WO2009010298A2 - Paullone derivatives and its use - Google Patents

Paullone derivatives and its use Download PDF

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WO2009010298A2
WO2009010298A2 PCT/EP2008/005908 EP2008005908W WO2009010298A2 WO 2009010298 A2 WO2009010298 A2 WO 2009010298A2 EP 2008005908 W EP2008005908 W EP 2008005908W WO 2009010298 A2 WO2009010298 A2 WO 2009010298A2
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optionally substituted
alkyl
dihydroindolo
benzazepin
butyl
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PCT/EP2008/005908
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French (fr)
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WO2009010298A8 (en
WO2009010298A3 (en
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Conrad Kunick
Christina Reichwald
Ute Dunkel
Charles Jaffe
Orly Shimony
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Technische Universität Carolo-Wilhelmina Zu Braunschweig
Yissum Research Development Company Of The Hebrew University Of Jerusalem
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis

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  • the present invention relates to new paullone derivatives.
  • the present invention relates to new paullone derivatives having anti-protozoan activity, in particular, anti-leishmanial activity.
  • the present invention relates to pharmaceutical compositions containing said paullones.
  • Leishmaniasis is a disease caused by protozoan parasites that belong to the genus Leishmania and is transmitted by the bite of certain species of sandfly, including sandflies in the genus Lutzomyia and Phlebotomus.
  • Various names are known for leishmaniasis, like black fever, kala-azar, or sandfly fever, etc.
  • L. donovani complex with three species (L. donovani, L. infantum and L. chagasi); the L. mexicana complex with three main species (L. mexicana, L amazonensis and L. venezulensis); L. tropica, L. major, L aethopica, and subgenus viannia with forming species, namely L. (v) braziliensis, L. (v) guyanensis, L (v.) panamensis, and L. (v.) peruviana.
  • the different species are morphologically indistinguishable, but they can be differentiated by other markers, like DNA sequence analysis, or isoenzyme analysis.
  • a specific type of leishmaniasis is the visceral leishmaniasis (VL) also known as kala- azar or black fever.
  • VL visceral leishmaniasis
  • Other types of leishmaniasis include cutaneous leishmaniasis, mucocutaneous leishmaniasis and diffuse cutaneous leishmaniasis.
  • Visceral leishmaniasis is a disease caused by infection with human protozoan parasites belonging to the Leishmania donovani complex.
  • Leishmania exists in two developmental stages: the extracellular promastigote transmitted by the bite of the sandfly vector and the intracellular amastigote that is an obligate parasite of macrophages.
  • VL occurs in tropical, subtropical and temperate regions; however, approximately 90% of the cases occur in Bangladesh, Brazil, India, Nepal and the Sudan.
  • Symptoms of disease include hepatosplenomegaly, fever, anaemia, immunosupression, hypergammaglobulinemia and weight loss, and without early diagnosis and proper treatment the disease is fatal.
  • the paullones (7,12-dihydroindolo[3,2-d][1]benzazepin-6(5/-/)-ones) are a class of protein kinase inhibitors acting predominantly on cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 5 (CDK5), and glycogen synthase kinase-3.
  • CDK1 CDK1 , respectively.
  • the present invention is based on the finding that compounds derived from the chemical class of paullones attenuate the proliferation of axenic amastigotes while displaying low toxicity towards macrophages. That is, the compounds according to the present invention not only inhibit the growth of parasites in Leishmania infected macrophages but also of axenic amastigotes while not having cytotoxic effects on macrophages.
  • the present invention relates to new compounds of general formulas (Ia) or (Ib) formula Ia
  • R 1 is aryl, optionally substituted, heteroaryl, optionally substituted, cycloaliphatic group, optionally substituted, CO-aryl, optionally substituted, CO-heteroaryl, optionally substituted, CO-cycloaliphatic group, optionally substituted, or CN, wherein the substituents are independently selected from one or more of halo, CN, OH, 0-C 1 -C 6 alkyl; COOH, COO-CrC 6 alkyl, CONH 2 , CONH(Ci-C 6 )alkyl, CON(CrC 6 alkyl) 2 , aryl, heteroaryl, polyoxyethenyl or combinations thereof;
  • R 2 is an electron-donating group
  • R 3 is independently H, Ci-C 6 alkyl, optionally substituted, or CO-CrC 6 alkyl, optionally substituted, wherein the substituents are independently selected from one or more of halo, CN, OH, 0-CrC 6 alkyl; COOH, COO " C 1 -C 6 alkyl, CONH 2 , CONHCd-CeJalkyl, CON(Ci-C 6 alkyl) 2 , aryl, heteroaryl, polyoxyethenyl or combinations thereof; or an optical isomer, or a salt or solvate thereof.
  • the present inventors found that these paullone derivatives are particularly useful for the treatment of leishmaniasis.
  • the residue R 3 is H or CH 3 .
  • the compounds according to the present invention are compounds wherein R 1 is an CO-aryl group, optionally substituted, or CO- heteroaryl group, optionally substituted, wherein the substituents are selected from one or more of halo, OH, or 0-C 1 -C 6 alkyl.
  • the electron donating group R 2 is a group selected from C 1 -C 6 alkyl, like methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, terf-butyl, sec-butyl, isobutyl, pentane-2-yl.
  • pentane-3-yl isopentyl, neopentyl; or C 1 -C 6 cycloalkyl, like cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; or benzyl, or substituted benzyl, wherein the substituents are selected from one or more of halo, OH, 0-C 1 -C 6 alkyl; or O- C 1 -C 6 alkyl; or O-benzyl; or O-substituted benzyl, wherein the substituents are selected from one or more of halo, OH, 0-CrC 6 alkyl.
  • R 3 is a ferf-butyl group.
  • paullone derivatives are 9-terf-Butyl-2-[(1E)-3-oxo-3-phenyl-1-propenyl]-7,12-dihydroindolo[3,2- cf][1 ]benzazepin-6(5/-/)-one;
  • said compounds may be present as base compounds or in the form of salts or solvates thereof.
  • Pharmaceutical acceptable addition salts of the above compounds (Ia) and (Ib) include but are not limited to salts with physiologically acceptable cations or anions.
  • cations are alkaline metals and alkine earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminium salts or the like, as well as non toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like.
  • amines useful for the formation of base addition salts include benzacethine, dicyclohexylamine, hydrabine, N-methyl-D-glucamine, N-methyl-D- glucamide, t-butyl-amine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like and salts with amino acids, such as arginine, lysine or the like.
  • anions are inorganic anions such as chloride, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate etc. and organic anions, e.g. carboxylate, sulfate or sulfonate anions, such as acetate, lactate, tartrate, tosylate, mesylate etc.
  • prodrug refers to a) an inactive form of a drug that exerts its effect after metabolic processes with the body converts it to a usable or active form, or b) a substance that gives rise to a pharmacologically active metabolite, although not itself active (i.e. an inactive precursor).
  • Ci-C 6 -alkyl refers to a Ci-C 6 , preferably C 1 -C 5 straight or branched alkyl group, such as methyl, ethyl, propyl (iso-, n-), butyl (iso-, n-, tert-), pentyl, hexyl.
  • halo refers to a halogen atom selected from fluorine, chlorine, bromine, iodine, preferably fluorine and chlorine, most preferably chlorine.
  • aryl refers to mono- and polycyclic aromatic groups having 6 to 10 backbone carbon atoms, optionally fused to a carbocylic group, such as phenyl, 1- naphthyl, indenyl, indanyl, azulenyl, fluorenyl, etc.
  • heteroaryl groups are such as benzofuranyl, furyl, thienyl, benzothienyl, thiazol, imidazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, benzothiazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, pyrrolyl, pyranyl, tetrahydropyranyl, pyrazolyl, pyridyl, pyrimidinyl, quinolinyl, isoquinolynyl, purinyl, benzooxazolyl, benzamidazolyl, indolyl, isoindolyl, pyrazinyl, diazinyl, pyrazinyl, triazinyltriazine, tetrazinyl, tetrazolyl, benzothiophenyl, benzopyridyl, benzimi
  • cycloaliphatic group refers to a stable monocyclic or multicyclic group which is not an aryl or heteroaryl as defined above. That is, the "cycloaliphatic group” is a saturated or partially unsaturated cyclyl group,
  • the cycloaliphatic group may contain 1 to 4 hetero atoms selected from N, S and O, with the remainder of the ring atoms being carbon atoms and having preferably a number of ring atoms of 3 to 10, such as morpholino, pyrrolidino, piperidino, piperazino, N-alkylpiperazino, azepanyl, thiazinyl, tetrahydropyranyl, tetrahydrofuranyl.
  • polyoxyethylenyl refers to groups containing at least 2 e.g. 2 - 50 oxyethylenyl (-OCH 2 -CH 2 -) groups, such as polyoxyethylenyloxycarbonyl or polyoxyethylenylaminocarbonyl groups.
  • the present invention relates to pharmaceutical compositions comprising as an active ingredient the compounds of the present invention, optionally together with pharmaceutically acceptable carriers, diluents, and adjuvants.
  • compositions may consist of the active ingredient of the invention.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone or in combination with other agents, drugs or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-artehal, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of pancreatic cells or in animal models, usually mice, rabbits, dogs or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, which is sufficient for treating a specific condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 ⁇ g, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
  • the compounds of the present invention can be used for the prophylaxis or treatment of diseases caused by single celled parasites.
  • Single celled parasites include trypanosomes, leishmaniases, toxosplasma, pneumocytis etc. preferably, the single celled parasites are Leishmania.
  • the compounds according to the present invention are particularly useful for the prophylaxis or treatment of leishmaniasis.
  • the compounds according to the present invention are for the prophylaxis or treatment of visceral leishmaniasis, also known as Kala-azar and black fever.
  • mexicana (LmexCRKI and LmexCRK3) have been investigated in detail.
  • Subsequent tests with 4 showed that the compound was able to inhibit the L. donovani infection of peritoneal mouse macrophages with an ED 5O of 19.6 ⁇ M.
  • the compound was not further pursued in this study because it exhibited toxicity for the host cells at 10 ⁇ M (Grant, K.; et al., Antimicrob. Agents Chemother. 2004, 48, 3033- 42).
  • the effect of drugs on intracellular parasites survival can be measured rapidly and simply by adding an appropriate enzyme substrate and measuring luminescence in a microplate reader.
  • This assay can replace labor-intensive assays where infected macrophages are stained and the number of intracellular parasites and percentage of infected macrophages counted by light microscope. Finally, the toxicity of the compounds was determined on the human macrophage cell line using the Alamar Blue viability assay.
  • a small in- house compound collection of various paullone derivatives are screened on L donovani axenic amastigotes.
  • the structures were initially tested at a single concentration (50 ⁇ M). If > 80% inhibition of parasite growth was observed, the compounds were examined at lower concentrations (30 and/or 15 ⁇ M).
  • two compounds (1 and 2) belonging to the paullone structure class strongly inhibited parasite growth at 50 ⁇ M (91.2 and 100%) and were clearly superior to alterpaullone in this test system which exhibited ⁇ 80% inhibition of parasite growth at 50 ⁇ M.
  • novel paullone derivatives may be prepared by acid-catalyzed Fischer indol cyclization reaction from appropriate phenyl hydrazones. These precursors are synthesized from an appropriate commercially available phenyl hydrazine and 7- iodo-3,4-dihydro-1H-1-benzazepine-2,5-dione (5) which may be prepared following a method published by Kunick et al. (Kunick, C; et al., Bioorg. Med. Chem. Lett. 2000, 10, 567-569)).
  • the ketone Mannich bases 11 were heated with the 2-iodopaullones 7 in DMF in the presence of palladium acetate and triethylamine under nitrogen to furnish the expected 2-vinylpaullones 12a-m.
  • the reaction could be transferred to a parallel synthesis procedure in 20 ml_ vials employing a parallel reactor station.
  • the reaction worked well also in the absence of a phosphine ligand. This is the first report on the use of Mannich bases in Heck reactions.
  • the modest yields mentioned here (14 - 46 %) are the result of compound loss during the workup procedures and still bear optimization potential.
  • the phenylethynyl derivative 15 was prepared. Because the 2-iodopaullone 7e gave unsatisfactory results in an attempted Sonogashira reaction with the phenyl acetylene 13, the latter was reacted with 7- iodo-3,4-dihydro-1/-/-1-benzazepine-2,5-dione (5). The obtained cyclic ketone 14 was subsequently transformed to the paullone 15 by a Fischer indole ring closure reaction.
  • the present inventors found that 3 proved to be inferior to the paullones 1 and 2 in the more relevant assay with axenic amastigotes, though differences in the efficacy of compounds between leishmanial species have been observed.
  • L donovani (MHOM/SD/1962/1 S-CI2d) was used in all bioassays. Axenic amastigotes were grown at 37 0 C in a 5 % CO 2 incubator as described (Debrabant, A.; et al., Int. J. Parasitol. 2004, 34, 205-217) in complete RPMI 1640 containing 20 % fetal calf serum, pH 5.5.
  • Stably transfected L donovani promastigotes expressing the firefly luciferase gene ⁇ Ld:pSSU-int/LUC) were cultured in Medium-199 adjusted to pH 6.8 and supplemented with L-glutamine (2 mM), adenosine (100 ⁇ M), folic acid (23 ⁇ M), 1x BME vitamin mix, 10 % fetal calf serum, penicillin G (100 IU), streptomycin (100 ⁇ g/ml) and hygromycin B (100 ⁇ g/ml).
  • the human leukaemia monocyte cell line (THP-1) was cultured in complete RPMI- 1640 supplemented with antibiotics (100 IU penicillin G and 100 mg/ml streptomycin), 2 mM L-glutamine and fetal calf serum (10 % v/v).
  • Axenic amastigote viability assay Screening of the compounds for leishmanicidal activity was carried out using the alamarBlue (AbD Serotec, Oxford, UK) viability assay with axenic amastigotes as recently described (Shimony, O. and Jaffe, C. L., doi:10.1016/j.mimet.2008.05.026). Compounds to be assayed were diluted to twice the final concentration in the complete amastigote medium, containing 1% DMSO, and were aliquoted in triplicate (125 ⁇ l/well) into 96-well flat-bottom plates (Nunc, Roskilde, Denmark).
  • Amastigotes (5.0 x 10 5 cells/ml; 125 ⁇ l/well) were added to each well and incubated for 24 hrs at 37 0 C in a 5% CO 2 incubator.
  • Complete medium both with and without DMSO was used as negative controls (0% inhibition of amastigote growth).
  • Amphotericin B (Sigma-AIdrich, St. Louis MO), a drug used to treat VL, was included as a positive control on each plate and gave >90% inhibition of parasite growth at 1 ⁇ M.
  • THP-1 cells in the log-phase of growth were differentiated by incubation for 3 days in complete RPMI-1640 containing 1 ⁇ M retinoic acid (RA, Sigma-AIdrich, St. Louis, MO) (Hemmi, H.; Breitman, T. Jpn. J. Cancer Res. 1985, 76, 345-51). Excess RA was removed by washing the cells three times with RPMI-1640 (250 x g, 10', 4 0 C) and the treated macrophages suspended in complete RPMI-1640 and transferred to 75 ml tissue culture flasks (Costar Brand, NUNCTM, Denmark).
  • RA retinoic acid
  • Drugs diluted in complete RPMI-1640 containing 1% DMSO (10 ⁇ M, 50 ⁇ l/well) were added to the infected cells. The cultures were incubated for 48 hrs (37 0 C, 5% CO 2 ). Cells were lysed by the addition of Steady-Glo ® Luciferase Assay substrate (100 ⁇ l/well, Promega, MT, USA) to each well and the luminescence measured after 10 minutes using a microplate reader (Luminometer Mithras LB940, Berthold Technologies, Germany). Complete medium both with and without DMSO was used as negative controls (0% inhibition). Amphotericin B (Sigma-AIdrich, St. Louis MO) was included as a positive control on each plate and gave >90% inhibition at 1 ⁇ M.
  • Toxicity assay Effect of the compounds on human cells was assessed using the alamarBlue viability assay. Drugs to be tested were diluted in the complete medium containing 1% DMSO (10 ⁇ M) and aliqouted in triplicate (125 ⁇ l/well) into 96-well flat- bottom plates. THP-1 macrophages in complete RPMI-1640 were added (8 x 10 5 cells/ml, 125 ⁇ l/well) to the plates and incubated for 48 hrs (37 0 C, 5% CO 2 ). The viability indicator alamarBlue (25 ⁇ l) was added, the plates incubated for an additional 3 hrs and the fluorescence read as described above. Complete medium both with and without DMSO was used as negative controls (0% inhibition).
  • Elemental analyses CE Instruments FlashEA ® 1112 Elemental Analyzer (Thermo Quest). Results obtained were within ⁇ 0.4% unless indicated otherwise.
  • Thin-layer chromatography Polygram SiI G/UV 254 silica gel plates (Macherey-Nagel); 254 nm UV illumination.
  • Parallel synthesis Carousel 12 Place Reaction StationTM (Radley Discovery Technologies).
  • Compounds 5 and 7a were prepared according to published procedures.
  • the ketone Mannich base hydrochlorides were prepared according to a standard procedure (Blicke, F. F. Org. Reactions 1942, 1, 303-341).
  • the remaining silica gel/reaction product mixture is added onto a silica gel pad in a glass frit and is then eluted with ethyl acetate (200 ml_). After evaporation of the solvent the remaining solid is purified by crystallization from ethanol.
  • the procedure was adapted to the use of a parallel synthesis reactor. In this case, the reaction was carried out without addition of triphenylphosphine in 20 ml_ vials with 2 ml_ DMF as solvent.
  • the vessel reactor block temperature was set to 140 0 C.
  • the work up procedure was carried out as described above.
  • the reaction is outlined in scheme 2.
  • Table 1 Antileishmanial activity and in vitro toxicity of Paullones 1, 2, 10, 12 and 15 wherein R 3 is H
  • Injection volume 10 ⁇ l; Flow rate: 1.000 ml/min; Runtime: 15 min; Retention time for

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Abstract

The present invention relates to new paullone derivatives. In particular, the present invention relates to new paullone derivatives having anti-protozoan activity, in particular, anti-leishmanial activity. In a further aspect, the present invention relates to pharmaceutical compositions containing said paullones.

Description

Paullone derivatives and its use
The present invention relates to new paullone derivatives. In particular, the present invention relates to new paullone derivatives having anti-protozoan activity, in particular, anti-leishmanial activity. In a further aspect, the present invention relates to pharmaceutical compositions containing said paullones.
Prior art
Leishmaniasis is a disease caused by protozoan parasites that belong to the genus Leishmania and is transmitted by the bite of certain species of sandfly, including sandflies in the genus Lutzomyia and Phlebotomus. Various names are known for leishmaniasis, like black fever, kala-azar, or sandfly fever, etc.
Most forms of the disease are transmissible only from animals (zoonosis), but some can be spread between humans. These species that infect mammals include the L. donovani complex with three species (L. donovani, L. infantum and L. chagasi); the L. mexicana complex with three main species (L. mexicana, L amazonensis and L. venezulensis); L. tropica, L. major, L aethopica, and subgenus viannia with forming species, namely L. (v) braziliensis, L. (v) guyanensis, L (v.) panamensis, and L. (v.) peruviana. The different species are morphologically indistinguishable, but they can be differentiated by other markers, like DNA sequence analysis, or isoenzyme analysis.
A specific type of leishmaniasis is the visceral leishmaniasis (VL) also known as kala- azar or black fever. Other types of leishmaniasis include cutaneous leishmaniasis, mucocutaneous leishmaniasis and diffuse cutaneous leishmaniasis.
Visceral leishmaniasis (VL) is a disease caused by infection with human protozoan parasites belonging to the Leishmania donovani complex. Typically, Leishmania exists in two developmental stages: the extracellular promastigote transmitted by the bite of the sandfly vector and the intracellular amastigote that is an obligate parasite of macrophages. VL occurs in tropical, subtropical and temperate regions; however, approximately 90% of the cases occur in Bangladesh, Brazil, India, Nepal and the Sudan. Symptoms of disease include hepatosplenomegaly, fever, anaemia, immunosupression, hypergammaglobulinemia and weight loss, and without early diagnosis and proper treatment the disease is fatal.
Unfortunately, treatment options for leishmaniasis are very limited. The main drugs in use today were introduced over 50 years ago, and all drug regimes have major drawbacks. First-line treatment based on pentavalent antimony (meglumine antimonate and sodium stibogluconate) show severe, unwanted side-effects. Resistance to these compounds has emerged to such an extent in India that they can no longer be used in many regions. Similarly use of second-line drugs, pentamidine and amphotericin B, is limited by toxicity. Liposomal amphotericin B is a highly effective option; however, these drug formulations are very expensive limiting their use in most endemic regions. Miltefosine, a new drug recently registered for use in India is the first drug available for oral treatment of VL. However due to reproductive toxicity, females of childbearing age cannot be treated without efficient contraception. Although current clinical trials with injectable paromomycin are showing encouraging results, an expanded catalogue of new drugs for these parasites is required in order to prevent the development of resistance. The WHO has designated leishmaniasis a "neglected and emerging disease" and the need for novel drugs against the parasites that cause them has been recognized. Considering the situation of the population in the developing countries threatened by the disease new drugs should be selective, non-toxic, inexpensive and orally available. Therefore, there is still a need for innovative drugs based on new molecular scaffolds directed against novel biological targets.
Extracellular promastigotes have been used frequently to screen compounds for activity against Leishmania; however, the two parasite stages show significant metabolic differences. Compounds that kill one stage, as in the case of the pentavalent antimony derivatives that are only active against the amastigote stage, may not be effective with the other stage and vice versa. The paullones (7,12-dihydroindolo[3,2-d][1]benzazepin-6(5/-/)-ones) are a class of protein kinase inhibitors acting predominantly on cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 5 (CDK5), and glycogen synthase kinase-3. Members of the paullone family have been used as biochemical tools in such diverse fields as Alzheimer's disease(Phiel, C. J.; et al., Nature, 2003, 423, 435-439), juvenile diabetes (Mussmann, R.; et al., J. Biol. Chem. 2007, 282, 12030-12037), and development biology (Mϋller, W.; et al., Int. J. Dev. Biol 2004, 48, 9-15).
Distinct paullones like alsterpaullone have been investigated as potential anticancer agents due to their growth inhibitory activity for cancer cell lines (Lahusen, T.; et al.,
MoI. Carcinog. 2003, 36, 183-194). The structure-activity relationships (SAR) in the paullone class of compounds have been extensively studied. It has been shown that an electron-withdrawing substituent in the 9-position is favourable for the CDK inhibitory activity. According to this SAR, paullones 1 and 2, as shown in figure 1 are one or two orders of magnitude inferior to alsterpaullone regarding inhibition of
CDK1 , respectively.
In view of the above and the increasing resistance to conventional treatment, there is an ongoing need for new active agents for the prevention and/or treatment of single celled parasites based diseases, in particular, of leishmaniasis. The technical problem underlying the present invention was to provide for means and methods for treating single celled parasites based diseases, in particular, leishmaniasis. The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
The present invention is based on the finding that compounds derived from the chemical class of paullones attenuate the proliferation of axenic amastigotes while displaying low toxicity towards macrophages. That is, the compounds according to the present invention not only inhibit the growth of parasites in Leishmania infected macrophages but also of axenic amastigotes while not having cytotoxic effects on macrophages.
The present invention relates to new compounds of general formulas (Ia) or (Ib) formula Ia
Figure imgf000005_0001
formula Ib
Figure imgf000005_0002
wherein R1 is aryl, optionally substituted, heteroaryl, optionally substituted, cycloaliphatic group, optionally substituted, CO-aryl, optionally substituted, CO-heteroaryl, optionally substituted, CO-cycloaliphatic group, optionally substituted, or CN, wherein the substituents are independently selected from one or more of halo, CN, OH, 0-C1-C6 alkyl; COOH, COO-CrC6 alkyl, CONH2, CONH(Ci-C6)alkyl, CON(CrC6alkyl)2, aryl, heteroaryl, polyoxyethenyl or combinations thereof;
R2 is an electron-donating group;
R3 is independently H, Ci-C6 alkyl, optionally substituted, or CO-CrC6 alkyl, optionally substituted, wherein the substituents are independently selected from one or more of halo, CN, OH, 0-CrC6 alkyl; COOH, COO" C1-C6 alkyl, CONH2, CONHCd-CeJalkyl, CON(Ci-C6alkyl)2, aryl, heteroaryl, polyoxyethenyl or combinations thereof; or an optical isomer, or a salt or solvate thereof.
The present inventors found that these paullone derivatives are particularly useful for the treatment of leishmaniasis.
In a preferred embodiment, the residue R3 is H or CH3.
In another preferred embodiment, the compounds according to the present invention are compounds wherein R1 is an CO-aryl group, optionally substituted, or CO- heteroaryl group, optionally substituted, wherein the substituents are selected from one or more of halo, OH, or 0-C1-C6 alkyl.
In a further preferred embodiment, the electron donating group R2 is a group selected from C1-C6 alkyl, like methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, terf-butyl, sec-butyl, isobutyl, pentane-2-yl. pentane-3-yl, isopentyl, neopentyl; or C1-C6 cycloalkyl, like cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; or benzyl, or substituted benzyl, wherein the substituents are selected from one or more of halo, OH, 0-C1-C6 alkyl; or O- C1-C6 alkyl; or O-benzyl; or O-substituted benzyl, wherein the substituents are selected from one or more of halo, OH, 0-CrC6 alkyl. Particularly preferred, R3 is a ferf-butyl group.
Particularly preferred examples of suitable paullone derivatives are 9-terf-Butyl-2-[(1E)-3-oxo-3-phenyl-1-propenyl]-7,12-dihydroindolo[3,2- cf][1 ]benzazepin-6(5/-/)-one;
9-ferf-Butyl-2-[(1 £)-3-(4-methoxyphenyl)-3-oxo-1 -propenyl]-7, 12-dihydroindolo[3,2- d\[\ ]benzazepin-6(5H)-one;
9-teAt-Butyl-2[(1E)-3-(4-chlorophenyl)-3-oxo-1-propenyl]-7,12-dihydroindolo-[3,2- c/][1]benzazepin-6(5/-/)-one;
9-fert-Butyl-2[(1E)-3(2-furyl)-3-oxo-1-propenyl]-7,12-dihydroindolo[3,2- d\[\ ]benzazepin-6(5H)-one; 9-terf-Butyl-2-[(1 E)-3-oxo-3-(3-pyridinyl)-1 -propenyl]-7, 12-dihydroindolo[3,2- c/][1]benzazepin-6(5H)-one; or
9-fert-Butyl-2-[(1E)-3-(2,4-dimethoxylphenyl)-3-oxo-1-propenyl]-7,12- dihydroindolo[3,2-c(][1]benzazepin-6(5/-/)-one 9-ferf-Butyl-2-(phenylethynyl)-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one.
As indicated before, said compounds may be present as base compounds or in the form of salts or solvates thereof.
Pharmaceutical acceptable addition salts of the above compounds (Ia) and (Ib) include but are not limited to salts with physiologically acceptable cations or anions. Examples of cations are alkaline metals and alkine earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminium salts or the like, as well as non toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like. Other representative amines useful for the formation of base addition salts include benzacethine, dicyclohexylamine, hydrabine, N-methyl-D-glucamine, N-methyl-D- glucamide, t-butyl-amine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like and salts with amino acids, such as arginine, lysine or the like.
Examples of anions are inorganic anions such as chloride, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate etc. and organic anions, e.g. carboxylate, sulfate or sulfonate anions, such as acetate, lactate, tartrate, tosylate, mesylate etc.
The present invention comprises all tautomeric forms. Furthermore, the present invention also comprises all stereoisomers of the compounds according to the invention, including its enantiomers and diastereomers. Individual stereoisomers of a compound according to the invention can be substantially present pure of other isomers, in a mixture thereof or as racemates or as selected stereoisomers. The invention also relates to metabolites and prodrugs. As used herein, the term "metabolite" refers to a) a product of metabolism including intermediate and products, b) any substance and metabolism (either as a product of metabolism or as necessary metabolism), or c) any substance produced or used to metabolism. In particular, it refers to the end product that remains after metabolism.
As used herein, the term "prodrug" refers to a) an inactive form of a drug that exerts its effect after metabolic processes with the body converts it to a usable or active form, or b) a substance that gives rise to a pharmacologically active metabolite, although not itself active (i.e. an inactive precursor).
The term "Ci-C6-alkyl" as used herein refers to a Ci-C6, preferably C1-C5 straight or branched alkyl group, such as methyl, ethyl, propyl (iso-, n-), butyl (iso-, n-, tert-), pentyl, hexyl.
The term "halo" refers to a halogen atom selected from fluorine, chlorine, bromine, iodine, preferably fluorine and chlorine, most preferably chlorine.
The term "aryl" refers to mono- and polycyclic aromatic groups having 6 to 10 backbone carbon atoms, optionally fused to a carbocylic group, such as phenyl, 1- naphthyl, indenyl, indanyl, azulenyl, fluorenyl, etc.
The term "heteroaryl" refers to mono- or bicyclic aromatic groups with 1 to 4 hetero atoms selected from N, S and O, with the remainer of the ring atoms being carbon atoms and having preferably a total number of ring atoms of 5 to 10. Examples without limitation of heteroaryl groups are such as benzofuranyl, furyl, thienyl, benzothienyl, thiazol, imidazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, benzothiazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, pyrrolyl, pyranyl, tetrahydropyranyl, pyrazolyl, pyridyl, pyrimidinyl, quinolinyl, isoquinolynyl, purinyl, benzooxazolyl, benzamidazolyl, indolyl, isoindolyl, pyrazinyl, diazinyl, pyrazinyl, triazinyltriazine, tetrazinyl, tetrazolyl, benzothiophenyl, benzopyridyl, benzimidazolyl. The term "cycloaliphatic group" refers to a stable monocyclic or multicyclic group which is not an aryl or heteroaryl as defined above. That is, the "cycloaliphatic group" is a saturated or partially unsaturated cyclyl group, The cycloaliphatic group may contain 1 to 4 hetero atoms selected from N, S and O, with the remainder of the ring atoms being carbon atoms and having preferably a number of ring atoms of 3 to 10, such as morpholino, pyrrolidino, piperidino, piperazino, N-alkylpiperazino, azepanyl, thiazinyl, tetrahydropyranyl, tetrahydrofuranyl.
The term "polyoxyethylenyl" refers to groups containing at least 2 e.g. 2 - 50 oxyethylenyl (-OCH2-CH2-) groups, such as polyoxyethylenyloxycarbonyl or polyoxyethylenylaminocarbonyl groups.
In a further aspect, the present invention relates to pharmaceutical compositions comprising as an active ingredient the compounds of the present invention, optionally together with pharmaceutically acceptable carriers, diluents, and adjuvants.
That is, the compounds of the invention are intended for pharmaceutical applications and may comprise with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of the active ingredient of the invention. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone or in combination with other agents, drugs or hormones. The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-artehal, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of pancreatic cells or in animal models, usually mice, rabbits, dogs or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of active ingredient, which is sufficient for treating a specific condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 μg, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
The compounds of the present invention can be used for the prophylaxis or treatment of diseases caused by single celled parasites. Single celled parasites include trypanosomes, leishmaniases, toxosplasma, pneumocytis etc. preferably, the single celled parasites are Leishmania. Thus, the compounds according to the present invention are particularly useful for the prophylaxis or treatment of leishmaniasis. Particularly preferred, the compounds according to the present invention are for the prophylaxis or treatment of visceral leishmaniasis, also known as Kala-azar and black fever.
Scattered hints in the literature point to the fact that paullones have been considered as anti-leishmanial agents before It has been shown, that high doses (10 μM) of alsterpaullone killed Leishmania mexicana promastigotes after 5 days of incubation, presumably because of binding to the mitochondrial malate dehydrogenase of the parasites (Knockaert, M. et al., J. Biol. Chem. 2002, 277, 25493-25501). From the genome of the parasite Leishmania major, eleven cdc2 (= CDK1)-related kinases have been predicted, and the function of two putative cyclin-dependent kinases from L. mexicana (LmexCRKI and LmexCRK3) have been investigated in detail. A screening program directed to find inhibitors of the LmexCRK3 revealed 42 active compounds, among them 9-cyanopaullone (4, in the following, the numbering of the compounds refer to the numbering of the compounds provided in the figures and the examples) (Grant, K.; et al., Antimicrob. Agents Chemother. 2004, 48, 3033-42). Subsequent tests with 4 showed that the compound was able to inhibit the L. donovani infection of peritoneal mouse macrophages with an ED5O of 19.6 μM. The compound was not further pursued in this study because it exhibited toxicity for the host cells at 10 μM (Grant, K.; et al., Antimicrob. Agents Chemother. 2004, 48, 3033- 42).
For the testing of appropriate compounds, two assays suitable for medium to high throughput screening of compounds against amastigotes have been developed. Initial screening was carried out using a fluorescent viability microplate assay and L donovani axenic amastigotes cultured under conditions, at 370C and acidic pH, which mimic the environment of tissue amastigotes (Shimony, O. and Jaffe, C. L., doi:10.1016/j.mimet.2008.05.026). The second assay utilizes a human macrophage cell line (THP-1) infected with L donovani stably transfected with the firefly luciferase gene. The effect of drugs on intracellular parasites survival can be measured rapidly and simply by adding an appropriate enzyme substrate and measuring luminescence in a microplate reader. This assay can replace labor-intensive assays where infected macrophages are stained and the number of intracellular parasites and percentage of infected macrophages counted by light microscope. Finally, the toxicity of the compounds was determined on the human macrophage cell line using the Alamar Blue viability assay.
In order to identify new anti-leishmanial lead structures, in a first step, a small in- house compound collection of various paullone derivatives are screened on L donovani axenic amastigotes. The structures were initially tested at a single concentration (50 μM). If > 80% inhibition of parasite growth was observed, the compounds were examined at lower concentrations (30 and/or 15 μM). Interestingly two compounds (1 and 2) belonging to the paullone structure class strongly inhibited parasite growth at 50 μM (91.2 and 100%) and were clearly superior to alterpaullone in this test system which exhibited <80% inhibition of parasite growth at 50 μM.
Determination of the IC5O value showed that 2 was five times more potent compared to 1 (Table 1). On the other hand, paullone 2 showed a considerable cell killing activity in a preliminary cellular toxicity assay for host cells, namely THP-1 macrophages in vitro. Neither 1 or 2 were able to inhibit the growth of parasites in
/.e/sΛma/i/a-infected macrophages.
The novel paullone derivatives may be prepared by acid-catalyzed Fischer indol cyclization reaction from appropriate phenyl hydrazones. These precursors are synthesized from an appropriate commercially available phenyl hydrazine and 7- iodo-3,4-dihydro-1H-1-benzazepine-2,5-dione (5) which may be prepared following a method published by Kunick et al. (Kunick, C; et al., Bioorg. Med. Chem. Lett. 2000, 10, 567-569)). The acid-catalyzed Fischer indole cyclization reaction with appropriately substituted phenyl hydrazines 6a-e led from the cyclic ketone 5 (Kunick, C; et al., Bioorg. Med. Chem. Lett. 2000, 10, 567-569) to the novel 2-iodo-substituted paullones 7a-e. A Heck reaction with either methyl vinyl ketone 8 or acrylonitrile 9 catalyzed by a palladium acetate/triphenylphosphine system in DMF furnished the compounds 10a-f as analogues of 2.
For the preparation of the 2-(3-aryl-3-oxopropen-1-yl)paullones 12a-m a conventional Heck reaction would have required the use of aryl vinyl ketones as reaction partners for 7. However, aryl vinyl ketones are inconvenient to handle since they tend to polymerize at elevated temperatures. Consistent with this finding, examples for the synthesis of 1 ,3-diarylpropenones by Heck reaction procedures employing aryl vinyl ketones are rare. Therefore the ketone Mannich bases 11 as precursors are used which under the typical conditions of the Heck reaction readily lose dimethylamine and release aryl vinyl ketones. Hence, the ketone Mannich bases 11 were heated with the 2-iodopaullones 7 in DMF in the presence of palladium acetate and triethylamine under nitrogen to furnish the expected 2-vinylpaullones 12a-m. The reaction could be transferred to a parallel synthesis procedure in 20 ml_ vials employing a parallel reactor station. The reaction worked well also in the absence of a phosphine ligand. This is the first report on the use of Mannich bases in Heck reactions. The modest yields mentioned here (14 - 46 %) are the result of compound loss during the workup procedures and still bear optimization potential. Because of the readily available starting materials, the simple protocol, and the short reaction times the Heck reaction with ketone Mannich bases and iodo arenes described herein favourably complements the well established Claisen-Schmidt synthesis for 1 ,3-diarylpropenones using aromatic aldehydes and acetophenone derivatives as starting materials.
As a further structure modification of 2, the phenylethynyl derivative 15 was prepared. Because the 2-iodopaullone 7e gave unsatisfactory results in an attempted Sonogashira reaction with the phenyl acetylene 13, the latter was reacted with 7- iodo-3,4-dihydro-1/-/-1-benzazepine-2,5-dione (5). The obtained cyclic ketone 14 was subsequently transformed to the paullone 15 by a Fischer indole ring closure reaction.
The present inventors found that 3 proved to be inferior to the paullones 1 and 2 in the more relevant assay with axenic amastigotes, though differences in the efficacy of compounds between leishmanial species have been observed.
Based on the observations with 1 and 2 and the information from the literature a program for structure optimization was initialized. This program was directed to the development of paullone derivatives with improved potency for the protection of macrophages against L. donovani infection in vitro as well as minimized toxicity towards THP-1 macrophages. The structures designed and synthesized included the main feature of 2, namely the unsaturated side chain in position 2 of the paullone parent scaffold. Because in contrast to 2 and 4 the methyl derivative 1 lacked a toxic effect on macrophages derivatives with electron donating substituents in the 9- position are present in the series of new analogues.
Brief description of the drawings
In figure 1 the structures of paullones 1 - 4 are shown
In figure 2, the structures of chalcone 16 as analogue of 12d and structure of licochalcone is provided.
In figure 3, compounds 12a to 12m and 15 are shown.
In the following, the present invention will be illustrated in more detail by examples. It is clear that said examples are intended to illustrate the invention further without limiting the scope of the present invention.
Examples Data of the compounds synthesized as described herein are given in table 2 (IR data for compounds 7c-e; 10b-f ; 12b-m, 14, and 15), table 3 (1H-NMR data (D6-DMSO, 400 MHz, [ppm]) for compounds 7c-e; 10b-f, 12b-m, 14, 15), table 4 (13 C-NMR Data (D6-DMSO, 100.6 MHz, [ppm]) for Compounds 7d-e; 10b-f , 12b-m, 14, 15), table 5 (HPLC data), and table 6 (elemental analysis data)
General procedures
Parasite and cell culture: L donovani (MHOM/SD/1962/1 S-CI2d) was used in all bioassays. Axenic amastigotes were grown at 370C in a 5 % CO2 incubator as described (Debrabant, A.; et al., Int. J. Parasitol. 2004, 34, 205-217) in complete RPMI 1640 containing 20 % fetal calf serum, pH 5.5. Stably transfected L donovani promastigotes expressing the firefly luciferase gene {Ld:pSSU-int/LUC) were cultured in Medium-199 adjusted to pH 6.8 and supplemented with L-glutamine (2 mM), adenosine (100 μM), folic acid (23 μM), 1x BME vitamin mix, 10 % fetal calf serum, penicillin G (100 IU), streptomycin (100 μg/ml) and hygromycin B (100 μg/ml).
The human leukaemia monocyte cell line (THP-1) was cultured in complete RPMI- 1640 supplemented with antibiotics (100 IU penicillin G and 100 mg/ml streptomycin), 2 mM L-glutamine and fetal calf serum (10 % v/v).
Axenic amastigote viability assay: Screening of the compounds for leishmanicidal activity was carried out using the alamarBlue (AbD Serotec, Oxford, UK) viability assay with axenic amastigotes as recently described (Shimony, O. and Jaffe, C. L., doi:10.1016/j.mimet.2008.05.026). Compounds to be assayed were diluted to twice the final concentration in the complete amastigote medium, containing 1% DMSO, and were aliquoted in triplicate (125 μl/well) into 96-well flat-bottom plates (Nunc, Roskilde, Denmark). Amastigotes (5.0 x 105 cells/ml; 125 μl/well) were added to each well and incubated for 24 hrs at 37 0C in a 5% CO2 incubator. The alamarBlue viability indicator was added (25 μl/well) and the plates incubated for an additional 24 hrs at which time the fluorescence (μex = 544 nm; μem = 590 nm) was measured in a microplate reader (Fluoroskan Ascent FL, Finland). Complete medium both with and without DMSO was used as negative controls (0% inhibition of amastigote growth). Amphotericin B (Sigma-AIdrich, St. Louis MO), a drug used to treat VL, was included as a positive control on each plate and gave >90% inhibition of parasite growth at 1 μM.
Screening on infected macrophages: THP-1 cells in the log-phase of growth were differentiated by incubation for 3 days in complete RPMI-1640 containing 1 μM retinoic acid (RA, Sigma-AIdrich, St. Louis, MO) (Hemmi, H.; Breitman, T. Jpn. J. Cancer Res. 1985, 76, 345-51). Excess RA was removed by washing the cells three times with RPMI-1640 (250 x g, 10', 4 0C) and the treated macrophages suspended in complete RPMI-1640 and transferred to 75 ml tissue culture flasks (Costar Brand, NUNC™, Denmark). Stationary-phase Lcf:pSSU-int/LUC promastigotes were added to the treated macrophages (3:1 parasite : macrophage ratio) and incubated in a 5% CO2 incubator for 16 h at 37 0C to allow for infection and differentiation of the Leishmania into intracellular amastigotes. Any remaining extracellular parasites were removed by centrifugation 4 - 5 times (210 x g, 8\ 4 0C). This was validated by phase microscopy. Infected THP-1 cells in complete RPMI-1640 were counted and aliquoted (1 x 105 cells in 50 μl/well) in triplicate into opaque 96-well flat bottom plates (Costar Brand, NUNC™, Denmark). Drugs diluted in complete RPMI-1640 containing 1% DMSO (10 μM, 50 μl/well) were added to the infected cells. The cultures were incubated for 48 hrs (37 0C, 5% CO2). Cells were lysed by the addition of Steady-Glo® Luciferase Assay substrate (100 μl/well, Promega, MT, USA) to each well and the luminescence measured after 10 minutes using a microplate reader (Luminometer Mithras LB940, Berthold Technologies, Germany). Complete medium both with and without DMSO was used as negative controls (0% inhibition). Amphotericin B (Sigma-AIdrich, St. Louis MO) was included as a positive control on each plate and gave >90% inhibition at 1 μM.
Toxicity assay: Effect of the compounds on human cells was assessed using the alamarBlue viability assay. Drugs to be tested were diluted in the complete medium containing 1% DMSO (10 μM) and aliqouted in triplicate (125 μl/well) into 96-well flat- bottom plates. THP-1 macrophages in complete RPMI-1640 were added (8 x 105 cells/ml, 125 μl/well) to the plates and incubated for 48 hrs (37 0C, 5% CO2). The viability indicator alamarBlue (25 μl) was added, the plates incubated for an additional 3 hrs and the fluorescence read as described above. Complete medium both with and without DMSO was used as negative controls (0% inhibition).
Synthetic chemistry: Melting points: IA 9100 instrument (Barnstedt Electrothermal), not corrected; Infrared spectra: KBr pellets; Thermo FT-IR 200 (Thermo Nicolet). NMR: Bruker Avance DRX-400; solvent [D6]-DMSO; internal standard thmethylsilane; signals in ppm (μ scale). Mass spectrometry: Finnigan-MAT 90 instrument. Reverse-phase HPLC: Merck/Hitachi LaChrome Elite system and LiChroCART 125-4, LiChrospher 100 RP-18 (5 μM) column, eluent acetonitrile/water mixtures. Elemental analyses: CE Instruments FlashEA® 1112 Elemental Analyzer (Thermo Quest). Results obtained were within ±0.4% unless indicated otherwise. Thin-layer chromatography: Polygram SiI G/UV254 silica gel plates (Macherey-Nagel); 254 nm UV illumination. Parallel synthesis: Carousel 12 Place Reaction Station™ (Radley Discovery Technologies). Compounds 5 and 7a were prepared according to published procedures. The ketone Mannich base hydrochlorides were prepared according to a standard procedure (Blicke, F. F. Org. Reactions 1942, 1, 303-341).
General Procedure A for Preparation of Paullone Derivatives 7a-e by Acid- Catalyzed Fischer Indole Reaction:
A mixture of 7-iodo-3,4-dihydro-1H-benzazepine-2,5-dione (5) (452 mg, 1.5 mmol), an appropriate phenylhydrazine (2.0 mmol) [or the appropriate phenylhydrazine hydrochloride (2.0 mmol) and sodium acetate (164 mg, 2.0 mmol)] in glacial acetic acid (15 mL) is stirred at 70 0C for 1 h. Concentrated sulfuric acid (0.1 ml_) is added and stirring is continued for 1 h. After cooling to room temperature, the mixture is poured into 5 % aqueous sodium acetate solution (20 mL). A precipitate is formed which is filtered off with suction and purified by crystallization from the given solvent. The reaction is shown in scheme 1.
Example 1 2-lodo-9-methyl-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (7b) Preparation according to General Procedure A from 7-iodo-3,4-dihydro-1H- benzazepine-2,5-dione (5) and 4-methylphenylhydrazine hydrochloride yielded 67% of an orange solid mp 312 - 323 0C (dec) (EtOH); IR 3199 (NH), 1648 (C=O); 1H- NMR 2.41 (S1 3 H), 3.48 (s, 2 H), 7.00 - 7.05 (m, 2 H), 7.32 (d, 1 H, J = 8.3 Hz), 7.44 (s, 1 H)1 7.67 (dd, 1 H, J = 2.0/8.6 Hz), 8.06 (d, 1 H, J = 2.0 Hz), 10.14 (s, 1 H), 11.51 (s, 1 H); 13C-NMR 21.2 (prim C), 31.6 (sec.C) 111.2, 117.6, 124.2, 124.3, 134.7, 136.0 (tert. C) 87.6, 107.8, 125.2, 126.6, 127.8, 131.0, 135.0, 136.0, 171.3 (quat. C); Anal. (C17H13IN2O) C, H, N.
Example 2
9-Fluoro-2-iodo-7,12-dihydroindolo[3,2-c(][1]benzazepin-6(5H)-one (7c)
Preparation according to General Procedure A from 7-iodo-3,4-dihydro-1H- benzazepine-2,5-dione (5) and 4-fluorophenylhydrazine hydrochloride yielded 19% of a yellow solid, mp 310 0C (dec) (EtOH). Anal. (C16H10FIN2O x 0.10 EtOH) C, H, N.
Example 3 2-lodo-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepine-9-carbonitrile (7d)
Preparation according to General Procedure A from 7-iodo-3,4-dihydro-1/-/- benzazepine-2,5-dione (5) and 4-cyanophenylhydrazine hydrochloride yielded 31% of a yellow solid, mp 313°C (dec) (EtOH); (C17H10IN3O) HRMS (El) (m/z): Calcd [M+] = 398.98688; found [M+] = 398.98603.
Example 4 9-fert-Butyl-2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (7e)
Preparation according to General Procedure A from 7-iodo-3,4-dihydro-1/-/- benzazepine-2,5-dione (5) and 4-teAf-butylphenylhydrazine hydrochloride yielded 61 % of a yellow solid, mp 313°C (dec) (EtOH); Anal. (C20H19IN2O) C, H, N. General Procedure B for the preparation of 3-oxo-1-butenyl substituted compounds (10a-e):
The 9-substituted 2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (7a-e) (0.50 mmol), methyl vinyl ketone (0.4 ml_, 5 mmol), palladium acetate (23 mg, 0.10 mmol), triphenylphosphine (26 mg, 0.10 mmol) and triethylamine (1 ml_) were suspended in DMF (10 ml_) and stirred at 150 0C under nitrogen atmosphere. The mixture was filtered after 15 min. After addition of silica gel (1.5 g), the mixture was dried in vacuo. The remaining silica gel/reaction product mixture was added onto a silica gel pad in a glass frit and was then eluted with ethyl acetate (150 mL). After evaporation of the solvent the remaining solid was purified by crystallization from ethanol. The reaction is outlined in scheme 1.
Example 5 2-[(1E)-3-Oxo-1-butenyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (10a)
Preparation following General Procedure B from 2-iodo-7,12-dihydroindolo[3,2- cf][1]benzazepin-6(5H)-one (7a) yielded 54% of an orange solid, mp > 330 0C; IR 3306 (NH), 3192 (NH), 1671 (C=O); 1H-NMR 2.37 (s, 3 H), 3.57 (s, 2H), 6.88 (d, 1 H, J = 16.3 Hz), 7.01 - 7.11 (m, 1H), 7.18-7.22 (m, 1 H), 7.29 (d, 1 H, J = 8.5 Hz), 7.46 (d, 1 H, J = 8.1 Hz), 7.65 - 7.71 (m, 3 H), 8.11 (d, 1 H, J = 1.8 Hz), 10.30 (s, 1 H), 11.63 (s, 1 H); 13C-NMR 21.2 (prim. C), 31.7 (sec. C) 111.4, 118.1 , 119.2, 122.4, 122.6, 126.9, 128.1 , 129.7, 142.5 (tert. C) 107.6, 122.9, 126.5, 126.8, 131.9, 137.0, 137.5, 171.3, 198.0 (quat. C); (C20Hi6N2O2) HRMS (El) (m/z): Calcd for [M+] = 316.12119, found for [M+] = 316.12039.
Example 6
9-Methyl-2-[(1E)-3-oxo-1-butenyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)- one (10b)
Preparation following General Procedure B from 2-iodo-9-methyl-7,12- dihydroindolo[3,2-d][1]benzazepin-6(5/-/)-one (7b) yielded 41% of a yellow-orange solid, mp > 330 °C; (C2IHi8N2O2) HRMS (El) (m/z): [M+] calcd 330.13681 ; found 330.13566.
Example 7 9-Fluoro-2-[(1E)-3-oxo-1-butenyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)- one (10c)
Preparation following General Procedure B from 9-fluoro-2-iodo-7,12- dihydroindolo[3,2-d][1]benzazepin-6(5/-/)-one (7c) yielded 39% of a brown-yellow solid, mp 319 0C (dec); (C20H15FN2O2) HRMS (El) (m/z): [M+] Calcd 334.11176, found 334.11155.
Example 8
6-Oxo-2-[(1 E)-3-oxo-1 -butenyl]-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepine- 9-carbonitrile (10d)
Preparation following General Procedure B from 2-iodo-6-oxo-5,6,7,12- tetrahydroindolo[3,2-d][1]benzazepine-9-carbonitπle (7d) yielded 40% of a brown- yellow solid, mp > 330 0C; (C2iHi5N3O2) HRMS (El) (m/z): [M+] calcd 341.11642, found 341.11574. Example 9
9-te/t-Butyl-2-[(1£)-3-oxo-1-butenyl]-7,12-dihydroindolo[3,2-d][1]benzazepin- 6(5H)-one (10e)
Preparation following General Procedure B from 9-terf-butyl-2-iodo-7,12- dihydroindolo[3,2-c/][1]benzazepin-6(5/-/)-one (7e) yielded 47% of a yellow solid, mp > 330 0C; (C24H24N2O2) HRMS (El) (m/z): [M+] calcd 372.18378; found 372.18294.
Example 10 (2E)-3-(9-.erf-Butyl-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepine-2-yl)-2- propenenitrile (10f) A mixture of 9-tert-butyl-2-iodo-7,12-dihydroindolo[3,2-c(][1]benzazepin-6(5H)-one (7e) (215 mg, 0.50 mmol), acrylonitrile (9) (330 μl_, 5.0 mmol), palladium acetate (11 trig, 0.005 mmol), triphenylphosphine (13 mg, 0.050 mmol), and DMF (10 ml_) was stirred at 150 0C under nitrogen. The mixture was filtered after 3 h. After addition of silica gel (1.5 g) the mixture was dried in vacuo. The remaining silica gel/reaction product mixture was added onto a silica gel pad in a glass frit and was then eluted with ethyl acetate (150 ml_). After evaporation of the solvent the remaining solid was purified by crystallization from ethanol to yield 58 mg (32%) of a yellow solid, mp > 330 0C; Anal. (C23H21N3O) C, H, N.
General Procedure C for the preparation of 2-[(1£)-3-aryl-3-oxo-1-propenyl]- 7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-ones 12 by Heck-type reaction with ketone Mannich bases:
A mixture of a 9-substituted 2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5/-/)-one (0.50 mmol) (7a, b or e), a ketone Mannich base hydrochloride (0.55 mmol), palladium(ll)acetate (11 mg, 0.050 mmol), triphenylphosphine (13 mg, 0.050 mmol), triethylamine (2 ml_) and DMF (10 ml_) is stirred at 150 0C under nitrogen. After filtration, silica gel (1.5 g) is added to the filtrate and the solvent is evaporated. The remaining silica gel/reaction product mixture is added onto a silica gel pad in a glass frit and is then eluted with ethyl acetate (200 ml_). After evaporation of the solvent the remaining solid is purified by crystallization from ethanol. For the synthesis of the derivatives 12a-c and 12g-m the procedure was adapted to the use of a parallel synthesis reactor. In this case, the reaction was carried out without addition of triphenylphosphine in 20 ml_ vials with 2 ml_ DMF as solvent. The vessel reactor block temperature was set to 140 0C. The work up procedure was carried out as described above. The reaction is outlined in scheme 2.
Example 11 2-[(1E)-3-Oxo-3-phenyl-1-propenyl]-7,12-dihydroindolo[3,2-c/][1]benzazepin- 6(5H)-one (12a) Preparation following General Procedure C from 2-iodo-7,12-dihydroindolo[3,2- d][1]benzazepin-6(5/-/)-one (7a) and Λ/,Λ/-dimethyl-3-oxo-3-phenyl-1-propanaminium chloride 37 yielded 40% of a yellow solid, mp 256 0C (dec); IR 3309 (NH), 3222 (NH), 1652 (C=O); 1H-NMR 3.59 (s, 2 H), 7.10 (ddd, 1 H, J = 9.0/7.97 Hz), 7.21 (ddd, 1 H, J = 8.0/1.1 Hz), 7.33 (d, 2 H, J = 8.5 Hz ), 7.49 (d, 2 H, J = 8.1 Hz), 7.59 - 7.63 (m, 2 H), 7.68 - 7.72 (m, 2 H), 7.81 (d, 2 H, J = 15.6 Hz), 7.91 (dd, 1 H, J = 8.6/2.0 Hz), 7.98 (d, 1 H), 8.16 - 8.18 (m, 2 H), 8.25 (d, 1H, J = 1.9 Hz1), 10.34 (s, 1 H), 11.63 (s, 1 H); 13C-NMR 31.6 (sec. C), 111.4, 118.0, 119.1 , 121.4, 122.3, 122.4, 127.6, 128.3, 128.4 (2 C), 128.8 (2 C), 133.0, 143.3 (tert. C), 107.5, 122.8, 126.4, 129.8, 131.8, 137.1 , 137.4, 137.5, 171.2, 189.0 (quat. C); (C25Hi8N2O2) HRMS (El) (m/z) [M+]: calcd 378.13681 , found 378.13611.
Example 12
2-[(1 E)-3-(4-Chlorophenyl)-3-oxo-1 -propenyl]-9-methyl-7,12-dihydroindolo[3,2- d][1]benzazepin-6(5H)-one (12b)
Preparation following General Procedure C from 2-iodo-9-methyl-7,12- dihydroindolo[3,2-c(][1]benzazepin-6(5/-/)-one (7b) and (4-chlorophenyl)-Λ/,/V- dimethyl-3-oxo-1-propanaminium chloride yielded 14% of a orange-red solid, mp > 330 0C; (C26Hi9CIN2O2) HRMS (El) (m/z) [M+]: calcd 426.11349, found 426.11309.
Example 13
9-fert-Butyl-2-[(1E)-3-oxo-3-phenyl-1-propenyl]-7,12-dihydroindolo[3,2- cf][1]benzazepin-6(5H)-one (12c)
Preparation following General Procedure C from 9-ferf-butyl-2-iodo-7,12- dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (1b) (7e) and 3 Λ/,Λ/-dimethyl-3-oxo-3- phenyl-1-propanaminium chloride 3 yielded 46% of a yellow-orange solid, mp 276 0C; Anal. (C29H26N2O2) C, H, N.
Example 14
9-fert-Butyl-2-[(1E)-3-(4-methoxyphenyl)-3-oxo-1-propenyl]-7,12- dihydroindolo[3,2-of][1]benzazepin-6(5H)-one (12d)
Preparation following General Procedure C from 9-te/f-butyl-2-iodo-7,12- dihydroindolo[3,2-c(][1]benzazepin-6(5/-/)-one (7e) and 3-(4-methoxyphenyl)-Λ/,Λ/- dimethyl-3-oxo-1-propanaminium chloride yielded 43% of a yellow solid, mp 279 0C; (C30H28N2O3) HRMS (El) (m/z): [M+] calcd 464.20996; found 464.20938.
Example 15 9-terf-Butyl-2-[(1 E)-3-(4-chlorophenyl)-3-oxo-1 -propenyl]-7,12-dihydroindolo- [3,2-d][1]benzazepin-6(5H)-one (12e)
Preparation following General Procedure C from 9-terf-butyl-2-iodo-7,12- dihydroindolo[3,2-c/][1]benzazepin-6(5H)-one (7e) and 3-(4-chlorophenyl)-Λ/,Λ/- dimethyl-3-oxo-1-propanaminium chloride 2 yielded 46% of orange crystals, mp 285 0C (dec); Anal. (C29H25CIN2O2) C, H, N.
Example 16
9-te/t-Butyl-2-[(1£)-3-oxo-3-(2-thlenyl)-1-propenyl]-7,12-dihydroindolo[3,2- d][1]benzazepin-6(5H)-one (12f)
Preparation following General Procedure C from 9-te/f-butyl-2-iodo-7,12- dihydroindolo[3,2-c(][1]benzazepin-6(5H)-one (7e) and Λ/,Λ/-dimethyl-3-oxo-3-(2- thienyl)-1-propanaminium chloride yielded 34% of orange crystals, mp 278 °C (dec); Anal (C27H24N2O2S x 0.15 EtOH) C, H, N.
Example 17 9-tert-Butyl-2-[(1£)-3-(2-furyl)-3-oxo-1-propenyl]-7,12-dihydroindolo[3,2- d][1]benzazepin-6(5H)-one (12g)
Preparation following General Procedure D from 3-(2-furyl)-Λ/,Λ/-dimethyl-3-oxo-1- propanaminium chloride yielded 34% of an orange solid, mp 274 0C (dec); Anal. (C27H24N2O3) C, H, N.
Example 18
9-fert-Butyl-2-[(1E)-3-oxo-3-(3-pyridinyl)-1-propenyl]-7,12-dihydroindolo[3,2- d][1]benzazepin-6(5H)-one (12h)
Preparation following General Procedure D from Λ/,Λ/-dimethyl-3-oxo-3-(3-pyridinyl)- 1-propanaminium chloride yielded 38% of a yellow solid, mp 274 0C; Anal. (C28H25N3O2 x 0.10 EtOH) C, H, N.
Example 19
9-teAt-Butyl-2-[(1£)-3-(4-hydroxyphenyl)-3-oxo-1-propenyl]-7,12- dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (12i)
Preparation following General Procedure D from 3-(4-hydroxyphenyl)-Λ/,Λ/-dimethyl- 3-OXO-1 -propanaminium chloride yielded 28% of a yellow solid, mp 264 0C; Anal. (C29H26N2O3) C, H, N.
Example 20
9-fert-Butyl-2-[(1£)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propenyl]-7,12- dihydroindolo[3,2-of][1]benzazepin-6(5H)-one (12j) Preparation following General Procedure D from 3-(3,4,5-trimethoxyphenyl)-Λ/,Λ/- dimethyl-3-oxo-1-propanaminium chloride yielded 28% of yellow crystals, mp 277 0C; Anal. (C32H32N2O5) C, H, N. Example 21 9-tert-Butyl-2-[(1 E)-3-(2,5-dimethoxyphenyl)-3-oxo-1 -propenyl]-7,12- dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (12k)
Preparation following General Procedure D from 3-(2,5-dimethoxyphenyl)-Λ/,Λ/- dimethyl-3-oxo-1-propanaminium chloride yielded 14% yellow crystals, mp 251 0C; Anal. (C31H30N2O4) C, H, N.
Example 22
9-te/t-Butyl-2-[(1 £)-3-(3,4-dimethoxyphenyl)-3-oxo-1 -propenyl]-7,12- dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (12I)
Preparation following General Procedure D from 3-(3,4-dimethoxyphenyl)-Λ/,Λ/- dimethyl-3-oxo-1-propanaminium chloride yielded 15% of a yellow solid, mp 258 0C; (C3IH30N2O4) HRMS (El) (m/z) [M+] calcd 494.22052; found 494.22006.
Example 23
9-fert-Butyl-2-[(1 E)-3-(2,4-dimethoxyphenyl)-3-oxo-1 -propenyl]-7,12- dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (12m)
Preparation following General Procedure D from 3-(2,4-dimethoxyphenyl)-Λ/,Λ/- dimethyl-3-oxo-1-propanaminium chloride yielded 17% of a yellow solid, mp 223 0C; (C31H30N2O4) HRMS (El) (m/z) [M+] calcd 494.22052; found 494.22063.
Example 24 7-(Phenylethynyl)-3,4-dihydro-1H-1-benzazepine-2,5-dione (14)
A mixture of 7-iodo-3,4-dihydro-1H-1-benzazepine-2,5-dione (5) (1.2 g, 4.0 mmol), bis(triphenylphosphine)palladium(ll)dichloride (56 mg; 0.080 mmol) and copper(l)iodide (30 mg, 0.16 mmol) in 80 ml triethylamine is stirred at 50 0C under nitrogen. Phenylacetylene (0.8 ml_; 8.0 mmol) (13) is added slowly and stirring is continued until the educt 5 is no longer detectable by tic (requires approx. 10 min.). After addition of acetone (150 ml_), filtration, and evaporation of the filtrate the residue is crystallized from ethanol to yield 92% brown crystals; mp 218 - 220 0C (dec); (Ci8H13NO2) HRMS (El) (m/z): [M]+- calcd 275.0946; found 275.0944. The reaction is outlined in scheme 3.
Example 25
9-ferf-Butyl-2-(phenylethynyl)-7,12-dihydroindolo[3,2-c(][1]benzazepin-6(5H)-one (15)
A mixture of 7-(phenylethinyl)-3,4-dihydro-1H-1-benzazepine-2,5-dione (14, 165 mg, 0.60 mmol), 1-(4-te/f-butylphenyl)hydrazine hydrochloride (181 mg, 0.90 mmol), sodium acetate (74 mg, 0.90 mmol) and glacial acetic acid (6 ml_) is stirred at 70-80 0C for 1 h. Concentrated sulfuric acid (0.06 ml.) is added and stirring at 70-80 0C is continued for 1 h. After cooling to room temperature, the mixture is poured into a 5% aqueous sodium acetate solution (12 ml_).The precipitate is filtered off, washed with water and crystallized twice from ethanol to yield 28% yellow crystals, mp 291 0C (dec); Anal. (C28H24N2O) C, H, N. The reaction is outlined in scheme 3.
Tests on inhibition an AxA growth, inhibition of parasite growth in infected macrophages, and killing of macrophages are described in the general procedure part above. Compounds according to the present invention and reference compounds have been tested accordingly. The results are given below in table 1
Table 1 : Antileishmanial activity and in vitro toxicity of Paullones 1, 2, 10, 12 and 15 wherein R3 is H
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
*AxA - axenic amastigotes; § ND = not determined. Average ± standard error for values determined by duplicate or triplicate assays. Values reported without standard error were determined in a single assay.
Table 2: IR Data for compounds 7c-e; 10b-f, 12b-m, 14, 15
Compound v [cm -1] #
7c 3328 (NH), 1651 (C=O)
7d 3327 (NH), 3275 (NH), 2218 (CN), 1662 (C=O)
Figure imgf000030_0001
Table 3: 1H-NMR data (D6-DMSO, 400 MHz, [ppm]) for compounds 7c-e; 10b-f, 12b-m, 14, 15
ComSignals in ppm (δ scale) pound
#
7c 3.52 (s, 2 H), 7 00-7. 06 (m, 1 H), 7.42 (dd, 1 H1J = = 8 .8/4.5 Hz), 7. 50 (dd,
1H1 J = 9 9/2.5 Hz), 7 .69 (dd 1 H ,J = 8.5/2 .0 Hz), 8. 06 (d, 1 H1J = 2 .0
Hz), 10.ie 5(s, 1 H), 11 •74 (s, 1 H)
7d 3.62 (s, 2 H), 7. 08 (d, 1 H1J = 8.6 Hz), 7.53 (d , 1 H, J = 8.5/1.4 Hz) ,7. 58
Figure imgf000031_0001
Figure imgf000032_0001
12k 1.37 (s, 9 H), 3.57 (s, 2 H), 3.76 (s, 3 H), 3.82 (s, 3 H), 7.04 (d, 1 H, J = 3.1), 7.11 - 7.17 (m, 2 H), 7.27 - 7.30 (m, 2 H), 7.37 (dd, 1 H1 J = 8.6/0.4 Hz), 7.41 (d, 1 H1 J = 15.9 Hz), 7.52 (d, 1 H, J = 15.9 Hz)1 7.61 (d, 1 H1 J = 1.7 Hz)1 7.74 (dd, 1 H, J = 8.5/1.9 Hz)1 8.05 (d, 1 H1 J = 1.9 Hz), 10.31 (s, 1 H), 11.45 (s, 1 H)
121 1.38 (s, 9 H)1 3.59 (s, 2 H)1 3.87 (s, 3 H), 3.89 (s, 3 H), 7.14 (d, 1 H, J = 8.6), 7.29 - 7.32 (m, 2 H), 7.40 (dd, 1 H1 J = 8.6/0.5 Hz)1 7.63 (d, 1 H1 J = 2.0 Hz)1 7.78 (d, 1 H, J = 15.4 Hz)1 7.90 - 7.95 (m, 2 H)1 7.98 (d, 1 H, J = 15.7 Hz)1 8.19 (d, 2 H1 J = 1.8 Hz), 10.31 (s, 1 H), 11.48 (s, 1 H)
12m 1.37 (s, 9 H)1 3.57 (s, 2 H), 3.87 (s, 3 H), 3.91 (s, 3 H), 6.67 (dd, 1 H, J = 8.6/2.3 Hz), 6.72 (d, 1 H, J = 2.3), 7.27 - 7.30 (m, 2 H)1 7.38 (d, 1 H, J = 8.6), 7.57 (s, 2 H), 7.60-7.62 (m, 2 H)1 7.73 (dd, 1 H1 J = 8.5/2.0 Hz)1 8.05 (d, 1 H1 J = 1.9 Hz), 10.29 (s, 1 H)1 11.47 (s, 1 H)
14 2.70 - 2.73 (m, 2 H), 2.93 - 2.96 (m, 2 H), 7.22 (d, 1 H1 J = 8.6 Hz), 7.41 - 7.45 (m, 3 H), 7.56 - 7.58 (m, 2 H)1 7.71 (dd, 1 H, J = 2.0/8.6 Hz)1 7.96 (d, 1 H1 J = 2.0 Hz), 10.31 (s, 1 H)
15 1.37 (S1 9 H)1 3.58 (s, 2 H)1 7.28 - 7.30 (m, 2 H), 7.37 (d, 1 H, J = 8.6 Hz)1 7.41 - 7.48 (m, 3 H), 7.54 (dd, 1 H1 J = 1.8/8.6 Hz)1 7.56 - 7.59 (m, 2 H), 7.62 (d, 1 H, J = 1.5 Hz), 7.95 (d, 1 H, J = 1.8 Hz)1 10.30 (s, 1 H), 11.55 (s, 1 H)
Table 4: 13C-NMR Data (D6-DMSO, 100.6 MHz, [ppm]) for Compounds 7d-e; 10b- f, 12b-m, 14, 15
Figure imgf000033_0001
Figure imgf000034_0001
131.4(2C), 143.0 187.7
12e 31.7 (3 31 .6 110.9, 113.5, 34.2, 107.6, 123.0, 126.1,
C) 120.6, 121.0, 122.4, 129.7, 131.9, 135.6 ,136.1,
127.7, 128.1, 128.9 137.1, 138.0, 141.5 ,171.3,
(2C), 130.3(2C), 187.9
143.8
12f 31.7 (3 31 .6 111.0, 113.5, 120.6, 34.3, 107.6, 123.0, 126.2,
C) 121.2, 122.4, 127.8, 129.9, 131.9, 135.7 , 137.0,
128.0, 128.8, 133.5, 141.6, 145.5, 171.3 , 181.4
135.6, 142.4
12g 31.8 (3 31 .7 111.0, 112.8, 113.6, 34.4, 107.7, 123.1, 126.2,
C) 119.2, 120.7, 121.3, 129.68, 132.0, 135. 7,137.1,
122.5, 127.6, 128.0, 141.7, 153.0, 171.4 , 176.6
142.2, 148.3
12h 31.7 (3 31 .6 111.0, 113.5, 120.7, 34.3, 107.7, 122.5, 126.2,
C) 121.3, 123.0, 124.0, 129.7, 132.0, 132.9 , 135.7,
127.8, 128.2, 135.8, 137.2, 141.6, 171.3 , 189.3
144.1, 150.0,153.3
12i 31.8 (3 31 .6 111.0, 113.5, 115.4 34.4, 107.7, 123.5, 126.3,
C) (2C), 120.6,121.5, 129.2, 130.2, 132.0 , 135.7,
122.5, 127.3, 128.0, 136.8, 142.0, 162.2 ,171.3,
131.1 (2C), 141.6 187.0
12j 31.7 (3 31 .6 106.0(2C), 111.0, 34.3, 107.6, 122.9, 126.1,
C), 55.9 113.5, 120.6, 121.3, 128.9, 131.9, 133.0 , 135.6,
(2C) , 122.4, 127.6, 128.3, 136.9, 141.8, 143.2 , 152.8(2
56.1 141.5 C), 171.3, 187.7
12k 31.5 (3 31 .3 110.7, 113.3, 113.5 34.1,48.3, 107.4, 122.7,
C), 55.2, (2C), 117.8, 120.3, 125.9, 129.1, 129.4 131.6,
56.0 122.3, 126.1, 127.1, 135.4, 136.6, 141.3 151.3,
127.3, 142.3 152.7, 171.1
121 31.8 (3 31 .7 110.7, 110.9,111.0, 34.4, 107.7, 123.0. 126.3,
C), 55.6, 113.6,120.7,121.4, 130.1, 130.6, 132.0 135.7, 55.8 122.4, 123.3 , 127.6, 136.9, 141.7, 148.9, 153.3,
127.9, 142.4 171.4, 187.2 m 31.8(3 31 .7 99.7, 107.7, 111.0, 34.4, 106.0, 121.5, 123.1,
C), 55.6, 113.5, 120.6 , 122.6, 126.2, 130.1, 132.0, 135.7,
56.0 126.7, 127.2 , 127.4, 137.0, 141.7, 160.1, 163.8,
131.8, 140.8 171.4, 189.6
29 .1, 122.2, 128.7 (2C), 88.1,89.4, 116.9, 122.1,
37 .7 128.8, 131.3 (2C), 126.5, 139.3, 173.4, 197.6
133.4, 136.2
31.8(3 31 .6 111.1, 113.5 , 120.7, 34.3,89.0, 108.0, 117.2,
C) 122.5, 128.8 , 128.8 122.3, 123.2, 126.2, 131.5,
(2C), 129.7, 130.4, 135.4, 135.8, 141.7, 171.4
131.3(2C) (one quat. C not detected due to peak overlapping)
Table 5: HPLC Data
About 1 mg of each substance was dissolved in 1000 μl of DMSO1 filtered through a
Acrodisc® GHP-filter and diluted (1 plus 3) before injection.
Injection volume: 10 μl; Flow rate: 1.000 ml/min; Runtime: 15 min; Retention time for
DMSO: 1.03 min.
Figure imgf000037_0001
Figure imgf000038_0001
Table 6: Elemental Analysis Data
Figure imgf000038_0002

Claims

Claims
A compound of general formulas (Ia) or (Ib) formula Ia
formula Ib
Figure imgf000039_0001
wherein R1 is aryl, optionally substituted, heteroaryl, optionally substituted, cycloaliphatic group, optionally substituted, CO-aryl, optionally substituted, CO-heteroaryl, optionally substituted, CO-cycloalphatic group, optionally substituted, or CN, wherein the substituents are independently selected from one or more of halo, CN, OH, 0-C1-C6 alkyl; COOH, COO-C1-C6 alkyl, CONH2, CONHfd-CeOalkyl, CON(C1-C6alkyl)2, aryl.heteroaryl, polyoxyethenyl or combinations thereof;
R ϊ2 is an electron-donating group;
R3 is independently H, C1-C6 alkyl, optionally substituted, or CO-C1-C6 alkyl, optionally substituted, wherein the substituents are independently selected from one or more of halo, CN, OH, 0-C1-C6 alkyl; COOH, COO" C1-C6 alkyl, CONH2, CONH(C1-C6)alkyl, CON^-Cealkylk, aryl, heteroaryl, polyoxyethenyl or combinations thereof; or an optical isomer, or a salt or solvate thereof.
2. The compound according to claim 1 wherein R3 is H or CH3.
3. The compound according to any one of claim 1 or 2, wherein R1 is an CO- aryl group, optionally substituted, or CO-heteroaryl group, optionally substituted, wherein the substituents are selected from one or more of halo, OH, 0-Ci-C6 alkyl.
4. The compound of general formula (Ia) wherein R1 is CO-aryl, optionally substituted with one or more of halo, OH, OCH3 or a 5-membered or 6- membered CO-heteroaryl, optionally substituted, wherein the substituents are independently selected from one or more of halo, OH or OCH3.
5. The compound according to any one of the preceding claims, wherein R2 is selected from Ci-C6 alkyl, optionally substituted, CrC6 cycloalkyl, optionally substituted, benzyl, or substituted benzyl, wherein the substituents are selected from one or more of halo, OH, 0-C1-C6 alkyl.
6. The compound according to any one of the preceding claims wherein R2 is a tert-butyl-group.
7. The compound according to any one of the preceding claims which are selected from: 9-te/t-Butyl-2-[(1E)-3-oxo-3-phenyl-1-propenyl]-7,12-dihydroindolo[3,2- d][1]benzazepin-6(5/-/)-one;
9-ferf-Butyl-2-[(1 £)-3-(4-methoxyphenyl)-3-oxo-1 -propenyl]-7, 12- dihydroindolo[3,2-d][1]benzazepin-6(5/-/)-one;
9-terf-Butyl-2[(1£)-3-(4-chlorophenyl)-3-oxo-1-propenyl]-7,12- dihydroindolo-[3,2-d][1]benzazepin-6(5/-/)-one; 9-feAf-Butyl-2[(1£)-3(2-furyl)-3-oxo-1-propenyl]-7,12-dihydroindolo[3,2- d\[\ ]benzazepin-6(5H)-one;
9-tert-Butyl-2-[(1E)-3-oxo-3-(3-pyridinyl)-1-propenyl]-7,12- dihydroindolo[3,2-d][1 ]benzazepin-6(5H)-one; or 9-teAt-Butyl-2-[(1E)-3-(2,4-dimethoxylphenyl)-3-oxo-1-propenyl]-7,12- dihydroindolo[3,2-cfl[1]benzazepin-6(5H)-one.
8. The compound according to claim 1 , which is 9-ferf-Butyl-2-(phenylethynyl)- 7,12-dihydroindolo[3,2-d][1 ]benzazepin-6(5H)-one.
9. Pharmaceutical composition comprising as an active ingredient a compound of any one of claims 1 to 8 optionally together with pharmaceutically acceptable carriers, diluents and adjuvants.
10. The compound according to any one of claims 1 to 8 for the prophylaxis or treatment of diseases caused by single celled parasites.
11. The compound according to claim 10 for the prophylaxis or treatment of Leishmaniasis.
12. The compound according to claim 10 or 11 for the prophylaxis or treatment of Visceral leishmaniasis.
PCT/EP2008/005908 2007-07-18 2008-07-18 Paullone derivatives and its use WO2009010298A2 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102924462A (en) * 2012-10-24 2013-02-13 华东师范大学 Method for synthesis of 1,2,3,4,5,9-substituted benzazepine compound
US9572815B2 (en) 2013-03-15 2017-02-21 St. Jude Children's Research Hospital Methods and compositions of p27KIP1 transcriptional modulators
EP3786164A1 (en) 2015-06-18 2021-03-03 Ting Therapeutics LLC Methods and compositions for the prevention and treatment of hearing loss

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999065910A1 (en) * 1998-06-16 1999-12-23 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Fused azepinone cyclin dependent kinase inhibitors
US20030181439A1 (en) * 2000-02-15 2003-09-25 Laurent Meijer Use of paullone derivatives for making medicines
EP1757607A1 (en) * 2005-08-24 2007-02-28 Molisa GmbH N5-substituted benzo¬2,3|azepino¬4,5-b|indol-6-ones for treating tropical diseases

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999065910A1 (en) * 1998-06-16 1999-12-23 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Fused azepinone cyclin dependent kinase inhibitors
US20030181439A1 (en) * 2000-02-15 2003-09-25 Laurent Meijer Use of paullone derivatives for making medicines
EP1757607A1 (en) * 2005-08-24 2007-02-28 Molisa GmbH N5-substituted benzo¬2,3|azepino¬4,5-b|indol-6-ones for treating tropical diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEOST M ET AL: "PAULLONES ARE POTENT INHIBITORS OF GLYCOGEN SYNTHASE KINASE-3BETA AND CYCLIN-DEPENDENT KINASE 5/P25" EUROPEAN JOURNAL OF BIOCHEMISTRY, BERLIN; DE, vol. 267, no. 19, 1 October 2000 (2000-10-01), pages 5983-5994, XP000981256 ISSN: 0014-2956 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102924462A (en) * 2012-10-24 2013-02-13 华东师范大学 Method for synthesis of 1,2,3,4,5,9-substituted benzazepine compound
US9572815B2 (en) 2013-03-15 2017-02-21 St. Jude Children's Research Hospital Methods and compositions of p27KIP1 transcriptional modulators
EP3786164A1 (en) 2015-06-18 2021-03-03 Ting Therapeutics LLC Methods and compositions for the prevention and treatment of hearing loss
US11446308B2 (en) 2015-06-18 2022-09-20 St Jude Children's Research Hospital Methods and compositions for the prevention and treatment of hearing loss

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