MXPA98009706A - Methods to increase the synthesis of oxide nitr - Google Patents

Abstract

The present invention provides methods for increasing the concentration of nitric oxide (NO) in vascular endothelial cells, which comprises administering a compound of formula (I), wherein: R is hydrogen, -OH, -O (C1-C4 alkyl) , -OCO (C 1 -C 4 alkyl), or -OCOAr, where Ar is an optionally substituted phenyl or phenyl, R 1 is R, -Cl or -F, R 2 is 1-pyrrolidinyl, 1-piperidinyl or 1-hexamethyleneimino; a salt or solvate thereof pharmaceutically acceptable

Description

METHODS TO INCREASE THE SYNTHESIS OF NITRIC OXIDE FIELD OF THE INVENTION The present invention relates to the fields of organic chemistry and pharmacology and provides compounds and methods for increasing the synthesis of endothelial nitric oxide (NO), particularly in vascular endothelial cells.

BACKGROUND OF THE INVENTION Nitric oxide is a regulatory molecule that plays a vital role in the normal physiology of the cardiovascular, intestinal, central nervous and immune systems. Nitric oxide is synthesized from L-arginine by a family of enzymes known as NO synthase (NOS). Particularly related to the present invention is calcium-dependent NOS produced in vascular endothelial cells, known as eNOS. Recently, there have been numerous studies that have linked the regulation of the eNOS gene with the hormone estrogen. It has been shown that estrogen over-regulates the production of eNOS m-RNA and by REF: 28604 consequently, the synthesis of eNOS in endothelial cells. This increase in the amount of eNOS allows endothelial cells to produce more NO in response to the appropriate stimulus in the vascular system. In the vasculature, the NO endothelial derivative has several actions, among which are the inhibition of platelet aggregation, the adhesion of inflammatory cells and the proliferation of smooth muscle cells. The endothelial derived NO is an important regulator of vascular tone. . Flow-dependent dilation, a commonly used index of endothelial functions, is largely mediated by NO. The mechanism for regulating vascular tone by NO is initiated by stimuli, such as acetylcholine, bradykinin, shear stress, etc., on the endothelial lining cells. Endothelial cells respond by producing NO from L-arginine by eNOS. The NO produced leaves the endothelial cells and stimulates the activity of the guanylate cyclase in the adjacent smooth muscle cells. The activation of guanylate cyclase increases the level of cGMP and causes the smooth cells to relax, thus dilating the vessel and increasing blood flow. For other information, see: Moneada et a l. , New In g. J. Med. , 329, pp. 2002-2012 (1993), and Vallance, et al. L. , J. Royl. Coi l. Phys i ci a n London, 28, pp. 209-219 (1994). The generation of reduced endothelial NO can lead to altered vasodilation, abnormal vasospasm, increased platelet aggregation, and increased adhesion and infiltration of inflammatory cells. Altered endothelial NO and endothelial function are associated with the risk factors for coronary artery disease, including smoking, hypercholesterolemia, homocysteineuria, and diabetes. The alteration of the activities modulated by NO in the coronary arteries can contribute to worsen the coronary syndrome that gives rise to myocardial infarction. The alteration of the endothelial NO system and its resultant vasoconstriction have been implicated in the exacerbation of damage to neurons in cerebral ischemic events, such as stroke. Additionally, recent studies indicate that endothelial NO mediates vascular sensitivity to insulin, thus increased NO production may be useful in treating vascular effects of diabetes. In general, there is a large body of evidence, both experimental and clinical, which indicates that increasing NO concentrations in the vasculature is beneficial in many pathological conditions, such as diabetes, stroke, erosions and hypertension. The current therapy for increasing NO concentrations in the vasculature has been either to administer high doses of L-arginine (the eNOS substrate) or compounds such as nitroglycerin or sodium nitroprusside, which metabolically release NO. Although, these therapies can be effective, each one has shown undesirable side effects. Additionally, therapies suffer from their inability to maintain sustained release, from NO, due to their rapid elimination from the body. It would be of great benefit if a new therapy were available to increase NO concentrations in the vasculature.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods for increasing the endothelial derivative of NO which comprises administering to a human being in need thereof an effective amount of a compound of the formula I e-n wherein: R is hydrogen, -OH, -O (C1-C4 alkyl), -OCO (C-alkyl) -C), or -OCOAr, where Ar is a phenyl or optionally substituted phenyl; Ri is R, -Cl or -F; R 2 is 1-pyrrolidinyl, 1-piperidinyl or 1-h'exametiminimino; or a pharmaceutically acceptable salt or solvate thereof.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery that a select group of 2-aryl-benzo [b] thiophenes, the compounds of formula I, are useful for increasing the concentration of eNOS and NO of the endothelial derivative. The general terms used in the description of the compounds described herein carry their usual meanings. For example, "C?-C 4 alkyl" refers to straight or branched α-linear chains of 1 to 4 carbon atoms including methyl, ethyl, propyl, isopropyl, n-butyl and the like. The term "substituted phenyl" refers to a phenyl group which has one or two substituents selected from the group consisting of C-C4 alkyl, C? -C4 alkoxy, hydroxy, nitro, chloro, fluoro or tri (chloro) or fluoro) met ilo. "0 (C 1 -C 4 alkyl)" refers to a C 1 -C 4 alkyl group attached via an oxygen bridge such as methoxy, ethoxy, n-propoxy, iso-propoxy and the like. The term "pharmaceutically acceptable salt" refers to any acid or base addition salts that are known to be non-toxic and are commonly used in the pharmaceutical literature. Commonly used acid addition salts include: inorganic salts formed by the addition of sulfuric acid, nitric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, phosphorous acid and the like; or organic salts formed by the addition of acetic acid, formic acid, benzoic acid, citric acid, methanesulfonic acid and the like. Commonly used basic addition salts would include salts formed by alkali or alkaline earth metal hydroxides, ammonium hydroxide, alkyl or aromatic amines and the like. A preferred salt of this invention would be the hydrochloride salt. The phrase "inhibit a physiological condition associated with a lack of or need for nitric oxide (NO)" includes prohibiting, preventing, restricting, and decreasing, stopping, or reversing progression, severity, or a symptom or effect resulting from the physiological condition. . Such physiological conditions include. those mentioned in this application, such as the aggregation of pathological platelets, pathological vasoconstriction, vascular effects of diabetes, stroke, atherosclerosis and abnormal vasospasm.

The term "solvate" represents an aggregate comprising one or more molecules of a solute, such as a compound of formula I, with one or more molecules of a suitable solvent. Raloxifene hydrochloride, which is a preferred embodiment of this invention, is a compound of formula I, wherein R and Ri each are hydroxyl; R2 is 1-piperidinyl; and it's like its hydrochloride salt. Raloxifene is named: [2- (4-hydroxyphenyl) -6-hydroxybenzo [b] thien-3-yl] [4- [2- (1-piperidenyl) ethoxy] phenyl] methanone hydrochloride. Generally, at least one compound of formula I is formulated with common excipients, diluents or carriers and compressed into tablets, or formulated as elixirs or solutions for convenient oral administration, or administered intramuscularly or intravenously. The compounds can be administered transdermally, and can be formulated as sustained release dosage forms and the like. The compounds used in the methods of the present invention can be made in accordance with established procedures, such as those detailed in U.S. Patent Nos. 4., 133,814, 4,418,068, 4,380,635 and 5,393,763, all of which are incorporated by reference herein. In general, the process starts with a benzo [b] thiophene having a 6-hydroxyl group and a 2- (4-hydroxyphenyl) group. The initial compound is protected, acylated and deprotected to form the compounds of formula I. Examples of the preparation of such compounds are provided in the U.S. Patents discussed above. The compounds used in the methods of this invention form pharmaceutically acceptable acid and base addition salts with a wide variety of organic and inorganic acids and include the physiologically acceptable salts that are frequently used in pharmaceutical chemistry. Such salts are also part of this invention. Typical inorganic acids used to form salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric acids and the like. Salts derived from organic acids, such as mono- and dicarboxylic aliphatic acids, f.enyl-substituted alkanoic acids, hydroxyalkanoic and hydroxyalkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids can also be used. Such pharmaceutically acceptable salts of this form include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methybenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, ß-hydroxybutyl, but in-1, 4-dioate, hexin-1,4-dioate, caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, malate, maleate, hydroxyaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, fatalate, teraphthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite , bisulfite, sulfonate, benzene sulfonate, p-bromophenylsulphonate, chlorobenzenesulfonate, ethanesulphonate, 2-hydroxyethane sulfonate, methanesulphonate, naphthalene-1-sulfonate, naphthalene-2-sulfonat or, p-toluenesulfonate, xylene sulphonate, tartrate and the like. A preferred salt is the hydrochloride salt. The pharmaceutically acceptable acid addition salts are typically formed by reacting a compound of the formula I with an equimolar or excess amount of acid. The reagents are generally combined in a m.utuous solvent such as diethyl ether or benzene. The salt is usually precipitated from the solution within about one hour to 10 days and can be isolated by filtration or the solvent can be washed by conventional means. As used herein, the term "effective amount" means an amount of the compound of formula I that is capable of increasing the NO concentration of the endothelial derivative in a human being in need of such treatment. Humans in need of treatment would include, but are not limited to, those suffering from inappropriate v-asoconst ricción or platelet aggregation due to damage of the endothelial NO regulatory pathway by the risk factors of coronary heart disease, diabetes , and similar. As prevented in this invention, a compound of formula I would be useful in inhibiting, improving, reducing, limiting or preventing pathological sequelae due to damage of the endothelial NO regulatory pathway. The specific dose of a compound administered according to this invention will, of course, be determined by the surrounding circumstances particular to the case including, for example, the compound administered, the route of administration, the state in which the patient is, and the pathological condition that is treated. A typical daily dose will contain a non-toxic dose level from about 0.1 mg to about 1000 mg / day of a compound of the present invention, and more particularly will be from about 15 mg to about 80 mg '/ day from one to three times each day or as often as necessary for its effectiveness. In addition, the compounds of the formula I can be used at the same time or sequentially with other agents that interact with the endothelial NO pathway, for example, nitroglycerin, sodium nitroprusside, L-arginine and the like. By "pharmaceutically acceptable formulation" it is meant that the carrier, diluent, excipients and salt must be compatible with the active ingredient (a compound of formula I) of the formulation, and not be deleterious to the container of the same. The pharmaceutical formulations can be prepared by methods known in the art.

For example, the compounds of this invention can be formulated with common excipients, diluents or carriers and formed into tablets, capsules and the like. Examples of excipients, diluents and carriers that are suitable for such formulations include the following: relievers and expanders such as starch, sugars, mannitol and silicic derivatives; binding agents such as carboxymethylcellulose and other cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrating agents such as agar agar, calcium carbonate and sodium bicarbonate; agents for delayed dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; active surface agents such as alcohol. cetyl, glyceryl monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate and solid polyethylene glycols. The final pharmaceutical forms can be: pills, tablets, powders, pills, syrups, aerosols, sachets, seals, elixirs, suspensions, emulsions, ointments, suppositories, sterile injectable solutions, or sterile packaged powders, depending on the type of excipient used. Additionally, the compounds of this invention are very suitable for formulation as sustained release dosage forms. The formulations can also be constituted in such a way that they release only the active ingredient or preferably in a particular section of the intestinal tract, possibly for a period of time. Such formulations would involve coatings, envelopes or protective matrices that can be made from polymeric substances or waxes. The following formulations are given for purposes of illustration and are not intended to be limiting in any way. The total active ingredients in such formulations comprise from 0.1% to 99.9% by weight of the formulation. The term "active ingredient" means a compound of the formula I or a salt or solvate thereof.

Formulations Formulation 1: Gelatin Capsules Hard gelatin capsules are prepared using the following: Ingredient Quantity (mg / capsule) Active ingredient 0. 1 - - 1 0 0 0 Starch, NF 0 - - 6 5 0 Flowable starch powder 0 - - 6 5 0 Silicone fluid 350 centistokes 0 - 1 5 The ingredients are mixed, passed through a 45 U.S. mesh screen. and they are filled into hard gelatin capsules. The above formulations can be changed in accordance with the reasonable variations provided. A tablet formulation is prepared by using the following ingredients: Formulation 2: Tablets Ingredient Quantity (mg / tablet Active ingredient 2.5 1000 Microcrystalline cellulose 200 650 Gaseous silicon dioxide 10 650 Acid stearate 5 15 The components are mixed and compressed to form tablets. Alternatively, each of the tablets containing 2.5-1000 mg of active ingredient is manufactured as follows: Formulation 3: Tablets Ingredient "Amount (mg / tablet Active ingredient 25 - 1000 Starch 45 Microcrystalline cellulose 35 Polyvinylpyrrolidone 4 (as 10% solution in water) Sodium carboxymethylcellulose 4.5 Magnesium stearate 0.5 Talcum 1 The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. and mixes completely. The polyvinylpyrrolidone solution is mixed with the resultant powders which are then passed through a No. 14 U.S. mesh screen. The granules thus produced are dried at 50 ° -60 ° C and passed through a No. 18 U.S. mesh screen. Sodium carboxymethyl starch, magnesium stearate and talc are passed through a No. 60 U.S. mesh screen, then added to the granules which, after mixing, are compressed in a compressor machine to produce tablets. Each of the suspensions containing 0.1 - 1000 mg of medication per 5 ml of dose is made as follows: Formulation 4: Suspensions Ingredient Quantity (mg / 5 ml) Active ingredient 0.1 - 1000 mg Sodium carboxymethylcellulose 50 mg Syrup 1.25 mg Benzoic acid solution 0.10 ml Flavor c. v. C color . V Purified water for 5 ml The medication is passed through a No. 45 mesh U.S. and it is mixed with sodium carboxymethyl cellulose and syrup to form a soft paste. The benzoic acid solution, flavor and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume. An aerosol solution is prepared containing the following ingredients: Formulation 5: Aerosol Ingredient Amount (% by weight) Active ingredient 0.25 Ethanol 25.75 Propellant 22 (Clorodi f luoromethane) 70.00 The active ingredient is mixed with ethanol and the mixture is added to a portion of the propellant 22, cooled to 30 ° C, and transferred to a filling device. The required amount is then fed * to a stainless steel vessel and diluted with the remaining propellant. The valve units are adjusted to the container.

Suppositories are prepared as follows Formulation 6: Suppositories Ingredient Quantity (mg / suppository) Active ingredient 250 Saturated fatty acid glycerides 2,000 The active ingredient is passed through a No. 60 mesh U.S. and is suspended in the saturated fatty acid glycerides previously fused using the minimum necessary heat. The mixture is then emptied into a suppository mold of 2 nd.minal capacity and allowed to cool. An intravenous formulation is prepared as follows: Formulation 7: Intravenous solution Ingredient Amount Active ingredient 50 mg Isotonic saline solution 1 000 ml The solution of the above ingredients is administered intravenously to a patient at a rate of approximately 1 ml per minute.

TEST PROCEDURE As evidence of the utility of the compounds of the present invention for raising the concentration of NO in the endothelial tissue, the following test system is used.

Cell Cultures: Single-donor, cryopreserved human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics Corporation, San Diego CA. These cells are defined by Clonetics to be the 1st (first-pass cells). The cells are stored in liquid nitrogen and fresh aliquots are taken for each experiment. The cells are thawed and placed in a T-75 flask with 75 ml of medium. For all HUVEC cultures, all glassware (Corning) is coated with 0.2% gelatin (Sigma Chemical Co.) in M199 medium (Gibco) for twenty minutes at 37 ° C. Cells were cultured in M199 free phenol red, with 10% fetal bovine serum (Gibco), 50 ug / ml endothelial cell culture supplement (Collaborative Biochemical Products, Bedford MA), 100 ug / ml heparin pig (Gibco, 10 units / ml of penicillin, 10 ug / ml of streptomycin, and 0.2 mM of L-glutamine at 37 ° C and 5% of C02.) When the cells of the 2nd crop reach 70-90 % confluence, divided 1: 3 over T-75 flasks coated with gelatin with 15 ml of medium to produce 3rd crop cells, after which these cultures reached 70-90% confluence, typically 3-4 days, these 4th crop cells were divided into 1: 3 on 12-well plates coated in 1 ml of medium.All experiments were conducted with these cells from the fourth step.After 72 hours, the cells were confluent and the treatments were initiated with drug The above medium is removed by aspiration and 1 ml of medium is added to test the drug with the drug (15% gelatinized horse serum (GemJni Bioproducts, Calabasas CA, 10 units / ml penicillin, 10 ug / ml streptomycin, and 0.2 mM L-glutamine). Stock solutions of the test compounds of formula I or 17-β-est radiol were prepared in 10 mM in DMSO. The cells were treated with the drug for 48 hours at 37 ° C.

Induction of cGMP Nitric Oxide Dependent Three plates (one experimental) are removed from the incubator and placed on a paper towel to prevent cooling. One plate at a time, the medium is removed, and 1 ml of warm HBSS is added (Gibco) This is removed and replaced with 0.5 ml of balance buffer +/- 200 uL of L-NAME (N-nitro-L-arginine methyl ester, Sigma). The equilibrium buffer consists of HBSS, 10 mM HEPES, 1.2 mM CaCl2, 0.6 mM MgSO4 and 0.5 mM isobutyl ilmet il-xanthine (IBMX) and 10 uM L-arginine, which is added freshly prepared to each stock solution. I.BMX is prepared as a 200 mM stock solution in DMSO at 37 ° C. The cells are allowed to equilibrate for 30 minutes at 37 ° C in 5% C02. After incubation, 0.5 ml of "stimulation" buffer with stimulant is added for 10 minutes. The stimulation buffer for the controls consists of the equilibrium buffer plus a designated stimulant, which are: 1) negative control, 2) positive control, sodium nitroprusside 1 mM (Sigma), 3) 1 uM of A-23187 ( calcium ionophore) (Sigma), 4) 1 uM of A-23187 and 200 uM of L-NAME (This control group demonstrates that the observed effects (increased in cGMP) in this assay are due solely to the NO 'produced in the endothelial cells.) Test cells treated with the compounds of formula I are stimulated with 1 uM of A-23187. After 10 minutes, the buffers are removed and 200 ul of 0.01 N HCl are added, and the cGMP is extracted by rocking the cells for 30 minutes at 4 ° C. Each aliquot of 200 ul is placed in a tube containing 2 ul of 1 N NaOH. Samples are frozen at -20 ° C for storage. To each well of the plate, 250 ul of 0.5% SDS in 0.1 N NaOH are added to solubilize the bound cells. The plates were wrapped in plastic wrap and frozen at -20 ° C for a subsequent protein assay by the BCA method (Pierce Chemical Co.). (The total average amount of protein in mg is determined for all wells in an experiment and used to normalize the cGMP content.) The cGMP content is determined by enzyme immunoassay (Amersham Co, RPN.226) according to the manufacturer's instructions for the acetylation protocol. Assays are made according to the instructions, except that the acetylation of the 200 ul samples was with 20 ul of reagent instead of 100 ul for 1 ml samples. The assays were quantified in a 450 nM thermomax spectrophotometer.

The positive control of 17-ß-est radio! demonstrates the expected increase in the amount of NO-dependent cGMP. It is discovered that the compounds of formula I increase the concentration of NO and induce cGMP in these endothelial cells. In Table 1, the increase in NO-dependent cGMP for raloxifene hydrochloride is shown.

Table 1 Compound Concentration3 Level of cGMPb Control 1.30 Raloxifene 0.1 2.55 * 1..0 2.65 * a pM / mL b pM / mg protein \ P > - 01 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers. Having described the invention as above, property is claimed as contained in the following:

Claims (4)

1. The use of a compound of the formula characterized in that: R is hydrogen, -OH, -O (Cj.-C4 alkyl), -OCO (C? -C4 alkyl), or -OCOAr, where Ar is a phenyl or optionally substituted phenyl; Ri is R, -Cl or -F; R2 is 1-pyrrolidinyl, 1-piperidinyl or 1-hexamethyleneimino; or a pharmaceutically acceptable salt or solvate thereof, used in the preparation of a β-cation to increase the synthesis of nitric oxide (NO) in vascular endothelial cells in a human.
2. 2. The use according to claim 1, characterized in that the compound of formula I is [2- (4-hydroxyphenyl) -6-hydroxybenzo [b] thien-3-yl] [4- [2- (1-hydrochloride. -piperidinyl) ethoxy] phenyl] ethanone.
3. 3. The use of a compound of the formula (I) characterized in that: R is hydrogen, -OH, -O (C? -C4 alkyl), -OCO (d-C4 alkyl), or -OCOAr, where Ar is a phenyl or optionally substituted phenyl; Ri is R, -Cl or -F; R2 is 1-pyrrolidinyl, 1-piperidinyl or 1-hexamethyleneimino; or a pharmaceutically acceptable salt or solvate thereof used in the preparation of a medicament for inhibiting a physiological condition associated with a lack of or need for, nitric oxide (NO).
4. 4. The use according to claim 3, characterized in that the compound of the formula I is [2- (4-hydroxy phenyl) -6-hydroxy-benzo [b] thien-3-yl] [4- [2- (l-piperidinyl) ethoxy] phenyl] methanone.