WO2009076212A1 - Catalytic topoisomerase ii inhibitors - Google Patents

Catalytic topoisomerase ii inhibitors Download PDF

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Publication number
WO2009076212A1
WO2009076212A1 PCT/US2008/085697 US2008085697W WO2009076212A1 WO 2009076212 A1 WO2009076212 A1 WO 2009076212A1 US 2008085697 W US2008085697 W US 2008085697W WO 2009076212 A1 WO2009076212 A1 WO 2009076212A1
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Prior art keywords
alkyl
pyrrolidinyl
optionally substituted
morpholino
hydrogen
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PCT/US2008/085697
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French (fr)
Inventor
David M. Ferguson
John R. Goodell
Hiroshi Hiasa
Joel W. Slaton
Daniel D. Billadeau
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Regents Of The University Of Minnesota
Mayo Foundation For Medical Education And Research
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Publication of WO2009076212A1 publication Critical patent/WO2009076212A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Topoisomerase (topo) inhibitors have found widespread applications in treating various forms of cancer. Most topoisomerase II anticancer drugs are referred to as poisons because they function by trapping the enzyme-DNA cleavable complex, terminating DNA replication. Well known poisons include doxorubicin, etoposide, mitoxantrone, and amsacrine. While these compounds have proven their utility in the war on cancer, they are highly toxic and mutagenic, greatly limiting their use.
  • a series of acridine-based anti-herpes agents with topoisomerase activity was disclosed by Goodell et al. (Goodell, J. R.; Madhok, A. A.; Hiasa, H.; Ferguson, D. M. Synthesis and evaluation of acridine- and acridone-based anti-herpes agents with topoisomerase activity. Bioorg. Med. Chem. 2006, 14, 5467-80).
  • these antiviral acridines may also be useful as anti-cancer agents.
  • anticancer agents that act by unique mechanisms, have higher selectivity for their target, and/or that produce less severe side-effects following administration.
  • anticancer agents particularly topoisomerase II inhibitors, that have enhanced metabolic stability, prolonged half-lives or improved oral bioavailability in animals.
  • topoisomerase II inhibitors that can cross the blood brain barrier or distribute within the central nervous system including the brain.
  • the invention provides compounds that intercalate into DNA and inhibit the catalytic activity of hTopo II.
  • Representative compounds of formula I have been shown to block the formation of etoposide-induced covalent hTopo II-DNA complexes, suggesting that the compounds inhibit an early step of the hTopo II catalytic cycle by preventing binding of the enzyme to DNA.
  • the compounds function as catalytic inhibitors. Unlike poisons, these catalytic inhibitors do not induce DNA cleavage and fragmentation and hence show lower toxicities.
  • Representative compounds of formula I were also found to be selective inhibitors of topoisomerase II and exhibit little or no effect on topoisomerase I activity.
  • the invention provides a method to induce apoptosis in a cell comprising contacting the cell with a compound of formula I:
  • R 4 is -NR c R d , or -OR 6 ;
  • 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 piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-
  • Rc is hydrogen or (Ci-C 6 )alkyl
  • R 4 is -NR c R d , or -OR 5 ;
  • R 3 and R b are each independently hydrogen or (d-C 6 )alkyl, or R 3 and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C 1 - C 6 )alkyl;
  • R c is hydrogen or (Q-C ⁇ alkyl;
  • R 4 is -NR 0 Ra, or -OR 6 ;
  • R a and R b are each independently hydrogen or (Ci-C 6 )alkyl, or R a and R b together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C 1 - C 6 )alkyl;
  • R 4 is -NR c R d , or -OR 6 ;
  • R a and R b are each independently hydrogen or (d-C 6 )alkyl, or R a and R b together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C 1 - C 6 )alkyl;
  • R 4 is -NR 0 R d , or -OR 8 ;
  • R a and R b are each independently hydrogen or (Ci-C 6 )alkyl, or R a and R b together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C 1 - C 6 )alkyl;
  • R c is hydrogen or (C 1 -C 6 )alkyl
  • R a and R b are each independently hydrogen or (C]-C 6 )alkyl, or R a and R b together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C 1 - C 6 )alkyl;
  • R 4 is -NR 0 R d , or -OR 6 ;
  • R 3 and R b are each independently hydrogen or (CrC 6 )alkyl, or R 3 and R b together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C 1 -
  • R c is hydrogen or (CrC 6 )alkyl
  • C 6 cycloalkyl(C r C 6 )alkyl, (CrC 6 )alkoxy, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, (C r C 6 )alkanoyl, halo(C r C 6 )alkyl, hydroxy(C r C 6 )alkyl, (C r C 6 )alkoxycarbonyl, (C 1 -C 6 )alkylthio, aryl(C r
  • R 4 is -NRcR d , or -OR 6 ;
  • R a and R b are each independently hydrogen or (d-C 6 )alkyl, or R 3 and R b together with the nitrogen to which they are attached form a piperidine, thiomorphorine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci- C 6 )alkyl;
  • R 4 is -NR 0 R d , or -OR 8 ;
  • R 3 and R b are each independently hydrogen or or R a and R b together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C 1 - C 6 )alkyl;
  • R c is hydrogen or (d-C 6 )alkyl; and
  • R d is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (C !
  • R 4 is -NRcR d , or -OR 6 ;
  • R 3 and R b are each independently hydrogen or (C t -C ⁇ alkyl, or R a and R b together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C 1 - C 6 )alkyl;
  • R c is hydrogen or (CrC 6 )alkyl
  • representative compounds of the invention are capable of crossing the blood brain barrier. No other current topo drugs possess this beneficial property. Accordingly, such compounds may be particularly useful for treating certain specific cancers (e.g. recurrent pediatric cancers, cancers in the central nervous system).
  • certain embodiments of the invention provide methods, uses, and compositions comprising compounds of formula I, and salts thereof, for inducing apoptosis of a cell, inhibiting topoisomerase II in a cell, and/or treating cancer, in the central nervous system (e.g., the brain) of a mammal (e.g., a human in need of such treatment), by administering the compound of formula I, or a salt thereof, to the mammal to a location outside the central nervous system of the mammal.
  • the central nervous system e.g., the brain
  • a mammal e.g., a human in need of such treatment
  • the invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of formula (I) or salts thereof.
  • Representative compounds of formula I have been shown to inhibit pancreatic cancer cell growth, ultimately leading to apoptosis. The compounds have also been found to inhibit pancreatic cancer cell proliferation in mouse xenografts. Representative compounds of formula I were also found to inhibit human cancer growth in vitro including the cancer cell lines DU- 145 (Prostate), HCT-116 (colon), Hepa-lclc7 (liver), H460 (non-small lung), MCF-7 (breast), SU86.86 (Pancreatic), MEL (melanoma), OCL-3 (ovarian), and REH (kidney).
  • FIG. 1 illustrates the induction of apoptosis and PARP cleavage following treatment with compounds 1-4.
  • FIG. 2 illustrates in vivo inhibition of pancreatic tumor cell proliferation. Representative sections from SU86.86 xenografts treated with either diluent, compound 2 or 3 and stained for (A) BrdU or (B) Ki-67. One hundred tumor cells were counted per microscopic field. The percentage of positively stained cells from 5 randomly selected fields is graphically depicted.
  • FIG. 3 illustrates the results of the topoisomerase I assay from Example 7.
  • halo is fluoro, chloro, bromo, or iodo.
  • Alkyl, alkoxy, etc. denote both straight and branched groups; 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.
  • 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.
  • Heteroaryl encompasses a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C 1 - C 4 )alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms comprising one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X).
  • (Ci-C 6 )alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec- butyl, pentyl, 3-pentyl, or hexyl;
  • (C 3 -C 6 )cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
  • (C 3 -C 6 )cycloalkyl(CrC 6 )alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl;
  • (Ci-C 6 )alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, is
  • a specific value for R 3 and R b independently is (d-C 6 )alkyl.
  • a specific value for R a and for R b is ethyl.
  • R a and R b together with the nitrogen to which they are attached is a morpholino ring.
  • a specific value for R a and R b together with the nitrogen to which they are attached is a piperazine ring that is optionally substituted at the 4-position nitrogen with (d-C 6 )alkyl.
  • a specific value for R a and R b together with the nitrogen to which they are attached is a
  • R 4 A specific value for R 4 is -NR 0 R 11 .
  • R 0 is hydrogen or (d-C 6 )alkyl
  • R 4 is -NH 2
  • R 0 is hydrogen or (CrC 6 )alkyl
  • R c is hydrogen and for R d is piperidinyl, that is substituted with arylCCrCe ⁇ lkyl.
  • R 4 is l-benzylpiperidin-4-ylamino.
  • R ⁇ is hydrogen and for R d is (C r C 6 )alkyl that is substituted with one or more heteroaryl.
  • a specific value for R 0 is hydrogen and for Ra is (CrC 6 )alkyl that is substituted with an indolyl ring.
  • a specific value for R 4 is N-(2-indol-3-ylethyl)amino.
  • R 4 A specific value for R 4 is -OR e .
  • R e is phenyl
  • a specific value for each of R f and R g is hydrogen.
  • the cancer is pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, a leukemia, liver cancer, melanoma, brain cancer or kidney cancer,
  • the cancer is pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, or a leukemia.
  • the cancer is liver cancer, melanoma, kidney cancer, non-small lung cancer or non-refractory prostate cancer.
  • the cancer is liver cancer, melanoma, or kidney cancer.
  • the cancer is non-small lung cancer or non-refractory prostate cancer.
  • the cancer is refractory prostate cancer. In another embodiment the cancer is non-refractory prostate cancer.
  • the cancer is a cancer of the central nervous system (CNS).
  • CNS central nervous system
  • Another intermediate useful for preparing a compound of formula I is a compound of the following formula II
  • a salt of a compound of formula I can be useful as an intermediate for isolating or purifying a compound of formula I. Additionally, administration of a compound of formula I as a pharmaceutically acceptable acid or base salt 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, tartrate, succinate, benzoate, ascorbate, ⁇ - ketoglutarate, and ⁇ -glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • 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.
  • a sufficiently basic compound such as an amine
  • 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, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • the present compounds may be systemically administered, e.g., 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.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • 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.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • 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.
  • the ultimate dosage form should 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.
  • 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.
  • 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.
  • 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.
  • a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 1 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day. In one embodiment of the invention the dose is in the range of 1 to 10 mg/kg/day. In another embodiment of the invention the dose is in the range of 10 to 40 mg/kg/day
  • the compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
  • the invention provides a composition comprising a compound of the invention formulated in such a 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.
  • Compounds of the invention can also be administered in combination with other therapeutic agents, for example, other agents that are useful for the treatment of cancer. Examples of such agents include taxanes, cyclophosphamide, docetaxel, epirubicin, and 5-FU.
  • the invention also provides a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier.
  • the invention also provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging material, and instructions for administering the compound of formula I or the pharmaceutically acceptable salt thereof and the other therapeutic agent or agents to an animal to treat cancer.
  • the cancer treatment methods of the invention can also be used in conjunction with radioimmunotherapy, wherein a therapeutic radioisotope (e.g. iodine-131) is administered along with a targeting agent (e.g. a monoclonal antibody) to deliver radiation directly to cancer cells.
  • a therapeutic radioisotope e.g. iodine-131
  • a targeting agent e.g. a monoclonal antibody
  • pancreatic cancer cell lines MiaPaCa-2, SU86.86 and BXPC-3 were obtained from ATCC (Rockville, MD) and were grown in DMEM medium containing 10% fetal calf serum and L- glutamine.
  • MTT assays were carried out over the indicated timecourse in the presence or absence of varying concentrations of compounds as previously described (Ougolkov et al., Cancer Research, 2005, 65, 2076-81).
  • apoptosis assays the indicated cell lines were treated as described in the text, harvested, and nuclei scored for apoptosis as previously described (Ougolkov et al., Cancer Research, 2005, 65, 2076-81). A minimum of 300 nuclei was analyzed per sample.
  • the SU86.86 pancreatic cancer cell line was incubated with diluent or a range of concentrations of compounds 1-4.
  • pancreatic cancer cells were incubated for 36 hours with diluent or the indicated compounds 1 thru 4 (15 ⁇ M final concentration). Samples were then fixed in 50% ethanol, rehydrated, treated with RNase, stained with 50 ⁇ g/ml propidium iodide in 0.1% sodium citrate, and analyzed (at least 20,000 events per sample) using a Becton Dickinson FACScan flow cytometer. Very small debris (forward scatter less than 10% of intact cells) was excluded from the analysis by gating. Cell-cycle profiles were determined using Modfit Software (Verity, Topsham, ME) as previously described (Bible and Kaufmann, Cancer Research, 1997; 57, 3375- 80).
  • Figure 1 shows the results following treatment of BXPC-3 cells with diluent or the indicated concentrations of compounds 1-4. Forty-eight hours post exposure, cells were collected and stained with Hoechst. Three hundred nuclei were counted per field and scored for apoptotic changes (fragmentation of the nucleus into multiple discrete fragments) and are graphically represented (lower Panel). Cell lysates from the same treated cells were probed for
  • PARP cleavage (upper panel), a hallmark of apoptosis.
  • ⁇ -Actin was used as a loading control.
  • Subcutaneous flank xenografts of SU86.86 tumor cells were established in female athymic nude mice to determine the effect of compounds 2 and 3 on tumor cell proliferation.
  • Tumor bearing mice (2 in each group) were injected intraperitoneally (IP) with either 50 ⁇ L of diluent (DMSO) in 500 ⁇ l PBS, or compound 2 (5 mg/kg) or 3 (10 mg/kg) dissolved in 50 ⁇ L of diluent in 500 ⁇ L of PBS.
  • the injections were given twice daily (every 12 hrs) over two days for a total of 4 injections. Three hours prior to sacrifice, mice were injected with 1 mg of BrdU.
  • Tumors were subsequently removed from the mice, fixed in paraformaldehyde and paraffin- embedded. Tumor sections were analyzed for proliferating tumor cells by performing immunohistochemical staining for either BrdU or the nuclear-associated proliferation antigen, Ki-67. Consistent with our in vitro cell-based assays, compared to diluent-treated animals, administration of either compound 2 or compound 3 resulted in a significant loss of BrdU+ and Ki-67+ tumor cells ( Figure 2). These data indicate that compounds 2 and 3 can abrogate tumor cell proliferation in vivo.
  • Compounds 1-4 were evaluated utilizing a MTT assay against nine cancer cell lines in vitro to determine the EC 50 value for cell growth inhibition.
  • the cancer cell lines investigated were DU-145 (Prostate), HCT-116 (colon), Hepa-lclc7 (liver), H460 (non-small lung), MCF-7 (breast), SU86.86 (Pancreatic), MEL (melanoma), OCL-3 (ovarian), and REH (kidney).
  • the following table summarizes the activities of compounds 1-4. Cancer cell growth inhibition against human cancers. a compound DU- 145 HCT-116 Hepa H460 MCF-7 SU86.86 MEL REH OCL-3
  • reaction was terminated by the addition of 1 ⁇ l of 10% (w/v) SDS and 2 ⁇ l of 10 mg/ml proteinase K followed by an additional 15-minute incubation at 37 °C. After extraction with 1 : 1 phenol: chloroform, samples were applied to 1% (w/v) agarose gels in TPE buffer (36 mM Tris, 30 mM NaH 2 PO 4 , and 1 mM EDTA) containing 10 ⁇ g/ml chloroquine. Gels were run at 45 V for 4.5 h, stained with 0.5 ⁇ g/ml ethidium bromide, and photographed under UV light.
  • Example 8 Mouse Xenograft (PC3-LN4 non-refractory prostate tumors)
  • mice were treated with twice daily 10 mg/kg IP injections of compound 2 (dissolved in ethanol) for 7 days. Tumor sizes were approximately 0.5 cm3 at the start of the treatment. Tumors sizes were measured for the 7 day dosing period and 2 days after dosing was completed. Compound 2 treated mice displayed a significant reduction in tumor growth as compared to the control group over the 9 day measuring period.
  • Example 10 The following illustrate representative pharmaceutical dosage forms, containing a compound of formula I ('Compound X 1 ), for therapeutic or prophylactic use in humans.
  • compositions may be obtained by conventional procedures well known in the pharmaceutical art.
  • Representative compounds of formula (I) have been shown to be selective for topoisomerase II over topoisomerase I. Accordingly, these selective inhibitors may possess fewer side effects or less severe side effects than other topoisomerase inhibitors.
  • Compounds of formula (I) also displayed unexpected metabolic stability and unexpectedly long half-lives in vivo. These attributes contributed to an enhanced pharmacokinetic(PK) profile for representative compounds of formula (I). This enhanced PK profile may be a significant factor regarding the in vivo efficacy of these compounds as demonstrated in the mouse xenograft model of Example 5.
  • Representative compounds of formula (I) have also been shown to distribute within the central nervous system (CNS) including the brain.
  • CNS central nervous system
  • the invention includes a method of detecting the location of a tumor in an animal comprising administering a compound of formula (I) to the animal, irradiating the tissue in the animal with an appropriate irradiation source and detecting the resulting emission of the compound by a suitable method of emission detection to ascertain the location of the tumor.
  • the invention also provides a method in which a compound of formula (I) is used to both locate a tumor in an animal and to treat said tumor in the animal.

Abstract

The invention provides methods and compositions useful for treating cancer, for selectively inhibiting topoisomerase II, and for inducing apoptosis.

Description

CATALYTIC TOPOISOMERASE II INHIBITORS
Related Application(s) This patent document claims the benefit of priority under 35 USC 119(a) to U.S. application serial No. 60/992,621, filed December 5, 2007, which application is herein incorporated by reference.
Background of the Invention Topoisomerase (topo) inhibitors have found widespread applications in treating various forms of cancer. Most topoisomerase II anticancer drugs are referred to as poisons because they function by trapping the enzyme-DNA cleavable complex, terminating DNA replication. Well known poisons include doxorubicin, etoposide, mitoxantrone, and amsacrine. While these compounds have proven their utility in the war on cancer, they are highly toxic and mutagenic, greatly limiting their use.
A series of acridine-based anti-herpes agents with topoisomerase activity was disclosed by Goodell et al. (Goodell, J. R.; Madhok, A. A.; Hiasa, H.; Ferguson, D. M. Synthesis and evaluation of acridine- and acridone-based anti-herpes agents with topoisomerase activity. Bioorg. Med. Chem. 2006, 14, 5467-80). In addition, it was suggested that these antiviral acridines may also be useful as anti-cancer agents.
Currently, there is a need for novel anticancer agents that act by unique mechanisms, have higher selectivity for their target, and/or that produce less severe side-effects following administration. There is also a need for anticancer agents, particularly topoisomerase II inhibitors, that have enhanced metabolic stability, prolonged half-lives or improved oral bioavailability in animals. There is also a need for topoisomerase II inhibitors that can cross the blood brain barrier or distribute within the central nervous system including the brain.
Summary of the Invention
The invention provides compounds that intercalate into DNA and inhibit the catalytic activity of hTopo II. Representative compounds of formula I have been shown to block the formation of etoposide-induced covalent hTopo II-DNA complexes, suggesting that the compounds inhibit an early step of the hTopo II catalytic cycle by preventing binding of the enzyme to DNA. As a result, the compounds function as catalytic inhibitors. Unlike poisons, these catalytic inhibitors do not induce DNA cleavage and fragmentation and hence show lower toxicities. Representative compounds of formula I were also found to be selective inhibitors of topoisomerase II and exhibit little or no effect on topoisomerase I activity.
Accordingly, in one embodiment the invention provides a method to induce apoptosis in a cell comprising contacting the cell with a compound of formula I:
Figure imgf000004_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen; R4 is -NRcRd, or -OR6;
Ra and Rb are each independently hydrogen or (C1-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-
C6)alkyl; Rc is hydrogen or (Ci-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(CrC6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (d-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (d-C6)alkylthio, aryl(Cr
C6)alkyl, and (C2-C6)alkanoyloxy; or R0 and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-C6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(Ci- C6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(C1-C6)alkyl, hydroxy(CrC6)alkyl, (CrC6)alkoxycarbonyl, (CrC6)alkylthio, aryl(CrC6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (CpC6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (CrC6)alkyl; or a pharmaceutically acceptable salt thereof.
In another embodiment the invention provides a method to inhibit topoisomerase II in a mammal comprising administering to the mammal a compound of formula I:
Figure imgf000005_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen; R4 is -NRcRd, or -OR5;
R3 and Rb are each independently hydrogen or (d-C6)alkyl, or R3 and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C1- C6)alkyl; Rc is hydrogen or (Q-C^alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (C1-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (C1-C6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(CrC6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(C1-C6)alkyl, hydroxy(CrC6)alkyl, (CrC6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Cr C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl; Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(Ci- C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkanoyl, halo(d-C6)alkyl, hydroxy(CrC6)alkyl, (d-C6)alkoxycarbonyl, (CrC6)alkylthio, aryl(CrC6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (d-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (C]-C6)alkyl; or a pharmaceutically acceptable salt thereof. In another embodiment the invention provides a method to induce apoptosis of cells in a mammal comprising administering to the mammal a compound of formula I:
Figure imgf000006_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 is -NR0Ra, or -OR6;
Ra and Rb are each independently hydrogen or (Ci-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C1- C6)alkyl;
Rc is hydrogen or (d-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (d-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (C1-C6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(Ci-C6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (d-C6)alkanoyl, halo(C1-C6)alkyl, hydroxy(d-C6)alkyl, (C]-C6)alkoxycarbonyl, (d-C6)alkylthio, aryl(Cr C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl; Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d- C6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(d-C6)alkyl, (C1-C6)alkoxycarbonyl, (d-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl; or a pharmaceutically acceptable salt thereof. In another embodiment the invention provides a method to treat cancer, in a mammal comprising administering a therapeutically effective amount of a compound of formula I:
Figure imgf000007_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 is -NRcRd, or -OR6;
Ra and Rb are each independently hydrogen or (d-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C1- C6)alkyl;
Rc is hydrogen or (d-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (d-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rj is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(C1-C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(d-C6)alkyl, hydroxy(CrC6)alkyl, (d-C6)alkoxycarbonyl, (CrC6)alkylthio, aryl(d- C6)alkyl, and (C2-C6)alkanoyloxy; or R0 and R<j together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl; Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d- C6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkanoyl, halo(C1-C6)alkyl, hydroxy(d-C6)alkyl, (C1-C6)alkoxycarbonyl, (d-C6)alkylthio, aryl(d-C6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (d-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl; or a pharmaceutically acceptable salt thereof: to the mammal.
In another embodiment the invention provides a pharmaceutical composition comprising a compound of formula I:
Figure imgf000008_0001
(I) wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 is -NR0Rd, or -OR8; Ra and Rb are each independently hydrogen or (Ci-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C1- C6)alkyl;
Rc is hydrogen or (C1-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (d-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Ra is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(CrC6)alkyl, (d-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (Ci-C6)alkoxycarbonyl, (CrC6)alkylthio, aryl(Cr C6)alkyl, and (C2-C6)alkanoyloxy; or Rς and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (CrC6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (C1-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(C1- C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (CrC6)alkoxycarbonyl, (CrC6)alkylthio, aryl(CrC6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (C]-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (C!-C6)alkyl; or a pharmaceutically acceptable salt thereof; a pharmaceutically acceptable diluent or carrier; and another anticancer agent. In another embodiment the invention provides the use of a compound of formula I:
Figure imgf000010_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 Is -NRcRd5 Or -ORe;
Ra and Rb are each independently hydrogen or (C]-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C1- C6)alkyl;
Rc is hydrogen or (d-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-
C6)cycloalkyl(CrC6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (Ci-C6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(CrC6)alkyl, (CrC6)alkoxycarbonyl, (d-C6)alkylthio, aryl(Cr C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d- C6)alkyl, (C,-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (Ci-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (CrC6)alkoxycarbonyl, (d-C6)alkylthio, aryl(d-C6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (CrC6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (CrC6)alkyl; or a pharmaceutically acceptable salt thereof; to prepare a medicament for inducing apoptosis in mammalian cells.
In another embodiment the invention provides the use of a compound of formula I:
Figure imgf000011_0001
wherein: one OfR1, R2, and R3 is -Q=O)NR3Rb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 is -NR0Rd, or -OR6;
R3 and Rb are each independently hydrogen or (CrC6)alkyl, or R3 and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C1-
C6)alkyl;
Rc is hydrogen or (CrC6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (C1-C6)alkyl, (C3-C6)cycloalkyl, (C3-
C6)cycloalkyl(CrC6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (CrC6)alkoxycarbonyl, (C1-C6)alkylthio, aryl(Cr
C6)alkyl, and (C2-C6)alkanoyloxy; or R0 and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (CrC6)alkyl; Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (C1-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d- C6)alkyl, (d-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(C1-C6)alkyl, hydroxy(CrC6)alkyl, (CrC6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(CrC6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (d-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (C]-C6)alkyl; or a pharmaceutically acceptable salt thereof; to prepare a medicament for inhibiting topoisomerase II in a mammal. In another embodiment the invention provides the use of a compound of formula I:
Figure imgf000012_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 is -NRcRd, or -OR6;
Ra and Rb are each independently hydrogen or (d-C6)alkyl, or R3 and Rb together with the nitrogen to which they are attached form a piperidine, thiomorphorine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci- C6)alkyl;
Rc is hydrogen or (d-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (d-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(d-C6)alkyl, (d-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (d-C6)alkanoyl, 1IaIo(C1 -C6)alkyl, hydroxy(CrC6)alkyl, (C1-C6)alkoxycarbonyl, (CrC6)alkylthio, aryl(Cr C6)alkyl, and (C2-C6)alkanoyloxy; or R0 and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (C]-C6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(C1- C6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (d-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(Ci-C6)alkyl, (CrC6)alkoxycarbonyl, (C1-C6)alkylthio, aryl(CrC6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (CrC6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl; or a pharmaceutically acceptable salt thereof; to prepare a medicament for treating cancer in a mammal.
In another embodiment the invention provides a compound of formula I:
Figure imgf000013_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 is -NR0Rd, or -OR8;
R3 and Rb are each independently hydrogen or
Figure imgf000013_0002
or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C1- C6)alkyl; Rc is hydrogen or (d-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (C!-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Ra is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3-
C6)cycloalkyl(C1-C6)alkyl, (d-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(CrC6)alkyl, (C1-C6)alkoxycarbonyl, (CrC6)alkylthio, aryl(d- C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Ra together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d- C6)alkyl, (Ci-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (d-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(d-C6)alkyl, (d-C6)alkoxycarbonyl, (CrC6)alkylthio, aryl(C]-C6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (d-C6)alkyl; or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of cancer.
In another embodiment the invention provides a novel compound of formula I:
Figure imgf000014_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen; R4 is -NRcRd, or -OR6;
R3 and Rb are each independently hydrogen or (Ct-C^alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (C1- C6)alkyl;
Rc is hydrogen or (CrC6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (CrC6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-
C6)cycloalkyl(C1-C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (CrC6)alkoxycarbonyl, (C1-C6)alkylthio, aryl(Cr C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or piperazine ring is optionally substituted with one or more (CrC6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(Cr C6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(d-C6)alkyl, hydroxy(CrC6)alkyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2- C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (CrC6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-C6)alkyl; or a pharmaceutically acceptable salt thereof.
In addition to the other embodiments described herein, it is believed that representative compounds of the invention are capable of crossing the blood brain barrier. No other current topo drugs possess this beneficial property. Accordingly, such compounds may be particularly useful for treating certain specific cancers (e.g. recurrent pediatric cancers, cancers in the central nervous system). Further, certain embodiments of the invention provide methods, uses, and compositions comprising compounds of formula I, and salts thereof, for inducing apoptosis of a cell, inhibiting topoisomerase II in a cell, and/or treating cancer, in the central nervous system (e.g., the brain) of a mammal (e.g., a human in need of such treatment), by administering the compound of formula I, or a salt thereof, to the mammal to a location outside the central nervous system of the mammal.
The invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of formula (I) or salts thereof.
Representative compounds of formula I have been shown to inhibit pancreatic cancer cell growth, ultimately leading to apoptosis. The compounds have also been found to inhibit pancreatic cancer cell proliferation in mouse xenografts. Representative compounds of formula I were also found to inhibit human cancer growth in vitro including the cancer cell lines DU- 145 (Prostate), HCT-116 (colon), Hepa-lclc7 (liver), H460 (non-small lung), MCF-7 (breast), SU86.86 (Pancreatic), MEL (melanoma), OCL-3 (ovarian), and REH (kidney).
Brief Description of the FJRures FIG. 1 illustrates the induction of apoptosis and PARP cleavage following treatment with compounds 1-4.
FIG. 2 illustrates in vivo inhibition of pancreatic tumor cell proliferation. Representative sections from SU86.86 xenografts treated with either diluent, compound 2 or 3 and stained for (A) BrdU or (B) Ki-67. One hundred tumor cells were counted per microscopic field. The percentage of positively stained cells from 5 randomly selected fields is graphically depicted. FIG. 3 illustrates the results of the topoisomerase I assay from Example 7.
Detailed Description
The following definitions are used, unless otherwise described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straight and branched groups; 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. 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. Heteroaryl encompasses a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C1- C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms comprising one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X).
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).
Specific 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.
Specifically, (Ci-C6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec- butyl, pentyl, 3-pentyl, or hexyl; (C3-C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C6)cycloalkyl(CrC6)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (Ci-C6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso- butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2-C6)alkenyl can be vinyl, allyl, 1- propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1- hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2-C6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3- pentynyl, 4-pentynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl; (C1- C6)alkanoyl can be acetyl, propanoyl or butanoyl; ImIo(C1 -C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2- trifluoroethyl, or pentafluoroethyl; hydroxy(CrC6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4- hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (C]-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (CrC6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; aryl(d-C6)alkyl can be phenethyl or benzyl; (C2-C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N- oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).
A specific value for R1 is -C(=O)NRaRb. A specific value for R2 is -C(=O)NRaRb. A specific value for R3 is -C(=O)NRaRb. A specific value for R3 and Rb independently is (d-C6)alkyl. A specific value for Ra and for Rb is ethyl.
A specific value for Ra and Rb together with the nitrogen to which they are attached is a morpholino ring.
A specific value for Ra and Rb together with the nitrogen to which they are attached is a piperazine ring that is optionally substituted at the 4-position nitrogen with (d-C6)alkyl. A specific value for Ra and Rb together with the nitrogen to which they are attached is a
4-methylpiperazine.
A specific value for R4 is -NR0R11.
A specific value for R0 is hydrogen or (d-C6)alkyl; and for Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (d-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (C]-C6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(d-C6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (C1-C6)alkoxycarbonyl, (CrC6)alkylthio, aryl(Cr C6)alkyl, and (C2-C6)alkanoyloxy.
A specific value for R4 is -NH2
A specific value for R0 is hydrogen or (CrC6)alkyl; and for Rd is piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(C1-C6)alkyl, (CrC6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(d-C6)alkyl, (C1-C6)alkoxycarbonyl, (CrC6)alkylthio, aryl(Cr C6)alkyl, and (C2-C6)alkanoyloxy.
A specific value for Rc is hydrogen and for Rd is piperidinyl, that is substituted with arylCCrCe^lkyl. A specific value for R4 is l-benzylpiperidin-4-ylamino.
A specific value for Rς is hydrogen and for Rd is (CrC6)alkyl that is substituted with one or more heteroaryl.
A specific value for R0 is hydrogen and for Ra is (CrC6)alkyl that is substituted with an indolyl ring. A specific value for R4 is N-(2-indol-3-ylethyl)amino.
A specific value for R4 is -ORe.
A specific value for Re is phenyl.
A specific value for each of Rf and Rg is hydrogen.
In one embodiment the cancer is pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, a leukemia, liver cancer, melanoma, brain cancer or kidney cancer,
In another embodiment the cancer is pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, or a leukemia.
In another embodiment the cancer is liver cancer, melanoma, kidney cancer, non-small lung cancer or non-refractory prostate cancer.
In another embodiment the cancer is liver cancer, melanoma, or kidney cancer.
In another embodiment the cancer is non-small lung cancer or non-refractory prostate cancer.
In another embodiment the cancer is refractory prostate cancer. In another embodiment the cancer is non-refractory prostate cancer.
In another embodiment the cancer is a cancer of the central nervous system (CNS).
Processes for preparing compounds of formula I are provided as further embodiments of the invention and are illustrated by the following procedures in which the meanings of the generic radicals are as given above unless otherwise qualified. An intermediate useful for preparing a compound of formula I, is a compound of the following formula II
Figure imgf000020_0001
wherein: one OfR1, R2, and R3 is -C(=O)C1 and the remaining two OfR1, R2, and R3 are each hydrogen; and R4 is Cl.
Another intermediate useful for preparing a compound of formula I, is a compound of the following formula II
Figure imgf000020_0002
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen; and R4 is Cl. hi cases where compounds are sufficiently basic or acidic, a salt of a compound of formula I can be useful as an intermediate for isolating or purifying a compound of formula I. Additionally, administration of a compound of formula I as a pharmaceutically acceptable acid or base salt 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, tartrate, 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, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
Thus, the present compounds may be systemically administered, e.g., 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 should 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. 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, e.g., from about 1 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day. In one embodiment of the invention the dose is in the range of 1 to 10 mg/kg/day. In another embodiment of the invention the dose is in the range of 10 to 40 mg/kg/day
The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a 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. Compounds of the invention can also be administered in combination with other therapeutic agents, for example, other agents that are useful for the treatment of cancer. Examples of such agents include taxanes, cyclophosphamide, docetaxel, epirubicin, and 5-FU. Accordingly, in one embodiment the invention also provides a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier. The invention also provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging material, and instructions for administering the compound of formula I or the pharmaceutically acceptable salt thereof and the other therapeutic agent or agents to an animal to treat cancer.
The cancer treatment methods of the invention can also be used in conjunction with radioimmunotherapy, wherein a therapeutic radioisotope (e.g. iodine-131) is administered along with a targeting agent (e.g. a monoclonal antibody) to deliver radiation directly to cancer cells.
The invention will now be illustrated by the following non-limiting Examples.
Chemistry (general). All reagents were purchased from commercial suppliers such as Sigma-Aldrich Chemical Co., Lancaster Chemical Co, Acros Chemical Co., etc. ACS Reagent grade or better solvents were used without further purification unless otherwise noted. Water was purified by Millipore filtration system. Column chromatography was conducted using silica gel 60 (40-63 microns) and thin-layer chromatography was conducted using EMD Chemical silica gel 60 F2S4 on aluminum sheets. 1H Nuclear Magnetic Resonance spectra were collected on a Varian Mercury 300 MHz instrument and a Varian Mercury 600 MHz instrument using CDCl3 and DMSOd6 as solvents. High resolution mass spectra (HRMS) were collected from a TOF- ESI Agilant LC-MS and analyzed using the Analyst QS software.
Example 1. Preparation of Compounds 1-4
As shown in Scheme 1, the synthesis of acridine target compounds 1-4 began with a modified Ullmann-Goldberg coupling between bromoterephthalic acid (5) and aniline, producing the anthranilic intermediate 6. This reaction was catalyzed by copper, with CuI and pyridine as co- catalysts responsible for maintaining the catalytic cycle of Cu0, reducing the reaction time and increasing yields. In order to synthesize acridines substituted in the 4-positions, different starting reagents were required in the Ullmann-Goldberg coupling. Hence, the reaction between 2- chlorobenzoic acid (7) and the appropriate 2-aminobenzoic acid (8) generated the desired substitution. Cyclization of the anthranilic intermediate 6 or 9 with POCl3 generated the 9- chloroacridine intermediate 10 in addition to producing an acid chloride of the second carboxylic acid. The reaction of this intermediate with phenol under mild heating produced the stable 9- phenoxyacridine 11. The final step entailed reacting the phenoxy intermediate 11 with the desired amine producing the substituted 9-aminoacridine 12.
Figure imgf000026_0001
(1) (2) (3) (4)
Figure imgf000026_0002
Figure imgf000026_0003
Scheme 1. Synthesis of acridine compounds. Reagents and conditions: (a) Cu. CuI, pyridine, K2CO3 , H2O, reflux (b) POCl3, reflux, (c) phenol, 6O0C; (d; amine, 12O0C. Compound 1 (9-(2-(lJfir-Indol-3-vl)-ethvlamino)-acridig-4-vl)-(4-methvl-piperazin-l-vl)- methanone. (4-Methyl-piperazin-l-yl)-(9-phenoxy-acridin-4-yl)-methanone (10 mmol) and 2 g of tryptamine and heated to 120 0C (reflux) for 15 minutes. The reaction was cooled and run on a silica column to purify the product and remove excess amine. A solvent ratio of 15:1
CH2Cl2MeOH was used for the first 200 mL followed by 15:1:0.1 CH2Cl2:Me0H:NEt3 until the product eluted from the column. The fractions with product were collect and concentrated. The yellow product was dissolved in EtOAc and precipitated out with petroleum ether or hexanes. Yield = 71.2%; 1H NMR (600MHz, CDCl3) δ 2.18 (s br , IH), 2.32 (s, 3H), 2.43 (s br, IH), 2.58 (s br, IH), 2.69 (s br, IH), 3.25 (s br, 4H), 3.88 (s br, IH), 4.2 (s br, 3H), 5.28 (s br, IH), 7.07 (s br, IH), 7.15 (t, IH), 7.25 (m, 3H), 7.42 (d, IH), 7.61 (m, 2H), 7.64 (d, IH), 7.97 (m br, 3H), 8.14 (s br, IH); HRMS (C29H30N5O) [M+H]+: found m/z 464.2414, calcd 464.2445. Anal. (C29H29N5O-0.75H2O) C, H, N.
Compound 2 (9-(l-Benzyl-piperidin-4-ylamino)-acridin-3-yl)-(4-methyl-piperazin-l-yl)- methanone. Yield = 40.1%; 1H NMR (600MHz, CDCl3) δ 1.76 (m br, 2H), 2.08 (m br, 4H), 2.34 (s, 3H), 2.41 (s br, 2H), 2.54 (s br, 2H), 2.88 (s br, 2H), 3.52 (s, 2H), 3.61 (s br, 2H), 3.88 (s br, 3H), 4.74 (s br, IH), 7.26 (m, IH), 7.31 (m, 4H), 7.46 (m, 2H), 7.73 (t, IH), 8.07 (d, IH), 8.08 (s, IH), 8.12 (d, IH), 8.14 (d, IH); HRMS (C31H36N5O) [M+H]+: found m/z 494.2894, calcd 494.2914. Anal. (C31H35N5O-0.5H2O) C, H, N.
Comp_ound_3 (9-(2-(l/Mndol-3-yl)-ethylamino)-acridin-3-yl)-(4-methyl-piperazin-l-yl)- methanone. Yield = 29.6%; 1H NMR (600MHz, CDCl3) δ 2.33 (s, 3H), 2.39 (s br, 2H), 2.52 (s br, 2H), 3.25 (t, 2H), 3.56 (s br, 2H), 3.87 (s br, 2H), 4.23 (t, 2H), 7.09 (s, IH), 7.17 (t, IH), 7.26 (t, IH), 7.31 (m, 2H), 7.43 (d, IH), 7.67 (m, 2H), 7.92 (d, IH), 7.96 (d, IH), 8.02 (s, IH), 8.06 (d, IH), 8.21 (s br, IH); HRMS (C29H30N5O) [M+H]+: found m/z 464.2414, calcd 464.2445. Anal. (C29H29N5O-I^H2O) C, H, N.
Compound 4 9-(2-(l/ir-Indol-3-yl)-ethylamino)-acridine-3-carboxylic acid diethylamide. Yield = 45.5%; 1H NMR (600MHz, CDCl3) δ 1.14 (s br, 3H), 1.30 (s br, 3H), 3.32 (s br, 4H), 3.60 (s br, 2H), 4.29 (s br, 2H), 7.13 (m, 2H), 7.24 (m, 3H), 7.43 (d, IH), 7.59 (t, IH), 7.63 (d, IH), 7.84 (s br, IH), 7.94 (d, IH), 8.06 (d, IH), 8.10 (s, IH), 8.48 (s br, IH); HRMS (C28H29N4O) [M+H]+: found m/z 437.2308, calcd 437.2336. Anal. (C28H28N5O(USH2O) C, H, N.
Example 2. MTT Cell Proliferation and Apoptosis Assay
The ability of representative compounds of the invention to inhibit cell proliferation and to promote apoptosis was evaluated using the following MTT cell proliferation and apoptosis assay. The pancreatic cancer cell lines MiaPaCa-2, SU86.86 and BXPC-3 were obtained from ATCC (Rockville, MD) and were grown in DMEM medium containing 10% fetal calf serum and L- glutamine.
MTT assays were carried out over the indicated timecourse in the presence or absence of varying concentrations of compounds as previously described (Ougolkov et al., Cancer Research, 2005, 65, 2076-81). For apoptosis assays, the indicated cell lines were treated as described in the text, harvested, and nuclei scored for apoptosis as previously described (Ougolkov et al., Cancer Research, 2005, 65, 2076-81). A minimum of 300 nuclei was analyzed per sample. The SU86.86 pancreatic cancer cell line was incubated with diluent or a range of concentrations of compounds 1-4. Cell proliferation was measured 72 hrs following addition of compounds and % growth inhibition compared to diluent-treated cells was measured using the colorimetric MTT assay. This is a representative example of 3 independent experiments performed in triplicate. The following table shows the IC5os of compounds 1-4 for the MTT assay for three pancreatic cell lines.
IC5o's estimated from MTT assay.
Cell Line
MiaPaCa-2 SU86.86 BXPC-3
Compound IC50 (μM) IC50 (μM) IC50 (μM)
1 4 ± 0.7 10 ± 0.8 18 ± 2.8
2 1 ± 0.25 6 ± 0.4 7 ± 0.35 3 6 ± 1.3 8 ± 1.2 12 ± 1.5 4 11 ± 1.6 10 ± 1.5 14 ± 1.8
EExxaammpple 3. Cell Cycle Assay
The ability of representative compounds of formula (I) to effect cell cycles was evaluated using the following cell cycle analysis.
The indicated pancreatic cancer cells were incubated for 36 hours with diluent or the indicated compounds 1 thru 4 (15 μM final concentration). Samples were then fixed in 50% ethanol, rehydrated, treated with RNase, stained with 50 μg/ml propidium iodide in 0.1% sodium citrate, and analyzed (at least 20,000 events per sample) using a Becton Dickinson FACScan flow cytometer. Very small debris (forward scatter less than 10% of intact cells) was excluded from the analysis by gating. Cell-cycle profiles were determined using Modfit Software (Verity, Topsham, ME) as previously described (Bible and Kaufmann, Cancer Research, 1997; 57, 3375- 80).
Cell cycle arrest by acridine compounds 1-4. Results tabulated after 36 hr treatment.
Cell Line
MiaPaCa-2 SU86.86 BXPC-3 ampound G1/S/G2M G1/S/G2M G1/S/G2M
DMSO 46/36/17 39/42/19 50/37/13
1 32/47/21 56/27/17 83/10/7
2 28/52/20 53/29/18 77/16/7
3 29/51/20 50/26/24 81/12/7
4 32/50/18 60/24/16 76/20/4
Example 4. Induction of Apoptosis
The ability of representative compounds of the invention to induce apoptosis was evaluated using the following assay.
Figure 1 shows the results following treatment of BXPC-3 cells with diluent or the indicated concentrations of compounds 1-4. Forty-eight hours post exposure, cells were collected and stained with Hoechst. Three hundred nuclei were counted per field and scored for apoptotic changes (fragmentation of the nucleus into multiple discrete fragments) and are graphically represented (lower Panel). Cell lysates from the same treated cells were probed for
PARP cleavage (upper panel), a hallmark of apoptosis. β-Actin was used as a loading control.
The following table shows the percentages of cells in apoptosis in three different cell lines following 48 hour exposure to compounds 1-4. Cell Line
Compound MiaPaCa-2 SU86.86 BXPC-3
1 (50 μM) 29 ± 4 23 ± 6 32 ± 8
2 (25 μM) 36 ± 9 30 ± 5 52 ± 11 3 (15 μM) 20 ± 5 34 ± 7 44 ± 6 4 (15 μM) 42 ± 7 17 ± 3 40 ± 5
Example 5. Mouse Xenografts
The effects of representative compounds of formula (I) in a mouse xenograft model were evaluated using the following assay.
Subcutaneous flank xenografts of SU86.86 tumor cells were established in female athymic nude mice to determine the effect of compounds 2 and 3 on tumor cell proliferation. Tumor bearing mice (2 in each group) were injected intraperitoneally (IP) with either 50 μL of diluent (DMSO) in 500 μl PBS, or compound 2 (5 mg/kg) or 3 (10 mg/kg) dissolved in 50 μL of diluent in 500 μL of PBS. The injections were given twice daily (every 12 hrs) over two days for a total of 4 injections. Three hours prior to sacrifice, mice were injected with 1 mg of BrdU. Tumors were subsequently removed from the mice, fixed in paraformaldehyde and paraffin- embedded. Tumor sections were analyzed for proliferating tumor cells by performing immunohistochemical staining for either BrdU or the nuclear-associated proliferation antigen, Ki-67. Consistent with our in vitro cell-based assays, compared to diluent-treated animals, administration of either compound 2 or compound 3 resulted in a significant loss of BrdU+ and Ki-67+ tumor cells (Figure 2). These data indicate that compounds 2 and 3 can abrogate tumor cell proliferation in vivo.
Example 6. Cancer Cell Lines
Compounds 1-4 were evaluated utilizing a MTT assay against nine cancer cell lines in vitro to determine the EC50 value for cell growth inhibition. The cancer cell lines investigated were DU-145 (Prostate), HCT-116 (colon), Hepa-lclc7 (liver), H460 (non-small lung), MCF-7 (breast), SU86.86 (Pancreatic), MEL (melanoma), OCL-3 (ovarian), and REH (kidney). The following table summarizes the activities of compounds 1-4. Cancer cell growth inhibition against human cancers.a compound DU- 145 HCT-116 Hepa H460 MCF-7 SU86.86 MEL REH OCL-3
1 37 29 56 16 37 7 17 20 40
2 32 29 27 39 26 21 15 21 20
3 17 28 25 32 19 16 5 4 15
4 12 11 11 18 13 16 16 15 18 "Values reported are EC50 (μM).
Example 7. Topoisomerase I Assay
To investigate the effect of compounds 1, 2, 3, and 4 on topo I catalytic activity, 400 ng of supercoiled plasmid 06 was incubated for 30 minutes at 37 0C in a 25 μl reaction mixture containing 2 units of enzyme (one unit of topo I relaxes 50% of the input plasmid) and the indicated final concentration of compound 1, 2, 3, or 4 (added from a concentrated DMSO stock and diluted in topo dilution buffer) in reaction buffer consisting of 100 mM NaCl, 50 mM Tris- HCl (pH 7.5 at 21 0C), 0.5 mM EDTA, 0.5 mM DTT, and 50 μg/ml BSA. The reaction was terminated by the addition of 1 μl of 10% (w/v) SDS and 2 μl of 10 mg/ml proteinase K followed by an additional 15-minute incubation at 37 °C. After extraction with 1 : 1 phenol: chloroform, samples were applied to 1% (w/v) agarose gels in TPE buffer (36 mM Tris, 30 mM NaH2PO4, and 1 mM EDTA) containing 10 μg/ml chloroquine. Gels were run at 45 V for 4.5 h, stained with 0.5 μg/ml ethidium bromide, and photographed under UV light.
As shown in Figure 3 compounds 1, 2, 3 and 4 failed to inhibit topoisomerase I activity. Supercoiled plasmid was incubated with 2 units of topo I in the absence (lane 2) or presence of the positive control pyrazoloacridine (PA, 8 μM) or compounds 1, 2, 3 or 4 (lanes 4-11) at a final concentration of 50 or 100 μM as indicated. Lane 1 is DNA incubated without enzyme. Results are representative of 4 experiments. As can be seen in a comparison of lanes 3 and 4-11, compounds 1-4 failed to inhibit topo I relaxation of supercoiled DNA (SC, supercoiled DNA; R, relaxed DNA).
Example 8. Mouse Xenograft (PC3-LN4 non-refractory prostate tumors)
The effects of representative compounds of formula (I) in a mouse xenograft bearing PC3-LN4 non-refractory prostate tumors were evaluated. Animals were injected with human PC3-LN4 cells. After approximately two weeks, the mice were treated with twice daily 10 mg/kg IP injections of compound 2 (dissolved in ethanol) for 7 days. Tumor sizes were approximately 0.5 cm3 at the start of the treatment. Tumors sizes were measured for the 7 day dosing period and 2 days after dosing was completed. Compound 2 treated mice displayed a significant reduction in tumor growth as compared to the control group over the 9 day measuring period.
Example 9. Preparation of Representative Compounds of Formula (I)
The following representative compounds of formula (I) (18a-19q) were prepared using procedures similar to those described in Example 1. The synthesis and characterization of these compounds is further described in Goodell et al. (Goodell, J. R.; Madhok, A. A.; Hiasa, H.; Ferguson, D. M. Synthesis and evaluation of acridine- and acridone-based anti-herpes agents with topoisomerase activity. Bioorg. Med. Chem. 2006, 14, 5467-80).
Figure imgf000033_0001
Compounds 18a - 19q
Comp. X Ring Sub Ri R2
18a N 4 N-methylpiperazinyl phenoxy
18b N 4 morpholino phenoxy
18c N 4 diethylamino phenoxy
19a N 4 N-methylpiperazinyl 4-amino-N-benzylpiperdinyl
19b N 4 morpholino 4-amino-N-benzylpiperdinyl
19c N 4 diethylamino 4-amino-N-benzylpiperdinyl
19e N 4 morpholino tryptamino
19f N 4 diethylamino tryptamino
ISd N 3 N-methylpiperazinyl phenoxy
ISc N 3 diethylamino phenoxy
19h N 3 diethylamino 4-amino-N-benzylpiperdinyl
18f N 2 N-methylpiperazinyl phenoxy
18g N 2 diethylamino phenoxy
19k N 2 N-methylpiperazinyl 4-amino-N-benzylpiperdinyl
191 N 2 diethylamino 4-amino-N-benzylpiperdinyl
19m N 2 N-methylpiperazinyl tryptamino
19n N 2 diethylamino tryptamino
19o N 2 diethylamino amino
19p N 2 diethylamino N,N-dimethylethylenediamino
19q N 2 diethylamino N-methyl-piperazinyl
Example 10. The following illustrate representative pharmaceutical dosage forms, containing a compound of formula I ('Compound X1), for therapeutic or prophylactic use in humans.
Ci) Tablet 1 mg/tablet
Compound X= 100.0
Lactose 77.5
Povidone 15.0
Croscarmellose sodium 12.0
Microcrystalline cellulose 92.5
Magnesium stearate M
300.0 CiD Tablet 2 mg/tablet
Compound X= 20.0
Microcrystalline cellulose 410.0
Starch 50.0
Sodium starch glycolate 15.0
Magnesium stearate £0
500.0
(iii) Capsule mg/capsule Compound X= 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5
Pregelatinized starch 120.0 Magnesium stearate 3J)
600.0
Civ) Iniection I d mg/ml) mg/ml
Compound X= (free acid form) 1.0
Dibasic sodium phosphate 12.0
Monobasic sodium phosphate 0.7
Sodium chloride 4.5
1.0 N Sodium hydroxide solution
(pH adjustment to 7.0-7.5) q.s.
Water for injection q.s. ad 1 mL fv) Iniection 2 (10 mg/ml) mg/ml
Compound X= (free acid form) 10.0
Monobasic sodium phosphate 0.3
Dibasic sodium phosphate 1.1
Polyethylene glycol 400 200.0
01 N Sodium hydroxide solution
(pH adjustment to 7.0-7.5) q.s.
Water for injection q.s. ad 1 mL
(Vi) Aerosol mg/can
Compound X= 20.0
Oleic acid 10.0
Trichloromonofluoromethane 5,000.0
Dichlorodifluoromethane 10,000.0
Dichlorotetrafluoroethane 5,000.0
The above formulations may be obtained by conventional procedures well known in the pharmaceutical art. Representative compounds of formula (I) have been shown to be selective for topoisomerase II over topoisomerase I. Accordingly, these selective inhibitors may possess fewer side effects or less severe side effects than other topoisomerase inhibitors. Compounds of formula (I) also displayed unexpected metabolic stability and unexpectedly long half-lives in vivo. These attributes contributed to an enhanced pharmacokinetic(PK) profile for representative compounds of formula (I). This enhanced PK profile may be a significant factor regarding the in vivo efficacy of these compounds as demonstrated in the mouse xenograft model of Example 5. Representative compounds of formula (I) have also been shown to distribute within the central nervous system (CNS) including the brain. Accordingly, the ability of these compounds to cross the blood brain barrier and accumulate in organs such as the brain after oral dosing is unexpected as there are no clinically available topoisomerase inhibitors that treat cancers of the CNS (e.g. brain). Furthermore, spectroscopically detected emissions of compounds of formula (I) following dosing in an animal (e.g. oral, intravenous, intraperitoneal) have been detected in the animal tissue and thus may be useful in locating tumor cells in an animal. Accordingly, the invention includes a method of detecting the location of a tumor in an animal comprising administering a compound of formula (I) to the animal, irradiating the tissue in the animal with an appropriate irradiation source and detecting the resulting emission of the compound by a suitable method of emission detection to ascertain the location of the tumor. The invention also provides a method in which a compound of formula (I) is used to both locate a tumor in an animal and to treat said tumor in the animal.
All publications, patents, and patent documents 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

CLAIMSWhat is claimed is:
1. A method to induce apoptosis in a cell comprising contacting the cell with a compound of formula I:
Figure imgf000036_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two of Ri, R2, and R3 are each hydrogen;
R4 is -NR0Rd, or -OR6;
Ra and Rb are each independently hydrogen or (Ci-C6)alkyl, or R3 and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
R0 is hydrogen or (Ci-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d-C6)alkyl, (C1- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci- C6)alkyl; Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(Ci-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-C6)alkyl; or a pharmaceutically acceptable salt thereof.
2. A method to inhibit topoisomerase II in a mammal comprising administering to the mammal a compound of formula I:
Figure imgf000037_0001
wherein: one of Ri, R2, and R3 is -C(=O)NRaRb and the remaining two of Ri, R2, and R3 are each hydrogen;
R4 is -NRcRd, or -OR6;
Ra and Rb are each independently hydrogen or (Ci-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
Rc is hydrogen or (Ci-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (d-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d-C6)alkyl, (Cr C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C]-C6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (C1- C6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(Ci-C6)alkyl, (Cr C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(C1-C6)alkyl, (C1-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (C]-C6)alkyl; or a pharmaceutically acceptable salt thereof.
3. The method of claim 2 wherein topoisomerase II is inhibited at least 2-fold more than topoisomerase I.
4. The method of claim 2 wherein topoisomerase II is inhibited at least 5-fold more than topoisomerase I.
5. The method of claim 2 wherein topoisomerase II is inhibited at least 10-fold more than topoisomerase I.
6. The method of claim 2 wherein topoisomerase II is inhibited at least 50-fold more than topoisomerase I.
7. The method of claim 2 wherein topoisomerase II is inhibited at least 100-fold more than topoisomerase I.
8. The method of any one of claims 2-7 wherein topoisomerase II is inhibited catalytically.
9. A method to induce apoptosis of cells in a mammal comprising administering to the mammal a compound of formula I:
Figure imgf000039_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 is -NRcRd, or -OR6;
Ra and Rb are each independently hydrogen or (CrC6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
Rc is hydrogen or (Ci-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (d-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(C1-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; or R0 and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci- C6)alkyl; Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(C1-C6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-C6)alkyl; or a pharmaceutically acceptable salt thereof.
10. A method to treat cancer in a mammal comprising administering a therapeutically effective amount of a compound of formula I:
Figure imgf000040_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two of Ri, R2, and R3 are each hydrogen;
R4 is -NR0Rd, or -ORe;
R3 and Rb are each independently hydrogen or (Ci-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
Rc is hydrogen or (Ci-C6)alkyl; and Ra is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d-C6)alkyl, (C1- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(C!-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(C!-C6)alkyl, and (C2-C6)alkanoyloxy; or R0 and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci- C6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(Ci-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-C6)alkyl; or a pharmaceutically acceptable salt thereof: to the mammal.
11. The method of claim 10 wherein the cancer is pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, a leukemia, liver cancer, melanoma, brain cancer or kidney cancer.
12. The method of any one of claims 1-11 wherein Ri is -C(=O)NRaRb.
13. The method of any one of claims 1-11 wherein R2 is -C(=O)NRaRb.
14. The method of any one of claims 1-11 wherein R3 is -C(=O)NRaRb-
15. The method of any one of claims 12-14 wherein wherein R2 and Rb are each independently (Ci-C6)alkyl.
16. The method of any one of claims 12-14 wherein R3 and Rb are each independently ethyl.
17. The method of any one of claims 12-14 wherein R3 and Rb together with the nitrogen to which they are attached form a morpholino ring.
18. The method of any one of claims 12-14 wherein Ra and Rb together with the nitrogen to which they are attached form a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl.
19. The method of any one of claims 12-14 wherein Ra and Rb together with the nitrogen to which they are attached form a 4-methylpiperazine.
20. The method of any one of claims 12-19 wherein R4 is -NRcRd,
21. The method of claim 20 wherein R0 is hydrogen or (Ci-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3- C6)cycloalkyl(C1-C6)alkyl, (Ci-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(Cr C6)alkyl, hydroxy(Ci-C6)alkyl, (d-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2- Cδ)alkanoyloxy.
22. The method of claim 20 wherein R4 is -NH2
23. The method of claim 20 wherein R0 is hydrogen or (Ci-C6)alkyl; and Rd is piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (d-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(Ci-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(CrC6)alkyl, hydroxy(d-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy.
24. The method of claim 20 wherein R0 is hydrogen and Rd is piperidinyl, that is substituted aryl(C1-C6)alkyl.
25. The method of claim 20 wherein R4 is l-benzylpiperidin-4-ylamino.
26. The method of claim 20 wherein R0 is hydrogen and Rd is (C]-C6)alkyl that is substituted with one or more heteroaryl.
27. The method of claim 20 wherein R0 is hydrogen and Rd is (C]-C6)alkyl that is substituted with an indolyl ring.
28. The method of claim 20 wherein R4 is N-(2-indol-3-ylethyl)amino.
29. The method of any one of claims 12-19 wherein R4 is -ORe.
30. The method of claim 29 wherein R6 is phenyl.
31. A pharmaceutical composition comprising a compound of formula I:
Figure imgf000043_0001
wherein: one OfR1, R2, and R3 is -C(=O)NRaRb and the remaining two OfR1, R2, and R3 are each hydrogen;
R4 is -NR0Rd, or -ORe;
Ra and Rb are each independently hydrogen or (Ci-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
Rc is hydrogen or (Ci-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(CrC6)alkyl, (Cr C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (d-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci- C6)alkyl;
R6 is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(CrC6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (d-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (C]-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-Ce)alkyl; or a pharmaceutically acceptable salt thereof; a pharmaceutically acceptable diluent or carrier; and another anticancer agent.
32. The use of a compound of formula I:
Figure imgf000045_0001
wherein: one of Ri, R2, and R3 is -C(=O)NRaRb and the remaining two of Ri, R2, and R3 are each hydrogen;
R4 is -NRcRd, or -OR6;
R3 and Rb are each independently hydrogen or (Ci-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
Rc is hydrogen or (C]-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(CrC6)alkyl, (C1- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (Ci-C6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(CrC6)alkyl, (Ci-C6)alkoxycarbonyl, (C]-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci- C6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (Ci-C6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (C]-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-C6)alkyl; or a pharmaceutically acceptable salt thereof; to prepare a medicament for inducing apoptosis in mammalian cells.
33. The use of a compound of formula I:
Figure imgf000046_0001
wherein: one of Ri, R2, and R3 is -C(K))NR3Rb and the remaining two of Ri, R2, and R3 are each hydrogen;
R4 is -NRcRd, or -OR6;
R3 and Rb are each independently hydrogen or (Ci-C6)alkyl, or R3 and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
Rc is hydrogen or (Ci-C6)alkyl; and Rj is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(CrC6)alkyl, (Cr C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(d-C6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; or Rc and Rd together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Cp C6)alkyl; R6 is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(Ci-C6)alkyl, (C1- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(d-C6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (C]-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-C6)alkyl; or a pharmaceutically acceptable salt thereof; to prepare a medicament for inhibiting topoisomerase II in a mammal.
34. The use of claim 33 wherein topoisomerase II is inhibited at least 2-fold more than topoisomerase I.
35. The use of claim 33 wherein topoisomerase II is inhibited at least 5-fold more than topoisomerase I.
36. The use of claim 33 wherein topoisomerase II is inhibited at least 10-fold more than topoisomerase I.
37. The use of claim 33 wherein topoisomerase II is inhibited at least 50-fold more than topoisomerase I.
38. The use of claim 33 wherein topoisomerase II is inhibited at least 100-fold more than topoisomerase I.
39. The use of any one of claims 33-38 wherein topoisomerase II is inhibited catalytically.
40. The use of a compound of formula I:
Figure imgf000048_0001
wherein: one of Ri, R2, and R3 is -C(=O)NRaRb and the remaining two of Ri, R2, and R3 are each hydrogen;
R4 is -NRcRd, or -OR6;
Ra and Rb are each independently hydrogen or (Ci-C6)alkyl, or Ra and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
Rc is hydrogen or (C]-C6)alkyl; and Ra is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (Ci-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (Ci-C6)alkanoyl, halo(d-C6)alkyl, hydroxy(d-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; or R0 and Ri together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci- C6)alkyl;
R6 is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (Ci-C6)alkanoyl, halo(Ci-C6)alkyl, hydroxy(d-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (Ci-C6)alkyl; or a pharmaceutically acceptable salt thereof; to prepare a medicament for treating cancer in a mammal.
41. The use of claim 40 wherein the cancer is pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, a leukemia, liver cancer, melanoma, brain cancer or kidney cancer.
42. The use of a compound of formula I:
Figure imgf000049_0001
wherein: one of Ri, R2, and R3 is -C(=O)NRaRb and the remaining two of Ri, R2, and R3 are each hydrogen;
R4 is -NRcRd, or -OR6;
Ra and Rb are each independently hydrogen or (Ci-C6)alkyl, or R3 and Rb together with the nitrogen to which they are attached form a piperidine, thiomorpholine, pyrrolidine, morpholino, or a piperazine ring that is optionally substituted at the 4-position nitrogen with (Ci-C6)alkyl;
Rc is hydrogen or (Ci-C6)alkyl; and Rd is hydrogen, piperidinyl, pyrrolidinyl, piperazinyl, heteroaryl, or (Ci-C6)alkyl that is optionally substituted with one or more piperidinyl, pyrrolidinyl, piperazinyl, -NRfRg, or heteroaryl, wherein each piperidinyl, pyrrolidinyl, piperazinyl, and heteroaryl of Rd is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(C,-C6)alkyl, (Cr C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (CrC6)alkanoyl, halo(d-C6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(Ci-C6)alkyl, and (C2-C6)alkanoyloxy; or R0 and Rj together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (C1- C6)alkyl;
Re is aryl that is optionally substituted with one or more groups independently selected from oxo (=0), halo, cyano, nitro, (CrC6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl(d-C6)alkyl, (Ci- C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (d-C6)alkanoyl, halo(CrC6)alkyl, hydroxy(CrC6)alkyl, (Ci-C6)alkoxycarbonyl, (Ci-C6)alkylthio, aryl(CrC6)alkyl, and (C2-C6)alkanoyloxy; and each Rf and Rg are each independently hydrogen or (Ci-C6)alkyl, or Rf and Rg together with the nitrogen to which they are attached form a piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring, which piperidino, thiomorpholino, pyrrolidinyl, morpholino, or a piperazine ring is optionally substituted with one or more (CrC6)alkyl; or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of cancer.
43. The use of claim 42 wherein the cancer is pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, a leukemia, liver cancer, melanoma, brain cancer or kidney cancer.
44. The method of any one of claims 1-11 wherein the compound of formula (I) is (9-(l-benzyl- piperidin-4-ylamino)-acridin-3-yl)-(4-methyl-piperazin-l-yl)-methanone or (9-(2-(lH-indol-3-yl)- ethylamino)-acridin-3-yl)-(4-methyl-piperazin- 1 -yl)-methanone; or a pharmaceutically acceptable salt thereof.
45. The pharmaceutical composition of claim 31 wherein the compound of formula (I) is (9-(l- benzyl-piperidin-4-ylamino)-acridin-3-yl)-(4-methyl-piperazin- 1 -yl)-methanone or (9-(2-( lH-indol-3- yl)-ethylamino)-acridin-3-yl)-(4-methyl-piperazin-l-yl)-methanone; or a pharmaceutically acceptable salt thereof.
46. The use of any one of claims 32-43 wherein the compound of formula (I) is (9-(l-benzyl- piperidin-4-ylamino)-acridin-3-yl)-(4-methyl-piperazin-l-yl)-methanone or (9-(2-(lH-indol-3-yl)- ethylamino)-acridin-3-yl)-(4-methyl-piperazin- 1 -yl)-methanone; or a pharmaceutically acceptable salt thereof.
47. The method of any one of claims 2-30 wherein the compound of formula I is administered to the mammal orally.
48. The method of claim 11 wherein the cancer is brain cancer.
49. The method of claim 48 wherein the compound of formula I is administered to the mammal orally.
50. The method of any one of claims 47-49 wherein compound of formula I catalytically inhibits topoisomerase I.
51. The use of claim 41 or claim 43 wherein the cancer is brain cancer.
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