USOO8338416B2 (12) United States Patent (10) Patent No.: US 8,338.416 B2 Buggy et al. (45) Date of Patent: Dec. 25, 2012 (54) INDOLE DERIVATIVES AS INHIBITORS OF WO WO2007/048841 * 5/2007 HISTONE DEACETYLASE WO WO2O08060721 * 5/2008 WO WO 2008061160 * 5/2008 (75) Inventors: Joseph J. Buggy, Mountain View, CA OTHER PUBLICATIONS (US); Sriram Balasubramanian, San & 8 18 Carlos, CA (US); Erik Verner, San Chawla et al., "Challenges in Polymorphism of Pharmaceuticals'. Mateo, CA (US); Vincent W. F. Tai, San CRIPS vol. 5 Nolan-Mar 2004 ("Pages"." Mateo, CA (US); Chang-Sun Lee, Belle Newman et al., “Solid-state analysis of the active pharmaceutical s s s ingredient in drug products'. DDT vol. 8, No. 19, Oct. 2003, p. Mead, NJ (US) 898-905. Bhalla, K.N., “Epigenetic and Chromatin Modifiers as Targeted (73) Assignee: Pharmacylics, Inc., Sunnyvale, CA Therapy of Hematologic Malignancies,” J. Clin. Oncol. 23:3971 (US) 3993 (2005). Buggy et al., “Cloning and characteristics of a novel human histone (*) Notice: Subject to any disclaimer, the term of this deacetylase, HDAC8.” Biochem. J. 350 Pt. 1: 199-205 (2000). patent is extended or adjusted under 35 Carta et al., “Histone deacetylase inhibitors prevent exocytosis of U.S.C. 154(b) by 581 days. interleukin-13-containing Secretory lysosomes: role of microtubules.” Blood 108(5):1618-1626 (2006). (21) Appl. No.: 11/687.565 Fuino, L. et al., "Histone deacetylase inhibitor LAQ824 down-regu lates Her-2 and sensitizes human breast cancer cells to trastuzumab, (22) Filed: Mar 16, 2007 536 smiline and epothilone B.” Mol. Cancer Ther. 2:971 Jacobs et al., “Substituted 3-(Phenylmethyl)-1 H-indole-5- (65) Prior Publication Data carboxamides and 1-(Phenylmethyl)indole-6-carboxamides as US 2007/O28 1934 A1 Dec. 6, 2007 Potent, Selective, Orally Active Antagonists of the Peptidoleukotrienes.” J. Med. Chem. 36:394-409 (1993). Related U.S. Application Data Leoni et al., “The antitumor histone deacetylase inhibitor Suberoylanilide hydroxamic acid exhibits antiinflammatory proper (60) Provisional application No. 60/783.287, filed on Mar. ties via supression of cytokines.” PNAS USA99:2995-3000 (2002). 16, 2006. Miller et al., “Histone deacetylase inhibitors.” J. Med. Chem. 46(24):5097-5116 (2003). (51) Int. Cl. Somoza et al., “Structural Snapshots of Human HDAC8 Provide CO7D 40/02 (2006.01) Insights into the Class 1 Histone Deacytelases.” Structure 12:1325 CO7D 209/04 (2006.01) 1334 (2004). CO7D 413/02 (2006.01) Vannini et al., “Crystal structure of a eukaryotic zinc-dependent histone deacytelase, human HDAC8, complexed with a hydroxamic A6 IK3I/404 (2006.01) acid inhibitor.” PNAS 101(42): 15064-15069 (2004). A6 IK3 L/4523 (2006.01) Yoo, C.B. and Jones, P.A., “Epigenetic therapy of cancer: past, A 6LX3/5377 (2006.01) present and future.” Nat. Rev. Drug Discov. 5:37-50 (2006). A6IP35/00 (2006.01) PCT/US07/06714 Search Report dated May 27, 2008. A6IP35/02 (2006.01) (52) U.S. Cl. ..................... 514/235.2: 514/320: 514/415; * cited by examiner 544/143: 546/201: 548/452 (58) Field of Classification Search ........................ None Primary Examiner — Sun Jae Loewe See application file for complete search history. X: terney, Agent, or Firm — Wilson, Sonsini, Goodrich OSa1 (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS Described herein are compounds and pharmaceutical com 6,069,156 A 5, 2000 Oku et al. positions containing such compounds, which inhibit the 6,303,600 B1 10/2001 Cox et al. activity of histone deacetylase 8 (HDAC8). Also described 200 SE R 258 East al. herein are methods of using such HDAC8 inhibitors, alone 2008/01 12889 A1* 5/2008 Buggy et also 424.9.2 and in combination with other compounds, for treating dis eases or conditions that would benefit from inhibition of FOREIGN PATENT DOCUMENTS HDAC8 activity. WO WO-02-055O17 T 2002 WO WO-2003-013493 2, 2003 19 Claims, 18 Drawing Sheets U.S. Patent Dec. 25, 2012 Sheet 1 of 18 US 8,338.416 B2 Fig. 1A Representative tissue samples Brain Breast 1374019: Neurons and Glia 1374021: Duct 40X 40X Kidney 1374023: Mucosa 40X 1374027: Glomerulus 40X Liver 1374033: Portal Triad and : XXa Hepatocytes 40X 1374036: Bronchiole 40X ovary Pancreas & SS 1374043: Follicles and Stroma 40X 1374172: Adjacent Residual Islet 40X U.S. Patent Dec. 25, 2012 Sheet 2 of 18 US 8,338.416 B2 Fig. 1B Representative tissue samples Prostate Skel Muscle 1374050: Glandular Epithelium and Stroma 40X Skin 1374053: Squamous 1374055: Villi 40X Epithelium 40X Spleen Stomach 1374068: Muscularis 1374059: Red Pulp 40X P ropriaia 40X & 1374158: Epithelium and Lymphocytes 40X U.S. Patent Dec. 25, 2012 Sheet 3 of 18 US 8,338.416 B2 Fig. 2 HDAC8 in islet DELTA cells HDAC8 Ab 1 HDAC8 HDAC8 insulin :-SS & S & 1370559: HDAC8-Celera, 1370562: HDAC8-Celera + insulin, set 40X Set 40X HDAC8 Somatostatin RS ' ' 1370567 HDAC8-Celera - 1370626 HDAC8-Celera - Glucagon, Islet 40X Somatostatin, Islet 40X HDAC8 Ab #2 HDAC8 HDAC8 insulin 137O608 HDAC8 SC 11405, Islet 40X 137O611. HDAC8-SC-11405 - Insulin, Islet 40X HDAC8+ glucagon HDAC8 + somatostatin 137O614 HDACB-SC-11405 1370617: HDAC8-SC-11405 + Glucagon, Islet 40X Somatostatin, Islet 40X U.S. Patent Dec. 25, 2012 Sheet 4 of 18 US 8,338.416 B2 Fig. 3 HDAC8 in plasma cells 1374430: Malignant Cells 1374431: Lymphocytes and 40X Plasma Cells 60X S. S XSS & S & 1374159: Lymphocytes 40X 1374.325: Cardiac Myocytes 1374326: Plasma Cells 6OX 40X 'S "S 1374460: Malignant Cells 40X 1374461: Plasma CellS 6OX U.S. Patent Dec. 25, 2012 Sheet 5 of 18 US 8,338.416 B2 Fig. 4 HDAC8 protein is found in tumor cell line Ramos Raji DHL-4 Jurkat HuT78 DB K562 HDAC8 HSC 70 o A549 HCT-116 MCF-7 OVCR-3 PC3 RKO U87 K562 Jurkat HDAC8 HSC 70 U.S. Patent Dec. 25, 2012 Sheet 6 of 18 US 8,338.416 B2 Fig. 5 HDAC8 protein expression is modulated in a dose-dependent manner by both compound 23 and the pan-HDAC inhibitor PCI-24781 Compound 23 (uM) PCI-24781 (uM) Control 1 5 25 0.04 0.2 1 U.S. Patent Dec. 25, 2012 Sheet 7 of 18 US 8,338.416 B2 Fig. 6 HDAC8 knockdown leads to apoptosis Untreated SCrambled Á??A??oeasedseopaz||euuuoN ©Ns= ee U.S. Patent Dec. 25, 2012 Sheet 8 of 18 US 8,338.416 B2 Fig. 7 Apoptosis induced by compound 23 is blocked by a pan-Caspase inhibitor Annexin VIPl: Jurkat PCI-34051 CD 2 5 O Annexin V O n s O SS Control 10M qVD-OPh Compound 23 Compound 23 (5uM) (5uM) + 1 OuMcVD-OPh Treatment U.S. Patent Dec. 25, 2012 Sheet 9 of 18 US 8,338.416 B2 Fig. 8 Compound 23 does not inhibit growth of Selected Solid tumor lines F33 - A1, OVCAR-3, A Pa Ca-2, PANC is 8:338. 33333.3-liki 388x *.si $33, 33.38 Si Esi E::s: U.S. Patent Dec. 25, 2012 Sheet 10 of 18 US 8,338.416 B2 Fig. 9 Compound 23 Does Not Inhibit Growth Of HCT116 Or Human PBMCS HCT-11648-hr Alamar Blue Assay 120 ---.24781 ... S. 23 s d B L O -H O.O1 0.1 1 10 1OO Concentration (uM) PBMC 24-hr Alamar Blue Assay 140 12O 100 3 80 3 60 9 40 --24781 2O ... &. 23 O - O.O1 O.1 1 10 1OO Concentration (uM) U.S. Patent Dec. 25, 2012 Sheet 11 of 18 US 8,338.416 B2 Fig. 10 Compound 23 is cytotoxic to T-cell derived tumor cells ... T-Cell derived Cell lines -- B-Cell derived Cell lines Jurkat (Glso = 3 uM) DB (Glso >20 uM) HuT78 (Glso = 4 uM) K562 (Go 220 uM) 8/8 Solid tumor lines 4.O - Human normal PBMCs (24 hrs) Š d 3. O C Control 2 s Comp23 g 20 al x 1 O g U.S. Patent Dec. 25, 2012 Sheet 12 of 18 US 8,338.416 B2 Fig.11 Inhibition of IL-1b secretion in human PBMCs Dexamethasone & Compound 23 GSS Ne1 C ...O C C 1E-05 1E-04 1E-03 1E-02 1E-O1 1E-OO 1E--O1 Concentration (uM) U.S. Patent Dec. 25, 2012 Sheet 13 of 18 US 8,338.416 B2 Fig. 12 Compound 23 inhibits IL-1 b secretion in THP-1 monocyte cell line Effect of LPS and compound 23 on IL-1B secretion in THP-1 cells normalized to control DMSO+LPS O.5uMH-LPS 5.OUM-LPS Treatment, 24 hrs U.S. Patent Dec. 25, 2012 Sheet 14 of 18 US 8,338.416 B2 Fig. 13 Phospholipase C-gamma 1-deficient Jurkat derivative J.gamma1 cells are resistant to Compound 23-induced apoptosis but TCR signaling mutants are not 1 O O SS se Control 2 s 8 O CD compound 23 c e 6 O O ? 4 O e s& S S S X d 2 O SS e O S.S S & U.S. Patent Dec. 25, 2012 Sheet 15 of 18 US 8,338.416 B2 Fig. 14 Phospholipase inhibitor modulates compound 23-induced apoptosis 6 O Ss I d5 O c) 4 O 2 t O n 2 S X C) C C C O 0.8 0.1 0.2 0.4 0.8 0.1 0.2 0.4 U-73343 U-73122 (uM) U-73343 U-73122 (uM) (uM) (uM) Control Compound 23 (10uM) U.S. Patent Dec. 25, 2012 Sheet 16 of 18 US 8,338.416 B2 Fig. 15 Compound 23 induced apoptosis is enhanced by Ca2+ effector thapsigargin 35 in Jurkat cells but not in J.gamma cells 122 5O5 1 O Control Compound 23 thapsigargin Compound 23 + thapsigargin U.S.