EP1139972A1 - Dihydropyrimidines et leurs utilisations - Google Patents

Dihydropyrimidines et leurs utilisations

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Publication number
EP1139972A1
EP1139972A1 EP99967609A EP99967609A EP1139972A1 EP 1139972 A1 EP1139972 A1 EP 1139972A1 EP 99967609 A EP99967609 A EP 99967609A EP 99967609 A EP99967609 A EP 99967609A EP 1139972 A1 EP1139972 A1 EP 1139972A1
Authority
EP
European Patent Office
Prior art keywords
compound
amount
therapeutically effective
pharmaceutical composition
effective amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99967609A
Other languages
German (de)
English (en)
Other versions
EP1139972A4 (fr
Inventor
Donghui Cui
Margaret R. Davis
Hanumath P. Kari
Kamlesh P. Vyas
Kanyin Zhang
Benjamin Evans
Michael Dunn
Dhanapalan Nagarathnam
Bharat Lagu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Synaptic Pharmaceutical Corp
Original Assignee
Merck and Co Inc
Synaptic Pharmaceutical Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck and Co Inc, Synaptic Pharmaceutical Corp filed Critical Merck and Co Inc
Publication of EP1139972A1 publication Critical patent/EP1139972A1/fr
Publication of EP1139972A4 publication Critical patent/EP1139972A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/20—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D239/22—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems

Definitions

  • the designation " 1A” is the appellation recently approved by the IUPHAR Nomenclature Committee for the previously designated " lc " cloned subtype as outlined in the 1995 Receptor and Ion Channel Nomenclature Supplement (Watson and Girdlestone, 1995) .
  • the designation ⁇ 1A is used throughout this application and the supporting tables and figures to refer to this receptor subtype.
  • the receptor formerly designated ⁇ 1A was renamed ⁇ 1D
  • Stable cell lines expressing these receptors are described herein; however, these cell lines were deposited with the American
  • Benign Prostatic Hyperplasia also called Benign Prostatic Hypertrophy
  • Benign Prostatic Hypertrophy is a progressive condition which is characterized by a nodular enlargement of prostatic tissue resulting in obstruction of the urethra. This results in increased frequency of urination, nocturia, a poor urine stream and hesitancy or delay in starting the urine flow.
  • Chronic consequences of BPH can include hypertrophy of bladder smooth muscle, a decompensated bladder and an increased incidence of urinary tract infection.
  • the specific biochemical, histological and pharmacological properties of the prostate adenoma leading to the bladder outlet obstruction are not yet known. However, the development of BPH is considered to be an inescapable phenomenon for the aging male population.
  • BPH is observed in approximately 70% of males over the age of 70.
  • the method of choice for treating BPH is surgery (epor, H., Urol . Clinics North Amer. , 17, 651 (1990)). Over 400,000 prostatectomies are performed annually (data from 1986) .
  • a medicinal alternative to surgery is clearly very desirable.
  • the limitations of surgery for treating BPH include the morbidity rate of an operative procedure in elderly men, persistence or recurrence of obstructive and irritative symptoms, as well as the significant cost of surgery.
  • -Adrenergic receptors are specific neuroreceptor proteins located in the peripheral and central nervous systems on tissues and organs throughout the body. These receptors are important switches for controlling many physiological functions and, thus, represent important targets for drug development. In fact, many -adrenergic drugs have been developed over the past 40 years. Examples include clonidine, phenoxybenzamine and prazosin (treatment of hypertension) , naphazoline (nasal decongestant) , and apraclonidine (treating glaucoma) .
  • ⁇ -Adrenergic drugs can be broken down into two distinct classes : agonists (clonidine and naphazoline are agonists) , which mimic the receptor activation properties of the endogenous neurotrans itter norepinephrine, and antagonists (phenoxybenzamine and prazosin are antagonists) , which act to block the effects of norepinephrine. Many of these drugs are effective but also produce unwanted side effects (for example, clonidine produces dry mouth and sedation in addition to its antihypertensive effects) .
  • ⁇ -adrenergic receptors which exist throughout the central and peripheral nervous systems : ⁇ 1A (new nomenclature) , ⁇ 1B , ⁇ 1D (new nomenclature) , ⁇ 2A , ⁇ 2B and ⁇ 2c (Bylund, D.B., FASEB J., 6, 832 (1992)).
  • ⁇ 1A new nomenclature
  • ⁇ 1B new nomenclature
  • ⁇ 1D new nomenclature
  • ⁇ 1A antagonists may be effective agents for the treatment of BPH with decreased side effects. Further studies have indicated that the ⁇ 1A receptor may also be present in other lower urinary tract tissues, such as urethral smooth muscle (Ford et al . Br. J. Pharmacol., 114, 24P, (1995) ) .
  • This invention is directed to dihydropyrimidine compounds which are selective antagonists for cloned human ⁇ 1A receptors. This invention is also related to uses of these compounds for treating benign prostatic hyperplasia, and for the treatment of any disease where antagonism of the ⁇ 1A receptor may be useful .
  • This invention is directed to compounds having the structure:
  • R x is -OCH 3 or OH
  • R 2 is -CH 2 OH, -CH 2 OCH 3 , or -COOH;
  • R ⁇ and R 2 together form a 5-membered lactone ring
  • R 3 is selected from the group consisting of -(CH 2 ) 3 OH,
  • This invention is also directed to a compound having the structure :
  • This invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the compounds described herein and a pharmaceutically acceptable carrier.
  • This invention also provides a method of treating a subject suffering from benign prostatic hyperplasia which comprises administering to the subject an amount of any one of the compounds described herein effective to treat benign prostatic hyperplasia.
  • This invention provides a method of relaxing lower urinary tract tissue which comprises contacting the lower urinary tract tissue with an amount of any one of the compounds described herein effective to relax lower urinary tract tissue .
  • This invention provides a method of inhibiting contraction of prostatic tissue in a subject which comprises administering an amount of any one of the compounds described herein effective to inhibit contraction of prostatic tissue.
  • This invention provides a method of treating a disease which is susceptible to treatment by antagonism of the ⁇ 1A receptor which comprises administering to the subject an amount of any one of the compounds described herein effective to treat the disease .
  • This invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of any one of the compounds described herein in combination with a therapeutically effective amount of finasteride and a pharmaceutically acceptable carrier.
  • This invention provides a process of making a pharmaceutical composition comprising combining a therapeutically effective amount of any one of the compounds described herein and a pharmaceutically acceptable carrier.
  • Figure 1A -IC Figure 1A-1C show the structures of Compounds
  • the present invention is directed to compounds having the following structure:
  • R 1 is -0CH 3 or OH
  • R 2 is -CH 2 0H, -CH 2 OCH 3 , or -C00H;
  • R 2 and R 2 together form a 5-membered lactone ring
  • R 3 is selected from the group consisting of -(CH 2 ) 3 OH,
  • the present invention also provides for the (+) and (-) enantiomers of all of the compounds described herein.
  • the invention further provides for the cis and trans isomers of all of the compounds described herein. It is noted that the terms “cis” and “trans” correspond to relative stereochemistry, as determined, for example by NOE (Nuclear Overhauser Effect) experiments.
  • the compounds of the present invention may be present as enantiomers, diasteriomers, isomers or two or more of the compounds may be present to form a ra emic mixture. Furthermore, the compounds of the present invention are preferably at least 80% pure, more preferably 90% pure, and most preferably 95% pure.
  • R 3 is selected from the group consisting of
  • the present invention also provides a compound having the structure :
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of any of the compounds described herein and a pharmaceutically acceptable carrier.
  • a "therapeutically effective amount” is any amount of a compound which, when administered to a subject suffering from a disease against which the compounds are effective, causes reduction, remission, or regression of the disease.
  • the therapeutically effective amount is an amount from about 0.01 mg per subject per day to about 500 mg per subject per day, preferably from about 0.1 mg per subject per day to about 60 mg per subject per day, more preferably from about 1 mg per subject per day to about 30 mg per subject per day.
  • the "pharmaceutically acceptable carrier” is any physiological carrier known to those of ordinary skill in the art useful in formulating pharmaceutical compositions .
  • the pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution.
  • the pharmaceutically acceptable carrier is a solid and the composition is in the form of a tablet or powder.
  • the pharmaceutical carrier is a gel and the composition is in the form of a suppository or a cream.
  • the compound may be formulated as a part of a pharmaceutically acceptable transdermal patch.
  • a solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet -disintegrating agents; it can also be an encapsulating material.
  • the carrier is a finely divided solid which is in admixture with the finely divided active ingredient.
  • the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient.
  • Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins .
  • Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators .
  • suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g.
  • cellulose derivatives preferably sodium carboxymethyl cellulose solution
  • alcohols including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil)
  • oils e.g. fractionated coconut oil and arachis oil
  • the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.
  • the liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellent.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously.
  • the compounds may be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes and coatings.
  • the compound can be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents, for example, enough saline or glucose to make the solution isotonic, bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • a sterile solution or suspension containing other solutes or suspending agents, for example, enough saline or glucose to make the solution isotonic, bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular compound in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
  • any one of the compounds described herein additionally does not cause a fall in blood pressure at dosages effective to alleviate benign prostatic hyperplasia.
  • This invention provides a method of treating a subject suffering from benign prostatic hyperplasia (BPH) which comprises administering to the subject any one of the compounds described herein effective to treat BPH.
  • BPH benign prostatic hyperplasia
  • the invention further provides that the compound additionally does not cause a fall in blood pressure at dosages effective to alleviate BPH.
  • the compound effects treatment of BPH by relaxing lower urinary tract tissue and in particular where the lower urinary tract tissue is urethral smooth muscle.
  • lower urinary tract tissue is used to include prostatic capsule, prostate urethra, bladderneck, urethral smooth muscle and prostatic smooth muscle.
  • This invention also provides a method of treating a subject suffering from benign prostatic hyperplasia, which comprises administering to the subject one of the compounds described herein in combination with a 5 -alpha reductase inhibitor effective to treat benign prostatic hyperplasia.
  • a 5-alpha reductase inhibitor is finasteride.
  • This invention further provides a method of relaxing lower urinary tract tissue which comprises contacting the lower urinary tract tissue with an amount of one of the compounds described herein effective to relax lower urinary tract tissue.
  • the lower urinary tract tissue is urethral smooth muscle.
  • the compound additionally does not cause a fall in blood pressure when it is effective to relax lower urinary tract tissue.
  • the invention further provides for a method of inhibiting contraction of prostatic tissue, which comprises administering to the subject an amount of any of the compounds described herein effective to inhibit contraction of prostatic tissue.
  • the prostatic tissue is prostatic smooth muscle and the compound additionally does not cause a fall in blood pressure.
  • This invention also provides a method of treating a disease which is susceptible to treatment by antagonism of the ⁇ 1A receptor which comprises administering to the subject one of the compounds describe herein effective to treat the disease .
  • the invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the compounds described herein in combination with a therapeutically effective amount of finasteride and a pharmaceutically acceptable carrier.
  • This invention also provides for a pharmaceutical composition comprising any one of the compounds described herein present in an amount from about O.Olmg to about 500mg and the therapeutically effective amount of the finasteride is about 5mg.
  • the therapeutically effective amount of the compound is an amount from about 0.1 mg to about 60 mg and the therapeutically effective amount of the finasteride is about 5 mg.
  • the therapeutically effective amount of the compound is an amount from about 1 mg to about 30 mg and the therapeutically effective amount of the finasteride is about 5 mg.
  • This invention provides a pharmaceutical composition made by combining a therapeutically effective amount of any of the compounds described herein and a pharmaceutically acceptable carrier.
  • This invention also provides a pharmaceutical composition made by combining a therapeutically effective amount of any of the compounds described herein with a therapeutically acceptable amount of finasteride and a pharmaceutically acceptable carrier.
  • the salts include, but are not limited to, the following acids and bases : Inorganic acids which include hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and boric acid; organic acids which include acetic acid, trifluoroacetic acid, formic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzoic acid, glycolic acid, lactic acid, and mandelic acid; inorganic bases include ammonia and hydrazine; and organic bases which include methylamine, ethylamine, hydroxyethylamine, propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, ethylenediamine, hydroethylamine, morph
  • the present invention includes within its scope prodrugs of the compounds of this inventions.
  • prodrugs will be functional derivatives of the compounds of the invention which are readily convertible in vivo into the required compound.
  • the term "administering" shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient.
  • Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.
  • the present invention further includes metabolites of the compounds of the present invention.
  • Metabolites include active species produced upon introduction of compounds of this invention into the biological milieu.
  • Methyl trimethylsilylethyl oxaloacetate (4.13 g, 16.78 mmole), 3 , 4-difluorobenzaldehyde (1.85 ml, 16.78 mmole), piperidine (66 ml, 0.67 mmole), and acetic acid (201ml, 3.52 mmole) were mixed in 50 ml of benzene at ambient temperature.
  • the reaction mixture was then refluxed for 2.5 hr in a Dean- Stark apparatus.
  • the reaction mixture was cooled, diluted with 100 ml of ethyl acetate, and washed with water and brine.
  • the reaction was stirred for 1 hr more, warmed to room temperature and partitioned between 100 ml of ethyl acetate and 50 ml of IN sodium carbonate solution. The organic phase was washed once more with sodium carbonate and then with brine . The combined organic extracts were dried (sodium sulfate) and concentrated to give a yellow semi -solid.
  • the crude product was flash chromatographed on silica gel (chloroform-acetone elution, 9:1, v/v) to give the title compound as a pale yellow solid.
  • (+) -5-Methoxycarbonyl-4-methyoxymethyl-l, 2,3, 6-tetrahydro-2- oxo - 6 ( S ) - ( 3 , 4 - di fluorophenyl ) - 1 - [ ( 2 - ethoxycarbonylethylamino) carbonyl] pyrimidine (168mg, 0.37mmol) was dissolved in 5mL of methanol. A 2.0M solution of aqueous sodium hydroxide (400mL, O. ⁇ Ommol) was added dropwise. The mixture was stirred at ambient temperature for 4.5 hours, then concentrated in vacuo.
  • (+) -5-Methoxycarbonyl-4 -methoxymethyl-1 ,2,3, 6-tetrahydro-2- oxo - 6 ( S ) - ( 3 , 4 - di f luorophenyl ) - 1 - [ ( 2 - carboxyethylamino) carbonyl] pyrimidine (308mg, 0.72mmol) was dissolved in 5mL of dichloromethane, and then cooled to -78°C in a dry ice/acetone bath while stirring under a nitrogen atmosphere. A 1.0M solution of boron tribromide in dichloromethane (3.25mL, 3.3mmol) was then added.
  • the mixture was stirred at -78°C for 45 minutes, then slowly warmed to ambient temperature over 1 hour. 15mL of sat ' d sodium bicarbonate was added until a neutral pH was achieved. The mixture was stirred at ambient temperature for 10 minutes. Volatile components were removed by evaporation. The resulting aqueous solution was stirred at ambient temperature for 18 hours. Aqueous solution was then acidified with concentrated hydrochloric acid, precipitating a white solid which was filtered. The resulting white solid was purified by preparative reverse phase HPLC using an H 2 0 : CH 3 CN gradient containing 0.1% TFA. The product fractions were evaporated to dryness to give the title compound as a white solid.
  • an oral composition of a compound of this invention 100 mg of one of the compounds described herein is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gel capsule.
  • Adrenergic Receptors Adrenergic Receptors .
  • Binding affinities were measured for selected compounds of the invention at six cloned human x and ⁇ 2 receptor subtypes, as well as at the L-type calcium channel. The protocols for these experiments are given below.
  • ⁇ ld Human Adrenergic Receptor The entire ceding region of ⁇ ld (1719 bp) , including 150 base pairs of 5' untranslated sequence (5 1 UT) and 300 bp of 3' untranslated sequence (3' UT) , was cloned into the BamHI and Clal sites of the polylinker-modified eukaryotic expression vector pCEXV-3, called EXJ.HR.
  • the construct involved the ligation of partial overlapping human lymphocyte genomic and hippocampal cDNA clones 5' sequence were contained on a 1.2 kb Smal- Xhol genomic fragment (the vector-derived BamHI site was used for subcloning instead of the internal insert-derived Smal site) and 3' sequences were contained on an 1.3 kb Xhol-Clal cDNA fragment (the Clal site was from the vector polylinker) .
  • Stable cell lines were obtained by cotransfection with the plasmid ⁇ lA/EXJ (expression vector containing the ⁇ la receptor gene (old nomenclature) ) and the plasmid pGCcos3neo
  • LM(tk-) cells using calcium phosphate technique.
  • the cells were grown, in a controlled environment (37°C, 5% C0 2 ) , as monolayers in Dulbecco ' s modified Eagle's Medium (GIBCO, Grand Island, NY) containing 25 mM glucose and supplemented with 10% bovine calf serum, 100 units/ml penicillin g, and lOO ⁇ g/ml streptomycin sulfate. Stable clones were then selected for resistance to the antibiotic G-418 (lmg/ml) , and membranes were harvested and assayed for their ability to bind [ 3 H] prazosin as described below (see "Radioligand Binding assays”) .
  • the cell line expressing the human ⁇ ld receptor used herein was designated L- ⁇ 1A (old nomenclature) and was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
  • the cell line expressing the human ⁇ ld receptor was accorded ATCC Accession No. CRL 11138, and was deposited on September 25, 1992.
  • ⁇ lb Human Adrenergic Receptor The entire coding region of ⁇ lb (1563 bp) , including 200 base pairs and 5' untranslated sequence (5' UT) and 600 bp of 3 ' untranslated sequence (3' UT) , was cloned into the EcoRI site of pCEXV-3 eukaryotic expression vector. The construct involved ligating the full- length containing EcoRI brainstem cDNA fragment from ⁇ ZapII into the expression vector. Stable cell lines were selected as described above. The cell line used herein was designated L- ⁇ 1B and was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The cell line L- ⁇ 1B was accorded ATCC Accession No. CR 11139, on September 29, 1992.
  • ⁇ la Human Adrenergic Receptor The entire coding region of ⁇ la (1401 bp) , including 400 base pairs of 5' untranslated sequence (5 1 UT) and 200 bp of 3' untranslated sequence (3 ' UT) , was cloned into the Kpnl site of the polylinker-modified pCEXV-3 -derived eukaryotic expression vector, EXJ.RH.
  • the construct involved ligating three partial overlapping fragments: a 5' 0.6 kb Hindi genomic clone, a central 1.8 EcoRI hippocampal cDNA clone, and a 3' 0.6Kb Pstl genomic clone.
  • the hippocampal cDNA fragment overlaps with the 5' and 3 ' genomic clones so t at the Hindi and Pstl sites at the 5' and 3' ends of the cDNA clone, respectively, were utilized for ligation.
  • This full-length clone was cloned into the Kpnl site of the expression vector, using the 5' and 3' Kpnl sites of the fragment, derived from vector (i.e., pBluescript) and 3 ' -untranslated sequences, respectively.
  • Stable cell lines were selected as described above.
  • the stable cell line expressing the human ⁇ la receptor used herein was designated L- ⁇ lc (old nomenclature) and was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
  • the cell line expressing the human ⁇ la receptor was accorded Accession No. CR 11140, on September 25, 1992.
  • Radioligand Binding Assays for ⁇ receptors Transfected cells from culture flasks were scraped into 5 ml of 5 mM Tris-HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. The cell lysates were centrifuged at 1000 rpm for 5 min at 4°C, and the supernatant was centrifuged at 30,000 x g for 20 min at 4°C. The pellet was suspended in 50 mM Tris-HCl, 1 mM MgCl 2 , and 0.1% ascorbic acid at pH 7.5.
  • Binding of the ⁇ antagonist [ 3 H] prazosin (0.5 nM, specific activity 76.2 Ci/mmol) to membrane preparations of LM(tk-) cells was done in a final volume of 0.25 ml and incubated at 37°C for 20 min. Nonspecific binding was determined in the presence of 10 ⁇ M phentolamine . The reaction was stopped by filtration through GF/B filters using a cell harvester. Inhibition experiments, routinely consisting of 7 concentrations of the tested compounds, were analyzed using a non-linear regression curve-fitting computer program to obtain Ki values.
  • ⁇ 2 Human Adrenergic Receptors To determine the potency of cx 1 antagonists at the ⁇ 2 receptors, LM(tk-) cell lines stably transfected with the genes encoding the ⁇ 2a , ⁇ 2b , and ⁇ 2c receptors were used. The cell line expressing the ⁇ 2a receptor is designated L- ⁇ 2A , and was deposited on November
  • the cell line expressing the ⁇ 2b receptor is designated L-NGC- ⁇ 2B , and was deposited on October 25, 1989 under ATCC Accession No.
  • the cell line expressing the ⁇ 2c receptor is designated L- ⁇ 2c , and was deposited on November 6, 1992 under
  • the potency of x antagonists at calcium channels may be determined in competition binding assays of [3H] nitrendipine to membrane fragments of rat cardiac muscle, essentially as described by Glossman and Ferry (Methods in Enzymology 109:513-550, 1985). Briefly, the tissue is minced and homogenized in 50 mM Tris-HCl (pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride. The homogenates are centrifuged at 1000 g for 15 minutes, and the resulting supernatant centrifuged at 45,000 g for 15 minutes. The 45,000 g pellet is suspended in buffer and centrifuged a second time.
  • the compounds described above were assayed using cloned human alpha adrenergic receptors .
  • the preferred compounds were found to be selective ⁇ la antagonists.
  • the binding affinities of several compounds are illustrated in the following table.
  • Binding affinities of selected compounds of the present invention at cloned human ⁇ ld , ⁇ lb , ⁇ la , ⁇ 2a , ⁇ 2b , and ⁇ 2c receptors (h human) .

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Abstract

La présente invention concerne des dihydropyrimidines constituant des antagonistes sélectifs des récepteurs humains α1a. L'invention concerne également des utilisations de ces composés visant à détendre les tissus des voies urinaires inférieures, à traiter l'hyperplasie prostatique bénigne et toute maladie dans laquelle l'antagonisme du récepteur α1a peut être utile. L'invention concerne enfin une composition pharmaceutique renfermant une quantité thérapeutiquement efficace des composés définis ci-dessus ainsi qu'un excipient acceptable d'un point de vue pharmaceutique.
EP99967609A 1998-12-23 1999-12-23 Dihydropyrimidines et leurs utilisations Withdrawn EP1139972A4 (fr)

Applications Claiming Priority (5)

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US221668 1988-07-20
US11361298P 1998-12-23 1998-12-23
US22166898A 1998-12-23 1998-12-23
US113612P 1998-12-23
PCT/US1999/030868 WO2000037026A1 (fr) 1998-12-23 1999-12-23 Dihydropyrimidines et leurs utilisations

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EP1139972A1 true EP1139972A1 (fr) 2001-10-10
EP1139972A4 EP1139972A4 (fr) 2002-03-27

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JP (1) JP2002532525A (fr)
AU (1) AU2386800A (fr)
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US7166603B2 (en) 2003-07-23 2007-01-23 Bristol-Myers Squibb Co. Dihydropyrimidone inhibitors of calcium channel function
WO2005007124A2 (fr) 2003-07-23 2005-01-27 Bristol-Myers Squibb Company Inhibiteurs dihydropyrimidine substituee de la fonction canaux calciques

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1996014846A1 (fr) * 1994-11-16 1996-05-23 Synaptic Pharmaceutical Corporation Dihydropyrimidines et leurs utilisations

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EP1021185A4 (fr) * 1996-05-16 2005-09-07 Synaptic Pharma Corp Dihydropyrimidines et leurs emplois
US6245773B1 (en) * 1996-05-16 2001-06-12 Synaptic Pharmaceutical Corporation 5-(heterocyclic alkyl)-6-aryl-dihydropyrimidines
WO1999007695A2 (fr) * 1997-08-05 1999-02-18 Merck & Co., Inc. ANTAGONISTE DE RECEPTEUR ADRENERGIQUE ALPHA 1a
AU3011299A (en) * 1998-03-23 1999-10-18 Merck & Co., Inc. Combination therapy for the treatment of benign prostatic hyperplasia

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Publication number Priority date Publication date Assignee Title
WO1996014846A1 (fr) * 1994-11-16 1996-05-23 Synaptic Pharmaceutical Corporation Dihydropyrimidines et leurs utilisations

Non-Patent Citations (2)

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Title
"Medicinal Chemistry" 1979, WILEY *
See also references of WO0037026A1 *

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JP2002532525A (ja) 2002-10-02
AU2386800A (en) 2000-07-12
WO2000037026A1 (fr) 2000-06-29
EP1139972A4 (fr) 2002-03-27
CA2355848A1 (fr) 2000-06-29

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