AU713317B2 - Trisbenzimidazoles useful as topoisomerase I inhibitors - Google Patents

Description

WO 96/36612 PCT/US96/06853 TRISBENZIMIDAZOLES USEFUL AS TOPOISOMERASE I INHIBITORS Background of the Invention DNA topoisomerases are nuclear enzymes that control and modify the topological states of DNA by catalyzing the concerted breaking and rejoining of DNA strands. See, for example, D'Arpa et al., Biochim. Biophys. Acta, 989, 163 (1989). Topoisomerase II enzymes alter the topological state of DNA by means of a double strand break in the DNA. Mammalian topoisomerase II represents an effective pharmacological target for the development of cancer chemotherapeutics.

Y. Chen et al., Annu. Rev. Pharmacol. Toxicol., 34, 191 (1994)). Among the clinical agents in use which are recognized as topoisomerase II inhibitors are etoposide (VP-16), teniposide (VM-26), mitoxantrone, m-AMSA, adriamycin (doxorubicin), ellipticine and daunomycin.

In comparison to topoisomerase II inhibitors, there are relatively few known topoisomerase I inhibitors. Camptothecin represents the most extensively studied mammalian topoisomerase I inhibitor. See R. C. Gallo et al., J. Natl.

Cancer Inst., 46, 789 (1971) and B. C. Giovanella et al., Cancer Res., 51, 3052 (1991). The broad spectrum of potent antineoplastic activity observed for camptothecin has prompted further efforts to identify other agents which can effectively poison mammalian topoisomerase I.

It has recently been demonstrated that Hoechst 33342 ethoxyphenyl)-5-(4-methyl- 1-piperazinyl)-2,5'-bi-1H-benzimidazole, is an inhibitor of topoisomerase I.

WO 96/36612 PCTIUS96/06853 H'JqN

N.

This agent, which binds to the minor groove of DNA, traps the reversible cleavable complex derived from DNA and topoisomerase I and produces a limited number of highly specific single-strand DNA breaks. For example, see A.Y. Chen et al., Cancer Res., 53, 1332 (1993) and A. Chen et al., PNAS, 90, 8131 (1993). A limitation of Hoechst 33342 as an anticancer agent is the previously reported observation that it is not effective against tumor cell lines which overexpress MDR1. While KB 3-1 cells are known to be quite sensitive to Hoechst 33342, with an IC 5 o of approximately 9 nM, this compound is approximately 130fold less cytotoxic to KB V-1 cells, which are known to overexpress MDR1.

Recently, several analogs of this bisbenzimidazole have been synthesized, to further investigate the structure activity relationships associated with their potency as topoisomerase I inhibitors and the related cytotoxicity. For example, Q. Sun et al., Biorg. and Med. Chem. Lett., 4, 2871 (1994) disclosed the preparation ofbisbenzimidazoles of formula where n is 0, 1, 2, or 3. However, these compounds were found to be about one order of magnitude less cytotoxic than Hoechst 33342. Therefore, a continuing need exists for new compounds that can induce DNA cleavage in the presence of mammalian topoisomerase

I.

Summary of the Invention The present invention provides a compound of the formula: 4 Y N/ Z* N .0Y 0

*S

*wherein Ar is (C 6

-C

12 )aryl, mono or bis-(C 1

-C

4 )alkyl-substituted (C 6

-C

12 )aryl, (6-

C

12 )aryl(0 1 -O4)alkyl, mono or bis-(C 1

-C

4 )alkyl-substituted (C 6

-C

12 )aryl (C 1

-C

4 )alkyl, or to 12-membered)heteroaryl comprising 1-3 N, S or non-peroxide 0, wherein N is 00 unsubstituted or is substituted with (C 1

-C

4 )alkyl, (C 3 -0 4 )cycloalkyl, or cyclopropylmethyl, 15 said Ar being optionally fused to the benzo moiety; X is CN, CHO, OH, acetyl, CF 3

O(C

1

-C

4 )alkyl, O(C 3

-C

4 )cycloalkyl, or cyclopropylmethoxy, NO 2

NH

2 halogen, halo(C 1 0000 C4)alkyl, halo(C 3 -O4)cycloalkyl, or halocyclopropylmethyl; each Y is individually H, (Cl- 0 C 4 )alkyl, (Ca-C4)cycloalkyl, cyclopropylmethyl, or aralkyl; Y' is H, (C 1

-C

4 )alkyl, (C3- 0 6 C 4 )cycloalkyl, or cyclopropylmethyl; each Z is individually H, (C 1

-C

4 )alkyl, (C3-

C

4 )cycloalkyl, cyclopropylmethyl, halogen, halo(C 1 -C4)alkyl, halo (C 3 -O4)cycloalkyl, or halocyclopropylmethyl; n is 1; or a pharmaceutically acceptable salt thereof.

Preferably, Ar is at the 5-position. Ar may also be a 5,6-membered heteroaryl comprising 1-2 N, S or non-peroxide O atoms.

Ar may also be benzo, phenyl or pyridyl. When Ar is pyridyl, it may be 2pyridyl, 3-pyridyl or 4-pyridyl. When Ar is benzo, it is preferably 4, 5-benzo or 5,6-benzo.

Y' may be varied and preferably is H. When Ar is at the 5-position and is phenyl or pyridyl, including 2-pyridyl, 3-pyridyl or 4-pyridyl, each Y is preferably H.

When Ar is at the 5-position and is phenyl, X is preferably a halogen such as Cl. More preferably, when Ar is phenyl at the 5-position and X is CI, X-Ar is preferably p-chlorophenyl. In this preferred form, each Y and each Z, is preferably H.

The present invention also provides a compound of the formula: N

N

4 Y N wherein X is CN, CHO, OH, acetyl, CF 3

O(C

1

-C

4 )alkyl, O(C3-C4) cycloalkyl, or cyclopropylmethoxy, N0 2

NH

2 halogen, halo(C1-C4)alkyl, halo(C3-C4)cycloalkyl, or halocyclopropylmethyl; each Y is individually H, (C1-C 4 )alkyl, (C 3

-C

4 )cycloalkyl, cyclopropylmethyl, or aralkyl; Y' is H, (C1-C 4 )alkyl, (C 3

-C

4 )cycloalkyl, or 15 cyclopropylmethyl; each Z is individually H, (C,-C 4 )alkyl, (C 3

-C

4 )cycloalkyl, cyclopropylmethyl, halogen, halo(C1-C4)alkyl, halo (C3-C 4 )cycloalkyl, or halocyclopropylmethyl; or a pharmaceutically acceptable salt thereof.

Preferably, X is CHO, CN or a halogen such as Cl or Br. When X is CHO So** or CN, Y' is preferably H. When X is Cl or Br, each Y and each Z are preferably H.

Preferably Z is H, F, CH 3 or CF 3 Compounds of the present invention are inhibitors of topoisomerase I, as demonstrated by their ability to promote DNA cleavage in the presence of topoisomerase I. Furthermore, compounds of the present invention also are cytotoxic to mammalian tumor cells, including camptothecin-sensitive and camptothecin-resistant tumor cells and tumor cell lines exhibiting multi-drug resistance due to expression of the P-glycoprotein.

Therefore, the present invention also provides a method for the inhibition of mammalian tumor cell growth, comprising contacting a susceptible population of tumor cells with an effective growth-inhibiting amount of a compound of the present invention, preferably in combination with a pharmaceutically acceptable carrier. In particular, the present invention provides a method of inhibiting the growth of a mammalian tumor cell comprising contacting a tumor cell susceptible thereto, with an effective inhibitory amount of a compound of the present invention in combination with a pharmaceutically acceptable carrier.

The growth of the tumor cells can be inhibited in vitro, or vivo, by S 10 administering the compound of the present invention to a mammal in need of such Ong• otreatment, such as a human cancer patient afflicted with a leukemia or solid tumor.

The compounds of the present invention can also be used to evaluate the boo: activity of topoisomerase I obtained from different sources, and are expected to exhibit .oo at least some of the other bioactivities observed for topoisomerase inhibitors, such as 15 antibacterial, antifungal, antiprotozoal, anthelmintic and/or antiviral activity. For example, compound 14, shown on Figure 1, exhibits antifungal activity.

The compounds of the present invention may be formulated into 0 therapeutic compositions. Thus, the present invention also provides a therapeutic 4000 0* composition comprising the compound of the present invention in combination with a 20 pharmaceutically acceptable carrier.

066@ Sa Brief Description of the Drawings 0 04 0 Figure 1 is a schematic depiction of the synthesis of compounds 10-16.

Figure 2 is a schematic depiction of the preparation of intermediates 4-8 used to prepare compounds of the invention.

WO 96/36612 PCT/US96/06853 Figure 3 is a schematic depiction of the preparation of intermediate 9.

Figure 4 is a schematic depiction of the synthesis of compounds JSKIV-68, 37 and -47.

Figure 5 is a schematic depiction of the preparation of intermediate JSKIV- 44.

Figure 6 is a schematic depiction of the preparation of analogs modified on the central benzimidazole moiety.

Figure 7 is a schematic depiction of the preparation of analogs modified on the terminal benzimidazole moiety.

Detailed Description of the Invention The aryl groups (Ar) useful in the present compounds comprise

(C

6

-C

1 8 )aryl, preferably (C 6

-C

1 4 aryl, systems containing aromatic rings, which systems comprise a total of 6 to 12 carbon atoms. Thus, as used herein, the term "aryl" includes mono- or bis-(Cl-C 4 )alkyl-substituted aryl, such as tolyl and xylyl; ar(C 1

-C

4 )alkyl, such as benzyl or phenethyl; and alkaralkyl. Preferably aryl is phenyl, benzyl or naphthyl.

Heteroaromatic rings include aromatic rings containing up to 3 ring heteroatoms such as N, S or non-peroxide 0, and up to 12 ring atoms.

Representative aromatic rings include thiophene, benzothiophene, naphthothiophene, trianthrene, furan, benzofuran, isobenzofuran, pyran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, pyridine, pyrazine, triazole, tetrazole, pyrazine, triazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, phenazine, isothiazole, phenothiazine, oxazole, isoxazole, furazan, phenoxazine and the like. Preferred heteroaromatic rings have a 5- or 6membered heteroaromatic ring which may or may not be fused to an aromatic ring such as a benzo ring, the preferred 3- or 4-pyridyl substituents.

WO 96/36612 PCTIUS96/06853 6 The term "alkyl" includes straight-chain or branched alkyl, as well as cycloalkyl and (cyloalkyl)alkyl, methyl, ethyl, i-propyl, cyclopropyl or cyclopropylmethyl.

Pharmaceutically acceptable salts include the acid addition salts of basic NH with organic or inorganic acids, hydrochloride, carbonate, sulfate, acetate, phosphate, tartarate, citrate, malate, maleate, propionate, and the like.

The preparation of representative substituted trisbenzimidazoles is outlined in Figure 1. With the exception of phenylenediamine which was commercially available, the appropriately substituted phenylenediamines were synthesized by catalytic hydrogenation of the respective o-nitroaniline derivatives.

These phenylenediamines were then coupled with 5-formyl-2-(benzimidazo-5'yl)benzimidazole, 9, by heating in nitrobenzene at 150 0 C to provide the various trisbenzimidazoles, 10-16, in yields ranging from 43-96%, employing the general methodologies ofM. P. Singh et al., Chem. Res. Toxicol., 5, 597 (1992) and Y.

Bathini et al., Synth Comm., 20, 955 (1990).

The requisite nitroanilines, as outlined in Figure 1, with the exception of 3 which was commercially available, were synthesized from 4-bromo- 2-nitroaniline, 17. Compound 17 was prepared from o-nitroaniline in good yield, 94%, using 2,4,4,6-tetrabromo-2,5-cyclohexadienone as the bromination reagent.

G. J. Fox et al., Org. Syn., 55, 20 (1973). While allyltributyltin and phenyltributyltin are commercially available, the pyridyltributyltin derivatives were prepared from tributyltin chloride and and 4-bromopyridine, respectively.

See D. Peters et al., Heterocyclic Chem., 27, 2165 (1990). These tributyltin derivatives were then coupled with 4-bromo-2-nitroaniline using PdCl 2 (PPh 3 2 as the catalyst in DMF as outlined in Figure 2 to provide compounds 4, 5, 6, 7, and 8, respectively, in accord with the methodology of M. Iwao et al., Heterocycles, 36, 1483 (1993). This methodology can generally be applied to prepare 5- or 6aryl- and heteroaryl-substituted 2-nitroanilines from the corresponding bromonitroanilines.

The preparation of 5-formyl-2-(benzimidazo-5'-yl)benzimidazole.

9, was accomplished as outlined in Figure 3. Reduction of benzimidazolecarboxylic acid to 5-hydroxymethylbenzimidazole was accomplished using LiAlH 4 Oxidation of the resulting crude benzylic alcohol with tetrapropylammonium perruthenate (TPAP) and N-methylmorpholine

N-

oxide provided in two steps the desired 5-formylbenzimidazole in 32% an overall yield. See, A. Cherif et al., J. Med. Chem., 35, 3208 (1992). Coupling of with 4-cyano-1,2-phenylenediamine provided 5-cyano- 2 which when treated with Ni-Al catalyst in the presence of aqueous formic acid gave 5-formyl- 2 yl)benzimidazole, 9, in 65% yield. R. Pipier et al., J. Med. Chem,, 1 2164 (1988)).

The compounds of the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human cancer patient, in a variety of forms adapted to the chosen route of administration, orally or parenterally, by intravenously, intramuscularly or subcutaneous routes.

Thus, the present compounds may be orally administered, for example, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful 30 compositions is such that an effective dosage level will be obtained.

AMENDED

SHEET

IPEAIEP

WO 96/36612 PCT/US96/06853 8 The tablets, troches, pills, capsules, and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion use can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusable solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier WO 96/36612 PCT/US96/06853 or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersion or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens. chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freezedrying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

Useful dosages of the compounds of 1 can be determined by comparing their in vitro activity, and in vivo activity in animal models, to that of an equivalent dosage of camptothecin (see, for example, B. C. Giovanella et al., Cancer Res., 51, 3052 (1991)) or Hoechst 33342 (see, A. Y. Chen et al., Cancer Res., 53, 1332 (1993)). Methods for the extrapolation of effective anti-tumor dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

The present analogs can be used to treat cancers known to be susceptible to topoisomerase I inhibitors, including, but not limited to, Burkitt's tumor, chronic lymphocytic leukemia, multiple myeloma, squamous cell and large WO 96/36612 PCTIUS96/06853 cell anaplastic carcinomas, adenocarcinoma of the lung, Ewing's sarcoma, non- Hodgkins lymphoma, breast tumor, colon tumor, stomach tumor, oat-cell bronchogenic carcinoma, squamous cell carcinoma of the cervix, ovarian tumors, bladder tumors, testicular tumors, endometrial tumors, malignant melanoma and acute lymphocytic leukemia, and prostatic carcinoma. The present compounds can be administered as single agents, or in combination with other antineoplastic drugs commonly employed to treat these cancers.

The invention will be further described by reference to the following detailed examples, wherein melting points were determined with a Thomas-Hoover unimelt capillary melting point apparatus. Infrared spectral data (IR) were obtained on a Perkin-Elmer 1600 Fourier transform spectrophotometer and are reported in cm Proton NMR) and carbon NMR) nuclear magnetic resonance were recorded on a Varian Gemini-200 Fourier Transform spectrometer. NMR spectra (200 MHz 'H and 50 MHz were recorded in CDCI 3 (unless otherwise noted) with chemical shifts reported in 6 units downfield from tetramethylsilane (TMS).

Coupling constants are reported in hertz. Mass spectra were obtained from Midwest Center for Mass Spectrometry within the Department of Chemistry at the University of Nebraska-Lincoln. Combustion analyses were performed by Atlantic Microlabs, Inc., Norcross, GA, and were with in THF was freshly distilled from sodium and benzophenone prior to use. Allyltributyltin and phenyltributyltin were purchased from Aldrich Chemical Company.

Example 1. General Procedure for PdCl,(PPhb3-catalyzed Coupling Reaction of 4-Bromo-2-nitroaniline (13) with Tin Compounds.

4-Phenyl-2-nitroaniline A solution of 4-bromo-2-nitroaniline 17 g, 4.67 mmol), tributylphenyl tin (2.2 g, 6.07 mmol), bis(triphenylphosphine)palladium (11) chloride (164 mg, 0.234 mmol), and triphenylphosphine (613 mg, 2.34 mmol) in DMF (15 ml) was heated under N 2 at 120"C overnight. After the solution was cooled to room temperature, the reaction WO 96/36612 WO 96/66 12PCTIUS96/06853 11I mixture was directly chromatographed on silica gel eluting with EtOAc/Hexane to give 752 mg of 5 as a yellow solid: mp 169-17 1 LB.

(CHCl 3 3517, 3398, 3022, 1635, 1525, 1250; 'H NMR 8 8.38 (1H, d, J1= 7.66 (IH, dd, J= 8.7, 7.59-7.54 (2H, in), 7.49-7.34 (3H, mn), 6.90 (1H, d, J= 8.8), 6.13 (NH, brs); 1 3 CNMIR6 144.2, 139.3, 135.0, 130.9, 129.5, 127.8, 126.8, 124.4, 119.8, 112.8;- Anal. Cal cd for C 1 2

HION

2 0 2 C, 67.28; H, 4.70; N, 13.08. Found: C, 67.38, H, 4.76; N, 13.01.

4-AIlyI-2-nitroaniline Prepared from 4-bromo-2-nitroaniline 17 (1.70 g, 7.84 minol) and allyltributyltin (3.38 g, 10.2 inmol) as a yellow solid in 96% yield as described above for 5: mp 29-31 lB. (KBr) 3490, 3374, 1638, 1518, 1341, 1253; 1H NMR 8 7.90 (1H, d, J 7.19 (1H, dd, J 6.77 (1H, d, J 6.05 (NH, brs), 6.00-5.80 (1H, in), 5.11 (IH, dd, J 1.4, 5.04 (I1H, ddd, J 3.0, 3.28 (1HK d, J 1 3 CNMR 8 143.81, 137.13, 129.34, 125.59, 119.49, 116.95, 39.18; HRMS (El) calcd for C 9

H,

0

N

2 0 2 178.0742, found 178.0746.

4-(2'-Pyridyl)-2-nitroaniline Prepared from 4-broino-2nitroaniline 17 (597 mng, 2.75 mmol) and 2-tributylstannylpyridine (1.01 g, 2.75 inmol) as a yellow solid in 52% yield as described above for 5: mp 146-148 IR

(CHCI

3 3516, 3397, 3020, 1634, 1524, 1341, 1250; 'H NMR 6 8.74 (1H, d, J= 8.63 (1H, dd, J 4.9, 8.13 (1H, dd, J 8.8, 7.78-7.66 (2H, in), 7.20 (1H, ddd, J 4.8, 4.7, 6.92 (1H, d, J= 6.37 (NH, brs); I'C NIVR 6 155.6, 150.1, 145.6, 137.4, 134.5, 129.1, 124.7, 122.4, 119.8, 119.7; Anal. Calcd for

CIIH

9

N

3 0 2 C, 61.39; H, 4.21; N, 19.53. Found: C, 61.29; H, 4.23; N, 19.43.

4-(3'-Pyridy)-2-nitroafife Prepared from 4-broino-2nitroaniline 17 (1.42 g, 6.53 iniol) and 3-tributylstannylpyridine (3.60 g, 9.79 minol) as a yellow solid in 32% yield as described above for 5: mp 177-179 IR

(CHC

3 3515, 3399, 3052, 2983, 1638, 1524, 1341, 1259; 'H NMR 8 8.68 (1H, d, J 8.42 (1H, dd. J1=4.8, 8.22 (1H, 7.74 (1H, ddd, J 7.9, 2.4, 1. 7.5 0 (1 H, dd, J= 8.7, 7.23 (1 H, ddd, J 8.0, 4.8, 6.92 (1 H, d, J= WO 96/36612 PTU9/65 PCTIUS96/06853 12 6.56 (NH, brs); 3 C NMR 6 148.7, 147.8, 145.4, 135.0, 134.4, 133.8, 126.5, 124.4, 124.0, 120.4; Anal. Calcd for C 11 HqN 3 0 2 C, 61.39; H, 4.21; N, 19.53.

Found: C, 61.28; H, 4.16; N, 19.40.

4-(4'-Pyridyl)-2-nitroaniline Prepared from 4-bromo-2nitroaniline 17 (165 mg, 0.76 mmol) and 4-tributyistannylpyridine (280 mg, 0.76 mmol) as a yellow solid in 25% yield as described above for 5: mp 23 0-232 JR

(CHC

3 3518, 3398, 3032, 1636, 1528, 1344; IH NMR (CD 3 OD) 6 8.55 (2H, d,J= 8.5 2 (1 H, d, J 7.84 (1 H, dd, J1= 8.9, 7.71 d, J 7.13 01H, d, J= 1 3 C NMIR (CD 3 OD) 6 149.4, 13 3.4, 124.0, 120.7, 120.0;- HRMS (El) calcd for CIIH 9

N

3 0 2 215.0695, found 215.0698.

Example 2. 5-Formyl-2-(benzi midazol-5 '-yl)benzi mid azole A mixture of 5-cyano-2-(benzimidazol-5'-yl)benzimidazole 19 (148 mg, 0.57 mmol), Ni-Al catalyst (500 mg), formic acid (7 ml) and water (3 ml) was heated under refluxed under N 2 for 4h. The hot reaction mixture was immediately filtered through a plug of celite, and evaporated to give a yellow solid. The yellow solid was then dissolved in hot water (5 ml), and the solution was neutralized to pH 9 by 2N NaOH. The solid precipitated was collected by suction filtration and further purified by flash chromatography on silica gel (15% MeOHIEtOAc) to give 142 mg of 9as awhite solid: mp >275 IR (KBr) 3106, 2835, 1685, 1618, 1432, 1293; 'H NMIR (CD 3 OD) 6 10.01 (1H, 8.39 (lH, 8.35 (1H1, 8.13 (IH, s), 8.06 (1 H, dd, J 8.6, 7.83 (1 H, dd, J 8.4, 7.77 (1 H, d, J 7.71 (1H, d, J= HERMS (FAB) calcd for C 15 1

N

4 0 263.0933, found 263.0932.

Example 3. General Procedures for Preparing trisbenzimidazoles.

2-1[2 -(1enzimidazol-5"-yI)benzi midazoI-5 -y'1 benzi mid azole A mixture of 5-formnyl-2-(benzimidazol-5'-yl)benzimidazole 9 (121 mg, 0.46 mmol) and phenylenediamine (60 mg, 0.55 mmol) in nitrobenzene (8 ml) was WO 96/366 12 PCTJUS96/06853 13 heated at 150 0 C under N 2 overnight. The mixture was cooled to room temperature and chromatographed on silica gel (0-20% MeOHj'EtOAc) to afford 155 mg (96%) of 10as asolid: mp >275 *C;IR (KBr) 3400, 3157, 1630, 1542, 1438, 1294; 'H NMIR (DMSO-d 6 3 drops of CF 3 COOH) 6 9.71 (1H, 8.75 (1H, 8.65 (1H, d, J 1. 8.4 8 (1 H, dd, J 8.7, 8.21 (1 H, dd, J 8. 6, 8.14 (1 H, d, J1= 8.08 (1H, d, J= 7.90 (2H, dd, J 7.61 (2H, dd,J 3. 1); 3 C NMvR (DMSO-d 6 3 drops of CF 3 COOH) 6 154.4, 149.8. 133.2, 132.0, 131.7, 126.2, 125.5, 125.4, 123.9, 123.6, 116.3, 115.9, 114.23, 114.17, 114.13; HiRMS (FAB) calcd for C 2 1 H1 5

N

6 351.1358, found 351.1367.

5-Cyan o-2- t2 '-(benzi midazol-5"I-yI)benzi mid azol-5 yllbenzimidazole Hydrogenation of 3 (70 mg, 0.43 mmol) was accomplished at 40 psi H 2 at room temperature for 1 h using 10% Pd-C (3 0 mg) in EtOAc (10 ml).

The reaction mixture was filtered and concentrated in vacuo to afford a solid. The solution of this solid and 9 (87 mg, 0.33 mmol) in nitrobenzene (5 ml) was heated at 150 *C under N 2 overnight. The mixture was cooled to room temperature, and chromatographed directly on silica gel 10% MeOHEtOAc) to give 107 mg of 11 as a solid; mp 280 IR (KBr) 3416, 3148, 2222, 1626, 1553, 1441, 1292; 'H NMR (DMSO-d 6 3 drops of CF 3 COOH) 6 8.50 (1H, 8.46 (IH, 8.40 (1H, 8.18-8.11 (3H, in), 7.81-7.75 (3H, in), 7.62 (1H, dd, J 8.3, HRMS (FAB) calcd for C 22

HI

3 N7 3 76.13 10, found 3 76.13 09.

5-Propyl-2- -(benzi midazol-5"-yI)benzi mid azol-5 yllbenzimidazole Prepared from 4-allyl-2-nitroaniline 4 (312 mg, 1.75 mmol) and 5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 (121 mg, 0.46 mmol) in 79% yield as described above for 11: solid; mp 270 LR (KBr) 3421, 3068, 2957, 1434; 'H NMIR (DMSO-d 6 3 drops of CF 3 COOH) 6 9.66 (1 H, 8.73 (1 H, 8.59 (1H, 8.48 (IH, dd, J 8.13 (1H, dd, J 8.7, 8.11 (1H, d, J 8.02 1 1H, d, J 7.79 I1H, d, J 7.66 (1 H, 7.45 (1 H, dd, J= 2.80 (2H, t, J 1.70 (2H, in), 0.96 (3H, t, J ITC NMIR (DMSO-d 6 3 drops of CF 3 COOH) 6 153.84, 149.74, 141.64, 141.01, 139.37, WO 96/36612 PCTIUS96/06853 14 133.10, 132.26, 131.99, 130.34, 127.08, 126.26, 125.14, 141.64, 141.01, 139.37, 133.10, 132.26, 131.99, 130.34, 127.08, 126.26, 125.14, 122.91, 117.52, 116.32, 116.06, 115.76, 113.78, 112.99, 37.45, 24.73, 13.74; 5-Phenyl-2-[2'(benzimidazol-5"-yl)benzimidazol-5'yl]benzimidazole Prepared from 4-phenyl-2-nitroaniline 5 (247 mg, 1.15 mmol) and 5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 (201 mg, 0.77 mmol) in 89% yield as described for 11: solid; mp 262-164 DC dec; IR (KBr) 3402, 3104, 1627, 1552, 1442, 1290; 'H NMR (DMSO-d 6 3 drops of CF 3 COOH) 6 9.66 (1H, 8.74 (1H, 8.65 (1H, 8.50 (1H, dd, J= 8.8, 8.21 (1H, dd, J= 8.7, 1.4), 8.12 (1H, d, J= 8.06 8.05 (1H, d, J= 7.97 (1H, d, J= 7.89 (1H, dd, J= 8.7, 7.80 (2H, d, J= 7.61-7.47 (3H, min); HRMS (FAB) calcd for C 2 7

H

9

N

6 427.1671, found 427.1666.

5-(2-Pyridyl)-2-[2'-(benzimidazol-5"-yl)benzimidazol-5'yl]benzimidazole Prepared from 4-(2'-pyridyl)-2-nitroaniline, 6 (110 mg, 0.50 mmol), and 5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 (51 mg, 0.25 mmol) in 84% yield as described above for 11: solid; mp 275 IR (KBr) 3411, 3157, 1630, 1593, 1432; 'H NMR (CD 3 OD) 6 8.59 (1H, d, J= 8.35 (1H, s), 8.31-8.25 (2H, 8.10 (1H, 8.04-7.94 (2H, min), 7.85-7.77 (3H, min), 7.72 (1H, d, J= 7.68 (1H, d, J= 7.64 (1H, d, J= 7.30 (1H, min); HRMS (FAB) calcd for C 2 6

H

18

N

7 428.1624, found 428.1611.

5-(3-Pyridyl)-2-[2'-(benzimidazol-5"-yl)benzimidazol-5'yl]benzimidazole Prepared from 4-(3'-pyridyl)-2-nitroaniline 7 (183 mg, 0.85 mmol) and 5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 in 46% yield as described above for 11: solid; mp 275 IR (KBr) 3400, 3070, 2836, 1438, 1289; '1H NMR (CD 3 OD) 6 8.83 (1H, d, J= 8.49 (1H, dd, J= 4.9, 8.38 (1H, d, J= 8.31 (1H, d, J= 8.29 (1H, 8.11 (1H, ddd, J= 8.0, 2.3, 1.6), 8.05 (1H, dd, J= 8.5, 8.00 (1H, dd, J= 8.5, 7.81 (1H, d, J= 7.77- 7.68 (3H, 7.55-7.47 (2H, min); HRMS (FAB) calcd for C 26

H,,

18

N

7 428.1624, found 428.1612.

WO 96/366 12 PTU9/6S PCTIUS96/06853 5-(4-Pyridyl)-2- -(benzimidazol-5"-yI)benzi midazol-5 yljbenzimidazole Prepared from 4-(4'-pyridyl)-2-nitroaniline 8 (35 mg, 0. 16 mmol) and 5-formyl-2-(benzimidazol-5 '-yl)benzimidazole 9 (50 mg, 0. 19 mmol) in 43% yield as described above for 11: solid; mp 280 IR (KBr) 3411, 3118, 1600, 1552, 1439, 1290; 1 1HNMR (CD 3 OD) 6 8.51 d, J= 8.33 (IH, d, J= 1. 8.2 7 (1 H, 8.2 5 (1IH, d, J 8. 01 (1 H, dd, J 7.96 (1 H, dd, J 8.9, 7.87 (1H, d, J 7.74-7.56 (611, in); FIRMS (FAB) calcd for

C

26

H

18

N

7 428.1624, found 428.1625.

Example 4. 4-Bromo-2-nitroaniline (17).

A solution of 2-nitroaniline (5 g, 3 6.2 mmol) in CH 2

C

2 (100 MI) Was cooled to 10 and treated by 90% 2,4,4,6-tetrabromo-2,5-cyclohexadienone (19.8 g, 43.5 m mol) in 5 portions. The mixture was stirred at 10 *C-0 *C for 1 hr. After being warmed to room temperature, the reaction mixture was washed by 2N NaOH (60 ml) and brine (50 ml), dried over Na 2

SO

4 and evaporated. Flash chromatography on silica gel EtOAc/Hexane) gave 7.40 g of 17 as a yellow solid: mp 109-110 (lit. mp 112-113 1H NNM 6 8.27 (1 H, d, J 7.43 (1lH, dd, J 8.9, 6.73 (1 H, d, J1= 6.09 (NH, brs).

Example 5. 5-Formyl benzi mid azole (18).

A suspension of 5-benzimidazolecarboxylic acid (1.57 g, 9.7 mmol) in dry THIF (5 0 ml) was cooled to -78 *C under N 2 and treated with LiAlH 4 (73 6 mg, 19.4 inmol). After the addition, the mixture was allowed to warm slowly to room temperature and then stirred at r.t. overnight. The mixture was quenched by MeOH and H 2 0 cautiously, and passed through a short silica gel column eluting with MeOHIEtOAc. The eluate was concentrated to give 876 mng crude alcohol as a solid. The crude alcohol (876 mg) was dissolved in a mixture of DMF (3 ml), TF ml) and CH 2 C1 2 (40 ml). 4-Methylmorpholine N-oxide (2.25 g, 19.2 mmol), 4A molecular sieves (5 and TPAP (169 mng, 0.48 minol) were subsequently added to WO 96/36612 PCTIUS96/06853 16 the crude alcohol solution. The mixture was stirred at room temperature overnight, and filtered through a pad of silica gel eluting with 10% MeOH/EtOAc. The elute was concentrated and further purified by flash chromatography on silica gel eluting with 0-10% MeOH/EtOAc to give 452 mg 2 steps) of 17 as a white solid: mp 164-166 IR (KBr) 3087, 2818, 1690, 1292; 'H NMR (CD 3 OD) 6 9.95 (1H, s), 8.34 (1H, 8.08 (1H, d, J= 7.74 (1H, dd, J= 8.4, 7.63 (1H, d, J= 8.4); 1 3 C NMR (CD 3 OD) 6 194.2, 146.0, 143.0, 139.8, 133.6, 124.9, 120.7, 116.6; Anal.

Calcd for CH 6

N

2 0: C, 65.75; H, 4.14; N, 19.17. Found: C, 65.60; H, 4.17; N, 19.08.

Example 6. 5-Cyano-2-(benzimidazol-5'-vl)benzimidazole (19).

A mixture of 5-formylbenzimidazole 18 (211 mg, 1.44 mmol) and 4-cyano- 1,2-phenylenediamine (230 mg, 1.73 mmol) in nitrobenzene (10 ml) was heated at 150"C under N 2 overnight. The mixture was cooled to room temperature and directly chromatographed on silica gel eluting with 0-15% MeOH/EtOAc to give 244 mg of 18 as a solid: mp >270 IR (KBr) 3110, 2826, 2224, 1627, 1426, 1294; 'H NMR (CD 3 OD) 6 8.41 (1H, 8.33 (1H, 8.07 (1H, dd, J= 8.6, 7.98 (1H, 7.78 (1H, d, J= 7.73 (1H, d, J= 7.56 (1H, dd, J 8.4, 3 C NMR (DMSO-d 6 3 drops of CF 3 COOH) 6 153.4, 140.4, 138.3, 132.9, 131.6, 127.0, 125.8, 125.3, 120.8, 119.8, 116.0, 115.8, 113.9, 105.5; HRMS (FAB) calcd for C 1

,H

0 iN 5 260.0936, found 260.0935.

Example 7.

5-Bromo-2-[2'-(benzimidazol-5"-yl)benzimidazol-5'-yl]benzimidazole (JSK IV-37) A mixture of 5-formyl-2-(benzimidazol-5'yl)benzimidazole (118.8 mg, 0.45 mmol) and 5-bromophenylenediamine (169.6 mg, 0.90 mmol) in nitrobenzene (5 mL) was heated at 150"C under N 2 overnight.

The mixture was cooled to room temperature and chromatographed using 0-10% methanol/ethylacetate to afford 127.3 mg of brownish yellow solid: WO 96/36612 PTU9/65 PCT/US96/06853 17 mp>280 1k (KBr) 3101, 1626, 1547, 1440; 'H NM (DMSO-d 6 6 7.34 (dd, III, J=7.0, 7.57 1H, 7.71-7.80 (in, 3H), 8.04-8.18 (in, 2H), 8.39 (s, 2H), 8.50 1W; 1 3 C NMR (DMSO-d 6 3 drops CF 3 COOH) 6 114.1 115.8, 116.2, 116.4, 11-7.0, 118.6, 123.5, 125.3, 126.2, 128.7, 128.9, 131.8, 132.0, 132.3, 133.1, 134.4, 138.3, 140.6, 151.1, 153.4.

5-Chloro-2- [2'-(benzi mid azol-5 "-yI)benzi mid azol-5'-yIj benzimidazole(JSK 1V-68) A mixture of 5-formyl-2-(benzimidazol-5'yl)benzimidazole (160 mg, 0.61 minol) and 5-chlorophenylenediamine, (174 mg, 1.22 inmol) in nitrobenzene (5 mL) was heated at 150*C under N 2 overnight. The mixture was cooled to room temperature and chromatographed using 0- methanol/ethylacetate to afford 167 mg of brownish yellow solid: inp>280*C; JR (KBr) 3103, 2826, 1427, 1293; 'H NMR (DMSO-d 6 6 7.24 (dd, I1H, J=8.5, 7.60-7.81 (in, 4H), 8.07-8.17 (in, 2H), 8.40 2H), 8. 50 I1H); I'C NMvR(DMSO-d 6 -i+3 drops CF 3 COOH) 6 114.3, 114.4,115.3, 115.5, 115.6, 116.2, 118.5, 123.1, 125.4, 125.5, 125.6, 129.4, 132.4, 132.9, 133.0, 135.2, 138.9, 140.9, 151.8, 153.5.

5-(p-Chlorophenyl)- 2- [2'-(benzimidazol-5"-yI)benzimidazol-5'-yI benzimidazole (JSK IV-47) A mixture of 5-forinyl-2-(benziinidazol-5'yl)benzimidazole (99 mg, 0.38 iniol) and (154 mg, 0.71 mmol) in nitrobenzene (5 inL) was heated at 150'C under N 2 overnight. The mixture was cooled to room temperature and chromatographed using 0-10% inethanol/ethylacetate to afford 85 ing of brownish yellow solid: mp>280*C; IR (KBr) 3046, 2820, 1426, 1282; 'H NMvR (DMSO-d 6 3 drops

CF

3 COOH) 6 7.56 2H, 7.82 2H, 7.88-8.2 1 (in, 6H), 8.48 (d, 1H, 8.63 1H) 8.72 11H), 9.69 1H); 3 C NMIR (DMSO-d 6 3 drops

CF

3 COOH) 6 111.8, 113.8, 114.7, 115.8, 116.1, 117.7, 123.0, 124.1, 125.2, 125.3, 129.2, 129.3, 131.9, 132.1, 133.0, 133.1, 137.2, 138,5, 139.3, 141.6, 150.8, 153.8.

WO 96/36612 PCT/US96/06853 18 4-Bromophenylenediamine (JSK IV-35) To 2-nitro-4-bromoaniline (340 mg, 1.57 mmol) in absolute ethanol (20mL) was added SnCl 2 (1.50g, 7.91 mmol) and refluxed overnight. The reaction mixture was then basified to pH 11 with 2N NaOH and extracted with other to give 275 mg of product. This product was used without further purification for the synthesis of JSK IV-37.

4-Chlorophenylenediamine (JSK IV-67) To (304 mg, 1.76 mmol) in absolute ethanol (20 mL) was added SnCl 2 (1.68g, 8.86 mmol) and refluxed overnight. The reaction mixture was then basified to pH 11 with 2N NaOH and extracted with ether to give 250 mg (quantitative yield) of product. This product was used without further purification for the synthesis of JSK IV-68.

p-Chlorotributylphenyltin (JSK IV-42) 4-Bromochlorobenzene (3.2 g, 16.62 mmol) was dissolved in dry THF (20mL). After bringing the reaction temperature down to -78C with an acetone/dry ice bath, nBuLi(15.58 mL, 1.6M, equiv.) was added slowly and stirred at -78°C for 30 min. Tributyltinchloride (6.77 mL, 1.5 equiv.) was added and stirred overnight while bringing the reaction to room temperature. Reaction mixture was quenched by stirring the reaction flask open in air for 1 hour after which THF was rotavaporated off. Product was obtained as an oil (7.35g, 97%) after passing the mixture through a quick silicagel column eluting with 100% hexanes.

2-Nitro-5-(p-chlorophenyl)aniline (JSK IV-44) To JSK IV-42 (2.02 g, 5.04 mmol) and 2-nitro-4-bromoaniline (730 mg, 3.36 mmol) in DMF (18 mL) was added Pd(PPh 3 2 C1 2 (117.9 mg, 0.17 mmol) and PPh 3 (440.2 mg, 1.70 mmol) and heated at 120"C overnight. DMF was rotavaporated off and the mixture was separated on a silicagel column eluting with 5-10% ethylacetate/hexanes to give 270 mg of reddish solid.

WO 96/36612 PCT/US96/06853 19 4-(p-Chlorophenyl)phenylenediamine (JSK IV-46) JSK IV-44 (190 mg, 0.77 mmol) was dissolved in ethylacetate (100 mL) and after adding 10% Pd-C mg) was reduced by hydrogenation (45 psi). Product (quantitative yield) was used in JSK IV-47 without further purification.

Example 8. Bioassays A. Topoisomerase I-Mediated DNA Cleavage Assays DNA topoisomerase I was purified from calf thymus gland as reported previously by B. D. Halligan et al., J. Biol. Chem., 260, 2475 (1985). Plasmid YEpG was also purified by the alkali lysis method followed by phenol deproteination and CsCl/ethidium isopycnic centrifugation as described by T.

Mariatis et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Labs, NY (1982) at pages 149-185. The end-labeling of the plasmid was accomplished as previously described by L. F. Liu et al., J. Biol. Chem., 258, 15365 (1983). The cleavage assays were performed as previously reported by A. Y. Chen et al., Cancer Res., 53, 1332 (1993). Human topoisomerase was isolated as a recombinant fusion protein using a T7 expression system.

B. Cytotoxicity assay The cytotoxicity was determined using the as MTT-microtiter plate tetrazolinium cytotoxicity assay (MTA) following the procedures of F. Denizot et al., J. Immunol. Methods, 89, 271 (1986); J. Carmichael et al., Cancer Res., 47, 936 (1987) and T. J. Mosmann et al., Immunol. Methods. 65, 55 (1983). The human lymphoblast RPMI 8402 and its camptothecin-resistant variant cell line, were provided by Dr. Toshiwo Andoh (Aichi Cancer Center Research Institute, Nagoya, Japan). See, for example, T. Andoh et al., Adv. Pharmacol., 29B, 93 (1994). The cytotoxicity assay was performed using 96-well microtiter plates.

Cells were grown in suspension at 37 "C in 5% CO 2 and maintained by regular WO 96/36612 PCT/US96/06853 passage in RPMI medium supplemented with 10% heat inactivated fetal bovine serum, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (0.1 mg/ml).

For determination of ICso, cells were exposed continuously with varying concentrations of drug concentrations and MTT assays were performed at the end of the fourth day.

The drug sensitive human epidermoid carcinoma KB3-1 cell line (S.

Aliyama et al., Somatic Cell Mol. Genet., 11, 117 (1985)) and its vinblastineselected multidrug-resistant variant KBV-1 cells W. Shen et al., Science, 32, 643 (1986)) were provided by Dr. Michael Gottesmann (National Cancer Institute, Bethesda, ML). These cells were grown as monolayer cultures at in 5% CO 2 and maintained by regular passage in Dulbecco's minimal essential medium supplemented with 10% heat inactivated fetal bovine serum. KBV-1 cells were similarly maintained except they were grown in the presence of 1 /,g/ml vinblastine.

C. Results As shown on Table 1, comparison of compounds 10-16 with Hoechst 33342 as inhibitors of topoisomerase I demonstrated that several of these trisbenzimidazoles had similar potency.

WO 96/36612 PCT/US96/06853 21 Table 1.

Topoisomerase I-mediated DNA Cleavage and Cytotoxicity of Bis- and Trisbenzimidazoles Cytotoxicity IC 50 a (zM) Topo I- Cell Lines mediated Compound DNA cleavage b RPMI CPT-K5 KB3-1 KBV-1 Hoechst 33342 1 0.03 0.9 0.01 1.2 1.1 14 28 N.D. N.D.

11 1 25c 25 c N.D. N.D.

12 100 7.6 20 N.D. N.D.

13 2 0.09 0.58 0.58 0.35 14 3.3 0.16 5.8 0.05 0.09 2 0.035 2.5 0.02 0.02 16 2 0.035 2.5 0.02 0.01 19 1000 25c N.D. N.D. N.D.

JSKIV-37 1 1.40 1.40 JSKIV-47 10 0.09 0.20 JSKIV-68 1 1.04 0.65 a) IC 5 0 has been calculated after 4 days of continuous drug exposure. N.D. Not determined.

b) Topoisomerase I cleavage values are reported as REC, Relative Effective Concentration, i.e. concentrations relative to Hoechst 33342, whose value is arbitrarily assumed as 1, that are able to produce the same cleavage on the plasmid DNA in the presence of calf thymus topoisomerase I. Cleavage is calculated from the intensity of the strongest Hoechst specific band.

c) No indication of cytotoxicity were considered indicative of IC 50 values substantially greater than the highest doses assayed.

0 While 10 and 11 exhibited similar potency in their inhibition of topoisomerase I as observed with Hoechst 33342, both of these compounds failed to exhibit significant cytotoxicity towards the human lymphoblast cell line, RPMI 8402. However, this may be due to the inability of the pure compound to penetrate the target cells, which may be overcome by selection of a suitable carrier, such as liposomes. The 5-phenyl substituted trisbenzimidazole, 13, was approximately onehalf as potent as Hoechst 33342 as a topoisomerase I inhibitor. In contrast to and 11, however, it had significant cytotoxicity towards the human lymphoblast cell line, RPMI 8402 cells. As observed with Hoechst 33342, 13 was also effective

I

WO 96/36612 PCTIUS96/06853 22 against camptothecin-resistant CPT-K5 cells. The relative resistance of Hoechst 33342 and 13, expressed as the ratio of the IC 50 values of the resistant verses the drug sensitive cell line, is approximately 30 fold as compared to the relative resistance of camptothecin which is 2,500 fold, as reported by A. Y. Chen et al., Cancer Res, 53, 1332 (1993). A similar effect was observed in another pair of cell lines; 13 has an IC 0 s of 0.015 /g/ml in the human ovarian tumor cell line, A2780, relative to an ICo 5 of 0.03 /.g/ml in CPT-2000, a variant of A2780 selected for camptothecin-resistance and known to contain a mutant camptothecin-resistant topoisomerase I. The 5-n-propyl trisbenzimidazole derivative, 12, was much less active than either 10, 11, or 13 as an inhibitor of topoisomerase I. Its weak activity as a topoisomerase I inhibitor correlated with its weak cytotoxicity. The activity of several of these compounds were also evaluated using recombinant human topoisomerase I. Several of these analogs induced similar DNA cleavage in the presence of human topoisomerase I as compared to that observed with topoisomerase I isolated from calf thymus.

The cytotoxic activity of Hoechst 33342 and 13 was also evaluated against KB 3-1 and KB V-1 cells. The primary difference between these cell lines is in the degree to which human MDRI (P-glycoprotein) is expressed. Recent studies have demonstrated that antineoplastic agents which are cationic at physiological pH are more likely to serve as substrates for MDR1 and, therefore, are likely to be less effective against cells that overexpress P-glycoprotein. In view of the fact that Hoechst 33342 is extensively protonated at physiological pH, it is not surprising that the IC 5 0 differs by approximately two-orders of magnitude for KB 3- 1 as compared to KB V-l cells, as reported by A.Y. Chen et al., Adv. Pharmacol., 245, 29B (1994). In contrast to Hoechst 33342, there is little difference between the

IC

5 0 values observed for 13 in these two cell lines. Thus, 13 appears not to be a substrate for human MDR1. This data indicate that these trisbenzimidazole derivatives may have significant chemotherapeutic advantages as compared to WO 96/36612 PCT/US96/06853 23 Hoechst 33342 or pibenzimol (Hoechst 33258), 2'-(4-hydroxyphenyl)-5-(4-methyl- 1-piperazinyl)- 2 ,5'-bi-1H-benzimidazole.

These data indicate that substitution of these trisbenzimidazole with a substituent can yield derivatives which are active as topoisomerase I inhibitors and cytotoxic to tumor cells. Trisbenzimidazoles substituted at the 5- position with either a or 4-pyridyl group, 14-16, were evaluated for their potency as topoisomerase I inhibitors and for cytotoxicity as summarized in Table 1. These analogs, similar to 13, have activity as topoisomerase I inhibitors. The 3- and 4pyridyl analogs, 15 and 16, are somewhat more active than the 2-pyridyl derivative, 14, as topoisomerase I inhibitors as well as cytotoxic agents. As was observed with 13, these pyridyl-substituted tribenzimidazoles had similar cytotoxicity to KB 3-1 cells as well as to KB V-l cells which overexpress MDR1. A principal advantage of these heteroaryl substituted trisbenzimidazoles as compared to Hoechst 33342 is their efficacy against cell lines which express MDR1.

All publications and patents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques.

However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims (20)

1. A compound of the formula: 54 X-(Ar)n z 1 14 wherein Ar is (C 6 -C, 2 )aryl, mono or bis-(C 1 -C 4 )alkyl-substituted(C 6 C1 2 )aryl, (C 6 -C 12 )arYl(C 1 -C 4 )alkyl, mono or bis-(C 1 -C 4 )alkyl- SUbstituted(C 6 -C 1 2 )arYl(CI-C 4 )alkyl, or to 12-membered)heteroaryl comprising 1-3 N, S or non-peroxide 0, wherein N is unsubstituted or is substituted with (CI-C 4 )alkyl, (CX- 4 )cycloalkyl, or cyclopropylmethyl, said Ar being optionally fused to the benzo moiety; X is CN, CHO, OH, acetyl, CF 3 O(C 1 -C 4 )allcyl, O(C 3 -C 4 )CYClOalll or cyclopropylmethoxy, NO 2 NH 2 halogen, halo(C 1 -C 4 )alkyl, halo(C 3 -C 4 )cycloalkyl, or halocyclopropylmethyl; each Y is individually H, (C 1 -C 4 )alkyl, (C 3 C 4 )cycloalkyl, cyclopropylmethyl, or arailkyl; Y' is H, -C 4 )alkyl, (C 3 C 4 )cycloalkyl, or cyclopropylmethyl; each Z is individually H, (CI- C 4 )alkyl, (C 3 -C 4 )cycloalkyl, cyclopropylmethyl, halogen, halo(Cl- C 4 )alkyl, halo(C 3 -C 4 )cycloallcyl, or halocyclopropyhnethyl; n is 1; or a pharmaceutically acceptable salt thereof. The compound of cli 1 wherein Ar is at the The compound of claim I wherein Ar is a 5-6 membered heteroaryl. :30 comprising 1-2 N, S or non-peroxide 0 atoms.

4. The compound of claim 2 wherein Ar is phenyl or pyridyl. The compound of claim 4 wherein pyridyl is 2-pyridyl, 3-pyridyl or 4- pyridyl.

6. The compound of claim 1 wherein Y' is H.

7. The compound of claims 4 or 5 wherein each Y is H.

8. The compound of claim 4 wherein Ar is phenyl.

9. The compound of claim 8 wherein X is halogen. The compound of claim 9 wherein X is Cl.

11. The compound of claim 10 wherein X-Ar is p-chlorophenyl.

12. The compound of claim 11 wherein Y' is H; each Y is H; and each Z is H.

13. The compound of claim 1 wherein Ar is benzo.

14. The compound of claim 13 wherein Ar is i: 25 15. The compound of claim 13 wherein Ar is 5,6-benzo.

16. A compound of the fornula: 4 Y wherein X is CN, CHO, OH, acetyl, CF 3 O(C 1 -C 4 )allcyl, O(C 3 -C 4 )cycloallcyl, or cyclopropylmethoxy, NO 2 NH 2 halogen, halo(C,-C 4 )alkyl, halo(C 3 C 4 )cycloalkyl, or halocyclopropylmethyl; each Y is individually H, (C 1 -C 4 )alkyl, (C 3 -C 4 )cycloalkyl, cyclopropylmethyl, or aralkyl; Y' is H, (CI -C 4 )alkyl, (C 3 C 4 )cycloalkyl, or cyclopropylmethyl; each Z is individually H, (Cj-C 4 )alkyl, (C 3 C 4 )cycloallyl, cyclopropylmethyl, halogen, halo(C 1 -C 4 )alkyl, halo(C 3 C 4 )cycloailkyl, or halocyclopropylmethyl; or a pharmaceutically acceptable salt thereof.

17. The compound of claim 16 wherein X is CHO or CN.

18. The compound of claim 17 wherein Y' is H.

19. The compound of claim 16 wherein X is halogen. 25 20. The compound of claim 19 wherein X is Cl.

21. The compound of claim 19 wherein X is Br. S S S S. *1 S

22. The compound of claims 20 or 21 wherein Y' is H; each Y is H; and each Z is H. 30 27

23. The compound of claim 16 wherein Z is H, F, CH 3 or CF 3

24. A compound according to claim 1 substantially as herein described with reference to any one of the examples. A method of inhibiting the growth of a mammalian tumor cell comprising contacting a tumor cell susceptible thereto, with an effective inhibitory amount of a compound of any one of claims 1 to 25 in combination with a pharmaceutically acceptable carrier.

26. A therapeutic composition comprising the compound of any one of claims 1 to in combination with a pharmaceutically acceptable carrier. JO 0 0 S.. Oa a 0 0 SOC 0000 S a *e 0 06

650. a S 0e a 6 600O 0 o 0 o S S o 17 k