WO2006011750A1 - Tetrahydro-beta-carbolinone derivatives and process for preparing the same - Google Patents

Tetrahydro-beta-carbolinone derivatives and process for preparing the same Download PDF

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Publication number
WO2006011750A1
WO2006011750A1 PCT/KR2005/002433 KR2005002433W WO2006011750A1 WO 2006011750 A1 WO2006011750 A1 WO 2006011750A1 KR 2005002433 W KR2005002433 W KR 2005002433W WO 2006011750 A1 WO2006011750 A1 WO 2006011750A1
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compound
tetrahydro
bromo
carbolin
disease
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PCT/KR2005/002433
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French (fr)
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Jae-Ki Min
Bu-Young Choi
Mee-Hyun Lee
Bon-Am Koo
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C & C Research Laboratories
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to a new tetrahydro-beta-carbolinone derivative having antagonist effect for cyclin dependent kinase ('CDK 3 ' below) as well as an agonist effect for the expression of endogenous tumor suppressor protein.
  • the present invention also relates to a composition for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy- induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection, comprising the tetrahydro-beta-carbolinone derivative as an active ingredient.
  • the anticancer drugs According to the aging of modern society, the risk caused by cancer is getting more serious.
  • the anticancer drugs have not seemed to show any prominent improvements for tumorigenesis because most anticancer drugs except Taxol rely upon the spontaneous cell death mechanism evoked by the abnormality of DNA in cancer cells which can be induced by a direct injury of DNA or the perturbation of DNA replication process.
  • the most serious drawback of these anticancer drugs is that the cancer cells rapidly process to resistance against the drugs.
  • the radiotherapy or anticancer chemotherapy also relies on the same mechanism of DNA injury, the drug resistant cancer cells can resist to other anticancer therapies.
  • CDK is a major enzyme that regulates the eukaryotic cell cycle.
  • extracellular growth signal promotes the cell proliferation following the sequential cell cycle of Gl phase (gap 1 phase) ⁇ S phase (synthesis phase) ⁇ G2 phase (gap 2 phase) ⁇ M phase (mitosis phase).
  • CDK is activated by binding with specific cyclin, and regulate the cell cycle. For example, in Gl phase it is decided whether the cell is to proliferate or not. In a cell that is decided to proliferate, cyclin D/CDK4,6 complex phosphorylates pRB, and cyclin E/CDK2 complex hyperphosphorylates pRB.
  • a transcription factor E2F is dissociated from pRB and promotes the cell into S phase where DNA replication is carried out.
  • the transfer into G2 phase is stimulated by cyclin AJCDKl complex.
  • G2 phase the completion of DNA replication is confirmed and the timing of cell division is decided.
  • M phase equal distribution of completely replicated chromosomes and subsequent cell division is accomplished, and the cell returns to Gl phase again.
  • Most normal cells cease their division at Gl phase and enter the dormant GO phase where protein synthesis or energy metabolism is suspended, while the cancer cells maintain the rapid and anarchic cell cycle by the continual signal.
  • the present inventors have tried to develop a new anticancer agent which exhibits anticancer activity through a mechanism of inhibiting cell cycle regulators to overcome the drug resistance and various side effects of the existing inhibitors against DNA synthesis.
  • a tetrahydro-beta-carbolinone derivative of the formula (1) shows a selective and potent inhibitory activity against CDK and can be used as a new orally administrable CDK inhibitor, and completed the present invention.
  • the tetrahydro-beta-carbolinone derivative of the present invention has the effect of enhancing the expression of such endogenous tumor suppressor proteins as pi 6 and p21, as well as the inhibitory activity against CDK.
  • the compound of the present invention shows an improved anticancer effect (inhibitory effect against cell growth) compared with R-roscovitine, and can be used for the treatment of autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection, in addition to the cancer.
  • It is still another object of the present invention to provide a composition comprising the above compound as an active ingredient- together with a pharmaceutically acceptable carrier, for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection.
  • Figure 1 represents the Western blotting result showing the enhancing effect of the compound according to the present invention for the expression of pl6 and p21.
  • the present invention relates to a new tetrahydro-beta-carbolinone derivative of the following formula (1): [Formula 1] in which
  • R represents -(CH 2 ) H -R 1 , -(CO)-R 2 , or -(SO 2 )-R 3 , wherein n denotes an integer of 0 to 5, R 1 represents hydrogen, Q-C ⁇ -alkoxy, d-Ce-hydroxyalkyl, C 2 -C 6 -alkoxycarbonyl, carboxy, carbamoyl, or phenyl,
  • R 2 represents C 2 -C 6 -alkoxyalkyl, d-C 6 -alkyl, halogeno-Q-Ce-alkyl, d ⁇ C 6 -alkoxy, d-Q-alkylcarbonyloxyalkyl, C 3 -C 7 -cycloalkyl, or phenyl,
  • R 3 represents Ci-C 6 -alkyl
  • X represents halogen, d-C 6 -alkoxy, or aminosulfonyl, pharmaceutically acceptable salt, or stereoisomer thereof.
  • alkyl in the present invention may be linear or branched.
  • Preferred compounds among the compound of formula (1) above useful as inhibitory agent against CDK or enhancing agent for the expression of pi 6 and p21 are those wherein R represents -(CO)-R 2 , pharmaceutically acceptable salt, or stereoisomer thereof.
  • More preferred compounds are those wherein X represents bromine, pharmaceutically acceptable salt, or stereoisomer thereof.
  • Most preferred compounds are those wherein R represents -(CO)-R , wherein R represents d-C ⁇ -alkoxyalkyl or halogeno-d-C 6 -alkyl, and X represents bromine, pharmaceutically acceptable salt, or stereoisomer thereof.
  • Typical compounds among the compound of formula (1) are those selected from the group consisting of:
  • Particularly preferred compounds among the above typical compounds are those selected from the group consisting of: 6-Bromo-9-(2-chloro-acetyl)-2,3,4,9-tetrahydro- ⁇ -carbolin-l-one (Compound 3); 9-Acetyl-6-bromo-2,3,4,9-te1xahydro-/?-carbolm-l-one (Compound 6); 6-Bromo-9-(2-methoxy-acetyl)-2,3,4,9-tetrahydro- / 9-carbolin-l-one (Compound 12); and Acetic acid 2-(6-bromo-l-oxo-l,2,3,4-tetrahydro-/ ⁇ carbolm-9-yl)-2-oxo-ethyl ester (Compound 13).
  • the compound of formula (1) according to the present invention can also form a pharmaceutically acceptable salt.
  • Such salt includes non-toxic acid addition salt containing pharmaceutically acceptable anion, for example, a salt with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydriodic acid, etc., a salt with organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc., or a salt with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.
  • a salt with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid,
  • the compound of formula (I) can also form a salt with alkaline metals such as sodium, potassium, etc., and a salt with other acids or bases which have been well-known and widely used in the technical field to which the tetrahydro-beta-carbolinone derivative belongs.
  • These salts can be prepared according to any of the conventional conversion methods.
  • the compound of the present invention may have asymmetric carbon atoms in the structure depending on the kind of substituents, and so may exist in the form of R or S isomer, or mixtures thereof including racemates.
  • the pure stereoisomers may be obtained by the conventional resolution method known in this field.
  • the present invention also includes all of these stereoisomers and their mixtures in its scope.
  • the compound of the present invention may be prepared according to the process explained below, and so the present invention also provides such process for preparing the compound of formula (1).
  • the compound of formula (1) defined above, pharmaceutically acceptable salt, or stereoisomer thereof may be prepared by the process which comprises
  • the above process (a) of reacting the compound of formula (2) with the compound of formula (3) is preferably carried out in a solvent and in the presence of a base.
  • a solvent one or more can be selected from the group consisting of acetonitrile, dimethylsulfoxide, N,N-dimethylforrnamide, and tetrahydrofuran
  • the base one or more can be selected from the group consisting of sodium hydroxide, potassium fluoride, potassium carbonate, and sodium hydride.
  • reaction aids as aluminum oxide may be further used. It is economic in the aspect of yield, etc.
  • the compound of formula (1) according to the present invention may be advantageously used as an anticancer agent due to its excellent inhibitory activity against CDKs. Therefore, the present invention relates to an anticancer composition comprising the compound of formula (1), pharmaceutically acceptable salt, or stereoisomer thereof as an active ingredient together with the pharmaceutically acceptable carrier.
  • the compound of the present invention may also be advantageously used for the preparation of therapeutic agents for treating autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection besides cancer.
  • the cancer is defined as solid tumor and leukemia;
  • the autoimmune disease is defined as psoriasis, alopecia, and multiple sclerosis;
  • the cardio-vascular disease is defined as stenosis, arteriosclerosis, and recurrent stricture;
  • the infection disease is defined as those caused by unicellular parasites;
  • the kidney disease is defined as glomerulonephritis;
  • the chronic neurodegenerative disease is defined as Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, AIDS, dementia, and Alzheimer's disease;
  • the acute neurodegenerative disease is defined as cerebral ischemia and neurotrauma;
  • the viral infection is defined as the infections by giant cell, herpes, hepatitis B or C, and HIV.
  • the compound of the present invention When the compound of the present invention is used for clinical purpose, it is preferable to administer it to the subject patient in an amount ranging from 0.5 to lOOOmg, preferably 50 to 200mg, per day.
  • the total daily dosage may be administered once or over several times.
  • specific administration dosage for an individual patient can be varied depending on specific compound used, body weight, gender, hygienic condition, diet of subject patient, time or method of administration, excretion rate, mixing ratio of agent, severity of disease to be treated, etc.
  • composition according to the present invention As a result of administering the composition according to the present invention in the amount of lOOmg/kg to 10 rats and observing their conditions in 1 day, there was no rat which died or showed serious disease. Thus, it was identified that the compound of the present invention is not toxic.
  • the compound of the present invention may be formulated as a parenteral injection or oral preparation, depending on its application purpose.
  • aqueous or oily suspension for sterilized injection can be prepared according to the known procedure using suitable dispersing agent, wetting agent, or suspending agent.
  • Solvents that can be used for preparing injections include water, Ringer's fluid, and isotonic NaCl solution, and also sterilized fixing oil may be conveniently used as the solvent or suspending media. Any non-irritating fixing oil including mono- or di-glyceride may be used for this purpose.
  • Fatty acid such as oleic acid may also be used for injections.
  • the orally administrable solid preparations include capsules, tablets, pills, powders, and granules, capsules and tablets among which are preferable. It is desirable for tablets and pills to be formulated into enteric-coated preparation.
  • the solid preparations are prepared by mixing the active compound of formula (1) with one or more carriers selected from inert diluents such as sucrose, lactose, starch, etc., lubricants such as magnesium stearate, disintegrating agents, and binding agents.
  • the active compound of formula (1) may be administered together with one or more known anticancer agents.
  • the anticancer agents to be mixed and administered together with the compound of the present invention in this manner include 5-fluorouracil, cisplatin, doxorubicin, taxol, Gemcitabine, etc.
  • the preparations comprising the compound of the present invention which expect several pharmacological effects including the anticancer activity, are not restricted to those explained above, but may include any preparations useful for the prevention and treatment of diseases.
  • the extract was dried over MgSO 4 , filtered, and distilled under reduced pressure.
  • the residue was dissolved in dichloromethane (1OmA), and trifluoroacetic acid (0.032mA, 0.411mmol) was slowly added at 0 ° C, and stirred for 1 h.
  • the reaction mixture was distilled under reduced pressure, diluted with water, neutralized with aqueous NaHCO 3 solution, and extracted twice with ethyl acetate (3OmA).
  • the extract was dried over MgSO 4 , filtered, and distilled under reduced pressure.
  • the extract was dried over MgSO 4 , filtered, and distilled under reduced pressure.
  • the residue was dissolved in dichloromethane (10m£), and trifluoroacetic acid (0.032m£, 0.411mmol) was slowly added at 0 ° C, and stirred for 1 h.
  • the reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO 3 solution, and extracted twice with ethyl acetate (30ml).
  • the extract was dried over MgSO 4 , filtered, and distilled under reduced pressure.
  • the reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO 3 solution, and extracted twice with ethyl acetate (40m£).
  • the extract was dried over MgSO 4 , filtered, and distilled under reduced pressure.
  • n-hexane 5in£, which was stirred for 10 min at room temperature and filtered to give the title compound (80mg, 86.9%) as white solid.
  • the reaction mixture was diluted with water (5ra£), saturated aqueous NaHCO 3 solution was added, and the mixture was extracted twice with ethyl acetate (20m£).
  • the extract was dried over MgSO 4 , filtered, and distilled under reduced pressure.
  • the residue was dissolved in dichloromethane (5m£), and trifluoroacetic acid (0.032m£, 0.411mmol) was slowly added at 0 ° C, and stirred for 1 h.
  • the reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO 3 solution, and extracted twice with ethyl acetate (20m ⁇ ).
  • the extract was dried over MgSO 4 , filtered, and distilled under reduced pressure.
  • the reaction mixture was diluted with water (5ml), saturated aqueous NaHCO 3 solution was added, and the mixture was extracted twice with ethyl acetate (20ml). The extract was dried over MgSO 4 , filtered, and distilled under reduced pressure. The residue was dissolved in dichloromethane (5ml), and trifluoroacetic acid (0.032m£, 0.411mmol) was slowly added at 0 ° C, and stirred for 1 h. The reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO 3 solution, and extracted twice with ethyl acetate (20M). The extract was dried over MgSO 4 , filtered, and distilled under reduced pressure.
  • CDK2/cyclin A and CDK2/cyclin E complexes were prepared by using Baculovirus recombinant system and insect cell S£21 purchased from Invitrogen Co.
  • the substrate Histone (Cat. # 382150) was purchased from Calbiochem Co.
  • Test samples (0 ⁇ M and 0.032 ⁇ M-IOO ⁇ M) diluted in various concentrations in 2X reaction buffer [2OmM Tris/HCl (pH8.0), 1OmM MgCl 2 , lOO ⁇ M NaVO 3 , 5mM glycerophosphate, ImM DTT] were added to a 96-well filter plate (Cat. # MHVBN4550, Millipore).
  • 2X reaction buffer 2OmM Tris/HCl (pH8.0), 1OmM MgCl 2 , lOO ⁇ M NaVO 3 , 5mM glycerophosphate, ImM DTT
  • the mixture of substrate and ATP [3 ⁇ M cold ATP/well (0.3/ ⁇ /well, stock ImM), 0.3 ⁇ Ci [ ⁇ - 32 P]ATP/well (0.03/ ⁇ /well, stock 10 ⁇ Ci/ ' ⁇ ), Histone (10/ ⁇ /well, stock lmgM), 20/ ⁇ dH 2 O] was added and subjected to enzymatic reaction for 30 min at 30 ° C.
  • the reaction was stopped by adding 100 ⁇ i of 70% ice-cold TCA.
  • the reaction mixture was washed five times with 200 ⁇ of 25% ice-cold TCA and dried for 1 h at 60 ° C.
  • MTS one solution was purchased from Promega, and PA-I (ovarian) and HeLa(cervix) cells were purchased from ATCC. The cells were cultured in MEM media containing 10% FBS at 37 "C in a CO 2 incubator.
  • the cells cultured in T75 flask were diluted with growth media and distributed into a plate in a population of 5000 cells per well in 100 ⁇ l of media. After incubating for 24 h in a CO 2 incubator at 37 "C, 100 ⁇ i of test samples diluted with growth media in various concentrations (0 ⁇ M and 0.5 ⁇ M-100 ⁇ M; the final concentration of DMSO was
  • Thermo-MAX Microplate Reader (Molecular Devices Co.).
  • the absorbance data were treated on Microsoft Excel, with using the value from the well treated only by MTS one solution as blanc, and adopting the value from 0 ⁇ M well as 100% activity.
  • the relative values were calculated for each well, and the IC 50 values were determined as presented in Table 2 below.
  • the antibody against pl6 was purchased from NeoMarkers Co.; the antibodies against p21 (C-19, sc-397) and actin (1-19, sc-1616) were from Santacruz Co.; the secondary antibodies, HRP-goat-anti-mouse IgG, HRP-goat-anti-rabbit IgG, and HRP-donkey-anti-goat IgG, were from ZYMED Co. and Santacruz Co.; and Chemiluminscent ECL plus detection reagent (RPN2132) was from Amersharm biosciences Co.
  • HeLa cells were distributed into 60mm dishes in a population of 2x10 5 cells per dish in 5ml of MEM containing 10% FBS, and cultured at 37 "C for about 24 h in a CO 2 incubator. The media was replaced with fresh one (5ml), and the test samples were added in various concentrations (0 ⁇ M and 0.5 ⁇ M-IO ⁇ M). The cells were incubated for 48 h, lysis buffer was added in order to obtain cell lysate, and the amount of protein was determined.
  • the membrane to which the protein was transferred was blocked with the blocking solution [5% Non-fat dry milk in IX TBS-T (Tris buffered saline, 1OmM Tris-Cl, pH 7.6, 15OmM NaCl)-0.1% T(Tween-20)] for 1 h, washed three times with IX 0.1% TBS-T for 10 min, and treated with 4 ⁇ g of the primary antibody (pl6, p21, actin) diluted with 10ml of IX TBS-T.
  • the blocking solution [5% Non-fat dry milk in IX TBS-T (Tris buffered saline, 1OmM Tris-Cl, pH 7.6, 15OmM NaCl)-0.1% T(Tween-20)] for 1 h, washed three times with IX 0.1% TBS-T for 10 min, and treated with 4 ⁇ g of the primary antibody (pl6, p21, actin) diluted with 10ml of IX TBS-T.
  • the membrane was washed three times with IX TBS-T, and treated with the secondary antibody (HRP-goat-anti-mouse IgG, HRP-goat-anti-rabbit IgG, HRP-donkey-anti-goat IgG) diluted with 10mA of IX TBS-T for the ratio of 1:5000. After 1 hrs incubation, the membrane was washed four times with IX TBS-T for 10 min. Each band was visualized on a X-ray film using Chemiluminescent ECL plus detection reagent, and the results are presented in Figure 1.
  • the new tetrahydro-beta-carboline derivative provided by the present invention shows the inhibitory activity against cyclin dependent kinases as well as the activity for enhancing the expression of endogenous tumor suppressive proteins, and so can be used for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection.

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Abstract

The present invention relates to a new tetrahydro-beta-carbolinone derivative having antagonist effect for cyclin dependent kinase as well as an agonist effect for the expression of endogenous tumor suppressor proteins. The present invention also relates to a composition for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection, comprising the tetrahydro-beta-carbolinone derivative as an active ingredient.

Description

TETRAHYDRO-BETA-CARBOLINONE DERIVATIVES AND PROCESS FOR
PREPARING THE SAME
TECHNICAL FIELD
The present invention relates to a new tetrahydro-beta-carbolinone derivative having antagonist effect for cyclin dependent kinase ('CDK3' below) as well as an agonist effect for the expression of endogenous tumor suppressor protein. The present invention also relates to a composition for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy- induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection, comprising the tetrahydro-beta-carbolinone derivative as an active ingredient.
BACKGROUND ART
According to the aging of modern society, the risk caused by cancer is getting more serious. However, at the clinical results of the past 30 years, the anticancer drugs have not seemed to show any prominent improvements for tumorigenesis because most anticancer drugs except Taxol rely upon the spontaneous cell death mechanism evoked by the abnormality of DNA in cancer cells which can be induced by a direct injury of DNA or the perturbation of DNA replication process. In particular, the most serious drawback of these anticancer drugs is that the cancer cells rapidly process to resistance against the drugs. Furthermore, since the radiotherapy or anticancer chemotherapy also relies on the same mechanism of DNA injury, the drug resistant cancer cells can resist to other anticancer therapies. Recently, NCI reported that p53 gene which plays a crucial role in the induction of cell death is mutated and loses its function in 50-70% of drug resistant cancer cells. This report addressed that the affected function of p53 gene is directly related to the development of resistance against anticancer drugs {Cancer Res., 1997, 57, 4285-4300). For these reasons, the survival probability of cancer patients still remains at 20%, which is not a clear improvement in comparison with that of early 1970s. These situations still ask for the development of new anticancer drugs which function through a novel mechanism distinct from the conventional mechanism of DNA injury. Under these circumstances, CDK inhibitor began to gather attention in 1980s-1990s as a new anticancer drug based on the basic biological knowledge about the generation and development of cancer cells. CDK is a major enzyme that regulates the eukaryotic cell cycle. In normal eukaryotic cells, through the signal transduction pathway, extracellular growth signal promotes the cell proliferation following the sequential cell cycle of Gl phase (gap 1 phase) → S phase (synthesis phase) → G2 phase (gap 2 phase) → M phase (mitosis phase). CDK is activated by binding with specific cyclin, and regulate the cell cycle. For example, in Gl phase it is decided whether the cell is to proliferate or not. In a cell that is decided to proliferate, cyclin D/CDK4,6 complex phosphorylates pRB, and cyclin E/CDK2 complex hyperphosphorylates pRB. As a result of these phosphorylation processes, a transcription factor E2F is dissociated from pRB and promotes the cell into S phase where DNA replication is carried out. The transfer into G2 phase is stimulated by cyclin AJCDKl complex. In G2 phase, the completion of DNA replication is confirmed and the timing of cell division is decided. When the cell reaches M phase, equal distribution of completely replicated chromosomes and subsequent cell division is accomplished, and the cell returns to Gl phase again. Most normal cells cease their division at Gl phase and enter the dormant GO phase where protein synthesis or energy metabolism is suspended, while the cancer cells maintain the rapid and anarchic cell cycle by the continual signal. For these reasons, the regulation of cyclin and CDK which play critical roles in the control of entire cell cycle became a new target research for cancer treatment. Several CDK inhibitors are already under the clinical application, as represented by a CDK inhibitor R-Roscovitine (Oral administration lOOmg/bid; Used for the treatment of breast cancer and lung cancer in combination with standard chemotherapeutic agents; Average cytotoxicity for tumor cell lines IC50( U M)=I 5).
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have tried to develop a new anticancer agent which exhibits anticancer activity through a mechanism of inhibiting cell cycle regulators to overcome the drug resistance and various side effects of the existing inhibitors against DNA synthesis. As a result, they identified that a tetrahydro-beta-carbolinone derivative of the formula (1) shows a selective and potent inhibitory activity against CDK and can be used as a new orally administrable CDK inhibitor, and completed the present invention. Particularly, the tetrahydro-beta-carbolinone derivative of the present invention has the effect of enhancing the expression of such endogenous tumor suppressor proteins as pi 6 and p21, as well as the inhibitory activity against CDK. According to these mechanisms, the compound of the present invention shows an improved anticancer effect (inhibitory effect against cell growth) compared with R-roscovitine, and can be used for the treatment of autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection, in addition to the cancer.
Therefore, it is an object of the present invention to provide a new tetrahydro-beta-carbolinone derivative, pharmaceutically acceptable salt, or stereoisomer thereof, having excellent pharmacological activities including the anticancer activity.
It is another object of the present invention to provide a process for preparing the above compound.
It is still another object of the present invention to provide a composition comprising the above compound as an active ingredient- together with a pharmaceutically acceptable carrier, for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 represents the Western blotting result showing the enhancing effect of the compound according to the present invention for the expression of pl6 and p21.
BEST MODES FOR CARRYING OUT THE INVENTION
The present invention relates to a new tetrahydro-beta-carbolinone derivative of the following formula (1): [Formula 1]
Figure imgf000006_0001
in which
R represents -(CH2)H-R1, -(CO)-R2, or -(SO2)-R3, wherein n denotes an integer of 0 to 5, R1 represents hydrogen, Q-Cδ-alkoxy, d-Ce-hydroxyalkyl, C2-C6-alkoxycarbonyl, carboxy, carbamoyl, or phenyl,
R2 represents C2-C6-alkoxyalkyl, d-C6-alkyl, halogeno-Q-Ce-alkyl, dτC6-alkoxy, d-Q-alkylcarbonyloxyalkyl, C3-C7-cycloalkyl, or phenyl,
R3 represents Ci-C6-alkyl, and X represents halogen, d-C6-alkoxy, or aminosulfonyl, pharmaceutically acceptable salt, or stereoisomer thereof.
Without any special mention, the alkyl in the present invention may be linear or branched.
Preferred compounds among the compound of formula (1) above useful as inhibitory agent against CDK or enhancing agent for the expression of pi 6 and p21 are those wherein R represents -(CO)-R2, pharmaceutically acceptable salt, or stereoisomer thereof.
More preferred compounds are those wherein X represents bromine, pharmaceutically acceptable salt, or stereoisomer thereof. Most preferred compounds are those wherein R represents -(CO)-R , wherein R represents d-Cβ-alkoxyalkyl or halogeno-d-C6-alkyl, and X represents bromine, pharmaceutically acceptable salt, or stereoisomer thereof. Typical compounds among the compound of formula (1) are those selected from the group consisting of:
6-Bromo-9-methyl-2,3,4,9-tetrahydro-y9-carbolin-l-one (Compound 1); 9-Benzyl-6-bromo-2,3,4,9-tetrahydro1ff-carbolm-l-one (Compound 2); 6-Bromo-9-(2-chloro-acetyl)-2,3,4,9-tetrahydro-^-carbolin-l-one (Compound 3);
6-Bromo-9-(2-methoxy-ethyl)-2,3,4,9-tetrahydro-j-?-carbolin-l-one (Compound
4);
6-Bromo-9-(2-hydroxy-ethyl)-2,3,4,9-tetrahydro-/?-carbolin-l -one (Compound 5);
9-Acetyl-6-bromo-2,3,4,9-tetrahydro-/?-carbolin-l-one (Compound 6); , 6-Bromo-l-oxo-l,2,3,4-tetrahydro-/?-carboline-9-carboxylic acid tert-butyl ester
(Compound 7);
6-Bromo-9-methanesulfonyl-2,3,4,9-tetrahydro-^-carbolin-l-one (Compound 8);
(6-Bromo-l-oxo-l,2,3,4-tetrahydro-/?-carbolin-9-yl)-acetic acid methyl ester (Compound 9); (6-Bromo-l-oxo-l,2,3,4-tetrahydro-^-carbolin-9-yl)-acetic acid (Compound 10);
2-(6-Bromo-l-oxo-l,2,3,4-tetrahydro-^-carbolin-9-yl)-acetamide (Compound 11);
6-Bromo-9-(2-methoxy-acetyl)-2,3 ,4,9-tetrahydro-/?-carbolin- 1 -one (Compound
12);
Acetic acid 2-(6-bromo- 1 -oxo- 1 ,2,3 ,4-tetrahydro-/?-carbolin-9-yl)-2-oxo-ethyl ester (Compound 13); and
6-Bromo-9-cyclohexanecarbonyl-2,3,4,9-tetrahydro-^-carbolin-l-one (Compound 14).
Particularly preferred compounds among the above typical compounds are those selected from the group consisting of: 6-Bromo-9-(2-chloro-acetyl)-2,3,4,9-tetrahydro-^-carbolin-l-one (Compound 3); 9-Acetyl-6-bromo-2,3,4,9-te1xahydro-/?-carbolm-l-one (Compound 6); 6-Bromo-9-(2-methoxy-acetyl)-2,3,4,9-tetrahydro-/9-carbolin-l-one (Compound 12); and Acetic acid 2-(6-bromo-l-oxo-l,2,3,4-tetrahydro-/^carbolm-9-yl)-2-oxo-ethyl ester (Compound 13).
The compound of formula (1) according to the present invention can also form a pharmaceutically acceptable salt. Such salt includes non-toxic acid addition salt containing pharmaceutically acceptable anion, for example, a salt with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydriodic acid, etc., a salt with organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc., or a salt with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc. The compound of formula (I) can also form a salt with alkaline metals such as sodium, potassium, etc., and a salt with other acids or bases which have been well-known and widely used in the technical field to which the tetrahydro-beta-carbolinone derivative belongs. These salts can be prepared according to any of the conventional conversion methods. Furthermore, the compound of the present invention may have asymmetric carbon atoms in the structure depending on the kind of substituents, and so may exist in the form of R or S isomer, or mixtures thereof including racemates. The pure stereoisomers may be obtained by the conventional resolution method known in this field. Thus, the present invention also includes all of these stereoisomers and their mixtures in its scope. The compound of the present invention may be prepared according to the process explained below, and so the present invention also provides such process for preparing the compound of formula (1).
Specifically, the compound of formula (1) defined above, pharmaceutically acceptable salt, or stereoisomer thereof may be prepared by the process which comprises
(a) reacting a compound of the following formula (2): [Formula 2]
in which X is as defined above, with a compound of the following formula (3): [Formula 3]
R-L in which R is as defined above, and L represents a leaving group, preferably halogen, to give the compound of formula (1), or
(b) reacting a compound of the following formula (4): [Formula 4]
Figure imgf000009_0002
in which X is as defined above, and P represents an amino-protecting group, preferably t-butoxycarbonyl, with the compound of formula (3) to give a compound of the following formula (5): [Formula 5]
Figure imgf000010_0001
in which R, X, and P are as defined above, and deprotecting the compound of formula (5) to give the compound of formula (1).
The reaction conditions for the above process are explained below in more detail. The above process (a) of reacting the compound of formula (2) with the compound of formula (3) is preferably carried out in a solvent and in the presence of a base. As the solvent, one or more can be selected from the group consisting of acetonitrile, dimethylsulfoxide, N,N-dimethylforrnamide, and tetrahydrofuran, and as the base, one or more can be selected from the group consisting of sodium hydroxide, potassium fluoride, potassium carbonate, and sodium hydride. In order to facilitate the reaction, such reaction aids as aluminum oxide may be further used. It is economic in the aspect of yield, etc. to use the compound of formula (3) in an amount of 1 to 3 equivalents, preferably 1.5 to 2 equivalents, with respect to the compound of formula (2). The reaction is usually carried out at a temperature between 0 and 25 °C for 0.5 to 4 h. The step of reacting the compound of formula (4) with the compound of formula
(3) in the process (b) may proceed under the same conditions as explained for the above process (a). And, the deprotection of the compound of formula (5) may be carried out by applying the conventional conditions well-known in this filed. Preferably, 3 to 10 molar equivalents of trifluoroacetic acid with respect to the compound of formula (5) is added, and the mixture is stirred at room temperature for about 1 h to give the desired compound of formula (1).
The above processes will be more specifically explained by the following examples. However, the processes for preparing the compound according to the present invention are not limited to those explained above, and can be easily selected by optionally combining various synthetic methods described in the present specification or known in the art. And, such a combination may be easily carried out by one of ordinary skill in the art. After the completion of reaction, the product may be separated and purified by conventional work-up processes such as chromatography, recrystallization, etc.
The compound of formula (1) according to the present invention may be advantageously used as an anticancer agent due to its excellent inhibitory activity against CDKs. Therefore, the present invention relates to an anticancer composition comprising the compound of formula (1), pharmaceutically acceptable salt, or stereoisomer thereof as an active ingredient together with the pharmaceutically acceptable carrier.
Due to its excellent inhibitory activity against CDKs, the compound of the present invention may also be advantageously used for the preparation of therapeutic agents for treating autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection besides cancer. In the present invention, the cancer is defined as solid tumor and leukemia; the autoimmune disease is defined as psoriasis, alopecia, and multiple sclerosis; the cardio-vascular disease is defined as stenosis, arteriosclerosis, and recurrent stricture; the infection disease is defined as those caused by unicellular parasites; the kidney disease is defined as glomerulonephritis; the chronic neurodegenerative disease is defined as Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, AIDS, dementia, and Alzheimer's disease; the acute neurodegenerative disease is defined as cerebral ischemia and neurotrauma; and the viral infection is defined as the infections by giant cell, herpes, hepatitis B or C, and HIV. When the compound of the present invention is used for clinical purpose, it is preferable to administer it to the subject patient in an amount ranging from 0.5 to lOOOmg, preferably 50 to 200mg, per day. The total daily dosage may be administered once or over several times. However, specific administration dosage for an individual patient can be varied depending on specific compound used, body weight, gender, hygienic condition, diet of subject patient, time or method of administration, excretion rate, mixing ratio of agent, severity of disease to be treated, etc.
As a result of administering the composition according to the present invention in the amount of lOOmg/kg to 10 rats and observing their conditions in 1 day, there was no rat which died or showed serious disease. Thus, it was identified that the compound of the present invention is not toxic.
The compound of the present invention may be formulated as a parenteral injection or oral preparation, depending on its application purpose.
Parenteral Injections, for example, aqueous or oily suspension for sterilized injection, can be prepared according to the known procedure using suitable dispersing agent, wetting agent, or suspending agent. Solvents that can be used for preparing injections include water, Ringer's fluid, and isotonic NaCl solution, and also sterilized fixing oil may be conveniently used as the solvent or suspending media. Any non-irritating fixing oil including mono- or di-glyceride may be used for this purpose. Fatty acid such as oleic acid may also be used for injections.
The orally administrable solid preparations include capsules, tablets, pills, powders, and granules, capsules and tablets among which are preferable. It is desirable for tablets and pills to be formulated into enteric-coated preparation. The solid preparations are prepared by mixing the active compound of formula (1) with one or more carriers selected from inert diluents such as sucrose, lactose, starch, etc., lubricants such as magnesium stearate, disintegrating agents, and binding agents.
In case that the compound of the present invention is clinically administered to obtain the desired anticancer effect, the active compound of formula (1) may be administered together with one or more known anticancer agents. The anticancer agents to be mixed and administered together with the compound of the present invention in this manner include 5-fluorouracil, cisplatin, doxorubicin, taxol, Gemcitabine, etc.
However, the preparations comprising the compound of the present invention, which expect several pharmacological effects including the anticancer activity, are not restricted to those explained above, but may include any preparations useful for the prevention and treatment of diseases.
The present invention will be more specifically explained by the following preparations, examples, and experiments. However, it should be understood that these preparations, examples, and experiments are intended to illustrate the present invention but not in any manner to limit the scope of the present invention.
Example 1
Synthesis of 6-bromo-9-methvI-2,3,4,9-tetrahydro-^-carbolin-l-one
(Compound 1) In a lOOiM flask, the commonly used compound 6-bromo-2,3,4,9-tetrahydroz
^carbolin-1-one (0.5g, 1.88mmol) was dissolved in acetone (2Om-U), and DMSO (OΛSml, 1.88 mmol) was added at room temperature, and then NaOH (O.lg, 2.5mmol) in water (5m£) solution was added. The mixture was stirred for 8 h at room temperature, and the reaction mixture was extracted twice with water (50ml) and dichloromethane (7Om-U). The extract was dried over Na2SO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of dichloromethane/methanol=30/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (0.5g, 95.0%) as brown solid. 1H-NMR(CDCl3); δ = 7.73(d, J= 1.8 Hz, IH), 7.42(d, J= 8.6 Hz, IH)5 7.25(d, J=
8.6 Hz, IH), 5.56(s, IH), 4.09(s, 3H), 3.65(td, J= 6.7, 2.6 Hz, 2H), 3.00(t, J= 6.7 Hz, 2H)
Example 2
Synthesis of 9-benzyl-6-bromo-2,3;4,9-tetrahvdro-/?-carboIin-l-one (Compound 2)
Into a 100m£ flask under nitrogen atmosphere was introduced acetonitrile (20ml), and potassium fluoride (O.lg, 1.88mmol) and aluminum oxide (0.2g, 1.88mmol) were added at room temperature. To this mixture were added 6-bromo-2,3,4,9-tetrahydro-/?-carbolin-l- one (0.5g, 1.88mmol) and benzyl bromide (0.2ml, 1.88mmol). The mixture was stirred for 3 h under heat-reflux condition, cooled to room temperature, and stirred overnight. The reaction mixture was extracted twice with water (50ml) and dichloromethane (70ml). The extract was dried over Na2SO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of dichloromethane/methanol=30/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (0.4g, 60.0%). as brown solid.
1H-NMR(CDCl3); δ = 7.74(d, J = 1.8 Hz, IH), 7.34(d, J = 8.6 Hz, IH), 7.26-7.11(m, 6H), 5.87(s, 2H), 5.54(bs, IH), 3.67(td, J= 6.7, 2.6 Hz, 2H), 3.04(t, J= 6.7 Hz, 2H) Example 3
Synthesis of ό-bromo^-te-chloro-acety^^Ag-tetrahydro-ff-carboIin-l- one (Compound 3) In a IOOIM flask, the commonly used compound 6-bromo-2,3,4,9-tetrahydro-
/?-carbolin-l-one (0.56g, 2.1mmol) was dissolved in N,N-dimethylformamide (DMF) (10m£), potassium carbonate (K2CO3) (0.58g, 4.2mmol) and sodium hydride (NaH) (0.17g, 4.2 mmol) were added at room temperature, and the mixture was stirred for 20 min. The reaction mixture was cooled to 0°C, and chloro acetyl' chloride (0.34m£, 4.2mmol) was added, and stirred for 4 h. The reaction mixture was diluted with water (20m£), and extracted twice with dichloromethane (70m£). The extract was dried over Na2SO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of n-hexane/ethyl acetate=l/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (0.36g, 50.2%) as brown solid.
1H-NMR(DMSOdO) ; δ = 8.29(s, IH), 8.06(s, IH), 8.04(d, J = 5.7 Hz, IH), 7.69(dd, J= 2.2, 8.7 Hz, IH,), 5.17(s, 2H), 3.55(2H, m), 2.99(2H, t, J= 6.8 Hz)
Example 4 Synthesis of 6-bromo-9-(2-methoxy-ethyl)-2,3,4.,9-tetrahydro- ff-carbolin-
1-one (Compound 4)
In a 100m£ flask, the commonly used compound 6-bromo-2,3,4,9-tetrahydro-/?- carbolin-1-one (0.5g, 1.88mmol) was dissolved in N,N-dimethylformamide (DMF) (10m£), and sodium hydride (NaH) (0.15g, 3.76mmol) was added at 0°C , and stirred for 30 min. 2-Bromoethyl methyl ether (BrCH2CH2OCH3) (0.35m£, 3.76mmol) was slowly added dropwise at 0°C . And, the reaction mixture was stirred for 4 h at room temperature, diluted with water (50m£), and extracted twice with dichloromethane (7(M). The extract was dried over Na2SO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of dichloromethane/methanol=30/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (0.38g, 62.2%) as white solid.
1H-NMR(DMSO-d6); δ = 7.84(d, J= 1.9 Hz, IH), 7.71(bs, IH), 7.54(d, J= 8.7 Hz, IH), 7.37(dd, J= 8.7, 1.9 Hz, IH), 4.69(t, J= 5.3 Hz, 2H); 3.61(t, J= 5.3 Hz, 2H), 3.45(td, J = 6.8, 2.6 Hz, 2H), 3.15(s, 3H), 2.91(t, J= 6.8 Hz, 2H)
Example 5
Synthesis of 6-bromo-9-(2-hvdroxy-ethyl)-2,3,4,9-tetrahydro-jg-carboIin-l- one (Compound 5) In a 50mi flask, 6-bromo-9-(2-methoxy-ethyl)-2,3,4,9-tetrahydro-j3-carbolin-l- one (Compound 4) prepared in Example 4 (O.lg, 0.30mmol) was dissolved in 48% aqueous HBr solution (10m£), which was then stirred for 1O h under heat-reflux condition. The reaction mixture was distilled under reduced pressure, and the residue was purified by silica gel column chromatography eluting with a solvent mixture of dichloromethane/ methano 1=20/1 (v/v). The fractions containing the product were combined and evaporated to give the title compound (68mg, 72.3%) as pale yellow solid.
1H-NMR(MeOH-d4); δ = 7.86(d, J = 1.9 Hz, IH), 7.57(d, J = 8.7 Hz, IH), 7.48(dd, J = 8.7, 1.9 Hz, IH), 4.75(t, J = 5.7 Hz, 2H), 3.96(t, J = 5.7 Hz, 2H), 3.69(t, J = 6.8 Hz, IH), 3.08(t, J = 6.8 Hz, 2H) Example 6
Synthesis of 9-acetyI-6-bromo-2,3<4,9-tetrahydro-/?-carbolin-l-one
(Compound 6) a) Synthesis of β-bromo-l-oxo-l^AΘ-tetiahydro-β-carbolme^-carboxylic acid tert-butyl ester
In a 10Om-C flask, the commonly used compound 6-bromo-2,3,4,9-tetrahydro- β-carbolin-1-one (0.61g, 2.32mmol) was dissolved in anhydrous THF (30ml), and 2.0M LDA (1.17IIL£, 2.32mmol) was slowly added dropwise at -78 °C, and stirred for 10 min. To this reaction mixture was slowly added di-tert-butyldicarbonate (0.76g, 3.48mmol) dissolved in dichloromethane (10m£), which was then warmed to room temperature and stirred for 1 h at this temperature. The reaction mixture was diluted with water (50m£), and extracted twice with ethyl acetate (70m#). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of n-hexane/ethyl acetate=l/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (0.36g, 42.5%) as brown solid.
1H-NMR(CDCl3) ; δ = 9.25(bs, IH), 7.76(s, IH), 7.42(d, J= 8.8 Hz, IH), 7.35(d, J= 8.8 Hz, IH), 4.21(t, J=6.8 Hz, 2H), 3.15(t, J=6.8 Hz, 2H), 1.59(s, 9H)
b) Synthesis of 9-acetyl-6-bromo-2,3,4,9-tetrahydro-/^carbolin-l-one In a 50IE£ flask, ό-bromo-l-oxo-l^^^-tetrahydro-^-carboline^-carboxylic acid tert-butyl ester (50mg, 0.137mmol) was dissolved in dimethylformamide (5iu£), and NaH (8mg, 0.206mmol) and acetic anhydride (0.016m£, 0.164mmol) were added at 0°C, and stirred for 30 min. The reaction mixture was diluted with water (1OmA), and extracted twice with ethyl acetate (5OmA). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was dissolved in dichloromethane (1OmA), and trifluoroacetic acid (0.032mA, 0.411mmol) was slowly added at 0°C, and stirred for 1 h. The reaction mixture was distilled under reduced pressure, diluted with water, neutralized with aqueous NaHCO3 solution, and extracted twice with ethyl acetate (3OmA). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of n-hexane/ethyl acetate=l/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (34mg, 80.9%) as white solid.
1H-NMR(DMSO^O) ; δ = 8.12(bs, IH), 8.03(d, J=9.2 Hz, IH), 7.98(s, IH), 7.61(dd, J=9.2, 1.9 Hz, IH), 3.50(m, 2H), 2.92(t, J=6.5 Hz, 2H), 2.65(s, 3H)
Example 7 Synthesis of ό-bromo-l-oxo-l^^^-tetrahydro-jg-carboline-g-carboxylic acid tert-butyl ester (Compound 7)
The commonly used compound 6-brorno-2,3,4,9-tetrahydro-/?-carbolin-l-one
(0.61g, 2.32mmol) was dissolved in anhydrous THF (3OmA) and reacted with 2.0M LDA
(1.17mA, 2.32mmol) and di-tert-butyldicarbonate (0.76g, 3.48mmol) at -78 °C according to the same procedure as Step a) of Example 6 to give the title compound (O.lg, 11.8%) as brown solid.
1H-NMR(CDCl3) ; δ = 7.98(d, J=9.0 Hz, IH), 7.67(d, J=I.8 Hz, IH), 7.51(dd, J=8.8, 1.8 Hz, IH), 5.88(bs, IH), 3.66(m, 2H), 2.91(t, J=4.3 Hz, 2H), 1.64(s, 9H) Example 8
Synthesis of 6-bromo-9-methanesulfonyl-23A9-tetrahydro-/?-carbolin-l-one (Compound 8)
In a 50ml flask, 6-bromo-l-oxo-l,3,4,9-tetrahydro-/^carbolme-2-carboxylic acid tert-butyl ester (50mg, 0.137mmol) prepared in Step a) of Example 6 was dissolved in anhydrous THF (5m£), and NaH (8mg, 0.206mmol) was added at 0°C, and stirred for 30 min. To the reaction mixture was added methanesulfonyl chloride (0.016ml, 0.206mrnol) at 0°C, which was then stirred for 10 min. The reaction mixture was diluted with water (5ml) and extracted twice with ethyl acetate (20ml). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was dissolved in dichloromethane (10m£), and trifluoroacetic acid (0.032m£, 0.411mmol) was slowly added at 0°C, and stirred for 1 h. The reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO3 solution, and extracted twice with ethyl acetate (30ml). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of n-hexane/ethyl acetate=l/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (35mg, 74.5%) as white solid.
1H-NMR(CDCl3) ; δ = 8.02(d, J=9.1 Hz, IH), 7.70(d, J=I .8 Hz, IH), 7.53(dd, J=9.1, 1.8 Hz, IH), 5.85(bs, IH), 3.87(s, 3H)3 3.68(m, 2H), 2.95(t, J=6.3 Hz, 2H)
Example 9
Synthesis of (ό-bromo-l-oxo-l^^^-tetrahydro-ff-carbolin^-vD-acetic acid methyl ester (Compound 9) In a 50in£ flask, 6-bromo-l-oxo-l,3,4,9-tetrahydro-/β-carboline-2-carboxylic acid tert-butyl ester prepared in Step a) of Example 6 (O.lg, 0.274mmol) was dissolved in anhydrous THF (10m£), and NaH (16mg, 0.412mmol) was added at 0°C, and stirred for 30 min. To the reaction mixture was added methylbromo acetate (0.04ml, 0.412mmol) at 0°C, which was then stirred for 10 min and stirred again for 1 h at room temperature. The reaction mixture was diluted with water (10ml), saturated aqueous ammonium chloride solution was added, and the mixture was extracted twice with ethyl acetate (50mi). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was dissolved in dichloromethane (10ml), and trifluoroacetic acid (0.064in£, 0.822mmol) was slowly added at 0°C, and stirred for 1 h. The reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO3 solution, and extracted twice with ethyl acetate (40m£). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. To the residue was added n-hexane (5in£), which was stirred for 10 min at room temperature and filtered to give the title compound (80mg, 86.9%) as white solid.
1H-NMR(CDCl3) ; δ = 7.75(d, J=I .9 Hz, IH), 7.42(dd, J=I.9, 8.7 Hz, IH), 7.14(d, J=8.8 Hz, IH), 5.51(bs, IH), 5.39(s, 2H), 3.74(s, 3H), 3.68(m, 2H), 3.04(t, J=7.1 Hz, 2H)
Example 10 Synthesis of (ό-bromo-l-oxo-l^^^-tetrahydro-jg-carbolin-g-vD-acetic acid
(Compound 10)
Li a 50m£ flask, (6-bromo-l-oxo-l,2,354-tetrahydro-/?-carbolin-9-yl)-acetic acid methyl ester prepared in Example 9 (70mg, 0.206mmol) was dissolved in a solvent mixture of methanol (4m4) and THF (4m£), and IN NaOH (0.88m£) was added, and stirred for 2 h at room temperature. The reaction mixture was distilled under reduced pressure to remove the solvent, cooled with using an ice-bath, diluted with water (1OmA), and neutralized with IN HCl. Thus neutralized reaction product was extracted twice with ethyl acetate (4OmA). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. To the residue was added a solvent mixture of n-hexane/ethyl acetate=l/l(5mA), which was stirred for 10 min at room temperature and filtered to give the title compound (60mg, 90.4%) as white solid.
1H-NMR(DMSO-d6) ; δ = 12.79(bs, IH), 7.88(d, J=2.0 Hz, IH), 7.52(d, J=8.8 Hz, IH), 7.39(dd, J=9.1, 1.9 Hz, IH), 5.32(s, 2H), 3.47(m, 2H), 2.93(t, J=6.8 Hz, 2H)
Example 11
Synthesis of 2-(6-bromo-l-oxo-l,2,3,4-tetrahydro-jg-carbolin-9-yI)-acetamide (Compound 11)
In a 5OmA flask, (6-bromo-l-oxo-l,2,3,4-tetrahydro-^-carbolin-9-yl)-acetic acid prepared in Example 10 (35mg, 0.108mmol) was dissolved in anhydrous THF (5m-£), and triethylamine (0.03OmC, 0.216mmol) and methylchloroformate (0.013mA, 0.162 mmol) were added at 0°C, and stirred for 20 min. The reaction mixture was stirred at 0°C while bubbling ammonia gas through the solution, and then the reaction mixture was distilled under reduced pressure. The residue was diluted with water (1OmA) and extracted twice with ethyl acetate (2OmA). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of dichloromethane/methanol=10/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (30mg, 86.2%) as
white solid. 1H-NMR(DMSO-d6) ; δ = 7.87(d, J=I.1 Hz3 IH), 7.70(bs, IH), 7.45(bs, IH), 7.38(s, IH), 7.09(bs, IH), 5.21(s, 2H), 3.46(m, 2H)3 2.93 (t, J=6.98 Hz3 2H)
Example 12 Synthesis of 6-bromo-£-(2-methoxy-acetγI)-2,3A9-tetrahvdro-/^carboIin-l- one (Compound 12)
In a 5OmU. flask, 6-bromo-l-oxo-l,3,4,9-tetrahydro-/?-carboline-2-carboxylic acid tert-butyl ester prepared in Step a) of Example 6 (50mg, 0.137mmol) was dissolved in anhydrous THF (5mA), and NaH (17mg, 0.411mmol) was added at 0°C, and stirred for 30 min. To the reaction mixture was added methoxyacetylchloride (0.038m£, 0.411mmol) at 0°C , and the mixture was stirred for 30 min. The reaction mixture was diluted with water (5ra£), saturated aqueous NaHCO3 solution was added, and the mixture was extracted twice with ethyl acetate (20m£). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was dissolved in dichloromethane (5m£), and trifluoroacetic acid (0.032m£, 0.411mmol) was slowly added at 0°C, and stirred for 1 h. The reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO3 solution, and extracted twice with ethyl acetate (20mβ). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of dichloromethane/methanol=30/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (25mg, 54.3%) as white solid.
1H-NMR(CDCl3) ; δ = 8.18(d, J=8.7 Hz, IH)3 7.69(d, J=1.9 Hz, IH), 7.55(dd, J=8.7, 1.9 Hz3 IH), 6.04(bs, IH), 4.72(s, 2H), 3.69(m, 2H), 3.30(s, 3H), 2.95(t, J=6.8 Hz,
2H) Example 13
Synthesis of acetic acid 2-(6-bromo-l-oxo-l,2,3.l4-tetrahvdro-ig-carboIin-9-vI)- 2-oxo-ethvI ester (Compound 13) In a 50inϋ, flask, 6-bromo-l-oxo-l,3,4,9-tetrahydro-/?-carboline-2-carboxylic acid tert-butyl ester prepared in Step a) of Example 6 (50mg, 0.137mmol) was dissolved in anhydrous THF (5ml), and NaH (17mg, 0.41 lmmol) was added at -78 °C , and stirred for 30 min. To the reaction mixture was added acetoacetylchloride (0.044m£, 0.41 lmmol) at -78 °C, and stirred for 30 min. The reaction mixture was diluted with water (5ml), saturated aqueous NaHCO3 solution was added, and the mixture was extracted twice with ethyl acetate (20m£). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was dissolved in dichloromethane (5m£), trifluoroacetic acid (0.032m£, 0.41 lmmol) was slowly added at 0°C, and the mixture was stirred for 1 h. The reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO3 solution, and extracted twice with ethyl acetate (20m£). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of dichloromethane/methanol=30/l(v/v). The fractions containing the product were combined and evaporated to give the title compound (26mg, 54.2%) as white solid. 1H-NMR(CDCl3); δ = 8.20(d, J=8.4Hz, IH), 7.69(d, J=I.9 Hz, IH), 7.57(dd, J=8.7,
1.9 Hz, IH), 6.05(bs, IH), 5.35(s, 2H), 3.70(m, 2H), 2.97(t, J=6.8 Hz, 2H), 2.13(s, 3H)
Example 14
Synthesis of 6-bromo-9-cvcIohexanecarbonyl-2.,3,4,9-tetrahvdro-ig-carbolin-l- one (Compound 14)
In a 50ml flask, 6-bromo-l-oxo-l,3,4,9-tetrahydro-/^carboline-2-carboxylic acid tert-butyl ester prepared in Step a) of Example 6 (50mg, 0.137mmol) was dissolved in anhydrous THF (5ml), and NaH (17mg, 0.411mmol) was added at -78 °C, and stirred for 30 min. To the reaction mixture was added cyclohexanecarbonylchloride (0.054m-(J, 0.411mmol) at -78 °C, and stirred for 30 min. The reaction mixture was diluted with water (5ml), saturated aqueous NaHCO3 solution was added, and the mixture was extracted twice with ethyl acetate (20ml). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was dissolved in dichloromethane (5ml), and trifluoroacetic acid (0.032m£, 0.411mmol) was slowly added at 0°C, and stirred for 1 h. The reaction mixture was distilled under reduced pressure, diluted with water, neutralized with saturated aqueous NaHCO3 solution, and extracted twice with ethyl acetate (20M). The extract was dried over MgSO4, filtered, and distilled under reduced pressure. The residue was purified by silica gel column chromatography eluting with a solvent mixture of n-hexane/ethyl
Figure imgf000024_0001
The fractions containing the product were combined and evaporated to give the title compound (36mg, 70.0%) as white solid.
1H-NMR(CDCl3); δ = 7.95(d, J=8.7Hz, IH), 7.69(d, J=1.9 Hz, IH), 7.51(dd, J=8.7, 1.9 Hz, IH), 5.80(bs, IH), 3.72(td, 1=6.8, 3.4 Hz, 2H), 3.26(tt, J=ILO, 3.4 Hz, 2H), 2.98(t, J=6.8 Hz, 2H), 1.92-1.87(m, 2H), 1.76(m, 2H), 1.66(m, IH), 1.54-1.45(m, 2H), 1.34-1.20(m, 3H)
Experiment 1
Inhibitory activity against CDK2/cyclin A/cycIin E kinases
CDK2/cyclin A and CDK2/cyclin E complexes were prepared by using Baculovirus recombinant system and insect cell S£21 purchased from Invitrogen Co. The substrate Histone (Cat. # 382150) was purchased from Calbiochem Co.
Test samples (0 μ M and 0.032 μ M-IOO μ M) diluted in various concentrations in 2X reaction buffer [2OmM Tris/HCl (pH8.0), 1OmM MgCl2, lOOμM NaVO3, 5mM glycerophosphate, ImM DTT] were added to a 96-well filter plate (Cat. # MHVBN4550, Millipore). Each of purified CDK2/cyclin A and CDK2/cyclin E enzyme complexes diluted with dH2O in concentrations of 20ng/well for CDK2/cyclin A and 25ng/well for CDK2/cyclin E was added thereto, and pre-incubated for 15 min at 30°C . Then, the mixture of substrate and ATP [3 μ M cold ATP/well (0.3/^/well, stock ImM), 0.3μCi [γ-32P]ATP/well (0.03/^/well, stock 10 μ Ci/ '≠), Histone (10/^/well, stock lmgM), 20/^ dH2O] was added and subjected to enzymatic reaction for 30 min at 30°C. The reaction was stopped by adding 100 μi of 70% ice-cold TCA. The reaction mixture was washed five times with 200μβ of 25% ice-cold TCA and dried for 1 h at 60 °C. 50/^ of LSC-cocktail (Wallac Corp.) was distributed to each well, and the activity (CPM) was measured by Micro-scintillation counter (Wallac 1450). Collected data were treated with Prism Software for the determination of IC50 values, which are shown in the following Table 1. [Table 1 ]
Figure imgf000025_0001
Experiment 2
Assay for inhibition of cell growth
MTS one solution was purchased from Promega, and PA-I (ovarian) and HeLa(cervix) cells were purchased from ATCC. The cells were cultured in MEM media containing 10% FBS at 37 "C in a CO2 incubator.
The cells cultured in T75 flask were diluted with growth media and distributed into a plate in a population of 5000 cells per well in 100 μl of media. After incubating for 24 h in a CO2 incubator at 37 "C, 100 μi of test samples diluted with growth media in various concentrations (0 μ M and 0.5 μ M-100 μ M; the final concentration of DMSO was
0.5%; triplicated) were added. The cells treated with the test samples were incubated at
37 °C for 72 h in a CO2 incubator. 20μβ of MTS one solution was distributed to each well by using a dispenser, and allowed to stand at 37°C for about 2 h in a CO2 incubator.
10 μJL of 10% SDS was added for the complete lysis of cells. The inhibitory activity against cell growth was determined by measuring the absorbance at 490nm by using
Thermo-MAX Microplate Reader (Molecular Devices Co.). The absorbance data were treated on Microsoft Excel, with using the value from the well treated only by MTS one solution as blanc, and adopting the value from 0 μ M well as 100% activity. The relative values were calculated for each well, and the IC50 values were determined as presented in Table 2 below.
[Table 2]
Figure imgf000027_0001
Experiment 3 Western Blotting Assay HeLa cell was purchased from ATCC and cultured in MEM media containing 10%
FBS at 37 °C in a CO2 incubator.
The antibody against pl6 (MS-887-P1) was purchased from NeoMarkers Co.; the antibodies against p21 (C-19, sc-397) and actin (1-19, sc-1616) were from Santacruz Co.; the secondary antibodies, HRP-goat-anti-mouse IgG, HRP-goat-anti-rabbit IgG, and HRP-donkey-anti-goat IgG, were from ZYMED Co. and Santacruz Co.; and Chemiluminscent ECL plus detection reagent (RPN2132) was from Amersharm biosciences Co.
HeLa cells were distributed into 60mm dishes in a population of 2x105 cells per dish in 5ml of MEM containing 10% FBS, and cultured at 37 "C for about 24 h in a CO2 incubator. The media was replaced with fresh one (5ml), and the test samples were added in various concentrations (0 μ M and 0.5 μ M-IO μ M). The cells were incubated for 48 h, lysis buffer was added in order to obtain cell lysate, and the amount of protein was determined. to a 1.5 mi tube were added 30μg of the quantified protein and 4X sample buffer (25OmM Tris-Cl, ρH6.8, 8% SDS, 40% glycerol, 20% 2-mercaptoethanol, 2% bromophenol blue) for denature the protein, which was then loaded to 14% Tris-Glycine pre-casted gel (invitrogne corp.). The sample was developed in the gel at HOV for about 2 h, and transferred to PVDF (polyvinlyidene difluoride, 0.2 μ m, invitrogen corp.) membrane (18OmA, 40min). The membrane to which the protein was transferred was blocked with the blocking solution [5% Non-fat dry milk in IX TBS-T (Tris buffered saline, 1OmM Tris-Cl, pH 7.6, 15OmM NaCl)-0.1% T(Tween-20)] for 1 h, washed three times with IX 0.1% TBS-T for 10 min, and treated with 4μg of the primary antibody (pl6, p21, actin) diluted with 10ml of IX TBS-T. After 2 h's incubation, the membrane was washed three times with IX TBS-T, and treated with the secondary antibody (HRP-goat-anti-mouse IgG, HRP-goat-anti-rabbit IgG, HRP-donkey-anti-goat IgG) diluted with 10mA of IX TBS-T for the ratio of 1:5000. After 1 hrs incubation, the membrane was washed four times with IX TBS-T for 10 min. Each band was visualized on a X-ray film using Chemiluminescent ECL plus detection reagent, and the results are presented in Figure 1.
INDUSTRIAL APPLICABILITY
As explained above, the new tetrahydro-beta-carboline derivative provided by the present invention shows the inhibitory activity against cyclin dependent kinases as well as the activity for enhancing the expression of endogenous tumor suppressive proteins, and so can be used for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection.

Claims

WHAT IS CLAIMED IS
1. A tetrahydro-beta-carbolinone derivative of the following formula ( 1 ) : [Formula 1]
Figure imgf000029_0001
in which
R represents -(CH2VR1, -(CO)-R2, or -(SO2)-R3,' wherein n denotes an integer of 0 to 5,
R1 represents hydrogen, Ci-C6-alkoxy, CrCβ-hydroxyalkyl, C2-C6-alkoxycarbonyl, carboxy, carbamoyl, or phenyl,
R2 represents C2-C6-alkoxyalkyl, Q-Q-alkyl, halogeno-Ci-C6-alkyl, CrCβ-alkoxy,
Cs-Ce-alkylcarbonyloxyalkyl, C3-C7-cycloalkyl, or phenyl,
R3 represents d-C6-alkyl, and
X represents halogen, d-C6-alkoxy, or aminosulfonyl, pharmaceutically acceptable salt, or stereoisomer thereof.
2. The compound of claim 1 wherein R represents -(CO)-R2, pharmaceutically acceptable salt, or stereoisomer thereof.
3. The compound of claim 1 wherein X represents bromine, pharmaceutically acceptable salt, or stereoisomer thereof.
4. The compound of claim 1 wherein R represents -(CO)-R2, R2 represents C2-C6-alkoxyalkyl or halogeno-d-Cό-alkyl, and X represents bromine, pharmaceutically acceptable salt, or stereoisomer thereof.
5. The compound of claim 1 which is selected from the group consisting of:
6-Bromo-9-methyl-2,3,4,9-tetrahydro-/5-carbolin-l-one (Compound 1);
9-Benzyl-6-bromo-2,3,4,9-tetrahydro-^-carbolin-l-one (Compound 2);
6-Bromo-9-(2-chloro-acetyl)-2,3,4,9-tetrahydro-^-carbolin-l-one (Compound 3);
6-Bromo-9-(2-methoxy-ethyl)-2,3,4,9-tetrahydro-j5-carbolin-l-one (Compound 4); 6-Bromo-9-(2-hydroxy-ethyl)-2,3,4,9-tetrahydro-/?-carbolin-l-one (Compound 5);
9-Acetyl-6-bromo-2,3,4,9-tetrahydro-/?-carbolin-l-one (Compound 6);
6-Bromo-l-oxo-l,2,3,4-tetrahydro-y?-carboline-9-carboxylic acid tert-butyl ester
(Compound 7);
6-Bromo-9-methanesulfonyl-2,3 ,4,9-tetrahydro-/?-carbolin- 1 -one
(Compound 8); (6-Bromo-l-oxo-l,2,3,4-te1xahydro-/?-carbolm-9-yl)-acetic acid methyl ester
(Compound 9);
(6-Bromo-l-oxo-l,2,3,4-tetrahydro-yff-carbolin-9-yl)-acetic acid
(Compound 10);
2-(6-Bromo-l-oxo-l,2,3,4-tetrahydro-^-carbolin-9-yl)-acetamide
(Compound 11);
6-Bromo-9-(2-methoxy-acetyl)-2,3,4,9-tetrahydro-y5-carbolin-l -one
(Compound 12); Acetic acid 2-(6-bromo-l-oxo-l,2,3,4-tetrahydro-^-carbolin-9-yl)-2-oxo-ethyl ester
(Compound 13); and
6-Bromo-9-cyclohexanecarbonyl-2,3,4,9-tetrahydro-^-carbolin-l-one (Compound 14), pharmaceutically acceptable salt, or stereoisomer thereof. 6. The compound of claim 5 which is selected from the following group:
6-Bromo-9-(2-chloro-acetyl)-2,3 ,4,9-tetahydro-/?-carbolin- 1 -one (Compound 3); 9-Acetyl-6-bromo-2,3,4,9-tetrahydro-y9-carbolin-l-one (Compound 6); 6-Bromo-9-(2-methoxy-acetyl)-2,3,4,9-tetrahydro-^-carbolin-l-one (Compound 12); and
Acetic acid 2-(6-bromo-l-oxo-l,2,3,4-tetrahydro-/?-carbolin-9-yl)-2-oxo-ethyl ester (Compound 13), pharmaceutically acceptable salt, or stereoisomer thereof.
7. A process for preparing the compound of formula (1) defined in1 claim 1, pharmaceutically acceptable salt, or stereoisomer thereof, comprising
(a) reacting a compound of the following formula (2): [Formula 2]
Figure imgf000031_0001
in which X is as defined in claim 1, with a compound of the following formula (3): [Formula 3]
R-L in which R is as defined in claim 1, and L represents a leaving group, or
(b) reacting a compound of the following formula (4):
[Formula 4]
Figure imgf000031_0002
in which X is as defined in claim 1, and P represents an amino-protecting group, with the compound of formula (3) to give a compound of the following formula (5): [Formula 5]
Figure imgf000032_0001
in which R and X are as defined in claim 1 and P is as defined above, and deprotecting the compound of formula (5).
8. A composition for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection, comprising an effective amount of the compound of formula (1) defined in claim 1, pharmaceutically acceptable salt, or stereoisomer thereof as an active ingredient together with a pharmaceutically acceptable carrier.
9. A method for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection, comprising administering an effective amount of the compound of formula (1), pharmaceutically acceptable salt, or stereoisomer thereof as defined in claim 1 to a patient.
10. A process for preparing a composition for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection, comprising admixing the compound of formula (1) defined in claim 1, pharmaceutically acceptable salt, or stereoisomer thereof with a pharmaceutically acceptable carrier.
11. Use of the compound of formula (1) defined in claim 1, pharmaceutically acceptable salt, or stereoisomer thereof for the treatment of cancer, autoimmune disease, cardio-vascular disease, chemotherapy-induced alopecia and mucositis, infection disease, kidney disease, chronic and acute neurodegenerative disease, and viral infection.
PCT/KR2005/002433 2004-07-27 2005-07-27 Tetrahydro-beta-carbolinone derivatives and process for preparing the same WO2006011750A1 (en)

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CN108623585A (en) * 2018-05-22 2018-10-09 中国人民解放军第二军医大学 Beta-tetrahydro carboline class antifungal drug and its preparation method and application

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