Indolecarboxamides, pharmaceutical compositions and methods of inhibiting calpain

Pharmaceutical compositions and methods of inhibiting calpain using novel indolecarboxamides are disclosed.

SUMMARY OF THE INVENTION
 This invention relates to novel chemical compounds which are
 indolecarboxamides. The claimed pharmaceutical compositions and methods
 use those compounds as active ingredients to inhibit calpain and thus are
 useful in the treatment of, for example, neurodegenerative disorders,
 strokes and traumatic brain injury.
 BACKGROUND OF THE INVENTION
 Calpains are calcium--dependent cysteine proteases present in a variety of
 tissues and cells. Excessive activation of calpain provides a molecular
 link between ischaemia or injury induced by increases in intraneuronal
 calcium and pathological neuronal degeneration. If the elevated calcium
 levels are left uncontrolled, serious structural damage to neurons may
 result. Recent research has suggested that calpain activation may
 represent a final common pathway in many types of brain damage. Selective
 inhibition of calpain would, therefore, be an attractive therapeutic
 approach in the treatment of neurodegenerative diseases. Exemplary of
 these diseases would be myocardial ischaemia, cerebral ischaemia, muscular
 dystrophy, stroke, Alzheimer's disease, or traumatic brain injury. The
 compounds of this invention may also be useful in the treatment of
 cataracts and platelet aggregation.
 DETAILED DESCRIPTION OF THE INVENTION
 The compounds which are the active ingredients of the pharmaceutical
 compositions and methods of this invention are represented by the
 following formula:
 ##STR1##
 in which:
 R.sub.1 is CH.sub.2 Ph, CH.sub.2 CH(CH.sub.3).sub.2, or CH.sub.2 CH.sub.2
 CH.sub.2 CH.sub.2 NR.sub.6,R.sub.7 ;
 Z is CHO, COCH.sub.2 F, COCOOH, COCONH-alkyl, COCOO-alkyl,
 COCO(CH.sub.2).sub.n -aryl, COCONHCH(R.sub.1)COOH, or COCH.sub.2
 O-(3-phenylisoxazol-5-yl);
 n=1-6;
 R.sub.2 is H, CH.sub.3, CH.sub.2 Ph, CH.sub.2 -pyridine, CH.sub.3 SO.sub.2,
 CF.sub.3 SO.sub.2, or PhSO.sub.2 ;
 R.sub.3 is H, CH.sub.3, or lower alkyl;
 R.sub.4 and R.sub.5 are independently H, halo, lower alkyl, lower alkoxy,
 or benzyloxy;
 R.sub.6 is COOCH.sub.2 Ph, COOCH.sub.2 -pyridine, CO-aryl, SO.sub.2
 CH.sub.3, SO.sub.2 CF.sub.3, SO.sub.2 -aryl, H, or lower alkyl; and
 R.sub.7 is H, or lower alkyl, provided that when Z is CHO and R.sub.3 is
 other than H, then R.sub.2 is not H, or a pharmaceutically acceptable salt
 thereof.
 Preferred compounds are those where the sterochemistry at the R.sub.1 group
 corresponds to that of the naturally occurring amino acids. Also preferred
 are those compounds where R.sub.1 is CH.sub.2 Ph and Z is CHO.
 The following preferred compounds are representative of the compounds of
 the invention:
 (S)-N-(1-formyl-2-phenylethyl)1-methyl-2-indolecarboxamide;
 (S)-N-(1-formyl-2-phenylethyl)-2-indolecaboxamide;
 (S)-N-(1-formyl-2-phenylethyl)-5-methoxy-6-(phenylmethoxy)-2-indolecarboxam
 ide;
 (S)-5-bromo-N-(1-formyl-2-phenylethyl)-2-indolecarboxamide;
 (S)-N-(1-formyl-2-phenylethyl)-1-(methylsulfonyl)-2-indolecarboxamide;
 (S)-N-(1-formyl-2-phenylethyl)-1-phenylmethyl)-2-indolecarboxamide;
 (S)-N-(1-formyl-2-phenylethyl)-6-methoxy-2-indolecarboxamide; and
 (S)-N-(1-formyl-2-phenylethyl)-3-methyl-1-(phenylmethyl)-2-indolecarboxamid
 e.
 Compounds of Formula I are prepared by the methods illustrated in Schemes 1
 and 2.
 ##STR2##
 The indole-2-carboxylic acids 1 (Scheme 1), whether prepared or
 commercially obtained, are converted to the amide alcohols 2 by standard
 coupling conditions (e.g., (S)-(-)-2-amino-3-phenylpropanol,
 1-hydroxybenzotriazole hydrate (HOBT),
 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC),
 N-methylmorpholine (NMM), methylene chloride). Oxidation of 2 (the
 Dess-Martin reagent in methylene chloride is preferred, but not limiting)
 affords the aldehydes 3. This procedure can be repeated with a wide
 variety of substituted indole-2-carboxylic acids and with a wide variety
 of amino alcohol derivatives to obtain compounds with varying R.sub.1
 substituents.
 Compounds of Formula I wherein the indole carboxylic acid is not
 commercially available are prepared by the method described in Scheme 2.
 ##STR3##
 The commercially available indole esters 4 are treated with a base such as
 NaH to form the indole anion. This is then reacted with either an
 alkylating agent such as, but not limited to, benzyl bromide or a
 sulfonylating agent such as, but not limited to, methanesulfonyl chloride
 to provide the N-protected compounds 5. The ester is then hydrolyzed under
 standard conditions using NaOH to provide the desired indole 2-carboxylic
 acids 1. The acids 1 are then converted to the desired final compounds
 according to Scheme 1. For the cases where the indole ester 5 is
 commercially available, these compounds are converted directly to the
 acids 1 as described above.
 Although these methods illustrate the preparation of compounds for which Z
 is CHO, alternative "enzyme reactive groups" can be substituted as has
 been extensively described in the literature (J. Med. Chem., 1994, 37,
 2918-2929; J. Med. Chem., 1993, 36, 3472-3480; J. Med. Chem., 1990, 33,
 11-13; Biochem. J., 1986, 239, 633-640; J. Med. Chem., 1992, 35, 216-220).
 In addition, these methods are not intended to limit the scope of the
 possible R.sub.1 groups which can be readily derived from any amino
 alcohol or amino acid by methods well known in the art.
 Also included in the scope of the present invention are pharmaceutically
 acceptable salts of the compounds of Formula I. Preferred salts include,
 but are not limited to, hydrochloride, hydrobromide, citrate, tartrate,
 malate, maleate, lactate, gluctose 1,6-diphosphate, phosphate, succinate,
 sulfate, aspartate, adipate, methanesulfonate, lauryl sulfate, diguaiacyl
 phosphate, diacetyl sulfate, glutamate, edetate, ethylene diamine, sodium,
 potassium, calcium and ethanolamine salts. Such salts are prepared
 according to standard procedures well known in the art.
 The pharmaceutical activity of the compounds of this invention is
 demonstrated by inhibition of calpain in vitro by the assay procedure
 described by Sasaki et al., J. Biol. Chem. 1984, 259, 12489-12494. The
 assays were performed using synthetic fluorogenic substrates. Inhibition
 of enzyme activity was calculated on the percent decrease in the rate of
 substrate hydrolysis in the presence of inhibitor relative to the rate in
 its absence. IC.sub.50s (nM) were calculated. Table 1 demonstrates the
 results of testing representative compounds of Formula I.
 TABLE 1
 ##STR4##
 R.sub.1 Z R.sub.2 R.sub.3 R.sub.4 R.sub.5 IC.sub.50 (nM)
 Ph CHO CH.sub.3 H H H 500
 Ph CHO H H H H 600
 Ph CHO H H CH.sub.3 O PhCH.sub.2 O 135
 Ph CHO H H Br H 230
 Ph CHO CH.sub.3 SO.sub.2 H H H 600
 Ph CHO PhCH.sub.2 H H H 500
 Ph CHO H H H CH.sub.3 O 600
 Ph CHO PhCH.sub.2 CH.sub.3 H H 30
 The above results clearly indicate that all compounds tested exhibited
 significant inhibition of calpain.
 The pharmaceutical compositions of this invention employed to inhibit
 calpain comprise a pharmaceutical carrier and as the active ingredient a
 compound of Formula I. The active ingredient will be present in the
 compositions of this invention in an effective amount to inhibit calpain.
 Preferably, the compositions contain the active ingredient of Formula I in
 an amount of from about 0.1 mg to about 250 mg, advantageously from about
 25 mg to about 150 mg per dosage unit.
 The pharmaceutical carrier may be, for example, a solid or liquid.
 Exemplary of solid carriers are lactose, magnesium stearate, sucrose,
 talc, stearic acid, gelatin, agar or acacia. Exemplary of liquid carriers
 are syrups, peanut oil, olive oil, propylene glycol, polyethylene glycol
 and water.
 A wide variety of pharmaceutical forms may be employed. Thus, if a solid
 carrier is used, the preparation can be tabletted or placed in a hard
 gelatin capsule. If a liquid carrier is used, the preparation may be in
 the form of a soft gelatin capsule, placed in an ampule, a liquid
 suspension, syrup or suspension.
 Preferably, parenteral solutions or suspensions are employed. They comprise
 the active compound in a sterile aqueous or oil carrier such as, for
 example, peanut oil, polyethylene glycol or polyvinyl pyrolidone.
 Preferably, such solutions contain the active compound in the range of 0.1
 to 140 mg/kg of body weight of the patient to whom it will be
 administered. The sterile parenteral solutions may also contain additives
 such as, for example, preservatives such as benzyl alcohol and buffering
 agents to bring the injectable preparation to a satisfactory pH.
 Stabilizing agents such as ascorbic acid or sodium bisulfate may also be
 employed. DMSO or alcoholic solvents may be used to aid in the solubility
 and penetration of the calpain inhibitor.
 The sterile aqueous solutions can also be lyophilized and reconstituted
 prior to administration.
 The parenteral solution may be administered subcutaneously, intravenously,
 intramuscularly, interperitoneally, intrasternally or by intrathecal
 injection directly into the central nervous system.
 The pharmaceutical compositions are prepared by conventional techniques
 involving procedures such as mixing, granulating and compressing to
 dissolve the ingredients as appropriate to the desired preparation.
 The method of inhibiting calpain according to this invention comprises
 administering to an animal or human in an amount sufficient to inhibit
 calpain a compound of Formula I.
 Preferably the compounds of Formula I are administered in conventional
 dosage unit forms prepared by combining an appropriate dose of the
 compound with standard pharmaceutical carriers.
 Most preferably, the active ingredients of Formula I will be administered
 in a daily dosage regimen of from about 2.0 mg to about 1.0 g, most
 preferably from about 50 mg to about 400 mg. Advantageously, equal doses
 will be administered two to four times a day. When the administration is
 carried out as described above, inhibition of calpain is produced.
 The route of administration of the pharmaceutical compositions of this
 invention and in accordance with the methods of this invention is
 internal, more specifically either oral or preferably parenteral, in an
 amount sufficient to produce the desired biological activity.
 The following examples are not limiting but are illustrative of the
 compounds and compositions of this invention and the process for their
 preparation.

EXAMPLE 1
 Preparation of (S)-N-(1-formyl-2-phenylethyl)-1-methyl-2-indolecarboxamide
 a) (S)-N-(1-Hydroxymethyl-2-phenylethyl)-1-methyl-2-indolecarboxamide
 To a stirred solution of 1-methyl-2-indolecarboxylic acid (100 mg, 0.57
 mmol) in methylene chloride (1 mL) was added N-methylmorpholine (0.063 mL,
 0.57 mmol) followed by (S)-(-)-2-amino-3-phenyl-1-propanol (86 mg, 0.57
 mmol), 1-hydroxybenzotriazole hydrate (81 mg, 0.60 mmol), and
 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (120 mg, 0.63
 mmol). The resulting mixture was stirred at room temperature for 20 h. The
 reaction was diluted with methylene chloride and washed sequentially with
 10% citric acid, saturated NaHCO.sub.3, water, and brine. The organic
 layer was dried over Na.sub.2 SO.sub.4 and concentrated in vacuo to give a
 crude oil. Trituration of the crude oil with methanol and diethyl ether
 afforded the title compound (68%, 119.7 mg) as an off-white solid. MS (ES)
 m/e 309.4 [M+H].sup.+.
 b) (S)-N-(1-Formyl-2-phenylethyl)-1-methyl-2-indolecarboxamide
 To a solution of the compound of Example 1(a) (90 mg, 0.29 mmol) in
 methylene chloride (7 mL) was added
 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin
 periodinane) (129 mg, 0.3 mmol). The resulting mixture was stirred at room
 temperature for 1h and quenched with 10% sodium thiosulfate solution.
 After stirring for 10 minutes, the reaction was diluted with methylene
 chloride and washed with 10% sodium thiosulfate solution (3.times.),
 saturated NaHCO.sub.3, and brine. The organic layer was dried over
 Na.sub.2 SO.sub.4 and concentrated in vacuo to give a crude oil.
 Trituration of the crude oil with ethyl acetate/diethyl ether/petroleum
 ether afforded the title compound (49%, 43.1 mg) as a white solid MS (ES)
 m/e 307.4 [M+H].sup.+.
 EXAMPLE 2
 Preparation of (S)-N-(1-formyl-2-phenylethyl)-2-indolecarboxamide
 Following the procedures of Example 1(a) and 1(b), except substituting
 2-indolecarboxylic acid for 1-methyl-2-indolecarboxylic acid, the title
 compound was prepared as a white solid. MS (ES) m/e 293.3 [M+H].sup.+.
 EXAMPLE 3
 Preparation of
 (S)-N-(1-formyl-2-phenylethyl)-5-methoxy-6-(phenylmethoxy)-2-indolecarboxa
 mide
 Following the procedures of Example 1(a) and 1(b), except substituting
 5-methoxy-6-(phenylmethoxy)-2-indolecarboxylic acid for
 1-methyl-2-indole-carboxylic acid, the title compound was prepared as a
 yellow solid. MS (ES) m/e 429.3 [M+H].sup.+.
 EXAMPLE 4
 Preparation of (S)-5-bromo-N-(1-formyl-2-phenylethyl)-2-indolecarboxamide
 Following the procedures of Example 1(a) and 1(b), except substituting
 5-bromo-2-indolecarboxylic acid for 1-methyl-2-indolecarboxylic acid, the
 title compound was prepared as a tan solid. MS (ES) m/e 371.3 [M+H].sup.+.
 EXAMPLE 5
 Preparation of
 (S)-N-(1-formyl-2-phenylethyl)-1-(methylsulfonyl)-2-indolecarboxamide
 a) Ethyl 1-(methylsulfonyl)-2-indolecarboxylate
 A solution of ethyl-2-indolecarboxylate (500 mg, 2.64 mmol) in dry THF (5
 mL) under an argon atmosphere was cooled to 0.degree. C. and treated with
 sodium hydride (116 mg, 2.90 mmol). After stirring at room temperature for
 5 minutes, the reaction was cooled to 0.degree. C. and treated with
 methanesulfonyl chloride (0.23 mL, 2.90 mmol). The resulting mixture was
 gradually warmed to room temperature and stirred for 20 h. The reaction
 was quenched with saturated NaHCO.sub.3 and diluted with ethyl acetate.
 The organic layer was washed with water and brine, dried over Na.sub.2
 SO.sub.4, and concentrated in vacuo. The resulting golden yellow oil was
 chromatographed over silica gel eluting with 5%, 10%, and 15% ethyl
 acetate/hexane to provide the title compound (79%, 556.6 mg) as a cream
 solid. .sup.1 H NMR (400 MHz, CDCl.sub.3) .delta.8.05 (d, 1H), 7.65 (d,
 1H), 744 (t, 1H), 7.31 (m, 2H), 4.42 (q, 2H), 3.71 (s, 3H), 1.42 (t, 3H).
 b) 1-Methylsulfonyl)-2-indolecarboxylic Acid
 A solution of the compound of Example 5(a) (456.6 mg, 1.71 mmol) in THF (6
 mL) and methanol (3 mL), was treated with aqueous 1N NaOH (3.42 mL, 3.42
 mmol) and stirred at room temperature for 20 h. The reaction mixture was
 acidified with 10% HCl (to pH 3) and the solvent was removed at reduced
 pressure. The resulting slurry was diluted with methylene chloride and
 washed with brine. The organic layer was dried over Na.sub.2 SO.sub.4 and
 concentrated in vacuo to provide an off-white solid. Trituration with
 diethyl ether afforded the title compound (57%, 234 mg) as a white solid.
 MS (ES) m/e 239.1 [M].sup.+.
 c)
 (S)-N-(1-Hydroxymethyl-2-phenylethyl)-1-(methylsulfonyl)-2-indolecarboxami
 de
 Following the procedure of Example 1(a), except substituting the compound
 of Example 5(b) for 1-methyl-2-indolecarboxylic acid, the title compound
 was prepared as a viscous colorless oil. MS (ES) m/e 373.4 [M+H].sup.+.
 d) (S)-N-(1-Formyl-2-phenylethyl)-1-(methylsulfonyl)-2-indolecarboxamide
 Following the procedure of Example 1(b), except substituting the compound
 of Example 5(c) for the compound of Example 1(a), the title compound was
 prepared as an off-white solid. MS (ES) m/e 371.3 [M+H].sup.+.
 EXAMPLE 6
 Preparation of
 (S)-N-(1-formyl-2-phenylethyl)-1-(phenylmethyl)-2-indolecarboxamide
 Following the procedures of Examples 5(a)-(d) respectively, except
 substituting benzyl bromide for methanesulfonyl chloride, the title
 compound was prepared as a white solid. MS (ES) m/e 383.5 [M+H].sup.+.
 EXAMPLE 7
 Preparation of (S)-N-(1-formyl-2-phenylethyl)-6-methoxy-2-indolecarboxamide
 Following the procedures of Examples 5(b)-(d) respectively, except
 substituting methyl 6methoxy-2-indolecarboxylate for ethyl
 2-indolecarboxylate, the title compound was prepared as a beige solid. MS
 (ES) m/e 323.3 [M+H].sup.+.
 EXAMPLE 8
 Preparation of
 (S)-N-(1-formyl-2-phenylethyl)-3-methyl-1-(phenylmethyl)-2-indolecarboxami
 de
 Following the procedures of Examples 5(a)-(d) respectively, except
 substituting methyl 3-methyl-2-indolecarboxylate for ethyl
 2-indolecarboxylate and benzyl bromide for methanesulfonyl chloride, the
 title compound was prepared as a cream solid. MS (ES) m/e 397.3
 [M+H].sup.+.
 EXAMPLE 9

Ingredients Mg./Capsule
 (S)-N-(1-formyl-2-phenylethyl)-3-methyl-1- 250.00
 (phenylmethyl)-2-indolecarboxamide
 Magnesium Stearate 5.00
 Lactose 100.00
 The ingredients are thoroughly mixed and filled into a hard gelatin
 capsule.
 EXAMPLE 10

Ingredients Mg./Tablet
 (S)-N-(1-formyl-2-phenylethyl)-5-methoxy-6- 100.00
 (phenylmethoxy)-2-indolecarboxamide
 Lactose 250.00
 Starch 13.00
 Talc 5.00
 Magnesium Stearate 2.50
 The lactose and indolecarboxamide are mixed and granulated with hot 10%
 gelatin. The granules are dried and passed through a #20 mesh screen. The
 granules are then mixed with the starch, talc and magnesium stearate and
 compressed into a tablet.
 One tablet is administered four times a day to mammals for treatment of
 neurodegenerative diseases.
 EXAMPLE 11

Ingredients Amounts/Mg.
 (S)-N-(1-formyl-2-phenylethyl)-3-methyl-1- 75.00
 (phenylmethyl)-2-indolecarboxamide
 DMSO 500.00
 Sodium Chloride 375.00
 Sodium Bisulfite 100.00
 Water for Injection q.s. 100 ml
 The indolecarboxamide is dissolved in the DMSO and 50% of the water. The
 salts are thoroughly dissolved and the volume is brought up to 100 ml. The
 solution is then filtered and filled into ampules and autoclaved.
 EXAMPLE 12

Ingredients Amounts/Mg.
 (S)-N-(1-formyl-2-phenylethyl)-5-methoxy-6 150.00
 (phenylmethoxy)-2-indolecarboxamide
 Peanut Oil 300.00
 The ingredients are mixed to a thick slurry and filled into soft gelatin
 capsules. One capsule is administered orally to mammals for treatment of
 neurodegenerative diseases.