Bis-diazeniumdiolate compounds as anti-cancer agents

Abstract

A series of double-component, bis O.sup.2-aryl diazeniumdiolate derivatives are provided, of which each molecule can release up to four nitric oxide molecules. These compounds show cytotoxic activities to cancer cells, such as human leukemia, breast cancer and lung cancer. Among them, the compound 3 showed the highest specific activity against human leukemia cells.

Claims

1. A compound selected from formula I: ##STR00022## wherein: X is CH or N; and L is selected from C.sub.1-C.sub.10 alkyldiyl, C.sub.1-C.sub.10 alkyldiyl-(C.sub.3-C.sub.6 cycloalkyldiyl), and C.sub.1-C.sub.10 alkyldiyl-(C.sub.3-C.sub.6 cycloalkyldiyl)-C.sub.1-C.sub.10 alkyldiyl, where alkyldiyl and cycloalkyldiyl are independently and optionally substituted with one or more groups selected from halo, hydroxy, nitro, cyano, (C.sub.1-C.sub.6) alkoxy, and oxo (O); or a pharmaceutically-acceptable salt thereof.

2. The compound of claim 1 which is a compound of formulae Ia, Ib, or Ic: ##STR00023## where m and n are independently selected from 1, 2, and 3.

3. The compound of claim 1 wherein X is CH.

4. The compound of claim 1 wherein X is N.

5. The compound of claim 1 wherein L is optionally substituted C.sub.1-C.sub.10 alkyldiyl.

6. The compound of claim 1 wherein L is selected from CH.sub.2, CH.sub.2CH.sub.2, and CH.sub.2CH.sub.2CH.sub.2.

7. The compound of claim 1 wherein L is optionally substituted C.sub.1-C.sub.10 alkyldiyl-(C.sub.3-C.sub.6 cycloalkyldiyl).

8. The compound of claim 1 wherein L is optionally substituted C.sub.1-C.sub.10 alkyldiyl-(C.sub.3-C.sub.6 cycloalkyldiyl)-C.sub.1-C.sub.10 alkyldiyl.

9. A compound selected from compounds 1-4: ##STR00024##

10. A pharmaceutical composition comprising a compound of formula I as described in claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, glidant, or excipient.

11. A method for treating cancer in an animal in need thereof, comprising administering a therapeutically-effective amount of a compound of formula I as described in claim 1, or a pharmaceutically acceptable salt thereof, to the said animal; wherein the cancer is selected from pancreatic cancer, bile duct carcinoma, neuroblastoma, colon cancer, breast cancer, myeloma, gastric cancer, liver cancer, gliblastoma, ovarian cancer, colorectal cancer, non-Hodgkin lymphoma, lung cancer, prostate cancer, small-cell lung cancer, large cell lung cancer, kidney cancer, esophageal cancer, stomach cancer, cervical cancer or lymphoma tumors.

12. The method of claim 11 wherein the cancer is breast cancer.

13. A method for inhibiting cancer cell growth in an animal in need thereof, comprising administering an inhibitory effective amount of a compound of formula I as described in claim 1, or a pharmaceutically acceptable salt thereof, to the said animal; wherein the cancer is selected from pancreatic cancer, bile duct carcinoma, neuroblastoma, colon cancer, breast cancer, myeloma, gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, non-Hodgkin lymphoma, lung cancer, prostate cancer, small-cell lung cancer, large cell lung cancer, kidney cancer, esophageal cancer, stomach cancer, cervical cancer or lymphoma tumors.

14. The method of claim 13 wherein the cancer cell is a breast cancer cell.

Description

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(1) JS-K is activated by glutathione S-transferase (GST) for nitric oxide (NO) releasing, which is a part of the mechanism of its anticancer activity. The delivery efficiency of nitric oxide may be increased or otherwise optimized by linking two parts of JS-K together in a compound having formula I:

(2) ##STR00002##

(3) wherein:

(4) X is CH or N; and

(5) L is selected from C.sub.1-C.sub.10 alkyldiyl, C.sub.1-C.sub.10 alkyldiyl-(C.sub.3-C.sub.6 cycloalkyldiyl), and C.sub.1-C.sub.10 alkyldiyl-(C.sub.3-C.sub.6 cycloalkyldiyl)-C.sub.1-C.sub.10 alkyldiyl, where alkyldiyl and cycloalkyldiyl are independently and optionally substituted with one or more groups selected from halo, hydroxy, nitro, cyano, (C.sub.1-C.sub.6) alkoxy, and oxo (O);

(6) or a pharmaceutically-acceptable salt thereof.

(7) Exemplary embodiments of a formula I compound include formulae Ia, Ib, or Ic:

(8) ##STR00003##

(9) where m and n are independently selected from 1, 2, and 3.

(10) In one exemplary embodiment, X is CH.

(11) In one exemplary embodiment, X is N.

(12) In one exemplary embodiment, L is optionally substituted C.sub.1-C.sub.10 alkyldiyl.

(13) In one exemplary embodiment, L is selected from CH.sub.2, CH.sub.2CH.sub.2, and CH.sub.2CH.sub.2CH.sub.2.

(14) In one exemplary embodiment, L is optionally substituted C.sub.1-C.sub.10 alkyldiyl-(C.sub.3-C.sub.6 cycloalkyldiyl).

(15) In one exemplary embodiment, L is optionally substituted C.sub.1-C.sub.10 alkyldiyl-(C.sub.3-C.sub.6 cycloalkyldiyl)-C.sub.1-C.sub.10 alkyldiyl.

Preparation of Bis-Diazeniumdiolate Compounds of Formula I

(16) Formula I compounds were prepared by the methods, processes, intermediates, reactions, and reagents in Schemes 1-3 and the Examples. Two molecules of JS-K were coupled via an alkane linker in an exemplary method to synthesize the formula I, double-component, bis O.sup.2-aryl diazeniumdiolate compounds. A series of bis O.sup.2-aryl diazeniumdiolate compounds were designed and synthesized and their anti-cancer activities tested, including compounds 1-4:

(17) ##STR00004##

(18) ##STR00005##

(19) As shown in Scheme 1, tert-butyl piperazine-1-carboxylate 6 reacted with 1,2-dibromoethane (7) or 1,3-dibromopropane (8) to obtain compounds di-tert-butyl 4,4-(ethane-1,3-diyl)bis(piperazine-1-carboxylate) 9 and di-tert-butyl 4,4-(propane-1,3-diyl)bis(piperazine-1-carboxylate) 10 with a yield of 43% and 78%, respectively. The BOC-protection groups were removed by trifluoroacetic acid (TFA) to generate the intermediates 1,2-di(piperazin-1-yl)ethane 11 and 1,3-di(piperazin-1-yl)propane 12, which were treated with nitric oxide gas to obtain the intermediates disodium (1Z,1Z)-1,1-(ethane-1,2-diylbis(piperazine-4,1-diyl))bis(2-(11-oxidaneyl)diazene 1-oxide) 13 and disodium (1Z,1Z)-1,1-(propane-1,3-diylbis(piperazine-4,1-diyl))bis(2-(11-oxidaneyl)diazene 1-oxide) 14, respectively. The intermediates 13 and 14 were reacted with 2,4-dinitrofluorobenzene to obtain (1Z,1Z)-1,1-(ethane-1,2-diylbis(piperazine-4,1-diyl))bis(2-(2,4-dinitrophenoxy)diazene 1-oxide) 1 and (1Z,1Z)-1,1-(propane-1,3-diylbis(piperazine-4,1-diyl))bis(2-(2,4-dinitrophenoxy)diazene 1-oxide) 2 in a final yield of 10% and 17%, respectively, after three steps of reaction.

(20) ##STR00006##

(21) The synthesis of compound 3 was described as shown in Scheme 2. Commercially available, 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid 15 was treated with BH.sub.3 to obtain tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate 16 in a yield of 93% and which was reacted with NaI to generate tert-butyl 4-(iodomethyl)piperidine-1-carboxylate 17 in a yield of 50%. Intermediate 17 was coupled with tert-butyl piperazine-1-carboxylate 6 to obtain tert-butyl 4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methyl)piperazine-1-carboxylate 18 in a yield of 66%. The BOC-protection groups of 18 were removed by trifluoroacetic acid (TFA) to generate 1-(piperidin-4-ylmethyl)piperazine 19, which was treated with nitric oxide gas to obtain disodium (Z)-2-(4-((1-(2-(11-oxidaneyl)-1-oxidodiazen-1-yl)piperidin-4-yl)methyl)piperazin-1-yl)-1-(11-oxidaneyl)diazene 1-oxide 20. Intermediate 20 was reacted with 2,4-dinitrofluorobenzene to obtain (E)-1-(2,4-dinitrophenoxy)-2-(4-((1-((Z)-2-(2,4-dinitrophenoxy) 1-oxidodiazen-1-yl)piperidin-4-yl)methyl)piperazin-1-yl)diazene 1-oxide 3 in a final yield of 7.9% after three steps of reaction.

(22) ##STR00007##

(23) As shown in Scheme 3, the compound 4 was synthesized using a similar procedure as Schemes 1 and 2. Cyclohexane-1,4-diyldimethanol 21 is bis-mesylated with methanesulfonyl chloride in trimethylamine and dichloromethane followed by displacement with iodine by sodium iodide in acetonitrile to give 1,4-bis(iodomethyl)cyclohexane 22. Intermediate 22 was reacted with tert-butyl piperazine-1-carboxylate 6 and potassium carbonate in dimethylformamide at room temperature to give di-tert-butyl 4,4-(cyclohexane-1,4-diylbis(methylene))bis(piperazine-1-carboxylate) 23. Removal of the Boc groups from 23 with trifluoroacetic acid in dichloromethane gave 1,4-bis(piperazin-1-ylmethyl)cyclohexane 24. Treatment of 24 with nitric oxide gas, sodium methoxide in methanol and ether gave disodium (1Z,1Z)-1,1-((cyclohexane-1,4-diylbis(methylene))bis(piperazine-4,1-diyl))bis(2-(11-oxidaneyl)diazene 1-oxide) 25. Intermediate 25 was reacted with 2,4-dinitrofluorobenzene in aqueous sodium bicarbonate to give (1Z,1Z)-1,1-((cyclohexane-1,4-diylbis(methylene))bis(piperazine-4,1-diyl))bis(2-(2,4-dinitrophenoxy)diazene 1-oxide) 4.

(24) TABLE-US-00001 TABLE 1 Half maximal inhibitory concentration (IC.sub.50) of compounds 1-4 against different cancer cells. Half maximal inhibitory concentration (IC50, mol/L) Cell line Compound 1 Compound 2 Compound 3 Compound 4 JS-K CCRF-CEM 2.64 0.37 3.73 0.15 0.46 0.02 2.54 0.14 0.29 0.01 Jurkat E6-1 3.71 0.04 4.06 0.18 1.94 0.08 3.01 0.22 0.52 0.02 BT474 10.7 0.97 10.8 0.08 7.06 1.03 6.01 0.21 3.66 0.41 A549 17.1 1.02 18.8 0.21 11.3 1.61 18.5 0.15 2.11 0.15 MDA-MB-231 17.6 0.08 >20 17.86 1.01 16.4 0.53 7.93 0.47

(25) As shown in Table 1, the control compound JS-K had a high inhibitory effect on cellular proliferation against all four tested cancer cells including, breast cancer cell BT474, breast cancer cell MDA-MB-231, lung cancer cell A549, leukemia cell CCRF-CEM and leukemia Jurkat cells. The compounds of the invention 1-4 had comparable activity to JS-K against all these five tested cancer cells. Among them, the compound 3 showed higher selectivity than JS-K. It had a similar IC.sub.50 to JS-K against leukemia CEM cells (0.46 M: 0.29 M). However, the IC.sub.50 values for compound 3 against other four cancer cells were 1.9-5.4 fold higher (less cell killing potency) than for JS-K. Especially, it showed less efficiency to breast cancer and lung cancer. Compound 3 had a higher selectivity to leukemia cells than to breast cancer and lung cancer cells.

EXAMPLES

(26) ##STR00008##

Di-tert-butyl 4,4-(ethane-1,2-diyl)bis(piperazine-1-carboxylate) 9

(27) To a stirred solution of 200 mg (1.07 mmol) of mono Boc-piperazine 6 in 5 mL of dry DMF were added 47.0 L (101 mg, 0.54 mmol) of 1,2-dibromoethane 7 and 592 mg (4.28 mmol) of K.sub.2CO.sub.3 at room temperature. After 24 h, reaction mixture was diluted with 25 mL of water and extracted with two 50 mL portions of ethyl acetate. The organic phase was dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by chromatography on a silica gel column (102 cm). Elution with 19:1 ethyl acetate/methanol gave desired product 9 as a colorless solid: yield 183 mg (43%); silica gel TLC R.sub.f 0.27 (9:1 ethyl acetate/methanol); .sup.1H NMR (CDCl.sub.3) 1.47 (s, 18H), 2.33-2.40 (m, 12H) and 3.37-3.45 (m, 8H); .sup.13C NMR (CDCl.sub.3) 28.6, 53.5, 53.6, 56.1, 79.8 and 154.9.

(28) ##STR00009##

Di-tert-butyl 4,4-(propane-1,3-diyl)bis(piperazine-1-carboxylate) 10

(29) To a stirred solution of 300 mg (1.61 mmol) of mono Boc-piperazine 6 in 5 mL of dry DMF were added 82.0 L (163 mg, 0.80 mmol) of 1,3-dibromopropane 8 and 890 mg (6.44 mmol) of K.sub.2CO.sub.3 at room temperature. After 24 h, reaction mixture was diluted with 25 mL of water and extracted with two 50 mL portions of ethyl acetate. The organic phase was dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by chromatography on a silica gel column (102 cm). Elution with 19:1 ethyl acetate/methanol gave desired product 10 as a colorless solid: yield 521 g (78%); silica gel TLC R.sub.f 0.27 (9:1 ethyl acetate/methanol); .sup.1H NMR (CDCl.sub.3) 1.46 (s, 18H), 1.64-1.74 (m, 2H), 2.35-2.39 (m, 12H) and 3.41-3.44 (m, 8H); .sup.13C NMR (CDCl.sub.3) 28.4, 31.4, 36.4, 53.1, 56.6, 79.5 and 154.7.

(30) ##STR00010##

1,2-Di(piperazin-1-yl)ethane 11

(31) To a solution of 50 mg (0.13 mmol) of 9 in 500 L of CH.sub.2Cl.sub.2 was added 500 L of CF.sub.3COOH. The reaction mixture was stirred overnight and then concentrated under diminished pressure to obtain the deprotected amine, which was used in next step without further purification.

(32) ##STR00011##

1,3-Di(piperazin-1-yl)propane 12

(33) To a solution of 100 mg (0.24 mmol) of 10 in 500 L of CH.sub.2Cl.sub.2 was added 500 L of CF.sub.3COOH. The reaction mixture was stirred overnight and then concentrated under diminished pressure to obtain the deprotected amine, which was used in next step without further purification.

(34) ##STR00012##

(1Z,1Z)-1,1-(ethane-1,2-diylbis(piperazine-4,1-diyl))bis(2-(2,4-dinitrophenoxy)diazene 1-oxide) 1

(35) To a stirred solution of 25.0 mg (0.13 mmol) of amine 11 in 15 mL of 1:3 methanol-diethyl ether was added 0.38 mL (0.38 mmol) of 1M NaOMe solution in Parr bottle at room temperature. The reaction mixture was charged with 60 psi of nitric oxide and stirred at room temperature for 24 h. The white crystalline solid was collected by filtration and washed with diethyl ether and dried under vacuum.

(36) To an ice-cooled solution of 13 in 1 mL of aqueous 5% NaHCO.sub.3 solution was added a solution of 47.0 mg (0.25 mmol) of 2,4-dinitrofluorobenzene in 1 mL of 1,4-dioxane. The reaction mixture was allowed to ambient temperatures and stirred for overnight. The reaction mixture was diluted with 5 mL of water and extracted with two 10 mL portions of ethyl acetate. The organic phase was dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by chromatography on a silica gel column (101 cm). Elution with 15:1 ethyl acetate/methanol gave desired product 1 as a pale yellow solid: yield 8.0 mg (10%); silica gel TLC R.sub.f 0.19 (9:1 ethyl acetate/methanol); .sup.1H NMR (DMSO-d.sub.6) 2.33-2.38 (m, 12H) and 3.46-3.54 (m, 8H), 7.68 (d, 2H, J=8.8 Hz), 8.47 (dd, 2H, J=8.8 Hz and 2.4 Hz) and 8.87 (d, 2H, J=2.4 Hz); .sup.13C NMR (DMSO-d.sub.6) 52.1, 53.6, 55.3, 117.7, 122.2, 129.2, 137.3, 142.3 and 153.9.

(37) ##STR00013##

(1Z,1Z)-1,1-(propane-1,3-diylbis(piperazine-4,1-diyl))bis(2-(2,4-dinitrophenoxy)diazene 1-oxide) 2

(38) To a stirred solution of 51.5 mg (0.24 mmol) amine 12 in 15 mL of 1:3 methanol-diethyl ether was added 0.72 mL (0.72 mmol) of 1M sodium methoxide (NaOMe) solution in a Parr bottle at room temperature. The reaction mixture was charged with 60 psi of nitric oxide and stirred at room temperature for 24 h. The white crystalline solid was collected by filtration and washed with diethyl ether and dried under vacuum.

(39) To an ice-cooled solution of 14 in 1 mL of aqueous 5% NaHCO.sub.3 solution was added a solution of 89.3 mg (0.48 mmol) of 2,4-dinitrofluorobenzene in 1 mL of 1,4-dioxane. The reaction mixture was allowed to ambient temperatures and stirred for overnight. The reaction mixture was diluted with 5 mL of water and extracted with two 10 mL portions of ethyl acetate. The organic phase was dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by chromatography on a silica gel column (101 cm). Elution with 15:1 ethyl acetate/methanol gave desired product 2 as a pale yellow solid: yield 28 mg (17%); silica gel TLC R.sub.f 0.19 (9:1 ethyl acetate/methanol); .sup.1H NMR (DMSO-d.sub.6) 1.65-1.74 (m, 2H), 2.32-2.38 (m, 12H) and 3.44-3.51 (m, 8H), 7.69 (d, 2H, J=9.2 Hz), 8.47 (dd, 2H, J=9.2 Hz and 2.4 Hz) and 8.88 (d, 2H, J=2.4 Hz); .sup.1H NMR (DMSO-d.sub.6) 31.3, 36.5, 52.7, 56.5, 117.5, 122.2, 129.3, 137.4, 142.1 and 153.9.

(40) ##STR00014##

tert-Butyl 4-(Iodomethyl)piperidine-1-carboxylate 17

(41) The compound 16 was prepared using compound 15 as starting material in a yield of 93%. To an ice-cooled stirred solution of 400 mg (1.86 mmol) of compound 16 in 5 mL of CH.sub.2Cl.sub.2 was added 0.52 mL (375 mg, 3.72 mmol) of triethylamine and 0.17 mL (256 mg, 2.23 mmol) of methanesulfonyl chloride. The reaction mixture was allowed to warm to ambient temperature and stirred for 2 h. The reaction mixture was diluted with 35 mL of CH.sub.2Cl.sub.2, washed successively with 20 mL of water, 20 mL of satd. aq. NaHCO.sub.3, 20 mL of brine, dried (MgSO.sub.4) and concentrated under reduced pressure. The resulting crude product was dissolved in 2 mL of acetonitrile and 1.39 g (9.30 mmol) of NaI was added. The reaction mixture was stirred at r.t. for 12 h, and poured into 50 mL of water, extracted with two 30-mL portions of ethyl acetate. The combined organic extract was washed with 20 mL of brine, dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (152 cm). Elution with 2:3 hexanes-ethyl acetate afforded 17 as a colorless solid: yield 302 mg (50%); silica gel TLC R.sub.f 0.31 (2:3 hexanes-ethyl acetate); .sup.1H NMR (CDCl.sub.3) 1.11-1.19 (m, 2H), 1.45 (s, 9H), 1.59-1.62 (m, 1H), 1.82 (d, 2H, J=13.2 Hz), 2.66 (brs, 2H), 3.12 (d, 2H, J=6.8 Hz) and 4.10 (brs, 2H); .sup.13C NMR (CDCl.sub.3) 13.5, 28.4, 32.5, 38.4, 43.5, 79.3 and 154.5.

(42) ##STR00015##

tert-Butyl 4-((1-(tert-Butoxycarbonyl)piperidin-4-yl)methyl)piperazine-1-carboxylate 18

(43) To a stirred solution of 250 mg (0.77 mmol) of 17 in 2 mL of anhydrous DMF was added 319 mg (2.31 mmol) of K.sub.2CO.sub.3 followed by 0.14 mg (0.77 mmol) of boc-piperazine 25 at r.t. After 15 h, the reaction mixture was poured into 50 mL of water and extracted with three 30-mL portions of ethyl acetate. The combined organic extract was washed with 40 mL of brine, dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (152 cm). Elution with 1:1 hexanes-EtOAc afforded 18 as a colorless solid: yield 194 mg (66%); silica gel TLC R.sub.f 0.36 (1:1 hexanes-EtOAc); .sup.1H NMR (CDCl.sub.3) 1.01-1.11 (m, 2H), 1.452 (s, 9H), 1.456 (s, 9H), 1.59-1.67 (m, 1H), 1.73 (d, 2H, J=13.6 Hz), 2.17 (d, 2H, J=7.2 Hz), 2.33 (t, 4H, J=4.8 Hz), 2.68 (t, 2H, J=11.6 Hz), 3.40 (t, 4H, J=4.8 Hz) and 4.09 (brs, 2H); .sup.13C NMR (CDCl.sub.3) 28.30, 28.34, 30.6, 33.4, 43.6, 43.8, 53.3, 64.4, 79.0, 79.2, 154.6 and 154.7.

(44) ##STR00016##

1-(Piperidin-4-ylmethyl)piperazine 19

(45) To a solution of 152 mg (0.40 mmol) of 18 in 500 L of CH.sub.2Cl.sub.2 was added 500 L of CF.sub.3COOH. The reaction mixture was stirred overnight and then concentrated under diminished pressure to obtain the deprotected amine, which was used in next step without further purification.

(46) ##STR00017##

(E)-1-(2,4-dinitrophenoxy)-2-(4-((1-((Z)-2-(2,4-dinitrophenoxy)-1-oxidodiazen-1-yl)piperidin-4-yl)methyl)piperazin-1-yl)diazene 1-oxide 3

(47) To a stirred solution of 100 mg (0.40 mmol) of amine 19 in 15 mL of 1:3 methanol-diethyl ether was added 1.19 mL (1.19 mmol) of 1M NaOMe solution in Parr bottle at room temperature. The reaction mixture was charged with 60 psi of nitric oxide and stirred at room temperature for 24 h. The white crystalline solid of 20 was collected by filtration and washed with diethyl ether and dried under vacuum.

(48) To an ice-cooled solution of the above obtained solid in 1 mL of aqueous 5% NaHCO.sub.3 solution was added a solution of 47.0 mg (0.25 mmol) of 2,4-dinitrofluorobenzene in 1 mL of 1,4-dioxane. The reaction mixture was allowed to ambient temperatures and stirred for overnight. The reaction mixture was diluted with 5 mL of water and extracted with two 10 mL portions of ethyl acetate. The organic phase was dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by chromatography on a silica gel column (101 cm). Elution with 15:1 ethyl acetate/methanol gave desired product 3 as a colorless solid: yield 7.2 mg (7.9%); silica gel TLC R.sub.f 0.19 (2:1 ethyl acetate/hexanes); .sup.1H NMR (DMSO-d.sub.6) 1.01-1.10 (m, 2H), 1.59-1.69 (m, 1H), 1.77 (d, 2H, J=12.4 Hz), 2.17 (d, 2H, J=6.8 Hz), 2.30 (t, 4H, J=4.8 Hz), 2.67 (t, 2H, J=11.6 Hz), 3.44 (t, 4H, J=4.8 Hz), 4.06 (brs, 2H), 7.63 (d, 1H, J=8.4 Hz), 7.65 (d, 1H, J=8.8 Hz), 7.40-7.46 (m, 2H), 8.83 (d, 1H, J=2.4 Hz) and 8.85 (d, 1H, J=2.8 Hz); .sup.13C NMR (CDCl.sub.3) 30.5, 33.4, 43.1, 43.9, 53.8, 64.3, 169.8, 117.2, 122.2, 122.3, 129.3, 129.6, 136.8, 136.9, 141.4, 141.7, 154.5 and 154.7.

(49) ##STR00018##

1,4-Bis(iodomethyl)cyclohexane 22

(50) To an ice-cooled stirred solution of 500 mg (1.86 mmol) of compound 21 in 10 mL of CH.sub.2Cl.sub.2 was added 1.20 mL (875 mg, 8.67 mmol) of triethylamine and 0.32 mL (477 mg, 4.16 mmol) of methanesulfonyl chloride. The reaction mixture was allowed to warm to ambient temperature and stirred for 2 h. The reaction mixture was diluted with 35 mL of CH.sub.2Cl.sub.2, washed successively with 20 mL of water, 20 mL of satd. aq. NaHCO.sub.3, 20 mL of brine, dried (MgSO.sub.4) and concentrated under reduced pressure. The resulting crude product was dissolved in 2 mL of acetonitrile and 2.60 g (17.3 mmol) of NaI was added. The reaction mixture was stirred at r.t. for 12 h, and poured into 50 mL of water, extracted with two 30-mL portions of ethyl acetate. The combined organic extract was washed with 20 mL of brine, dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (152 cm). Elution with 1:4 hexanes-ethyl acetate afforded 22 as a colorless liquid: yield 706 mg (56%); silica gel TLC R.sub.f 0.43 (3:2 hexanes-ethyl acetate); .sup.1H NMR (CDCl.sub.3) 1.45-1.54 (m, 4H), 1.57-1.67 (m, 4H), 1.70-1.75 (m, 2H) and 3.20 (d, 2H, J=7.2 Hz); .sup.13C NMR (CDCl.sub.3) 13.2, 28.6 and 37.9.

(51) ##STR00019##

Di-tert-butyl 4,4-(cyclohexane-1,4-diylbis(methylene))bis(piperazine-1-carboxylate) 23

(52) To a stirred solution of 320 mg (0.88 mmol) of 22 in 2 mL of anhydrous DMF was added 486 mg (3.52 mmol) of K.sub.2CO.sub.3 followed by 327 mg (1.76 mmol) of 6 at r.t. After 15 h, the reaction mixture was poured into 50 mL of water and extracted with three 30-mL portions of ethyl acetate. The combined organic extract was washed with 40 mL of brine, dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by flash chromatography on a silica gel column (152 cm). Elution with 1:1 hexanes-EtOAc afforded 23 as a colorless liquid: yield 317 mg (75%); silica gel TLC R.sub.f 0.22 (1:1 hexanes-EtOAc); .sup.1H NMR (CDCl.sub.3) 1.29-1.36 (m, 4H), 1.46-1.51 (m, 4H), 1.69 (m, 2H), 2.20 (d, 4H, J=7.6 Hz), 2.31 (t, 8H, J=4.0 Hz) and 3.40 (t, 8H, J=2.6 Hz); .sup.13C NMR (CDCl.sub.3) 27.1, 28.4, 31.4, 32.3, 53.4, 62.5, 79.2 and 154.6.

(53) ##STR00020##

1,4-Bis(piperazin-1-ylmethyl)cyclohexane 24

(54) To a solution of 200 mg (0.42 mmol) of 23 in 500 L of CH.sub.2Cl.sub.2 was added 500 L of CF.sub.3COOH. The reaction mixture was stirred overnight and then concentrated under diminished pressure to obtain the deprotected amine, which was used in next step without further purification.

(55) ##STR00021##

(1Z,1Z)-1,1-((cyclohexane-1,4-diylbis(methylene))bis(piperazine-4,1-diyl))bis(2-(2,4-dinitrophenoxy)diazene 1-oxide) 4

(56) To a stirred solution of 85 mg (0.30 mmol) of amine 24 in 15 mL of 1:3 methanol-diethyl ether was added 0.91 mL (0.91 mmol) of 1M NaOMe solution in Parr bottle at room temperature. The reaction mixture was charged with 60 psi of nitric oxide and stirred at room temperature for 24 h. The white crystalline solid was collected by filtration and washed with diethyl ether and dried under vacuum; yield 21.3 mg (16%).

(57) To an ice-cooled solution of 21.3 mg (0.05 mmol) of 25 in 1 mL of aqueous 5% NaHCO.sub.3 solution was added a solution of 20.8 mg (0.11 mmol) of 2,4-dinitrofluorobenzene in 1 mL of 1,4-dioxane. The reaction mixture was allowed to ambient temperatures and stirred for overnight. The reaction mixture was diluted with 5 mL of water and extracted with two 10 mL portions of ethyl acetate. The organic phase was dried (MgSO.sub.4) and concentrated under diminished pressure. The residue was purified by chromatography on a silica gel column (101 cm). Elution with 19:1 ethyl acetate/methanol gave desired product 4 as a pale yellow solid: yield 8.4 mg (24%); silica gel TLC R.sub.f 0.13 (19:1 ethyl acetate/methanol); .sup.1H NMR (CDCl.sub.3) 1.29-1.35 (m, 4H), 1.46 (s, 18H), 1.48-1.54 (m, 4H), 1.69 (m, 2H), 2.19 (d, 4H, J=6.8 Hz), 2.31 (t, 8H, J=4.4 Hz) and 3.44 (t, 8H, J=2.6 Hz), 7.64 (d, 2H, J=8.0 Hz), 8.40 (dd, 2H, J=9.2 Hz and 2.4 Hz) and 8.43 (d, 1H, J=2.8 Hz); .sup.13C NMR (CDCl.sub.3) 26.7, 31.8, 32.1, 53.3, 62.1, 117.2, 122.3, 129.3, 136.7, 141.5 and 154.3.

(58) In Vitro Cellular Proliferation Assay: BT474 breast cancer cells (ATCC HTB-20, overexpression of HER2), A549 lung cancer cells, leukemia cell CCRF-CEM (ATCC# CCL-119), and leukemia Jurkat cell were cultured at 37 C. in a 5% CO.sub.2 atmosphere and grown in Gibco RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimitotic mix antibiotic supplement before use.

(59) Exponentially growing cells were harvested and plated in 96-well plates at a concentration of 110.sup.4 cells/well. After incubation at 37 C. for 24 h, the cells were treated with analogues of JS-K at different concentrations for an additional 24, 48 or 72 h. Then 20 L of MTT (5 mg/mL) or MTS was added to each well and the plates were incubated at 37 C. for 4 h. The supernatant was discarded for MTT assay, and 100 L of DMSO was added to each well. For the MTS assay, this step is not necessary. The absorbance was recorded at 490 nm after 15 min. Inhibition of cell growth was obtained by the following formula: Inhibition of cell growth (%)=(OD.sub.negative controlOD.sub.treatment)100%/(OD.sub.negative controlOD.sub.background). Data are reported as the mean of three independent experiments, each run in quintuplicate.

(60) Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of the invention as defined by the claims that follow. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.