Nitro and amino substituted topoisomerase agents

This application claims priority to U.S. Provisional Patent Application No. 60/402,138, filed Aug. 09, 2002.

The invention described herein was made with government support under Grant Numbers CA39662 and CA077433 from the National Cancer Institute. The United States Government has certain rights in the invention.

DNA-topoisomerases are enzymes which are present in the nuclei of cells where they catalyze the breaking and rejoining of DNA strands, which control the topological state of DNA. Recent studies also suggest that topoisomerases are also involved in regulating template supercoiling during RNA transcription. There are two major classes of mammalian topoisomerases. DNA-topoisomerase-I catalyzes changes in the topological state of duplex DNA by performing transient single-strand breakage-union cycles. In contrast, mammalian topoisomerase II alters the topology of DNA by causing a transient enzyme bridged double-strand break, followed by strand passing and resealing. Mammalian topoisomerase II has been further classified as Type II α and Type II β. The antitumor activity associated with agents that are topoisomerase poisons is associated with their ability to stabilize the enzyme-DNA cleavable complex. This drug-induced stabilization of the enzyme-DNA cleavable complex effectively converts the enzyme into a cellular poison.

Several antitumor agents in clinical use have potent activity as mammalian topoisomerase II poisons. These include adriamycin, actinomycin D, daunomycin, VP-16, and VM-26 (teniposide or epipodophyllotoxin). In contrast to the number of clinical and experimental drugs which act as topoisomerase II poisons, there are currently only a limited number of agents which have been identified as topoisomerase I poisons. Camptothecin and its structurally-related analogs are among the most extensively studied topoisomerase I poisons. Recently, bi- and terbenzimidazoles (Chen et al., Cancer Res. 1993, 53, 1332–1335; Sun et al., J. Med. Chem. 1995, 38, 3638–3644; Kim et al., J. Med. Chem. 1996, 39, 992–998), certain benzo[c]phenanthridine and protoberberine alkaloids and their synthetic analogs (Makhey et al., Med. Chem. Res. 1995, 5, 1–12; Janin et al., J. Med. Chem. 1975, 18, 708–713; Makhey et al., Bioorg. & Med. Chem. 1996, 4, 781–791), as well as the fungal metabolites, bulgarein (Fujii et al., J. Biol. Chem. 1993, 268, 13160–13165) and saintopin (Yamashita et al., Biochemistry 1991, 30, 5838–5845) and indolocarbazoles (Yamashita et al., Biochemistry 1992, 31, 12069–12075) have been identified as topoisomerase I poisons. Other topoisomerase poisons have been identified including certain benzo[i]phenanthridine and cinnoline compounds (see LaVoie et al., U.S. Pat. No. 6,140,328 and WO 01/32631). Despite these reports there is currently a need for additional agents that are useful for treating cancer.

Applicant has discovered compounds that show inhibitory activity against topoisomerase I and/or topoisomerase II, and compounds that are effective cytotoxic agents against cancer cells, including drug-resistant cancer cells. Accordingly, the invention provides a compound of the invention which is a compound of formula I:

wherein:

one of R 1 , R 2 and R 3 is nitro or NR a R b ; and the remaining R 1 , R 2 , and R 3 are each independently hydrogen, (C 1 –C 6 )alkyl, (C 3 –C 6 )cycloalkyl, NR a R b , COOR c , OR d ; or R 1 and R 2 , or R 2 and R 3 taken together are methylenedioxy, 1,2-ethylenedioxy, or 1,3-propylenedioxy; R 4 is hydrogen, hydroxy, or fluoro; each R 5 , R 6 , R 7 , and R 8 is independently hydrogen, (C 1 –C 6 )alkyl, (C 3 –C 6 )cycloalkyl, NR a R b , COOR c , OR d ; or R 5 and R 6 , R 6 and R 7 , or R 7 and R 8 taken together are methylenedioxy, 1,2-ethylenedioxy, or 1,3-propylenedioxy; R 9 is (C 1 –C 6 )alkyl substituted with one or more (e.g. 1, 2, 3, or 4) solubilizing groups; W is N or CH; X is two hydrogens, ═O, ═S, or ═NR e ; R a and R b are each independently hydrogen or (C 1 –C 6 )alkyl, or R a and R b together with the nitrogen to which they are attached form a pyrrolidino, piperidino or morpholino ring; each R c is hydrogen, (C 1 –C 6 )alkyl, aryl, or aryl(C 1 –C 6 )alkyl; each R d is hydrogen, (C 1 –C 6 )alkyl, (C 1 –C 6 )alkanoyl, aryl, or aryl(C 1 –C 6 )alkyl; and R e is hydrogen, (C 1 –C6)alkyl, aryl, or aryl(C 1 –C 6 )alkyl; or a pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising a effective amount of a compound of the invention in combination with a pharmaceutically acceptable diluent or carrier.

The invention also provides a method of inhibiting cancer cell growth, comprising administering to a mammal afflicted with cancer, an amount of a compound of the invention, effective to inhibit the growth of said cancer cells.

The invention also provides a method comprising inhibiting cancer cell growth by contacting said cancer cell in vitro or in vivo with an amount of a compound of the invention, effective to inhibit the growth of said cancer cell.

The invention also provides a compound of the invention for use in medical therapy, preferably for use in treating cancer, for example, solid tumors, as well as the use of a compound of the invention for the manufacture of a medicament useful for the treatment of cancer, for example, solid tumors.

The invention also provides processes and novel intermediates disclosed herein which are useful for preparing compounds of the invention. Some of the compounds of formula I are useful to prepare other compounds of formula I.

FIG. 1 shows the structure of compounds of the invention (4a, 4b, 5a, 5b, 6a, and 6b) as well as compounds 1, 2, and 3.

FIG. 2 illustrates the synthesis of representative compounds of formula I (4a, 4b, 5a, 5b, 6a, and 6b) and compound 3.

The following definitions are used, unless otherwise described.

“(C 1 –C 6 )alkyl” denotes both straight and branched carbon chains with 1, 2, 3, 4, 5, or 6, carbon atoms, but reference to an individual radical such as “propyl” embraces only the straight chain radical, a branched chain isomer such as “isopropyl” being specifically referred to. “(C 3 –C 6 )cycloalkyl” denotes a carbocyclic ring with 3, 4, 5, or 6, carbon atoms. “Aryl” denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Examples of aryl include phenyl, indenyl, and naphthyl. “Aryl(C 1 –C 6 )alkyl” refers to a group of the formula aryl-(C 1 –C 6 )alkyl-, where aryl and (C 1 –C 6 )alkyl are as defined herein. 1. “Solubilizing group (R z )” is a substituent that increases the water solubility of the compound of formula I compared to the corresponding compound lacking the R z substituent (i.e. wherein R z is hydrogen). Examples of solubilizing groups include (C 1 –C 6 )alkoxycarbonyl (e.g.—CO 2 Me), cyano, halo, hydroxy, mercapto, oxo (═O), carboxy (COOH), nitro, pyrrolidinyl, piperidinyl, imidazolidinyl, imidazolinyl, piperazinyl, morpholinyl, thiomorpholinyl, and —NR f R g , wherein R f and R g may be the same or different and are chosen from hydrogen, (C 1 –C 6 )alkyl, and (C 3 –C 6 )cycloalkyl.

Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.

A specific value for W is N.

Another specific value for W is CH.

A specific value for R 1 is nitro.

Another specific value for R 1 is NR a R b .

A specific compound of formula (I) is a compound wherein R 2 and R 3 are each independently hydrogen, or OR d , wherein each R d is hydrogen or (C 1 –C 6 )alkyl.

A specific compound of formula (I) is a compound wherein R 2 and R 3 are each hydrogen.

A specific value for R 2 is nitro or NR a R b .

Another specific value for R 2 is nitro.

Another specific value for R 2 is NR a R b .

A specific compound of formula (I) is a compound wherein R 1 and R 3 are each independently hydrogen, or OR d , wherein each R d is hydrogen or (C 1 –C 6 )alkyl.

A specific compound of formula (I) is a compound wherein R 1 and R 3 are each hydrogen.

A specific value for R 3 is nitro or NR a R b .

Another specific value for R 3 is nitro.

Another specific value for R 3 is NR a R b .

A specific compound of formula (I) is a compound wherein R 1 and R 2 are each independently hydrogen, or OR d , wherein each R d is hydrogen or (C 1 –C 6 )alkyl.

A specific compound of formula (I) is a compound wherein R 1 and R 2 are each hydrogen.

A specific value for R 4 is hydrogen.

Another specific value for R 4 is fluoro or hydroxy.

A specific compound of formula (I) is a compound wherein one of R 2 and R 3 is nitro or NR a R b .

A specific compound of formula (I) is a compound wherein each R 5 , R 6 , R 7 , and R 8 is independently hydrogen, or OR d .

A specific value for R 5 is hydrogen.

A specific value for R 6 OR d .

A specific value for R 7 OR d .

A specific value for R 8 is hydrogen.

A specific compound of formula (I) is a compound wherein or R 5 and R 6 taken together are methylenedioxy, 1,2-ethylenedioxy, or 1,3-propylenedioxy.

A specific compound of formula (I) is a compound wherein or R 6 and R 7 taken together are methylenedioxy, 1,2-ethylenedioxy, or 1,3-propylenedioxy.

A specific compound of formula (I) is a compound wherein R 7 and R 8 taken together are methylenedioxy, 1,2-ethylenedioxy, or 1,3-propylenedioxy.

A specific value for R 9 is (C 1 –C 6 )alkyl substituted with one or more hydroxy groups.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one hydroxy group.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one or more mercapto groups.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one mercapto group.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one or more carboxy groups.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one carboxy group.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one or more NR f R g groups.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one NR f R g group.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one or more NH 2 groups.

Another specific value for R 9 is a (C 1 –C 6 )alkyl substituted with one NH 2 group.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one or more N(CH 3 ) 2 groups.

Another specific value for R 9 is a (C 1 –C 6 )alkyl substituted with one N(CH 3 ) 2 group.

Another specific value for R 9 is (C 1 –C 6 )alkyl substituted with one or more N(CH 2 CH 3 ) 2 groups.

Another specific value for R 9 is a (C 1 –C 6 )alkyl substituted with one N(CH 2 CH 3 ) 2 group.

Another specific value for R 9 is a (C 1 –C 6 )alkyl substituted with one or more (C 1 –C 6 )alkoxycarbonyl, cyano, halo, hydroxy, mercapto, oxo, carboxy, nitro, pyrrolidinyl, piperidinyl, imidazolidinyl, imidazolinyl, piperazinyl, morpholinyl, thiomorpholinyl, or —NR f R g groups.

Another specific value for R 9 is a (C 2 –C 4 )alkyl substituted with one or two groups selected from hydroxy, mercapto, carboxy, amino, methylamino, ethylamino, dimethylamino, and diethylamino.

Another specific value for R 9 is 2-hydroxyethyl.

Another specific value for R 9 is 3-hydroxypropyl.

Another specific value for R 9 is 2-hydroxypropyl.

Another specific value for R 9 is —CH 2 CH 2 —NR f R g wherein R f and R g are each independently hydrogen or (C 1 –C 6 )alkyl.

Another specific value for R 9 is —CH 2 CH 2 —NR f R g wherein R f and R g are each independently methyl or ethyl.

A specific compound is any one of compounds 4a, 4b, 5a, 5b, 6a, and 6b; or a pharmaceutically acceptable salt thereof.

Another specific compound is a compound of formula (I) wherein R 1 is hydrogen; R 2 is nitro; R 3 is hydrogen; R 4 is hydrogen; R 5 is hydrogen; R 6 and R 7 taken together are methylenedioxy; R 8 is hydrogen; R 9 is 2-(N,N-dimethylamino)ethyl or 2-(N,N-diethylamino)ethyl; W is N or CH; and X is ═O; or a pharmaceutically acceptable salt thereof.

Another specific compound is a compound of formula (I) wherein R 1 is hydrogen; R 2 is hydrogen; R 3 is nitro; R 4 is hydrogen; R 5 is hydrogen; R 6 and R 7 taken together are methylenedioxy; R 8 is hydrogen; R 9 is 2-(N,N-dimethylamino)ethyl or 2-(N,N-diethylamino)ethyl; W is N or CH; and X is ═O; or a pharmaceutically acceptable salt thereof.

A compound of formula I can be prepared as described in the Examples below and as illustrated in FIG. 2 . Reaction of a 4-chloroquinoline with the requisite amine provides compound 7. Amide formation provides compounds 8b–8d, which can be cyclized to provide compounds 4a–6a. Subsequent reduction of the nitro group in compounds 4a–6a provides aryl amines 4b–6b.

The starting materials employed in the synthetic methods described herein are commercially available, have been reported in the scientific literature, or can be prepared from readily available starting materials using procedures known in the field. It may be desirable to optionally use a protecting group during all or portions of the above described synthetic procedures. Such protecting groups and methods for their introduction and removal are well known in the art. See Greene, T. W.; Wutz, P. G. M. “Protecting Groups In Organic Synthesis” second edition, 1991, New York, John Wiley & Sons, Inc.

It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine topoisomerase inhibition activity or cytotoxic activity using the standard tests described herein, or using other similar tests which are well known in the art.

In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal, for example, sodium, potassium or lithium, or alkaline earth metal, for example calcium, salts of carboxylic acids can also be made.

The compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, that is, orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, for example, orally, 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 compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders 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, fructose, lactose or aspartame 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 or fructose 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 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 infusible 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 or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, 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 dispersions 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 freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of formula I in a liquid composition, such as a lotion, will be from about 0.1–25 wt-%, preferably from about 0.5–10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1–5 wt-%, preferably about 0.5–2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg per day, e.g., from about 1 to about 60 mg/kg of body weight per day or about 2 to 50 mg/kg per day.

The compound may conveniently be administered in unit dosage form; for example, containing 5 to 1,000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

The ability of a compound of the invention to effect topoisomerase I or II mediated DNA cleavage can be determined using pharmacological models that are well known to the art, for example, using a model like Test A described below.

Human topoisomerase I was expressed in E. Coli and isolated as a recombinant fusion protein using a T7 expression system as described by Gatto, B., et al., Cancer Res. 1996, 56, 2795–2800. Plasmid YepG was purified by the alkali lysis method followed by phenol deproteination and CsCl/ethidium isopycnic centrifugation method as described by Maniatis, T., et al., J. Molecular Cloning, a Laboratory Manual ; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. 1982; pp 149–185. The end-labeling of the plasmid was accomplished by digestion with a restriction enzyme followed by end-filling with Klenow polymerase as previously described by Tewey, K. M., et al., Science 1984, 226, 466–468. The cleavage assays were performed as previously reported (see Gatto, B., et al., Cancer Res. 1996, 56, 2795–2800; and Wang, H., et al., Biochemistry 2001, 40; 3316–3323). The drug and the DNA in presence of topoisomerase I was incubated for 30 minutes at 37° C. The reactions were terminated by the addition of 5 μl of 5% SDS and 1 mg/ml protein kinase K with an additional 1 hour of incubation at 37° C. Samples were then alkali denatured by the addition of NaOH, EDTA, sucrose, and bromophenol blue to final concentrations of 75 mM, 2.5%, and 0.05 mg/ml, respectively, prior to loading onto a neutral agarose gel. After development of the gels, typically 24-hour exposure was used to obtain autoradiograms outlining the extent of DNA fragmentation. Topoisomerase I-mediated DNA cleavage values are reported as REC, Relative Effective Concentration, i.e. concentrations relative to topotecan, whose value is arbitrarily assumed as 1.0, that are able to produce the same cleavage on the plasmid DNA in the presence of human topoisomerase I. Data for representative compounds of the invention is provided in Table 1 below.

The cytotoxic effects of a compound of the invention can be determined using pharmacological models that are well known to the art, for example, using a model like Test B described below.

Test B. Inhibition of Cell Growth: MTT-Microtiter Plate Tetrazolinium Cytotoxicity Assay (RPMI 8402, CPT-K5 Cells)

The cytotoxicity was determined using the MTT-microtiter plate tetrazolinium cytotoxicity assay (MTA—See Mosmann, T., J. Immunol. Methods 1983, 65, 55–63; Carmichael, J., et al., Cancer Res. 1987, 47, 936–942; and Denizot, F., et al., J. Immunol. Methods 1986, 89, 271–277). The human lymphoblast RPMI8402 and its camptothecin-resistant variant cell line, CPT-K5 were provided by Dr. Toshiwo Andoh (Aichi Cancer Center Research Institute, Nagoya, Japan—See Andoh, T., et al., Adv. in Pharmacology 1994, 29B, 93–103). 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 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). Each well was plated with either 2,000 RPMI8402 cells or 4,000 CPT-K5 cells. For determination of IC 50 , cells were exposed continuously for 4 days to varying concentrations of drug, and MTT assays were performed at the end of the fourth day. Each assay was performed with a control that did not contain any drug. All assays were performed at least twice in 6 replicate wells. Data for representative compounds is provided in Table 1 below.

The compounds of the invention can function as cytotoxic agents against tumor cell lines, including multi-drug resistant tumor cell lines. Thus, the compounds are useful to treat cancer and can be used to treat tumors that are resistant to other specific chemotherapeutic agents.

Topoisomerase inhibitors are also known to possess antibacterial, antifungal, antiprotozoal, antihelmetic, antipsoriatic, and antiviral activity. Accordingly, the topoisomerase inhibitors of the invention may also be useful as antibacterial, antifungal, antiprotozoal, antihelmetic, antipsoritic, or antiviral agents. In particular, compounds of the invention that demonstrate little or no activity as mammalian topoisomerase I poisons, because of the possibility of similar molecular mechanism of action, could be highly active and selective antibacterial, antifungal, antiprotozoal, antihelmetic, antipsoriatic, or antiviral agents. Thus, certain compounds of the invention may be particularly useful as systemic antibacterial, antifungal, antiprotozoal, antihelmetic, antipsoriatic, or antiviral agents in mammals. The invention also provides the use of a compound of the invention for the manufacture of a medicament useful for producing an antibacterial, antifungal, antiprotozoal, antihelmetic, antipsoriatic, or antiviral effect in a mammal.

As used herein, the term “solid mammalian tumors” include cancers of the head and neck, lung, mesothelioma, mediastinum, esophagus, stomach, pancreas, hepatobiliary system, small intestine, colon, rectum, anus, kidney, ureter, bladder, prostate, urethra, penis, testis, gynecological organs, ovarian, breast, endocrine system, skin central nervous system; sarcomas of the soft tissue and bone; and melanoma of cutaneous and intraocular origin. The term “hematological malignancies” includes childhood leukemia and lymphomas, Hodgkin's disease, lymphomas of lymphocytic and cutaneous origin, acute and chronic leukemia, plasma cell neoplasm and cancers associated with AIDS. The preferred mammalian species for treatment are humans and domesticated animals.

The invention will now be illustrated by the following non-limiting Examples.

General Experimental

Melting points were determined with a Thomas-Hoover Unimelt capillary melting point apparatus. Column chromatography refers to flash chromatography conducted on SiliTech 32–63 μm, (ICN Biomedicals, Eschwegge, Ger.) using the solvent systems indicated. Infrared spectral data (IR) were obtained on a Perkin-Elmer 1600 Fourier transform spectrophotometer and are reported in cm −1 . Proton ( 1 H NMR) and carbon ( 13 C NMR) nuclear magnetic resonance were recorded on a Varian Gemini-200 Fourier Transform spectrometer. NMR spectra (200 MHz 1 H and 50 MHz 13 C) were recorded in the deuterated solvent indicated with chemical shifts reported in δ units downfield from tetramethylsilane (TMS). Coupling constants are reported in hertz (Hz). Mass spectra were obtained from Washington University Resource for Biomedical and Bio-organic Mass Spectrometry within the Department of Chemistry at Washington University, St. Louis, Mo.