USOO6992O79B2 (12) United States Patent (10) Patent No.: US 6,992,079 B2 Brugnara et al. (45) Date of Patent: *Jan. 31, 2006

(54) SUBSTITUTED 4,006,023 A 2/1977 McLaughlin et al...... 96/90 R 11-PHENYL-DBENZAZEPINE COMPOUNDS 4,806,685 A 2/1989 Abraham et al...... 563/324 USEFUL FOR THE TREATMENT OR 5,273,992 A 12/1993 Brugnara et al...... 514/398

5,358,959 A 10/1994 Halperin et al...... 514/396 ERR2E, RMAL cell 5,430,062. A 7/1995 Cushman et al...... 514/646 5,512.563 A * 4/1996 Albright et al...... 514/217 PROLIFERATION 6,063,921 A 5/2000 Moussa et al. (75) Inventors: Carlo Brugnara, Newton Highlands, 6,291,4496,201,120 B1 9/20013/2001 Ms t alal. MA (US); Jose Halperin, Brookline, 6,534,497 B1 * 3/2003 Brugnara et al...... 514/217 MA (US); Rudolf Fluckiger, Brookline, 2001/0007030 A1 7/2001 Moussa et al. MA (US); Emile M. Bellott, Jr., Beverly, MA (US); Richard John FOREIGN PATENT DOCUMENTS Lombardy, Littleton, MA (US); John EP O 323 740 A2 7/1989 J. Clifford, Arlington, MA (US); EP O 583 665 A2 2/1994 Ying-Duo Gao, Edison, NJ (US); Reem EP O 636 608 A1 2/1995 M. Haidar, Woburn, MA (US); Eugene JP OS 58894 3/1993 W. Kelleher, Medford, MA (US); Adel WO WO95/26720 10/1995 M. Moussa, Burlington, MA (US); WO WO 96/08240 3/1996 Yesh P. Sachdeva, Concord, MA (US); W W 't f1. Minghua Sun, Libertyville, IL (US); Heather N. Taft, Littleton, MA (US); WO WO 99/26928 6/1999 Michael H. Zeldin, Cambridge, MA OTHER PUBLICATIONS Starnes, Jr., Chem. Abstract 69:43555. 1968.* (73) ASSignees: restry's sole Koelsch. Chem. Abstract 55:9360. 1961. Melileen Corporation, Boston, Gagnon, Beilstein Reg. No. 4924895. 1929.* MA (US); NuChem Pharmaceuticals Dakkouri et al., Chem. Abstract 125:305256, 1997.* Inc., Toronto Enokida et al., Chem. Abstract 125:39835, 1996.* (*)* ) Notice: Sub.E. disclai"E"ME h f thi BanliManning et al., et al.,Chem. Chem. Abstract Abstract 107:58774, 94.102538, 1987.* 1981." U.S.C. 154(b) by 0 days. Omenn, Cancer Prevention, Cecil Textbook of Medicine, 20th Edition, vol. 2, pp. 1008-1010, 1996.* Sasakura et al. Synthesis of 11-phenyl-5 clTh Air tentent IS Subjectbiect tto a tterminal 1 dis-dIS 6-dihydro-11H-dibenz(b,e)azepines derivatives, Heteros cycles, vol. 15, No. 1, pp. 421-425, 1981.* Simone, Oncology: Introduction, Cecil Textbook of Medi (21)21) Appl. No.: 10/013,10/013,308 cine, 20ths Edition, vol. 1, pp. 1004–1008,s 1996.* (22) Filed: Dec. 7, 2001 Brugnara, C., et al., “Oral Administration of Clotrimazole (65) Prior Publication Data and Blockade o Human Erytrocyte Ca++-Activated K+ Channel: the Imidazole Ring is not Required for Inhibitory US 2004/0002486 A1 Jan. 1, 2004 Activity,” JPRT 273:266–272 (1995). O O Skehan, P., et al., “New Colorimetric Cytotoxicity Assay for Related U.S. Application Data Anticancer-Drug Screening.” J. Natl. Cancer InSt. (63) Continuation-in-part of application No. 09/554,848, filed as 82:1107–1112 (1990). application No. PCT/US98/24787 on Nov. 20, 1998, now Pat. No. 6,534,497, which is a continuation of application (Continued) No. 08/975,592, filed on Nov. 20, 1997, now abandoned. (51) Int. Cl. Primary Examiner Deepak Rao A6 IK3I/55 (2006.01) (74) Attorney, Agent, or Firm-Fish & Richardson P.C. C07D 233/18 (2006.01) (57) ABSTRACT (52) U.S. Cl...... 514/217; 540/587. The present invention provides Substituted 11-phenyl (58) Field of Classification Search ...... 540,587; dibenzazepine compounds which are specific, potent and 514/217 safe inhibitors of mammalian cell proliferation. The com See application file for complete Search history. pounds can be used to inhibit mammalian cell proliferation in Situ as a therapeutic approach towards the treatment or (56) References Cited prevention of diseases characterized by abnormal cell proliferation, Such as cancer. U.S. PATENT DOCUMENTS 3.546,155 A * 12/1970 Morgan ...... 260/47 22 Claims, 51 Drawing Sheets US 6,992,079 B2 Page 2

OTHER PUBLICATIONS Brugnara, C., et al., “Erythrocyte Gardos Channel and Sickle Cell Disease: Pathophysiology and Clinical Signifi Aldrich Catalog, 1994–1995: Compounds 13, 484-8; cance, Advanced Course and International Symposium On T8,380-1; 10.130–3; D21,320-9; 32.543–0; 35,890-8; "Membrane Transporters and Channels' Program and T8.160-4; 25,657-9; T8.470-0; T8.417–7; T8,360-7; Abstracts, Lecture 34 (1995). S36.533–5; S50,949-3; 37.411-3; 37,152–1; 30.151-5; Brugnara, C., et al., “Therapy with Oral Clotrimazole 27,520-4; 34.182-7, 35,889–4; 584.240–0; S79.226-8; Induces Inhibition of the Gardos Channel and Reduction of 27,519-0, S64,640; S81,477-6; 36,005-8; S70.627-2; S34, Erythrocyte Dehydration in Patients with Sickle Cell Dis 966–6; S87.739–5; 11,500-2; S83,881-0; S58.110-0; ease,” J. Clin. Invest., 97(5):1227–1234 (1996). 21.560–0; S91,408-8; S60,147-0; S83,571-4; S82,654–5; Burgess, M.A., et al., “Clotrimazole (Bay b 5097): In Vitro S8,388-2; S94,371-1; S87,649-6; S83,719-9; S83,042–9; and Clinical Pharmacological Studies' Antimicrobial Agents S823,162-4; S81,468-7, S82,290–6; S79,387. and Chemotherapy, 2(6):423-426 (1972). Ando, W., et al., “A Novel Reduction of C.-Aryl Alkanols by Byers, H.R., et al., “Organ-Specific Metastases in Immu Diiododimethylsilane,” Tetrahedron Letters, 51:4941-4942 nodeficient Mice Injected with Human Melanoma Cells: a (1979). Quantative Pathological Analysis,” Melanoma Research, Barton, D. H. R., et al., “Pentavalent Organobismuth 3:247-253 (1993). Reagents. Part 3. Phenylation of Enols and of Enolate and Carter, A. J., et al., “Morphologic Characteristics of Lesion Other Anions.” J. Chem. Soc. 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Brugnara, C., et al., “Inhibition of Gardos Channel and Red Epstein, R. J., et al., “Corneal Neovascularization,” Cornea, Cell Dehydration by Oral Administration of Clotrimazole in 6(4):250–257 (1987). Sickle Cell Disease,” Blood, 220a:865 (1994). Ethridge, D. E., et al., “Economic Weed Control in High Brugnara, C., et al., “Inhibition of Gardos Channel and Red Plains Cotton,” J. Prod. Agric., 3(2):246-252 (1990). Cell Dehydration by Oral Administration of Clotrimazole in Fahim, M., et al., “Cardiovascular Effects and Pharmacoki Sickle Cell Disease.” The 20" Annual Meeting of the netics of the Carboxylic Ionophore Monensin in Dogs and National Sickle Cell Program, p. 138 (1995). Rabbits,” Life Sciences, 29:1959–1966 (1981). US 6,992,079 B2 Page 3

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U.S. Patent Jan. 31, 2006 Sheet 2 of 51 US 6,992,079 B2

U.S. Patent US 6,992,079 B2

009 (uu)?009 azSIOun ?|-001 |0

U.S. Patent Jan. 31, 2006 Sheet 5 of 51 US 6,992,079 B2

Growth of Human lung large Cell Carcinoma (NCI-H460) in CD-1 Nude Mice 2500 -A- Vehicle n=10 2000 || -- CompoundES5 28

ea2 1500 / E. 1000 - 5 lo11 500 2-1 Day 10 Day 13 Day 7 Day 22 Days after Tumor injection F.G. 5

Growth of Human Melanoma (A2058) in CD-1 Nude Mice 1400 --- Control 1200 -o- Compound 28 P=0.0002 1 OOO 800 E. 2 600 E 400 200

Day 5 Day 10 Day 13 Day 17 Day 19 Day 21 Days after Tumor injection F.G. 6 U.S. Patent Jan. 31, 2006 Sheet 6 of 51 US 6,992,079 B2

Number of MM-RUMelanoma lung Metastases

Control Compound 28 Compound 28 CLT S.C. 9.0. The number of maCrOSCOpic pleural metastases in vehicle Control, COmpOund 28 S.C., COmpOUnd 28 p.O., and CLT groups. Each grOup COntained 8 mice. Treatments were started at One day after turnOrinoculation F.G. 7 U.S. Patent Jan. 31, 2006 Sheet 7 of 51 US 6,992,079 B2

Polysome profile (32P)-eF2 80S 60S 40s aamamma

- (-) CLT CLT S s . . . . PKR-K296

eIF2-51A

O 200 400 600 Time (seconds)

FIG. 8 U.S. Patent Jan. 31, 2006 Sheet 8 of 51 US 6,992,079 B2

+ ~); ****

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i?* 00900?[==ŒGEOEFST) U.S. Patent Jan. 31, 2006 Sheet 9 of 51 US 6,992,079 B2

3.0

2.5

CC 2.0

15 Compound O T 28 TG O 100 200 300 Time (seconds) F.G. 10

2

i Compound 28 s co scy

250 TG Time (Sec) F.G. 11 U.S. Patent Jan. 31, 2006 Sheet 10 Of 51 US 6,992,079 B2

DOMINANT NEGATIVE PKR REEMEN CELLS ARE RESISTANT TO CLTAND COMPOUND 28 CLT COMPOUND 28 0.6 0.5 0.4 0.3 s 5 0.2 O. O O BABE PKR 1. BABE PKR -f- Resistance of PKR-/- Cells indicates that the Compound inhibits Cell growth at the level Of translation initiation

FIG. 12 U.S. Patent Jan. 31, 2006 Sheet 11 of 51 US 6,992,079 B2

DNA content analysis of cells treated with DMSO and COMPOUND 28

1000 1200 800 has 900 6OO E 2 2 2 600 400 200 300

O O O 50 100 150 200 O 50 OO 150 200 DNA Content DNA content DMSO COMPOUND 28 GO-G1: 52.61% GO-G1: 65.00% S: 34.61% S: 25.29% G2-M: 12.79% G2-M: 9.71% FIG. 13

U.S. Patent Jan. 31, 2006 Sheet 13 Of 51 US 6,992,079 B2

XTT assay for PC-3 human prostate tumor cells treated with Compound 28

-o- DMSO -o- C = 1. Oum -v- C = 1 OUm -v- (C) = 25um -- C=50um

O 10 20 30 40 50 60 70 80 Time (hr)

FIG. 15 U.S. Patent Jan. 31, 2006 Sheet 14 Of 51 US 6,992,079 B2

00Z gulu) 82SiOuJnl. U.S. Patent Jan. 31, 2006 Sheet 15 Of 51 US 6,992,079 B2

Cyclin D1

Ing deX

FIG. 17 U.S. Patent Jan. 31, 2006 Sheet 16 of 51 US 6,992,079 B2 leukemia 100

50 s dis ge, 0 E-50

-100 -9 -8 -7 -6 -5 -4 LOg 10 of Sample Concentration (Molar) CCRF-CEM-8- HL-60(TB)---0--- K-562 ---4--- MOLT-4 ....a.... RPM-8226---0---. SR---4---. FIG. 18A

100 Non-Small Cell lung Cancer

5 O

O

-50

-9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) A549/ATCC-B- EKVX ---0--- HOP-62 ------. HOP-92 ....o.o... NC-H226 ---0--- NC-H23------NC-H322M-...--A-...- NC-H460 ------NC-H522 ---a--- F.G. 18B U.S. Patent Jan. 31, 2006 Sheet 17 of 51 US 6,992,079 B2

Colon Cancer 100

50

O

-9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) COL0205-8- HCT-116------, HCT-15---4--- HT29.0.... KM12 ---o----, SW-620 ------. FIG. 18C

Log 10 of Sample Concentration (Molar) CF-268 -s- SF-295---e---. SF-539 ---4---- SNB-19 ----0---- SNB-75 ---o--- U251 ----0--- F.G. 18D U.S. Patent Jan. 31, 2006 Sheet 18 of 51 US 6,992,079 B2

OO Melanoma

5 0.

O

50

-100 -9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) LOX IMV. -a- MALME-3M------. M14 --- A -- SK-MEL-2 ----0---- SK-MEL-28 ---o--- SK-MEL-5 ------UACC-257 ---A- - UACC-62 ------

s - w Sss S n co ------SO m

-50

-100 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) GROW -e- OWCAR-3------. OWCAR-4--- a--- OWCAR-5'-O---- OWCAR-8 ---o--- SK-OV-3------FIG 18F U.S. Patent Jan. 31, 2006 Sheet 19 of 51 US 6,992,079 B2 Renai Cancer 100

5 O

O

50

-100 -8 -7 -4 LOg 10 of Sample Concentration (Molar) 186-0 -s A498------. ACHN ---4--- CAK-1 ----0---- RXF393 ---0--- SN12C------TK-10 -., -a-...- U0-31 -----...-

Prostate Cancer 00 run N N Ya s N----- SO es

50

-100 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) PC-3-e- DU-145 ---e--- F.G. 18H U.S. Patent Jan. 31, 2006 Sheet 20 of 51 US 6,992,079 B2

Breast Cancer 100

5 O

O

-9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) MCF7 - a - NCI/ADR-RES------. MDA-MB-2311---4--- HS578T.. --O--.. MDA-N ---0----- BT-549 ------T-47D-...--A-...-- FIG. 18 U.S. Patent Jan. 31, 2006 Sheet 21 of 51 US 6,992,079 B2

100 O50

-5 O

Log 10 of Sample Concentration (Molar) FIG. 19 U.S. Patent Jan. 31, 2006 Sheet 22 of 51 US 6,992,079 B2

Mean Graphs Units: Molar

Panel/Cell Line L0g G150 G150 Leukemia CCRF-CEM -7, HL-60(TB) -6. K-562 -6, : MOLT-4 -6. RPM-8226 -6. SR -6. Non-Small Cellung Cancer ...... A-549/ATCC 6. EKWX -5. HOP-62 4. HOP-92 -5. NC-H226 -5. NC-H23 -5. NC-H322M o 6. NC-H460 6. NC-H522 6. COOn Cancer COLO 2. anorPu LOX IMW MALME-3M M14 SK-ME-2 SK-ME-28 SK-MEL-5 es UACC-257 UACC-62 OVarian Cancer e s a as s us s a a b a as as a s is us is a p so a so o s is so up r s to GROW OWCAR-3 OWCAR-4 OWCAR-5 OWCAR-8 SK-OV-3

FIG. 20A U.S. Patent Jan. 31, 2006 Sheet 23 of 51 US 6,992,079 B2

Mean Graphs Units: Molar

RXF393 SN12C TK-10 U0-31 PrOState Cancer PC-3 DU-145 BreaSt Cancer MCF7 NC/ADR-RES MOA-MB-231/ATCC HS 578T MDA-N BT-549

MG MID Delta Range

FIG. 20A (Cont.) U.S. Patent Jan. 31, 2006 Sheet 24 of 51 US 6,992,079 B2 Mean Graphs Units: Molar Panel/Cell Line LOQ-TGl TG Leukemia CCRP-CEM -5.53 HL-60(TB) -4,11 K-562 > -4.00 MOLT-4 > -4.00 RPM-8226 > -4.00 SR > -4.00 Non-Small Cellung Cance ...... A-549/ATCC > -4.00 EKVX > -4.00 HOP-62 > -4.00 HOP-92 > -4.00 NC-H226 > -4.00 NC-H23 > -4.00 NC-H322M > -4.00 NC-H460 > -4.00 NC-H522 -4.57 Colon Canoer ...... e. e. a a as up w a to a s a to so in is a in a a COLO 205 -485 HCT-116 > -4.00 HCT-15 > -400 HT29 > -4.00 KM12 > -4.00 SW-620 > -4.00 CNS Canoer as a a so a so up to a w SF-268 > -4.00 SF-295 > -4.00 SF-539 > -4.00 SNB-19 > -4.00 SNB-75 > -4.00 U251 > -4.00 MeanOma so se su u is s as a s p s son or as w r ------LOX MW > -4.00 MALME-3M > -4.00 M4 -4.66 SK-ME-2 -4.96 SK-MEL-28 > -4.00 SK-MEL-5 -4.75 UACC-257 > -400 UACC-62 -401 OMarian Canoer ------as IGROV1 > -4.00 OWCAR-3 > -4.00 OWCAR-4 > -4.00 OVCAR-5 > -4.00 OWCAR-8 > -4.00 SK-OV-3 > -4.00 sap use m sm a us b- sums - -- s sa v arm r -- - - - U.S. Patent Jan. 31, 2006 Sheet 25 Of 51 US 6,992,079 B2

Mean Graphs Units: Molar Panel/Celine L09-10 TGl TGI Rena Canoer T ...... 786-0 A498 ACHN CAK RXF393 SN12C TK-10

U0-31 ssa PrOState Cancer --...------...------PC-3 DU-145 Breast Cancer a ...... as as a as a s is a as MCF7 NC/ADR-RES MDA-MB-231/ATCC HS 578T MDA-N BT-549

is ...... us is ...... s s p a a as a up

MG MID Delta Range

FIG. 20B (Cont.) U.S. Patent Jan. 31, 2006 Sheet 26 of 51 US 6,992,079 B2 Mean Graphs Units: Molar

Panel/Celine l09 LC50 LC50 Leukemia CCRP-CEM X HL-60(TB) X K-562 > - MOLT-4 X RPM-8226 > SR X A-549/ATCC > -4 EKWX > -4 HOP-62 > -4 HOP-92 > -4 NC-H226 > -4. NC-H23 > -4 NC-H322M > -4 NC-H460 > -4 NC-H522 > -4 Colon Canoer a a P - 8 up ur w w w - is us as as a a up on s : COLO 205 -4 HCT-116 > -4 HCT-15 > -4. HCT-29 > -4. KM12 > -4 SW-620 > -4 CNS Canoer ...... s. s is so so a a on as a as a sar h m n e o o vess is de v ------SF-268 S

UACC-62 OMarian Canoer ------...------GROW > -4 OWCAR-3 > -4 OWCAR-4 > -4. OWCAR-8OWCAR-5 > -4:

m m r with v w r- a w is a mm a P. Put U.S. Patent Jan. 31, 2006 Sheet 27 of 51 US 6,992,079 B2

Mean Graphs Units: Molar Panel/Cell Line L09 in LC50 C50

RXF393 SN12C TK-10 - U0-31 PrOState CanCar PC-3 DU-145 X s Breast Cancer MCF7 NC/ADR-RES MDA-MB-231/ATCC O HS 578T MDA-N BT-549

MGMID Delta Range

FIG. 20c (Cont.) U.S. Patent Jan. 31, 2006 Sheet 28 of 51 US 6,992,079 B2 LEUKEMA 100 SS - - C-5:S Aca. SSS SS-st - 50 N S. r s iss-Se-testic d s.sysics: O

-50

-100 mammom -9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) CCRF-CEM -e-...- HL-60(TB) ---o---. K562 --- a--- MOLT-4 ...... RPM-8226 ---0--- SR ------FIG. 21A

Non-Small Cell lung Cancer 100

- 50 s s ap O - A

50

-100 -9 -8 -7 -6 -5 -4 LOg 10 of Sample Concentration (Molar) A549/ATCC -a- EKVX------HOP-62 ---A--- HOP-92-0. NC-H226 ---0---- NC-H23------NC-H322M--4--- NC-H460 ------NC-H522 ---G - - - FIG. 21B U.S. Patent Jan. 31, 2006 Sheet 29 of 51 US 6,992,079 B2

1OO

5 O

50 O

-100 re -9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) COLO205 -- HCT-116 ------, HCT-15 ---4---- HT29.- .... KM12 ---0---, SW-620 ------FIG. 21C

CNS Cancer 100 e Sel

5 O Šs A.

5 O

-100 -9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) SF-268 - SF-295 ------. SF539 ---4---. SNB-19 - - -0'- SNB-75 ---0----- U251 ------. F.G. 21D U.S. Patent Jan. 31, 2006 Sheet 30 of 51 US 6,992,079 B2 Melanoma 100

5 O

O

50

-100s -8 -7 -6 T -4 LOg10 of Sample ConCentration (Molar) LOX FMV -e- MALME-3M------. M14 --- a---. SK-MEL-2------SK-MEL-28 ---0--- SK-MEL-5------. UACC-257 -...-A-...- UACC-62-...-----.

Ovarian Cancer

es S---. "...... ---... is 50 Snecess 0 -s--see

50

-100; -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) IGROV1 -e- OVCAR-3 ------. OWCAR-4--- a--- OVCAR-50" OVCAR-8 ---0--- SK-OV-3------FIG. 21 F U.S. Patent Jan. 31, 2006 Sheet 31 of 51 US 6,992,079 B2

Renal Cancer 1CO SSgar

5 O

O

-50

-9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) 786-0 -e- A498------. ACHN ---4--- CAK-1 0". RXF393 ---0--- SN2C ------TK-10 -...-A-...-. U0-31 - - - - F.G. 21G

Prostate Cancer 1 OO He as see n. n N Ya s N 50 Y----

5 O

-1005 -8 -7 -6 -5 -4 LOg 10 Of Sample Concentration (Molar) PC-3-8- DU-145 --0---- FIG 21H U.S. Patent Jan. 31, 2006 Sheet 32 of 51 US 6,992,079 B2

Breast Cancer

's s sa SSS - - a = 50 SS Ees s ta. S. .. d -- a-- O

50 dam-on

-100 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) MCFI -8- NC/ADR-RES ---0--- MDA-MB-231/ --- a---. HS578T----0---- MDA-N ---0--- BT-549 ---0--- T-47D -...-A-...-- FIG. 21 U.S. Patent Jan. 31, 2006 Sheet 33 Of 51 US 6,992,079 B2

100

5 O

O

-5 O

-9 -8 -7 -6 -5 -4 Logo of Sample Concentration (Molar) FIG. 22 U.S. Patent Jan. 31, 2006 Sheet 34 of 51 US 6,992,079 B2 Mean Graphs Units: Molar

Panel/Cell Line L09 G150 G150 Leukemia CCRF-CEM HL-60(TB) K-562 MOLT-4 RPM-8226 SR Non-Small Cell LUg Cancer a as up us us up u v s a 0 do a vo up us up a a p q w A-549/ATCC EKWX HOP-62 HOP-92 i NC-H226 NC-H23 > er NC-H322M NC-H460 n NC-H522 Colon Cancer a as w w w - us un uur ul opes tP e o P - www COO 205 HCT-116 EEHCT-29 KM12 SW-620 CNS Cancer sess see a sara aas a a o p a s - F - P - a is a let ------SF-268 SF-295 SF-539 SNB-19 SNB-75 U25 MeanOr?a ...... s a as s as a a a a a a e a a LOX MV MALME-3M M14 es SK-MEL-2 SK-MEL-28 SK-ME-5 UACC-257 UACG-62 OMarian Cancer ...... a no e s a e du u so e - a ------GROW OWCAR-3 OVCAR-4 OVCAR-5 X OWCAR-8 susup SK-OV-3 is ------w m -- U.S. Patent Jan. 31, 2006 Sheet 35 of 51 US 6,992,079 B2

Mean Graphs Units: Molar Pane/Cell Line L09 G150 G150

RXF393 SN12C TK-10 U0-31 Prostate Cancer PC-3 DU-145 Breast Cancer MCF7 NCADR-RES MDA-MB-231/ATCC HS 578T MDA-N BT-549

MG MID Delta Range

FIG. 23A (Cont.) U.S. Patent Jan. 31, 2006 Sheet 36 of 51 US 6,992,079 B2 Mean Graphs Units: Molar

Panet/Cell Line LOQ-TG1 TG Leukemia CCRP-CEM > -4 HL-60(TB) > -4 K-562 > -4. MOLT-4 > -4. RPM-8226 > -4 SR > -4 A-549/ATCC > EKVX > HOP-62 > HOP-92 > - NC-H226 X NC-H23 d NC-H.323M d NC-H460 X NC-H522 Colon Cancer a a we w a a us it up us u ss C00205 X HCT-116 X HCT-15 X HT-29 > - KM12 X SW-620 > CNS Cancer a a as a a as A as a D H is a a m is a A s SF-268 X - SF-295 X - SF-539 X - SNB-19 X - SNB-75 > U25 o MeanOma LOX MV > a MALME-3M M14 X SK-MEL-2 X SK-MEL-28 > - SK-MEL-5 > UACC-257 X UACC-62 X OMarian Canoer ...... ------GROW > -4 OWCAR-3 > -4 OWCAR-4 -5. OWCAR-5 X 4 OVCAR-8 X - 4 SK-OV-3 X 4 --- v - unit - or rom ------m------U.S. Patent Jan. 31, 2006 Sheet 37 of 51 US 6,992,079 B2

Mean Graphs Units: Molar Panel/Cell Line LOgn TG1 TG

Rena Can TT...... m lur w was a humb s mass mu + units s is a so u q a 786-0 > -4.00

A498 > -4.00 ACHN > -4.00 CAK-1 > -4.00 RXF393 > -4.00 SN12C > -4.00 TK-10 > -4.00

U0-31 > -4.00 PrOState Cancer ...... ------PC-3 > -4.00

DU-145 > -4.00 Breast Canoer ...... ------MCF7 > -4.00

NC/ADR-RES > -4.00 MDA-MB-231/ATCC > -4.00 HS 578T >-400 MDA-N > -4.00 BT-549 > -4.00 T-47D > -4.00 a a a suss us as s us as us s a s h is is a a - - - - - PP

MG MID Delta Range

FIG. 23B (Cont.)

U.S. Patent Jan. 31, 2006 Sheet 39 of 51 US 6,992,079 B2

Mean Graphs Units: Molar Panel/Cell Line Ogn LC50 LC50

In a a is a ......

> -4.00 > -4.00 > -4.00 > -4.00 RXF393 > -4.00 SN12C > -4.00 TK-10 > -4.00 U0-31 > -4.00 PrOState Carcer ...... ------...------

PC-3 > -4.00 DU-145 > -4.00 Breast Canoer ...... ------MCF7 i. NC/ADR-RES MDA-MB-231/ATCC HS 578T MDA-N

BT-549

as a a as up a a a up is is a v p a ......

MG MID Delta Range

FIG. 23C (Cont.) U.S. Patent Jan. 31, 2006 Sheet 40 of 51 US 6,992,079 B2

Leukemia OO O50

-5 O

-100 8 -7 -6 -5 -4 LOg 10 of Sample Concentration (Molar) CCRP-CEM-8- HL-60(TB) ---0--- K-562 --- a---. MOLT-4 ....a.... RPM-8226 ---O--- SR ---4---- FIG. 24A Non-Small Cell Lung Cancer 100 SeaSts. Ya SS ------A. SYa, -- S. SS------50 =ac =a&SAS as as-S. s Ye -- S.S.-NNN’s c N- to-NSNR 5old O th O old S2 d O 50

-100-9 -8 7 6 -5 -4 Log 10 of Sample Concentration (Molar) A549/ATCC -8- EKVX ---0---. HOP-62 ---A---. HOP-92 - - - -o---- NC-H226 ---0---. NC-H23 ------. NC-H322M-...-a-...-. NC-H460 - - - - HC-H522 - -a-- FIG. 24B U.S. Patent Jan. 31, 2006 Sheet 41 of 51 US 6,992,079 B2

Colon Cancer

-9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) HCC-2998 -a-, HCT-16 ------. HCT-15 ------HC29 --0---- KM12 ---0---, SW-620 ---4--- FIG. 24C

CNS Cancer 100 r. Nr. 50 p. ess s .. N ars --- O - su

50

-100; -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) SF-268 -8- SF-295 ---0--- SNB-19 --- a---. SNB-75 ------FIG. 24D U.S. Patent Jan. 31, 2006 Sheet 42 of 51 US 6,992,079 B2

100

5 O

O

-50

-1005 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) - LOX MV a MALME-3M M14 ---A--- SK-MEL-2----0---- SK-MEL-28 ---0--- SK-ME-5---4--- UACC-257 -...-A-...- UACC-62 ------FIG. 24E

Ovarian Cancer

5 O

-100-9 -8 -7 -6 -5 Log 10 of Sample Concentration (Molar) GROW -e- OVCAR-4---0--- OVCAR-5 ---4--- OVCAR-8 ------SK-OV-3 - - -0---. FG. 24F U.S. Patent Jan. 31, 2006 Sheet 43 of 51 US 6,992,079 B2 Renal Cancer 100

5 O

-50

-100.5 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) 786-0-a- A498 ------. ACHN --- a---. CAK-1 ------RXF393 ---O---. SN12C------TK-O-...--A-...- U0-31 - - - - - FG.24G

Prostate Cancer 100

50

-50

-9 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) PC-3-8- DU-145.-----. FIG. 24H U.S. Patent Jan. 31, 2006 Sheet 44 of 51 US 6,992,079 B2

Breast Cancer 100

5 O

5 O

-100 -8 -7 -6 -5 -4 Log 10 of Sample Concentration (Molar) MCF7 -e- NC/ADR-RES ------. HS578T---4---- MDA-MB-435-0---- MDA-N ---O--- BT-549 ------T-47D -...--A-...-. FIG. 24 U.S. Patent Jan. 31, 2006 Sheet 45 of 51 US 6,992,079 B2

100

50

O

-9 -8 -7 -6 -5 -4 Logo of Sample Concentration (Molar) FIG. 25 U.S. Patent Jan. 31, 2006 Sheet 46 of 51 US 6,992,079 B2 Mean Graphs Units: Molar Pane/Cet Line LOC10 G150 G150 Leukemia CCRF-CEM 5.8 H-60(TB) -6.2 K-562 -6.3 MOLT-4 -6.4 RPM-8226 6.4 S R 6.8U. Non-Small Cellung Cancer ...... A-549/ATCC EKVX HOP-62 HOP-92 NC-H226 NC-H23 NC-H.322M NC-H460 NC-H522 Colon Cancer s so a s s us as a s a b d e is a bus s an or e s our o as a to a sa e o up be a r u u

a see as a sa as a s so a o su e s m ms up a Po to up - s - P - 9 to Pre - LOX MV MALME-3M M14 SK-MEL-28SME; SK-ME-5 s UACC-257 UACC-62 OVarian Canoer ...... ------GROW OWCAR-4 OWCAR-5 OWCAR-8 SK-OV-3 > - U.S. Patent Jan. 31, 2006 Sheet 47 of 51 US 6,992,079 B2

Mean Graphs Units: Molar Panel/Celi Line L0910 G150 G150 Renal Cancer ...... I...... 786-0 -5.86

A498 -5.92 ACHN -6.10 CAK-1 -605 RXF393 -488 SN12C -5.16 TK-10 -4.77 U0-31 -5.74

PrOState Cancer ...... - a to O.------PC-3 -6.48

DU-145 -516 Breast Cancer ... ------...-- is ...... MCF7 -609 NC/ADR-RES -5.4

MOA-MB-435 -6.04 MDA-N -5.71 BT-549 -5.42

T-47D -5.53

a as a s a a m in a a v a a a 'n on in a a MG MID Delta Range

FIG. 26A (Cont.) U.S. Patent Jan. 31, 2006 Sheet 48 of 51 US 6,992,079 B2

Mean Graphs Units: Molar Pane/Cell Line LOgio TG1 TG Leukemia CCRF-CEM > -4.00 HL-60(TB) -486 K-562 > -4.00 MOLT-4 > -4.00 RPM-8226 > -4.00 SR > -4.00 Non-Small Cell Ling Cancer ...... ------A-549/ATCC > -400 EKWX > -4.00 HOP-62 > -4.00 HOP-92 > -4.00 NC-H226 > -4.00 NC-H23 > -4.00 NC-H322M > -4.00 NC-H460 -4.62 NC-H522 > -4.00 Colon Cancer is s as a a use a ea o os e i u o is o a a to a to by w P to r u v up to u O P. HCC-2998 > -4.00 HCT-116 > -4.00 E.HCT29 > -4.00-4.00 KM12 > -4.00 SW-620 > -4.00 CNS Cancer ...... a. s. . . a as s a on s is a o as up s up u in up we a a in a s s as no - - - r w w a the SF-268 > -4.00 SF-295 > -4.00 SNB-19 -4.25 SNB-75 -4.19 MeanOma ...... a w y a a in a but v LOX MV > -4.00 MALME-3M > -4.00 M4 > -4.00 SK-MEL-2 > -4.00 SKMEL-28 > -4.00 SK-MEL-5 > -4.00 UACC-257 > -4.00 UACG-62 > -4.00 Ovarian Cancer ...... as up up to a p a to is GROW > -4.00 OWCAR-4 -4.08 OWCAR-5 > -4.00 OVCAR-8 > -4.00 SK-OV-3 . . .I. 0------

------FIG. 26B +3 +2 +1 O - -2 -3 U.S. Patent Jan. 31, 2006 Sheet 49 of 51 US 6,992,079 B2

Mean Graphs Units: Molar Panel/Cell Line L094n TG1 TG

Rena Cancer s a on u e s p r a to a as a f is op p up a is a la In us was us as s so a as 786-0 > -4.00 A498 -5.16 ACHN > -4.00 CAK-1 > -4.00 RXF393 > -4.00 SN12C > -4.00 TK-10 > -4.00

U0-31 > -4.00 PrOState Cancer ...... PC-3 -5.14 DU-145 > -4.00 BreaSt Ca?car w r as a was us s p q MCF7 > -4.00 NC/ADR-RES > -4.00 HS 578T > -4.00 MDA-MB-435 > -4.00 MDA-N > -4.00 BT-549 > -4.00

T-47D > -4.00

a so as a up s as a w w a sub is a s h m s as a so o up us as up or u v . . . . m .

MG MID Delta s

Range

FIG. 26B (Cont.) U.S. Patent Jan. 31, 2006 Sheet 50 of 51 US 6,992,079 B2

Mean Graphs Units: Molar

Panel/Cell Line LOGOLC50 LC50 Leukemia CCRF-CEM > -4.00 HL-60(TB) > -4.00 K-562 > -4.00 MOL-4 > -4.00 RPM-8226 > -4.00 SR > -4.00 Non-Small Cellung Ca?car ...... A-549/ATCC > -4.00 EKWX > -4.00 HOP-62 > -4.00 HOP-92 > -4.00 NC-H226 > -4.00 NC-H23 > -4.00 NC-H.323M > -4.00 NC-H460 > -4.00 NC-H522 > -4.00 Colon Cancer as a as a s as a as a a s a us s- a us up a e s or up us - P - P - a a HCC-2998 > -4.00 HC-116 > -4.00 HCT-15 > -400 HCT-29 > -4.00 KM12 > -4.00 SW-620 > -4.00 CNS Cancer ...... g . a a s m a to is a a SF-268 > -400 SF-295 > -4.00 SNB-19 > -4.00 SNB-75 > -4.00 LOX MV > -4.00 MALME-3M > -4.00 M14 > -4.00 SK-MEL-2 > -4.00 SK-ME-28 > -4.00 SK-MEL-5 > -4.00 UACC-257 > -4.00 UACC-62 > -4.00 OMa?an Canoer ...... ------GROW > -4.00 OWCAR-4 > -400 OWCAR-5 > -4.00 OWCAR-8 > -4.00 --SKOV3 ----|2400-----.

L------FIG. 26C +3 +2 +1 O -1 -2 -3 U.S. Patent Jan. 31, 2006 Sheet 51 of 51 US 6,992,079 B2

Mean Graphs Units: Molar Panel/Cell Line LOg10 IC50 c50

RXF393 SN12C TK-10 U0-3 POState Cancer PC-3 DU-145 Breast Cancer MCF7 NC/ADR-RES HS 578 MD-AMB-435 MDA-N BT-549 T-47D MG MID Delta Range

FIG. 26C (Cont.) US 6,992,079 B2 1 2 SUBSTITUTED namically impossible for intracellular gelation to take place, 11-PHENYL-DBENZAZEPINE COMPOUNDS or if the delay time at venous oxygen pressures is longer than USEFUL FOR THE TREATMENT OR about 15 Seconds, cell Sickling will not occur. Alternatively, PREVENTION OF DISEASES if the delay time is between about 1 and 15 seconds, the red CHARACTERIZED BY ABNORMAL CELL cell will likely sickle in the veins. However, if the delay time is less than about 1 second, red cells will sickle within the PROLIFERATION capillaries. RELATED APPLICATIONS For red cells that sickle within the capillaries, a number of possible consequent events exist, ranging from no effect on This application is a continuation-in-part of Ser. No. transit time, to transient occlusion of the capillary, to a more 09/554,848 filed Sep. 22, 2000 now U.S. Pat. No. 6,534,497 permanent blockage that may ultimately result in ischemia which is a 371 of PCT/US98/24787 filed Nov. 20, 1998 or infarction of the Surrounding cells and in destruction of which is a continuation of Ser. No. 08/975,592 filed Nov. 20, the red cell. 1997 now abandoned. It has long been recognized that the cytoplasm of the 15 normal erythrocyte comprises approximately 70% water. 1. FIELD OF THE INVENTION Water crosses a normal erythrocyte membrane in millisec The present invention relates to aromatic organic com onds; however, the loSS of cell water causes an exponential pounds which are specific, potent and Safe inhibitors of the increase in cytoplasmic Viscosity as the mean cell hemoglo Ca"-activated potassium channel (Gardos channel) of bin concentration (MCHC) rises above about 32 g/dl. Since erythrocytes and/or of mammalian cell proliferation. More cytoplasmic Viscosity is a major determinate of erythrocyte particularly, the invention relates to Substituted 11-phenyl deformability and sickling, the dehydration of the erythro dibenzazepine compounds capable of inhibiting the GardoS cyte has Substantial rheological and pathological conse channel of Sickle erythrocytes and/or mitogen-induced quences. Thus, the physiological mechanisms that maintain mammalian cell proliferation. The compounds can be used the water content of normal erythrocytes and the pathologi to reduce Sickle erythrocyte dehydration and/or delay the 25 cal conditions that cause loSS of water from erythrocytes in occurrence of erythrocyte Sickling or deformation in Situ as the blood circulation are critically important. Not a therapeutic approach towards the treatment or prevention Surprisingly, regulation of erythrocyte dehydration has been of Sickle cell disease. The compounds can also be used to recognized as an important therapeutic approach towards the inhibit mammalian cell proliferation in Situ as a therapeutic treatment of sickle cell disease. Since cell water will follow approach towards the treatment or prevention of diseases any osmotic change in the intracellular concentration of characterized by abnormal cell proliferation. ions, the maintenance of the red cell's potassium concen tration is of particular importance (Stuart and Ellory, 1988, 2. BACKGROUND OF THE INVENTION Brit J. Haematol. 69:1-4). Many attempts and approaches to therapeutically treating Sickle cell disease has been recognized within West 35 dehydrated Sickle cells (and thus decreasing polymerization Africa for Several centuries. Sickle cell anemia and the of hemoglobin S by lowering the osmolality of plasma) have existence of sickle hemoglobin (Hb-S) was the first genetic been tried with limited Success, including the following disease to be understood at the molecular level. It is recog approaches: intravenous infusion of distilled water (Gye et nized today as the morphological and clinical result of a al., 1973, Am. J. Med. Sci. 266:267–277); administration of glycine to valine substitution at the No. 6 position of the beta 40 the antidiuretic hormone vasopressin together with a high globin chain (Ingram, 1956, Nature 178:792–794). The fluid intake and salt restriction (Rosa et al., 1980, M. Eng.J. origin of the amino acid change and of the disease State is the Med. 303:1138-1143; Charache and Walker, 1981, Blood consequence of a single nucleotide Substitution (Marotta et 58:892-896); the use of monensin to increase the cation al., 1977, J. Biol. Chem. 252:5040–5053). content of the sickle cell (Clark et al., 1982, J. Clin. Invest. The major Source of morbidity and mortality of patients 45 70:1074-1080; Fahim and Pressman, 1981, Life Sciences Suffering from Sickle cell disease is vascular occlusion 29:1959-1966); intravenous administration of cetiedil cit caused by Sickled erythrocytes, which causes repeated epi rate (Benjamin et al., 1986, Blood 67: 1442-1447; Berkowitz Sodes of pain in both acute and chronic form and also causes and Orringer, 1984, Am. J. Hematol. 17:217-223; Stuartet ongoing organ damage with the passage of time. It has long al., 1987, J. Clin. Pathol. 40: 1182-1186); and the use of been recognized and accepted that the deformation and 50 oxpentifylline (Stuart et al., 1987, J. Clin. Pathol. distortion, of Sickle cell erythrocytes upon complete deoxy 40: 1182-1186). genation is caused by polymerization and intracellular gela Another approach towards therapeutically treating dehy tion of sickle hemoglobin, hemoglobin S (Hb S). The drated Sickle cells involves the administration of imidazole, phenomenon is well reviewed and discussed by Eaton and nitroimidazole and triazole antimycotic agents Such as Clo Hofrichter, 1987, Blood 70:1245. The intracellular gelation 55 trimazole (U.S. Pat. No. 5,273,992 to Brugnara et al.). and polymerization of Hb S can occur at any time during Clotrimazole, an imidazole-containing antimycotic agent, erythrocyte's journey through the vasculature. Thus, eryth has been shown to be a specific, potent inhibitor of the rocytes in patients with Sickle cell disease containing no Gardos channel of normal and Sickle erythrocytes, and to polymerized hemoglobin S may pass through the microcir prevent Cat"-dependent dehydration of sickle cells both in culation and return to the lungs without Sickling, may sickle 60 vitro and in vivo (Brugnara et al., 1993, J. Clin. Invest. in the veins or may sickle in the capillaries. 92:520–526; De Franceschi et al., 1994, J. Clin. Invest. The probability of each of these events occurring is 93: 1670–1676). When combined with a compound which determined by the delay time for intracellular gelation stabilizes the oxyconformation of Hb S, Clotrimazole relative to the appropriate capillary transit time (Eaton et al., induces an additive reduction in the clogging rate of a 1976, Blood 47:621). In turn, the delay time is dependent 65 micropore filter and may attenuate the formation of irrevers upon the Oxygenation State of the hemoglobin, with deoxy ibly sickled cells (Stuart et al., 1994, J. Haematol. genation shortening the delay time. Thus, if it is thermody 86:820–823). Other compounds that contain a heteroaryl US 6,992,079 B2 3 4 imidazole-like moiety believed to be useful in reducing proliferation are highly desirable, and are therefore also an sickle erythrocyte dehydration via Gardos channel inhibition object of the present invention. include miconazole, econazole, butoconazole, Oxiconazole and Sulconazole. Each of these compounds is a known 3. SUMMARY OF THE INVENTION antimycotic. Other imidazole-containing compounds have These and other objects are provided by the present been found to be incapable of inhibiting the Gardos channel invention, which in one aspect provides a novel class of and preventing loSS of potassium. organic compounds which are potent, Selective and Safe AS can be seen from the above discussion, reducing Sickle inhibitors of the Ca"-activated potassium channel (Gardos erythrocyte dehydration via blockade of the Gardos channel channel) of erythrocytes, particularly sickle erythrocytes, is a powerful therapeutic approach towards the treatment and/or of mammalian cell proliferation. The compounds are and/or prevention of Sickle cell disease. Compounds capable generally Substituted 11-phenyl-dibenzazepine compounds. of inhibiting the Gardos channel as a means of reducing In one illustrative embodiment, the compounds capable of Sickle cell dehydration are highly desirable, and are there inhibiting the Gardos channel and/or mammalian cell pro fore an object of the present invention. liferation according to the invention are compounds having Cell proliferation is a normal part of mammalian 15 the structural formula: existence, necessary for life itself. However, cell prolifera tion is not always desirable, and has recently been shown to (I) be the root of many life-threatening diseases Such as cancer, certain Skin disorders, inflammatory diseases, fibrotic con ditions and arteriosclerotic conditions. Cell proliferation is critically dependent on the regulated movement of ions acroSS Various cellular compartments, and is associated with the synthesis of DNA. Binding of specific polypeptide growth factors to specific receptors in growth arrested cells triggers an array of early ionic Signals that are 25 critical in the cascade of mitogenic events eventually leading to DNA synthesis (Rozengurt, 1986, Science 234:161-164). These include: (1) a rapid increase in cystolic Ca", mostly due to rapid release of Ca" from intracellular stores; (2) capacitative Ca" influx in response to opening of ligand bound and hyperpolarization sensitive Ca" channels in the plasma membrane that contribute further to increased intra or pharmaceutically acceptable Salts of hydrates thereof, cellular Ca" concentration (Tsien and Tsien, 1990, Annu. wherein: Rev. Cell Biol. 6:715–760; Peppelenbosch et al., 1991, J. Biol. Chem. 266:19938–19944); and (3) activation of Cat 35 R is -R', (C-C-o) aryl or Substituted (C-C) aryl; dependent K channels in the plasma membrane with R is -R', -OR', -SR', halogen or trihalomethyl; increased K' conductance and membrane hyperpolarization R is -R', -OR', -SR', halogen or trihalomethyl or, (Magni et al., 1991, J. Biol. Chem. 261:9321-9327). These when taken together with R, is (C-C) aryleno; mitogen-induced early ionic changes, considered critical R is -R', -OR', -SR', halogen or trihalomethyl or, 40 when taken together with R, is (C-C-o) aryleno; events in the Signal transduction pathways, are powerful each of Rs, R6, R7, Rs. Ro, Rio, R11 R12, R1s and R14, is therapeutic targets for inhibition of cell proliferation in independently Selected from the group consisting of -R', normal and malignant cells. halogen and trihalomethyl; One therapeutic approach towards the treatment of dis Rs is -R", -C(O)R", -C(S)R", -C(O)OR", -C(S) eases characterized by unwanted or abnormal cell prolifera 45 OR", -C(O)SR", -C(S)SR", —C(O)N(R"), —C(S)N tion via alteration of the ionic fluxes associated with early (R"), —C(O)C(O)R", -C(S)C(O)R", —C(O)C(S)R", mitogenic signals involves the administration of Clotrima —C(S)C(S)R",—C(O)C(O)OR", —C(S)C(O)OR", —C(O) Zole. AS discussed above, Clotrimazole has been shown to C(S)OR", –C(O)C(O)SR", –C(S)C(S)OR", –C(S)C(O) inhibit the Ca"-activated potassium channel of erythro SR", –C(O)C(S)SR", - C(S) C(S) SR", cytes. In addition, Clotrimazole inhibits Voltage- and ligand 50 -C(O)C(O)N(R"), —C(S)C(O)N(R"), —C(O)C(S)N stimulated Cat" influx mechanisms in nucleated cells (R") or -C(S)C(S)N(R"); (Villalobos et al., 1992, FASEB J. 6:2742–2747; Montero et each R" is independently Selected from the group consist al., 1991, Biochem. J. 277:73–79) and inhibits cell prolif ing of -H, (C-C) alkyl, (C-C) alkenyl and (C-C) eration both in vitro and in vivo (Benzaquen et al., 1995, alkynyl, Nature Medicine 1:534–540). Recently, Clotrimazole and 55 each R" is independently Selected from the group con other imidazole-containing antimycotic agents capable of sisting of -H, (C-C) alkyl, (C-C) alkenyl, (C-C) inhibiting Ca"-activated potassium channels have been alkynyl, (C-C) aryl, Substituted (C-C) aryl, (C-C) shown to be useful in the treatment of arteriosclerosis (U.S. alkaryl and Substituted (C-C) alkaryl; and Pat. No. 5,358,959 to Halperin et al.), as well as other the aryl and alkaryl Substituents are each independently disorders characterized by unwanted or abnormal cell pro 60 selected from the group consisting of -CN, -OR', -SR', liferation. -NO, -NR'R', halogen, (C-C) alkyl, (C-C) alkenyl, AS can be seen from the above discussion, inhibiting (C-C) alkynyl and trihalomethyl. mammalian cell proliferation via alteration of ionic fluxes In another aspect, the present invention provides pharma asSociated with early mitogenic Signals is a powerful thera ceutical compositions comprising one or more compounds peutic approach towards the treatment and/or prevention of 65 according to the invention in admixture with a pharmaceu diseases characterized by unwanted or abnormal cell prolif tically acceptable carrier, excipient or diluent. Such a prepa eration. Compounds capable of inhibiting mammalian cell ration can be administered in the methods of the invention. US 6,992,079 B2 S 6 In Still another aspect, the invention provides a method for preferred embodiments, the alkyl groups are (C-C) alkyl, reducing Sickle erythrocyte dehydration and/or delaying the with (C-C) being particularly preferred. occurrence of erythrocyte Sickling or deformation in situ. “Substituted Alkyl:” refers to an alkyl radical wherein one The method involves contacting a Sickle erythrocyte in Situ or more hydrogen atoms are each independently replaced with an amount of at least one compound according to the with other substituents. Typical substituents include, but are invention, or a pharmaceutical composition thereof, effec not limited to, -OR, -SR, -NRR, CN, -NO, tive to reduce Sickle erythrocyte dehydration and/or delay -halogen and -trihalomethyl, where each R is independently the occurrence of erythrocyte Sickling or deformation. In a -H, alkyl, alkenyl, alkynyl, aryl or alkaryl as defined preferred embodiment, the Sickle cell dehydration is reduced herein. and erythrocyte deformation is delayed in a Sickle erythro “Alkenyl:” refers to an unsaturated branched, Straight cyte that is within the microcirculation vasculature of a chain or cyclic hydrocarbon radical having at least one Subject, thereby preventing or reducing the vaSo-occlusion carbon-carbon double bond. The radical may be in either the and consequent adverse effects that are commonly caused by cis or trans conformation about the double bond(s). Typical Sickled cells. alkenyl groups include, but are not limited to, ethenyl, In Still another aspect, the invention provides a method for 15 propenyl, isopropenyl, butenyl, isobutenyl, tertbutenyl, the treatment and/or prevention of Sickle cell disease in a pentenyl, hexenyl and the like. In preferred embodiments, Subject, Such as a human. The method involves administer the alkenyl group is (C-C) alkenyl, with (C-C) being ing a prophylactically or therapeutically effective amount of particularly preferred. at least one compound according to the invention, or a “Substituted Alkenyl:” refers to an alkenyl radical pharmaceutical composition thereof, to a patient Suffering wherein one or more hydrogen atoms are each indepen from Sickle cell disease. The patient may be Suffering from dently replaced with other substituents. Typical substituents either acute Sickle crisis or chronic Sickle cell episodes. include, but are not limited to, -OR, -SR, -NRR, -CN, In yet another aspect, the invention provides a method for -NO, -halogen and -trihalomethyl, where each R is inde inhibiting mammalian cell proliferation in situ. The method pendently -H, alkyl, alkenyl, alkynyl, aryl or alkaryl as involves contacting a mammalian cell in Situ with an amount 25 defined herein. of at least one compound according to the invention, or a “Alkynyl:” refers to an unsaturated branched, Straight pharmaceutical composition thereof, effective to inhibit cell chain or cyclic hydrocarbon radical having at least one proliferation. The compound or composition may act either carbon-carbon triple bond. Typical alkynyl groups include, cytostatically, cytotoxically or a by a combination of both but are not limited to, ethynyl, propynyl, butynyl, mechanisms to inhibit proliferation. Mammalian cells that isobutynyl, pentynyl, heXynyl and the like. In preferred can be treated in this manner include vascular Smooth embodiments, the alkynyl group is (C-C) alkynyl, with muscle cells, fibroblasts, endothelial cells, various types of (C-C) being particularly preferred. pre-cancer cells and various types of cancer cells. “Substituted Alkynyl:” refers to an alkynyl radical In Still another aspect, the invention provides a method for wherein one or more hydrogen atoms are each indepen treating and/or preventing unwanted or abnormal cell pro 35 dently replaced with other substituents. Typical substituents liferation in a Subject, Such as a human. In the method, at include, but are not limited to, -OR, -SR, -NRR, -CN, least one compound according to the invention, or a phar -NO, -halogen and -trihalomethyl, where each R is inde maceutical composition thereof, is administered to a Subject pendently -H, alkyl, alkenyl, alkynyl, aryl or alkaryl as in need of Such treatment in an amount effective to inhibit defined herein. the unwanted or abnormal mammalian cell proliferation. 40 “Aryl:” refers to an unsaturated cyclic hydrocarbon radi The compound and/or composition may be applied locally to cal having a conjugated at electron System. Typical aryl the proliferating cells, or may be administered to the Subject groups include, but are not limited to, penta-2,4-diene, Systemically. Preferably, the compound and/or composition phenyl, naphthyl, anthracyl, azulenyl, indacenyl, and the is administered to a Subject that has a disorder characterized like. In preferred embodiments, the aryl group is (C5-Co) by unwanted or abnormal cell proliferation. Such disorders 45 aryl, with (Cs-Co) being particularly preferred. include, but are not limited to, cancer, epithelial precancer “Substituted Aryl:” refers to an aryl radical wherein one ous lesions, non-cancerous angiogenic conditions or arte or more hydrogen atoms are each independently replaced riosclerosis. with other substituents. Typical substituents include, but are In a final aspect, the invention provides a method for the not limited to, -OR, -SR, -NRR, CN, -NO, treatment and/or prevention of diseases that are character 50 -halogen and -trihalomethyl where each R is independently ized by unwanted and/or abnormal mammalian cell prolif -H, alkyl, alkenyl, alkynyl, aryl or alkaryl as defined eration. The method involves administering a prophylacti herein. cally or therapeutically effective amount of at least one “Aryleno:” refers to an aryl radical that is capable of compound according to the invention, or a pharmaceutical fusing to another aryl group. Typical aryleno groups include, composition thereof, to a Subject in need of Such treatment. 55 but are not limited to, benzeno, naphthaleno, anthracaleno Diseases that are characterized by abnormal mammalian cell and the like. In preferred embodiments, the aryleno group is proliferation which can be treated or prevented by way of the (Co-Co) aryleno. methods of the invention include, but are not limited to, “Substituted Aryleno:” refers to an aryleno group wherein cancer, blood vessel proliferative disorders, fibrotic disor one or more hydrogen atoms are each independently derS and arteriosclerotic conditions. 60 replaced with other Substituents. Typical Substituents 3.1 Definitions include, but are not limited to, -OR, -SR, -NRR, -CN, As used herein, the following terms shall have the fol -NO, -halogen and -trihalomethyl, where each R is inde lowing meanings: pendently -H, alkyl, alkenyl, alkynyl, aryl or alkaryl as “Alkyl:” refers to a Saturated branched, Straight chain or defined herein. cyclic hydrocarbon radical. Typical alkyl groups include, but 65 “Alkaryl:” refers to a Straight-chain alkyl, alkenyl or are not limited to, methyl, ethyl, propyl, isopropyl, butyl, alkynyl group wherein one of the hydrogen atoms bonded to isobutyl, t-butyl, pentyl, isopentyl, hexyl, and the like. In a terminal carbon is replaced with an aryl moiety. Typical US 6,992,079 B2 7 8 alkaryl groups include, but are not limited to, benzyl, FIG. 18 shows Compound 28 dose response curves for benzylidene, benzylidyne, benzenobenzyl, naphthalenoben various cancers, melanoma and leukemia. Zyl and the like. In preferred embodiments, the alkaryl group FIG. 19 shows Compound 28 dose response curves for all is (C-C) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety cell lines. of the alkaryl group is (C-C) and the aryl moiety is (Cs-Co). In particularly preferred embodiments the alkaryl FIG. 20 shows the Compound 28 mean values for all cell group is (C-C), i.e., the alkyl, alkenyl or alkynyl moiety lines. of the alkaryl group is (C-C) and the aryl moiety is FIG. 21 shows Compound 30 dose response curves for (Cs-Co). various cancers, melanoma and leukemia. “Substituted Alkaryl:” refers to an alkaryl radical wherein FIG.22 shows Compound 30 dose response curves for all one or more hydrogen atoms on the aryl moiety of the cell lines. alkaryl group are each independently replaced with other FIG. 23 shows the Compound 30 mean values for all cell Substituents. Typical Substituents include, but are not limited lines. to, -OR, -SR, -NRR, -CN, -NO, -halogen and FIG. 24 shows Compound 31 dose response curves for -trihalomethyl, where each R is independently -H, alkyl, 15 various cancers, melanoma and leukemia. alkenyl, alkynyl, aryl or alkaryl as defined herein. FIG.25 shows Compound 31 dose response curves for all “In Situ:' refers to and includes the terms “in vivo,” “ex cell lines. Vivo, and “in vitro” as these terms are commonly recog nized and understood by perSons ordinarily skilled in the art. FIG. 26 shows the Compound 31 mean values for all cell Moreover, the phrase “in situ' is employed herein in its lines. broadest connotative and denotative contexts to identify an 5. DETAILED DESCRIPTION OF THE entity, cell or tissue as found or in place, without regard to INTENTION its Source or origin, its condition or Status or its duration or longevity at that location or position. AS discussed in the Background Section, blockade of 25 sickle dehydration via inhibition of the Gardos channel is a 4 BRIEF DESCRIPTION OF THE DRAWINGS powerful therapeutic approach towards the treatment and/or prevention of Sickle cell disease. Studies have shown that FIG. 1 provides a reaction Scheme for Synthesizing antimycotic agents such as Clotrimazole block Ca"- 11-aryl-5,6-dihydro-11H-dibenzb,eazepine compounds activated K' transport and reduce cell dehydration in Sickle according to the invention; and erythrocytes in vitro (Brugnara et al., 1993, J. Clin. Invest. FIG. 2 provides a reaction Scheme for Synthesizing 92:520–526), and also inhibit the Gardos channel of 11-aryl-11-substituted-5,6-dihydro-dibenzb,eazepine erythrocytes, increase red cell K" content, decrease the mean compounds according to the invention. cell hemoglobin concentration (MCHC) and decrease red FIG.3 shows the pre-clinical efficacy of Compound 28 on cell density in Vivo in a transgenic mouse model for Sickle Human Pancreatic Carcinoma in nude mice. 35 cell disease (SAD mouse, Trudel et al., 1991, EMBO J. FIG. 4 shows the pre-clinical efficacy of Compound 28 on 11:3157-3165; De Franceschi et al., 1994, J. Clin. Invest. Human Small Lung Carcinoma in SCID mice. 93: 1670–1676). Moreover, therapy with oral Clotrimazole induces inhibition of the Gardos channel and reduces eryth FIG. 5 shows the pre-clinical efficacy of Compound 28 on rocyte dehydration in human patients with Sickle cell disease Human Lung Large Cell Carcinoma in CD-1 nude mice. (Brugnara et al., 1996, J. Clin. Invest. 97: 1227–1234). Other FIG. 6 shows the pre-clinical efficacy of Compound 28 on 40 antimycotic agents which inhibit the GardoS channel in Vitro Human Melanoma in CD-1 nude mice. include miconazole, econazole, butoconazole, oxiconazole FIG. 7 shows the number of macroscopic pleural and Sulconazole (U.S. Pat. No. 5,273,992 to Brugnara et al.). metastases in four different test groups. All of these compounds contain an imidazole-like ring, i.e., FIG. 8 shows a polysome profile. 45 a heteroaryl ring containing two or more nitrogens. FIG. 9 shows preferential inhibition of translation and Also as discussed in the Background Section, the modul expression of G1 cyclins. lation of early ionic mitogenic Signals and inhibition of cell FIG. 10 is an absorbance profile for Compound 28. proliferation are powerful therapeutic approaches towards FIG. 11 is an absorbance profile for Compound 28. the treatment and/or prevention of disorders characterized FIG. 12 shows the resistance of dominant negative PKR 50 by abnormal cell proliferation. It has been shown that mutant cells to CLT and Compound 28, indicating that Clotrimazole, in addition to inhibiting the Gardos channel of Compound 28 inhibits cell growth at the level of translation erythrocytes, also modulates ionic mitogenic Signals and initiation. inhibits cell proliferation both in vitro and in vivo. For example, Clotrimazole inhibits the rate of cell pro FIG. 13 shows the DNA content analysis of cells treated 55 liferation of normal and cancer cell lines in a reversible and with DMSO and Compound 28. dose-dependent manner in vitro (Benzaquen et al., 1995 FIG. 14 shows the results of tumor cells that were Nature Medicine 1:534–540). Clotrimazole also depletes the synchronized for 72 hours, then treated with DMSO or 75 intracellular Cat" stores and prevents the rise in cystolic uM NC381 for various times. The cell lysates were resolved Ca" that normally follows mitogenic stimulation. on SDS-PAGE gels and immunoblotted with antibodies 60 Moreover, in mice with Severe combined immunodeficiency against total Rb, and phosphorylated Rb S780. disease (SCID) and inoculated with MM-RU human mela FIG. 15 shows an XTT assay for PC-3 Human Prostate noma cells, daily administration of Clotrimazole resulted in Tumor cells treated with Compound 28. a Significant reduction in the number of lung metastases FIG. 16 shows growth of Human Prostate Carcinoma observed (Benzaquen et al., Supra). (PC-3) in SCID mice treated with Compound 28. 65 It has now been discovered that substituted 11-phenyl FIG. 17 shows a comparison of signal for Compound 28 dibenzazepine compounds also inhibit the Gardos channel of as compared to vehicle. erythrocytes and/or mammalian cell proliferation. Thus, in US 6,992,079 B2 10 one aspect, the present invention provides a new class of cytostatically, cytotoxically, or by a combination of both organic compounds that are capable of inhibiting the Ca"- mechanisms to inhibit cell proliferation. Mammalian cells activated potassium channel (Gardos channel) of treatable in this manner include vascular Smooth muscle erythrocytes, particularly Sickle erythrocytes and/or of cells, fibroblasts, endothelial cells, various pre-cancer cells inhibiting mammalian cell proliferation, particularly and various cancer cells. In a preferred embodiment, cell mitogen-induced cell proliferation. proliferation is inhibited in a Subject Suffering from a dis The discovery that 11-phenyl dibenzazepine compounds order that is characterized by unwanted or abnormal cell inhibit the Gardos channel and/or mammalian cell prolif proliferation. Such diseases are described more fully below. eration was quite Surprising. Significantly, the compounds of Based in part on the Surmised role of mammalian cell the invention do not contain an imidazole or imidazole-like proliferation in certain diseases, the invention is also moiety. The imidazole or imidazole-like moiety is well directed to methods of treating or preventing diseases char recognized as the essential functionality underlying the acterized by abnormal cell proliferation. In the method, an antimycotic and other biological activities of Clotrimazole effective amount of at least one compound according to the and the other above-mentioned anti-mycotic agents. Thus, invention, or a pharmaceutical composition thereof, is the 11-phenyl dibenzazepine compounds of the invention 15 administered to a patient Suffering from a disorder that is provide an entirely new class of compounds that are capable characterized by abnormal cell proliferation. While not of effecting inhibition of the Gardos channel and/or mam intending to be bound by any particular theory, it is believed malian cell proliferation and that are therefore useful for the that administration of an appropriate amount of a compound treatment of Sickle cell disease and/or diseaseS related to according to the invention to a Subject inhibits cell prolif abnormal or unwanted cell proliferation. eration by altering the ionic fluxes associated with early In another aspect, the invention provides a method of mitogenic signals. Such alteration of ionic fluxes is thought reducing Sickle cell dehydration and/or delaying the occur to be due to the ability of the compounds of the invention to rence of erythrocyte Sickling in Situ as a therapeutic inhibit potassium channels of cells, particularly Ca"- approach towards the treatment of Sickle cell disease. In its activated potassium channels. The method can be used broadest Sense, the method involves only a Single step-the 25 prophylactically to prevent unwanted or abnormal cell administration of at least one pharmacologically active proliferation, or may be used therapeutically to reduce or compound of the invention, or a composition thereof, to a arrest proliferation of abnormally proliferating cells. The Sickle erythrocyte in Situ in an amount effective to reduce compound, or a pharmaceutical formulation thereof, can be dehydration and/or delay the occurrence of cell Sickling or applied locally to proliferating cells to arrest or inhibit deformation. proliferation at a desired time, or may be administered to a While not intending to be bound by any particular theory, Subject Systemically to arrest or inhibit cell proliferation. it is believed that administration of the active compounds Diseases which are characterized by abnormal cell pro described herein in appropriate amounts to Sickle erythro liferation that can be treated or prevented by means of the cytes in Situ causes nearly complete inhibition of the GardoS present invention include blood vessel proliferative channel of Sickle cells, thereby reducing the dehydration of 35 disorders, fibrotic disorders, arteriosclerotic disorders and Sickle cells and/or delaying the occurrence of cell Sickling or various cancers. deformation. In a preferred embodiment, the dehydration of Blood vessel proliferation disorders refer to angiogenic a sickle cell is reduced and/or the occurrence of Sickling is and vasculogenic disorders generally resulting in abnormal delayed in a Sickle cell that is within the microcirculation proliferation of blood vessels. The formation and spreading vasculature of the Subject, thereby reducing or eliminating 40 of blood vessels, or vasculogenesis and angiogenesis, the vaso-occlusion that is commonly caused by Sickled cells. respectively, play important roles in a variety of physiologi Based in part on the Surmised importance of the GardoS cal processes Such as embryonic development, corpus channel as a therapeutic target in the treatment of Sickle cell luteum formation, wound healing and organ regeneration. disease, the invention is also directed to methods of treating 45 They also play a pivotal role in cancer development. Other or preventing Sickle cell disease. In the method, an effective examples of blood vessel proliferative disorders include amount of one or more compounds according to the arthritis, where new capillary blood vessels invade the joint invention, or a pharmaceutical composition thereof, is and destroy cartilage and ocular diseases Such as diabetic administered to a patient Suffering from Sickle cell disease. retinopathy, where new capillaries in the retina invade the The methods may be used to treat Sickle cell disease 50 Vitreous, bleed and cause blindness and neovascular glau prophylactically to decrease intracellular Hb Sconcentration CO. and/or polymerization, and thus diminish the time and Another example of abnormal neovascularization is that duration of red cell Sickling and Vaso-occlusion in the blood associated with Solid tumors. It is now established that circulation. The methods may also be used therapeutically in unrestricted growth of tumors is dependent upon angiogen patients with acute Sickle cell crisis, and in patients Suffering 55 esis and that induction of angiogenesis by liberation of chronic Sickle cell episodes to control both the frequency angiogenic factors can be an important Step in carcinogen and duration of the crises. esis. For example, basic fibroblast growth factor (bFGF) is The compounds of the invention are also potent, Specific liberated by Several cancer cells and plays a crucial role in inhibitors of mammalian cell proliferation. Thus, in another cancer angiogenesis. The demonstration that certain animal aspect, the invention provides methods of inhibiting mam 60 tumors regreSS when angiogenesis is inhibited has provided malian cell proliferation as a therapeutic approach towards the most compelling evidence for the role of angiogenesis in the treatment or prevention of diseases characterized by tumor growth. Other cancers that are associated with unwanted or abnormal cell proliferation. In its broadest neovascularization include he mangioendotheliomas, Sense, the method involves only a Single Step-the admin hemangiomas and Kaposi's Sarcoma. istration of an effective amount of at least one pharmaco 65 Proliferation of endothelial and vascular Smooth muscle logically active compound according to the invention to a cells is the main feature of neovascularization. The invention mammalian cell in situ. The compound may act is useful in inhibiting Such proliferation, and therefore in US 6,992,079 B2 11 12 inhibiting or arresting altogether the progression of the venting various types of neoplastic diseases. Neoplastic angiogenic condition which depends in whole or in part diseases which can be treated by means of the present upon Such neovascularization. The invention is particularly invention include, but are not limited to, biliary tract cancer, useful when the condition has an additional element of brain cancer, including glioblastomas and medulloblasto endothelial or vascular Smooth muscle cell proliferation that mas, breast cancer; cervical cancer, choriocarcinoma; colon is not necessarily associated with neovascularization. For cancer, endometrial cancer, esophageal cancer, gastric can example, psoriasis may additionally involve endothelial cell cer, hematological neoplasms, including acute and chronic proliferation that is independent of the endothelial cell lymphocytic and myelogenous leukemia, multiple myeloma, proliferation associated with neovascularization. Likewise, a AIDS associated leukemias and adult T-cell leukemia lym Solid tumor which requires neovascularization for continued phoma; intraepithelial neoplasms, including Bowen's dis growth may also be a tumor of endothelial or vascular ease and Paget’s disease; liver cancer; lung cancer, Smooth muscle cells. In this case, growth of the tumor cells lymphomas, including Hodgkin's disease and lymphocytic themselves, as well as the neovascularization, is inhibited by lymphomas, neuroblastomas, oral cancer, including Squa the compounds described herein. mous cell carcinoma, Ovarian cancer, including those arising from epithelial cells, Stromal cells, germ cells and mesen The invention is also useful for the treatment of fibrotic 15 chymal cells, pancreas cancer, prostate cancer, rectal cancer, disorderS Such as fibrosis and other medical complications of Sarcomas, including leiomyosarcoma, rhabdomyosarcoma, fibrosis which result in whole or in part from the prolifera liposarcoma, fibrosarcoma and osteosarcoma; Skin cancer, tion of fibroblasts. Medical conditions involving fibrosis including melanoma, Kaposi's Sarcoma, basocellular cancer (other than atherosclerosis, discussed below) include unde and Squamous cell cancer, testicular cancer, including ger Sirable tissue adhesion resulting from Surgery or injury. minal tumors (seminoma, non-Seminoma (teratomas, Other cell proliferative disorders which can be treated by choriocarcinomas)), Stromal tumors and germ cell tumors; means of the invention include arteriosclerotic conditions. thyroid cancer, including thyroid adenocarcinoma and med Arteriosclerosis is a term used to describe a thickening and ullar carcinoma, and renal cancer including adenocarcinoma hardening of the arterial wall. An arteriosclerotic condition 25 and Wilms tumor. as used herein means classical atherosclerosis, accelerated The compounds of the invention are useful with hormone atherosclerosis, atherosclerotic lesions and any other arte dependent and also with nonhormone dependent cancers. riosclerotic conditions characterized by undesirable endot They also are useful with prostate and breast cancers. They helial and/or vascular Smooth muscle cell proliferation, further are useful with multidrug resistant Strains of cancer. including vascular complications of diabetes. In addition to the particular disorders enumerated above, Proliferation of vascular Smooth muscle cells is a main the invention is also useful in treating or preventing derma pathological feature in classical atherosclerosis. It is tological diseases including keloids, hypertrophic Scars, believed that liberation of growth factors from endothelial Seborrheic dermatosis, papilloma virus infection (e.g., pro cells stimulates the proliferation of Subintimal smooth ducing Verruca Vulgaris, Verruca plantaris, Verruca plan, muscle which, in turn, reduces the caliber and finally 35 condylomata, etc.), eczema and epithelial precancerous obstructs the artery. The invention is useful in inhibiting lesions Such as actinic keratosis, other inflammatory dis Such proliferation, and therefore in delaying the onset of, eases including proliferative glomerulonephritis, lupus inhibiting the progression of, or even halting the progression erythematosus, Scleroderma; temporal arthritis, throm of Such proliferation and the associated atherosclerotic con boangiitis obliterans, mucocutaneous lymph node Syn dition. 40 drome; and other pathologies mediated by growth factors Proliferation of vascular Smooth muscle cells produces including uterine leiomyomas. accelerated atherosclerosis, which is the main reason for The compounds and methods of the invention provide failure of heart transplants that are not rejected. This pro myriad advantages over agents and methods commonly used liferation is also believed to be mediated by growth factors, to treat Sickle cell disease and/or cell proliferative disorders. and can ultimately result in obstruction of the coronary 45 The compounds and methods of the invention also provide arteries. The invention is useful in inhibiting Such obstruc myriad advantages over the treatment of Sickle cell disease tion and reducing the risk of, or even preventing, Such and/or cell proliferative disorders with Clotrimazole or other failures. antimycotic agents. For example, many of the compounds of Vascular injury can also result in endothelial and vascular the invention are more potent than Clotrimazole in in Vitro Smooth muscle cell proliferation. The injury can be caused 50 tests, and therefore may provide consequential therapeutic by any number of traumatic events or interventions, includ advantages in clinical Settings. ing vascular Surgery and balloon angioplasty. Restenosis is Most significantly, the compounds of the invention have the main complication of Successful balloon angioplasty of reduced toxicity as compared with Clotrimazole and other the coronary arteries. It is believed to be caused by the antimycotic agents. For Clotrimazole, it is well-known that release of growth factors as a result of mechanical injury to 55 the imidazole moiety is responsible for inhibiting a wide the endothelial cells lining the coronary arteries. Thus, by range of cytochrome P-450 isozyme catalyzed reactions, inhibiting unwanted endothelial and Smooth muscle cell which constitutes their main toxicological effects (Pappas proliferation, the compounds described herein can be used to and Franklin, 1993, Toxicology 80:27-35; Matsuura et al., delay, or even avoid, the onset of restenosis. 1991, Biochemical Pharmacology 41:1949–1956). Ana Other atherosclerotic conditions which can be treated or 60 logues and metabolites of Clotrimazole do not induce cyto prevented by means of the present invention include dis chrome P-450 (Matsuura et al., 1991, Biochemical Pharma eases of the arterial walls that involve proliferation of cology 41: 1949-1956), and therefore do not share endothelial and/or vascular Smooth muscle cells, Such as Clotrimazole's toxicity. complications of diabetes, diabetic glomerulosclerosis and 5.1 The Compounds diabetic retinopathy. 65 The compounds which are capable of inhibiting the The compounds described herein are also potent antine Gardos channel and/or mammalian cell proliferation accord oplastic agents and are therefore useful in treating or pre ing to the invention are generally Substituted 11-phenyl US 6,992,079 B2 13 14 dibenzazepine compounds. In one illustrative embodiment, In another preferred embodiment of the invention, the the compounds capable of inhibiting the GardoS channel compounds are those of structural formula (I) wherein: and/or mammalian cell proliferation according to the inven R is -R' or (C-C) aryl; tion are compounds having the Structural formula: R is -R' or -OR'; R is -R' or -OR' or, when taken together with R, is (I) (Co-Co) aryleno; R15 R is -R' or -OR' or, when taken together with R, is (Co-Co) aryleno; each of Rs, Re, R7, Rs. Ro, Rio, R1, R12, R1a and R1 is independently Selected from the group consisting of -R and halogen; Rs is -R", -COO)R", -C(O)OR", -C(O)N(R"), —C(O)C(O)R", —C(O)C(O)OR", or -C(O)C(O)N(R"); each R" is independently Selected from the group consist 15 ing of -H, (C-C) alkyl, (C-C) alkenyl and (C-C) alkynyl, each R" is independently Selected from the group con sisting of -H, (C-C) alkyl, (C-C) alkenyl, (C-C) alkynyl, (C-C) aryl, Substituted (C-C-o) aryl, (C-C) alkaryl and Substituted (C-C) alkaryl; and the aryl and alkaryl Substituents are each independently wherein: selected from the group consisting of -CN, -OR', -NO, R is -R', (C-C-o) aryl or Substituted (C-C-o) aryl; -NR'R', halogen, (C-C) alkyl, (C-C) alkenyl and R is -R', -OR', -SR', halogen or trihalomethyl; (C-C) alkynyl. R is -R', -OR', -SR', halogen or trihalomethyl or, 25 In another preferred embodiment, the compounds are when taken together with R, is (C-C) aryleno; those of formula (I) wherein: R is -R', -OR', -SR', halogen or trihalomethyl or, R is -R' or (Co-Co) aryl; when taken together with R, is (C-C-o) aryleno; R is -R' or -OR'; each of Rs. R6, R7, Rs, Ro, Rio, R11, R12, R1s and R4 is R is -R' or -OR' or, when taken together with R, is independently Selected from the group consisting of -R', (C-Clio) aryleno; halogen and trihalomethyl; R is -R' or -OR' or, when taken together with R, is R is -R", —C(O)R", —C(S)R", —C(O)OR", -C(S) (C-Clio) aryleno; OR", -C(O)SR", -C(S)SR", —C(O)N(R"), —C(S)N each of Rs, R. and R, is -H, (R"), —C(O)C(O)R", -C(S)C(O)R", —C(O)C(S)R", Rs is -R', -F, -Cl, -Br or -I; -C(S)C(S)R",—C(O)C(O)OR", —C(S)C(O)OR", —C(O) 35 each of Ro, Ro and R is -H, C(S)OR", –C(O)C(O)SR", –C(S)C(S)OR", –C(S)C(O) R is -R', -F, -Cl, -Br or -I; SR", –C(O)C(S)SR", - C(S) C(S) SR", R is -H, -C(O)C(O)N(R"), —C(S)C(O)N(R"), —C(O)C(S)N R is -R', -F, -Cl, -Br or -I; (R") or -C(S)C(S)N(R"); Rs is -R", -COO)R", -C(O)OR", -C(O)N(R"), each R" is independently Selected from the group consist 40 —C(O)C(O)R", —C(O)C(O)OR" or -C(O)C(O)N(R"); ing of -H, (C-C) alkyl, (C-C) alkenyl and (C-C) each R" is independently Selected from the group consist alkynyl, ing of -H, (C-C) alkyl, (C-C) alkenyl and (C-C) each R" is independently Selected from the group con alkynyl, sisting of -H, (C-C) alkyl, (C-C) alkenyl, (C-C) each R" is independently Selected from the group con alkynyl, (C-C) aryl, (C-C) Substituted aryl, (C-C) 45 sisting of -H, (C-C) alkyl, (C-C) alkenyl, (C-C) alkaryl and Substituted (C-C) alkaryl; and alkynyl, (C-Co) aryl, Substituted (C-Co) aryl, (C-C) the aryl and alkaryl Substituents are each independently alkaryl or Substituted (C-C) alkaryl; and selected from the group consisting of -CN, -OR', -SR', the aryl and alkaryl Substituents are each independently -NO, -NR'R', halogen, (C-C) alkyl, (C-C) alkenyl, Selected from the group consisting of -OR', -NO, (C-C) alkynyl and trihalomethyl. 50 NR'R', -F, -Cl, -Br, -I, (C-C) alkyl, (C-C) In a preferred embodiment of the invention, the com alkenyl and (C-C) alkynyl. pounds are those of structure (I) wherein the chalcogens are In Still another preferred embodiment, the compounds are each oxygen. those of structural formula (I) wherein: In another preferred embodiment, the compounds are R is -R' or phenyl; those of Structure (I) wherein the halogens are each inde 55 R is -R' or -OR'; pendently -F, -Cl, -Br or -I. R is -R' or -OR' or, when taken together with R, is In another preferred embodiment, the alkyl, alkenyl and benzeno; alkynyl groups are each independently (C-C) and/or the R is -R' or -OR' or, when taken together with R, is aryl groups are phenyl and/or the aryleno groups are ben benzeno; ZCO. 60 each of Rs, Re and R7 is -H, In another preferred embodiment, Rs, Re, R7, Ro, Rio, R. Rs is -R', -Cl or -Br; and R are each independently -R'. each of Ro, Ro and R is -H, In another preferred embodiment, the Substituted aryland R is -R', -F or -Cl; alkaryl are mono-Substituted. R is -H, In another preferred embodiment, Risis-R", -C(O)R", 65 R is -R' or -Cl; -C(O)OR", —C(O)N(R"), —C(O)C(O)R", -C(O)C(O) R is -R", -C(O)OR", -C(O)OR", —C(O)NHR", OR", or -C(O)C(O)N(R"). —C(O)C(O)R" or -C(O)C(O)OR"; US 6,992,079 B2 15 16 each R" is independently Selected from the group consist -continued ing of -H, (C-C) alkyl, (C-C) alkenyl and (C-C) (6) alkynyl, each R" is independently Selected from the group con sisting of -H, (C-C) alkyl, (C-C) alkenyl, (C-C) alkynyl, (C-Co) aryl, mono-Substituted (C-Co) aryl, (C-C) alkaryl or mono-Substituted (C-C) alkaryl; and the aryl and alkaryl Substituents are each independently Selected from the group consisting of -OR', -NO, -NR'R', -Cl, (C-C) alkyl, (C-C) alkenyl and (C-C) alkynyl. In Still another preferred embodiment, the compounds of the invention are as follows: (1) (7) 15

HC (2) * V N (8) 25

(3)

35

(9) 40 (4)

45

50 (10) (5)

55

60 US 6,992,079 B2 17 18 -continued -continued (11) (16) HO

15 (12) (17)

25 (13)

3O (18)

35

40 (19)

(14)

45

50 (20)

55 (15)

60

65 US 6,992,079 B2 19 20 -continued (21) -continued (25)

15

(22) (26)

25

(23) (27) 35

40

45

(24) so NO2 (28)28

55

60 US 6,992,079 B2 21 22 -continued -continued

(33) (29) 5

1O

15

(30) NO2

(34) 25

35 (31) NO2

40

(35)

45

50

(32) 55

In yet another preferred embodiment, the compounds are those of structural formula (I), with the proviso that when R. 60 and Rs are each -R', at least one of R, R2, R, R, Rs, R, R7, Ro, Rio, R, R2, R1s or R1 is other than -R', Rs is other than -R' or halogen and at least three of R2, R. R. and Rs are other than -OR'. 65 In yet another preferred embodiment, the compounds are those of structural formula (I), with the proviso that when R. and Rs are each -H, at least one of R, R., R., R., Rs, R, US 6,992,079 B2 23 24 R7, Ro, Rio, R, R2, R1 or R is other than -H, Rs is and/or about 25% inhibition of mammalian cell proliferation other than-H or -Cl and at least three of R, R, R and (measured at about 10 um), as measured using in vitro Rs are other than-OCH. assays that are commonly known in the art (See, e.g., In a final preferred embodiment, the compounds of the Brugnara et al., 1993, J. Biol. Chem. 268(12):8760-8768; invention are not 11-phenyl-5,6-dihydro-11H-dibenzbe Benzaquen et al., 1995, Nature Medicine 1:534–540). azepine, 11-phenyl-9-halo-5,6-dihydro-11H-dibenzb,e Alternatively, or in addition, the active compounds of the azepine, 11-phenyl-9-chloro-5,6-dihydro-11H-dibenzb,e invention generally will have an ICso (concentration of azepine, 11-phenyl-5,6-dihydro-1,2,3-trialkoxy-11H-dibenz compound that yields 50% inhibition) for inhibition of the beazepine, 11-phenyl-5,6-dihydro-1,2,3-trimethoxy-11H Gardos channel of less than about 10 M and/or an ICs for inhibition of cell proliferation of less than about 10 uM, as dibenzb,eazepine, 11-phenyl-5,6-dihydro-2,3,4-trialkoxy measured using in vitro assays that are commonly known in 11H-dibenzb,eazepine and/or 11-phenyl-5,6-dihydro-2,3, the art (see, e.g., Brugnara et al., 1993, J. Biol. Chem. 4-trimethoxy-11H-dibenzb,eazepine. 268(12):8760-8768; Benzaquen et al., 1995, Nature Medi The chemical formulae referred to herein may exhibit the cine 1:534–540). phenomena of tautomerism, conformational isomerism, Ste Representative active compounds according to the inven reo isomerism or geometric isomerism. AS the formulae 15 tion include Compounds 1 through 35, as illustrated above. drawings within this Specification can represent only one of In certain embodiments of the invention, compounds the possible tautomeric, conformational isomeric, enantio which exhibit only one pharmacological activity, or a higher meric or geometric isomeric forms, it should be understood degree of one activity, may be preferred. Thus, when the that the invention encompasses any tautomeric, conforma compound is to be used in methods to treat or prevent Sickle tional isomeric, enantiomeric or geometric isomeric forms cell disease, or in methods to reduce Sickle cell dehydration which exhibit biological or pharmacological activity as and/or delay the occurrence of erythrocyte Sickling or defor described herein. mation in situ, it is preferred that the compound exhibit at The compounds of the invention may be in the form of least about 75% Gardos channel inhibition (measured at free acids, free bases or pharmaceutically effective Salts about 10 uM) and/or have an ICso for Gardos channel thereof. Such Salts can be readily prepared by treating a 25 inhibition of less than about 1 uM, with at least about 90% compound with an appropriate acid. Such acids include, by inhibition and/or an ICs of less than about 0.1 uM being way of example and not limitation, inorganic acids Such as particularly preferred. Even more preferred are compounds hydrohalic acids (hydrochloric, hydrobromic, etc.), Sulfuric which meet both the % inhibition and ICs criteria. acid, nitric acid, phosphoric acid, etc., and organic acids Exemplary preferred compounds for use in methods Such as acetic acid, propanoic acid, 2-hydroxyacetic acid, related to Gardos channel inhibition and Sickle cell disease 2-hydroxypropanoic acid, 2-oxopropanoic acid, propandioic include Compounds 1, 2, 3, 4, 6, 9, 18, 29 and 35, with acid, butandioic acid, etc. Conversely, the salt can be con Compounds 2, 3, 4, 6, 9, 29 and 35 being particularly verted into the free base form by treatment with alkali. preferred. In addition to the above-described compounds and their When the compound is to be used in methods to treat or pharmaceutically acceptable Salts, the invention may 35 prevent disorders characterized by abnormal cell prolifera employ, where applicable, Solvated as well as unsolvated tion or in methods to inhibit cell proliferation in situ, it is forms of the compounds (e.g. hydrated forms). preferable that the compound exhibit at least about 75% The compounds described herein may be prepared by any inhibition of mitogen-induced cell proliferation (measured processes known to be applicable to the preparation of at about 10 uM) and/or have an ICso of cell proliferation of chemical compounds. Suitable processes are well known in 40 less than about 3.5 uM, with at least about 90% inhibition the art. Preferred processes are illustrated by the represen and/or an ICso of less than about 1 uM being particularly tative examples. Additional methods are described in preferred. Even more preferred are compounds which meet copending application Ser. No. 08/974,472, entitled “SYN both the % inhibition and ICso criteria. THESIS OF 11-ARYL-5,6-DIHYDRO-11 Hb,e) Exemplary preferred compounds for use in methods of AZEPINES, filed concurrently herewith, which is incorpo 45 inhibiting mammalian cell proliferation or for the treatment rated herein by reference in its entirety. Necessary Starting or prevention of diseases characterized by abnormal cell materials may be obtained commercially or by Standard proliferation include Compounds 2, 3, 4, 7, 8, 9, 13, 14, 16, procedures of organic chemistry. Moreover, many of the 17, 18, 19, 20, 21, 22, 26, 27, 28, 29, 30, 31, 32, 33 and 34, compounds are commercially available. with Compounds 14, 26, 28, 29, 30 and 31 being particularly An individual compound's relevant activity and potency 50 preferred. as an agent to affect Sickle cell dehydration or deformation 5.2 Formulation and Routes of Administration and/or mammalian cell proliferation may be determined The compounds described herein, or pharmaceutically using Standard techniques. Preferentially, a compound is acceptable addition Salts or hydrates thereof, can be deliv Subject to a Series of Screens to determine its pharmacologi ered to a patient using a wide variety of routes or modes of cal activity. 55 administration. Suitable routes of administration include, In most cases, the active compounds of the invention but are not limited to, inhalation, transdermal, oral, rectal, exhibit two pharmacological activities: inhibition of the transmucosal, intestinal and parenteral administration, Gardos channel of erythrocytes and inhibition of mamma including intramuscular, Subcutaneous and intravenous lian cell proliferation. However, in Some cases, the com injections. pounds of the invention may exhibit only one of these 60 The compounds described herein, or pharmaceutically pharmacological activities. Any compound encompassed by acceptable Salts and/or hydrates thereof, may be adminis formula (I) which exhibits at least one of these pharmaco tered Singly, in combination with other compounds of the logical activities is considered to be within the Scope of the invention, and/or in cocktails combined with other thera present invention. peutic agents. Of course, the choice of therapeutic agents In general, the active compounds of the invention are 65 that can be co-administered with the compounds of the those which induce at least about 25% inhibition of the invention will depend, in part, on the condition being Gardos channel of erythrocytes (measured at about 10 uM) treated. US 6,992,079 B2 25 26 For example, when administered to patients Suffering compatible bufferS Such as Hanks's Solution, Ringer's from Sickle cell disease, the compounds of the invention can Solution, or physiological Saline buffer. For transmucosal be administered in cocktails containing agents used to treat administration, penetrants appropriate to the barrier to be the pain, infection and other Symptoms and Side effects permeated are used in the formulation. Such penetrants are commonly associated with Sickle cell disease. Such agents generally known in the art. include, e.g., analgesics, antibiotics, etc. The compounds For oral administration, the compounds can be formulated can also be administered in cocktails containing other agents readily by combining the active compound(s) with pharma that are commonly used to treat Sickle cell disease, including ceutically acceptable carriers well known in the art. Such butyrate and butyrate derivatives (Perrine et al., 1993, N. carriers enable the compounds of the invention to be for Engl. J. Med. 328(2):81–86); hydroxyurea (Charache et al., mulated as tablets, pills, dragees, capsules, liquids, gels, 1995, N. Engl. J. Med. 323(20): 1317-1322); erythropoietin Syrups, Slurries, Suspensions and the like, for oral ingestion (Goldberg et al., 1990, N. Engl. J. Med. 323(6): 366-372); by a patient to be treated. Pharmaceutical preparations for and dietary Salts Such as magnesium (De Franceschi et al., oral use can be obtained Solid excipient, optionally grinding 1996, Blood 88(648a):2580). a resulting mixture, and processing the mixture of granules, When administered to a patient undergoing cancer after adding Suitable auxiliaries, if desired, to obtain tablets treatment, the compounds may be administered in cocktails 15 or dragee cores. Suitable excipients are, in particular, fillers containing other anti-cancer agents and/or Supplementary Such as Sugars, including lactose, Sucrose, mannitol, or potentiating agents. The compounds may also be adminis Sorbitol, cellulose preparations Such as, for example, maize tered in cocktails containing agents that treat the Side-effects Starch, wheat Starch, rice Starch, potato Starch, gelatin, gum of radiation therapy, Such as anti-emetics, radiation tragacanth, methyl cellulose, hydroxypropylmethyl protectants, etc. cellulose, Sodium carboxymethylcellulose, and/or polyvi Anti-cancer drugs that can be co-administered with the nylpyrrolidone (PVP). If desired, disintegrating agents may compounds of the invention include, e.g., Aminoglutethim be added, Such as the cross-linked polyvinyl pyrrolidone, ide, Asparaginase; Bleomycin; BuSulfan, Carboplatin; Car agar, or alginic acid or a Salt thereof Such as Sodium alginate. mustine (BCNU); Chlorambucil; Cisplatin (cis-DDP); Dragee cores are provided with Suitable coatings. For this Cyclophosphamide; Cytarabine HCl; Dacarbazine; Dactino 25 purpose, concentrated Sugar Solutions may be used, which mycin; Daunorubicin HCl; Doxorubicin HCl; Estramustine may optionally contain gum arabic, talc, polyvinyl phosphate sodium; Etoposide (VP-16); Floxuridine; Fluo pyrrollidone, carbopol gel, polyethylene glycol, and/or tita rou racil (5-FU); Flutamide; Hydroxyu rea nium dioxide, lacquer Solutions, and Suitable organic Sol (hydroxycarbamide); Ifosfamide; Interferon Alfa-2a, Alfa vents or Solvent mixtures. Dyestuffs or pigments may be 2b, Lueprolide acetate (LHRH-releasing factor analogue); added to the tablets or dragee coatings for identification or Lomustine (CCNU); Mechlorethamine HCl (nitrogen to characterize different combinations of active compound mustard); Melphalan; Mercaptopurine; Mesna; Methotrex doses. ate (MTX); Mitomycin; Mitotane (op'-DDD); Mitoxantrone Pharmaceutical preparations which can be used orally HCl; Octreotide; Plicamycin; Procarbazine HCl; Streptozo include push-fit capsules made of gelatin, as well as Soft, cin; Tamoxifen citrate; Thioguanine; Thiotepa, Vinblastine 35 Sealed capsules made of gelatin and a plasticizer, Such as sulfate; Vincristine sulfate; Amsacrine (m-AMSA); Azaciti glycerol or Sorbitol. The push-fit capsules can contain the dine; Hexamethylmelamine (HMM); Interleukin 2; active ingredients in admixture with filler Such as lactose, MitoguaZone (methyl-GAG; methyl glyoxal bis binderS Such as Starches, and/or lubricants Such as talc or guanylhydrazone; MGBG); Pentostatin; Semustine (methyl magnesium Stearate and, optionally, Stabilizers. In Soft CCNU); Teniposide (VM-26); paclitaxel and other taxanes; 40 capsules, the active compounds may be dissolved or SuS and Vindesline Sulfate. pended in Suitable liquids, Such as fatty oils, liquid paraffin, Supplementary potentiating agents that can be or liquid polyethylene glycols. In addition, Stabilizers may co-administered with the compounds of the invention be added. All formulations for oral administration should be include, e.g., Tricyclic anti-depressant drugs (e.g., in dosages Suitable for Such administration. imipramine, desipramine, amitriptyline, clomipramine, 45 For buccal administration, the compositions may take the trimipramine, doxepin, nortriptyline, protriptyline, amoxap form of tablets or lozenges formulated in conventional ine and maprotiline); non-tricyclic and anti-depressant drugs C. (e.g., Sertraline, trazodone and citalopram); Ca" antagonists For administration by inhalation, the compounds for use (e.g., Verapamil, nifedipine, nitrendipine and caroverine); according to the present invention are conveniently deliv Amphotericin (e.g., Tween 80 and perhexiline maleate); 50 ered in the form of an aeroSol Spray presentation from Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs preSSurized packs or a nebulizer, with the use of a Suitable (e.g., quinidine); antihypertensive drugs (e.g., reserpine); propellant, e.g., dichloro difluoro me thane, Thiol depleters (e.g., buthionine and Sulfoximine); and cal trichlorofluoromethane, dichlorotetrafluoroethane, carbon cium leucovorin. dioxide or other Suitable gas. In the case of a pressurized The active compound(s) may be administered perse or in 55 aeroSol the dosage unit may be determined by providing a the form of a pharmaceutical composition wherein the active Valve to deliver a metered amount. Capsules and cartridges compound(s) is in admixture with one or more pharmaceu of e.g. gelatin for use in an inhaler or insufflator may be tically acceptable carriers, excipients or diluents. Pharma formulated containing a powder mix of the compound and a ceutical compositions for use in accordance with the present Suitable powder base Such as lactose or Starch. invention may be formulated in conventional manner using 60 The compounds may be formulated for parenteral admin one or more physiologically acceptable carriers comprising istration by injection, e.g., by bolus injection or continuous excipients and auxiliaries which facilitate processing of the infusion. Formulations for injection may be presented in unit active compounds into preparations which can be used dosage form, e.g., in ampoules or in multi-dose containers, pharmaceutically. Proper formulation is dependent upon the with an added preservative. The compositions may take Such route of administration chosen. 65 forms as Suspensions, Solutions or emulsions in oily or For injection, the agents of the invention may be formu aqueous vehicles, and may contain formulatory agents Such lated in aqueous Solutions, preferably in physiologically as Suspending, Stabilizing and/or dispersing agents. US 6,992,079 B2 27 28 Pharmaceutical formulations for parenteral administra channel and/or cell proliferation in cell culture assays are tion include aqueous Solutions of the active compounds in preferred. The percentage of inhibition of the Gardos chan water-Soluble form. Additionally, Suspensions of the active nel and/or cell proliferation in the patient can be monitored compounds may be prepared as appropriate oily injection to assess the appropriateness of the plasma drug concentra Suspensions. Suitable lipophilic Solvents or vehicles include tion achieved, and the dosage can be adjusted upwards or fatty oils. Such as Sesame oil, or Synthetic fatty acid esters, downwards to achieve the desired percentage of inhibition. Such as ethyl oleate or triglycerides, or liposomes. Aqueous Therapeutically effective amounts for use in humans can injection Suspensions may contain Substances which also be determined from animal models. For example, a dose increase the Viscosity of the Suspension, Such as Sodium for humans can be formulated to achieve a circulating carboxymethyl cellulose, Sorbitol, or dextran. Optionally, concentration that has been found to be effective in animals. the Suspension may also contain Suitable Stabilizers or A particularly useful animal model for Sickle cell disease is agents which increase the Solubility of the compounds to the SAD mouse model (Trudel et al., 1991, EMBO J. allow for the preparation of highly concentrated Solutions. 11:3157-3165). Useful animal models for diseases charac Alternatively, the active ingredient may be in powder terized by abnormal cell proliferation are well-known in the form for constitution with a Suitable vehicle, e.g., Sterile art. In particular, the following references provide Suitable pyrogen-free water, before use. 15 animal models for cancer xenografts (Corbett et al., 1996, J. The compounds may also be formulated in rectal com Exp. Ther. Oncol. 1:95-108; Dykes et al., 1992, Contrib. positions Such as Suppositories or retention enemas, e.g., Oncol. Basel. Karger 42:1–22), restenosis (Carter et al., containing conventional Suppository bases Such as cocoa 1994, J. Am. Coll. Cardiol. 24(5):1398–1405), atheroscle butter or other glycerides. rosis (Zhu et al., 1994, Cardiology 85(6):370–377) and In addition to the formulations described previously, the neovascularization (Epstein et al., 1987, Cornea 6(4): compounds may also be formulated as a depot preparation. 250–257). The dosage in humans can be adjusted by moni Such long acting formulations may be administered by toring Gardos channel inhibition and/or inhibition of cell implantation or transcutaneous delivery (for example Sub proliferation and adjusting the dosage upwards or cutaneously or intramuscularly), intramuscular injection or a downwards, as described above. transdermal patch. Thus, for example, the compounds may A therapeutically effective dose can also be determined be formulated with suitable polymeric or hydrophobic mate 25 from human data for compounds which are known to exhibit rials (for example as an emulsion in an acceptable oil) or ion Similar pharmacological activities, Such as Clotrimazole and eXchange resins, or as sparingly Soluble derivatives, for other antimycotic agents (see, e.g., Brugnara et al., 1995, example, as a sparingly Soluble Salt. JPET 273:266–272; Benzaquen et al., 1995, Nature Medi The pharmaceutical compositions also may comprise Suit cine 1:534-540; Brugnara et al., 1996, J. Clin. Invest. able Solid or gel phase carriers or excipients. Examples of 97(5):1227-1234). The applied dose can be adjusted based Such carriers or excipients include but are not limited to on the relative bioavailability and potency of the adminis calcium carbonate, calcium phosphate, Various Sugars, tered compound as compared with Clotrimazole. Starches, cellulose derivatives, gelatin, and polymerS Such as Adjusting the dose to achieve maximal efficacy in humans polyethylene glycols. based on the methods described above and other methods as 5.3 Effective Dosages 35 are well-known in the art is well within the capabilities of Pharmaceutical compositions suitable for use with the the ordinarily skilled artisan. present invention include compositions wherein the active Of course, in the case of local administration, the Systemic ingredient is contained in a therapeutically effective amount, circulating concentration of administered compound will not i.e., in an amount effective to achieve its intended purpose. be of particular importance. In Such instances, the compound Of course, the actual amount effective for a particular is administered So as to achieve a concentration at the local application will depend, inter alia, on the condition being 40 area effective to achieve the intended result. treated. For example, when administered in methods to For use in the prophylaxis and/or treatment of Sickle cell reduce Sickle cell dehydration and/or delay the occurrence of disease, including both chronic Sickle cell episodes and erythrocyte Sickling or distortion in Situ, Such compositions acute Sickle cell crisis, a circulating concentration of admin will contain an amount of active ingredient effective to istered compound of about 0.001 uM to 20 uM is considered achieve this result. When administered in methods to inhibit 45 to be effective, with about 0.1 uM to 5 uM being preferred. cell proliferation, Such compositions will contain an amount Patient doses for oral administration of the compounds of active ingredient effective to achieve this result. When described herein, which is the preferred mode of adminis administered to patients Suffering from Sickle cell disease or tration for prophylaxis and for treatment of chronic Sickle disorders characterized by abnormal cell proliferation, Such cell episodes, typically range from about 80 mg/day to compositions will contain an amount of active ingredient 50 16,000 mg/day, more typically from about 800 mg/day to effective to, inter alia, prevent the development of or alle 8000 mg/day, and most typically from about 800 mg/day to Viate the existing Symptoms of, or prolong the Survival of, 4000 mg/day. Stated in terms of patient body weight, typical the patient being treated. For use in the treatment of cancer, dosages range from about 1 to 200 mg/kg/day, more typi a therapeutically effective amount further includes that cally from about 10 to 100 mg/kg/day, and most typically amount of compound which arrests or regresses the growth 55 from about 10 to 50 mg/kg/day. Stated in terms of patient of a tumor. Determination of an effective amount is well body Surface areas, typical dosages range from about 40 to within the capabilities of those skilled in the art, especially 8000 mg/ml/day, more typically from about 400 to 4000 in light of the detailed disclosure herein. mg/m/day, and most typically from about 400 to 2000 For any compound described herein the therapeutically mg/m/day. effective amount can be initially determined from cell cul For use in the treatment of disorders characterized by ture arrayS. Target plasma concentrations will be those 60 abnormal cell proliferation, including cancer, arteriosclero concentrations of active compound(s) that are capable of sis and angiogenic conditions Such as restenosis, a circulat inducing at least about 25% inhibition of the Gardos channel ing concentration of administered compound of about 0.001 and/or at least about 25% inhibition of cell proliferation in luM to 20 uM is considered to be effective, with about 0.1 uM cell culture assays, depending, of course, on the particular to 5 uM being preferred. desired application. Target plasma concentrations of active 65 Patient doses for oral administration of the compounds compound(s) that are capable of inducing at least about described herein for the treatment or prevention of cell 50%, 75%, or even 90% or higher inhibition of the Gardos proliferative disorders typically range from about 80 mg/day US 6,992,079 B2 29 30 to 16,000 mg/day, more typically from about 800 mg/day to methods for Synthesizing certain exemplary compounds of 8000 mg/day, and most typically from about 800 mg/day to the invention. In all the reaction Schemes described herein, 4000 mg/day. Stated in terms of patient body weight, typical Suitable starting materials are either commercially available dosages range from about 1 to 200 mg/kg/day, more typi or readily obtainable using Standard techniques of organic cally from about 10 to 100 mg/kg/day, and most typically Synthesis. Where necessary, Suitable groups and Schemes for from about 10 to 50 mg/kg/day. Stated in terms of patient body Surface areas, typical dosages range from about 40 to protecting the various functionalities are well-known in the 8000 mg/m°/day, more typically from about 400 to 4000 art, and can be found, for example, in Kocienski, Protecting mg/m/day, and most typically from about 400 to 2000 Groups, Georg Thieme Verlag, New York, 1994 and Greene mg/m/day. & Wuts, Protective Groups in Organic Chemistry, John For other modes of administration, dosage amount and Wiley & Sons, New York, 1991. interval can be adjusted individually to provide plasma In FIGS. 1 and 2, the various Substituents are as defined levels of the administered compound effective for the par for structure (I). ticular clinical indication being treated. For example, if acute Sickle crises are the most dominant clinical 6.1 Synthesis of 11-Aryl-5,6-dihydro-11H-dibenzb, manifestation, a compound according to the invention can elazepines be administered in relatively high concentrations multiple 15 times per day. Alternatively, if the patient exhibits only This example provides a general method for Synthesizing periodic Sickle cell crises on an infrequent or periodic or substituted 11-aryl-5,6-dihydro-11H-dibenzb,eazepine irregular basis, it may be more desirable to administer a compounds according to the invention. A general reaction compound of the invention at minimal effective concentra scheme is provided in FIG. 1. In FIG. 1, R-Rs are as tions and to use a leSS frequent regimen of administration. previously defined for structural formula (I). This will provide a therapeutic regimen that is commensu Referring to FIG. 1, a mixture of an appropriately Sub rate with the Severity of the Sickle cell disease State. Stituted 2-aminobenzophenone 100 (1 equivalent), an appro For use in the treatment of tumorigenic cancers, the priately substituted benzyl chloride 102 (1 equivalent), compounds can be administered before, during or after potassium carbonate (2 equivalents) and Sodium iodide (1 Surgical removal of the tumor. For example, the compounds equivalent) in acetonitrile is refluxed for 12 hours. The can be administered to the tumor via injection into the tumor 25 reaction mixture is cooled to room temperature and water mass prior to Surgery in a Single or Several doses. The tumor, or as much as possible of the tumor, may then be removed added. The mixture is extracted with ethyl acetate. The Surgically. Further dosages of the drug at the tumor site can combined ethyl acetate extracts are washed with water then be applied post removal. Alternatively, Surgical removal of dried over Sodium sulfate. Evaporation of the solvent fol as much as possible of the tumor can precede administration lowed by column chromatography gives the Substituted of the compounds at the tumor site. N-alkyl-2-aminobenzophenone derivative 104 in about Combined with the teachings provided herein, by choos 55–80% yield. ing among the various active compounds and weighing The substituted N-alkyl-2-aminobenzophenone derivative factorS Such as potency, relative bioavailability, patient body 104 (1 equivalent) is dissolved in a 3:1 mixture of tetrahy weight, Severity of adverse side-effects and preferred mode drofuran:methanol. Sodium borohydride (10 equivalents) is of administration, an effective prophylactic or therapeutic 35 Slowly added and the reaction mixture is stirred at room treatment regimen can be planned which does not cause temperature for 12 hours. The reaction is quenched by Substantial toxicity and yet is entirely effective to treat the adding 2 Naqueous hydrochloric acid Solution. The reaction clinical Symptoms demonstrated by the particular patient. Of mixture is neutralized by adding 4 N aqueous Sodium course, many factors are important in determining a thera hydroxide solution and extracted with ethyl acetate. The peutic regimen Suitable for a particular indication or patient. 40 combined ethyl acetate extracts are dried over Sodium Severe indications Such as cancer may warrant administra sulfate. Evaporation of the solvent followed by column tion of higher dosages as compared with less Severe indi chromatography gives the Substituted N-alkyl-2-amino cations Such as Sickle cell disease. benzyl alcohol derivative 106 in about 40-60% yield. 5.4 Toxicity A mixture of the substituted N-alkyl-2-amino The ratio between toxicity and therapeutic effect for a 45 benzylalcohol derivative 106 (1 equivalent), phosphorous particular compound is its therapeutic indeX and can be pentoxide (5 equivalents) and methaneSulfonic acid (5 expressed as the ratio between LDso (the amount of com equivalents) in dichloromethane is stirred at room tempera pound lethal in 50% of the population) and EDs (the ture for 12 hours. The mixture is neutralized by adding amount of compound effective in 50% of the population). aqueous Sodium carbonate and then extracted with dichlo Compounds which exhibit high therapeutic indices are pre romethane. The organic Solution is dried over Sodium Sul ferred. Therapeutic index data obtained from cell culture 50 fate. Evaporation of the solvent followed by column chro assays and/or animal Studies can be used in formulating a matography gives the Substituted 11-aryl-5,6-dihydro-11H range of dosages for use in humans. The dosage of Such dibenzb,eazepine derivative 108 in about 45-70% yield. compounds preferably lies within a range of plasma con The substituted 11-aryl-5,6-dihydro-11H-dibenzb,e centrations that include the EDso, with little or no toxicity. azepine derivative 108 (1 equivalent) is combined with The dosage may vary within this range depending upon the 55 potassium carbonate (3.5 equivalents) and alkyl or acyl dosage form employed and the route of administration halide (3 equivalents) in acetonitrile and stirred at room utilized. The exact formulation, route of administration and temperature for two days. Water is added and the mixture is dosage can be chosen by the individual physician in View of Stirred for 15 min. at room temperature and extracted with the patient's condition. (See e.g. Finglet al., 1975, In. The ethyl acetate. Evaporation of the Solvent gives the crude Pharmacological Basis of Therapeutics, Ch. 1 p 1). 60 product as an oil. Tituration of the product from ethanol The invention having been described, the following followed by Washing with hexane gives the pure examples are intended to illustrate, not limit, the invention. N-Substituted 11-aryl-5,6-dihydro-11H-dibenzb,eazepine 6. EXAMPLE product 110 as a white solid in 30-80% yield. Alternatively, the substituted 11-aryl-5,6-dihydro-11H Compound Syntheses 65 dibenzb,eazepines can be synthesized from appropriate This Example demonstrates general methods for Synthe Starting materials according to the methods described in sizing the compounds of the invention, as well as preferred Sasakura and Sugasawa, 1981, Heterocycles 15:421-425. US 6,992,079 B2 31 32 6.2 Synthesis of 11-Aryl-11-substituted-5,6- extracted with ethyl acetate (2x35 mL). The organic layer dihydro-dibenzb,eazepines was dried over magnesium Sulfate. Evaporation gave the This example provides a general method for Synthesizing crude product as an oily material. Trituration of the obtained 11-aryl-11-substituted-5,6-dihydro-dibenzb,eazepine oil with ethanol then washing it with hexane gave 0.285 g compounds according to the invention. A general reaction (80% yield) of a white solid having a melting point of scheme is provided in FIG. 2. In FIG. 2, R-Rs are as 155-165 C. previously defined for structural formula (I). The product gave the following analytical data: NMR Referring to FIG. 2, the substituted N-alkyl-2- (CDC1) & 4.46 ppm (1H, d, J=7 Hz, CHN); & 5.28 ppm aminobenzophenone derivative 104 is prepared as described (1H, s, CH); 85.69 ppm (1H, d, J=7 HZ, CHN); 8 6.52 ppm in Section 6.1, Supra. To a solution (0.25 M) of an appro (2H, m, aryl); 8 6.98 ppm (2H, m, aryl); 8 7.14-7.42 ppm priate grignard reagent in diethyl ether at -40 C. is added (13H, m, aryl); 8 7.58 (1H, m, aryl). a solution (0.1 M) of the substituted N-alkyl-2- aminobenzophenone derivative 104 in diethyl ether. The mixture is stirred at -40° C. for 30 min., warmed to room 6.5 Synthesis of N-Phenoxycarbonyl-11-(2- temperature and quenched with water. Extraction with ethyl 15 chlorophenyl)-5,6-dihydro-11H-dibenzb,elazepine acetate and evaporation of the Solvent gives the crude (Compound 26) alcohol product 112 as an oil. The alcohol product 112 is purified by column chromatography to give the pure alcohol A preferred method of synthesis of N-phenoxycarbonyl product 112 as a white solid in 85-90% yield. 11-(2-chlorophenyl)-5,6-dihydro-11H-dibenzb,elazepine Compound 112 (1 equivalent) is dissolved in dichlo (Compound 26) is as follows: A mixture of 0.2 g (0.00065 romethane to 0.5-1.0 M. Phosphorous pentoxide (4 mole) of 11-(2-chlorophenyl)-5,6-dihydro-11H-dibenzbe equivalents) and methane Sulfonic acid (4 equivalents) are azepine, 0.18 g (0.0013 mole) of potassium carbonate and added and the mixture is stirred at room temperature for 2 0.204 g (0.0013 mole) of phenyl chloroformate in 10 mL of hours. The reaction is quenched with Saturated aqueous acetonitrile, was stirred at room temperature for 2 days. The Sodium bicarbonate and extracted with ethyl acetate. Evapo 25 mixture was stirred with 15 mL of water for 10 minutes and ration of the Solvent followed by column chromatography then extracted with ethyl acetate (2x35 mL). The organic gives the 11-aryl-11-substituted-5,6-dihydro-dibenzb,e layer was dried over magnesium Sulfate, filtered and the azepine 114 in about 90% yield. solvent was evaporated. Trituration of the obtained residue The 11-aryl-11-substituted-5,6-dihydro-dibenzbe with ethanol then washing it with hexane gave 0.082 g (30% aZepine 114 can be converted to the corresponding yield) of a white solid having a melting point of 95-99 C. N-substituted-11-aryl-11-substituted-5,6-dihydro-dibenzb, The product gave the following analytical data: NMR eazepine 116 as described in Section 6.1, Supra. (CDC1): 8 4.50 ppm (1H, d, J=7 Hz, CHN); 8 5.58 ppm 6.3 Synthesis of N-Methoxycarbonyl-11-(2'- (1H, s, CH); 85.80 ppm (1H, d, J=7 HZ, CHN); 8 6.52 ppm chlorophenyl)-5,6-dihydro-11H-dibenzb,elazepine (2H, m, aryl); 8 7.06–7.38 ppm (14H, m, aryl); 8 7.79 ppm (Compound 9) 35 (1H, m, aryl). A preferred method of synthesis of N-methoxycarbonyl 11-(2-chlorophenyl)-5,6-dihydro-11H-dibenzb,elazepine 6.6 Synthesis of N-(4-Nitrobenzoyl)-11-(2- (Compound 9) is as follows: A mixture of 0.3 g (0.00098 chlorophenyl)-5,6-dihydro-11H-dibenzb,elazepine mole) of 11-(2-chlorophenyl)-5,6-dihydro-11H-dibenzbe 40 (Compound 28) azepine, 1.08 g (0.0078 mole) of potassium carbonate and 1.54 g (0.016 mole) of methyl chloroformate in 10 mL of preferred method of synthesis of N-(4-nitrobenzoyl)-11 acetonitrile, was refluxed for 12 hours. The mixture was then (2'-chlorophenyl)-5,6-dihydro-1H-dibenzb,eazepine allowed to cool to room temperature and stirred with 15 mL (Compound 28) is as follows: A mixture of 0.2 g (0.00065 of water for 10 minutes. The reaction mixture was extracted mole) of 11-(2-chlorophenyl)-5,6-dihydro-11H-dibenzbe with ethyl acetate (2x15 mL). The organic layer was dried 45 azepine, 0.179 g (0.0013 mole) of potassium carbonate and over magnesium Sulfate. Evaporation gave the crude product 0.133 g (0.00072 mole) of 4-nitrobenzoyl chloride in 10 mL as a brown solid. Trituration of the crude product with of acetonitrile, was Stirred at room temperature for 12 hours. ethanol and washing the obtained Solid with hexane gave The mixture was stirred with 15 mL of water for 10 minutes. 0.172 g (48% yield) of a white solid having a melting point The reaction mixture was extracted with ethyl acetate (2x15 of 159-161° C. 50 mL). The organic layer was dried over magnesium Sulfate. The product gave the following analytical data: NMR Evaporation gave the crude product as a Sticky Solid. Tritu (CDC1): 8 3.10 ppm (3H, s, OCH); & 4.45 ppm (1H, d, ration of the crude product with ethanol and washing the J=10 HZ, CHN); 8 5.48 ppm (1H, s, CH); 8 5.82 ppm (1H, obtained solid with hexane gave 0.148 g (50% yield) of a d, J-10 HZ, CHN); 8 6.94 ppm (1H, m, aryl); 8 7.10 ppm white solid having a melting point of 178-181 C. (4H, m, aryl); 8 7.28 ppm (6H, m, aryl); 8 7.69 ppm (1H, m, 55 The product gave the following analytical data: NMR aryl). (CDC1): 8 4.42 ppm (1H, d, J=7 Hz, CHN); 8 5.68 ppm (1H, s, CH); 8 6.36 ppm (1H, d, J=7 HZ, CHN); 8 6.52 ppm 6.4 Synthesis of N-Phenoxycarbonyl-11-phenyl-5,6- (3H, m, aryl); 8 7.06 ppm (3H, m, aryl); 8 7.12 ppm (1H, m, dihydro-11H-dibenzb.elazepine (Compound 14) aryl); 8 7.26 ppm (6H, m, aryl); 8 7.79 ppm (3H, m, aryl). A preferred method of synthesis of N-phenoxycarbonyl 60 11-phenyl-5,6-dihydro-11H-dibenzb,elazepine (Compound 6.7 Other Compounds 14) is as follows: A mixture of 0.25 g (0.00092 mole) of 11-phenyl-5,6-dihydro-11H-dibenzb,eazepine, 0.318 g Other compounds of the invention can be synthesized by (0.0023 mole) of potassium carbonate and 0.318 g (0.002 routine modification of the above-described syntheses, or by mole) of phenyl chloroformate in 10 mL of acetonitrile, was 65 other methods that are well known in the art. Appropriate Stirred at room temperature for 2 days. The mixture was Starting materials are commercially available or can be stirred with 15 mL of water for 15 minutes and then Synthesized using routine methods. US 6,992,079 B2 33 34 7. EXAMPLE variety of cancer cell lines. The assays are generally appli In Vitro Activity cable for demonstrating the antiproliferative activity of other This Example demonstrates the ability of several exem compounds of formula (I). plary compounds of formula (I) to inhibit the Gardos chan nel of erythrocytes (Gardos channel assay) and/or mitogen induced cell proliferation (mitogenic assay) in Vitro. The assays are generally applicable for demonstrating the in vitro activity of other compounds of formula (I). 8.1 Growth of Cells 7.1 Experimental Protocol The percent inhibition of the Gardos channel (10 uM 1O compound) and the ICso were determined as described in The antiproliferative assays described herein were per Brugnara et al., 1993, J. Biol. Chem. 268(12):8760-8768. The percent inhibition of mitogen-induced cell proliferation formed using Standard aseptic procedures and universal (10 uM compound) and the ICso were determined as precautions for the use of tissues. Cells were propagated described in Benzaquen et al. (1995, Nature Medicine using RPMI 1640 media (Gibco) containing 2% or 5% fetal 1:534–540) with NIH 3T3 mouse fibroblast cells (ATCC No. 15 calf serum (FCS) (Biowhittaker) at 37° C., 5% CO and 95% CRL 1658). Other cell lines, e.g., cancer cells, endothelial humidity. The cells were passaged using Trypsin (Gibco). cells and fibroblasts, as well as many others, may be used in Prior to addition of test compound, the cells were harvested, the cell proliferation assay. Selection of a particular cell line the cell number counted and seeded at 10,000 cells/well in will depend in part on the desired application, and is well 100 ul 15% fetal calf serum (FCS) containing RPMI within the capabilities of an ordinarily Skilled artisan. medium in 96-well plates and incubated overnight at 37 C., 7.2 Results 5% CO and 95% humidity. The results of the experiment are provided in TABLE 1, below. Clotrimazole is reported for purposes of comparison. 25 On the day of the treatment, stock solutions of the test TABLE 1. compounds (10 mM compound/DMSO) were added in 100 INVITRO DATA FOR EXEMPLARY COMPOUNDS All FCS containing medium to a final concentration of 10-0.125 uM and the cells were incubated for 2, 3 or 5 days Mitogenic Assay Gardos Channel ASSay at 37° C., 5% CO and 95% humidity. ICso Inhibition ICso Inhibition Compound (uM) (%) (uM) (%) Clotrimazole O.626 93.0 O.O46 99.3 Following incubation, the cellular protein was determined (1) 56.O O.775 75.2 with the Sulforhodamine B (SRB) assay (Skehan et al., (2) 5.20 99.0 1.30 99.0 (3) 2.40 99.0 O886 97.4 35 1990, J. Natl. Cancer Inst. 82:1107–1112). Growth (4) 1.5 89.0 O384 98.1 inhibition, reported as the concentration of test compound (5) 91.O >1O.O 14.4 which inhibited 50% of cell proliferation (ICs) was deter (6) 87.O O.236 97.5 mined by curve fitting. (7) 1.60 99.0 >1O.O 35.8 (8) 2.20 84.O O (9) 2.10 99.0 O.O850-0093 97.3 40 (10) 53.0 1.533-1940 63.0 (11) 32.O >1O.O 9.5 Values for VP-16, a Standard anti-cancer agent, are pro (12) 13.0 >1O.O 54.8 Vided for comparison. (13) 1.7 97.0 O (14) O.04 98.0 >1O.O 14.8 (15) 40.O >1O.O 9.50 (16) 1.7 99.0 >1O.O O.45 (17) 1.6 99.0 >1O.O 20.6 Except for MMRU cells, all cancer cell lines tested were (18) 2.6 99.0 0.502-0.692 81.5 obtained from the American Type Culture Collection (19) 1.6 99.0 >1O.O 52.O (ATCC, Rockville, Md.). The ATCC assession numbers (20) 1.7 95.0 >1O.O 13.6 were as follows: HeLa (CCL-2); CaSki (CRL-1550); (21) 2.7 93.0 >1O.O 2.1 MDAMB-231 (HTB-26); MCF-7 (HTB-22); A549 (CCL (22) 3.6 99.0 >1O.O 14.9 50 (23) 55.0 >1O.O 18.2 185); HTB-174 (HTB-174); HEPG2 (HB-8065), DU-145 (24) 89.0 >1O.O 32-55 (HTB81); SK-MEL-28 (HTB-72); HT-29 (HTB-38); HCT (25) 75.0 >1O.O 8.5 15 (CCL-225); ACHN(CRL-1611); U-118MG (HTB-15); (26) O.04-0.90 99.0 >1O.O O.8 SKOV-3 (HTB-77). (27) 2.20 99.0 >1O.O 3.0 (28) 0.04-0.50 99.0 O 55 (29) O.800 99.0 0.414-0.433 95.1 (30) O.6OO 99.0 >10 14.6 (31) O.400 99.0 >10 12.3 MMRU cells (Stender et al., 1993, J. Dermatology (32) 1.1OO 99.0 O 20:611-617) were a gift of one of the authors. (33) 2.400 99.0 >10 67.5 (34) 4.00 99.0 >10 12.0 60 (35) O O.O71-O.O99 98.3 8.2 Results 8. EXAMPLE Activity In Cancer Cell Lines 65 This Example demonstrates the antiproliferative effect of The results of the cell culture assays are presented in Several exemplary compounds of formula (I) against a TABLES 2 and 3, below. US 6,992,079 B2 35 36

TABLE 2 RESULTS OF SRB ASSAY 5% FCS/5 DAY INCUBATION Cancer Cell Test Compound ICs (IM Type Line VP-16 13 16 17 20 21 22 23 27 28 3O 31 33 34 35 Cervical HeLa <1.25 O6 O.2 O.3 O.S. O.2 CaSki 8 O.9 O.9 O.S. O.7 O.S Breast MDA-MB-23 <1.25 >1 >1 (0.5 0.9 0.4 MCF7 <1.25 2.7 4.2 4.51 3.5 1.8 3.1 0.5 0.6 1.3 0.7 2.1 2 2.4 Lung A549 <1.25 3.1 >5 >5 >1 4.2 1.9 3.4 1.1 (0.8 1.5 0.6 3.1 >5 >5 HTB174 <1.25 1.4 2.6 3.6 0.7 1.9 1.4 1.8 0.3 0.1 0.5 0.2 1.4 0.9 4 Hepatocel HEPG2 <1.25 1.8 3.4 2.6 0.91.9 1.4 2.3 0.4 (0.3 0.9 0.4 1.4 1.5 4.5 Prostate DU-145 <1.25 >1 >1 O.9-1. O.9 Melanoma SK-MEL-28 <1.25 O.9 O.7 O.4 O.6 0.3 MMRU 1.25 15 2.7 2.5 OS 1.5 O.9 2.2 O.2 O.2 0.5 O.2 13 1.2 3.1 Colon HT29 <1.25 1.5 2.6 1.7 0.7 1.8 0.7 2.1 0.4 (0.3 0.6 0.2 1.2 1.6 4.8 HCT-15 3 O.9 0.4 0.4 0.5 0.3 Renal ACHN <1.25 >1 >1 O.7>1 O.8 CNS U118MG 2.2 O.9 0.4 0.5 0.8 0.4 Ovary SK-OV-3 >1 >1 >1 >1 0.6 Normal HUVEC <1.25 >1 >1 O.7>1 O.8 human GM .4 >1 >1 (0.9-1 >1 3T3 >1 >1 >1 >1 >1 OSC L929 <1.25 O.9 >1 O.6-1. O.9

TABLE 3

RESULTS OF SRB ASSAY Conditions Test Compound ICs (IM) Value in Cell Lines Compound % FCS/days A549 HT29 MMRU MCF7 HEPG2 U118MG VP-16 2%/3 days 2.3 2O <2.5 <2.5 4 2%/3 days 11.4 9.7 4.6 1.7 5 5%/2 days 1.4 1. >10 6 5%/2 days >10 >10 1O 7 5%/2 days >10 >10 >10 14 5%/2 days >10 1O >10 26 5%/3 days 7 7.2 4.9 5.9 5.4 5.7 27 5%/3 days <1.25 <1.25 <1.25 <1.25 <1.25 1.9 28 5%/3 days 1.8 <1.25 <1.25 2.1 <1.25 1.3 29 5%/2 days >10 >10 >10 3O 5%/3 days 1.7 <1.25 <1.25 1.6 <1.25 <1.25 31 5%/3 days <1.25 <1.25 <1.25 <1.25 <1.25 <1.25 32 5%/3 days 6.9 6.3 4.9 5.4 6.2 6.8 33 5%/3 days 6.9 6.3 4.9 5.4 6.2 6.8 34 5%/2 days >10 6.8 >10 35 5%/2 days >10 1O >10

AS can be seen in Tables 2 and 3, compounds which exhibited Significant activity in the mitogenic assay described in Section 7, Supra, exhibit significant antiprolif 50 erative activity against a variety of cancer cell lines and Active Compound 60 mg cancer types (ICs of less than about 10 uM). Starch 45 mg Many of the compounds exhibit comparable or even Microcrystalline 45 mg Cellulose greater antiproliferative activity against a variety of cancer Sodium carboxymethyl 4.5 mg cell types than VP-16, a known anti-cancer agent. 55 starch 9. EXAMPLE Talc 1 mg Polyvinylpyrrolidone 4 mg Formulations (10% in water) Magnesium Stearate 0.5 mg The following examples provide exemplary, not limiting, 150 mg formulations for administering the compounds of the inven 60 tion to mammalian, especially human, patients. Any of the compounds described herein, or pharmaceutical Salts or The active ingredient, Starch and cellulose are passed hydrates thereof, may be formulated as provided in the through a No. 45 mesh U.S. sieve and mixed thoroughly. following examples. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 9.1 Tablet Formulation 65 mesh U.S. sieve. The granules are dried at 50-60 C. and Tablets each containing 60 mg of active ingredient are passed through a No. 18 mesh U.S. sieve. The Sodium made up as follows: carboxymethyl Starch, magnesium Stearate and talc, previ US 6,992,079 B2 37 38 ously passed through a No. 60 mesh U.S. sieve, are then lulose and Syrup to form a Smooth paste. The benzoic acid added to the granules, which, after mixing are compressed Solution, flavor and Some color are diluted with Some of the by a tablet machine to yield tablets each weighing 150 mg. water and added, with Stirring Sufficient water is then added Tablets can be prepared from the ingredients listed by wet to produce the required Volume. granulation followed by compression. The foregoing written Specification is considered to be Sufficient to enable one skilled in the art to practice the 9.2 Gelatin Capsules invention various modifications of the above-described modes for carrying out the invention which are obvious to Hard gelatin capsules are prepared using the following those skilled in the pharmaceutical arts or related fields are ingredients: intended to be within the scope of the following claims. All cited references are hereby incorporated in their entireties by reference herein. Clotrimazole (CLT), a drug marketed to fight fungal Active Compound 250 mg/capsule infections, inhibits cell proliferation by blocking the cell Starch dried 200 mg/capsule cycle specifically in G1 through Ca" store-mediated inhi Magnesium Stearate 10 mg/capsule bition of translation initiation. Although CLT may represent 15 a promising anti-mitogenic therapeutic entity, it is known to be hepatotoxic via inhibition of cytochrome P450. The The above ingredients are mixed and filled into hard instant application relates to more effective and Safer com gelatin capsules in 460 mg quantities. pounds inhibit the growth of tumour cells via Similar mecha nism to that of CLT. The exemplary compound 28 has shown 9.3 Aerosol Solution anti-proliferative activity in human cancer cell lines and An aeroSol Solution is prepared containing the following XTT growth inhibition assays, for example, inhibits PC-3 components: prostate cancer cell growth. In vitro Studies indicated that compound 28, like CLT, releases Ca" from intracellular pools and inhibits capacitative Cat" influx into the cell. The 25 activation of PKR or non-phosphorylation of eIF2C. was also Active Compound 0.25% (w?w) demonstrated using cells expressing a dominant negative Ethanol 29.75% (w/w) PKR or non-phosphorylation of eIF2C. In addition, com Propellant 22 77.00% (w/w) pound 28 was shown to inhibit cyclin D1 and E expression (Chlorodifluoromethane) in vitro, which resulted in the induction of Go-Gi cell cycle arrest. In vivo efficacy studies exhibited significant inhibi The active compound is mixed with ethanol and the tory effects on the growth of PC-3 human prostate carcinoma mixture added to a portion of the propellant 22, cooled to in mouse Xenograph With no sign of apparent toxicity. -30 C. and transferred to a filling device. The required Preliminary Toxicology: amount is then fed to a StainleSS Steel container and diluted The toxicological effects of CLT, and compounds 28, 30 and 31 were tested in CD-1 female mice. A dose of 80 with the remainder of the propellant. The valve units are then 35 fitted to the container. mg/kg/day was given to each mouse via Subcutaneous injection for a period of 14 dayS. Preliminary toxicological 9.4 Suppositories data was determined by measuring body weight, grOSS pathology of organs blood chemistry and histology. The Suppositories each containing 225 mg of active ingredient vehicle used was DMF 12%+2% Tween 20 88%. are made as follows: 40 In Vitro Screening Exemplary compounds 28, 30 and 31 have been screened, using Sulforhodamine B (SRB) assay to Screen the antipro Active Compound 225 mg liferative effects of the compounds against a panel of human Saturated fatty acid 2,000 mg cancer cells. The panel comprises 60 human cancer cell glycerides 45 lines. The data obtained in his Study was evaluated using the COMPARE algorithm. The results from these studies have shown that the above exemplary compounds exhibited activ The active ingredient is passed through a No. 60 mesh ity against the human cell panel. U.S. Sieve and Suspended in the Saturated fatty acid glycer In Vivo Mouse Models of Human Tumors Cell Line ides previously melted using the minimum heat necessary. 50 NCI-A2058 human melanoma cell line was grown as The mixture is then poured into a Suppository mold of monolayer culture in Minimum essential medium (D-MEM) nominal 2 g capacity and allowed to cool. supplemented with 10% fetal bovine serum (FBS), 0.1 mM non-essential amino acid, 1.0 mM sodium pyruvate, 100U/ 9.5 Suspensions ml penicillin, 100 lug/ml Streptomycin and 0.25 ug/ml Suspensions each containing 50 mg of medicament per 5 55 amphotericin B and L-alanyl-1-glutamine (2 mM) at 37 C. mL dose are made as follows: in an atmosphere of 5% CO in air. The tumor cells were routinely subcultured twice weekly by trypsin-EDTA treat Active Compound 50 mg ment. The cells were harvested from Subconfluent logarith Sodium 50 mg mically growing culture by treatment with typsin-EDTA and carboxymethylcellulose counted for tumor inoculation. Syrup 1.25 mL. 60 Tumor Inoculation Benzoic acid solution O.10 mL. An acclimation period of at least 7 days were allowed Flavor G.V. between animal receipt and commence of tumor inoculation. Color G.V. When the female CD-1 nude mice were more than 6-7 Purified water to 5 mL. weeks of age (20–25 g), each mouse was Subcutaneously 65 injected with 5.0x106 NCI-A2058 human melanoma cells in The active ingredient is passed through a No. 45 mesh 0.1 ml of PBS solution at the right flank of the mice to U.S. sieve and mixed with the Sodium carboxymethyl cel induce tumor growth. US 6,992,079 B2 39 40 Results Where; All compounds were well tolerated and incompletely Mean OD=The average of optical density measurements absorbed with a varying degree of deposition exhibiting the SRB-derived color just before exposure of cells to the test following rank order, compound 28=31<30. No toxicities to compound. the hemopoesis and renal function were found but there was mild toxicity in regard to liver function. Histopathological Mean OD=The average of optical density measurement data showed no Systemic toxicity but there were the normal of SRB-derived color after 48 hours exposure of cells to Signs of skin rash caused by the injection of mildly irritating the test compound. material. Mean OD=The average of optical density measurements The highest accumulation of each compound occurring at of SRB-derived color after 48 hours with no exposure of the site of injection were in the rank order of 30>31>28. cells to the test compound. There were no significant gains or losses to overall body weight in mice injected with CLT and CLT derivatives The Data Sheet: compared to age matched control animals. No differences This page of the data package presents the experimental were found between animals injected with CLT derivatives data collected against each cell line. The first two columns and control animals upon groSS pathological examination of 15 describe the Subpanel (e.g. leukemia) and cell line (e.g. all mice and no Systemic toxicity was observed between CCRF-CEM) involved. The next two columns list the Mean control and experimental animals. OD, and Mean OD, the next five columns list the Mean WBC, RBC, BUN and creatine levels were measured, and OD for each of five different concentrations. Each con again, no differences were found between animals injected centration is expressed as the logo (molar or ug/ml). The with compounds 28, 30 and 31 (Table 4). next five columns list the calculated PGs for each concen There was a slight elevation of SGPT and SGOT in tration. The response parameters GI50, TGI, and LC50 are animals injected with compounds CLT derivatives but these interpolated values representing the concentrations at which increases were not significant when compared to control the PG is +50.0, and -50, respectively. Sometimes these animals (Table 5). response parameters cannot be obtained by interpolation. If, Preclinical Efficacy 25 for instance, all of the PGs in a given row exceed +50, then The in vivo inhibition of tumor growth by the compounds none of the three parameters can be obtained by interpola in the dibenzazepine Series compounds 28, 30 and 31, was tion. In Such a case, the value given for each response determined in Xenograft models of human cancer, Specifi parameter is the highest concentration tested and is preceded cally in a murine mouse model using Squamous cell carci by a “c” sign. This practice is extended similarly to the other noma (KLN-205) cells. Using this model, CLT and com possible situations where a response parameter cannot be pounds 28, 30 and 31 significantly inhibited tumor growth obtained by interpolation. after either Subcutaneous injection or oral administration of Dose-Response Curves: 80 mg/kg/day for 30 days. In addition these studies con firmed the purposed mechanism of action showing that CLT The dose-response curve page of the data package is and compound 28 abrogated cyclin D1 and PCNA expres created by plotting the PGS against the logo of the corre sion in vivo in the KLN induced tumors. Furthermore, lung 35 sponding concentration for every cell line. The cell line metastases were also significantly reduced in the animals curves are grouped by Subpanel. Horizontal lines are pro used the KLN-205 study. In addition lung metastases were vided at the PG values of +50.0, and -50. The concentrations Significantly reduced in nude mice inoculated with human corresponding to points where the curves croSS these lines melanoma MM-RU cells following oral administration or are the GI50, TGI, and LC50, respectively. Subcutaneous injection of compound 28. 40 The Mean Graphs: Efficacy Studies using mouse Xenograft models showed Mean graphs facilitate Visual Scanning of data for poten that compound 28 significantly inhibited tumor growth of tial patterns of Selectivity for particular cell lines or for human cance r S Such as SU.86.86 pancreatic particular Subpanels with respect to a Selected response adenocarcinoma, H209 Small lung cell carcinoma, H460 parameter. Differences in apparent Selectivity patterns may large cell lung carcinoma and A2058 metastatic melanoma. 45 occur for the same compound against the same cell lines Although compounds 30 and 31 also showed inhibition of when different parameters are compared. The mean graphs tumor growth in these models, the efficacy of compounds 30 page of the data package shows mean graphs at each of the and 31 was not significantly different from compound 28. In principal response parameters: GI50, TGI, and LC50. Bars addition, preliminary toxicity Studies in mice and rats indi extending to the right represent Sensitivity of cells line to the cated that no obvious Systemic toxicity was observed in the 50 test agent in excess of the average Sensitivity of all tested animals treated with compound 28. Whether administered cell lines. Since the bar Scale is logarithmic a bar 2 units to intraperitoneally, orally or Subcutaneously, compound 28, the right implies the compound achieved the response did not cause significant body weight losses in treated parameter (e.g. GI50) for the cell line at a concentration animals and no abnormalities were detected upon groSS one-hundredth the mean concentration required over all cell observations of brain, heart, lungs, liver, Spleen, kidney and lines, and-thus the cell line is usually Sensitive to that gastrointestinal tract. 55 compound. Bars extending to the left correspondingly imply Sensitivity less than the mean. If for a particular drug and cell Figure & Table Legend line it was not possible to determine the desired response The Calculated Measurement of Effect: Percentage parameter by interpolation, the bar length shown is either the Growth (PG) highest concentration tested (and the listed logo of the The measured effect of the compound on a cell line is 60 response parameter will be preceded by a “s”) or the lowest currently calculated according to one or the other of the concentration tested (and the listed logo will be preceded by following two expressions: a “4”). The values at either limit (>or <) are also calculated in the If (Mean OD-Mean OD)20, then PG=100x(Mean OD mean used for the mean graph. Therefore, the mean used in Mean OD)/(Mean OD-Mean OD tzero) 65 the mean graph may not be the actual mean of the G150 for If (Mean OD-Mean OD)<0, then PG=100x(Mean OD instance. For is reason, we shall refer to his value as the MG Mean OD)/Mean OD, MID (for mean graph midpoint).

US 6,992,079 B2 S1 52 -SR', -NO, -NR'R'', halogen, (C-C)alkyl, -continued (C-C) alkenyl, (C-C) alkynyl and trihalomethyl, (9) wherein Said disorder is a Solid tumor or a hematological OCH neoplasm or an angiogenic disorder or a vasculogenic dis order. 2. The method of claim 1, wherein said subject is human. N 3. The method of claim 1, wherein the compound is Selected from the group of (2) 1O

15 (13)

(3)

25

HC (4) 3 V N

35 (14)

cL

40 HC (7) * V O 45 B

50 (16) (8)

55 C O 5C 60 C C O 65 US 6,992,079 B2 S3 S4 -continued -continued (17) (22)

15

(18) (26)

C

(19) 25

(20) (27) 35 OCH

40

45

(21) 50 (28) OCH3

55

60

65 US 6,992,079 B2 SS S6 -continued -continued (29) HC (33) v H s O

N

(30) 15 :

(34) NH2

25

(31) 35 O

40 and combinations thereof.

4. The method of claim 3, wherein the compound is 45 Selected from the group of

(14)

50

(32) 55

60

65 US 6,992,079 B2 57 -continued -continued (31) (26) NO2

O

os( N

Cl C 15 O Cl N ce CH (28) and combinations thereof. NO2 5. The method of claim 1, wherein said disorder is a solid tumor. 6. The method of claim 5, wherein said Solid tumor is a 25 metastatic cancer. 7. The method of claim 5, wherein said Solid tumor is Selected from the group of cervical cancer, breast cancer, N lung cancer, liver cancer, prostate cancer, colon cancer, renal cancer, ovarian cancer, cancer of the central nervous System and melanoma. 8. The method of claim 5, wherein said lung cancer is : Small cell lung carcinoma. 9. The method of claim 5, wherein said lung cancer is 35 large cell lung carcinoma. 10. The method of claim 6, wherein said cancer is metastatic melanoma. (29) HC 11. The method of claim 6, wherein said cancer is 40 metastatic lung cancer. 12. The method of claim 5, wherein said Solid tumor is a carcinoma, or a Sarcoma or lymphoma. 13. The method of claim 12, wherein said carcinoma is an 45 adenocarcinoma. Cl 14. The method of claim 12, wherein said carcinoma is a Squamous cell carcinoma. 15. The method of claim 13, wherein said adenocarci ce (30) 50 noma is pancreatic cancer. NO2 16. The method of claim 1, wherein said disorder is a hematological neoplasm. 17. The method of claim 16, wherein said hematological 55 neoplasm is a leukemia. 18. The method of claim 16, wherein hematological neoplasm is Selected from acute lymphocytic leukemia, chronic lymphocytic leukemia, myelogenous leukemia, / s. Y.NN. O n multiple myeloma, an AIDS-associated leukemia, T-cell CH 60 leukemia, and T-cell lymphoma. 19. The method of claim 1, wherein said disorder is an C angiogenic disorder or a vasculogenic disorder. C 20. The method of claim 19, wherein said disorder is an 65 angiogenic disorder. 21. The compound of claim 20, wherein said compound is Selected from the group of: US 6,992,079 B2 59 60 -continued (28) and NO2 (31) NO2

O

N 1O

Cl O 15 (30) NO

and combinations thereof.

25 22. The method of claim 1, wherein said administration is oral, parenteral or intravenous. O O O YCH, 3O C C UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,992,079 B2 Page 1 of 1 APPLICATIONNO. : 10/013308 DATED : January 21, 2006 INVENTOR(S) : Brugnara et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Title page. Item *) Notice, delete “by 0 days and insert -- by 123 days --.

Signed and Sealed this Fourth Day of July, 2006 WDJ

JON. W. DUDAS Director of the United States Patent and Trademark Office UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,992,079 B2 Page 1 of 1 APPLICATIONNO. : 10/013308 DATED : January 31, 2006 INVENTOR(S) : Brugnara et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Title page. Item *) Notice, delete “by 0 days and insert -- by 123 days --.

This certificate supersedes certificate of correction issued July 4, 2006.

Signed and Sealed this Twenty-ninth Day of August, 2006 WDJ

JON. W. DUDAS Director of the United States Patent and Trademark Office