WO2008136631A1 - 3,4-dihydroquinazoline derivatives - Google Patents

3,4-dihydroquinazoline derivatives Download PDF

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Publication number
WO2008136631A1
WO2008136631A1 PCT/KR2008/002529 KR2008002529W WO2008136631A1 WO 2008136631 A1 WO2008136631 A1 WO 2008136631A1 KR 2008002529 W KR2008002529 W KR 2008002529W WO 2008136631 A1 WO2008136631 A1 WO 2008136631A1
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compound
pharmaceutically acceptable
mmol
solution
biphenylyl
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PCT/KR2008/002529
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French (fr)
Inventor
Jae Yeol Lee
Dong Joon Choo
Young Deuk Kim
Chun Rim Oh
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Dongwoo Syntech Co., Ltd.
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Priority to US12/598,734 priority Critical patent/US8168646B2/en
Priority to AT08753326T priority patent/ATE556062T1/en
Priority to CN2008800151697A priority patent/CN101679312B/en
Priority to JP2010507318A priority patent/JP5330377B2/en
Priority to EP08753326A priority patent/EP2142518B1/en
Publication of WO2008136631A1 publication Critical patent/WO2008136631A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/78—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 2
    • C07D239/84—Nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to 3,4-dihydroquinazoline derivatives having excellent
  • T-type calcium channel blocking effect and anti-cancer activity a process of preparing them and a pharmaceutical composition including them.
  • T-type calcium channel blockers (CCBs) inhibited cellular proliferation [McCalmont, W. F., et al., Bioorg. Med. Chem. Lett. 2004, 14, 3691-3695; McCalmont, W. F., et al., Bioorg. Med. Chem. 2005, 13, 3821-3839].
  • L-type CCBs as anti-hypertensive agents may be related to the risk of cancer in the elderly and promote growth of pre-existing cancer cells in human by inhibition of apoptosis [La Vecchia, C. et al., Eur. J. Cancer 2003, 39, 7-8]. Therefore, selective T-type CCBs could be another tool to treat cancer where the cell cycle has become aberrant.
  • T-type calcium channel may cause epilepsy (Tsakiridou, E. et al., J. Neurosci. 1995, 75, 3110- 3117), high blood pressure (Self, D. A. et al., J Vacs. Res. 1994, 31, 359-366), ventricular hypertrophy (Nuss, H. B. et al. , Circ Res. 1995, 73, 111- 7825), pain (Shin, H. S. et al., Science 2003, 302, 117-119) and angina pectoris (Van der Vring, J. A. et al., Am. J. Ther. 1999, 6, 229- 233). Therefore, selective T-type calcium channel blockers could be used for preventing or treating epilepsy, high blood pressure, ventricular hypertrophy, pain and angina pectoris.
  • the present inventors have extensively studied to develop a novel compound for treating overgrowth diseases such as cancer via T-type calcium channel blocking. As a result, they discovered that a 3,4-dihydroquinazoline derivative of the following formula (I) has excellent T-type calcium channel blocking effect and anti-cancer activity.
  • R 1 , R 2 and R 3 are defined below.
  • An object of the present invention is, therefore, to provide a compound of the above formula (I) or pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the compound of the above formula (I) or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.
  • Ri is NR 4 (CHz) n NR 5 R 6 , wherein R 4 is hydrogen or C r C 5 alkyl, n is 4, 5 or 6, R 5 and
  • R 6 are each independently hydrogen or Ci-C 5 alkyl, or taken together with the nitrogen atom to which they are attached form a 4 to 6-membered heterocycle; [18] R 2 is 4-biphenylyl;
  • R 3 is benzylamino, 4-aminobenzylamino or
  • R 4 is preferably Ci-C 5 alkyl, more preferably methyl or ethyl, and n is most preferably 5.
  • R 5 and R 6 are preferably identical and hydrogen or Ci-C 5 alkyl, more preferably methyl or ethyl; or taken together with the nitrogen atom to which they are attached form pyrrolidine or piperidine, more preferably pyrrolidine.
  • Ci-C 5 alkyl as used herein means a straight or branched hydrocarbon having 1 to 5 carbon atoms, which includes methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, etc. but is not limited thereto.
  • the most preferable compound among the compounds of the present invention is selected from the following group.
  • [24] 4- (W-benzylacetamino)- 3 - (4-biphenylyl) -2- [N- (5 -pyrrolidin- 1 -ylpentyl) -/V-methylami no] - 3 ,4-dihyrdoquinazoline ;
  • [25] 4-(N-benzylacetamino)-3-(4-biphenylyl)-2-[N-(5-N',N'-dimethylaminopentyl)-N-met hylamino]-3,4-dihyrdoquinazoline; and [26] 4-(N-benzylacetamino)-3-(4-biphenylyl)-2-[N-(5-aminopentyl)-N-methylamino]-3,4- dihyrdoquinazoline.
  • the compound (4) is reacted with di-t-butyl dicarbonate to give t-butyl (5-pyrrolidin-l -ylpentyl) carbamate (5). Then, the compound (5) is reacted with lithium aluminum hydride in a suitable solvent to give JV-methyl-/V - [5-(pyrrolidin- l-yl)pentyl] amine (6).
  • compound (14) according to the present invention can be prepared using ⁇ f-methyl- ⁇ f-[5-(pyrrolidin-l-yl)pentyl]amine (6) obtained according to the above Reaction Scheme 1.
  • 2-nitrocinnamic acid (7) is reacted with methanol under acid catalyst to give methyl 2-nitrocinnamate (8), of which the nitro group is reduced preferably with SnCl 2 2H 2 O to provide methyl 2-aminocinnamate (9).
  • the compound (9) is reacted with triphen- ylphosphine, preferably in the presence of hexachloroethane and triethylamine to give methyl 3-[2-(triphenylphosphineimino)phenyl]acrylate (10).
  • the compound (10) is reacted with 4-biphenylyl isocyanate in a suitable solvent to give methyl 3-[2-(4-biphenylyliminomethyleneamino)phenyl]acrylate (11).
  • the compound (11) is reacted with ⁇ f-methyl- ⁇ f-[5-(pyrrolidin-l-yl)pentyl]amine (6) obtained according to the above Reaction Scheme 1 in a suitable solvent to give 3-(4-biphenylyl)-2-[N-methyl-N-(5-pyrrolidin-l-ylpentyl)amino]-4-methoxycarbonylm ethyl-3,4-dihydroquinazoline (12).
  • This compound (12) is hydrolized preferably using LiOH/H 2 O to provide compound (13), which is reacted with benzylamine, preferably in the presence of 1-hydroxybenzotriazole (HOBT) and l-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloric acid (EDC) to give 4-( N-benzylacetamino)-3-(4-biphenylyl)-2-[N-(5-pyrrolidin-l-ylpentyl)-N-methylamino]- 3 ,4-dihyrdoquinazoline (14).
  • HOBT 1-hydroxybenzotriazole
  • EDC l-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloric acid
  • compound (18) can be prepared as follows. [42] 1,5-Diaminopentane (15) is reacted with di-t-butyl dicarbonate in a suitable solvent to give t-butyl (5-aminopentyl) carbamate (16), which is reacted with benzyl bromide in the presence of base to give t-butyl (5-dibenzylaminopentyl) carbamate (17). The obtained compound (17) is reacted with lithium aluminum hydride in a suitable solvent to give N,N-dibenzyl-N'-methylpentane-l,5-diamine (18).
  • compound (19) is hydrolized preferably using LiOH/H 2 O to give compound (21), which is reacted with benzylamine, preferably in the presence of 1-hydroxybenzotriazole (HOBT) and l-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloric acid (EDC) to give N - benzyl-3-(4-biphenylyl)-2-[N-(5-N',N'-dibenzylaminopentyl)-N-methylamino]-3,4-dihy rdoquinazolin-4-ylacetamide (22).
  • HOBT 1-hydroxybenzotriazole
  • EDC l-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloric acid
  • the compound (22) is hydrogenated in the presence of Pd(C) and formalin solution to give 4-(N -ben- zylacetamino)-3-(4-biphenylyl)-2-[N-(5-N',N'-dimethylaminopentyl)-N-methylamino]- 3,4-dihyrdoquinazoline (23).
  • compound (24) and compound (25) can be prepared using N,N-dibenzyl-N'-methylpentane-l,5-diamine (18) obtained according to the above Reaction Scheme 3 and methyl
  • compositions comprising the compound of the above formula (I) or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be used for treating cancer, especially colon cancer; or for preventing or treating epilepsy, high blood pressure, ventricular hypertrophy, pain or angina pectoris.
  • the pharmaceutical composition according to the present invention can be ad- ministered orally, e.g., ingestion or inhalation; or parenterally, e.g., injection, deposition, implantation or suppositories.
  • the injection can be, for example, intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal.
  • the pharmaceutical composition of the present invention may be formulated as tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injection solutions, emulsions, suspensions, syrups, aerosols, etc.
  • composition of the present invention can be prepared in a manner well known in the art using a pharmaceutically acceptable carrier(s) which are usually used for each form.
  • pharmaceutically acceptable carriers include excipient, binder, disintegrating agent, lubricant, preservative, antioxidant, isotonic agent, buffer, coating agent, sweetening agent, dissolvent, base, dispersing agent, wetting agent, suspending agent, stabilizer, colorant, etc.
  • the pharmaceutical composition of the present invention contains 0.01 to 100 wt% of a 3,4-dihydroquinazoline derivative of the above formula (I) or pharmaceutically acceptable salt thereof depending on the form thereof.
  • the specific dosage of the present pharmaceutical composition can be varied with species of mammals including a human-being, body weight, gender, severity of disease, judgment of doctor, etc. It is preferable that 0.01 to 50 mg of the active ingredient is administered per kg of body weight a day for oral use, while 0.01 to 10 mg of the active ingredient is administered per kg of body weight a day for parenteral use.
  • the total daily dosage can be administered once or over several times depending on the severity of disease, judgment of doctor, etc.
  • the 3,4-dihydroquinazoline derivatives of the present invention have excellent T- type calcium channel blocking effect and anti-cancer activity. Therefore, the compounds of the present invention can be used as new chemical therapy for treating overgrowth diseases such as cancer via T-type calcium channel blocking. Also, the compounds of the present invention can be used for preventing or treating epilepsy, high blood pressure, ventricular hypertrophy, pain or angina pectoris.
  • NC 4 H 8 2.51 (s, 4H, -NC 2 H 4 C 2 H 4 ), 2.45-2.41 (m, 2H, -(CH 2 ) 4 -CH_ 2 -NC 2 H 4 C 2 H 4 ), 1.76 (s, 4H, -(CHz) 5 -NC 2 H 4 C 2 H. ! ), 1.54-1.28 (m, 15H, -CH 2 -CH 2 -CH 2 -CHa-CH 2 -NC 2 H 4 C 2 H 45 -C(CH 3- I 3 ).
  • Example 11 Preparation of 4-( N -benzylacetamino)-3-(4-biphenylyl)-2-[ N - (5-pyrrolidin-l-ylpentyl)- N -methylamino]-3,4-dihyrdoquinazoline (14) (KYS05080)
  • Example 15 Preparation of 3-(4-biphenylyl)-2-[ N -(5-dibenzylaminopentyl)- N -methylamino]-4-methoxycarbonylmethyl-3,4-dihyrdoquinazoline (19) [126] To a solution of methyl 3-[2-(4-biphenylyliminomethyleneamino)phenyl]acrylate (11) (1.12 g, 3.15 mmol) in toluene (100 ml) was added N,N-dibenzy ⁇ -N'- methylpentane- 1,5 -diamine (18) (1.87 g, 6.30 mmol).
  • Example 17 Preparation of N -benzyl-3-(4-biphenylyl)-2-[ N -(5- JVJT- diben- zylaminopentyl)- N -methylamino]-3,4-dihyrdoquinazolin-4-ylacetamide (22) [136] To a solution of 3-(4-biphenylyl)-2-[ ⁇ 5-dibenzylaminopentyl)-/V - methylamino]-4-methoxycarbonylmethyl-3,4-dihyrdoquinazoline (19) (2.56 g, 3.93 mmol) in dihydrofuran (50 ml) and water (50 ml) was added LiOH/H 2 O (0.825 g, 19.7 mmol), and the reaction mixture was stirred at 70 0 C for 2 hours.
  • LiOH/H 2 O 0.825 g, 19.7 mmol
  • Example 18 Preparation of 4-(7V -benzylacetamino)-3-(4-biphenylyl)-2-[7V-(5- N' JV- dimethylaminopentyl)- N -methylamino]-3,4-dihyrdoquinazoline (23) (KYS05090)
  • Example 20 Preparation of 4-( N -benzylacetamino)-3-(4-biphenylyl)-2-[ N - (5-aminopentyl)- N -methylamino]-3,4-dihyrdoquinazoline (25) (KYS05097) [152] To a solution of N-benzyl-3-(4-biphenylyl)-2-[N-(5-N',N'-dibenzylaminopentyl)-N - methylamino]-3,4-dihyrdoquinazolin-4-ylacetamide (22) (1.01 mmol) in methanol (20 ml) was added 10% Pd(C) (183 mg), and the reaction mixture was stirred under hydrogen atmosphere for 24 hours.
  • Pd(C) 183 mg
  • T-type calcium channel blocking effects of the compounds of the present invention were tested according to an electrophysiological whole-cell patch-clamp method, which uses mammalian HEK293 cell line selectively expressing ⁇ 1G among T-type calcium channel encoding genes, wherein the ⁇ 1G is mainly expressed at nerve cells (derived from human kidney cancer cells), as described in Korean Patent No. 0610731 and Monteil, A., et al., J. Biol. Chem. 2000, 275, 6090-6100. Also, cytotoxicities of the compounds according to the present invention were tested according to a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • SRB sulforhodamine B
  • HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (v/v) in 36.5 0 C humidified incubator (95% air-5% CO 2 ).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin/streptomycin v/v
  • the cells used for T-type calcium channel activity assay were cultured on a cover slip coated with poly-L- lysine (0.5 mg/mL) whenever sub-cultured, and their calcium channel activity was recorded 2 to 7 days after the cultivation.
  • Current of the T-type calcium channel at a single cell level was measured according to an electrophysiological whole-cell patch-clamp method using EPC-9 amplifier (HEKA, Germany).
  • a cell exterior solution [NaCl 140 mM, CaCl 2 2 mM, and HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) 10 mM (pH 7.4)] and a cell interior solution [KCl 130 mM, HEPES 10 mM, EGTA (ethylene glycol tetraacetic acid) 11 mM, and MgATP 5 mM (pH 7.4)] were employed.
  • [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was conducted as follows.
  • the cultured HEK293 cells were treated with each compound at a concentration of 10 and 100 ⁇ M, respectively.
  • the cells treated with a solvent, that is, 0.1% DMSO were used as a negative control and the cells treated with H 2 O 2 (125 ⁇ M) inducing cytotoxicity were used as a positive control.
  • the cells were treated with 50 ⁇ L MTT (1 mg/mL) dissolved in PBS (phosphate buffered saline) for 4 h.
  • the reaction mixture was centrifuged to remove a supernatant, and formazan crystals thus obtained were dissolved in 100 ⁇ L DMSO.
  • the solution's absorbance was measured at 560 nm with an automated spectro- photometric plate reader.
  • the compounds of the present invention did not show any cytotoxicity at a concentration of 100 ⁇ M.
  • the compounds according to the present invention were evaluated for their growth inhibition against five cancer cell lines, including human lung carcinoma (A-549), human prostate cancer (DU 145), human colon cancer (HT-29), human malignant melanoma (SK-MEL-2) and human ovarian cancer (SK-OV-3) using sulforhodamine B (SRB) assay. All cell lines were grown in RPMI 1640 (Gibco BRL) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and maintained at 37 0 C in a humidified atmosphere with 5% CO 2 . The cells were seeded into 96- well plate.
  • b GI 50 value was determined from dose-response curve and obtained from three independent experiments.

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Abstract

The present invention relates to 3,4-dihydroquinazoline derivatives, a process of preparing them and a pharmaceutical composition including them. The 3,4-dihydroquinazoline derivatives of the present invention have excellent T-type calcium channel blocking effect and anti-cancer activity.

Description

Description
3,4-Dihydroquinazoline Derivatives Technical Field
[1] The present invention relates to 3,4-dihydroquinazoline derivatives having excellent
T-type calcium channel blocking effect and anti-cancer activity, a process of preparing them and a pharmaceutical composition including them.
[2]
Background Art
[3] Calcium plays a critical role as an intracellular signal, and controls many different cell processes, of which calcium appears to play an important role in cell growth [Berridge, M. J., et al., Nat. Rev. MoI. Cell Biol. 2003, 4, 517-529]. For example, it has also been shown that calcium signaling is required for cell cycle progression from Gl/S phase through mitosis. It has been demonstrated that depletion of intracellular calcium arrests the cell cycle in the G0/G1 and S interphases [Clapham, D. E. Cell 1995, 80, 259-268]. Regulation of the changes in intracellular calcium has been proposed to be via a T-type calcium channel. Lined with this proposition, it has recently been reported that T-type calcium channel blockers (CCBs) inhibited cellular proliferation [McCalmont, W. F., et al., Bioorg. Med. Chem. Lett. 2004, 14, 3691-3695; McCalmont, W. F., et al., Bioorg. Med. Chem. 2005, 13, 3821-3839]. Opposed to T-type CCBs, however, it remains to discussion that some L-type CCBs as anti-hypertensive agents may be related to the risk of cancer in the elderly and promote growth of pre-existing cancer cells in human by inhibition of apoptosis [La Vecchia, C. et al., Eur. J. Cancer 2003, 39, 7-8]. Therefore, selective T-type CCBs could be another tool to treat cancer where the cell cycle has become aberrant.
[4] On the other hand, it has been reported that over-expression of T-type calcium channel may cause epilepsy (Tsakiridou, E. et al., J. Neurosci. 1995, 75, 3110- 3117), high blood pressure (Self, D. A. et al., J Vacs. Res. 1994, 31, 359-366), ventricular hypertrophy (Nuss, H. B. et al. , Circ Res. 1995, 73, 111- 7825), pain (Shin, H. S. et al., Science 2003, 302, 117-119) and angina pectoris (Van der Vring, J. A. et al., Am. J. Ther. 1999, 6, 229- 233). Therefore, selective T-type calcium channel blockers could be used for preventing or treating epilepsy, high blood pressure, ventricular hypertrophy, pain and angina pectoris.
[5]
Disclosure of Invention Technical Problem
[6] The present inventors have extensively studied to develop a novel compound for treating overgrowth diseases such as cancer via T-type calcium channel blocking. As a result, they discovered that a 3,4-dihydroquinazoline derivative of the following formula (I) has excellent T-type calcium channel blocking effect and anti-cancer activity.
[7]
Figure imgf000003_0001
(I) [9]
[10] wherein, R1, R2 and R3 are defined below.
[11] An object of the present invention is, therefore, to provide a compound of the above formula (I) or pharmaceutically acceptable salt thereof. [12] Another object of the present invention is to provide a pharmaceutical composition comprising the compound of the above formula (I) or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. [13]
Technical Solution [14] One aspect of the present invention relates to a compound of the following formula
(I) or pharmaceutically acceptable salt thereof.
Figure imgf000003_0002
(I)
[16] wherein,
[17] Ri is NR4(CHz)nNR5R6, wherein R4 is hydrogen or CrC5 alkyl, n is 4, 5 or 6, R5 and
R6 are each independently hydrogen or Ci-C5 alkyl, or taken together with the nitrogen atom to which they are attached form a 4 to 6-membered heterocycle; [18] R2 is 4-biphenylyl;
[19] R3 is benzylamino, 4-aminobenzylamino or
4-fluorobenzenesulfoneaminobenzylamino. [20] In the above formula (I), R4 is preferably Ci-C5 alkyl, more preferably methyl or ethyl, and n is most preferably 5. [21] R5 and R6 are preferably identical and hydrogen or Ci-C5 alkyl, more preferably methyl or ethyl; or taken together with the nitrogen atom to which they are attached form pyrrolidine or piperidine, more preferably pyrrolidine.
[22] The term "Ci-C5 alkyl" as used herein means a straight or branched hydrocarbon having 1 to 5 carbon atoms, which includes methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, etc. but is not limited thereto.
[23] The most preferable compound among the compounds of the present invention is selected from the following group. [24] 4- (W-benzylacetamino)- 3 - (4-biphenylyl) -2- [N- (5 -pyrrolidin- 1 -ylpentyl) -/V-methylami no] - 3 ,4-dihyrdoquinazoline ; [25] 4-(N-benzylacetamino)-3-(4-biphenylyl)-2-[N-(5-N',N'-dimethylaminopentyl)-N-met hylamino]-3,4-dihyrdoquinazoline; and [26] 4-(N-benzylacetamino)-3-(4-biphenylyl)-2-[N-(5-aminopentyl)-N-methylamino]-3,4- dihyrdoquinazoline.
[27] [28] The processes for preparing the compounds according to the present invention are depicted in the following Reaction Schemes 1 to 5. However, those illustrated in the following Reaction Schemes represent only typical processes used in the present invention. The manipulation order, reagents, reaction conditions, etc. may be changed without limit.
[29] [30] [Reaction Scheme 1] H2O
Figure imgf000004_0001
LlAIH4
Figure imgf000004_0002
Figure imgf000004_0003
[32] As shown in the above Reaction Scheme 1, 1,5-dibromopentane is reacted with potassium phthalimide in a suitable solvent to give JV-(5-bromopentyl)phthalimide (2). The compound (2) is reacted with pyrrolidine in the presence of potassium carbonate and sodium iodide to give JV-(5-pyrrolidin-l-ylpentyl)phthalimide (3). The compound (3) is reacted with hydrazine to provide 5-(pyrrolidin-l-yl)pentylamine (4). The compound (4) is reacted with di-t-butyl dicarbonate to give t-butyl (5-pyrrolidin-l -ylpentyl) carbamate (5). Then, the compound (5) is reacted with lithium aluminum hydride in a suitable solvent to give JV-methyl-/V - [5-(pyrrolidin- l-yl)pentyl] amine (6).
[33] [34] [Reaction Scheme 2]
Figure imgf000005_0001
Figure imgf000005_0002
[36] As shown in Reaction Scheme 2, compound (14) according to the present invention can be prepared using Λf-methyl-Λf-[5-(pyrrolidin-l-yl)pentyl]amine (6) obtained according to the above Reaction Scheme 1.
[37] 2-nitrocinnamic acid (7) is reacted with methanol under acid catalyst to give methyl 2-nitrocinnamate (8), of which the nitro group is reduced preferably with SnCl22H2O to provide methyl 2-aminocinnamate (9). The compound (9) is reacted with triphen- ylphosphine, preferably in the presence of hexachloroethane and triethylamine to give methyl 3-[2-(triphenylphosphineimino)phenyl]acrylate (10). The compound (10) is reacted with 4-biphenylyl isocyanate in a suitable solvent to give methyl 3-[2-(4-biphenylyliminomethyleneamino)phenyl]acrylate (11). Then, the compound (11) is reacted with Λf-methyl-Λf-[5-(pyrrolidin-l-yl)pentyl]amine (6) obtained according to the above Reaction Scheme 1 in a suitable solvent to give 3-(4-biphenylyl)-2-[N-methyl-N-(5-pyrrolidin-l-ylpentyl)amino]-4-methoxycarbonylm ethyl-3,4-dihydroquinazoline (12). This compound (12) is hydrolized preferably using LiOH/H2O to provide compound (13), which is reacted with benzylamine, preferably in the presence of 1-hydroxybenzotriazole (HOBT) and l-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloric acid (EDC) to give 4-( N-benzylacetamino)-3-(4-biphenylyl)-2-[N-(5-pyrrolidin-l-ylpentyl)-N-methylamino]- 3 ,4-dihyrdoquinazoline (14).
[38] [39] [Reaction Scheme 3]
Figure imgf000006_0001
LiAIH4
Figure imgf000006_0002
18
[41] As shown in Reaction Scheme 3, compound (18) can be prepared as follows. [42] 1,5-Diaminopentane (15) is reacted with di-t-butyl dicarbonate in a suitable solvent to give t-butyl (5-aminopentyl) carbamate (16), which is reacted with benzyl bromide in the presence of base to give t-butyl (5-dibenzylaminopentyl) carbamate (17). The obtained compound (17) is reacted with lithium aluminum hydride in a suitable solvent to give N,N-dibenzyl-N'-methylpentane-l,5-diamine (18).
[43] [44] [Reaction Scheme 4]
Figure imgf000006_0003
[46] As shown in Reaction Scheme 4, compound (20) and compound (23) can be prepared using Λf,Λf-dibenzyl-/V'-methylpentane-l,5-diamine (18) obtained according to the above Reaction Scheme 3 and methyl
3-[2-(4-biphenylyliminomethyleneamino)phenyl]acrylate (11) obtained according to the above Reaction Scheme 2.
[47] Compound (11) is reacted with compound (18) in a suitable solvent to give 3-(4-biphenylyl)-2-[N-(5-dibenzylaminopentyl)-N-methylamino]-4-methoxycarbonylm ethyl-3,4-dihyrdoquinazoline (19). Then, this compound (19) is hydrogenated in the presence of Pd(C) and formalin solution to give 2-[N-(5-N',N'-dimethylaminopentyl)-N -methylamino]-3-(4-biphenylyl)-4-methoxycarbonylmethyl-3,4-dihyrdoquinazoline (20). [48] On the other hand, compound (19) is hydrolized preferably using LiOH/H2O to give compound (21), which is reacted with benzylamine, preferably in the presence of 1-hydroxybenzotriazole (HOBT) and l-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloric acid (EDC) to give N - benzyl-3-(4-biphenylyl)-2-[N-(5-N',N'-dibenzylaminopentyl)-N-methylamino]-3,4-dihy rdoquinazolin-4-ylacetamide (22). Then, the compound (22) is hydrogenated in the presence of Pd(C) and formalin solution to give 4-(N -ben- zylacetamino)-3-(4-biphenylyl)-2-[N-(5-N',N'-dimethylaminopentyl)-N-methylamino]- 3,4-dihyrdoquinazoline (23).
[49] [50] [Reaction Scheme 5]
Figure imgf000007_0001
[52] As shown in Reaction Scheme 5, compound (24) and compound (25) can be prepared using N,N-dibenzyl-N'-methylpentane-l,5-diamine (18) obtained according to the above Reaction Scheme 3 and methyl
3-[2-(4-biphenylyliminomethyleneamino)phenyl]acrylate (11) obtained according to the above Reaction Scheme 2. The specific process is the same as the above Reaction Scheme 4 except for using only Pd(C) catalyst instead of Pd(C) and formalin solution.
[53] [54] Another aspect of the present invention relates to a pharmaceutical composition comprising the compound of the above formula (I) or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. In particular, the pharmaceutical composition of the present invention can be used for treating cancer, especially colon cancer; or for preventing or treating epilepsy, high blood pressure, ventricular hypertrophy, pain or angina pectoris.
[55] [56] The pharmaceutical composition according to the present invention can be ad- ministered orally, e.g., ingestion or inhalation; or parenterally, e.g., injection, deposition, implantation or suppositories. The injection can be, for example, intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal. Depending on the route of administration, the pharmaceutical composition of the present invention may be formulated as tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injection solutions, emulsions, suspensions, syrups, aerosols, etc. The above various forms of the pharmaceutical composition of the present invention can be prepared in a manner well known in the art using a pharmaceutically acceptable carrier(s) which are usually used for each form. Examples of the pharmaceutically acceptable carriers include excipient, binder, disintegrating agent, lubricant, preservative, antioxidant, isotonic agent, buffer, coating agent, sweetening agent, dissolvent, base, dispersing agent, wetting agent, suspending agent, stabilizer, colorant, etc.
[57] The pharmaceutical composition of the present invention contains 0.01 to 100 wt% of a 3,4-dihydroquinazoline derivative of the above formula (I) or pharmaceutically acceptable salt thereof depending on the form thereof.
[58] The specific dosage of the present pharmaceutical composition can be varied with species of mammals including a human-being, body weight, gender, severity of disease, judgment of doctor, etc. It is preferable that 0.01 to 50 mg of the active ingredient is administered per kg of body weight a day for oral use, while 0.01 to 10 mg of the active ingredient is administered per kg of body weight a day for parenteral use. The total daily dosage can be administered once or over several times depending on the severity of disease, judgment of doctor, etc.
[59]
Advantageous Effects
[60] The 3,4-dihydroquinazoline derivatives of the present invention have excellent T- type calcium channel blocking effect and anti-cancer activity. Therefore, the compounds of the present invention can be used as new chemical therapy for treating overgrowth diseases such as cancer via T-type calcium channel blocking. Also, the compounds of the present invention can be used for preventing or treating epilepsy, high blood pressure, ventricular hypertrophy, pain or angina pectoris.
[61]
Best Mode for Carrying Out the Invention
[62] The present invention is further illustrated by the following examples, which are not to be construed to limit the scope of the invention.
[63]
[64] Example 1: Preparation of N -(5-bromopentyl)phthalimide (2) [65] To a solution of 1,5-dibromopentane (9.00 ml, 65.22 mmol) dissolved in DMF (100 ml) was added potassium phthalimide (12.08 g, 65.22 mmol) at room temperature, and the reaction mixture was stirred for 24 hours. After the reaction was completed, distilled water was added, and the resulting reaction mixture was stirred for 10 minutes and extracted with ethyl acetate three times. Then, the organic phase was washed with a saline solution, dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: ethyl acetate:hexane = 1:3) to give the target compound (10.6 g, 55 %).
[66] 1H-NMR (400 MHz, CDCl3) δ 7.85-7.70 (m, 4H, aromatic), 3.71-3.67 (t, 2H, -CH2 -
CH2-CH2-CH2-CH2-Br), 3.4-3.38 (t, 2H, -CH^-CH2-CH2-CH2-Qi2-Br), 1.94-1.87 (m, 2H, -CH2-CIl2-CH2-CH2-CH2-Br), 1.73-1.67 (m, 2H, -CH2-CH2-CH2-CH2-CH2-Br), 1.53-1.47 (m, 2H, -CH2-CH2-CH2-CH2-CH2-Br).
[67]
[68] Example 2: Preparation of N -(5-pyrrolidin-l-ylpentyl)phthalimide (3)
[69] Potassium carbonate (13.57 g, 98.20 mmol), pyrrolidine (4.47 ml, 54.01 mmol) and sodium iodide (7.359 g, 49.098 mmol) were added to ethanol/acetone (1:1) at room temperature and stirred for 1 hour. To the reaction mixture was slowly added N - (5-bromopentyl)phthalimide (2) (10.60 g, 49.10 mmol), and the resulting reaction mixture was refluxed at 50 0C for 24 hours. After the reaction was completed, the resulting white solid was filtered and the solvent was evaporated under reduced pressure. Afterwards, the obtained residue was extracted with a saturated IN NaOH aqueous solution and dichloromethane three times. Then, the organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane :methnol: ammonia water = 100:9:1) to give the target compound (3.27 g, 45 %).
[70] 1H-NMR (400 MHz, CDCl3) δ 7.81-7.66 (m, 4H, aromatic), 3.66-3.63 (t, 2H, -CH2 -
(CH2)4-NC4H8), 2.51-2.49 (t, 4H, -(CH2)S-NC2H4C2H4), 2.45-2.41 (m, 4H, -(CH2)5-NC2 H4C2H4), 1.77-1.74 (m, 2H, -(CH2VCH2-NC4H8), 1.68-1.55 (m, 4H, -CH2-CU2-CU2 - CH2-CH2-NC4H8), 1.36-1.32 (m, 2H, -CH2-CH2-CH2 -CH2-CH2-NC4H8).
[71]
[72] Example 3: Preparation of 5-(pyrrolidin-l-yl)pentylamine (4)
[73] To a solution of N-(5-pyrrolidin-l-ylpentyl)phthalimide (3) (3.27 g, 11.42 mmol) dissolved in ethanol (100 ml) was added NH2NH2-H2O (hydrazine monohydrate) (5.54 ml, 114.19 mmol) at room temperature, and the reaction mixture was refluxed for 24 hours. After the reaction was completed, the resulting reaction mixture was filtered under reduced pressure to give the target compound (1.50 g, 84 %).
[74] 1H-NMR (400 MHz, CDCl3) δ 4.28 (br s, 2H, H2N-), 2.69-2.66 (t, 2H, -CH_2-(CH2)4 - NC4H8), 2.51 (s, 4H, -(CH2)S-NC2H4C2H4), 2.46-2.42 (t, 2H, -(CH2)4-CH_2 -NC4H8), 1.73 (s, 4H, -(CH2)S-NC2H4C2H4), 1.52-1.43 (m, 4H, -CH2-CH2-CH2-Qi2-CH2-NC4H s), 1.33-1.25 (m, 2H, -CH2-CH2-Qi2-CH2-CH2-NC4H8).
[75] 13C-NMR (CDCl3) δ 56.18, 54.06, 41.30, 31.92, 28.22, 24.68, 23.31.
[76]
[77] Example 4: Preparation of t-butyl (5-pyrrolidin-l-ylpentyl) carbamate (5)
[78] 5-(pyrrolidin-l-yl)pentylamine (4) (1.50 g, 9.60 mmol) was added to 50 ml of flask, which was then evacuated and equipped with a nitrogen balloon. Afterwards, di- chloromethane (30 ml) was added to dissolve the compound (4) and di-t-butyl di- carbonate (2.64 ml, 11.52 mmol) was slowly added at 0 0C. Then, dichloromethane (20 ml) was further added and stirred for 24 hours. After the reaction was completed, the reaction mixture was basified with a saturated IN NaOH aqueous solution to pH 10-11 and extracted with dichloromethane three times. Then, the organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane :methnol: ammonia water = 40:7:1) to give the target compound (1.5 g, 65 %).
[79] 1H-NMR (400 MHz, CDCl3) δ 4.63 (br s, IH, -NH-), 3.07-3.06 (t, 2H, -C£b-(CH2)4 -
NC4H8), 2.51 (s, 4H, -NC2H4C2H4), 2.45-2.41 (m, 2H, -(CH2)4-CH_2 -NC2H4C2H4), 1.76 (s, 4H, -(CHz)5-NC2H4C2H.!), 1.54-1.28 (m, 15H, -CH2-CH2-CH2-CHa-CH2-NC2H4C2 H45 -C(CH3-I3 ).
[80]
[81] Example 5: Preparation of N -methyl- N -[5-(pyrrolidin-l-yl)pentyl]amine (6)
[82] To a solution of t-butyl (5-pyrrolidin-l-ylpentyl) carbamate (5) (1.59 g, 6.20 mmol) dissolved in dihydrofuran (100 ml) was added LiAlH4 (lithium aluminum hydride) (2.35 g, 62.00 mmol), and the reaction mixture was stirred for 24 hours. To the resulting reaction mixture was added sodium potassium tartrate (17.5 g, 62.0 mmol) for quenching, and the mixture was filtered with celite. The solvent was evaporated under reduced pressure to give the target compound (0.7 g, 66 %).
[83] 1H-NMR (400 MHz, CDCl3) δ 2.63-2.47 (m, 8H, -CH2 -(CH^3-Qi2-NC2H4 C2H4),
2.45 (s, 3H, -NH-QL ), 1.80-1.79 (m, 4H, -(CH2)S-NC2H4C2H4), 1.56-1.53 (m ,4H, - CH2-Qi2-CH2-CH2-CH2-NC4H8), 1.41-1.33 (m, 2H, -(CH2)2-CH_2-(CH2)2-NC4H8).
[84] 13C-NMR(CDCl3) δ 56.43, 54.05, 51.79, 36.14, 29.48, 28.70, 25.27, 23.19.
[85]
[86] Example 6: Preparation of methyl 2-nitrocinnamate (8)
[87] To a solution of 2-nitrocinnamic acid (7) (10.38 g, 53.76 mmol) dissolved in methanol (200 ml) was dropped a small amount of concentrated sulfuric acid at room temperature, and the reaction mixture was stirred at 70 0C for 12 hours. After the reaction was completed, the resulting solution was basified with a saturated aqueous solution of sodium bicarbonate to weak basicity and extracted with dichloromethane three times. Then, the organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: ethyl acetate:hexane = 1:5) to give the target compound (11.03 g, 99 %).
[88] 1H-NMR (400 MHz, CDCl3) δ 8.16-8.12 (d, IH, -C-CH=CH-), 8.09-7.58 (m, 4H, aromatic), 6.43-6.37 (d, IH, -C-CH=CH-), 3.86 (s, 3H, -OCH3).
[89]
[90] Example 7: Preparation of methyl 2-aminocinnamate (9)
[91] To a solution of methyl 2-nitrocinnamate (8) (11.03 g, 53.24 mmol) in ethyl acetate
(200 ml) was added SnCl2-H2O (60.06 g, 266.19 mmol) at room temperature, and the resulting solution was stirred at 70 0C for 60 minutes. After the reaction was completed, the solution was allowed to cool to room temperature, basified with a saturated aqueous solution of sodium bicarbonate to weak basicity, and filtered with celite. The aqueous phase was extracted with ethyl acetate three times, and the combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: ethyl acetate:hexane = 1:5) to give the target compound (8.50 g, 90 %).
[92] 1H-NMR (400 MHz, CDCl3) δ 7.92-7.86 (d, IH, -C-CH=CH-), 7.44-6.76 (m, 4H, aromatic), 6.42-6.36 (d, IH, -C-CH=CH-), 3.83 (s, 3H, -OCH3).
[93]
[94] Example 8: Preparation of methyl
3- [2-(triphenylphosphineimino)phenyl]acrylate (10)
[95] To a solution of methyl 2-aminocinnamate (9) (8.93 g, 50.40 mmol) in toluene (200 ml) was dropped triphenylphosphine (19.83 g, 75.59 mmol) and hexachloroethane (17.90 g, 75.59 mmol). Then, to the reaction mixture was slowly dropped triethylamine (3.24 ml, 23.3 mmol) at room temperature, and the resulting reaction mixture was refluxed for 3 hours. After the reaction was completed, the solution was allowed to cool to room temperature and filtered with celite. The resulting solution was extracted with dichlormethane three times, and the combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: ethyl acetate:hexane:dichloromethane = 1:3:1) to give the target compound (22.05 g, quantitative yield).
[96] 1H-NMR (400 MHz, CDCl3) δ 8.78-8.81 (d, IH, -C-CH=CH-), 7.84-6.69 (m, 19H, aromatic), 6.42-6.53 (d, IH, -C-CH=CH-), 3.84 (s, 3H, -OCH3). [97]
[98] Example 9: Preparation of
3-[2-(4-biphenylyliminomethyleneamino)phenyl]acrylate (ll)
[99] To a solution of methyl 3-[2-(triphenylphosphineimino)phenyl]acrylate (10) (22.05 g, 50.40 mmol) in toluene (300 ml) was added 4-biphenylyl isocyanate (9.84 g, 50.40 mmol). The reaction mixture was stirred at room temperature for 12 hours, distilled under reduced pressure, and recrystallized with methanol to give the target compound as a white solid (6.95 g, 64 %).
[100] 1H-NMR (400 MHz, CDCl3) δ 8.17-8.13 (d, IH, -C-CH=CH-), 7.61-7.25 (m, 13H, aromatic), 6.54-6.50 (d, IH, -C-CH=CH-), 3.81 (s, 3H, -OCH3).
[101]
[102] Example 10: Preparation of 3-(4-biphenylyl)-2-[ N -methyl- N -
(5-pyrrolidin-l-ylpentyl)amino]-4-methoxycarbonylmethyl-3,4-dihydroquinazolin e (12)
[103] To a solution of 3-[2-(4-biphenylyliminomethyleneamino)phenyl]acrylate (11) (0.83 g, 2.35 mmol) in toluene (50 ml) was slowly added N-methy\-N - [5-(pyrrolidin-l-yl)pentyl]amine (6) obtained in Example 5 (0.80 g, 4.70 mmol) at 0 0C, and the reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the resulting reaction mixture was extracted with a saturated IN NaOH aqueous solution and dichloromethane three times. Then, the combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane:methnol:ammonia water = 100:9:1) to give the target compound (0.63 g, 52 %).
[104] 1H-NMR (400 MHz, CDCl3) δ 7.51-6.89 (m, 13H, aromatic), 5.1.(dd, J= 4.5 and 10 Hz, IH, -NH-CH-), 3.76 (s, 3H, -OCH3), 2.85-2.48 (m, HH, -N-CH3, -CH^CH^-C H2-NC2H4C2H4), 1.88 (s, 4H, -(CH2)S-NC2H4C2H4), 1.66 (s, 2H, -CH2-CH2 -(CH2), - NC2H4C2H4), 1.53 (s, 2H, -(CH^3-Qi2-CH2-NC2H4C2H4), 1.26-1.22 (m, 2H, -CH2-CH 2-CH2 -CH2-CH2-NC2H4C2H4).
[105] 13C-NMR (CDCl3) δ 171.88, 153.38, 145.38, 144.15, 140.30, 136.77, 128.76, 128.36, 127.85, 127.08, 126.73, 124.75, 122.86, 122.59, 122.12, 61.21, 56.30, 54.06, 53.45, 51.89, 39.54, 35.40, 30.90, 28.21, 27.16, 24.91, 23.37.
[106]
[107] Example 11: Preparation of 4-( N -benzylacetamino)-3-(4-biphenylyl)-2-[ N - (5-pyrrolidin-l-ylpentyl)- N -methylamino]-3,4-dihyrdoquinazoline (14) (KYS05080)
[108] To a solution of 3-(4-biphenylyl)-2-[iV-methyl-iV -
(5-pyrrolidin-l-ylpentyl)amino]-4-methoxycarbonylmethyl-3,4-dihydroquinazoline (12) (0.53 g, 1.01 mmol) in dihydrofuran (20 ml) and water (20 ml) was added LiOH/ H2O (0.21 g, 5.05 mmol), and the reaction mixture was stirred at 70 0C for 2 hours. After the reaction was completed, the solution was allowed to cool to room temperature and the solvent was evaporated under reduced pressure. Then, the reaction mixture was acidified with 3N HCl to pH 3-4 and extracted with dichloromethane three times. The combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure to give compound (13) as a white solid. To a solution of compound (13) and 1-hydroxybenzotriazole (HOBT) (0.881 g, 6.52 mmol) in dichloromethane/tetrahydrofuran (1:1, 40 ml) was dropped benzylamine (0.71 ml, 6.52 mmol) at 0 0C, and the reaction mixture was stirred at 0 0C for 1 hour. Then, l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloric acid (EDC) (1.0 g, 1.20 mmol) was added and stirred at room temperature for 12 hours. After the reaction was completed, the resulting reaction mixture was distilled under reduced pressure and dissolved in dichloromethane. Afterwards, the resulting solution was extracted with a 0.5M hydrochloric acid aqueous solution two times, a saturated aqueous solution of sodium bicarbonate two times and water one time, and washed with a saline solution. The organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane :methnol: ammonia water = 100:9:1) to give the target compound as a yellow liquid (0.33 g, 64 %).
[109] 1H-NMR (400 MHz, CDCl3) δ 7.55-6.97 (m, 18H, aromatic), 5.33-5.29 (dd, J = 5.3 and 9.4 Hz, IH, -NH-CH-), 4.52-4.50 (m, 2H, C6H5-CH2-), 3.41-3.16 (d, 2H, -CH2 - CH-), 2.70-2.62 (m, 4H, -(CHz)5-NC2H4C2H4), 2.46-2.37 (m, 7H, -CH2-CH2-CH2-CH2 -CH2-NC4H8 1-NCIi2), 1.96-1.95 (m, 4H, -(CH2^-NC2H4C2H4), 1.74-1.25 (m, 6H, - CH2-(CH2I2 -CH2-NC4H8).
[110] 13C-NMR(CDCl3) δ 170.26, 154.01, 145.47, 143.89, 140.41, 138.53, 136.75, 128.77, 128.58, 128.05, 127.78, 127.40, 127.07, 126.77, 126.35, 125.08, 122.97, 122.33, 122.21, 61.17, 56.55, 55.44, 54.23, 49.81, 43.72, 41.63, 35.46, 28.66, 27.06, 25.02, 23.38.
[I l l]
[112] Example 12: Preparation of t-butyl (5-aminopentyl) carbamate (16)
[113] To a solution of 1,5-diaminopentane (15) (1.14 ml, 9.79 mmol) in dichloromethane (100 ml) was added di-tert-butyl dicarbonate (1.12 ml, 4.90 mmol) at 0 0C, and the resulting solution was stirred at room temperature for 12 hours. After the reaction was completed, H2O was added to remove solid residue. The reaction mixture was basified with IN NaOH aqueous solution to pH 10-11 and extracted with dichloromethane three times. Then, the combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane:methnol: ammonia water = 100:9:1) to give the target compound as a yellow liquid (1.00 g, 51 %).
[114] 1H-NMR (400 MHz, CDCl3) δ 4.57 (br, IH, -NH-), 3.11-3.09 (d, J = 6.2 Hz, 2H, - NH-CH2-), 2.71-2.67 (t, J = 6.9 Hz, -CH2-(Bn)2), 1.78 (br, 2H, -NH2), 1.51-1.44 (m, 4H, -NH-CH2-CH2-, -Cu2- CH2- N-(Bn)2), 1.42 (s, 9H, -O(CH3)3), 1.37-1.31 (m, 2H, -CH2-CH2-CH2-).
[115]
[116] Example 13: Preparation of t-butyl (5-dibenzylaminopentyl) carbamate (17)
[117] To a solution of t-butyl (5-aminopentyl) carbamate (16) (1.00 g, 4.94 mmol) in di- chloromethane (100 ml) was slowly dropped benzyl bromide (1.99 ml, 16.81 mmol) and sodium carbonate (2.24 g, 26.69 mmol) at room temperature. The resulting reaction mixture was stirred at 40 0C for 12 hours and extracted with dichloromethane three times. Then, the combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: ethyl acetate :hexane: dichloromethane = 1:10:1) to give the target compound as a yellow liquid (0.92 g, 49 %).
[118] 1H NMR (400 MHz, CDCl3) δ 7.40-7.23 (m, 1OH, aromatic), 3.57 (s, 4H, -(Bn)2), 3.09-3.07 (m, 2H, HN-CH2-), 2.44 (t, J = 7.05 Hz, 2H, -CH2-N-), 1.58-1.51 (m, 2H, HN-CH2-CH2-), 1.48 (s, 9H, (CHs )3C-O), 1.41-1.27 (m, 4H, HN-CH2-CH2-CH2-CH2 -CH2-).
[119]
[120] Example 14: Preparation of NJV -dibenzyl- N'- methylpentane-l,5-diamine (18)
[121] To a solution of t-butyl (5-dibenzylaminopentyl) carbamate (17) (0.92 g, 2.41 mmol) in dihydrofuran (100 ml) was added lithium aluminum hydride (0.91 g, 24.1 mmol) at room temperature, and the reaction mixture was stirred at 70 0C for 12 hours. After the reaction was completed, the resulting solution was allowed to cool to room temperature and Rochelle's salt (6.79 g, 24.1 mmol) was added. Then, the reaction mixture was stirred for 8 hours and filtered with celite. The filtrate was extracted with dichloromethane three times, dried with anhydrous magnesium sulfate, and distilled under reduced pressure to give the target compound as a yellow liquid (0.42 g, 60 %).
[122] 1H NMR (400 MHz, CDCl3) δ 7.42-7.22 (m, 1OH, aromatic), 3.58 (s, 4H, -(Bn)2),
2.52-2.44 (m, 5H, HN-CH2 -CH2-CH2-CH2-CH2 -), 2.38 (s, 3H, -NH-CHa), 1.57-1.53 (m, 2H, HN-CH2-CH2-CH2-), 1.42-1.31 (m, 4H, HN-CH2-CH2-CH2-CH2 -CH2-).
[123] 13C NMR (400 MHz, CDCl3) δ 139.9, 128.8, 128.1, 126.7, 58.4, 53.2, 51.7, 35.9, 29.1, 26.9, 24.8.
[124]
[125] Example 15: Preparation of 3-(4-biphenylyl)-2-[ N -(5-dibenzylaminopentyl)- N -methylamino]-4-methoxycarbonylmethyl-3,4-dihyrdoquinazoline (19) [126] To a solution of methyl 3-[2-(4-biphenylyliminomethyleneamino)phenyl]acrylate (11) (1.12 g, 3.15 mmol) in toluene (100 ml) was added N,N-dibenzy\-N'- methylpentane- 1,5 -diamine (18) (1.87 g, 6.30 mmol). Then, the reaction mixture was stirred for 2 hours and extracted with dichloromethane three times. The combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: ethyl acetate:hexane:dichloromethane = 1:2:1) to give the target compound as a yellow liquid (2.56 g, quantitative yield).
[127] 1H-NMR (400 MHz, CDCl3) δ 7.51-7.01 (m, 23H, aromatic), 5.14-5.10 (dd, J = 4.7 and 10.3 Hz, IH, -CH2-CH-N-), 3.75-3.71 (m, 5H, -OCH2, -3HCN-CH2-), 3.57-3.58 (m, 4H, -(Bn)2), 3.27-2.52(m, 7H, -CO-CH2-, -NCH2, -CH2-N(Bn)2), 2.41 (m, 2H, - CH2-CH2-(CH2)S-N(Bn)2), 1.51-1.45 (m, 4H, -(CH2)S-CH2-CH2-N(Bn)2).
[128]
[129] Example 16: Preparation of 2-[ N -(5- JVVV- dimethylaminopentyl)- N - methylamino]-3-(4-biphenylyl)-4-methoxycarbonylmethyl-3,4-dihyrdoquinazoline (20) (KYS05089)
[130] To a solution of 3-(4-biphenylyl)-2-[N-(5-dibenzylaminopentyl)-N - methylamino]-4-methoxycarbonylmethyl-3,4-dihyrdoquinazoline (19) (430 mg, 0.33 mmol) and formalin solution (37%, 0.3 ml) in methanol (20 ml) was added 10% Pd(C) (110 mg), and the reaction mixture was stirred under hydrogen atmosphere for 24 hours. After the reaction was completed, the resulting reaction mixture was filtered with celite, extracted with dichloromethane three times and washed with a saline solution. The combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane : methanol: ammonia water = 100:9:1) to give the target compound as a yellow liquid (274 mg, 60 %).
[131] 1H-NMR (400 MHz, CDCl3) δ 7.52-6.88 (13H, m, Ph), 5.11 (IH, dd, J = 10.4 and 4.5 Hz, COCH2CH), 3.75 (3H, s, OCH3), 3.50 (IH, m, CH3N-CH), 3.15 (IH, m, CH3 N-CH), 2.88-2.82 (4H, m, CHj-NandCOCH), 2.51 (IH, dd, J = 15.5 and 4.6 Hz, COC H), 2.25-2.23 (2H, m, -NCH2), 2.20 (6H, s, 2 x N-CH3), 1.52-1.45 (4H, m, 2 x CH2), 1.24-1.23 (2H, m, CH2).
[132] 13C NMR (400 MHz, CDCl3) δ 171.9, 153.4, 145.4, 144.2, 140.3, 136.8, 128.8,
128.4, 127.9, 127.1, 126.8, 125.4, 124.8, 122.9, 122.6, 122.1, 61.2, 59.7, 49.5, 45.2, 43.8, 41.8, 35.3, 27.1, 27.0, 24.7.
[133] HRMS (FAB+) calcd for C3iH39N4O2: [M+H]+= 499.3073, found = 499.3060.
[134]
[135] Example 17: Preparation of N -benzyl-3-(4-biphenylyl)-2-[ N -(5- JVJT- diben- zylaminopentyl)- N -methylamino]-3,4-dihyrdoquinazolin-4-ylacetamide (22) [136] To a solution of 3-(4-biphenylyl)-2-[Λ^5-dibenzylaminopentyl)-/V - methylamino]-4-methoxycarbonylmethyl-3,4-dihyrdoquinazoline (19) (2.56 g, 3.93 mmol) in dihydrofuran (50 ml) and water (50 ml) was added LiOH/H2O (0.825 g, 19.7 mmol), and the reaction mixture was stirred at 70 0C for 2 hours. After the reaction was completed, the resulting solution was allowed to cool to room temperature and the solvent was evaporated under reduced pressure. Then, the resulting reaction mixture was acidified with 3N HCl to pH 3-4 and extracted with dichloromethane three times. The combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure to give compound (21) as a white solid. To a solution of compound (21) and 1-hydroxybenzotriazole (HOBT) (1.00 g, 7.41 mmol) in di- chloromethane/tetrahydrofuran (1:1, 40 ml) was dropped benzylamine (0.804 ml, 7.41 mmol) at 0 0C, and the reaction mixture was stirred at 0 0C for 1 hour. Then, l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloric acid (EDC) (1.21 g, 6.30 mmol) was added and stirred at room temperature for 12 hours. After the reaction was completed, the reaction mixture was distilled under reduced pressure and dissolved in dichloromethane. Afterwards, the resulting solution was extracted with a 0.5M hydrochloric acid aqueous solution two times, a saturated aqueous solution of sodium bicarbonate two times and water one time, and washed with a saline solution. The organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane :methnol = 15:1) to give the target compound as a yellow liquid (2.13 g, 79 %).
[137] 1H-NMR (400 MHz, CDCl3) δ 7.63-7.05 (m, 28H, aromatic), 5.42-5.38 (dd, J = 5.6 and 9.0 Hz, IH, -CH2-CH-N-), 4.68-4.64 (m, 2H, Bn-NH-), 3.62 (s, 4H, -N-(Bn) A 2.95 (br, IH, -CH2-CH-N-), 2.74-2.69 (m, 4H, -3HCN-CH2-, -CH2-N(Bn)2), 2.46-2.41 (m, 3H, -NCH2), 1.57-1.19 (m, 7H, -CH2-CH-N-, -CH)-(CH-^ -CH,-).
[138]
[139] Example 18: Preparation of 4-(7V -benzylacetamino)-3-(4-biphenylyl)-2-[7V-(5- N' JV- dimethylaminopentyl)- N -methylamino]-3,4-dihyrdoquinazoline (23) (KYS05090)
[140] To a solution of iV-benzyl-3-(4-biphenylyl)-2-[iV-(5-iV', N'-dibenzylaminopentyl)-J/V - methylamino]-3,4-dihyrdoquinazolin-4-ylacetamide (22) (530 mg, 0.73 mmol) and formalin solution (37%, 0.27 ml) in methanol (10 ml) was added 10% Pd(C) (120 mg), and the reaction mixture was stirred under hydrogen atmosphere for 24 hours. After the reaction was completed, the resulting reaction mixture was filtered with celite, extracted with dichloromethane three times, and washed with a saline solution. The combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chro- matography (eluent: dichloromethane: methanol: ammonia water = 100:9:1) to give the target compound as a yellow liquid (260 mg, 62 %).
[141] 1H-NMR (400 MHz, CDCl3) δ 7.72-6.98 (18H, m, Ph), 5.32 (IH, dd, J = 9.1 and 4.6 Hz, COCH2CH), 4.50 (2H, d, J= 5.8 Hz, PhCH2 -), 3.50-3.20 (2H, m, CH3N-CH2 ), 2.71-2.33 (4H, CHj-NandCOCH), 2.44 (IH, dd, J = 14.5 and 5.2 Hz, COCH), 2.30-2.29 (2H, m, -NCH2), 2.21 (6H, s, 2 x N-CH3), 1.65-1.35(4H, m, 2 x CH2), 1.25-1.10 (2H, m, CH2).
[142] 13C NMR (400 MHz, CDCl3) δ 170.1, 153.9, 145.3, 143.8, 140.4, 138.4, 136.7, 128.8, 128.6, 128.1, 128.0, 127.8, 127.4, 127.1, 127.0, 126.2, 125.1, 122.8, 122.4, 122.2, 61.2, 59.7, 49.5, 45.2, 43.8, 41.8, 35.3, 27.1, 27.0, 24.7.
[143] MS (FAB+), m/z (relative intensity, %) 596.7([M+Na]+, 100), 574.7([M+H]+, 30); MS (FAB-), m/z (relative intensity, %) 572.7([M-H]+, 100);
[144] HRMS (FAB+) calcd for C37H44N5O: [M+H]+= 574.3546, found = 574.3516.
[145]
[146] Example 19: Preparation of 2-[ N -(5-aminopentyl)- N - methylamino]-3-(4-biphenylyl)-4-inethoxycarbonylinethyl-3,4-dihyrdoquinazoline (24) (KYS05096)
[147] To a solution of 3-(4-biphenylyl)-2-[N-(5-dibenzylaminopentyl)-N - methylamino]-4-methoxycarbonylmethyl-3,4-dihyrdoquinazoline (19) (362 mg, 0.556 mmol) in methanol (20 ml) was added 10% Pd(C) (883 mg), and the reaction mixture was stirred under hydrogen atmosphere for 24 hours. After the reaction was completed, the resulting reaction mixture was filtered with celite, extracted with dichloromethane three times and washed with a saline solution. The combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane: methanol: ammonia water = 100:9:1) to give the target compound as a yellow liquid (110 mg, 42 %).
[148] 1H-NMR (400 MHz, CDCl3) δ 7.52-6.88 (m, 13H, aromatic), 5.13-5.09 (dd, J = 4.5 and 10.4 Hz, IH, -CH2-CH-N-), 3.75 (s, 3H, -OCH2), 3.15 (br , IH, -CH2-CH-N-), 2.88-2.82 (m, 4H, -3HCN-CH2-, -CH2-NH2), 2.54-2.49 (dd, J = 3.1 and 15.5 Hz, IH, - CH2-CH-N-), 2.25-2.23 (m, 3H, -NCFL3 ), 1.52-1.46 (m, 4H, -CH2-CH2-CH2 CH2-CH 2-NH2), 1.26-1.23 (m, 2H, -(CH2VCH2 -(CH2)2-NH2).
[149] 13C NMR (400 MHz, CDCl3) δ 171.9, 153.4, 145.4, 144.2, 140.4, 136.8, 128.8,
128.4, 127.9, 127.1, 126.8, 125.4, 124.8, 122.9, 122.6, 122.1, 61.2, 59.6, 51.9, 49.9, 45.3, 39.6, 35.5, 27.3, 24.8.
[150]
[151] Example 20: Preparation of 4-( N -benzylacetamino)-3-(4-biphenylyl)-2-[ N - (5-aminopentyl)- N -methylamino]-3,4-dihyrdoquinazoline (25) (KYS05097) [152] To a solution of N-benzyl-3-(4-biphenylyl)-2-[N-(5-N',N'-dibenzylaminopentyl)-N - methylamino]-3,4-dihyrdoquinazolin-4-ylacetamide (22) (1.01 mmol) in methanol (20 ml) was added 10% Pd(C) (183 mg), and the reaction mixture was stirred under hydrogen atmosphere for 24 hours. After the reaction was completed, the resulting reaction mixture was filtered with celite, extracted with dichloromethane three times and washed with a saline solution. The combined organic phase was dried with anhydrous magnesium sulfate and distilled under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: dichloromethane: methanol: ammonia water = 100:9:1) to give the target compound as a yellow liquid (304 mg, 55 %).
[153] 1H-NMR (400 MHz, CDCl3) δ 7.72-6.98 (m, 18H, aromatic), 5.34-5.30 (dd, J = 4.6 and 9.1 Hz, IH, -CH2-CH-N-), 4.51-4.49 (d, J = 5.8 Hz, 2H, Bn-NH-), 2.72-2.66 (m, 4H, -3HCN-CH2-, -CH2-NH2), 2.47-2.42 (dd, J = 5.1 and 14.5 Hz, IH, -CH2-CH-N-), 2.30-2.28 (m, 3H, -NCH2), 1.47-1.24 (m, 7H, -CH2-CH-N-, -CH2-(CiLl2-CH2-).
[154] 13C NMR (400 MHz, CDCl3) δ 170.1, 153.9, 145.3, 143.8, 140.4, 138.4, 136.7, 128.8, 128.6, 128.1, 128.0, 127.8, 127.4, 127.1, 127.0, 126.2, 125.1, 122.8, 122.4, 122.2, 61.2, 60.0, 49.5, 45.2, 43.8, 41.8, 35.3, 27.1, 24.7.
[155]
[156] The chemical structures and physiochemical properties of the representative compounds of the present invention which were prepared in the above Examples are summarized in Table 1.
[157]
[158] Table 1
[159]
Figure imgf000019_0001
[ 160] Biological Activity Test
[161] T-type calcium channel blocking effects of the compounds of the present invention were tested according to an electrophysiological whole-cell patch-clamp method, which uses mammalian HEK293 cell line selectively expressing α1G among T-type calcium channel encoding genes, wherein the α1G is mainly expressed at nerve cells (derived from human kidney cancer cells), as described in Korean Patent No. 0610731 and Monteil, A., et al., J. Biol. Chem. 2000, 275, 6090-6100. Also, cytotoxicities of the compounds according to the present invention were tested according to a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, growth inhibition effects against cancer cell lines of the compounds according to the present invention were tested according to a sulforhodamine B (SRB) assay, as disclosed in Skehan, P. et al., J. Natl Cancer Inst. 1990, 82, 1107-1112.
[162]
[163] Experiment 1: Methods for culturing HEK293 cells and measuring T-type calcium channel activity using an electrophysiological method
[164] HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (v/v) in 36.5 0C humidified incubator (95% air-5% CO2). The culture solution was replaced with a fresh medium every 3 to 4 days, and the cultured cells were subjected to subculture every week. At this time, the culture solution was treated with G-418 (0.5 mg/ mL) solution so that only HEK293 cells expressing αiG T-type calcium channel can grow. The cells used for T-type calcium channel activity assay were cultured on a cover slip coated with poly-L- lysine (0.5 mg/mL) whenever sub-cultured, and their calcium channel activity was recorded 2 to 7 days after the cultivation. Current of the T-type calcium channel at a single cell level was measured according to an electrophysiological whole-cell patch-clamp method using EPC-9 amplifier (HEKA, Germany). At this time, a cell exterior solution [NaCl 140 mM, CaCl2 2 mM, and HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) 10 mM (pH 7.4)] and a cell interior solution [KCl 130 mM, HEPES 10 mM, EGTA (ethylene glycol tetraacetic acid) 11 mM, and MgATP 5 mM (pH 7.4)] were employed. Inward current caused by the T-type calcium channel activation which occurred when the cells were converted into a whole-cell recording mode by stabbing a microglass electrode having 3-4 MΩ resistance which was filled with the cell interior solution into a single cell and depolarized at -30 mV (50 ms duration period) every 10 s with fixing membrane potential to -100 mV was measured according to a T-type calcium channel protocol activated at low current.
[165]
[166] Experiment 2: Analysis for cytotoxicities of T-type calcium channel blockers using MTT assay
[167] In order to analyze cytotoxicities of the compounds according to the present invention in HEK293 cells, MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was conducted as follows. The cultured HEK293 cells were treated with each compound at a concentration of 10 and 100 μM, respectively. At this time, the cells treated with a solvent, that is, 0.1% DMSO, were used as a negative control and the cells treated with H2O2 (125 μM) inducing cytotoxicity were used as a positive control. Six hours after the drug treatment, the cells were treated with 50 μL MTT (1 mg/mL) dissolved in PBS (phosphate buffered saline) for 4 h. Then, the reaction mixture was centrifuged to remove a supernatant, and formazan crystals thus obtained were dissolved in 100 μL DMSO. The solution's absorbance was measured at 560 nm with an automated spectro- photometric plate reader. As a result, the compounds of the present invention did not show any cytotoxicity at a concentration of 100 μM.
[168]
[169] Experiment 3: Analysis for growth inhibition against cancer cell lines using SRB assay
[170] The compounds according to the present invention were evaluated for their growth inhibition against five cancer cell lines, including human lung carcinoma (A-549), human prostate cancer (DU 145), human colon cancer (HT-29), human malignant melanoma (SK-MEL-2) and human ovarian cancer (SK-OV-3) using sulforhodamine B (SRB) assay. All cell lines were grown in RPMI 1640 (Gibco BRL) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and maintained at 37 0C in a humidified atmosphere with 5% CO2. The cells were seeded into 96- well plate. Various concentrations of compounds were added to each well in triplicate and then incubated at 37 0C with 5% CO2 for 72 hours such that cells are in the exponential phase of growth at the time of drug addition. After incubation, 100 μL of formalin solution was gently added to the wells. Microplates were left for 30 min at room temperature and washed five times with tap water. One hundred microliters of 0.4% SRB solution was added to each well and left at room temperature for 30 min. SRB was removed and the plates were washed five times with 1% acetic acid before air-drying. Bound SRB was solubilized with 100 μL of 1OmM unbuffered Tris-base solution (Sigma) and plates were left on a plate shaker for at least 10 min. The optical density was measured using a microplate reader (Versamax, Molecular Devices) with a 520 nm wavelength and the growth inhibition concentration was expressed as GI50.
[171]
[172] The T-type calcium channel blocking effects and growth inhibition effects against cancer cell lines of the compounds according to the present invention, which were analyzed in the above Experiments, are summarized in Table 2.
[173]
[174] Table 2
[175] Channel blocking effect Growth inhibition of cancer cell: GI50 (μM)
Compound (IC50: μM)a
T-type (α1G) A-549C DU 145" HT-29e SK-MEL-2f SK-OV-3g
(KYS05042) 0.11 ±0.06 1.93 ±0.13 1.71 ±0.16 1.71 ±0.17 1.73 ±0.28 1.95 ± 0.23
(KYS05043) 0.30 ±0.09 2.90 ±0.38 4.09 ±0.71 1.08 ±0.28 1.80 ±0.48 3.99 ± 1.07
(KYS05046) 0.68 ±0.18 3.83 ±0.55 3.83 ±1.08 2.08 ±0.43 4.31 ±2.56 4.61 ± 0.30
(KYS05047) 0.17 ±0.03 1.87 ±0.05 1.79 ±0.04 1.70 ±0.16 1.59 ±0.27 2.01 ± 0.32
(KYS05048) 0.16 ±0.02 1.83 ±0.22 1.64 ±0.09 1.67 ±0.18 1.48 ±0.31 1.61 ± 0.09
(KYS05055) 0.35 ± 0.07 >100 >100 22.31 ± 1.0031.62 ± 3.05 >100
(KYS05056) 0.38 ±0.15 >100 >100 28.28 ± 1.4243.95 ± 7.68 >100
(KYS05057) 0.63 ±0.04 14.27 ±1.05 6.52 ±1.00 3.15 ±0.66 8.53 ±1.65 15.61 ±1.14
(KYS05065) 1.04 ± 0.25 24.46 ± 5.7917.21 ± 1.3913.94 ± 1.1317.03 ± 1.5626.14 ± 6.08
(KYS05080) 0.26 ±0.01 0.87 ±0.18 2.49 ±0.53 0.61 ±0.12 0.69 ±0.15 2.76 ± 0.76
(KYS05085) 0.57 ±0.05 7.74 ± 2.60 4.26 ±0.56 3.18 ±0.52 2.02 ±0.40 7.18 ±1.18
(KYS05089) 0.23 ±0.03 1.77 ±0.16 1.78 ±0.17 0.52 ±0.24 1.91 ±0.11 1.95 ± 0.04
(KYS05090) 0.041 ±0.001 0.17 ±0.02 0.19 ±0.02 0.04 ± 0.01 0.48 ±0.13 0.66 ±0.13
Doxorubicin NDh 0.16 ±0.01 0.06 ±0.01 0.21 ±0.04 0.11 ±0.01 0.12±0.02
[176] a Value was determined from dose-response curve and obtained from three independent experiments.
[177] b GI50 value was determined from dose-response curve and obtained from three independent experiments.
[178] c Human lung carcinoma (A-549). [179] d Human prostate cancer (DU 145). [180] e Human colon cancer (HT-29). [181] f Human malignant melanoma (SK-MEL-2). [182] g Human ovarian cancer (SK-OV-3). [183] h ND: not determined [184] [185] The biological activities of doxorubicin and KYS05042 disclosed in Korean Patent No.0610731 were evaluated as references, and the results are indicated in Table 2. As shown in Table 2, a compound of the present invention, KYS05090 showed nearly equipotent growth inhibition and exhibited about 5 times stronger activity against human colon cancer cell line, compared to doxorubicin. Moreover, KYS05090 showed about 10 times stronger growth inhibition and exhibited about 40 times stronger activity against human colon cancer cell line, compared to KYS05042.

Claims

Claims
[1] A compound of formula (I) or pharmaceutically acceptable salt thereof:
Figure imgf000024_0001
(I) wherein,
R1 is NR4(CH2)DNR5R6, wherein R4 is hydrogen or Ci-C5 alkyl, n is 4,
5 or 6, R5 and R6 are each independently hydrogen or Ci-C5 alkyl, or taken together with the nitrogen atom to which they are attached form a 4 to 6-membered het- erocycle;
R2 is 4-biphenylyl;
R3 is benzylamino, 4-aminobenzylamino or
4-fluorobenzenesulfoneaminobenzylamino. [2] The compound according to claim 1 or pharmaceutically acceptable salt thereof, wherein R4 is Ci-C5 alkyl, n is 5, and R5 and R6 are identical and hydrogen or Ci -
C5 alkyl, or taken together with the nitrogen atom to which they are attached form pyrrolidine or piperidine. [3] The compound according to claim 1 or pharmaceutically acceptable salt thereof, wherein R4 is Ci-C5 alkyl, n is 5, and R5 and R6 are identical and Ci-C5 alkyl. [4] The compound according to claim 1 or pharmaceutically acceptable salt thereof, wherein R4 is Ci-C5 alkyl, n is 5, and R5 and R6 taken together with the nitrogen atom to which they are attached form pyrrolidine. [5] The compound according to claim 1 or pharmaceutically acceptable salt thereof, wherein the compound is 4-(Λf-benzylacetamino)-3-(4-biphenylyl)-2-[./V -
(5-pyrrolidin-l-ylpentyl)-Λf-methylamino]-3,4-dihyrdoquinazoline.
[6] The compound according to claim 1 or pharmaceutically acceptable salt thereof, wherein the compound is 4-(Λf-benzylacetamino)-3-(4-biphenylyl)-2-[Λf-(5-/V',./V'- dimethylaminopentyl)-./V-methylamino] -3 ,4-dihyrdoquinazoline.
[7] The compound according to claim 1 or pharmaceutically acceptable salt thereof, wherein the compound is 4-(Λf-benzylacetamino)-3-(4-biphenylyl)-2-[./V -
(5 - aminopentyl) -,/V-methylamino] - 3 ,4-dihyrdoquinazoline
[8] A pharmaceutical composition comprising the compound of formula (I) according to any one of claims 1 to 7 or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.
[9] The pharmaceutical composition according to claim 8 for treating cancer.
[10] The pharmaceutical composition according to claim 8 for treating colon cancer.
[11] The pharmaceutical composition according to claim 8 for preventing or treating epilepsy, high blood pressure, ventricular hypertrophy, pain, or angina pectoris.
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CN110003121A (en) * 2019-04-30 2019-07-12 郑州大学 A kind of 3,4- dihydroquinazoline derivatives and preparation method thereof, application

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US8168646B2 (en) 2012-05-01
US20100120803A1 (en) 2010-05-13
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KR100902145B1 (en) 2009-06-10
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CN101679312A (en) 2010-03-24
EP2142518A4 (en) 2010-08-04

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