MXPA97009651A - Methods for modulating the nf transcription factor - Google Patents

Abstract

The present invention relates to a method for modulating the transcription factor NF-KB, which comprises administering to a human in need, an effective amount of a compound of the formula (I), wherein R1 and R3 are independently an of hydrogen, a radical -CH3, a radical (a) or a radical (b), wherein Ar is optionally a substituted phenyl radical, R2 is selected from the group consisting of pyrrolidino, hexamethyleneamino, and piperidino radicals; pharmaceutically acceptable solvate

Description

METHODS FOR MODULATING THE TRANSCRIPTION FACTOR NF-KB BACKGROUND OF THE INVENTION The control of all biological processes is the result of a balance between several factors that act positively and negatively, which interact with regulatory elements of DNA and with each other. These protein factors can play a critical role in the control of protein expression and, thus, are essential for both normal processes and pathological processes. Understanding these protein factors and how they modulate gene expression is a key strategy for the development of agents to control the onset and progression of diseases. A number of these important transactive regulatory proteins have been described in the scientific literature and have been shown to play a role in pathological processes. One such factor is NF-lB, which is a member of the Rei family of eukaryotic transcription factors. The Reí family of proteins controls a wide variety of cellular responses. For example, they are key regulatory molecules for inducing responses to gene expression signals, REF: 26248 host defense responses and growth responses. The ability to specifically modulate the binding of NF-kB and other members of the Reí family would be useful for the treatment of a wide variety of disorders, ranging from septic shock, graft vs. blood reactions. host, acute inflammatory disorders, systemic inflammatory responses, acute phase responses, vascular coagulation, ischemic reperfusion injury, atherosclerosis, HIV infections, even cancer. Another important transcription factor is BEF-1, which is a member of the NF-1 family of transcription regulators. BEF-1 was identified for the first time as a transcriptional repressor within the human BK virus enhancer. The binding site for this ubiquitous transcription factor is present in the regulatory regions of a number of human genes. For example, it has been shown that BEF-1 controls the expression of human apolipoprotein E, which is a major component of plasma lipoprotein that functions in the transport and redistribution of lipids (reverse cholesterol transport). ApoE probably also plays an important role in inhibiting the development and / or progress of atherosclerosis. It has been shown that both the concentration and the binding activity of BEF-1 are regulated by intracellular signals, as demonstrated by the effects mediated by the Ela viral oncogene, by cytokines and also by tyrosine phosphorylation. . It has been found that the BEF-1 binding sites can overlap with the NF-kB binding sites (eg, the vascular adhesion molecule-1: VCAM-1). Conversely, BEF-1 binding sites, such as in the ApoE promoter, can bind to ReI proteins. Thus, both BEF-1 and NF-kB can compete to join the same site. In addition, compounds that modulate the activity levels of BEF-1 can be effective not only for modulating genes controlled by BEF-1, but also those controlled by NF-kB. BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods for modulating the transcription factor NF-kB, comprising administering to a human in need thereof, an effective amount of a compound of Formula I: (i) wherein R 1 and R 3 are independently a 0 0 II i hydrogen atom, a radical -CH 3, -C- ("aikio of qa Cg ^ Q -c-Ar, where Ar is an optionally substituted phenyl 2 R is selected from the group consisting of pyrrolidino, hexamethylene imino and piperidino radicals, and pharmaceutically acceptable salts and solvates thereof DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery that a select group of 2-phenyl-3 -aroylbenzothiophenes (benzothiophenes), those of Formula I, are useful for modulating the transcription factor NF-kB. The methods of use provided by the present invention are practiced by administering to a human in need, a dose of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof, which is effective to modulate the NF transcription factor. -kB. As such, the method herein includes both therapeutic and prophylactic administration, as appropriate. Raloxifene, a compound of the present invention which is the hydrochloride salt of a compound of the Formula I, wherein R 1 and R 3 are hydrogen atoms and R 2 is a 1-piperidinyl radical, is a nuclear regulatory molecule. It has been shown that raloxifene binds to the estrogen receptor and was originally thought to be a molecule whose function and pharmacology were those of an antiestrogen, because it blocked the ability of estrogen to activate estrogen-dependent cancers of the uterine and breast tissue. In fact, raloxifene blocks the action of estrogen in some cells; however, in other cell types, raloxifene activates the same genes as estrogen and demonstrates the same pharmacology, for example, osteoporosis, hyperlipidemia. As a result, raloxifene has been considered as an anti-estrogen agent with mixed agonist-antagonist properties. The unique profile that demonstrates raloxifene and that differs from that demonstrated by estrogen, is thought to be due to the activation and / or single suppression of several gene functions by the raloxifene-estrogen receptor complex, in a manner contrary to activation and / or suppression of genes by the estrogen-estrogen receptor complex. Therefore, although raloxifene and estrogen use the same receptor and compete for it, the pharmacological result of gene regulation of the two can not be easily predicted and is unique to each of them. In general, the compound is formulated with common excipients, diluents or vehicles and compressed to form tablets or formulated into 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 prepared in accordance with established procedures, such as those detailed in US Pat. Nos. 4,133,814; 4,418,068 and 4,380,635, which are incorporated herein by reference. In general, the process starts with a benzo [b] thiophene having a 6-hydroxyl radical and a 2- (4-hydroxyphenyl) radical. The starting compound is protected, acylated and deprotected, to form the compounds of Formula I. Examples of the preparation of such compounds are provided in the US Patents described above. Optionally substituted phenyl groups include phenyl and phenyl groups substituted once or twice with alkyl radicals of 1 to 6 carbon atoms, alkoxy of 1 to 4 carbon atoms, hydroxy, nitro, chloro, fluoro or tri (chloro or fluoro) methyl . The compounds used in the methods of the present invention form pharmaceutically acceptable acid and base addition salts with a wide variety of organic and inorganic acids and bases and include the physiologically acceptable salts often used in pharmaceutical chemistry. Such salts are also part of the present invention. Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric acids and the like. Salts derived from organic acids, such as monoaliphatic acids and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic and hydroxyalkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids can also be used. Thus, these pharmaceutically acceptable salts include salts of acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, β- hydroxybutyrate, butyne-1,4-dioate, hexin-1,4-dioate, caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, malate, maleate, hydroxyalate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogen phosphate (acid phosphate), dihydrogen phosphate (diacid phosphate), metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite , bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylene sulfonate, 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 Formula I with an equimolar amount or an excess of the acid. The reactants are usually combined in a mutual solvent, such as diethyl ether or benzene. The salt is usually precipitated from the solution in a period of about one hour to ten days and can be isolated by filtration, or the solvent can be removed by conventional means. Commonly used bases for the formation of salts include ammonium hydroxide and alkali and alkaline earth metal hydroxides, carbonates, as well as primary, secondary and tertiary aliphatic amines, as well as aliphatic diamines. Bases especially useful in the preparation of the addition salts include ammonium hydroxide, potassium carbonate, methylamine, diethylamine, ethylenediamine and cyclohexylamine. The pharmaceutically acceptable salts generally have better solubility characteristics as compared to the compounds from which they are derived and, therefore, are often more likely to be formulated in the form of liquids or emulsions. Pharmaceutical formulations can be prepared by methods known in the art. For example, the compounds can be formulated with common excipients, diluents or vehicles and given the form of tablets, capsules, suspensions, powders and the like. Examples of excipients, diluents and vehicles that are suitable for such formulations include the following: fillers and extenders such as starch, sugars, mannitol and silicic derivatives; binding or binding agents such as carboxymethylcellulose and other cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrating agents such as calcium carbonate and sodium carbonate; agents for delaying dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surfactants such as cetyl alcohol, glycerol monostearate; adsorptive agents such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate and solid polyethylglycols. The compounds can also be formulated in the form of elixirs or solutions for convenient oral administration, or in the form of solutions suitable for parenteral administration, for example by intramuscular, subcutaneous or intravenous route. Additionally, the compounds are suitable to be formulated as sustained release dosage forms and the like. The formulations may also be constituted so as to release the active ingredient only or preferably in a particular part of the intestinal tract, possibly in a period. The coatings, envelopes and protective matrices can be prepared, for example, from polymeric substances or waxes. The particular dose of a compound of the Formula I necessary to modulate the transcription factor NF-kB or any other described herein, and in accordance with the present invention, will depend on the severity of the disorder, the route of administration and related factors that will be decided by the physician that attends the patient. In general, accepted and effective doses will be from about 0.1 to about 1000 mg / day and more typically from about 50 to about 200 mg / day. Such doses will be administered to a subject in need, once to three times a day or more frequently, as necessary to effectively modulate the transcription factor NF-kB, or for any other use described herein. It is generally preferred to administer a compound of Formula I in the form of an acid addition salt, as is customary in the administration of pharmaceutical products that are carriers of a basic group, such as a piperidino ring. It is also advantageous to administer such compound orally. For such purposes oral dosage forms are available. Formulation »» In the following formulations, the term "active ingredient" means a compound of Formula I. Formulation 1: Gelatin capsules. Hard gelatin capsules were prepared using the following: Ingredient Quantity (mg / capsule) Active ingredient 0.1 - 1000 Starch, NF 0 - 650 Starch powder with 0 - 650 flow characteristics Silicone fluid 350 0 - 15 centistokes The ingredients are mixed, passed through a No. 45 U.S. mesh screen. and hard gelatin capsules are filled. Examples of formulations of raloxifene-specific capsules that have been prepared include those shown below: Formulation 2: Raloxifene capsule Ingredient Quantity (mg / capsule) Raloxifene 1 Starch, NF 112 Starch powder with 225.3 flow characteristics Silicone fluid 350 1.7 centistokes Formulation 3: Raloxifene capsules Ingredient Quantity (mg / capsule) Raloxifene 5 Starch, NF 108 Starch powder with 225.3 flow characteristics Silicone fluid 350 1.7 centistokes Formulation 4: Raloxifene capsule Ingredient Quantity (mg / capsule) Raloxifene 10 Starch, NF 103 Starch powder with 225.3 flow characteristics Silicone fluid 350 1.7 centistokes Formulation 5: Raloxifene capsule Ingredient Quantity (mg / capsule) Raloxifene 50 Starch, NF 150 Starch powder with 397 flow characteristics Silicone fluid 350 3.0 centistokes The above specific formulations can be changed within reasonable limits. A tablet formulation is prepared using the following ingredients: Formulation 6: Tablets Ingredient Quantity (mg / tablet) Active ingredient 0.1 1000 Microcrystalline cellulose 0 650 Smoked silicon dioxide 0 650 Stearic acid 0 15 The components are mixed and compressed to form tablets. Alternatively, each tablet is prepared containing 0.1 to 1000 mg of the active ingredient, as follows: Formulation 7: Tablets Ingredient Quantity (mg / tablet) Active ingredient 0.1 - 1000 Starch 45 Microcrystalline cellulose 35 Polyvinylpyrrolidone (in 4 form of 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 they mix well. The polyvinylpyrrolidone solution is mixed with the resulting powders, which are then passed through a No. 14 U.S. mesh screen. The granules thus produced are dehydrated at 50-60 ° C and passed through a No. 18 U.S. mesh screen. Sodium carboxymethylstarch, magnesium stearate and talc, previously passed through a No. 60 US mesh screen, are added to the granules, and this granulate, after being mixed, is compressed in a tabletting machine to produce tablets Suspensions are prepared each containing 0.1 to 1000 mg of the drug for every 5 ml of dose: Formulation 8: Suspensions Ingredient Quantity (mg / 5 ml) Active ingredient 0. 1 - - 1000 mg Carboxymethylcellulose sodium 50 mg Syrup 1 .25 mg Benzoic acid solution 0. 10 mi Flavoring c. b. p. Coloring c. b. p. Purified water up to 5 mi The medication is passed through a No. 45 mesh U.S. and it is mixed with the sodium carboxymethyl cellulose and the syrup to form a light paste. Then the benzoic acid solution, the flavor and the dye are added, diluted in a little water, with stirring. Afterwards, enough water is added until the required volume is obtained. Treatment of cells with Compound A * Human HepG2 cells were cultured in a 3: 1 v / v mixture of Dulbecco's modified Eagle medium and Ham F12 nutrient mixture, supplemented with 10 nM selenium, 50 μM 2-aminoethanol, HEPES 20 nM and gentamicin 50 μg / ml and 2.5% fetal bovine serum. When the cells reached confluence, the monolayers were rinsed once with HBSS and fed with the above medium without bovine fetal serum (FBS), but with 100 μg / ml of fatty acid-free BSA, 0.8 μg / ml of oleic acid and with or without the addition of Compound A 10 nM. After incubation for 24 hours, the cells were processed to obtain nuclear extracts and the nuclear factor assays were carried out in the manner described below, by the method of Reifel-Miller et al. (Reifel-Miller, A.E., Berg, D.T. and Grinnel, B.G. (1991) The Journal of Biological Chemistry 266, 13873-13882). Union of the Nuclear Factor by Gel Mobility Delay Test. Nuclear extracts were prepared in the manner described by Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. (1983) Nucleic Acids Res, 11, 1475-1489. For the Gel Mobility Delay Test, the nuclear factor binding site for NF-kB was from VCAM-1 5'-CCTTGAAGGGATTTCCCTCCGCCT-3 'and for BEF-1 it was the prototype sequence 5'-AGTGCATGACTGGGCAGCCAGCCAGTGGCAG-3' . The oligonucleotides were labeled at their 5 'ends with T4 polynucleotide kinase (Bethesda Research Laboratories, Inc.) and (g-32P) ATP (Du Pont-New England Nuclear). Nuclear extracts either containing BEF-1 or purified human NF-kB proteins (p49 and p50), were incubated with 0.2 to 0.3 pmol of the probe (approximately 10,000 cpm) at 25 ° C for 30 minutes. The 10 μl of the reaction mixture also contained 200 ng of poly (di-dC) -poly (d? -dC) (Pharmacia), 15% glycerol, 20 mM HEPES (pH 7.9), 100 mM KCl, MgCl2. mM, 0.2 mM EDTA and 0.5 mM dithiothreitol. After 30 minutes of incubation, the samples were subjected to electrophoresis in a 4% polyacrylamide gel of low ionic strength, in the manner described by Fried, M. and Crothers, D.M. (1981) Nucleic Acids Res. 9, 6505-6525. After drying the gel, the specific protein-DNA complexes were visualized by autoradiography and quantified using a Betascope 603 Blot Analyzer or a phosphoimager (Molecular Dynamics). The results of the quantification using the Betascope 603 Blot Analyzer were expressed as the amount of labeled probe bound / amount of free probe, per microgram of protein. The phosphoimager data were analyzed with TP Label gel software (version 1.5, Signal Analytics Corp.) and expressed as pixel intensity units (PU). Results Using the binding assays of the transcription factor, the ability of BEF-1 of the present, in cell extracts, to bind to the VCAM NF-kB binding site was evaluated and, conversely, the binding of the purified proteins NF-kB p49 and p50 to BEF-1 binding site. As shown in Table 1, using two levels of nuclear extract containing BEF-1, a concentration-dependent binding of BEF-1 to the binding site VCAM-1 NF-kB was obtained. As shown in Table 2, the purified NF-kB proteins (p50 and p49: Promega) bound to the BEF-1 prototype binding site in a concentration-dependent manner. As expected, these two NF-kB proteins also bound to the VCAM NF-kB binding site. The effect of Compound A on the concentration of intracellular BEF-1 was determined by measuring BEF-1 binding activity in nuclear extracts prepared from human HepG2 cells, either treated with Compound A, or untreated. As shown in Table 3, in four separate experiments, treatment of the cells with Compound A 10 nM produced an increase of 193 to 293% (average of 253 +/- 42) in the concentration of BEF-1. Thus, Compound A increases the levels or concentration of the intracellular repressor BEF-1 and, in this way, can modulate the activity of the genes controlled by BEF-1. In addition, the ability of BEF-1 to interact at NF-kB binding sites indicates that Compound A could be effective in blocking the action of the important cellular regulatory molecule NF-kB.

Table 1. BEF-1 binding to an NF-kB Binding Site Nuclear extract BEF-1 complex binding site / BEF-1 containing binding site (PIU) None VCAM1 (NF-kB) 0 1.4 μg VCAM1 ( NF-kB) 8.63 2. 8 μg VCAM1 (NF-kB) 15.03 Table 2. Union of Purified NF-kB to BEF-1 and NF-kB Binding Sites. Proteins NF-kB Complex binding site NF-kB / binding sites (PIU) None BEF-1 0.88 p50 56 pg BEF-1 4.46 p50 112 pg BEF-1 11.16 p49 74 pg BEF-1 42.66 p49 148 pg BEF- 1 77.73 None VCAM1 (NF-kB) -0.04 p50 112 pg VCAM1 (NF- • kB) 46.19 p49 148 pg VCAM1 (NF-kB) 17.02 Table 3. Effect of Compound A on the Intracellular Level or Concentration of BEF-1.

Experiment Treatment with Percent Activity of No. Compound A binding (bound / change with free per μg) treatment 1 - 0.12 + 0.33 292 2 - 0.14 + 0.27 195 3 - 0.12 + 0.33 275 4 - 0.12 + 0.30 250 * Compound A is a compound of Formula I wherein R1 and RJ are hydroxy radicals and R1 is a pyrrolidino radical.

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 to which it relates. Having described the invention as an antecedent, what is contained in the following is claimed as property.

Claims (3)

CLAIMS 1. Use of a compound of formula (I) for the manufacture of a medicament for modulating the transcription factor NF-kB comprises administering to a human being in need thereof, an effective amount of said compound:
1. (I) where R and R are independently a hydrogen atom, a radical -CH3, Q
2. Or I -C-Ar, wherein Ar is optionally a substituted phenyl radical; 2 R is selected from the group consisting of pyrrolidino, hexamethyleneimino and piperidino radicals; or a pharmaceutically acceptable salt or solvate thereof. 2. The use according to claim 1, characterized in that the compound is the hydrochloride salt thereof.
3. 3. The use according to claim 1, characterized in that the compound is as follows: or the hydrochloride salt thereof.