming Zhuanli Shenqing (2009), 26pp. CODEN: CNXXEV CN 10134.3268 A 20090114 CAN 150: 191511 AN 2009: 65234 CAPLUS Efros, L. S.; Davidenkov, L. S. Benzothiazole derivatives, Preparation of 1 benzothiazolyl-3-methyl-5(4H)-pyrazolone. Zhurnal Obshchei Khimii (1951), 21 2046-50. CODEN: ZOKHA4 ISSN: OO44-46OX. CAN 46: 48.608 AN 1952:48.608 CAPLUS Patel, Satyen P.; Joshi, Ashutosh M.: Hirapara, Ketan V.; Parekh, Hansa H. Synthesis and biological evaluation of some new pyrazolones and imidazolinones. Oriental Journal of Chemistry (2003), 19(2), 435-440. CODEN: OJCHEGISSN: 0970-02OX. CAN 140: 287320 AN 2003: 876733 CAPLUS Emandi, Anca; Maior, Ovidiu; Negoiu, Maria; Lazar, Laurentiu. 1-(2-Benzothiazolyl)-3- methyl-5-pyrazolone-based dyes. Revistade Chimie (Bucharest, Romania) (1994), 45(3), 179-82. CODEN: RCBUAUISSN: 0034-7752. CAN 122: 83682 AN 1995: 17782 CAPLUS Mahesh, V. K.; Maheshwari, Mamta; Kumar, Virendra. Separation of some closely related 1-(2-benzothiazolyl)-3-methyl-4-arylhydrazono-pyrazoline-5-one derivatives by thin layer chromatography. Fresenius' Zeitschrift fuer Analytische Chemie (1981), 309(5), 4O4. CODEN: ZACFAUISSN: OO16-1152. CAN96: 144438 AN 1982: 144438 CAPLUS Wang, Renxiao; Ma, Dawei, Li, Xun; Sun, Wei; Zhou, Bingcheng; Shi, Zhimin: Zhang, Xinglong; Zhu, Cuixia; Li, Wenwen, Preparation of thiazolylpyrazolone derivatives as Bcl-2 family proteins antagonists. Faming Zhuanli Shenqing (2009), 26pp. CODEN: CNXXEV CN 10134.3268 A 20090114 CAN 150: 191511 AN 2009: 65234 CAPLUS Mamedov, V.A.; Mustakimova, L. V. Gubaidullin, A.T.: Litvinov, I.A.: Levin, Ya. A. Reactions of Isomeric Arylchloropyruvates and Glycidates with Hydrazines. Russian Journal of Organic Chemistry (2005), 41(5), 694-702. CODEN: RJOCEQISSN: 1070-4280. CAN 144: 232964 AN 2005: S84176 CAPLUS Goldfarb, David Scott. Method using lifespan-altering compounds for altering the lifespan of eukaryotic organisms, and screening for Such compounds. U.S. Pat. Appl. Publ. (2009), 57 pp. CODEN: USXXCO US 20090163545 A1 20090625 CAN 151: 115084AN 2009: 875996 CAPLUS Westman, Jacob: Kull, Bjoern; Stenberg, Patric. Hydrazono-5-oxo-4,5-dihydropyrazole-1- carbothioic acid amide derivatives, and use thereof in the treatment of prostaglandin E synthase-related diseases. PCT Int. Appl. (2009), 3Opp. CODEN: PIXXD2 WO 2009130242A1 20091029 CAN 151:4853SOAN 2009: 1330996 CAPLUS Wang, Renxiao; Ma, Dawei, Li, Xun; Sun, Wei; Zhou, Bingcheng; Shi, Zhimin: Zhang, Xinglong; Zhu, Cuixia; Li, Wenwen. Preparation of thiazolylpyrazolone derivatives as Bcl-2 family proteins antagonists. Faming Zhuanli Shenqing (2009), 26pp. CODEN: CNXXEV CN 10134.3268 A 20090114 CAN 150: 191511 AN 2009: 65234 CAPLUS Baell, Jonathan B.: Holloway, Georgina A. New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. Journal of Medicinal Chemistry (2010), 53 (7), 2719-2740. CODEN: JMCMARISSN: 0022-2623. CAN 152: 326153 AN 2010: 159922 CAPLUS hi, Un; Hudson, Andrew R.: Van Oeveren, Cornelis A.; Roach, Steven L.: Pickens, Jason C.: Shen, Yixing; Cuervo, Catalina; Valdez, Lino J.; Basinger, Jillian; Grant, Virgina H. Preparation of Small molecule hematopoietic growth factor mimetic compounds that activate hematopoietic growth factor receptors. U.S. Pat. Appl. Publ. (2011), 40pp. CODEN: USXXCO US 2011 OOO3851A1 201101.06 CAN 154: 1096O1 AN 2011: 20083 CAPLUS El-Haty, M. T. A coordination and stability study on some heterocyclic azopyrazolin-5- ones with yttrium(III), lanthanum(III), cerium(III) and uranyl(2+) ions. Journal of the Electrochemical Society of India (1991), 40(3), 113-18. CODEN: JESIA5 ISSN: 0013-466X. CAN 118: 176931 AN 1993: 176931 CAPLUS El-Haty, M.T.: Adam, F. A.; Amrallah, A. H.; Abdalla, N. A. Structure of some new aZopyrazolones derived from heterocyclic amines. Bulletin of the Faculty of Science, Assiut University (1989), 18(1), 23-33. CODEN: BSAUDW ISSN: 0366-4740. CAN 14: 23321 AN 1991: 23321 CAPLUS Atta, Aly H. Reactions of 1-(2-benzothiazolyl)-4-(dicyanomethylene)-3-methyl-2- pyrazolin-5-one towards amines. Afinidad (1999), 56(483), 303-306. CODEN: AFINAE SSN: OOO1-9704. CAN 132: 785O1 AN 1999: 71913 OCAPLUS Adam, F. A.; El-Haty, M. T. Synthesis and studies of thorium(IV) and cerium(III) complexes with some azopyrazolone compounds. Delta Journal of Science (1987), 1(3), 1089-103. CODEN: DISCES ISSN: 1012-5965. CAN 111: 145745 AN 1989: 545745 US 9,303,024 B2 13 14 -continued

CAPLUS Atta, Aly H. Reactions of 1-(2-benzothiazolyl)-4-(dicyanomethylene)-3-methyl-2- pyrazolin-5-one towards amines. Heterocyclic Communications (1999), 5(3), 243-247. CODEN: HCOMEXISSN: O793-0283. CAN 131: 2S7477 AN 1999: SO8741 CAPLUS Gehring, Reinhold; Lindig, Markus; Wroblowsky, Heinz Juergen; Santel, Hans Joachim; Schmidt, Robert R.; Brandes, Wilhelm; Strang, Harry. Preparation of 4-(aminomethylene)- 2-pyrazolin-5-ones as herbicides and fungicides. Ger. Offen. (1988), 155 pp. CODEN: GWXXBXDE 3728278 A1198.80623 CAN 110: 23881 AN 1989: 23881 CAPLUS Goldfarb, David Scott. Method using lifespan-altering compounds for altering the lifespan of eukaryotic organisms, and screening for Such compounds. U.S. Pat. Appl. Publ. (2009),57pp. CODEN: USXXCO US 20090163545 A1 20090625 CAN 151: 115085 AN 2009: 875997 CAPLUS Parija, K. Nayak, A.; Rout, Mahendra K. Preparation of azamerocyanines and evaluation of the effect of introduction of nitrogen atom in the chromophoric chain. Journal of the Indian Chemical Society (1970), 47(12), 1129-34. CODEN: JICSAH ISSN: 0019-4522. CAN 74: 113196 AN 1971: 113196 CAPLUS Rout, M. K. Effect of structural changes on absorption. I. Merocyanine dyes and aza analogs of merocyanines, unsymmetrical cyanines, and p-dialkylaminostyryl dyes. Proceedings of the Institution of Chemists (India) (1963), 35(Pt. 3), 11730. CODEN: PCHLA2 ISSN: 0369-8599. CAN 59: 82634 AN 1963: 482634 CAPLUS Zhi, Lin; Hudson, Andrew R.: Van Oeveren, Cornelis A.; Roach, Steven L.: Pickens, Jason C.: Shen, Yixing; Cuervo, Catalina; Valdez, Lino J.; Basinger, Jillian; Grant, Virgina H. Preparation of Small molecule hematopoietic growth factor mimetic compounds that activate hematopoietic growth factor receptors. U.S. Pat. Appl. Publ. (2011), 40pp. CODEN: USXXCO US 2011OOO3851A1 2011 O106 CAN 154: 109601 AN 2011: 20083 CAPLUS Goldfarb, David Scott. Method using lifespan-altering compounds for altering the ifespan of eukaryotic organisms, and screening for Such compounds. U.S. Pat. Appl. Publ. (2009),57pp. CODEN: USXXCO US 20090163545 A1 20090625 CAN S1: 115085 AN 2009: 875997 CAPLUS Bondock, Samir, El-Azap, Hossam; Kandeel, Ez-Eldin M.; Metwally, Mohamed A. Eco riendly solvent-free synthesis of thiazolylpyrazole derivatives. Monatshefte fuer Chemie (2008), 139(11), 1329-1335. CODEN: MOCMB7 ISSN: 0026-9247. CAN 51: 33471 AN 2008: 13212O3 CAPLUS Naylor, Edmund; Arredouani, Abdelilah; Vasudevan, Sridhar R.: Lewis, Alexander M.: Parkesh, Raman; Mizote, Akiko; Rosen, Daniel; Thomas, Justyn M.: Izumi, Minoru; Ganesan, A.; Galione, Antony; Churchill, Grant C. Identification of a chemical probe for NAADP by virtual screening. Nature Chemical Biology (2009), 5(4), 220-226. CODEN: NCBABT SSN: 1552-44SO. CAN 150: 369.168 AN 2009: 216734 CAPLUS Wang, Renxiao; Ma, Dawei; Li, Xun; Sun, Wei; Zhou, Bingcheng; Shi, Zhimin: Zhang, Xinglong; Zhu, Cuixia; Li, Wenwen. Preparation of thiazolylpyrazolone derivatives as Bcl-2 family proteins antagonists. Faming Zhuanli Shenqing (2009), 26pp. CODEN: CNXXEV CN 101343268 A 20090114 CAN 150: 191511 AN 2009: 65234 CAPLUS brahim, M. K. A.; Elghandour, A. H. H.; Abdel-Sayed, G. S. M.; Abdel Fattah, A. S. M. Synthesis of pyrazoles and fused pyrazoles. Novel synthesis of pyrano 2,3-cpyrazole, hieno 2,3-cpyrazole and pyrazolo3.4-bipyridine derivatives. Journal of the Indian Chemical Society (1997), 74(3), 206-208. CODEN: JICSAH ISSN: 0019-4522. CAN 27: SO36 AN 1997: 260589 CAPLUS Zhi, Lin; Hudson, Andrew R.: Van Oeveren, Cornelis A.; Roach, Steven L.: Pickens, Jason C.: Shen, Yixing; Cuervo, Catalina; Valdez, Lino J.; Basinger, Jillian; Grant, Virgina H. Preparation of Small molecule hematopoietic growth factor mimetic compounds that activate hematopoietic growth factor receptors. U.S. Pat. Appl. Publ. (2011), 40pp. CODEN: USXXCO US 2011OOO3851A1 2011 O106 CAN 154: 109601 AN 2011: 20083 CAPLUS Kalluraya, Balakrishna; Gunaga, Prashantha; Ramana, M. V. Synthesis of some triheterocyclic thiazole derivatives of biological interest. Indian Journal of Heterocyclic Chemistry (1999), 8(3), 241-242. CODEN: IJCHEI ISSN: 0971-1627. CAN 131:87864 AN 999: 285,731 CAPLUS Goldfarb, David Scott. Method using lifespan-altering compounds for altering the ifespan of eukaryotic organisms, and screening for Such compounds. U.S. Pat. Appl. Publ. (2009),57pp. CODEN: USXXCO US 20090163545 A1 20090625 CAN S1: 115085 AN 2009: 875997 CAPLUS Commercially available compounds.

In some embodiments, the foregoing exclusions can be X can have formula X-1: combined with any one or more of the exclusions described in section E above. P Embodiments can include any one or more of the fol- to (X-1) lowing features. X it X can contain 2 ring atoms independently selected from N. N NH, N(C-C alkyl), O, and S; e.g., N and S or N and NH. f \ One of the two ring atoms can be independently selected X X" from N, NH, and N(C-C alkyl)(e.g., N), and the other ring 65 atom is independently selected from N, NH, N(C-C alkyl). O, and S (e.g., S or NH, e.g., S). US 9,303,024 B2 15 16 in which: a bond or C-C alkylene (e.g., a bond); e.g., R is X" is NH, O, or S; and optionally substituted morpholino or optionally substi one of X" and X" is Y, and the other of X" and X" is H or tuted piperazinyl; and R. phenyl or heteroaryl containing from 5-6 ring atoms, X can be S. wherein from 1-2 of the ring atoms of the heteroaryl is X can be NH. independently selected from N. NH, N(C-C alkyl), O, X" can be Y. and S.; wherein each of said phenyl and heteroaryl is X" can be H or R. optionally substituted with from 1-3 substituents inde X" can be H. pendently selected from halo: hydroxyl; cyano. —C(O) X" can be R. In embodiments, R' can be C-C alkyl (e.g., 10 (C-C alkyl); C(O)OH: —C(O)C(C-C alkyl); nitro: CH). In other embodiments, R' can be phenyl that is option —NH; —NH(C-C alkyl), N(C-C alkyl). —NHC ally substituted with from 1-4 R; or C-C cycloalkyl which (O) (C-C alkyl), C-C alkoxy; C-C haloalkoxy; is optionally substituted with from 1-4 independently C-C thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, selected C-C alkyl groups. and C-C haloalkyl, wherein said alkyl or alkyl portion Y can be Co-Co aryl (e.g., phenyl), which is optionally 15 is optionally substituted with —OH, -NH, or —SH. substituted with from 1-5 independently selected R. In In embodiments, R can be C-C alkoxy (e.g., ethoxy; e.g., embodiments, Y can be unsubstituted phenyl. containing branched alkyl, Such a iso-propoxy). R" can be Co-Co aryl, which is optionally substituted with In embodiments, R can be C-C alkoxy (e.g., C-C, from 1-5 independently selected R. alkoxy, e.g., ethoxy; e.g., containing branched alkyl. Such a R" can be phenyl, which is substituted with from 1-5 (e.g., iso-propoxy). 1-3, 1-2, or 1) independently selected R. In embodiments, R can be C-C alkoxy (e.g., C-C, Each occurrence of R can be, independently, selected from alkoxy, e.g., ethoxy; e.g., containing branched alkyl. Such a any one the Substituents delineated collectively in (aaa), iso-propoxy) that is substituted with —NH. (bbb), (ccc), and (ddd) below: R" can have formula A: (aaa) C-C alkoxy, C-C alkyl, C-C haloalkyl, -NH 25 (C-C alkyl), N(C-C alkyl), or—NHC(O)(C-C alkyl). each of which is optionally substituted with —OH, -NH, or (A) - SH: (bbb) C(O)OH: –C(O)O(C-C alkyl); OC(O)(C-C, alkyl); —SONH; SONHC-C alkyl); or - SON(C- 30 Co alkyl); or -C(O)O-(CH2), is C(O)-(phenyl optionally substituted as defined in (ddd) below (e.g., —C(O) O—CH C(O)-(phenyl); (ccc) C-C cycloalkoxy or L-heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of the ring atoms of the 35 heterocyclyl is independently selected from N. NH, N(C-C, alkyl), NC(O)(C-C alkyl), NC(O)O(C-C alkyl), O, and S; and each of which is optionally substituted with from 1-3 The following definitions apply to any formula described independently selected C-C alkyl groups; and wherein L is herein that contains formula (A). a bond or C-C alkylene; and 40 One or two of R', R', and R'' is(are) an independently (ddd) phenyl or heteroaryl containing from 5-6 ring atoms, selected R, and the other(s) is(are) hydrogen. wherein from 1-2 of the ring atoms of the heteroaryl is inde R' can be R (as defined anywhere herein). pendently selected from N, NH, N(C-C alkyl). O, and S; R' can be H. wherein each of said phenyl and heteroaryl is optionally R" can be H. substituted with from 1-3 substituents independently selected 45 R" can be R. from halo: hydroxyl; cyano; —C(O)(C-C alkyl); C(O)OH: R" can be R (as defined anywhere herein), and each of R' —C(O)C(C-C alkyl); nitro; NH; NH(C-C alkyl). and R' can be H. N(C-C alkyl), NHC(O)(C-C alkyl), C-C alkoxy; R' can be R (as defined anywhere herein), and each of R' C-C haloalkoxy: C-C thioalkoxy: C-C thiohaloalkoxy: and R' can be H. C-C alkyl, and C-Chaloalkyl, wherein said alkyl or alkyl 50 In embodiments, R can be C-C alkoxy (e.g., ethoxy; e.g., portion is optionally substituted with —OH, -NH2 or containing branched alkyl, Such a iso-propoxy). SH In embodiments, R can be C-C alkoxy (e.g., C-C, In embodiments, each occurrence of R is, independently, alkoxy, e.g., ethoxy; e.g., containing branched alkyl. Such a selected from: iso-propoxy). C-C alkoxy; 55 In embodiments, R can be C-C alkoxy (e.g., C-C, C-C alkoxy (e.g., C-C alkoxy, e.g., ethoxy); or C-C, alkoxy, e.g., ethoxy; e.g., containing branched alkyl. Such a alkoxy (e.g., C-C alkoxy, e.g., ethoxy) that is Substi iso-propoxy) that is substituted with —NH. tuted with —NH; R can be –C(O)OH. C-C alkyl; R can be L-heterocyclyl containing from 5-7 (e.g., 6) ring - NHC(O)(C-C alkyl) 60 atoms, wherein from 1-2 of the ring atoms of the heterocyclyl C(O)OH: is independently selected from N, NH, N(C-C alkyl). L-heterocyclyl containing from 5-7 ring atoms, wherein NC(O)(C-C alkyl), NC(O)O(C-C alkyl), O, and S; and from 1-2 of the ring atoms of the heterocyclyl is inde each of which is optionally substituted with from 1-3 inde pendently selected from N. NH, N(C-C alkyl), NC(O) pendently selected C-C alkyl groups; and wherein L is a (C-C alkyl), NC(O)O(C-C alkyl), O, and S; and each 65 bond or C-C alkylene (e.g., a bond); e.g., R is morpholino of which is optionally substituted with from 1-3 inde or piperazinyl. In embodiments, L can be a bond or CH (e.g., pendently selected C-C alkyl groups; and wherein L is L can be a bond). US 9,303,024 B2 17 18 R" can be H, and each of R'' and R' can be R (each R" is Co-Co aryl, which is optionally substituted with from independently as defined anywhere herein). 1-5 independently selected R (e.g., R' can be phenyl, which One of R'' and R' can be C-C alkoxy (e.g., ethoxy), and is substituted with from 1-5 (e.g., 1-3, 1-2, or 1) indepen the other of R'' and R' can be independently selected from: dently selected R). C-C alkoxy: 5 In certain embodiments, the compound can have formula C-C alkyl; I-A: C(O)OH: —NHC(O)(C-C alkyl); L-heterocyclyl containing from 5-7 ring atoms, wherein (I-A) 10 R13 from 1-2 of the ring atoms of the heterocyclyl is inde R 14 pendently selected from N, NH, N(C-C alkyl), NC(O) (C-C alkyl), NC(O)O(C-C alkyl), O, and S; and each R12 of which is optionally substituted with from 1-3 inde O pendently selected C-C alkyl groups; and wherein L is a bond or C-C alkylene (e.g., a bond); e.g., R is 15 NN N X it optionally substituted morpholino or optionally substi H S. M N -( i tuted piperazinyl; and R2 N X X" phenyl or heteroaryl containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms of the heteroaryl is 2O independently selected from N, NH, N(C-C alkyl), O, wherein: and S.; wherein each of said phenyl and heteroaryl is X is NH, O, or S; optionally substituted with from 1-3 substituents inde one of X" and X" is Y, and the other of X" and X" is H or pendently selected from halo: hydroxyl; cyano. —C(O) R; (C-C alkyl); C(O)OH: —C(O)C(C-C alkyl); nitro: 2s one or two of R', R', and R'' is(are) an independently —NH; NH(C-C alkyl), N(C-C alkyl). - NHC selected R, and the other(s) is(are) hydrogen; and (O) (C-C alkyl), C-C alkoxy; C-C haloalkoxy; R can be as defined anywhere herein. C-C thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, X can be S or NH (e.g., S), X" is Y, and X" is Hor R (e.g., and C-Chaloalkyl, wherein said alkyl or alkyl portion X" can be H or C-C alkyl, e.g., CH). 30 Y can be C-C aryl, which is optionally substituted with is optionally substituted with —OH, -NH2 or —SH. from 1-5 independently selected R' (e.g., Y can be unsubsti R" can be heteroaryl, which contains from 5-10 ring atoms, tuted phenyl). wherein from 1-4 of the ring atoms is independently selected R° can be R, and each of R and R is H; or R' is R, from N. NH, N(C-C alkyl). O, and S; and wherein said and each of R'' and R' is H. heteroaryl ring is optionally substituted with from 1-3 inde 35 Each occurrence of R is, independently, selected from: pendently selected R. C-C alkoxy; R" can be heteroaryl, which contains from 5-6 ring atoms, C-C alkoxy (e.g., C2-C alkoxy, e.g., ethoxy); or C-C, wherein from 1-4 (e.g., 1-2) of the ring atoms is indepen alkoxy (e.g., C-C alkoxy, e.g., ethoxy) that is Substi dently selected from N, NH, N(C-C alkyl), O, and S; and tuted with —NH; wherein said heteroaryl ring is optionally substituted with 40 C-C alkyl; from 1-3 (e.g., 1-2 or 1) independently selected R. For C(O)OH: example, R' can be thiazolyl. —NHC(O)(C-C alkyl); R" can be heteroaryl, which contains from 8-10 ring atoms, L-heterocyclyl containing from 5-7 (e.g., 6) ring atoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is indepen wherein from 1-2 of the ring atoms of the heterocyclyl is dently selected from N. NH, N(C-C alkyl), O, and S; and 45 independently selected from N, NH, N(C-C alkyl). wherein said heteroaryl ring is optionally substituted with NC(O)(C-C alkyl), NC(O)O(C-C alkyl). O, and S; from 1-3 (e.g., 1-2 or 1) independently selected R. For and each of which is optionally substituted with from example, R' can be indazolyl. 1-3 independently selected C-C alkyl groups; and R can be C-C alkyl. For example, R can be CH. wherein L is a bond or C-C alkylene (e.g., a bond): e.g., R can be phenyl that is optionally substituted with from 50 R is an optionally substituted morpholino or optionally 1-4 R. In embodiments, R can be unsubstituted phenyl. Substituted piperazinyl ring; and R can be C-Cs cycloalkyl which is optionally substituted phenyl or heteroaryl containing from 5-6 ring atoms, with from 1-4 independently selected C-C alkyl groups. wherein from 1-2 of the ring atoms of the heteroaryl is R is heteroaryl containing from 5-6 (e.g., 5) ring atoms, independently selected from N, NH, N(C-C alkyl), O, wherein from 1-4 of the ring atoms is independently selected 55 and S.; wherein each of said phenyl and heteroaryl is from N, NH, N(C-C alkyl), NC(O)(C-C alkyl). O, and S; optionally substituted with from 1-3 substituents inde and wherein said heteroaryl is optionally substituted with pendently selected from halo: hydroxyl; cyano. —C(O) from 1-3 R. For example, R can be furanyl, thienyl, or (C-C alkyl); C(O)OH: —C(O)C(C-C alkyl); nitro: thiazolyl. —NH; —NH(C-C alkyl), N(C-C alkyl). —NHC A can be N. 60 (O) (C-C alkyl), C-C alkoxy; C-C haloalkoxy; In some embodiments: C-C thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, A is N: and C-C haloalkyl, wherein said alkyl or alkyl portion X contains 2 ring atoms independently selected from N. is optionally substituted with —OH, -NH, or —SH. NH, N(C-C alkyl), O, and S, and one of the ring atoms is R can be C-C alkoxy (e.g., ethoxy or iso-propoxy); or R independently selected from N, NH, and N(C-C alkyl), and 65 can be —COOH, or R can be morpholino or piperazinyl. the other ring atom is independently selected from N. NH, R can be C-C alkoxy (e.g., C-C alkoxy, e.g., ethoxy or N(C-C alkyl). O, and S; and iso-propoxy) that is optionally substituted with —NH; or R US 9,303,024 B2 19 20 can be —COOH, or R can be an optionally substituted mor In certain embodiments, the compound can have formula pholino or optionally Substituted piperazinyl ring. I-B: R can be C-C alkoxy (e.g., C-C alkoxy, e.g., ethoxy or iso-propoxy) that is optionally substituted with —NH. R can be C-Cs alkyl (e.g., CH4). (I-B) Rican be phenylor heteroaryl containing from 5-6 (e.g.,5) O ring atoms, wherein from 1-4 of the ring atoms is indepen "N-N N - X" dently selected from N. NH, N(C-C alkyl), NC(O)(C-C, n alkyl), O, and S; and wherein said heteroaryl is optionally S. M N-( i substituted with from 1-3 R (e.g., R is heteroarylas defined 10 R2 N X X" above, e.g., thienyl, furanyl, or thiazolyl). X" can be H or CH. wherein: R can be C-C alkyl, such as CHs. R can be unsubsti X" is NH, O, or S; and tuted phenyl. R can be heteroaryl containing from 5-6 ring one of X" and X" is Y, and the other of X" and X" is H or atoms, wherein from 1-4 of the ring atoms is independently 15 selected from N, NH, N(C-C alkyl), NC(O)(C-C alkyl). R; O, and S; and wherein said heteroaryl is optionally substituted X' can be S, X" can be Y, and X" can be H or R. with from 1-3 R, such as thienyl, furanyl, or thiazolyl. X" can be H. R' can be C(O)OH. R' can be C-C alkoxy that is R" can be thiazolyl. optionally substituted with —NH, such as —OCHCH or Y can be C-C aryl, which is optionally substituted with —OCHCH-NH. R' can be heterocyclyl containing from from 1-5 independently selected R' (e.g., unsubstituted phe 5-7 ring atoms, wherein from 1-2 of the ring atoms of the nyl). heterocyclyl is independently selected from N, NH, N(C-C, R’ can be phenyl that is optionally substituted with from alkyl), NC(O)(C-C alkyl), NC(O)O(C-C alkyl), O, and S; 1-4 R; or heteroaryl containing from 5-6 (e.g., 5) ring atoms, and each of which is optionally substituted with from 1-3 25 wherein from 1-4 of the ring atoms is independently selected independently selected C-C alkyl groups, such as an option from N, NH, N(C-C alkyl), NC(O)(C-C alkyl), O, and S; ally Substituted piperazinyl ring. and wherein said heteroaryl is optionally substituted with X" can be H or CH; and R can be C-C alkyl, such as from 1-3 R. CHs; or R can be unsubstituted phenyl; or R can be het R can be unsubstituted phenyl. eroaryl containing from 5-6 ring atoms, wherein from 1-4 of 30 The compound can be selected from compounds delin the ring atoms is independently selected from N. NH, N(C- eated in FIGS. 11, 12, and 14. C alkyl). NC(O)(C-C alkyl). O, and S; and wherein said The contacting can be in vitro. heteroaryl is optionally substituted with from 1-3 R, such as The contacting is in vivo. thienyl, furanyl, or thiazolyl. In some embodiments, any compound, composition, or X" can be H or CH; and 35 method described herein can also include or further include Rican be C-C alkyl, such as CH; or R can be unsub any one or more of the other features delineated in the detailed stituted phenyl; or R can be heteroaryl containing from 5-6 description and/or in the claims. ring atoms, wherein from 1-4 of the ring atoms is indepen IQ Definitions dently selected from N. NH, N(C-C alkyl), NC(O)(C-C, The term “mammal’ includes organisms, which include alkyl), O, and S; and wherein said heteroaryl is optionally 40 mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, substituted with from 1-3 R, such as thienyl, furanyl, or dogs, cats, and humans. thiazolyl; and In embodiments, an amount of a compound of formula (I) R' can be –C(O)OH: or R' can be C-C alkoxy that is or salt thereof can be an effective amount. An effective optionally substituted with —NH, such as —OCHCH or amount refers to an amount of a compound that confers a —OCH2CH-NH2 or R' can be heterocyclyl containing 45 therapeutic effect (e.g., treats, e.g., controls, relieves, amelio from 5-7 ring atoms, wherein from 1-2 of the ring atoms of the rates, alleviates, or slows the progression of or prevents, e.g., heterocyclyl is independently selected from N, NH, N(C-C, delays the onset of or reduces the risk of developing, a dis alkyl), NC(O)(C-C alkyl), NC(O)O(C-C alkyl), O, and S; ease, disorder, or condition or symptoms thereof) on the and each of which is optionally substituted with from 1-3 treated subject. The therapeutic effect may be objective (i.e., independently selected C-C alkyl groups, such as an option 50 measurable by some test or marker) or subjective (i.e., Subject ally Substituted piperazinyl ring. gives an indication of or feels an effect). An effective amount X" can be H or CH; and of the compound described above may range from about 0.01 R can be C-C alkyl, such as CH; and mg/kg to about 1000 mg/kg, (e.g., from about 0.1 mg/kg to R' can be C-C alkoxy that is optionally substituted with about 100 mg/kg, from about 1 mg/kg to about 100 mg/kg). —NH, such as —OCHCH or —OCHCH-NH2: 55 Effective doses will also vary depending on route of admin In some embodiments: istration, as well as the possibility of co-usage with other A is N: agents. X contains 2 ring atoms independently selected from N. The term “halo' or “halogen refers to any radical of fluo NH, N(C-C alkyl), O, and S, and one of the ring atoms is rine, chlorine, bromine or iodine. independently selected from N, NH, and N(C-C alkyl), and 60 In general, and unless otherwise indicated, Substituent the other ring atom is independently selected from N. NH, (radical) prefix names are derived from the parent hydride by N(C-C alkyl). O, and S; and either (i) replacing the “ane' in the parent hydride with the R" is heteroaryl, which contains from 5-10 ring atoms, suffixes “yl,”“diyl.”“triyl.”“tetrayl,” etc.; or (ii) replacing the wherein from 1-4 of the ring atoms is independently selected “e' in the parent hydride with the suffixes “yl,” “diyl.”“triyl.” from N. NH, N(C-C alkyl). O, and S; and wherein said 65 “tetrayl, etc. (Here the atom(s) with the free valence, when heteroaryl ring is optionally substituted with from 1-3 inde specified, is (are) given numbers as low as is consistent with pendently selected R. any established numbering of the parent hydride). Accepted US 9,303,024 B2 21 22 contracted names, e.g., adamantyl, naphthyl, anthryl, additional groups may be present to complete the nitrogen phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and pip valence and/or form a salt), or S. The heteroatom or ring eridyl, and trivial names, e.g., vinyl, allyl, phenyl, and thienyl carbon can be the point of attachment of the heterocyclyl are also used herein throughout. Conventional numbering/ Substituent to another moiety. Any atom can be optionally lettering systems are also adhered to for Substituent number 5 Substituted, e.g., by one or more Substituents. Heterocyclyl ing and the nomenclature of fused, bicyclic, tricyclic, and groups can include groups such as tetrahydrofuryl, tetrahy polycyclic rings. dropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl The following definitions are used unless otherwise (morpholino), pyrrolinyl, and pyrrolidinyl. By way of described. Specific and general values listed below for radi example, a phrase Such as "heterocyclic ring containing from cals, Substituents, and ranges are for illustration only. They do 10 not exclude other defined values or other values within 5-6 ring atoms', wherein from 1-2 of the ring atoms is inde defined ranges for the radicals and Substituents. Unless oth pendently selected from N, NH, N(C-C alkyl), NC(O)(C- erwise indicated, alkyl, alkylene, alkoxy, alkenyl, and the like C alkyl), O, and S; and wherein said heterocyclic ring is denote both straight and branched groups. optionally substituted with from 1-3 independently selected The term “alkyl refers to a saturated hydrocarbon chain 15 R" would include (but not be limited to) tetrahydrofuryl, that may be a straight chain or branched chain, containing the tetrahydropyranyl, piperidyl (piperidino), piperazinyl, mor indicated number of carbon atoms. For example, C-C alkyl pholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. indicates that the group may have from 1 to 6 (inclusive) The term “cycloalkyl refers to a fully saturated monocy carbon atoms in it. Any atom can be optionally Substituted, clic, bicyclic, tricyclic, or other polycyclic hydrocarbon e.g., by one or more Substitutents. Examples of alkyl groups group. Any atom can be optionally substituted, e.g., by one or include, without limitation, methyl, ethyl, n-propyl, isopro more Substituents. A ring carbon serves as the point of attach pyl, and tert-butyl. ment of a cycloalkyl group to another moiety. Cycloalkyl As used herein, the term "C., alkylene.” employed alone moieties can include groups such as cyclopropyl, cyclobutyl, or in combination with other terms, refers to a non-branched cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and nor divalent alkyl linking group having n to m carbon atoms. 25 bornyl (bicyclo[2.2.1]heptyl). The term “haloalkyl refers to an alkyl group in which at The term “cycloalkenyl refers to partially unsaturated least one hydrogenatom is replaced by halo. In some embodi monocyclic, bicyclic, tricyclic, or other polycyclic hydrocar ments, more than one hydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, bon groups. A ring carbon (e.g., Saturated or unsaturated) is 9, 10, 11, 12, 13, or 14) is replaced by halo. In these embodi the point of attachment of the cycloalkenyl substituent. Any ments, the hydrogenatoms can each be replaced by the same 30 atom can be optionally Substituted e.g., by one or more Sub halogen (e.g., fluoro) or the hydrogen atoms can be replaced stituents. Cycloalkenyl moieties can include, e.g., cyclohex by a combination of different halogens (e.g., fluoro and enyl, cyclohexadienyl, or norbornenyl. chloro). “Haloalkyl also includes alkyl moieties in which all hydrogens have been replaced by halo (sometimes referred to The term “aryl” refers to an aromatic monocyclic, bicyclic herein as perhaloalkyl, e.g., perfluoroalkyl. Such as trifluo 35 (2 fused rings), tricyclic (3 fused rings), or polycyclic (>3 romethyl). Any atom can be optionally Substituted, e.g., by fused rings) hydrocarbon ring system. One or more ring one or more substituents. atoms can be optionally Substituted by one or more substitu As referred to herein, the term “alkoxy' refers to a group of ents for example. Aryl moieties include groups such as phenyl formula —O(alkyl). Alkoxy can be, for example, methoxy and naphthyl. (—OCH), ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, 40 The term "heteroaryl” refers to an aromatic monocyclic, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, 3-pentoxy, or bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic hexyloxy. Likewise, the term “thioalkoxy' refers to a group of (>3 fused rings) hydrocarbon groups having one or more formula —S(alkyl). The terms “haloalkoxy” and “thio-ha heteroatom ring atoms independently selected from O, N (it is loalkoxy' refer to —O(haloalkyl) and —S(haloalkyl). understood that one or two additional groups may be present respectively. Finally, the terms “cycloalkoxy” and "heterocy 45 to complete the nitrogen Valence and/or form a salt), or S. One clyloxy' refer to a group of the formula—O(cycloalkyl) and or more ring atoms can be optionally substituted, e.g., by one —O(heterocyclyl), respectively. or more Substituents. Examples of heteroaryl groups include, The term “alkenyl refers to a straight or branched hydro but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinoliz carbon chain containing the indicated number of carbon inyl, acridinyl, benzobthienyl, benzothiazolyl, B-carboli atoms and having one or more carbon-carbon double bonds. 50 nyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo Any atom can be optionally Substituted, e.g., by one or more b.dfuranyl, furazanyl, furyl, imidazolyl, imidazolyl, Substituents. Alkenyl groups can include, e.g., vinyl, allyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, 1-butenyl, and 2-hexenyl. One of the double bond carbons can isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidi optionally be the point of attachment of the alkenyl substitu nyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phena ent. 55 Zinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, The term “alkynyl refers to a straight or branched hydro phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyra carbon chain containing the indicated number of carbon Zolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazoli atoms and having one or more carbon-carbon triple bonds. nyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiaz Alkynyl groups can be optionally Substituted, e.g., by one or olyl, thienyl, triazolyl, and Xanthenyl. more Substituents. Alkynyl groups can include groups such as 60 As used herein, the descriptor “ CN represents the ethynyl, propargyl, and 3-hexynyl. One of the triple bond cyano group (and Vice versa), wherein the carbon and nitro carbons can optionally be the point of attachment of the genatoms are bound together by a triple bond. As used herein, alkynyl Substituent. the descriptor “ OH represents the hydroxy group (and The term "heterocyclyl refers to a fully saturated mono vice versa). The descriptors “C=O or “C(O)” refers to a cyclic, bicyclic, tricyclic or other polycyclic ring system hav 65 carbon atom that is doubly bonded to an oxygen atom. ing one or more constituent heteroatom ring atoms indepen In general, when a definition for a particular variable dently selected from O, N (it is understood that one or two includes hydrogen and non-hydrogen (halo, alkyl, aryl, etc.) US 9,303,024 B2 23 24 possibilities, the term “substituent(s) other than hydrogen BAX structures. This screening yielded a panel of 100 can refers collectively to the non-hydrogen possibilities for that didate BAX activator molecules (BAMs). particular variable. FIG. 3B is a compilation of the docked structures and The term “substituent” refers to a group “substituted on demonstrates how candidate BAMs occupy the topographic groups such as an alkyl, haloalkyl, cycloakyl, heterocyclyl, 5 landscape of the BAX trigger site. aryl, or heteroaryl group at any atom of that group. In one FIG. 4A is a graph showing the direct binding interaction aspect, the Substituent(s) on a group are independently any between FITC-BIM SAHB and recombinant full-length one single or any combination of two or more of the permis BAX. This formed the basis for developing a competitive sible atoms or groups of atoms delineated for that Substituent. fluorescence polarization binding assay to screen for BAMs. 10 FIG. 4B is a graph showing that acetylated BIM SAHB In another aspect, a substituent may itself be substituted with (Ac-BIM SAHB), which effectively competed with FITC any one of the above substituents. BIMSAHB for BAX binding, served as a positive control for Further, as used herein, the phrase “optionally substituted the assay. means unsubstituted (e.g., Substituted with hydrogen (H)) or FIG. 4C is a bar graph showing that eleven molecules substituted. As used herein, the term "substituted” means that 15 achieved >55% displacement of FITC-BIMSAHB at the 100 a hydrogen atom is removed and replaced by a Substituent. It M Screening dose. These eleven compounds were advanced is understood that Substitution at a given atom is limited by to dose-responsive competitive binding analysis. *, no detect Valency. able FITC-BIMSAHB binding. Descriptors such as "C-Clo aryl that is optionally Substi FIG. 4D is a graph that shows that a compound sometimes tuted with from 1-4 independently selected R' (and the like) referred to herein as BAM7 emerged as the most effective of is intended to include both an unsubstituted C-Caryl group the tested Small molecule competitors, displaying an ICs of and a Co-Co aryl group that is Substituted with from 1-4 3.3 uM. Data are mean and S.d. for experiments performed in independently selected R. The use of a substituent (radical) at least triplicate. prefix name such as alkyl without the modifier “optionally FIG.4E shows the chemical structure of BAM7, which was substituted” or “substituted' is understood to mean that the 25 confirmed by NMR and mass spectrometry, synthesized de particular substituent is unsubstituted. However, the use of novo, and found to have a similar ICs (4.4LM) upon retesting “haloalkyl without the modifier “optionally substituted” or by competitive FPA. “Substituted” is still understood to mean an alkyl group, in FIG. 4F shows the 'H-NMR spectrum of BAM7. which at least one hydrogen atom is replaced by halo. FIG. 5A is a graph showing that in addition to engaging R Unless otherwise defined, all technical and scientific 30 BAX, FITC-BIM SAHB binds to the broad range of anti terms used herein have the same meaning as commonly apoptotic targets. understood by one of ordinary skill in the art to which this FIGS. SB-5D are graphs summarizing the following data. invention belongs. Methods and materials are described The specificity of BAM7 for the BH3 binding site on BAX hereinforuse in the present invention; other, suitable methods was examined by competitive FPA employing FITC-BIM and materials known in the art can also be used. The materials, 35 SAHB and anti-apoptotic BCL-X, MCL-1, and BFL-1/A1. methods, and examples are illustrative only and not intended Whereas Ac-BIM SAHB effectively competed with FITC to be limiting. All publications, patent applications, patents, BIM SAHB for binding to a diversity of anti-apoptotic tar sequences, database entries, and other references mentioned gets, BAM7 demonstrated little to no capacity to compete herein are incorporated by reference in their entirety. In case with FITC-BIM SAHB for interaction at the BH3 binding of conflict, the present specification, including definitions, 40 sites of anti-apoptotic proteins. Data are mean and S.d. for will control. experiments performed in at least triplicate. Other features and advantages of the invention will be FIG. 6A shows (i) a graph in which the measured chemical apparent from the following detailed description and figures, shift changes of 'N-BAX upon BAM7 titration up to a ratio and from the claims. of 1:1 BAX:BAM7 are plotted as a function of BAXresidue 45 number and (ii) a ribbon diagram. Residues with significant DESCRIPTION OF DRAWINGS backbone amide chemical shift change are concentrated in the region of the trigger site (C.1, C.6; magenta). The Caatoms FIG. 1A is a structural depiction of BAX, which shows that of affected residues are represented as spheres in the ribbon the BIM BH3 trigger site on pro-apoptotic BAX (left hand diagram and lighter shaded bars in the graph (calculated side of structural depiction) localizes to the N-terminal face of 50 significance threshold >0.009 p.p.m.). the protein. In contrast, the canonical BH3 binding pocket of FIG. 6B is a structural depiction of the docked structure of anti-apoptotic proteins (right hand side of structural depic BAM7 at the trigger site and predicts that the pyrazolone core tion) maps to the opposite side of BAX and remains occupied of BAM7 sits at the base of the 6A7 activation epitope (amino by the C-terminal helix 9 (yellow) when the protein is in the acids 12-24), with the molecule's carbonyl group engaged in inactive, monomeric state. 55 hydrogen bonding interactions with K21. The ethoxyphenyl FIG.1B is a structural depiction of BAX, which shows that group is positioned at the confluence of residues comprising the BH3 trigger site is comprised of a hydrophobic groove the C.1-C2 loop's C-terminus and the N-terminiD of C.1 and with a perimeter of charged and hydrophilic residues from C2, a presumed hinge region for loop opening upon initiation C.-helices 1 and 6. BAX is oriented to demonstrate its N-ter of direct BAX activation. The methyl and phenylthiazol R minal face and the individual amino acids that comprise the 60 groups are predicted to make hydrophobic contact with ali trigger site, with the unstructured loop between C.1 and C2 phatic residues of C1 and O.6, which also form a portion of the depicted in the open position. BIM BH3-binding groove. FIGS. 2A-2I are reaction schemes showing the syntheses FIG. 7 includes a ribbon diagram and a graph. BAM7 used to prepare a variety of compounds of formula (I). induces allosteric changes in key functional domains impli FIG. 3A is a flow diagram showing the results of a com 65 cated in functional BAX activation. Upon increasing the ratio putational Screening algorithm employing an in silico library of BAM7:'N-BAX from 1:1 to 2:1, a series of chemical shift of 750,000 small molecules docked on averaged minimized changes become more prominent in the C1-C2 loop, C2 US 9,303,024 B2 25 26 (BH3), and C.9, three regions previously implicated in BIM FIG. 16 is a graph that compares the anti-leukemic activity BH3-triggered N-terminal loop opening, BAX BH3 expo of BAM7 to compounds 165-93, 165-60, and 172-90 (see sure, and C-terminal helix mobilization, respectively'. FIG. 14). These BAM7-induced allosteric changes reflect a major con FIG. 17 is a graph that demonstrates the broad anti-leuke formational change that has been linked to functional BAX mic activity of compound 172-90. activation. Ca atoms of affected residues are represented as FIGS. 18A and 18B are graphs that demonstrate that com spheres in the ribbon diagram and lighter shaded bars in the pound 172-90 overcomes the apoptotic resistance conferred plot (calculated significance threshold >0.011 p.p.m.). The by BCL-2 family anti-apoptotic members BCL-XL and C1-C2 loop, C2 (BH3), and C.9 are highlighted in pink, cyan, MCL-1; whereas the BCL-2/BCL-XL selective inhibitor and yellow, respectively. 10 ABT-737 induces cell death of the BCL-XL-dependent leu FIG. 8A is a graph showing that Co-incubation of BAM7 kemia cell line, significant resistance to ABT-737 is manifest (10, 20, and 30 uM) and monomeric BAX(5M) induces dose in the isogenic MCL-1 dependent leukemia cell line. In con and time-responsive BAX oligomerization, as monitored by trast, FIGS. 18C and 18D are graphs that demonstrate that size exclusion chromatography. 15 compound 172-90 induces dose-responsive caspase 3/7 activ FIG.8B is a graph showing that in the presence of ANTS/ ity and cell death in both cell lines, overcoming formidable DPX-loaded liposomes, BAM7 treatment triggers dose-re apoptotic resistance. sponsive BAX-mediated liposomal release. The exposure of FIGS. 19 A-19F are reaction schemes showing the synthe liposomes to BAM7 or BAX alone had no such effect. ses used to prepare a variety of compounds of formula (I), FIG. 8C is a bar graph that shows BAM7 selectively such as those delineated in FIG. 14. impaired the viability of Bak MEFs, but had no effect on MEFs that lack BAX(Bax) or both BAX and BAK (Bax DETAILED DESCRIPTION Bak'). FIG.8D shows that Bax Bak MEF's reconstituted with This application features pyrazol-3-one compounds that EGFP-BAX (-60% EGFP-positive cells) display dose-re 25 activate a pro-apoptotic function of BAX, making them thera sponsive BAX translocation upon exposure to BAM7, as peutically useful for treating (e.g., controlling, relieving, evidenced by the conversion of EGFP-BAX localization from ameliorating, alleviating, or slowing the progression of) or a diffuse pattern to a mitochondrion-localized distribution. preventing (e.g., delaying the onset of or reducing the risk of EGFP-BAX, green; Mitotracker, red; Colocalization, yellow: developing) diseases, disorders, and conditions associated BAM7.30puM;Vehicle, 0.3% DMSO. Data are mean and s.d. 30 with deregulated apoptosis of cells (e.g., diseased or damaged for experiments performed in quadruplicate. cells; e.g., insufficient apoptosis of diseased or damaged FIG. 8E shows Bak MEFs that contain endogenous cells; or lack of or reduced apoptosis of diseased or damaged BAX exhibiting the morphologic features of apoptosis in cells). Examples of Such diseases, disorders, and conditions response to BAM7 treatment (15uM). The time lapse images include (but are not limited to) those associated with reveal progressive cellular shrinkage, membrane blebbing, 35 and the formation of apoptotic bodies. 1, 20 min; 2, 6 h; 3, 12 blockade(s) of cell death pathways (e.g., over-expression of h; 4, 12.5 h; 5, 13.5 h, 6, 14.5 h; 7, 16.5 h; 8, 17.5 h. anti-apoptotic BCL-2 proteins), e.g., hyperproliferative dis FIG. 9A-9D are bar graphs that shows formula (I) com eases, such as cancer. While not wishing to be bound by pounds selectively impair the viability of Bak MEFs, but theory, it is believed that the compounds described herein had no effect on MEFs that lack BAX (Bax) or both BAX 40 induce and increase apoptosis in target cells (e.g., pathogenic and BAK (Bax Bak'). For each triplet of bars centered on cells including, but not limited to, cancer cells), thereby Sup the indicated X-axis value, the BAK knockout result is the pressing tumor growth and/or proliferation. It is further left-most bar; the BAX knockout result is the middle bar; and believed that increasing apoptosis in said target cells reestab the double knockout result is the right-most bar. lishes the normal apoptotic control that, during homeostasis, FIG. 10A shows that BAM7 dose-responsively impairs the 45 is associated with a regulated balance between pro- and anti viability of DHL5 diffuse large B-cell lymphoma (DLBCL) apoptotic protein functions. cells and the addition of BCL-2/BCL-XL inhibitor ABT-737 Compounds further sensitizes the cells to BAM7 anti-cancer activity. This application features pyrazol-3-one compounds hav FIGS. 10B and 10C are bargraphs showing that BAM7 can ing formula (I) below as well as compositions and methods sensitize DHL5 DLBCL cells to ABT-737, which is other 50 that include the formula (I) compounds. wise less effective in DHL5 cells due to the expression of anti-apoptotic proteins that lie outside its binding spectrum. In FIGS. 10A-10C, for each triplet of bars centered on the (I) indicated x-axis value, the BAM7 (10 uM) is the left-most R O bar: the ABT-737 is the middle bar; and BAM7 (10 uM) plus 55 HN-A ABT-737 is the right-most bar. n FIG. 11 includes the chemical structures and binding data N-X for formula (I) compounds. R2 N Y FIG. 12 includes the chemical structures of some formula (I) compounds. 60 FIG. 13 includes data that supplements the screening data Here and throughout this specification, the attendant vari provided in FIG. 4C. ables R', R, A, X, and Y (and any sub-variables) can be as FIG. 14 includes the chemical structures of some formula defined anywhere herein. (I) compounds that are encompassed by formula (I-A) or It is appreciated that certain features of the invention, (I-B). 65 which are, for clarity, described in the context of separate FIGS. 15A-15H are graphs that demonstrate the anti-leu embodiments, can also be provided in combination in a single kemic activity of BAM7. embodiment. Conversely, various features of the invention US 9,303,024 B2 27 28 which are, for brevity, described in the context of a single X" is Y, which can be as defined anywhere herein; and X" embodiment, can also be provided separately or in any Suit is Hor R. In embodiments, X is S or NH (e.g., S). able sub-combination. Variable Y Thus, for ease of exposition, it is also understood that In some embodiments, Y is: where in this specification, a variable (e.g., R') is defined by (i) C-C aryl, which is optionally substituted with from “as defined anywhere herein” (or the like), the definitions for 1-5 independently selected R'; or that particular variable include the first occurring and broad (ii) heteroaryl, which contains from 5-10 ring atoms, est generic definition as well as any Sub-generic and specific wherein from 1-4 of the ring atoms is independently selected definitions delineated anywhere in this specification. from N, NH, N(C-C alkyl), O, and S; and wherein said Variable X 10 heteroaryl ring is optionally substituted with from 1-3 inde In some embodiments, X is heteroaryl, which contains 5 pendently selected R'; or ring atoms, wherein from 1-2 of the ring atoms is/are inde (iii) C-C alkyl or C-C haloalkyl, each of which is pendently selected from N, NH, N(C-C alkyl). O, and S; optionally substituted with —OH, -NH2 or —SH. wherein: In some embodiments. Y is C-C aryl, which is optionally 15 substituted with from 1-5 independently selected R. In cer X is connected to the pyrazolone nitrogen via a ring carbon tain embodiments, Y is phenyl, which is optionally substi atom in X; and tuted with from 1-5 independently selected R. In certain X is optionally further substituted with 1 R: embodiments, Y is unsubstituted phenyl. O In some embodiments, Y is heteroaryl, which contains X is phenyl optionally substituted with from 1-5 R'. from 5-10 ring atoms, wherein from 1-4 of the ring atoms is In some embodiments, X is heteroaryl, which contains 5 independently selected from N, NH, N(C-C alkyl). O, and ring atoms, wherein from 1-2 of the ring atoms is/are inde S; and wherein said heteroaryl ring is optionally substituted pendently selected from N, NH, N(C-C alkyl). O, and S; with from 1-3 independently selected R. wherein: In some embodiments, Y is heteroaryl, which contains X is connected to the pyrazolone nitrogen via a ring carbon 25 from 5-6 ring atoms, wherein from 1-4 (e.g., 1-2) of the ring atom in X; and atoms is independently selected from N, NH, N(C-C alkyl). X is optionally further substituted with 1 R: O, and S; and wherein said heteroaryl ring is optionally Sub In some embodiments, X contains 2 ring atoms indepen stituted with from 1-3 independently selected R. dently selected from N, NH, N(C-C alkyl), O, and S (e.g., In Some embodiments, Y is C-C alkyl or C-Chaloalkyl, 30 each of which is optionally substituted with —OH, -NH, or selected from N and S; e.g., selected from N and NH). —SH (e.g., C-C alkyl, which is optionally substituted with In certain embodiments, one of the two ring atoms is inde —OH, -NH2 or —SH; e.g., C-C alkyl, such as CH). pendently selected from N, NH, and N(C-C alkyl)(e.g., N), Variable R' and the other ring atom is independently selected from N, NH, R" is: N(C-C alkyl). O, and S (e.g., the other ring atom is O or S, 35 (i) C-C aryl, which is optionally substituted with from e.g., S.; or e.g., NH). 1-5 independently selected R; or As an example, X can have formula X-1: (ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N. NH, N(C-C alkyl). O, and S; and wherein said (X-1) X it 40 heteroaryl ring is optionally substituted with from 1-3 inde pendently selected R; or (iii) —C(O)—(C-C aryl or heteroaryl, which contains from 5-10 ring atoms as defined in R' definition (i) and (ii), respectively); or 45 (iv) hydrogen. In some embodiments, R' is any one, two, or three of the above (e.g., (i) and/or (ii) and one of (iii) or (iv); e.g., (i) and/or in which: (ii); e.g., (iii) and/or (iv)). X" is NH, O, or S; and In some embodiments, R' is Co-Co aryl, which is option one of X" and X" is Y, and the other of X" and X" is H or 50 ally substituted with from 1-5 independently selected R. R. In certain embodiments, R' is phenyl, which is substituted Embodiments in which X has formula X-1 can include one with from 1-5 (e.g., 1-3, 1-2, or 1) independently selected R. or more of the following features. In certain embodiments, each occurrence of R is, indepen X is S. X" is NH. dently, selected from any one the substituents delineated col X" is Y, which can be as defined anywhere herein, and X" 55 lectively in (aaa), (bbb), (ccc), and (ddd) below: is H or R. (aaa) C-C alkoxy, C-C alkyl, C-C haloalkyl, -NH X is S; and X" is Y, which can be as defined anywhere (C-C alkyl), N(C-C alkyl), or—NHC(O)(C-C alkyl). herein, and X" is Hor R' (e.g., X" is H). each of which is optionally substituted with —OH, -NH, or X is NH; and X" is Y, which can be as defined anywhere - SH: herein, and X" is Hor R' (e.g., X" is H). 60 (bbb) C(O)OH: –C(O)O(C-C alkyl); OC(O)(C-C, X" is H. alkyl); —SONH; SONHC-C alkyl); or - SON(C- X" is R'. In certain embodiments, R is C-Cs (e.g., C-C, Co alkyl); or -C(O)C-(CH2) is C(O)-(phenyl C-C) alkyl (e.g., CH). In other embodiments, R is phenyl optionally substituted as defined in (ddd) below (e.g., —C(O) that is optionally substituted with from 1-4 R. In still other O—CH2—C(O)-(phenyl); embodiments, R is or C-C cycloalkyl which is optionally 65 (ccc) C-C cycloalkoxy or L-heterocyclyl containing substituted with from 1-4 independently selected C-C alkyl from 5-7 ring atoms, wherein from 1-2 of the ring atoms of the groups. heterocyclyl is independently selected from N, NH, N(C-C, US 9,303,024 B2 29 30 alkyl), NC(O)(C-C alkyl), NC(O)O(C-C alkyl), O, and S; Embodiments in which R' has formula A can include one and each of which is optionally substituted with from 1-3 or more of the following features. independently selected C-C alkyl groups; and wherein L is R2 is R. a bond or C-C alkylene (e.g., a bond): e.g., R is morpholino R" is H. or piperazinyl; and R is H. (ddd) phenyl or heteroaryl containing from 5-6 ring atoms, R is R. wherein from 1-2 of the ring atoms of the heteroaryl is inde R" is R, and each of R'' and R'' is H. pendently selected from N, NH, N(C-C alkyl). O, and S; R" is R, and each of R'' and R is H. wherein each of said phenyl and heteroaryl is optionally In some of the above described embodiments, R is C-C, substituted with from 1-3 substituents independently selected 10 from halo: hydroxyl; cyano; —C(O)(C-C alkyl); C(O)OH: alkoxy (e.g., ethoxy or iso-propoxy). —C(O)C(C-C alkyl); nitro; NH; NH(C-C alkyl). In embodiments, R can be C-C alkoxy (e.g., C-C, N(C-C alkyl), NHC(O)(C-C alkyl), C-C alkoxy; alkoxy, e.g., ethoxy; e.g., containing branched alkyl. Such a C-C haloalkoxy; C-C thioalkoxy; C-C thiohaloalkoxy; iso-propoxy). C-C alkyl, and C-Chaloalkyl, wherein said alkyl or alkyl In embodiments, R can be C-C alkoxy (e.g., C-C, portion is optionally substituted with —OH, -NH2 or 15 alkoxy, e.g., ethoxy; e.g., containing branched alkyl. Such a SH iso-propoxy) that is substituted with —NH. In certain embodiments, each occurrence of R is, indepen dently, selected from: In some of the above described embodiments, R is —C(O) C-C alkoxy; OH (or a salt thereof, e.g., sodium salt thereof). C-C alkoxy (e.g., C2-C alkoxy, e.g., ethoxy); or C-C, In some of the above described embodiments, R is L-het alkoxy (e.g., C-C alkoxy, e.g., ethoxy) that is Substi erocyclyl containing from 5-7 (e.g., 6) ring atoms, wherein tuted with NH; from 1-2 of the ring atoms of the heterocyclyl is indepen C-C alkyl, dently selected from N, NH, N(C-C alkyl), NC(O)(C-C, NHC(O)(C-C alkyl) alkyl), NC(O)O(C-C alkyl), O, and S; and each of which is C(O)OH: 25 optionally substituted with from 1-3 independently selected L-heterocyclyl containing from 5-7 (e.g., 5-6, or 6) ring C-C alkyl groups; and wherein L is a bond or C-C alky atoms, wherein from 1-2 of the ring atoms of the hetero lene (e.g., L is a bond or CH, e.g., a bond); e.g., R is cyclyl is independently selected from N, NH, N(C-C, morpholino or piperazinyl. alkyl), NC(O)(C-C alkyl), NC(O)O(C-C alkyl). O, R" is H, and each of R'' and R'' is R. In certain embodi and S; and each of which is optionally substituted with 30 ments, one of R'' and R'' is C-C alkoxy (e.g., ethoxy), and from 1-3 independently selected C-C alkyl groups; and wherein L is a bond or C-C alkylene (e.g., a bond); the other of R'' and R'' is independently selected from: e.g., R is morpholino or piperazinyl; and C-C alkoxy: phenyl or heteroaryl containing from 5-6 ring atoms, C-C alkoxy (e.g., C-C alkoxy, e.g., ethoxy); or C-C, wherein from 1-2 of the ring atoms of the heteroaryl is alkoxy (e.g., C-C alkoxy, e.g., ethoxy) that is Substi independently selected from N, NH, N(C-C alkyl), O, 35 tuted with NH; and S.; wherein each of said phenyl and heteroaryl is C-C alkyl; optionally substituted with from 1-3 substituents inde C(O)OH: pendently selected from halo: hydroxyl; cyano. —C(O) —NHC(O)(C-C alkyl); (C-C alkyl); C(O)OH: —C(O)C(C-C alkyl); nitro: L-heterocyclyl containing from 5-7 ring atoms, wherein —NH; —NH(C-C alkyl), N(C-C alkyl). —NHC 40 from 1-2 of the ring atoms of the heterocyclyl is inde (O) (C-C alkyl), C-C alkoxy; C-C haloalkoxy; pendently selected from N. NH, N(C-C alkyl), NC(O) C-C thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, (C-C alkyl), NC(O)O(C-C alkyl), O, and S; and each and C-Chaloalkyl, wherein said alkyl or alkyl portion of which is optionally substituted with from 1-3 inde is optionally substituted with —OH, -NH2 or —SH. pendently selected C-C alkyl groups; and wherein L is In certain embodiments, R is C-C alkoxy (e.g., ethoxy 45 a bond or C-C alkylene (e.g., a bond); e.g., R is mor e.g., containing branched alkyl. Such as iso-propoxy). pholino or piperazinyl; and In embodiments, R can be C-C alkoxy (e.g., C-C, alkoxy, e.g., ethoxy; e.g., containing branched alkyl. Such a phenyl or heteroaryl containing from 5-6 ring atoms, iso-propoxy). wherein from 1-2 of the ring atoms of the heteroaryl is In embodiments. R can be C-C alkoxy (e.g., C-C, independently selected from N, NH, N(C-C alkyl), O, alkoxy, e.g., ethoxy; e.g., containing branched alkyl. Such a 50 and S.; wherein each of said phenyl and heteroaryl is iso-propoxy) that is substituted with NH. optionally substituted with from 1-3 substituents inde In certain embodiments, R' has formula A: pendently selected from halo: hydroxyl; cyano. —C(O) (C-C alkyl); C(O)OH: —C(O)C(C-C alkyl); nitro: —NH; NH(C-C alkyl), N(C-C alkyl). - NHC 55 (O) (C-C alkyl), C-C alkoxy; C-C haloalkoxy; (A) C-C thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, and C-C haloalkyl, wherein said alkyl or alkyl portion is optionally substituted with —OH, -NH2 or —SH. In some embodiments, R is heteroaryl, which contains 60 from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C-C alkyl). O, and R13 S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected R. R 14 In certain embodiments, R' is heteroaryl, which contains 65 from 5-6 ring atoms, wherein from 1-4 (e.g., 1-2) of the ring wherein one or two of R', R', and R'' is(are) an inde atoms is independently selected from N, NH, N(C-C alkyl). pendently selected R. and the other(s) is(are) hydrogen. O, and S; and wherein said heteroaryl ring is optionally Sub US 9,303,024 B2 31 32 stituted with from 1-3 (e.g., 1-2 or 1) independently selected one of X" and X" is Y, and the other of X" and X" is H or R. For example, R' can be thiazolyl. R"; and In other embodiments, R is heteroaryl, which contains one or two of R', R', and R'' is(are) an independently from 8-10 ring atoms, wherein from 1-4 (e.g., 1-2) of the ring selected R, and the other(s) is(are) hydrogen; R can be as atoms is independently selected from N, NH, N(C-C alkyl). defined anywhere herein. O, and S; and wherein said heteroaryl ring is optionally Sub Embodiments in which the compound has formula I-A can stituted with from 1-3 (e.g., 1-2 or 1) independently selected include one or more of the following features. R. For example, R' can be indazolyl. X is S, X" is Y, and X" is Hor R' (e.g., X" is H). In some embodiments, R' is hydrogen. X is NH, X" is Y, and X" is Hor R (e.g., X" is H). 10 R is C-Cs alkyl (e.g., C-C alkyl, e.g., C-C alkyl, e.g., In some embodiments, R' is —C(O)—(Co-Co aryl or het CH). eroaryl, which contains from 5-10 ring atoms as defined in R' Y is C-C aryl, which is optionally substituted with from definition (i) and (ii), respectively); e.g., —C(O)-(heteroaryl, 1-5 independently selected R' (e.g., Y is unsubstituted phe which contains from 5-10 (e.g., 5-6, e.g., 5) ring atoms as nyl). defined in R' definition (i) and (ii), respectively), such as 15 R" is R, and each of R'' and R'' is H; or R'' is R, and —C(O)-(thiazolyl). each of R'' and R' is H. Variable R Each occurrence of R is, independently, selected from: In some embodiments, R is C-C alkyl (e.g., C-C alkyl, C-C alkoxy; e.g., C-C alkyl, e.g., CH). C-C alkoxy (e.g., C2-C alkoxy, e.g., ethoxy); or C-C, In some embodiments, R is phenyl that is optionally sub alkoxy (e.g., C-C alkoxy, e.g., ethoxy) that is Substi stituted with from 1-4 R. In certain embodiments, R is tuted with NH; unsubstituted phenyl. C-C alkyl; In some embodiments, R is heteroaryl containing from C(O)OH: 5-6 (e.g., 5) ring atoms, wherein from 1-4 of the ring atoms is —NHC(O)(C-C alkyl); independently selected from N, NH, N(C-C alkyl), NC(O) 25 L-heterocyclyl containing from 5-7 ring atoms, wherein (C-C alkyl). O, and S; and wherein said heteroaryl is option from 1-2 of the ring atoms of the heterocyclyl is inde ally substituted with from 1-3 R. In certain embodiments, R pendently selected from N. NH, N(C-C alkyl), NC(O) is heteroaryl containing from 5 ring atoms, wherein from 1-4 (C-C alkyl), NC(O)O(C-C alkyl), O, and S; and each of the ring atoms is independently selected from N, NH, of which is optionally substituted with from 1-3 inde 30 pendently selected C-C alkyl groups; and wherein L is N(C-C alkyl), NC(O)(C-C alkyl), O, and S; and wherein a bond or C-C alkylene (e.g., a bond); e.g., R is mor said heteroaryl is optionally substituted with from 1-3 R: pholino or piperidinyl; and e.g., thienyl or furyl. phenyl or heteroaryl containing from 5-6 ring atoms, In some embodiments, R is C-Cs cycloalkyl which is wherein from 1-2 of the ring atoms of the heteroaryl is optionally substituted with from 1-4 independently selected 35 independently selected from N, NH, N(C-C alkyl), O, C-C alkyl groups. and S.; wherein each of said phenyl and heteroaryl is Variable A optionally substituted with from 1-3 substituents inde In some embodiments, A is N. pendently selected from halo: hydroxyl; cyano. —C(O) Non-Limiting Combinations of Attendant Variables (C-C alkyl); C(O)OH: —C(O)C(C-C alkyl); nitro: In some embodiments: 40 —NH; NH(C-C alkyl), N(C-C alkyl). - NHC A is N: (O) (C-C alkyl), C-C alkoxy; C-C haloalkoxy; X contains 2 ring atoms independently selected from N. C-C thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, NH, N(C-C alkyl), O, and S, and one of the ring atoms is and C-C haloalkyl, wherein said alkyl or alkyl portion independently selected from N, NH, and N(C-C alkyl)(e.g., is optionally substituted with —OH, -NH2 or —SH. N), and the other ring atom is independently selected from N. 45 R is C-C alkoxy (e.g., ethoxy; e.g., containing branched NH, N(C-C alkyl), O, and S (e.g., the other ring atom is Sor alkyl, Such as iso-propoxy). NH, e.g., S); and R is C-C alkoxy (e.g., C-C alkoxy, e.g., ethoxy; e.g., R" is C-Caryl, which is optionally substituted with from containing branched alkyl, Such a iso-propoxy). 1-5 independently selected R (e.g., R' is phenyl, which is R is C-C alkoxy (e.g., C-C alkoxy, e.g., ethoxy; e.g., substituted with from 1-5 (e.g., 1-3, 1-2, or 1) independently 50 containing branched alkyl, such aiso-propoxy) that is Substi selected R), which R can be as defined anywhere herein. tuted with —NH. In some embodiments the compound has formula I-A: R is C1-Cs alkyl (e.g. C1-C alkyl, e.g., C-C alkyl, e.g., CH). R is phenyl that is optionally substituted with from 1-4 R. R is heteroaryl containing from 5-6 (e.g. 5) ring atoms, (I-A) 55 wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C-C alkyl), NC(O)(C-C alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 R. O In some embodiments of formula I-A: 60 X is S or NH; N -Nis N X X" is Y, X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., S. /N-( i C-C alkyl, e.g., CH-): (e.g., X" is H or CH, e.g., X" is R2 N X X" H): 65 Y is C-C aryl, which is optionally substituted with from wherein: 1-5 independently selected R' (e.g., Y is unsubstituted X is NH, O, or S (e.g., S or NH, e.g., S); phenyl); and US 9,303,024 B2 33 34 R is C1-Cs alkyl (e.g., C-C alkyl, e.g., C-C alkyl, e.g., Y is C-C aryl, which is optionally substituted with from CH). 1-5 independently selected R' (e.g., Y is unsubstituted In certain of these formula I-A embodiments: phenyl); X is S or NH; R’ is phenyl that is optionally substituted with from 1-4 R X" is Y, (e.g., each occurrence of R is, independently, halo; X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., cyano; —C(O)(C-C alkyl); C(O)OH:—C(O)O(C-C, C-C alkyl, e.g., CH); (e.g., X" is H or CH, e.g., X" is alkyl); nitro; NH; NH(C-C alkyl), N(C-C, H): alkyl). —NHC(O)(C-C alkyl), C-C alkoxy; C-C, Y is C-C aryl, which is optionally substituted with from haloalkoxy: C-C thioalkoxy; C-C thiohaloalkoxy: 1-5 independently selected R' (e.g., Y is unsubstituted 10 C-C alkyl, C-C cycloalkyl, and C-C haloalkyl; or phenyl); e.g., R is unsubstituted phenyl); and R is C1-Cs alkyl (e.g., C-C alkyl, e.g., C-C alkyl, e.g., R2 is CH, and R2 is: C-C alkoxy (e.g., C-C alkoxy, e.g., ethoxy; e.g., con 15 taining branched alkyl, Such a iso-propoxy) that is - C(O)OH; or optionally substituted with NH, such as C-C alkoxy (e.g., C2-C alkoxy, e.g., ethoxy; e.g., con –OCHCH or - OCHCH-NH; or taining branched alkyl, Such a iso-propoxy) that is heterocyclyl containing from 5-7 ring atoms, wherein from optionally substituted with —NH, such as 1-2 of the ring atoms of the heterocyclyl is indepen –OCHCH or - OCHCH-NH; or dently selected from N, NH, N(C-C alkyl), NC(O)(C- heterocyclyl containing from 5-7 (e.g., 6) ring atoms, C alkyl), NC(O)C(C-C alkyl), O, and S; and each of wherein from 1-2 of the ring atoms of the heterocyclyl is which is optionally substituted with from 1-3 indepen independently selected from N, NH, N(C-C alkyl). dently selected C-C alkyl groups, such as an optionally NC(O)(C-C alkyl), NC(O)C(C-C alkyl), O, and S; Substituted piperazinyl ring. and each of which is optionally substituted with from 25 In certain of these formula I-A embodiments: 1-3 independently selected C-C alkyl groups, such as X is S or NH; an optionally Substituted piperazinyl ring. X" is Y, In certain of these formula I-A embodiments: X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., X is S or NH; C-C alkyl, e.g., CH); (e.g., X" is H or CH, e.g., X" is 30 H): X" is Y, Y is C-C aryl, which is optionally substituted with from X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., 1-5 independently selected R' (e.g., Y is unsubstituted C-C alkyl, e.g., CH-): (e.g., X" is H or CH, e.g., X" is phenyl); H): R’ is phenyl that is optionally substituted with from 1-4 R Y is C-C aryl, which is optionally substituted with from 35 (e.g., each occurrence of R is, independently, halo; 1-5 independently selected R' (e.g., Y is unsubstituted cyano; —C(O)(C-C alkyl); C(O)OH:—C(O)O(C-C, phenyl); alkyl): nitro; NH; —NH(C-C alkyl), N(C-C, R is C1-Cs alkyl (e.g., C-C alkyl, e.g., C-C alkyl, e.g., alkyl). —NHC(O)(C-C alkyl), C-C alkoxy; C-C, CH, and haloalkoxy; C-C thioalkoxy; C-C thiohaloalkoxy; R" is C1-C alkoxy (e.g., C2-C alkoxy, e.g., ethoxy; e.g., 40 C-C alkyl, C-C cycloalkyl; and C-C haloalkyl; or containing branched alkyl. Such a iso-propoxy) that is e.g., R is unsubstituted phenyl); and optionally substituted with NH, such as R'' is —C(O)OH (see, e.g., compound 165-74). —OCHCH or —OCHCH-NH. (see e.g., com In some embodiments of formula I-A: pounds 172-90 and 165-93). X is S or NH; In some embodiments of formula I-A: 45 X" is Y, X is S or NH; X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., X" is Y, C-C alkyl, e.g., CH-): (e.g., X" is H or CH, e.g., X" is X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., H): C-C alkyl, e.g., CH-): (e.g., X" is H or CH, e.g., X" is Y is C-C aryl, which is optionally substituted with from H): 50 1-5 independently selected R' (e.g., Y is unsubstituted Y is C-C aryl, which is optionally substituted with from phenyl); and 1-5 independently selected R' (e.g., Y is unsubstituted R is heteroaryl containing from 5-6 (e.g., 5) ring atoms, phenyl); and wherein from 1-4 of the ring atoms is independently R’ is phenyl that is optionally substituted with from 1-4 R selected from N, NH, N(C-C alkyl), NC(O)(C-C, (e.g., each occurrence of R is, independently, halo; 55 alkyl), O, and S; and wherein said heteroaryl is option cyano;—C(O)(C-C alkyl); C(O)CH;—C(O)C(C-C, ally substituted with from 1-3 R, such as thienyl, fura alkyl): nitro; NH; —NH(C-C alkyl), N(C-C, nyl, or thiazolyl; (e.g., each occurrence of R is, inde alkyl). —NHC(O)(C-C alkyl), C-C alkoxy; C-C, pendently, halo; cyano; —C(O)(C-C alkyl); C(O)OH: haloalkoxy; C-C thioalkoxy; C-C thiohaloalkoxy; —C(O)C(C-C alkyl): nitro; —NH; NH(C-C, C-C alkyl, C-C cycloalkyl, and C-C haloalkyl; or 60 alkyl), N(C-C alkyl), NHC(O)(C-C alkyl), C-C, e.g., R is unsubstituted phenyl). alkoxy; C-C haloalkoxy: C-C thioalkoxy; C-C, In certain of these formula I-A embodiments: thiohaloalkoxy; C-C alkyl, C-C cycloalkyl; and X is S or NH; C-C haloalkyl; or e.g., R is unsubstituted heteroaryl, X" is Y, Such as unsubstituted thienyl, furanyl, or thiazolyl). X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., 65 In certain of these formula I-A embodiments: C-C alkyl, e.g., CH); (e.g., X" is H or CH, e.g., X" is X is S or NH; H): X" is Y, US 9,303,024 B2 35 36 X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., In some embodiments, the compound has formula I-B: C-C alkyl, e.g., CH-): (e.g., X" is H or CH, e.g., X" is H): Y is C-C aryl, which is optionally substituted with from (I-B) O 1-5 independently selected R' (e.g., Y is unsubstituted 5 R phenyl); N-N X it R is heteroaryl containing from 5-6 (e.g., 5) ring atoms, Ns N wherein from 1-4 of the ring atoms is independently S. MN-( i selected from N, NH, N(C-C alkyl), NC(O)(C-C, R2 N X X" alkyl), O, and S; and wherein said heteroaryl is option 10 ally substituted with from 1-3 R, such as thienyl, fura wherein: nyl, or thiazolyl; (e.g., each occurrence of R is, inde X" is NH, O, or S; and pendently, halo; cyano; —C(O)(C-C alkyl); C(O)OH: one of X" and X" is Y, and the other of X" and X" is H or —C(O)C(C-C alkyl); nitro; NH; NH(C-C, R. alkyl), N(C-C alkyl). —NHC(O)(C-C alkyl), C-C, 15 alkoxy; C-C haloalkoxy: C-C thioalkoxy; C-C, Embodiments in which the compound has formula I-B can thiohaloalkoxy; C-C alkyl, C-C cycloalkyl; and include one or more of the following features. C-C haloalkyl, or e.g., R is unsubstituted heteroaryl, X is S, X" is Y, and X" is Hor R' (e.g., X" is H). Such as unsubstituted thienyl, furanyl, or thiazolyl); and X is NH, X" is Y, and X" is Hor R (e.g., X" is H). R’ is R" is thiazolyl. - C(O)OH; or Y is C-C aryl, which is optionally substituted with from C-C alkoxy (e.g., C2-C alkoxy, e.g., ethoxy; e.g., con 1-5 independently selected R. R is phenyl that is optionally taining branched alkyl, Such a iso-propoxy) that is substituted with from 1-4 R. R. is unsubstituted phenyl. optionally substituted with —NH, such as Compound Forms and Salts –OCHCH or - OCHCH-NH; or 25 In some embodiments, the compounds described herein heterocyclyl containing from 5-7 ring atoms, wherein from may contain one or more asymmetric centers and thus occur 1-2 of the ring atoms of the heterocyclyl is indepen as racemates and racemic mixtures, enantiomerically dently selected from N, NH, N(C-C alkyl), NC(O)(C- enriched mixtures, single enantiomers, individual diastere C alkyl). NC(O)O(C-C alkyl), O, and S; and each of omers and diastereomeric mixtures (e.g., including (R)- and which is optionally substituted with from 1-3 indepen 30 (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, dently selected C-C alkyl groups, such as an optionally (+) (dextrorotatory) forms, (-) (levorotatory) forms, the race substituted piperazinyl ring. mic mixtures thereof, and other mixtures thereof). Additional In certain of these formula I-A embodiments: asymmetric carbon atoms may be present in a Substituent, X is S or NH; Such as an alkyl group. All Such isomeric forms, as well as X" is Y, 35 X" is H or R'; R is C-C alkyl (e.g., C-C alkyl, e.g., mixtures thereof, of these compounds are expressly included C-C alkyl, e.g., CH); (e.g., X" is H or CH, e.g., X" is in the present invention. The compounds described herein H): may also or further contain linkages wherein bond rotation is Y is C-C aryl, which is optionally substituted with from restricted about that particular linkage, e.g. restriction result 1-5 independently selected R' (e.g., Y is unsubstituted 40 ing from the presence of a ring or double bond (e.g., carbon phenyl); carbon bonds, carbon-nitrogen bonds such as amide bonds). R is heteroaryl containing from 5-6 (e.g., 5) ring atoms, Accordingly, all cis/trans and E/Z isomers and rotational iso wherein from 1-4 of the ring atoms is independently mers are expressly included in the present invention. The selected from N, NH, N(C-C alkyl), NC(O)(C-C, compounds of this invention may also be represented in mul alkyl), O, and S; and wherein said heteroaryl is option 45 tiple tautomeric forms; in Such instances, the invention ally substituted with from 1-3 R, such as thienyl, fura expressly includes all tautomeric forms of the compounds nyl, or thiazolyl; (e.g., each occurrence of R is, inde described herein, even though only a single tautomeric form pendently, halo; cyano; —C(O)(C-C alkyl); C(O)OH: may be represented. All Such isomeric forms of Such com —C(O)C(C-C alkyl); nitro; NH; NH(C-C, pounds are expressly included in the present invention. alkyl), N(C-C alkyl). —NHC(O)(C-C alkyl), C-C, 50 Unless otherwise mentioned or indicated, the chemical des alkoxy; C-C haloalkoxy; C-C thioalkoxy; C-C, ignation of a compound encompasses the mixture of all pos thiohaloalkoxy; C-C alkyl, C-C cycloalkyl; and sible stereochemically isomeric forms of that compound. C-C haloalkyl, or e.g., R is unsubstituted heteroaryl, Optical isomers can be obtained in pure form by standard Such as unsubstituted thienyl, furanyl, or thiazolyl); and procedures known to those skilled in the art, and include, but R" is C(O)OH (see, e.g., compound 165-87). 55 are not limited to, diastereomeric salt formation, kinetic reso In some embodiments: lution, and asymmetric synthesis. See, for example, Jacques, A is N: et al., Enantiomers, Racemates and Resolutions (Wiley Inter X contains 2 ring atoms independently selected from N. science, New York, 1981); Wilen, S. H., et al., Tetrahedron NH, N(C-C alkyl), O, and S, and one of the ring atoms is 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Com independently selected from N, NH, and N(C-C alkyl), and 60 pounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of the other ring atom is independently selected from N. NH, Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, N(C-C alkyl). O, and S; and Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972), R" is heteroaryl, which contains from 5-10 ring atoms, each of which is incorporated herein by reference in their wherein from 1-4 of the ring atoms is independently selected entireties. It is also understood that this invention encom from N. NH, N(C-C alkyl). O, and S; and wherein said 65 passes all possible regioisomers, and mixtures thereof, which heteroaryl ring is optionally substituted with from 1-3 inde can be obtained in pure form by standard separation proce pendently selected R. dures knownto those skilled in the art, and include, but are not US 9,303,024 B2 37 38 limited to, column chromatography, thin-layer chromatogra In addition to salt forms, the invention provides com phy, and high-performance liquid chromatography. pounds which are in a prodrug form. Prodrugs of the com The compounds of this invention include the compounds pounds described herein are those compounds that undergo themselves, as well as their salts and their prodrugs, if appli chemical changes under physiological conditions to provide cable. A salt, for example, can be formed between an anion the compounds of the invention. Additionally, prodrugs can and a positively charged substituent (e.g., amino) on a com be converted to the compounds of the invention by chemical pound described herein. Suitable anions include chloride, or biochemical methods in an ex vivo environment. For bromide, iodide, Sulfate, nitrate, phosphate, citrate, methane example, prodrugs can be slowly converted to the compounds Sulfonate, trifluoroacetate, and acetate. Likewise, a salt can of the invention when placed in a transdermal patch reservoir also be formed between a cation and a negatively charged 10 Substituent (e.g., carboxylate) on a compound described with a suitable enzyme or chemical reagent. Prodrugs are herein. Suitable cations include Sodium ion, potassium ion, often useful because, in Some situations, they may be easier to magnesium ion, calcium ion, and an ammonium cation Such administer than the parent drug. They may, for instance, be as tetramethylammonium ion. Examples of prodrugs include more bioavailable by oral administration than the parent drug. C. alkyl esters of carboxylic acid groups, which, upon 15 The prodrug may also have improved solubility in pharma administration to a subject, are capable of providing active cological compositions over the parent drug. A wide variety compounds. of prodrug derivatives are known in the art, Such as those that Pharmaceutically acceptable salts of the compounds of this rely on hydrolytic cleavage or oxidative activation of the invention include those derived from pharmaceutically prodrug. An example, without limitation, of a prodrug would acceptable inorganic and organic acids and bases. As used be a compound of the invention which is administered as an herein, the term “pharmaceutically acceptable salt” refers to a ester (the “prodrug), but then is metabolically hydrolyzed to salt formed by the addition of a pharmaceutically acceptable the carboxylic acid, the active entity. In embodiments, the acid or base to a compound disclosed herein. As used herein, ester can be an alkyl ester (e.g., C-C alkyl, e.g., CH or the phrase “pharmaceutically acceptable” refers to a sub CHCH, or C-C alkyl, e.g., C-C branched alkyl, e.g., stance that is acceptable for use in pharmaceutical applica 25 t-butyl, isopropyl, isobutyl). Additional examples include tions from a toxicological perspective and does not adversely peptidyl derivatives of a compound of the invention. interact with the active ingredient. The invention also includes various hydrate and solvate Examples of Suitable acid salts include acetate, adipate, forms of the compounds (and salts thereof) described herein. alginate, aspartate, benzoate, benzenesulfonate, bisulfate, The compounds of the invention may also contain unnatu butyrate, citrate, camphorate, camphorsulfonate, diglucon 30 ate, dodecylsulfate, ethanesulfonate, formate, fumarate, glu ral proportions of atomic isotopes at one or more of the atoms coheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, that constitute such compounds. For example, the compounds hydrochloride, hydrobromide, hydroiodide, 2-hydroxy may be radiolabeled with radioactive isotopes, such as for ethanesulfonate, lactate, maleate, malonate, methane example tritium (H), iodine-125 ('I) or carbon-14 (''C). Sulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, pal 35 All isotopic variations of the compounds of the invention, moate, pectinate, persulfate, 3-phenylpropionate, phosphate, whether radioactive or not, are intended to be encompassed picrate, pivalate, propionate, salicylate, Succinate, Sulfate, within the scope of the invention. tartrate, thiocyanate, tosylate and undecanoate. Other acids, Synthesis of Compounds Such as Oxalic, while not in themselves pharmaceutically The compounds described herein can be conveniently pre acceptable, may be employed in the preparation of salts use 40 pared in accordance with the procedures outlined herein, ful as intermediates in obtaining the compounds of the inven from commercially available starting materials, compounds tion and their pharmaceutically acceptable acid addition salts. known in the literature, or readily prepared intermediates, by Salts derived from appropriate bases include alkali metal employing standard synthetic methods and procedures (e.g., Sodium), alkaline earth metal (e.g., magnesium), known to those skilled in the art. Standard synthetic methods ammonium and N-(alkyl) salts. This invention also envi 45 and procedures for the preparation of organic molecules and sions the quaternization of any basic nitrogen-containing functional group transformations and manipulations can be groups of the compounds disclosed herein. Water or oil readily obtained from the relevant scientific literature or from soluble or dispersible products may be obtained by such standard textbooks in the field. It will be appreciated that quaternization. Salt forms of the compounds of any of the where typical or preferred process conditions (i.e., reaction formulae herein can be amino acid salts of carboxy groups 50 temperatures, times, mole ratios of reactants, solvents, pres (e.g. L-arginine, -lysine, -histidine salts). Sures, etc.) are given, other process conditions can also be Lists of suitable salts are found in Remington’s Pharma used unless otherwise Stated. Optimum reaction conditions ceutical Sciences, 17th ed., Mack Publishing Company, Eas may vary with the particular reactants or solvents used, but ton, Pa., 1985, p. 1418; Journal of Pharmaceutical Science, such conditions can be determined by one skilled in the art by 66, 2 (1977): “Pharmaceutical Salts: Properties, Selection, 55 routine optimization procedures. Those skilled in the art of and Use A Handbook; Wermuth, C. G. and Stahl, P. H. (eds.) organic synthesis will recognize that the nature and order of Verlag Helvetica Chimica Acta, Zurich, 2002 ISBN the synthetic steps presented may be varied for the purpose of 3-906390-26-8; and Berge et al. (1977) “Pharmaceutical optimizing the formation of the compounds described herein. Salts'. J. Pharm. Sci. 66:1-19; each of which is incorporated Synthetic chemistry transformations (including protecting herein by reference in its entirety. 60 group methodologies) useful in synthesizing the compounds The neutral forms of the compounds may be regenerated by described herein are known in the art and include, for contacting the salt with a base or acid and isolating the parent example, those Such as described in R. C. Larock, Compre compound in the conventional manner. The parent form of the hensive Organic Transformations, 2d. ed., Wiley-VCH Pub compound differs from the various Saltforms in certain physi lishers (1999); P. G. M. Wuts and T. W. Greene, Protective cal properties, such as Solubility in polar solvents, but other 65 Groups in Organic Synthesis, 4th Ed., John Wiley and Sons wise the salts are equivalent to the parent form of the com (2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents pound for the purposes of the invention. for Organic Synthesis, John Wiley and Sons (1994); and L. US 9,303,024 B2 39 40 Paquette, ed., Encyclopedia of Reagents for Organic Synthe commonly, however, the compositions are presented in unit sis, John Wiley and Sons (1995), and subsequent editions dosage forms to facilitate accurate dosing. The term “unit thereof. dosage forms’ refers to physically discrete units Suitable as The processes described herein can be monitored accord unitary dosages for human Subjects and other mammals, each ing to any Suitable method known in the art. For example, unit containing a predetermined quantity of active material product formation can be monitored by spectroscopic means, calculated to produce the desired therapeutic effect, in asso Such as nuclear magnetic resonance spectroscopy (e.g., "Hor ciation with a Suitable pharmaceutical excipient. Typical unit 'C), infrared spectroscopy (FT-IR), spectrophotometry (e.g., dosage forms include prefilled, premeasured ampules or UV-visible), or mass spectrometry (MS), or by chromatogra Syringes of the liquid compositions or pills, tablets, capsules, phy Such as high performance liquid chromatography 10 losenges or the like in the case of Solid compositions. In Such (HPLC) or thin layer chromatography (TLC). compositions, the compound is usually a minor component Preparation of compounds can involve the protection and (from about 0.1 to about 50% by weight or preferably from deprotection of various chemical groups. The need for pro about 1 to about 40% by weight) with the remainder being tection and deprotection, and the selection of appropriate various vehicles or carriers and processing aids helpful for protecting groups can be readily determined by one skilled in 15 forming the desired dosing form. the art. The chemistry of protecting groups can be found, for The amount administered depends on the compound for example, in Greene, et al., Protective Groups in Organic mulation, route of administration, etc. and is generally Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated empirically determined in routine trials, and variations will herein by reference in its entirety. necessarily occur depending on the target, the host, and the The reactions of the processes described herein can be route of administration, etc. Generally, the quantity of active carried out in suitable solvents which can be readily selected compound in a unit dose of preparation may be varied or by one of skill in the art of organic synthesis. Suitable solvents adjusted from about 1, 3, 10 or 30 to about 30, 100, 300 or can be substantially nonreactive with the starting materials 1000 mg, according to the particular application. In a particu (reactants), the intermediates, or products at the temperatures lar embodiment, unit dosage forms are packaged in a multi at which the reactions are carried out, i.e., temperatures which 25 packadapted for sequential use. Such as blisterpack, compris can range from the solvents freezing temperature to the ing sheets of at least 6, 9 or 12 unit dosage forms. The actual Solvent's boiling temperature. A given reaction can be carried dosage employed may be varied depending upon the require out in one solvent or a mixture of solvents. Depending on the ments of the patient and the severity of the condition being particular reaction step, Suitable solvents for aparticular reac treated. Determination of the proper dosage for a particular tion step can be selected. 30 situation is within the skill of the art. Generally, treatment is In some embodiments, the formula (I) compounds can be initiated with Smaller dosages which are less than the opti prepared using the reaction pathways and techniques as mum dose of the compound. Thereafter, the dosage is shown in FIGS. 2A-2I and 19A-19F. In FIG.2B, compounds increased by Small amounts until the optimum effect under 161-87 and 153-96 can be prepared using this route using the the circumstances is reached. For convenience, the total daily corresponding thiazolyl and phenyl Substituted reagents. 35 dosage may be divided and administered in portions during Pharmaceutical Compositions the day if desired. The term “pharmaceutically acceptable carrier” refers to a Use and Administration carrier or adjuvant that may be administered to a subject (e.g., This application features pyrazol-3-one compounds that a patient), together with a compound of this invention, and activate pro-apoptotic BAX, making them therapeutically which does not destroy the pharmacological activity thereof 40 useful for treating (e.g., controlling, relieving, ameliorating, and is nontoxic when administered in doses sufficient to alleviating, or slowing the progression of) or preventing (e.g., deliver a therapeutic amount of the compound. delaying the onset of or reducing the risk of developing) Pharmaceutically acceptable carriers, adjuvants and diseases, disorders, and conditions associated with deregu vehicles that may be used in the compositions of this inven lated apoptosis of (e.g., diseased or damaged cells; e.g., insuf tion include, but are not limited to, ion exchangers, alumina, 45 ficient apoptosis of diseased or damaged cells; or lack of or aluminum Stearate, lecithin, self-emulsifying drug delivery reduced apoptosis of diseased or damaged cells). Examples of systems (SEDDS) such as d-C-tocopherol polyethylenegly Such diseases, disorders, and conditions include (but are not col 1000 Succinate, Surfactants used in pharmaceutical dos limited to) those associated with blockade(s) of cell death age forms such as Tweens or other similar polymeric delivery pathways (e.g., over-expression of anti-apoptotic BCL-2 pro matrices, serum proteins, such as human serum albumin, 50 teins), e.g., hyperproliferative diseases, such as cancer. While buffer Substances Such as phosphates, glycine, Sorbic acid, not wishing to be bound by theory, it is believed that the potassium Sorbate, partial glyceride mixtures of Saturated compounds described herein induce and increase apoptosis in Vegetable fatty acids, water, salts, or electrolytes, such as target cells (e.g., pathogenic cells including, but not limited protamine Sulfate, disodium hydrogen phosphate, potassium to, cancer cells), thereby Suppressing tumor growth and/or hydrogen phosphate, Sodium chloride, Zinc salts, colloidal 55 proliferation. It is further believed that increasing apoptosis in silica, magnesium trisilicate, polyvinyl pyrrolidone, cellu said target cells reestablishes the normal apoptotic control lose-based substances, polyethylene glycol, Sodium car that, during homeostasis, is associated with a regulated bal boxymethylcellulose, polyacrylates, waxes, polyethylene ance between pro- and anti-apoptotic protein functions. polyoxypropylene-block polymers, polyethylene glycol and The compounds and compositions described herein can, wool fat. Cyclodextrins such as C-, 3-, and Y-cyclodextrin, or 60 for example, be administered orally, parenterally (e.g., Sub chemically modified derivatives such as hydroxyalkylcyclo cutaneously, intracutaneously, intravenously, intramuscu dextrins, including 2- and 3-hydroxypropyl-3-cyclodextrins, larly, intraarticularly, intraarterially, intrasynovially, or other solubilized derivatives may also be advantageously intrasternally, intrathecally, intralesionally and by intracra used to enhance delivery of compounds of the formulae nial injection or infusion techniques), by inhalation spray, described herein. 65 topically, rectally, nasally, buccally, vaginally, via an The compositions for administration can take the form of implanted reservoir, by injection, Subdermally, intraperito bulk liquid Solutions or Suspensions, or bulk powders. More neally, transmucosally, or in an ophthalmic preparation, with US 9,303,024 B2 41 42 a dosage ranging from about 0.01 mg/kg to about 1000 The compositions may be in the form of a sterile injectable mg/kg, (e.g., from about 0.01 to about 100 mg/kg, from about preparation, for example, as a sterile injectable aqueous or 0.1 to about 100 mg/kg, from about 1 to about 100 mg/kg, oleaginous Suspension. This Suspension may be formulated from about 1 to about 10 mg/kg) every 4 to 120 hours, or according to techniques known in the art using Suitable dis according to the requirements of the particular drug. The persing or wetting agents (such as, for example, Tween 80) interrelationship of dosages for animals and humans (based and Suspending agents. The sterile injectable preparation may on milligrams per meter squared of body Surface) is described also be a sterile injectable solution or Suspension in a non by Freireich et al., Cancer Chemother. Rep. 50, 219 (1966). toxic parenterally acceptable diluent or solvent, for example, Body Surface area may be approximately determined from as a solution in 1,3-butanediol. Among the acceptable height and weight of the patient. See, e.g., Scientific Tables, 10 Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970). In certain vehicles and solvents that may be employed are mannitol, embodiments, the compositions are administered by oral water, Ringer's Solution and isotonic sodium chloride solu administration or administration by injection. The methods tion. In addition, Sterile, fixed oils are conventionally herein contemplate administration of an effective amount of employed as a solvent or Suspending medium. For this pur compound or compound composition to achieve the desired 15 pose, any bland fixed oil may be employed including Syn or stated effect. Typically, the pharmaceutical compositions thetic mono- or diglycerides. Fatty acids, such as oleic acid of this invention will be administered from about 1 to about 6 and its glyceride derivatives are useful in the preparation of times per day or alternatively, as a continuous infusion. Such injectables, as are natural pharmaceutically-acceptable oils, administration can be used as a chronic or acute therapy. Such as olive oil or castor oil, especially in their polyoxyethy Lower or higher doses than those recited above may be lated versions. These oil solutions or Suspensions may also required. Specific dosage and treatment regimens for any contain a long-chain alcohol diluent or dispersant, or car particular patient will depend upon a variety of factors, boxymethyl cellulose or similar dispersing agents which are including the activity of the specific compound employed, the commonly used in the formulation of pharmaceutically age, body weight, general health status, sex, diet, time of acceptable dosage forms such as emulsions and or Suspen administration, rate of excretion, drug combination, the 25 sions. Other commonly used surfactants such as Tweens or severity and course of the disease, condition or symptoms, the Spans and/or other similar emulsifying agents or bioavailabil patient’s disposition to the disease, condition or symptoms, ity enhancers which are commonly used in the manufacture of and the judgment of the treating physician. pharmaceutically acceptable solid, liquid, or other dosage Upon improvement of a patient’s condition, a maintenance forms may also be used for the purposes of formulation. dose of a compound, composition or combination of this 30 invention may be administered, if necessary. Subsequently, The compositions of this invention may be orally admin the dosage or frequency of administration, or both, may be istered in any orally acceptable dosage form including, but reduced, as a function of the symptoms, to a level at which the not limited to, capsules, tablets, emulsions and aqueous Sus improved condition is retained when the symptoms have been pensions, dispersions and solutions. In the case of tablets for alleviated to the desired level. Patients may, however, require 35 oral use, carriers which are commonly used include lactose intermittent treatment on a long-term basis upon any recur and corn Starch. Lubricating agents, such as magnesium rence of disease symptoms. Stearate, are also typically added. For oral administration in a In some embodiments, the compounds described herein capsule form, useful diluents include lactose and dried corn can be coadministered with one or more other therapeutic starch. When aqueous Suspensions and/or emulsions are agents. In certain embodiments, the additional agents may be 40 administered orally, the active ingredient may be suspended administered separately, as part of a multiple dose regimen, or dissolved in an oily phase is combined with emulsifying from the compounds of this invention (e.g., sequentially, e.g., and/or Suspending agents. If desired, certain Sweetening and/ on different overlapping schedules with the administration of or flavoring and/or coloring agents may be added. one or more compounds of formula (I) (including any Sub The compositions of this invention may also be adminis genera or specific compounds thereof)). In other embodi 45 tered in the form of suppositories for rectal administration. ments, these agents may be part of a single dosage form, These compositions can be prepared by mixing a compound mixed together with the compounds of this invention in a of this invention with a suitable non-irritating excipient which single composition. In still another embodiment, these agents is solid at room temperature but liquid at the rectal tempera can be given as a separate dose that is administered at about ture and therefore will melt in the rectum to release the active the same time that one or more compounds of formula (I) 50 components. Such materials include, but are not limited to, (including any Subgenera or specific compounds thereof) are cocoa butter, beeswax and polyethylene glycols. administered (e.g., simultaneously with the administration of Topical administration of the compositions of this inven one or more compounds of formula (I) (including any Sub tion is useful when the desired treatment involves areas or genera or specific compounds thereof)). When the composi organs readily accessible by topical application. For applica tions of this invention include a combination of a compound 55 tion topically to the skin, the composition should be formu of the formulae described herein and one or more additional lated with a suitable ointment containing the active compo therapeutic or prophylactic agents, both the compound and nents suspended or dissolved in a carrier. Carriers for topical the additional agent can be present at dosage levels of administration of the compounds of this invention include, between about 1 to 100%, and more preferably between about but are not limited to, mineral oil, liquid petroleum, white 5 to 95% of the dosage normally administered in a mono 60 petroleum, propylene glycol, polyoxyethylene polyoxypro therapy regimen. pylene compound, emulsifying wax and water. Alternatively, The compositions of this invention may contain any con the composition can be formulated with a suitable lotion or ventional non-toxic pharmaceutically-acceptable carriers, cream containing the active compound Suspended or dis adjuvants or vehicles. In some cases, the pH of the formula solved in a carrier with suitable emulsifying agents. Suitable tion may be adjusted with pharmaceutically acceptable acids, 65 carriers include, but are not limited to, mineral oil, Sorbitan bases or buffers to enhance the stability of the formulated monostearate, polysorbate 60, cetyl esters wax, cetearyl alco compound or its delivery form. hol, 2-octyldodecanol, benzyl alcohol and water. The com US 9,303,024 B2 43 44 positions of this invention may also be topically applied to the (e.g., polymeric, liposomal) can also be used for delivery of lower intestinal tract by rectal Suppository formulation or in a the compounds and compositions delineated herein. Suitable enema formulation. In some embodiments, topical administration of the com EXAMPLES pounds and compositions described herein may be presented in the form of an aerosol, a semi-solid pharmaceutical com The invention will be further described in the following position, a powder, or a solution. By the term “a semi-solid examples. It should be understood that these examples are for composition' is meant an ointment, cream, salve, jelly, or illustrative purposes only and are not to be construed as lim other pharmaceutical composition of Substantially similar iting this invention in any manner. consistency Suitable for application to the skin. Examples of 10 semi-solid compositions are given in Chapter 17 of The Example 1 Theory and Practice of Industrial Pharmacy, Lachman, Lie Identification of Small Molecules that Bind the BAX berman and Kanig, published by Lea and Febiger (1970) and Trigger Site in Remington's Pharmaceutical Sciences, 21st Edition 15 (2005) published by Mack Publishing Company, which is We generated a diverse in silico compilation of 750,000 incorporated herein by reference in its entirety. small molecules from commercially available libraries and Topically-transdermal patches are also included in this docked the database of 3-dimensional molecules on average invention. Also within the invention is a patch to deliver active minimized BAX structures using Glide 4.0'' in standard chemotherapeutic combinations herein. A patch includes a precision mode (SPVS) (FIG. 3A). The top-ranked 20,000 material layer (e.g., polymeric, cloth, gauze, bandage) and the hits based on the Glidescore function for each BAX structural compound of the formulae herein as delineated herein. One model were selected and re-docked to the BAX structures side of the material layer can have a protective layer adhered using extra precision docking mode (XPVS). The top 1,000 to it to resist passage of the compounds or compositions. The hits from each docking calculation were visualized with the patch can additionally include an adhesive to hold the patch in 25 Glidepose viewer and analyzed for their interactions with key place on a subject. An adhesive is a composition, including BAX binding site residues. A subset of 100 molecules was those of either natural or synthetic origin, that when contacted selected for experimental analysis based on the presence of with the skin of a subject, temporarily adheres to the skin. It favorable hydrogen bonds, hydrophobic contacts, and can be water resistant. The adhesive can be placed on the molecular properties. Docking this compilation of putative patch to hold it in contact with the skin of the subject for an 30 BAX activator molecules (BAMs) demonstrates how the extended period of time. The adhesive can be made of a compounds blanket the surface of the BAX trigger site (FIG. tackiness, or adhesive strength, such that it holds the device in 3B). place Subject to incidental contact, however, upon an affirma To evaluate the capacity of candidate BAMs to bind BAX, tive act (e.g., ripping, peeling, or other intentional removal) we developed a screening competitive fluorescence polariza 35 tion assay (FPA) based on the interaction between recombi the adhesive gives way to the external pressure placed on the nant BAX and the fluoresceinated Stabilized Alpha-Helix of device or the adhesive itself, and allows for breaking of the BCL-2 domain (SAHB) modeled after BIMBH3 (EC.s 283 adhesion contact. The adhesive can be pressure sensitive, that nM) (FIG. 4A). Small molecules were then benchmarked is, it can allow for positioning of the adhesive (and the device against the displacement of FITC-BIMSAHB by N-terminal to be adhered to the skin) against the skin by the application 40 acetylated BIMSAHB (ICs, 314 nM) (FIG. 4B). Of the 78 of pressure (e.g., pushing, rubbing.) on the adhesive or device. molecules that lacked auto-fluorescence, 11 molecules The compositions of this invention may be administered by achieved >55% displacement of FITC-BIMSAHB at the 100 nasal aerosol or inhalation. Such compositions are prepared uM screening dose (FIG. 4C). The ability of BAMs 1-11 to according to techniques well-known in the art of pharmaceu dose-responsively compete with FITC-BIMSAHB for BAX tical formulation and may be prepared as Solutions in Saline, 45 binding was then examined by FPA. BAM7 emerged as the employing benzyl alcohol or other Suitable preservatives, most effective competitor, achieving an ICso of 3.3 LM, which absorption promoters to enhance bioavailability, fluorocar compared favorably with unlabeled BIMSAHB considering bons, and/or other solubilizing or dispersing agents known in that the molecule is only one-sixth the size of the BIMBH3 the art. C.-helical peptide (FIG. 4D). We verified the identity of A composition having the compound of the formulae 50 BAM7 by NMR, resynthesized it, and documented a similar herein and an additional agent (e.g., a therapeutic agent) can ICs value for competition with FITC-BIMSAHB (FIG.4D). be administered using any of the routes of administration The chemical structure of BAM7 (MW 405.5) is shown in described herein. In some embodiments, a composition hav FIG. 4E and its 'H-NMR spectra shown in FIG.4F. ing the compound of the formulae herein and an additional 55 Example 2 agent (e.g., a therapeutic agent) can be administered using an implantable device. Implantable devices and related technol BAM7 is Selective for the BH3-Binding Site on ogy are known in the art and are useful as delivery systems BAX where a continuous, or timed-release delivery of compounds or compositions delineated herein is desired. Additionally, 60 The BH3 binding pocket of anti-apoptotic targets shares the implantable device delivery system is useful for targeting topographic similarities with the BH3 trigger site on BAX, specific points of compound or composition delivery (e.g., including a hydrophobic groove that engages the hydropho localized sites, organs). Negrinet al., Biomaterials, 22(6):563 bic face of BH3 helices and a perimeter of similarly oriented (2001). Timed-release technology involving alternate deliv charged and polar residues that are complementary to discrete ery methods can also be used in this invention. For example, 65 residues of the hydrophilic BH3 interface. The two BH3 timed-release formulations based on polymer technologies, binding sites differ in their geographic location, pocket depth, Sustained-release techniques and encapsulation techniques and functionality. Whereas BIMBH3 is compatible with both US 9,303,024 B2 45 46 the BAXtrigger site and anti-apoptotic pockets, we examined conformational change upon BH3 triggering. We recently whether BAM7 was selective for BCL-2 family targets by demonstrated using correlative structural and biochemical competitive FPA. As demonstrated for BAX, direct FPA methods that these essential changes include “opening of the analyses documented high affinity interactions between C1/C2 loop, mobilization of the C-terminal C.9 for mitochon FITC-BIM SAHB and the anti-apoptotic proteins BCL-X, 5 drial translocation, and BAX BH3 exposure for propagating (ECs, 13.6 nM), MCL-1 (ECs, 19.8 nM), and BFL-1/A1 (ECso, 17.9 nM), which represent the structural diversity of BAX activation'. To determine if the selective binding inter the pro-survival arm of the BCL-2 family (FIG. 5A). Corre action we documented for BAM7 results in functional BAX spondingly, the N-terminal acetylated analogue of BIM activation, we performed a series of structural, biochemical, SAHB effectively competed with FITC-BIMSAHB forbind and cellular studies. First, we conducted an NMR analysis of ing to BCL-X (ICs, 572 nM), MCL-1 (ICs, 136 nM), and 10 'N-BAX upon titration with higher concentrations of BAM7 BFL-1/A1 (ICs, 603 nM) (FIGS. 5B-5D). These binding to examine secondary structural changes that ensue upon data highlight that BIMSAHB can readily engage the diver ligand binding. We observed that increasing the ratio of sity of apoptotic targets. In striking contrast, BAM7 exhibited BAM7/BAX from 1:1 to 2:1 caused additional chemical shift little to no anti-apoptotic binding interactions even at 50 LM 15 changes in the O. 1-C2 loop, the BH3 domain (O2), and in the dosing, revealing a remarkable selectivity of BAM7 for BAX C-terminal C.9 helix, three discrete regions that also undergo (FIGS. 4D and 5B-5D). allosteric changes in response to increased BIMSAHB expo Example 3 sure' (FIG. 7). To link these structural changes to the bio chemical conversion of BAX from monomer to oligomer, we Structural Analysis of the BAM7/BAX Interaction performed Solution-phase BAX oligomerization assays in To determine if BAM7 selectively competed with FITC which BAX is exposed to increasing quantities of triggering BIM SAHB for binding to BAX through a direct trigger site ligand followed by monitoring of BAX species by size-ex interaction or an indirect allosteric effect, we performed clusion chromatography over time. Like BIM SAHB', NMR analysis of 'N-BAX upon BAM7 titration. As BAM7 triggered the conversion of BAX from monomer to observed for BIM SAHB 7, the addition of BAM7 up to a 25 oligomer in a dose- and time-responsive manner (FIG. 8A). 1:1 ratio induced significant backbone amide chemical shift To confirm that the SEC-based detection of BAM7-induced changes in those BAXresidues concentrated in the region of BAX oligomerization reflects functional activation of BAX the C.1/O.6 trigger site (FIG. 6A). These data are consistent for its release activity, we performed liposomal assays that with a direct interaction between BAM7 and BAX at the very explicitly evaluate the capacity of BAM7 to directly trigger surface employed by the BIM BH3 helix to trigger BAX 30 activation. BAX pore formation in the absence of other factors. Whereas We next performed molecular docking analysis to examine treatment with BAX or BAM7 alone had no effect on the the predicted interactions between BAM7 and the BAX trig liposomes, the combination of BAM7 and BAXyielded dose ger site. Interestingly, BAM7 appears to insinuate itself along responsive liposomal release of entrapped fluorophore (FIG. a crevice formed by residues located at (1) the junction 35 8B). Thus, the direct interaction between BAM7 and BAX at between the C1-C2 loop's C-terminus and the N-terminus of the trigger site induces the characteristic structural changes C2, (2) the N-terminus of C1, and (3) the C-terminus of C.6 that yield functional BAX oligomerization. (FIG. 6B). This is an intriguing model of the complex from a Finally, we investigated whether this prototype BAX acti functional standpoint, as engagement of this region by BIM SAHB is believed to displace the O. 1-C 2 loop and expose an vator molecule that directly, selectively, and functionally acti epitope comprised of amino acids 12-24, which are recog 40 vates BAX in vitro could induce BAX-dependent cell death. nized by the 6A7 antibody only upon BAX activation'. For these studies, we employed genetically-defined mouse Indeed, the pyrazolone core of BAM7 sits at the base of the embryo fibroblasts (MEFs) that either express only BAX 6A7 activation epitope, with the carbonyl group engaged in hydrogen bonding interactions with K21, a key residue that (Bak), only BAK (Bax) or neither death effector (Bax participates in complementary charge-charge interactions 45 Bak'). Thus, to undergo apoptosis, Bak MEF's rely on with E158 of the BIM BH3 helix'''7. Whereas the ethox BAX and Bax MEF's rely on BAK, whereas Bax Bak yphenyl group abuts the confluence of residues at the C.1-C2 MEFs are profoundly resistant to apoptosis'. Strikingly, loop's C-terminus and the N-termini of C.1 and O2, a pre BAM7 dose-responsively impaired the viability of Bak Sumed hinge site for loop opening upon initiation of BAX MEFs that exclusively express BAX, but had no effect on activation, the methyl and phenylthiazol R groups make 50 hydrophobic contact with that portion of the BIMBH3-bind Bax MEFs that contain BAK but lack BAX (FIG. 8C). ing groove formed by aliphatic residues of C.1 and C.6. Thus, Similar specificity of action in Bak MEFs was also docking analysis positions BAM7 at a critical region of the observed for formula (I) compounds (FIG. 9A-D). BAM7 BAX trigger site implicated in ligand-induced C. 1-C2 loop treated Bak MEFs likewise exhibited characteristic micro displacement and resultant exposure of the 6A7 activation 55 scopic features of apoptosis, including cellular shrinkage and epitope. This binding region is geographically and function ally distinct from the canonical BH3-binding site located at membrane blebbing (FIG. 8E). Importantly, BAM7 did not the C-terminal face of anti-apoptotic BCL-2 family proteins, affect the viability of Bax Bak MEFs, further confirming and may account for the remarkable selectivity of BAM7 for its specificity of action (FIG. 8C). To evaluate the cytosolic BAX. vs. mitochondrial distribution of BAX in response to BAM7 60 Example 4 treatment, we transfected Bax/Bak MEFs with EGFP BAX, labeled mitochondria with MitoTracker, and then BAM7 Activates BAX and BAX-Dependent Cell monitored BAX translocation by confocal fluorescence Death microscopy. We observed a dose-responsive increase in BAX 65 translocation as evidenced by conversion of the diffuse, cyto In order to transform from an inactive cytosolic monomer solic EGFP-BAX pattern to a mitochondrion-localized dis into a toxic mitochondrial oligomer, BAX undergoes a major tribution (FIG. 8D). US 9,303,024 B2 47 48 Example 5 A1AC, or a chitin column (BioLabs) for Intein-BAX. On bead digestion of GST-tagged protein was accomplished by BAM7 Dose-Responsively Decreases Viability of overnight incubation at room temperature in the presence of DHL5 Diffuse Large B-Cell Lymphoma Cells thrombin (75units) in PBS (3 mL), whereas the intein tag was (DLBCL) and Synergizes with the BCL-2/BCL-XL 5 cleaved from BAX by overnight incubation of the chitin Inhibitor ABT-737 beads at 4°C. with 50 mMDTT. BCL-X, AC, MCL-1ANAC, and BFL-1/A1AC were purified by size exclusion chroma To assess the anti-cancer activity of BAM7, DHL5 DLBCL tography (SEC) using 150 mM. NaCl, 50 mM Tris, pH 7.4 cells, which are relatively resistant to ABT-737, were exposed to BAM7 and dose-responsive impairment of cell viability 10 buffer conditions, and full-length monomeric BAX protein was observed at 24 hours, as assessed by CellTilterGlo (FIG. isolated by SEC using a Superdex-75 column (GE Health 10A). Adding a subcytotoxic dose of the selective BCL-2/ care) and 20 mM Hepes pH 7.2, 150 mM KCl buffer condi BCL-XL inhibitor ABT-737 further sensitized the cells to tions. BAM7 (FIG. 10A). Conversely, BAM7 sensitized DHL5 Fluorescence Polarization Binding Assays. cells to ABT-737 (FIG. 10B, 10C), which is otherwise less 15 Fluorescence polarization assays (FPA) were performed as effective in DHL5 cells due to expression of anti-apoptotic previously described. Briefly, direct binding curves were proteins that lie outside its binding spectrum. first generated by incubating FITC-BIMSAHB (50 nM) with Thus, we find that BAM7 directly binds to the BAX trigger serial dilutions of full-length BAX, BCL-X, AC, MCL site and initiates the characteristic structural changes that lead 1ANAC, or BFL-1/A1AC and fluorescence polarization mea to functional BAX activation. When applied to genetically- 20 sured at 20 minutes on a SpectraMax M5 microplate reader defined MEFs, BAM7 only kills the cell line that contains (Molecular Devices). For competition assays, a serial dilution BAX, inducing the morphologic features of apoptosis; in the of small molecule or acetylated BIM SAHB (Ac-BIM context of imaging Bax Bak MEFs that express EGFP SAHB) was combined with FITC-BIM SAHB (50 nM), fol BAX, BAX translocation from cytosol to mitochondria, and lowed by the addition of recombinant protein at -EC, con attendant cellular shrinkage and membrane blebbing, is also 25 centration, as determined by the direct binding assay (BAX: observed. BAM7 impairs the viability of DHL5 lymphoma 500 nM; BCL-X, AC, MCL-1ANAC, BFL-1/A1AC: 200 cells and can sensitize the cells to the BCL-2/BCL-XL inhibi nM). Fluorescence polarization was measured at 20 minutes tor AB7-737. Taken together, these studies demonstrate the and ICso values calculated by nonlinear regression analysis of feasibility of targeting BAX with a selective small molecule competitive binding curves using Prism software (Graph to trigger its pro-apoptotic activity. 30 pad). Methods BAM7 Characterization by Mass Spectrometry and In Silico Screening. 'H-NMR Spectroscopy. A diverse in silico library was generated from the following 4-(2-(2-ethoxyphenyl)hydrazono)-3-methyl-1-(4-phe commercially available libraries downloaded from the ZINC nylthiazol-2-yl)-1H-pyrazol-5(4H)-one. LC-MS: ES+ 406 database': ACB Blocks, Asinex, Chembridge, Maybridge, 35 (M+1). H NMR (300 MHz, DMSO-d6)d 7.96 (d. 2H, J=8.1 Microsource, NCI, Peakdale, and FDA-approved. The in Hz), 7.86 (s, 1H), 7.75 (d. 1H, J–7.8 Hz), 7.46 (t, 2H), 737 silico library was filtered for drug-like features, ADME prop 7.33 (m. 1H), 7.25-7.20 (m. 2H), 7.13-7.07 (m, 1H), 4.29 erties, and appropriate functional groups with Qikprop. Mol 4.22 (q, 2H), 2.36 (s.3H), 1.48 (t,3H). ecules were converted to 3D all-atom structures, generating a NMR Samples and Spectroscopy. maximum of 4 stereoisomers, ionization states for pH 7.0 and 40 Uniformly N-labeled full-length human BAX was gen pH 2.0, and different tautomers with Ligprep. The database of erated as previously described'77. Protein samples were in silico 3D molecules totaled approximately 750,000 com prepared in 25 mM sodium phosphate, 50 mM. NaCl solution pounds. BAX structures for docking were prepared using an at pH 6.0 in 5% D.O. BAM7 (10 mM stock) was titrated into averaged BAX closed-loop structure and an averaged BAX a solution of 50 M BAX to achieve the indicated molar open-loop structure with GROMACS software. The two 45 ratios. Correlation "H-'N HSQC spectra were acquired at structures were generated in suitable format for docking with 25°C. on a Bruker 800 MHz NMR spectrometer equipped Maestro. Docking was performed using Glide, with the small with a cryogenic probe, processed using NMRPipe, and molecule database for each BAX structure in standard preci analyzed with NMRView'. The weighted average chemical sion mode (SPVS)'. The top 20,000 hits based on Glidescore shift difference A at the indicated molar ratio was calculated function were selected and redocked to the BAX structures 50 as W{(AH)+(AN/5)}/2 in pp.m. The absence of a bar indi using extra precision docking mode (XPVS)”. The top 1000 cates no chemical shift difference, or the presence of a proline hits from each docking calculation were visualized on the or residue that is overlapped or not assigned. BAXcross-peak structure and then analyzed for interactions with key BAX assignments were applied as previously reported. The sig residues, leading to selection of 100 compounds for experi nificance threshold for backbone amide chemical shift mental screening. 55 changes was calculated based on the average chemical shift BCL-2 Family Protein Production. across all residues plus the standard deviation, in accordance Recombinant and tagless full-length BAX, BCL-X, AC, with standard methods'. MCL-1ANAC, and BFL-1/A1AC were expressed and puri Structure Modeling. fied as previously reported''. Transformed Escherichia Docked structures of BAX and BAM7 were generated coli BL21 (DE3) were cultured in amplicillin-containing 60 using Glide 4.0'' (Schrodinger, 2006) and analyzed using Luria Broth and protein expression was induced with 0.5 mM PYMOL2. isopropyl B-D-1-thiogalactopyranoside (IPTG). The bacte BAX Oligomerization Assay. rial pellets were resuspended in buffer (250 mM. NaCl, 20 BAM7 was added to a 200 OL solution (20 mM Hepes/ mM Tris, complete protease inhibitor tablet, pH 7.2), soni KOH pH 7.2, 150 mM KC1, 0.5% CHAPS) containing size cated, and after centrifugation at 45,000xg for 45 min, the 65 exclusion chromatography (SEC)-purified, monomeric BAX Supernatants were applied to glutathione-agarose columns at the indicated BAM7:BAX ratios. The mixtures and BAX (Sigma) for GST-BCL-X, AC, MCL-1ANAC, and BFL-1/ monomer alone were incubated at 30° C. for the indicated US 9,303,024 B2 49 50 durations and then Subjected to analysis by SEC using an LipofectamineTM 2000 (Invitrogen) according to the manu SD75 column and 20 mM Hepes/KOH pH 7.2, 150 mM KCl facturer's protocol, using 750 ng of plasmid DNA and 1.0LL running buffer. The monomeric and oligomeric fractions of LipofectamineTM per well. Plasmid DNA was generated by elute at ~11.5-12.0 min and ~6.5-7.5 min, respectively. Pro cloning full-length BAX into the pEGFP-C3 plasmid (Clon tein standards (GE Healthcare) were used to calibrate the tech) using 5' PstI and 3' Xbal restriction sites. After overnight molecular weights of gel filtration peaks. Replicates were transfection, cells were treated with the indicated concentra performed using at least two independent preparations of tions of BAM7 or vehicle (0.3% DMSO) in supplemented freshly SEC-purified monomeric BAX protein. DMEM for 6 hours. Mitochondria were labeled with Liposomal Release Assay. MitoTracker R. Red CMXRos (Invitrogen) according to the Liposomes were prepared and release assays performed as 10 previously described. Liposomes were composed of the manufacturer's protocol using 20 nM probe in 500 uL supple following molar percentages of lipids (Avanti Polar Lipids): mented, phenol red-free DMEM for 15 minutes. The cells phosphatidylcholine, 48%; phosphatidylethanolamine, 28%; were then incubated in fresh media for an addition 15 minutes phosphatidylinositol, 10%; dioleoyl phosphatidylserine, prior to imaging. Confocal microscopy was performed using 10%; and tetraoleoyl cardiolipin, 4% and were loaded with 15 a Yokogawa spinning disk confocal microscope (Yokogawa ANTS/DPX (Molecular Probe) upon extrusion. BAX (400 Electric Corporation) equipped with a Nikon inverted Ti nM) was combined with BAM7 (200 nM,400 nM) in 96-well microscope. Solid state lasers set at 488 nm and 561 nm were format (Corning) and then liposomes were added (10 uL from used to visualize EGFP and MitoTracker R. Red CMXRos, 50 uM total lipid stock) in assay buffer (10 mM HEPES pH respectively. The plate temperature was maintained at 37°C. 7, 200 mMKC1, 5 mMMgCl, and 0.2 mM EDTA) to a final using an InVivo environmental chamber (InVivo Scientific). volume of 100 ul. Liposomal release was quantified based on Images were collected using an Andor iXon DU-897 EM the increase in fluorescence that occurs when the ANTS fluo CCD camera (Andor Technology) and analyzed with Image.J rophore is separated from the DPX quencher upon release software (NIH). Percent EGFP-positive cells was determined from the liposomes into the Supernatant. Fluorescence by counting EGFP-positive and Mitotracker-positive cells. (2355 nm and 520 nM) was measured for 2 hours at 25 Percent BAX translocation was calculated by dividing the 30°C. using a Tecan Infinite M1000 plate reader. To measure number of cells containing mitochondrion-localized BAX by maximal release, Triton X-100 was added to a final concen the total number of EGFP-positive cells. Each treatment was tration of 0.2% (v/v) after 2 hand fluorescence measured for performed in quadruplicate with >200 cells counted per well. an additional 10 min. The percentage release of ANTS/DPX is calculated as percentage release ((F-F)/(Foo-F))x100, 30 where Fo and Foo are baseline and maximal fluorescence, Example 6 respectively. Cell Viability Assay. Mouse embryonic fibroblasts (MEFs) cells were main Additional Biological Activity tained in DMEM high glucose (Invitrogen) supplemented 35 with 10% (v/v) FBS, 100 U/mL penicillin, 100 g/mL strep FIGS. 15A-15G are graphs that demonstrate the anti-leu tomycin, 2 mM L-glutamine, 50 mM HEPES, 0.1 mM MEM kemic activity of BAM7. non-essential amino acids and 50 uM B-mercaptoethanol. FIG. 16 is a graph that compares the anti-leukemic activity MEFs (2.5x10 cells/well) were seeded in 96-well opaque of BAM7 to compounds 165-93, 165-60, and 172-90 (see plates for 18-24 hours and then incubated with serial dilutions 40 FIG. 14). Other comparative data is provided in the tables of BAM7 or vehicle (0.15% DMSO) in DMEM at 37° C. in a final volume of 100 ul. DHL5 cells were cultured as described below: (Deng et al. Cancer Cell, 12, 171-185, 2007) and subjected to vehicle, BAM7, ABT-737, and combinations thereof at the FITC-BIMSAHB indicated doses. Cell viability was assayed at 24 hours by 45 addition of CellTiter-Glo reagent according to the manufac Compound Competition IC50 (M) turer's protocol (Promega), and luminescence measured BAM7 6 using a SpectraMax M5 microplate reader (Molecular 172-19 2.7 Devices). Viability assays were performed in at least triplicate 172-90 2 and the data normalized to vehicle-treated control wells. Leu 50 165-97 1.7 kemia cell viability and caspase 3/7 assays were performed as 172-11 1.5 described (Cohen et al., Chem Biol, 2012). Light Microscopy. MEFs (5,000 cells/well) were plated for 24 hours on glass FIG. 17 is a graph that demonstrates the broad anti-leuke bottom culture dishes (Mattek Corp., MA) and then incu 55 mic activity of compound 172-90. bated with BAM7 (15 M) or vehicle (0.15% DMSO). Live cell imaging was performed using a TE2000-E2 Nikon FIGS. 18A and 18B are graphs that demonstrate that com microscopy equipped with a temperature and CO-controlled pound 172-90 overcomes the apoptotic resistance conferred chamber that maintained an atmosphere of 3-5% humidified by BCL-2 family anti-apoptotic members BCL-XL and CO at 37° C. A Hamamatsu Orca ER digital CCD camera 60 MCL-1; whereas the BCL-2/BCL-XL selective inhibitor was used to capture images at 20x magnification for 24 hours ABT-737 induces cell death of the BCL-XL-dependent leu at 20 min intervals. Acquisition, hardware control, and image kemia cell line, significant resistance to ABT-737 is manifest analysis was performed using Nikon NIS-Elements software. in the isogenic MCL-1 dependent leukemia cell line. In con BAX Translocation Assay. trast, FIGS. 18C and 18D are graphs that demonstrate that MEFs were seeded on uncoated 24-well glass bottom 65 compound 172-90 induces dose-responsive caspase 3/7 activ plates at a density of 2.5x10 cells/well in 500 uL of supple ity and cell death in both cell lines, overcoming formidable mented DMEM. After 6 hours, cells were transfected using apoptotic resistance. US 9,303,024 B2 52 R’ is: FITC-BIMSAHB C(O)CH: Competition IC50 Compound (IM) C-C alkoxy that is optionally substituted with —NH; O BAM7 6 heterocyclyl containing from 5-7 ring atoms, wherein 161-79 6.2 183-50 2.5 from 1-2 of the ring atoms of the heterocyclyl is 165-60 O.9 independently selected from N, NH, N(C-C alkyl). BAM7 6 NC(O)(C-C alkyl), NC(O)C(C-C alkyl), O, and 16S-87 O.22 S; and each of which is optionally substituted with 165-74 O.1 10 from 1-3 independently selected C-C alkyl groups; each of RandR'' is H; and each occurrence of R is, independently, halo; cyano; —C(O)(C-C alkyl); C(O)OH:—C(O)C(C-C alkyl); FITC-BIMSABH —NH; —NH(C-C alkyl), N(C-C alkyl). —NHC Compound Competition IC50 (M) 15 (O) (C-C alkyl), C-C alkoxy; C-C haloalkoxy; BAM7 6 C-C thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, 161-87 2.4 C-C cycloalkyl; or C-C haloalkyl; 153-96 2 provided that R' cannot be –C(O)OH when R is unsub 165-93 O.S stituted phenyl; R' cannot be —OCHCH, when R is unsubstituted phenyl or CH; and provided that the com pound of formula (I-A) is not a compound of the follow OTHER EMBODIMENTS ing formula: In some embodiments, compounds can have the 5.6 heter oring structure that is present in compounds 165-90, 165-94, 25 O and 165-95 in FIG. 11. In embodiments, the nitrogen in the 6-membered ring adjacent to the carbonyl can be substituted with X-Y in which X and Y can be as defined anywhere N herein; and the remaining positions can be substituted with 30 N1 N N substituents as defined in R as defined anywhere herein. It is to be understood that while the invention has been H N /N-( described in conjunction with the detailed description N S thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the 35 2. A compound of formula (I-A), or a pharmaceutically Scope of the appended claims. Other aspects, advantages, and acceptable salt thereof: modifications are within the scope of the following claims.

What is claimed is: (I-A) 1. A compound of formula (I-A), or a pharmaceutically 40 acceptable salt thereof:

O (I-A) R13 45 N's N X it R 14 N / R12 R2 N CX X" O N-N N X 50 wherein: X is NH; H S. M N -( i X" is unsubstituted phenyl, R2 N X X" X" is H or C-C alkyl: R’ is: 55 C-C alkyl, or wherein: phenyl that is optionally substituted with from 1-4 R; or X is S: heteroaryl containing from 5-6 ring atoms, wherein X" is unsubstituted phenyl, from 1-4 of the ring atoms is independently selected X" is H or C-C alkyl: from N, NH, N(C-C alkyl), NC(O)(C-C alkyl), O, R’ is: 60 and S; and wherein said heteroaryl is optionally sub C-C alkyl; or stituted with from 1-3 R: phenyl that is optionally substituted with from 1-4 R; or R’ is: heteroaryl containing from 5-6 ring atoms, wherein C(O)CH: from 1-4 of the ring atoms is independently selected C-C alkoxy that is optionally substituted with —NH; from N, NH, N(C-C alkyl), NC(O)(C-C alkyl), O, 65 O and S; and wherein said heteroaryl is optionally sub heterocyclyl containing from 5-7 ring atoms, wherein stituted with from 1-3 R: from 1-2 of the ring atoms of the heterocyclyl is US 9,303,024 B2 53 54 independently selected from N, NH, N(C-C alkyl). -continued NC(O)(C-C alkyl), NC(O)C(C-C alkyl), O, and S; and each of which is optionally substituted with from 1-3 independently selected C-C alkyl groups; O HCI each of R' and R'' is H; and each occurrence of R is, independently, halo; cyano; —C(O)(C-C alkyl); C(O)OH: —C(O)O(C-C alkyl); nitro; NH; NH(C-C alkyl), N(C-C alkyl). —NHC(O)(C-C alkyl), C-C alkoxy; C-C, 10 haloalkoxy; C-C thioalkoxy; C-C thiohaloalkoxy; C-C alkyl, C-C cycloalkyl; or C-C haloalkyl. 3. The compound of claim 1, wherein R is C alkyl or heteroaryl containing from 5-6 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, 15 N(C-C alkyl), NC(O)(C-C alkyl), O, and S. 4. The compound of claim 1, wherein R is selected from the group consisting of methyl, ethyl, furanyl, thienyl, and thiazolyl. 5. The compound of claim 1, wherein R' is C(O)CH. 6. The compound of claim 1, wherein R' is C-C alkoxy that is optionally substituted with —NH. 7. The compound of claim 1, wherein R' is unsubstituted 25 isopropoxy or ethoxy that is optionally substituted with —NH. 8. The compound of claim 1, wherein R' is heterocyclyl s containing from 5-7 ring atoms, wherein from 1-2 of the ring 30 atoms of the heterocyclyl is independently selected from N. NH, N(C-C alkyl), NC(O)(C-C alkyl), NC(O)C)(C-C, alkyl), O, and S; and each of which is optionally substituted with from 1-3 independently selected C-C alkyl groups. 9. The compound of claim 1, wherein R' is selected from 35 the group consisting of piperazinyl and morpholinyl. 10. The compound of claim 1, wherein the compound is selected from the group consisting of:

O OEt N 50

H N MN-( N S

65 US 9,303,024 B2 55 56 -continued 15. The compound of claim 2, wherein the compound is:

O N N O N1 \ -(N s 10 N. HN or a pharmaceutically acceptable salt thereof. and 16. A pharmaceutical composition comprising a com pound of claim 1, or a pharmaceutically acceptable salt 15 thereof, and a pharmaceutically acceptable carrier. 17. A pharmaceutical composition comprising a com pound of claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 18. A method of treating cancer in a Subject, comprising administering the compound of claim 1, or a pharmaceuti OH O cally acceptable salt thereof, to a Subject in need of cancer treatment in an amount effective to treat the cancer, wherein N1's N s the cancer is leukemia. 19. The method of claim 18, wherein the leukemia is H N /N-( selected from the group consisting of acute lymphoblastic N N S leukemia (ALL) and acute myelogenous leukemia (AML). 20. A method of treating cancer in a Subject, comprising administering the compound of claim 2, or a pharmaceuti cally acceptable salt thereof, to a Subject in need of cancer 30 treatment in an amount effective to treat the cancer, wherein or a pharmaceutically acceptable salt thereof. the cancer is leukemia. 11. The compound of claim 2, wherein R is C, alkyl. 21. The method of claim 20, wherein the leukemia is 12. The compound of claim 2, wherein R is methyl. selected from the group consisting of acute lymphoblastic 13. The compound of claim 2, wherein R' is Calkoxy. leukemia (ALL) and acute myelogenous leukemia (AML). 14. The compound of claim 2, wherein R' is ethoxy. k k k k k