PT100976A - PHARMACEUTICAL COMPOSITION BASED ON RENIN INHIBITORS - Google Patents

Description

-2- (2-hydroxyethyl)

DESCRIPTIVE MEMORY

This is a continuation-in-part of U.S. Patent Application Serial No. 680,811, filed April 9, 1991, which is incorporated herein by reference. This is also a continuation in part of U.S. Patent Application Serial No. 683,663, filed April 15, 1991, which is incorporated herein by reference.

Technical Field A pharmaceutical composition for peptidomimetic compounds, which are renin inhibitors, is described. In particular, the composition comprises a tablet containing the renin inhibitor and a pharmaceutically acceptable organic polycarboxylic acid. The tablet may also contain one or more pharmaceutically acceptable nonionic surfactants.

BACKGROUND OF THE INVENTION The efficacy of orally administered peptides or therapeutic agents of the peptide genus has long been a goal of pharmaceutical research. For example, many efforts have been developed to develop an oral dosage form for insulin. Unfortunately, these efforts have not been successful.

The properties which make the peptides difficult to administer orally include their susceptibility to enzymatic degradation in the digestive tract and the fact that some peptides are not readily transported from the digestive system into the bloodstream. As a result of these problems, it is difficult to achieve the desired levels of peptides or therapeutic agents of the peptide genus in blood at relatively low oral doses and with a relatively low number of oral doses per day.

The methods used to overcome the capacity of the

peptides to be enzymatically degraded and to increase their uptake into the bloodstream from the digestive tract have included the production of analogs which are structurally less of the peptide genus and which are reduced in size (i.e., in molecular mass). Such methods are considered to be successful when the peptide analog allows for satisfactory blood levels after oral administration.

The aforementioned techniques have been applied in the preparation of analogs of the peptide substrate of the enzyme renin. Small peptide-like molecules have been prepared which have some efficacy in lowering blood pressure. For example, compound I (shown below) reduces blood pressure in dogs lacking salt after oral or intravenous administration. However, the oral bioavailability (for fasted dogs) of salts of compound I, in the form of a usual tablet or capsule-like compositions filled with a powder (see Example 12, compositions S1-S5) is about 9 to 44%. In order to be able to administer the compound at the lowest possible dosage and at the lowest dosing frequency, it would be preferable that the oral bioavailability of compound I and its pharmaceutically acceptable salts be greater than that presented by conventional tablets and capsules with a powder mentioned above.

(I)

DESCRIPTION OF THE INVENTION

According to the present invention there is a pharmaceutical tablet composition comprising a compound of formula

in which R 1 is 4-piperazinyl, 1-methyl-4-piperazinyl, 1-methyl-1-oxo-4-piperazinyl, 2-oxo-4-piperazinyl, 4-morpholinyl, 4-thiomorpholinyl or 1-methyl- 4-homopiperazinyl " R2 is benzyl, p-methoxybenzyl, 2-phenylethyl, 1-naphthylmethyl or 2-naphthylmethyl; R 3 is 4-thiazolyl, 2-amino-4-thiazolyl, 2-thiazolyl, 5-thiazolyl, 1-pyrazolyl, 3-pyrazolyl, 1-imidazolyl, n-propyl isopropyl, CH 3 S- or CH 3 SCH 2 -; R4 is lower alkyl or cyclopropyl; R 5 is hydrogen or lower alkyl; and X is CH2 or NH; or a pharmaceutically acceptable salt, ester or prodrug thereof, and a pharmaceutically acceptable organic polycarboxylic acid. Additionally, the tablet composition may further comprise one or more pharmaceutically acceptable nonionic surfactants. When formulated in the form of a tablet comprising a pharmaceutically acceptable polycarboxylic organic acid, or a pharmaceutically acceptable polycarboxylic organic acid and one or more pharmaceutically acceptable nonionic surfactants, the compound of formula (II) has an improved oral bioavailability, as compared to of the same compound administered in the form of a conventional tablet or capsule powder composition. The term " pharmaceutically acceptable organic polycarboxylic acid " as used herein, refers to organic compounds having two or more carboxylic acid substituents, such as citric acid, malonic acid, succinic acid,

tartaric acid, fumaric acid, maleic acid, malic acid, adipic acid, glutaric acid or oxalic acid and the like. Preferably, the organic polycarboxylic acid has a pKa of less than about 4.0. The term " pharmaceutically acceptable nonionic surfactant " as used herein, refers to monoesters of polyoxyethylene (20) sorbitan acids (for example Tweei® 20 (polyoxyethylene (20) sorbitan monolaurate), Tweei® 40 (polyoxyethylene (20) sorbitan monopalmitate), Tweer® 80 (polyoxyethylene sorbitan monooleate (20) and the like), polyoxyethylene ethers (for example Brij® 30 (polyoxyethylene (4) lauryl ether), Brij® 35 (polyoxyethylene lauryl ether (23) ), Brij® 58 (polyoxyethylene (20) cetyl ether), Brij® 78 (polyoxyethylene (20) stearyl ether), Brij® 99 (polyoxyethylene (20) oleyl ether) and the like), block copolymers of ethylene oxide and propylene oxide (for example Pluronic® L101, Pluronic® L31, Pluronic® L61, Pluronic® L7 2, Pluronic® 92 and the like), and ethoxylated fatty acids (for example, Emulphor® CO-5 and Emulphor® CO-25 (ethoxylated castor oil), Emulphor® MA-8, Emulphor® VN 610 and Emulphor® MS 8, Emulphor® COH 2 5 (hydrogenated and ethoxylated castor oil) and the like). The tablet pharmaceutical composition of the invention may also comprise excipients such as fillers (e.g., microcrystalline cellulose (Avicel®), starch, pregelatinized starch, lactose or dicalcium phosphate and the like), disintegrants (e.g., crospovidone , sodium croscarmellose, pregelatinized starch or sodium starch glycolate and the like), lubricants (for example magnesium stearate, searic acid, hydrogenated vegetable oils (for example Sterotej® K), talc or colloidal silica and others of the genus) and binders (for example, polyvinylpyrrolidone (povidone) or Klucel and the like). The pharmaceutical tablet composition of the invention may further comprise various additives such as anti-oxidants

(For example, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or tocopherol acetate (vitamin E) and the like) flavoring agents and coloring agents.

A typical tablet composition of the invention comprises a compound of Formula II, having a concentration of up to about 35% by weight, preferably about 4.0 to about 30% by weight, a pharmaceutically acceptable organic polycarboxylic acid from about 1% by weight to about 20% by weight, preferably from about 3.0% to about 15% by weight), a filler (from about 30.0% by weight to about 90.0% by weight). preferably from about 40.0 to about 85% by weight) and, optionally, one or more pharmaceutically acceptable nonionic surfactants (totaling from about 0% by weight to about 10.0% by weight, preferably from 0.3 to about 3.0% by weight).

A preferred tablet composition of the invention comprises a compound of formula (II) at a concentration from about 4.0 wt% to about 30 wt%, citric acid (from about 3.0 wt% to about 5.0% by weight) and Avicel (from about 40.0% by weight to about 80% by weight).

Another preferred composition of the invention comprises a compound of formula (II), having a concentration from about 4.0 wt% to about 30.0 wt%, citric acid (from about 3.0 wt% to about 5.0% by weight), one or more pharmaceutically acceptable nonionic surfactants (totaling from about 1% by weight to about 2.0% by weight) and Avicel ® (from about 40.0% by weight to about 80.0% by weight).

Another preferred composition of the invention comprises a compound of formula (III), or a pharmaceutically acceptable salt, ester or prodrug thereof, at a concentration from about 4.0 wt% to about 30.0 wt% , citric acid (from about 3.0 wt.% to about 5.0 wt.%) and Avicel® (from about 40.0 wt.% to about 80 wt.%).

-7-

NH, 74 357 4805.PG.03 or

II η 'h ο (III)

A more preferred composition of the invention comprises a compound of formula I or III, or a pharmaceutically acceptable salt, ester or prodrug thereof, having a concentration of about 4.0 wt.% To about 30 wt.%, citric acid (from about 3.0 wt% to about 5.0 wt%), Avicel (from about 40.0 wt% to about 80.0 wt%), crospovidone (from about 2.0 wt% to about 4.0 wt%), croscarmellose sodium (from about 2.0 wt% to about 4.0 wt%) and magnesium stearate (from about 0.1 wt% 5% by weight to about 1.5% by weight).

The compounds of formula I, II and III, contain two or more asymmetric carbon atoms, and thus pure diastereomers, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates may exist as pure diastereomers. The present invention includes in its scope all isomeric forms. The terms " R " and " S " used herein are those defined by IUPAC in 1974 in the Recommendations for, Section E, Fundamental Stereochemistry, Pure Appl. Chem. (1976) 45, 13-30. The term " lower alkyl " as used herein refers to straight or branched chain alkyl radicals having 1 to 7 carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, propyl, n-butyl, iso -butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-hexyl, and the like. The term " halogen " or " halide ", as used herein,

The term " haloalkyl ", as used herein, refers to a lower alkyl radical, wherein one or more of the hydrogen are substituted by a halogen, including but not limited to chloromethyl, trifluoromethyl, 1-chloro-2-fluoroethyl and the like.

The terms " alkoxide " and " thioalkoxide " as used herein refer to R 30 - and R 30 S -, respectively, wherein R 30 is a lower alkyl group or a benzyl. The term " haloalkoxy " as used herein refers to R 311-, wherein R 31 is a haloalkyl group. The term " aminocarbonyl " as used herein refers to -C (O) NH 2. The term " alkylaminocarbonyl " as used herein refers to -C (o) NHR 32, wherein R 32 is lower alkyl. The term " dialkylaminocarbonyl " as used herein refers to -C (O) NR 33 R 34, wherein R 33 and R 34 are independently selected from the lower alkyls. The term " alkoxycarbonyl " as used herein refers to -C (O) O R 35 wherein R 35 is lower alkyl.

The compounds of formula I, II or III may be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. Such salts include, but are not limited to, the following; acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, fumarate, hydrochloride, hydrobromide, dihydro-iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate (mesylate), myotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, phosphate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.

Examples of the acids that may be employed to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid and organic acids such as oxalic acid, maleic acid, succinic acid, methanesulfonic acid and citric acid.

The compounds of the present invention may also be used in the form of a prodrug, which includes esters. Examples of such esters include a hydroxyl-substituted compound of formula (II), which has been acetylated with a blocked or unblocked amino acid residue, a phosphate function, or a hemisuccinate residue. Esters of the amino acid of particular interest are glycine and lysine; however, other amino acid residues may also be used. These esters serve as prodrugs of the compounds of the present invention and serve to increase the solubility of these substances in the gastrointestinal tract. The prodrugs are metabolically converted in vivo into the parent compound of formula (II). The preparation of the prodrug esters is carried out by reacting a hydroxyl-substituted compound of formula (II) with an aminoacyl, phosphoryl or activated hemisuccinyl derivative. The resulting product is then deprotected to give the desired prodrug ester. Other prodrugs include a hydroxyl-substituted compound of Formula II, wherein the hydroxyl group is functionalized with a substituent of the formula -CH (R20) OC (O) R21 or -CH (R2q) OC (S) wherein R 21 is lower alkyl, haloalkyl, alkoxy, thioalkoxy or haloalkoxy and R 20 is hydrogen, lower alkyl, haloalkyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl or dialkylaminocarbonyl. These prodrugs can be prepared by condensation of the hydroxyl group with an activated aldehyde, followed by acylation of the intermediate hemiacetal.

74 357 4805.PG.03 -10-

Generally, the tablet formulation of this invention is prepared by mixing the compound of formula II with the filler and half the total amount of the polycarboxylic acid. This mixture is granulated with water or ethanol or an ethanol / water mixture. The granules are dried and sieved to the desired size. The resulting granules are then mixed with the remaining part of the polycarboxylic acid and any binders, lubricants and / or disintegrators or other additives. After mixing, the material is compressed into tablets.

The following examples will serve to further illustrate the invention.

Example IA

Composition for Tablet N2 1

Amount per tablet

Component% Compound I · HCl 107.2 30.6 Citric acid 50.0 14.3 Avicel 150.0 42.8 Crospovidone 40.0 HH Magnesium stearate 3.0 ..... Q -, - 9 350 , 200.0 Compound I (HCl salt, 5.4 g) was mixed with Avicel® (7.5 g) in citric acid (1.25 g) which was added. This mixture was granulated with ethanol (grade 200). The ethanol was added with continuous mixing either by dropwise addition or by spray in the powders. The granulated material was oven dried overnight at 50 ° C to 60 ° C. The granules were sieved through a 20 mesh mesh sieve. Crospovidone (2.0 g) and magnesium stearate (0.15 g) were added to the pellets and the remaining citric acid (1.25 g) was added. After mixing, the material was compressed into tablets. 74 357 4805.PG.03

Example 1B

Composition for Tablet NB 2

Amount per tablet

Component% Compound I · HCl 107.2 30.3 Citric acid 50.0 H · H Avicel® Brij® 35 150.0 42.4 2.4 0.7 Tween® 80 0.8 to Crospovidone 40, 0 11.3 Magnesium stearate 3.000 * 353.4 100.0 Compound I (HCl salt, 5.4 g) was mixed with (7.5 g) and citric acid (1.25 g) and sieved through a 20 mesh sieve. A solution of Brij® 35 (0.12 g) and Tween® 80 (0.04 g) in about 1.0 ml ethanol (grade 200) was added dropwise to the granulate. Alcohol (graduated ethanol 200) was added dropwise to complete the granulation. The granules were dried overnight at 60 ° C in an oven, and subsequently sifted through a 20 mesh mesh screen.

Crospovidone (2.0 g) and magnesium stearate (0.15 g) were added to the granules and the remaining citric acid (1.25 g) was added. After mixing, the material was compressed into tablets. (see Example 1C)

74 357 4805.PG.03 -12-

Example 1C

Composition for Na 3 tablet

Component Amount per tablet _mg_% Compound I · Flavored 120.5 20.1 Malonic acid 50.0 00 oj Avicel® 300.0 50.0 Brij® 35 H 00 0 3.0 Tween® 80 6.0 1.0 Emulphor ® 719 6.0 1/0 Crospovidone 50.0 8.3 Sodium starch glycolate 50.0 8.3 600.5 100.0 Compound I (mesylate salt, 1.45 g) was mixed with Avicel® (2.4 g ) and granulated with 1.8 ml of a solution of Brij® 35 (1.2 g), Tween® 80 (0.4 g) and Emulpho:? 719 (0.4 g) in 10 ml of 200 degree ethanol The granulate was dried at 50 ° C in a vacuum oven for at least 2 hours and then sieved through a 20 mesh mesh screen. Avicel (1.2 g), malonic acid (0.6 g), sodium starch glycolate (0.6 g) and crospovidone (0.6 g) were added to the granules and mixed. After mixing, this material was compressed into tablets.

Example 1D

Composition for Tablet NB 4

Amount per tablet

Component mg% Compound I · mesylate 120.5 20.2 Citric acid 75.0 12.6 Avicel ® 300.0 50.4 Crospovidone 0 LO 00 Sodium starch glycolate 50.0 8.4 595.5 100, 0

Compound I (mesylate salt, 1.45 g) was mixed with Avice] ® (3.6 g) and citric acid (anhydrous, 0.9 g) and dry blended a homogeneous mixture is obtained. Sodium starch glycolate (0.6 g) and crospovidone (0.6 g) were added and mixed. After mixing, this material was compressed into tablets.

Example 2A

Preparation of a tablet composition comprising the

compound III

Using the procedure of Example I, tablet compositions of compound III may be prepared.

Example 2B

Preparation of a tablet composition comprising the compound of Example 9

Using the process of Example 1, tablet compositions of the compound of Example 9 can be prepared.

Example 3 (2S) -2-Benzyl-3- (1-methyl-piperazin-4-ylsulfonyl] propionyl- (2S, 3R, 4S) -2-amino-1-cyclo -hexyl-3,4-dihydroxy-6-methylheptane

Example 3A Methyl 3-hydroxy-2-methylene-3-phenylpropionate

A mixture of benzaldehyde (82.1 mL, 0.81 mol), methyl acrylate (109.1 mL, 1.211 mol), 1,4-diazabicyclo (2,2,2) octane (13.6 g, 12 mol) and acetic acid (1.4 ml, 0.024 ml) was allowed to stir at 35øC for 60 h, at which time the reaction was found to be 70% complete,

74 357 4805.PG.03 -14 H-NMR. Methyl acrylate (20.9 ml, 0.23 mol) was then added to the solution and it was allowed to react at 35 ° C for a further 48 h. The mixture was diluted with diethyl ether (1.0 L) and washed with 2 x 200 ml portions of a pH 7 phosphate buffer. After concentration in vacuo, the remaining mixture was distilled under reduced pressure (12 mm) to give 6.5 g of unreacted benzaldehyde and 130.0 g (90%) of the desired product as a colorless oil: bp 130 ° C (12 mm); IR (film) 1718, 1440 cm -1; 1 H NMR (CDCl 3) δ 3.67 (s, 3H), 5.52 (br s, 1H), 5.83-5.85 (m, 1H), 6.29-6.31 (m, 1H) , 7.23-7.39 (m, 5H); 13 C NMR (75 MHz, CDCl 3) δ 51.8, 72.9, 125.8, 126.5, 127.7, 128.3, 141.2, 141.9, 166.6.

To a 3-necked Morton flask, 2 L, equipped with a thermometer, mechanical stirrer and addition funnel, was added 3-bromo-2-carbomethoxy-3-phenyl- the compound resulting from Example 3A (305.9 g, 1.585 mol) followed by addition of a portion of 48% HBr (505 ml, 4.46 mol). The flask was immersed in an ice-water bath, while concentrated sulfuric acid (460 mL, 8.62 mol) was added dropwise over 90 minutes, and the internal temperature of the reaction mixture was maintained at 23-27 ° C through the addition process. After removal of the ice-water bath, the mixture was allowed to stir at room temperature overnight. The solution was then transferred to a separatory stopper and the organic layer was allowed to separate from the acid layer. The acids were drained and the organic layer was diluted with 2 L of a 1: 1 solution of ethyl acetate / hexane, washed with a saturated aqueous solution of sodium bicarbonate (1 L), dried over sodium sulfate and concentrated, to give 400 g (99%) of the desired product as a yellow oil, which was used without any further purification: bp 180 ° C (12 mm); IR (film) 1718, 712 cm -1; 1 H NMR (CDCl 3) δ 3.89 (s, 3H), 4.40 (S, 2H), 7.38-7.45 (m, 3H), 7.56-7.60 (m, 2H), 7.83 (s, 1H); 13 C NMR (75 MHz, CDCl 3) δ 26.77, 52.47, 128.63, 128.87, 129.61, 134.20,

74 357 4805.PG, 03 142.95, 166.62.

Example 3C

(Z) -2-Carbomethoxy-3-phenyl-2-propene-1-sulfonic acid sodium salt To a three-necked 12 L round bottom flask equipped with a mechanical stirrer, thermometer and funnel of addition, the product resulting from Example 3B (400 g, 1.57 mol) and methanol (4 L) was added. The mixture was heated to 50 ° C, and a solution of sodium sulfite (199 g, 1.57 mol) dissolved in water (4 L) was added over 75 minutes while the internal temperature of the flask was maintained at 50 ° C. After the addition was complete, the clear solution was allowed to stir at 50 ° C for an additional 45 minutes. The reaction mixture in solution was taken to the next step without further purification. The compound may be isolated, by concentration, as an amorphous powder, which is contaminated with one equivalent of sodium bromide: IR (KBr) 1711, 1628, 1215 cm -1; 1 H NMR (DMSO D-6) δ 3.70 (s, 3H), 3.77 (s, 2H), 7.33-7.41 (m, 3H), 7.48 (s, 1H), 7 , 87-7.89 (m, 2H), * 13 C NMR (75 MHz, DMSO D-6) δ 49.88, 51.93, 127.36, 128.33, 128.91, 129.82, 134 , 75, 139.06, 168.60.

3D Example

Sodium salt of acid. 2-carbomethoxy-3-phenylpropane-1-sulfonic acid To the mixture of 1: 1 methanol / water containing the compound resulting from Example 3C, 60 g of Raney W-24 nickel were added. The resulting slurry was pressurized with 50 psi (344,000 Pa) hydrogen and allowed to stir on a Parr shaker for 24 h, at which time an additional 20 g of Raney nickel catalyst was added. After 6 h under hydrogen atmosphere at 50 psi (344,000 Pa), the catalyst was removed by filtration and the solution was concentrated to dryness. To the dry white solid,

C "

(61) and heptane (41) were added and the solution was vigorously stirred overnight with mechanical stirrer. The white suspension was removed by filtration, affording 530 g (88%) of the desired product as an amorphous powder which was contaminated as an amorphous powder which was contaminated with approximately one equivalent of NaBr. The compound was used without further purification: IR (KBr) 1740, 1215, 1050 cm -1. 1 H NMR (DMSO D-6) δ 2.48-2.54 (m, 1H), 2.74-2.87 (m, 2H), 2.91-3.04 (m, 2H) 48 (s, 3H), 7.12-7.32 (m, 5H); 13 C NMR (75 MHz, D 2 O / DMSO D-6) δ 38.18, 44.80, 52.67, 52.82, 127.42, 129.13, 129.34, 138.14, 176.84.

Example 3E

2-Carbomethoxy-3-phenyl-1-propanesulfonyl chloride To a 3 L round bottom flask was added the resultant compound of Example 3D (530 g, 1.39 mol) and toluene (520 ml), followed by addition of PC15 (317 g, 1.52 mol). The mixture was heated at 50 ° C with stirring for 45 minutes. This was then diluted with toluene (11) and filtered through celite. After concentration under vacuum, 371 g (96%) of the desired product were obtained as a light brown oil: IR (film) 1740, 1380, 1170 cm -1; 1 H NMR (CDCl 3) δ 2.92 (dd, 1H, J = 8.1, 14.0), 3.17 (dd, 1H, J = 6.6, 14.0) 1H), 3.67 (dd, 1H, J = 3.3, 14.3), 3.72 (s, 3H), 4.20 (dd, 1H, J = 8.8, 14). , 3), 7.15-7.18 (m, 2H), 7.25-7.35 (m, 3H); 13 C NMR (75 MHz, CDCl 3) δ 37.26, 42.88, 52.65, 64.89, 127.49, 128.87, 128.92, 135.61, 171.79.

To a 1 L round-bottomed flask, the compound resulting from Example 3E (84.5 g, 0.305 mol) and dichloromethane (305 g) were added. ml). The mixture was cooled to 0Â ° C in a water-ice bath and a solution of N, N-dimethylformamide

(35.5 ml, 32.1 g) dissolved in dichloromethane (305 ml) was added with vigorous stirring for 90 minutes. After the addition was complete, the ice-water bath was removed and the mixture was stirred for another 4 h while warming to room temperature. The solution was then poured into a settling boiler, counting 11 of 5% aqueous NaOH solution. The layers were partitioned and the organic phase was dried with potassium carbonate. Concentration in vacuo gave an oil, which was filtered through 200 g of silica gel using 4: 1 hexane / ethyl acetate as the eluent. Concentration gave 84.3 g (81%) of the desired product as a yellow oil: IR (film) 1735, 1165, 955 cm -1; % NMR (CDCl 3) δ 2.30 (s, 3H), 2.42 (t, 4H, J = 4.8), 2.88 (dd, 1H, J = 7.7, 14.0), 2. , 3.06 (dd, 1H, J = 7.0, 13.6), 3.18-3.27 (m, 5H), 3.43 (dd, 1H, J = 8.82, 13.9), 3.67 (s, 3H), 7.14-7.17 (m, 2H), 7.24-7.34 , 3H); 13 C NMR (75 MHz, CDCl 3) δ 37.91, 42.22, 45.36, 45.83, 49.61, 52.21, 54.36, 127.06, 128.66, 128.92, 129 , 06, 136.79, 173.33.

The racemic ester resulting from Example 3F (135 g, 397 mmol) was suspended in acetone (300 mL) and water (900 mL) in dichloromethane (3 mL) ). While stirring vigorously at a temperature of 35 ° C, a crude preparation of Cartilsberg Subtilisin (10 ml, Alcalase 241, Novo Laboratories) was added. Sodium hydroxide solution (6M) was used to maintain the reaction at pH 7.5-8.0. After 3 days, the acetone was removed under reduced pressure and the aqueous phase was extracted with CHCl3 (11) to remove the unreacted ester. The aqueous phase was adjusted to pH 7 with 3M HCl and was desalted by elution through a column of Ambertite XAD-16 (2 kg, pre-washed sequentially with water, methanol, and water) using a water to methanol / water gradient . Evaporation of the solvent gave 46 g (70%) of a white solid: mp 184.5 ° C; TLC (25% ethyl acetate / 25% water / 25% acetic acid / 25% n-butanol) Rf = (j) 74 357 4805.PG.03

0f 43;

Analysis (C 15 H 22 N 2 O 4 S-0.25 H 2 O)

Calculated: C, 54.44; H, 6.85; N, 8.47. Found: C, 54.77; H, 6.53; N, 8.39.

To a stirred mixture of diethyl acetamidomalonate (217 g, 1.0 mol) and 2,3-dibromopropene (240 g, 1.2 mol) in dry tetrahydrofuran (2 , 50 1) in a nitrogen atmosphere, sodium hydride (26.4 g, 1.1 mol) was added in several portions The reaction mixture was stirred at room temperature for 30 minutes, then heated under reflux. The resulting slurry was cooled to room temperature and filtered under suction through a small pad of silica gel The solid residue was washed with tetrahydrofuran (2 x 50 ml), and the filtrates were combined and concentrated The residue was dissolved in ethyl acetate (2.01), washed with water and brine, and then dried with MgSO4. Filtration and concentration gave a yellow oil which solidified on drying.The resulting solid was recrystallized from a warm ethyl acetate / hexane mixture, to give 301 g (89%) of the desired product: mp 85-87 ° C. 1 H NMR (300 MHz, CDCl 3) δ 1.28 (t, J = 7.4 Hz, 6H), 2.04 (s, 3H), 3.57 (s, 2H), 4.27 , 4H), 5.55 (bs, 1H), 5.61 (bs, 1H), 6.82 (br, 1H); IR (KBr) 1745, 1635 cm -1.

Calc'd for C 12 H 18 BrNO 5: C 42/87 'H / 5.40; Br, 23.77; N, 4.12. Found: C, 43.25; H, 5.56; Br, 22.97; N, 4.12.

Example 31 Diethyl (3-bromo-2-oxo-propyl) acetamidomalonate To a cold (0 ° C) and stirred solution of the resulting compound of Example 3H (280 g, 0.83 mol) in a 2: 1 mixture of 74 357 4805 .PG.03 -19-

acetonitrile / water (1.68 L) was added solid N-bromosuccinimide (193 g, 1.08 mol) in three portions over a period of 15 minutes. The resulting orange mixture was stirred at 0 ° C for an additional period of 1 h and then allowed to warm to room temperature. After 4 h, the reaction mixture was treated with a 10% aqueous solution of sodium thiosulfate, diluted with ethyl acetate, and sequentially washed with water, 10% aqueous NaHSO 4 (3 x), water and brine. Drying (MgSO4) and concentration gave a yellow solid which was recrystallized from a mixture of ethyl acetate and hexane to give 274 g (85%) of the desired compound as a white solid : mp 97-98.5 ° C. 1 H NMR (300 MHz, CDCl 3) δ 1.25 (t, J = 7.5 Hz, 6H), 2.01 (s, 3H), 3.87 (s, 2H), 3.93 (s, 2H ), 4.25 (q, J = 7.5 Hz, 4H), 7.0 (br, 1H); IR (KBr) 1760, 1732, 1634 and 1209 cm -1.

Calc'd for C 12 H 18 BrNO 6: C, 40.93; H, 5.15; Br, 22.62; N, 3.98. Found: C, 41.05; H, 5.23; Br, 23.28; N,. 3.93.

Example 3 Diethyl 3J (4-thiazolylmethyl) acetamidomalonate

In a three-necked, round-bottomed, 51-necked flask equipped with a mechanical stirrer, a plug and a drying tube was charged the resulting compound of Example 31 (325 g, 0.92 mol) and swamped with nitrogen. A freshly prepared solution of thioformamide in tetrahydrofuran (0.8 M, 1.25 L) was added in one portion. The reaction mixture was stirred at room temperature for 4 h. The resulting slurry was then diluted with ether (1.25 L) and cooled to 0 ° C. The solid was then collected by suction filtration and washed with cold ether (3x) to give the title compound as the hydrobromide salt. This material was transferred to a 4 1 separating funnel, slurried with ethyl acetate (21) and basified with the careful addition of a 2 M aqueous NaOH solution. The organic phase was separated, washed with

And brine, and then dried with MgSO4. Filtration and concentration gave a pale yellow oil which solidified on drying to give 242 g of the desired compound. This material was recrystallized from an ethyl acetate / hexane mixture to give 185.6 g (64%) of the pure material: mp 104-106 ° C.

Calc'd for C 13 H 18 N 2 O 5 S: C, 49.67; H, 5.77; N, 8.91; S, 10.20. Found: C, 49.90; H, 5.72; N, 8.97; S, 10.29.

To a stirred solution of the resultant compound of Example 3J (185.6 g, 0.59 mol) in a mixture of tetrahydrofuran (620 ml) and dichloromethane and ethanol (310 ml), a 2 M aqueous solution of LiOH (325 ml, 0.65 mol) was added dropwise over 20 min. After stirring at room temperature for 2.5 h, the reaction mixture was concentrated and the resulting aqueous mixture was extracted with ether (3 x 200 mL), adjusted to pH 3 with 3M HCl, and concentrated under reduced pressure. The residual water was removed by evaporation of portions of toluene (2 x 200 ml). The residue was diluted with toluene (1.5 L) and the resulting slurry was heated under reflux with separation of the residual water (Dean-Stark trap). After 3 h, the reaction mixture was cooled to room temperature, diluted with ethyl acetate (1.5 L) and filtered through suction through SiO 2 (60 g). The solids were washed with additional ethyl acetate (4 x 500 ml) and the combined organics were concentrated to give a pale yellow oil which solidified on drying (66.7 Pa) to give 119.6 g (84%) of the desired compound: mp 58-62 ° C.

Example 3 N-Acetyl-3- (4-thiazolyl) -L-alanine ethyl ester and N-acetyl-3- (4-thiazolyl) -D-alanine ethyl ester

A 5-neck round-bottomed three-necked flask,

(210 g, 0.87 mol), distilled water (1.6 L) and 1 M aqueous KCl solution (1 L) was added to the reaction mixture. 0.8 1). The homogenized solution was adjusted to pH 7.0 with 0.1 M NaOH and then treated with Cartilsberg Subtilisin (1.8 g) dissolved in a 0.1 H aqueous solution of KCl (25 ml). The reaction mixture was stirred at room temperature with 1.0 N NaOH, added as needed to maintain the pH at 6.25-7.25. After 4 h, 430 ml of the base had been consumed and the reaction was considered to be complete. The reaction mixture was then extracted with chloroform (4 x 1.5 L), the aqueous phase was carefully acidified to pH 4 with 2M HCl and was then concentrated under reduced pressure. The residual water was removed by sequential evaporation from toluene (3 x 500 ml) and ethanol (3 x 500 ml). The residue was taken up in warm ethanol and filtered by suction to remove the inorganic salts. The solids were washed with hot ethanol (3 x 400 mL) and the filtrates were concentrated to give 92.6 g (50%) of N-acetyl-3- (4-thiazolyl) -L-alanine as a white solid. a white solid: mp 186 ° C.

The chloroform fractions of the combined extractions were washed with saturated NaHCO 3 aqueous solution, water and brine and then dried with MgSO 4. Filtration and concentration gave 103 g (49%) of N-acetyl-3- (4-thiazolyl) -D-alanine ethyl ester. This material can be further purified by recrystallization from ethyl acetate / hexane: mp 79-80.5 ° C.

3M Exemnlo

Epimerization of N-acetyl-3- (4-thiazolyl) -D-alanine ethyl ester

A 2 L round bottom flask equipped with a magnetic stirrer, a reflux condenser, and a nitrogen inlet was charged with sodium (0.96 g, 0.045 mol) and ethanol (900 ml) and the mixture was left under reflux until sodium was

74 357 4805.PG.03 -22-consumed. The resulting sodium ethoxide solution was cooled slightly and N-acetyl-3- (4-thiazolyl) -D-alanine ethyl ester of Example 3L (102 g, 0.42 mol) was added. The reaction mixture was then heated to reflux. After 3 h, the solution was cooled to room temperature, quenched with glacial acetic acid (0.045 mol) and concentrated to remove ethanol. The residue was diluted with ethyl acetate, washed with water and brine and dried with MgSO4. Filtration and concentration gave a yellow oil which was purified by recrystallization from a mixture of hot ethyl acetate and hexane to give 89 g (87%) of material identical to that obtained in Example 3K.

Example 3N

3- (4-Thiazolyl) -L-alanine dihydrochloride

A round bottom flask equipped with a magnetic stirrer was charged with N-acetyl-3- (4-thiazolyl) -L-alanine from Example 3L (92.6 g, 0.43 mol) and HCl 6 Μ (11). The resulting solution was heated to reflux. After 3 h, the mixture was allowed to cool to room temperature. The solution was then concentrated under reduced pressure, evaporated from toluene (3 x 200 ml), and dried under vacuum overnight to give 120 g of a slightly wet solid. This material was used in the next reaction without further purification.

Example 30 N-Boc-3- (4-thiazolyl) -L-alanine

A 4 L Erlenmeyer flask equipped with a mechanical stirrer was charged with the resultant compound of Example 3N (125.9 g) and tetrahydrofuran (1.5 L), and the mixture was adjusted to pH 6.6 with saturated aqueous sodium bicarbonate solution. The resulting solution was then adjusted to pH 8.9 with 3M NaOH and a solution of di-tert-butyl dicarbonate

(117.8 g, 0.51 mol) in tetrahydrofuran 150 ml. The reaction mixture was stirred vigorously at room temperature for 40 h. The tetrahydrofuran was removed in vacuo, the pH of the residue was adjusted to 2.0 with 3M HCl and the mixture was extracted with ethyl acetate (3 x 300 mL). The combined extracts were dried over MgSO4, filtered, and concentrated to give 150 g of a white solid. Recrystallization from a warm 1: 1 ethyl acetate / hexane mixture (1.06 L) gave 107.6 g (82% of the resultant compound of Example 3L) of the desired compound: mp 115 ° C; [Î ±] D = +129.8 (c = 1.04, CHCl3).

Calc'd for C 16 H 16 N 2 O: C, 48.53; H, 5.88; N, 10.29. Found: C, 48.58; H, 5.91; N, 10.17.

Example 3P

(2S, 3R, 4S) -2-Amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane Boc-L- (4-thiazolyl) , 4S) -2 - [(tert -butyloxycarbonyl) amino] -1-cyclohexyl-3,4-dihydroxy-6-methylheptane (5.05 g, 14.7 mmol Luly et al. , Org. Chem. 1988, 53, 6109) in 4M HCl in ethanol was stirred for 90 minutes and then evaporated. Ether was added and evaporated 3 times, and the residue was dried under high vacuum. To this residue was added 1-hydroxybenzotriazole (5.57 g, 41.2 mmol), the acid resulting from Example 30 (4.00 g, 14.7 mmol), dimethylformamide (60 mL) and N-methylmorpholine 40 ml, 30.9 mmol). The mixture was cooled to -23 ° C, treated with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.03 g, 21.0 mmol). After 2 h at -23 ° C and 21 h at room temperature, the mixture was poured into saturated NaHCO 3 solution and extracted into ethyl acetate. The organic phase was washed with water and brine, then dried over Na 2 SO 4 and evaporated to give a white solid which was recrystallized from methylene chloride / ether 1:15 (v / v) (multiple crops) to give 6.28 g (86%) of the desired product as a white, flake solid: mp 159-160 ° C; TLC (15% CH 3 OH / 85% CHCl 3) Rf = 0.63; ¹ NMR (CDCl 3) δ 8.78 (1H, d), 7.14

(1H, br d), 4.44 (1H, dd), 4.27 (1H, m), 1.10 (1H, m), 3.37 (1H, dd), 3.30-3.12 (3H, m), 1.89 (1H, septet), 1.46 (9H, S), 0.94 (3H, d ), 0.88 (3H, d).

Calc'd for C 25 H 43 N 5 O 5 S: C, 60.33; H, 8.71; N, 8.44. Found: C, 60.43; H, 8.68; N, 8.51.

Example 30 (2S, 3R, 4S) -2-Amino-1-cyclohexyl-3,4-dihydroxy-6-methyl-heptane H-L- (4-thiazolyl)

Trifluoroacetic acid (50 mL) was slowly added via cannula to a solution of the compound resulting from Example 3P (6.27 g, 12.6 mmol) in methylene chloride (50 mL) at 0 ° C. The reaction mixture was stirred for 3 h at 0 ° C and concentrated in vacuo (in a 40 ° C bath) to an oil which was basified to pH 10-11 with an aqueous K 2 CO 3 solution. The product was extracted into chloroform, dried over Na2SO4, filtered and concentrated to a foam. Recrystallization from a 1: 4 (v / v) methylene chloride / hexane mixture gave 5.00 g (100%) of the desired product as a fluffy white solid: mp 111-112 ° C; TLC (15% CH 3 OH / 85% CHCl 3) Rf = 0.46; NMR (CDCl3) δ 8.77 (1H, d), 7.40 (1H, br d), 7.13 (1H, d), 4.54 (1H, m), 4.25 (1H, m) , 3.80 (1H, dd), 3.33 (1H, dd), 3.25-3.12 (3H, m), 0.95 (3H, d), 0.86 (3H, d).

Calc'd for C 20 H 35 N 3 O 3 Si: C, 60.42; H, 8.87; N, 10.57. Found: C, 60.05; H, 8.65; N, 10.42.

(2S, 3R, 4S) -2-Amino-1- (4-thiazolyl) (3S) -2-benzyl-3- (1-methylpiperazin-4-ylsulphonyl) propionyl- -cyclohexyl-3,4-dihydroxy-6-methyl-heptane

To the resultant acid of Example 3G (1,000 g, 3.064 mmol), to the resultant amine of Example 3Q (1.110 g, 2.792 mmol) and to 1-hydroxybenzotriazole (1.022 g, 7.563 mmol) in dimethylformamide

(20 mL) was added N-methylmorpholine (0.35 mL, 3.2 mmol). The mixture was cooled to -23 ° C and treated with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.760 g, 3.96 mmol). After 2 h and 23 h at room temperature, the reaction mixture was poured into saturated NaHCO 3 solution (100 mL) and extracted into ethyl acetate (2 x 50 mL) which was washed with water (2 x 50 mL ) and brine (50 ml) and dried with Na 2 SO 4 and evaporated to give 1.94 g. Recrystallization from ethanol (15 ml) / hexane (90 ml) gave 1.559 g (79%) of a white solid: mp 169-170Â ° C; tlc (10% CH3 OH / 90% CHCl3) Rf = 0.40; NMR (CDCl 3) δ 8.73 (1H, d), 7.43 (1H, d), 7.37-7.16 (6H, m), 6.23 (1H, d), 4.63 1H, dd), 2.30 (3H, s), 0.95 (3H, d), 0.87 (3H, d).

Calc'd for C 35 H 55 N 5 O 2 S 2 • 0.75H 2 O: C, 58.43; H, 7.91; N, 9.73. Found: C, 58.51; H, 7.74; N, 9.60.

Example 4

Alternative preparation of N-Boc-3- (4-thiazolyl) -L-alanine

To a solution of the product of Example 3H (3.36 g, 10.0 mmol) in dimethylformamide (10 mL) was added sodium chloride (586 mg, 10.0 mmol) in dichloromethane , water (360 μ, 20 mmol) and 4N hydrochloric acid in dioxane (0.12 mL, 0.5 mmol). The reaction vessel was placed under a nitrogen atmosphere under positive pressure. The reaction mixture was heated under reflux for 24 hours, and then was concentrated in vacuo. The obtained residue was diluted with water (5 ml) and extracted with ether (3 x 15 ml). The combined organic extracts were decolorized with charcoal (0.5 g), dried over magnesium sulfate, filtered and concentrated in vacuo to give the title product (2.51 g, 95%) as a yellow oil pale. 1 H NMR (300 MHz, CDCl 3) δ 1.29 (t, 3H), 2.04 (s, 3H), 2.99 (m, 2H), 4.22 (q, 2H), 4.79 1H), 5.53 (d, 1H), 5.68 (m, 1H), 6.44 (d, 1H); IR (film) 1195, 1220,

74 357 4805.PG.03 -26- 1370, 1540, 1660, 1740, 2990, 3050, and 3300 cm -1. MS (DCI / NH 3) m / e 264/266 (M + H) +, 281/283 (M + H + NH 3) +. Calc'd for C 9 H 14 NO 3 Br: C 40/92. H / 5/34; N, 5.30. Found: C, 42.04; N, 5.48; N, 5.26.

Example 4B N-Boc- (2-Bromoallyl) glycine

A slurry of the product of Example 4A (16.2 g, 61.3 mmol) in a 0.1 N potassium chloride solution (300 mL) containing 0.2 M phosphate buffer pH 7.0 (30 mL) was treated with a solution of Subtilisin Carlsberg (4 mg) a solution of 0.1 N potassium chloride (3 ml). OpH was maintained between 6.50 and 7.25 by addition of a 2.0 N sodium hydroxide solution via a pH controller (pH-Stat). After 25 minutes, the rate of hydrolysis slowed remarkably; and the D-ester which remained to be reacted was extracted with methylene chloride (3 x 150 ml). The resulting aqueous phase was treated with cobalt (II) acetate (6 mg) and Acylase I (80 mg). The reaction mixture was stirred for 4 hours and was found to have been completed. The pH of the reaction mixture was adjusted to 10 by addition of solid sodium carbonate. The resulting solution was treated with di-tert-butyl dicarbonate (6.55 g, 30 mmol) dissolved in THF (100 mL) and stirred vigorously for 16 hours. The aqueous solution was washed with hexane (200 mL), to remove any unreacted protecting agent. The aqueous phase was adjusted to pH 2.5 by the addition of solid potassium hydrogen sulfate, and extracted with ethyl acetate (2 x 200 ml). The combined organic phases were washed with brine (100 mL), dried over magnesium sulfate, and concentrated in vacuo to give the title compound (7.30 g, 81%) as a pale yellow crystalline solid. [Î ±] D at 25Â ° C = -9.86Â ° (MeOH), c = 1.085. 1 H NMR (300 MHz, CDCl 3) δ 1.48 (s, 9H), 2.91 (m, 2H), 4.52 (m, 1H), 5.19 (d, 0.5H), 5.53 (m, 1H), 5.71 (s, 1H), 6.79 (d, 0.5H), 11.3 (s, 1H); IR (CDCl 3) 1150, 1250, 1400, 1500, 1620, 1640, 1710, 3000, and 3520 cm -1. (DCI / NH 3) m / e 311/313

74 357 4805.PG.03 -27- (M + H + NH 3) +. Calc'd for C 10 H 16 NO 4 Br: C 42.22; H: 5.66; N, 4.91. Found: C, 41.38; H, 5.59; N, 4.75.

To a solution of the product of Example 4B (2.00 g, 6.80 mmol) in water (30 mL) and tetrahydrofuran (4 mL) (15 ml) cooled to 0 ° C was added N-bromosuccinimide (1.45 g, 8.16 mmol) in three portions over 20 minutes. After the addition was complete, the ice bath was removed and the solution was stirred for four hours. The tetrahydrofuran was removed in vacuo and the product was extracted with ethyl acetate (3 x 35 mL). The organic extracts were combined and washed with a solution of 5% sodium chloride (25 ml) and brine (25 ml), dried over magnesium sulfate, and concentrated in vacuo to give the title compound (1.70 g, 81%). 1 H NMR (300 MHz, CDCl 3) δ 1.45 (s, 9H), 3.30 (m, 2H), 3.93 (s, 2H), 4.61 (m, 1H), 5.51 (d , 1H). MS (DCI / NH 3) m / e 310/312 (M + H) +, 327/329 (M + H + NH 3) +.

To a solution of the product of Example 4C (91mg, 0.293mmol) in tetrahydrofuran (5ml) was added thioformamide (2R) -N-Boc-2-amino-3- (4-thiazolyl) propanoic acid 17.7 mg, 0.29 mmol). [The thioformamide was prepared by reacting a slight excess of phosphorus pentasulphite with formamide in tetrahydrofuran. The resulting solution was diluted with hexanes and filtered through a pad of silica gel and stored at -25 DEG C. The resulting solution was allowed to stand for 16 hours and then concentrated in vacuo to give a residue which was partitioned an aqueous solution of sodium bicarbonate was added dropwise between diethyl ether. The aqueous layer was washed with ether (2 x 10 mL) and methylene chloride (10 mL), adjusted to pH 2.3 with solid potassium hydrogen sulfate and extracted with ether (3 x 20 mL). The combined organic extracts were dried

74 357 4805.PG.03

with magnesium sulfate and concentrated in vacuo to give the title compound as a white crystalline solid (55 mg, 71%). NMR (300 MHz, CDCl 3) δ 1.48 (s, 9H), 3.48 (m, 2H), 4.52 (m, 1H), 5.61 (m, 1H), 7.18 (d, 1H), 8.91 (d, 1H).

Example 5

Alternative preparation of (2R) -N-Boc-2-amino-5-bromo-4-oxopentanoic acid

To a suspension of 95% sodium hydride (17.2 g, 680 mmol) in tetrahydrofuran (1.2 L) was added 2,3-dichloropropene (100 900 mmol), diethyl acetamidomalonate (146 g, 6.72 mmol) and tetrabutylammonium bromide (6.00 g). The resulting slurry was heated under reflux in a nitrogen atmosphere for 20 hours. The reaction mixture was concentrated in vacuo and the resulting residue partitioned between water (200 ml) and a mixture of ether (300 ml) and methylene chloride (100 ml). The organic phase was washed with 5% sodium chloride solution (200 mL) and brine (200 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. The resulting solid (195 g) was dissolved in hot hexanes (1300 ml) and allowed to cool to room temperature and then stand overnight to give the title compound as a crystalline solid (157 mg. g, 80%): mp 76.3 ° C. 1 H NMR (300 MHz, CDCl 3) δ 1.29 (t, 6H), 2.05 (s, 3H), 3.48 (s, 2H), 4.28 (m, 4H), 5.18 , 1H), 5.29 (m, 1H), 6.92 (bs, 1H); IR (CDCl 3) 1140, 1180, 1200, 1240, 1270, 1300, 1500, 1630, 1680, 1740, 2950, 2990, and 3300 cm -1. MS (DCI / NH 3) m / e 292/294 (M + H) +, 309/311 (M + H + NH 3) +. Calc'd for C 12 H 18 NO 5 Cl: C 49/41 H / 6/22; N, 4.80. Found: C, 49.18; H, 6.29; N, 4.75. 74 357 4805.PG.03

Example 5B (137 g, 500 mmol) was hydrolyzed and decarboxylated by the procedure described in Example 2A to give the title compound (105.4 g, 96%) as a white solid of a pale yellow oil which crystallized on standing. 1 H NMR (300 MHz, CDCl 3) δ 1.31 (t, 3H), 2.05 (s, 3H), 2.79 (m, 2H), 4.22 (q, 2H), 4.79 , 1H), 5.23 (m, 1H), 5.29 (m, 1H), 6.61 (m, 1H); IR 1200, 1220, 1280, 1300, 1370, 1440, 1550, 1638, 1659, 1740, 2890, 2990, 3050, and 3300 cm-1. MS (DCI / NH 3) m / e 220/222 (M + H) +, 237/239 (M + H + NH 3) +. Calc'd for C 19 H 14 NO 3 Cl: C, 49.21; H, 6.42; N, 6.38. Found: C, 46.58; H, 6.05; N, 6.02.

Example 5C (2R) -N-Boc-2-Amino-5-bromo-4-oxopentanoic acid The product of Example 5B is treated according to the procedure of Example 4B and 4C to provide the desired product.

Example 6

Alternative preparation of 2 (S) -benzyl-3- (1-methylpjperazin-4-ylsulfonyl) propionic acid

Example 6A

To a 0.3 M ethanolic solution of the product of Example 3B, (Z) -1-bromo-2-carbomethoxy-3-phenyl-2-propene (2-carbomethoxy-3-phenylpropane- 0.98 molar equivalents) was added a solution of 1.4 M sodium sulfite (1.0 molar equivalent) for one hour at 50 ° C. The mixture was stirred for 10 h at 50 ° C, and then the ethanol was removed under reduced pressure at 50 ° C. Acetic acid

(3kg per 1 kg of bromide) were added and the mixture was stirred for another 15 minutes and allowed to stand for 10 minutes. The phases were separated and the aqueous phase was washed with two additional aliquots of ethyl acetate (1 kg per 1 kg of bromide).

Raney nickel (1 kg per 1 kg of aqueous solution) was added to the aqueous solution which was then evacuated and purged with nitrogen, followed by hydrogen (3x) and placed in a hydrogen atmosphere at 40 psi for 6, 5 to 9.5 hours. The Raney nickel was removed by filtration using nitrogen pressure, and the filtrate was concentrated under reduced pressure at 55 ° C. A 10% aqueous solution of acetone (0.3 kg per 1 kg of starting bromide) was added to the residue obtained, and the mixture was heated at 50 ° C for 30 minutes. Additional acetone (3 kg per 1 kg of the starting bromide) was added slowly over one hour to effect crystallization of the product. After stirring for one hour the product was removed by filtration and washed with acetone to afford the title compound in about 60-65%: mp 255 ° C dec. . A second crop was obtained by addition of additional acetone (2.5 kg per 1 kg of the starting bromide) and cooling to -20 ° C for 10-12 hours and removal of the second crop by filtration. In this way an additional yield of 13-40% of the title compound was obtained.

Example 6B Methyl 2-benzyl-3- (1-methylpiperazin-4-ylsulfonyl) propionate The product of Example 6A (1 molar equivalent) was mixed with phosphorus pentachloride (1.5 molar equivalents) and heated to 70-75 ° C. C for 3-4 hours. The reaction mixture was cooled to room temperature and then diluted with toluene (16.7 molar equivalents) and added to a 10% aqueous sodium chloride solution (4 kg per 1 kg of phosphorus pentachloride) while maintaining the temperature below 40 ° C. The mixture was stirred for 5 minutes, allowed to stand for 15 minutes, and then the layers were separated. Washing with sodium chloride was repeated as described

74 357 4805.PG.03 -31- ago. The toluene phase was cooled to 5øC and N-methylpiperazine (3 molar equivalents in 3 molar equivalents of toluene) was added, maintaining the temperature below 15øC. The mixture was stirred for 4-6 hours and then washed with 8% aqueous sodium hydroxide solution (2 x 3.4 kg per 1 kg of phosphorus pentachloride). The combined organic phases were re-extracted with toluene (0.25 kg per 1 kg of the sodium hydroxide solution). The combined toluene extracts were washed with water (1 kg per 1 kg of phosphorus pentachloride), and the toluene was distilled off under reduced pressure to give the title compound (65-70%) as a white solid. viscous oil which crystallizes on standing. MS (DCI / NH 3) m / e 341 (M + H) +.

The product of Example 6B (69 kg, 20 mol) in acetone (420 kg) / water (960 kg) was adjusted to (2S) -2-benzyl-3-methylpiperazin-4-ylsulfonyl) pH 8.0 using 1N sodium hydroxide. Alcalase (Novo Industries, Denmark) (Subtilisin Carlsberg) (6.9 liters) was added and the pH was maintained between 7.9 and 8.4 by addition of 1N sodium hydroxide. When 80% of the theoretical amount of sodium hydroxide had been consumed, the reaction was quenched by the addition of ethyl acetate. The reaction mixture was concentrated to half the original volume under reduced pressure and was then washed with ethyl acetate (2 x 700 kg). The volume of the aqueous phase was concentrated to half and the pH adjusted to 5.2. The reaction mixture was treated with XAD-16 resin (50 kg), stirred for 18 hours and loaded onto a XAD-16 resin column (50 kg). The column was eluted with water (500 kg) and then with 35% ethanol in water (1000 kg) to provide a residue which was treated with isopropanol (270 kg) and heated to 75 ° C. Upon cooling to room temperature and subsequently to -5øC, a crystalline material was obtained. The solid was removed by filtration, washed with cold isopropanol (30 kg) and dried at 50 ° C to provide the title compound (13 kg, 49%). MS (DCI / NH 3) m / e 327 (M + H) +.

74 357 4805.PG.03 -32-

This compound can be recrystallized from a 1: 1 isopropanol / water mixture.

Example 7

Alternative preparation of (2S, 3R, 4S) -2-amino-1-cyclohexyl-4-ylsulfonyl) propionyl-L- (4-thiazolyl) hexyl-3,4-dihydroxy-6-methylheptane

Example 7A (2S, 3R, 4S) -2-Amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane

A 3.5% solution of 2S-t-butyloxycarbonylamino-1-cyclohexyl-3R, 4S-dihydroxy-6-methylheptane in 4N ethanolic hydrochloric acid at 0-5 ° C was prepared. After 4 hours at 0-5øC, nitrogen was bubbled through the reaction mixture to remove the dissolved hydrochloric acid. The solvent was removed under reduced pressure at 50 ° C to provide a solid which was dissolved in ethyl acetate and water. Potassium carbonate was added to bring the pH of the mixture to between 10 and 11, and the phases were separated. The aqueous phase was extracted with additional portions of ethyl acetate. The combined organic extracts were washed with water and brine, dried over magnesium sulfate, and concentrated under reduced pressure at 50 ° C to give a solid. The solid was crystallized by dissolving in a minimum amount of ethanol at 40 ° C and then slowly adding water until the ethanol / water ratio was 40/60 (w / w). The solution was cooled to 0-5 ° C for 2 hours and the product was collected by filtration. The solid was then dried under vacuum at 45 ° C to provide the title compound as a white crystalline solid (65-72%): mp 106-108 ° C. MS (DCI / NH 3) m / e 244 (M + H) +. 74 357 4805.PG.03 -33-

Example 7B

(2S, 3R, 4S) -2-Amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane Boc-L- (4-thiazolyl) ala-amide To a solution of the product of N-Boc-L- (4-thiazolyl) alanine (17.45 g, 64.4 mmol), and 1-hydroxybenzotriazole hydrate (HOBT) ( 9.86 g, 64.4 mmol) dissolved in dimethylformamide (DMF) (33 ml) and cooled to 0-5 ° C in an ice bath, was added dropwise, over 30 minutes, 3-dicyclohexylcarbodiimide (DCC) (14.5 g, 70.3 mmol), dissolved in DMF (27 ml). After 1 hour, the reaction mixture was allowed to warm to room temperature and stirred for 24 hours. The reaction was quenched by the addition of citric acid (1.14 g, 6.0 mmol) and ethanol (1.31 mL, 1.05 g, 22.0 mmol). The mixture was stirred for 1 hour and then ethyl acetate (285 ml) was added. After an additional 30 minutes, the solid by-product was removed by filtration and washed with ethyl acetate (48 ml). Additional ethyl acetate (1.9 L) was added and the organic phase was washed with 1% sodium chloride (713 ml), 5% citric acid containing 1% sodium chloride (2 x 713 ml), bicarbonate 8% sodium (2 x 713 ml) and 20% sodium chloride (2 x 713 ml) and concentrated under reduced pressure to provide a non-white solid. The solid was dissolved in isopropanol (200 ml), with heating, treated with charcoal to decolorize at 50øC for one hour, and filtered through Celite. The filtrate was diluted with isopropanol (50 ml) and stirred at room temperature with a mechanical stirrer for 15 hours. The solid suspension was cooled to 0-5 ° C with an ice bath and stirred at this temperature for 3 hours. The solid was removed by cold filtration, washed with a cold 1: 1 mixture of isopropanol / heptane (100 mL), and dried in a vacuum oven at 50 ° C for 48 hours to provide the title compound as a of a white solid in 85% yield: mp 156-158 ° C. MS (DCI / NH 3) m / e 498 (M + H) +. 74 357 4805.PG.03 -34-

Example 7C

(2S, 3R, 4S) -2-Amino-1-cyclohexyl-3,4-dihydroxy-6-methyl-heptane H-L- (4-thiazolyl)

A solution of 12% of the product of Example 7B in 3N aqueous hydrochloric acid was prepared at 15-25 ° C. After 4 hours at 15-25 ° C, the reaction mixture was quenched by being poured into a mixture of 4% sodium hydroxide / 15% sodium chloride / ethyl acetate. The pH of the mixture was brought to 10-12 by the addition of 10% sodium hydroxide. The phases were separated and the aqueous phase was extracted with ethyl acetate (2x). The combined organic extracts were washed with 25% sodium chloride (2x), dried over magnesium sulfate, treated with activated charcoal at 50 ° C for 1 hour, and filtered through Celite. The filtrate was concentrated to a solid under reduced pressure at 45 ° C. The solid was crystallized by dissolving in a minimal amount of ethyl acetate (5 x weight) and triturating with heptane until the ratio of ethyl acetate to heptane was 30/70 (w / w). The solution was cooled to 0-5 ° C and stirred for two hours and then filtered. The solid was dried in a vacuum oven at 45 ° C for 60 hours or until the weight loss on drying was less than 0.1%. The title compound was obtained as a white crystalline solid, in 70-82% yield. mp 109-112 ° C. MS (DCI / NH 3) m / e 398 (M + H) +.

Example 2 (2S) -3-Amino-1-cyclohexyl-3- (1-methylpiperazin-4-ylsulfonyl) propionyl-L- (4-thiazolyl) , 4-dihydroxy-6-methylheptane The product of Example 7C (3.00 g, 7.6 mmol), the product of Example 4C, 2S-benzyl-3- (1-methylpiperazin-4 (2.59 g, 7.9 mmol) and HOBT (1.27 g, 8.3 mmol) were dissolved in DMF (30 mL). After stirring at room temperature for 1 hour, the reaction mixture was cooled to

In an ice bath and treated by dropwise addition over 30 minutes of a solution of DCC (1.72 g, 8.3 mmol) in CH 2 Cl 2 mmol), dissolved in DMF (8 mL). After 1 hour, the reaction mixture was allowed to warm to room temperature and stirred for 24 hours. The reaction mixture was quenched with citric acid (0.15 g, 0.26 mmol) and ethanol (0.17 mL, 3.04 mmol) and stirred for 1 hour. Ethyl acetate (60 ml) was added and the mixture was stirred for an additional hour. The by-product was removed by filtration and washed with ethyl acetate (10 ml). The filtrate was diluted with ethyl acetate (400 ml) and washed with 5% sodium bicarbonate solution (2 x 100 ml), 1% sodium chloride solution (100 ml), and a solution of chloride 20% sodium hydroxide solution (100 ml). The solvent was removed under reduced pressure to provide an off-white solid. The solid was dissolved in isopropanol (80 ml) with heating, treated with decolorizing charcoal at 55øC for 1 hour, filtered through Celite, and stirred at room temperature with a mechanical stirrer for 12 hours. The suspension of the white solid was cooled to 0-5 ° C in an ice bath for 3 hours and filtered cold. The solid obtained was washed with a cold 1: 1 mixture of heptane / isopropanol (25 ml) and dried in a vacuum oven at 55 ° C for 48 hours to provide the title compound (4.32 g, 81%) as a white solid: mp 169-170 ° C. MS (DCI / NH 3) m / e 706 (M + H) +.

Example 8 (2S, 3S) -4-Amino-1-cyclohexyl-3,4-dihydroxy-6 -methylheptane The title compound can be prepared according to the procedure described in European Patent Application EP 399556, published November 28, 1990. 74 357 4805.PG.03

-36-

Example 9 (S) -Gr (S) -ar (t-butylsulfonyl) methyl] hydrocinnamidol-N '(1R, 2S, 3R) -1-cyclohexylmethyl-3-cyclopropyl-2,3-dihydroxypropynimidazol-4-ylpropionamide The title compound can be prepared according to the procedure described in Patent Application EP 332008, published September 13, 1989.

Example 10

(2S, 3R, 4S) -2-Amino-2- (2S) -2-benzyl-3- (1-methylpiperazin-4-ylsulfonyl) propionyl- (4-thiazolyl) -1-cyclohexyl-3,4-dihydroxy-6-methylheptane

Acetyl chloride (3.27 g, 41.65 mmol) was added to ethanol (150 mL) at 5 ° C. The mixture was stirred for two hours at 5 ° C and added to a suspension of the product of Example IR (30 g, 42.5 mmol) in ethanol (150 mL) at 5 ° C. After 2 hours, the mixture was filtered and the solvent removed in vacuo. The residue was dissolved in methylene chloride (300 ml) and the desired product precipitated by addition of 3: 1 heptane / ethyl acetate, either diethyl ether (1500 ml) or methyl t-butyl ether (1500 ml). Calc'd for C 35 H 56 N 5 O 6 S 2 ClO 0.5H 2 O: C, 55.94; H, 7.65; N, 9.32; Cl, 4.72; S, 8.53. Found: C, 56.06; H, 7.58; N, 9.30; Cl, 4.95; S, 8.17.

74 357 4805.PG.03 -37-

Example 11

Composition for tablet Na 4-6 5 25 125

Dosage of the tablet (in mg of compound I base)

Component mcf per tablet

Compound I (HCl salt) 5.3 26.3 131.4 Microcrystalline cellulose (Avicel ™ 99.5 105.0 314.0 Citric acid, USP, anhydrous 4.0 7.5 22.8 Crospovidone, NF 4, 6 o in OH Croscarmellose sodium, NF 4.6 5.0 o H Povidone, NF, K-30 6.0 n / an / a Magnesium stearate, (impalpable powder, NF) 1.0 -1.23. 8 Tablet Weight (mg) 125.0 150.0 500.0

A process for preparing the prior tablet formulations

The tablet compositions shown above may be prepared according to the following illustrative example, based on the 25 mg tablet.

To prepare 10 000 25 mg tablets: The HCl salt of Compound I (263 grams) was mixed with

Avicel (approximately 300 grams) and sieved through a 30 mesh mesh sieve. 2. Avicel (approximately 800 grams) was sieved through a 30 mesh mesh sieve and mixed with the mixture from step 1. 3. Citric acid (30 grams) was sieved through a 30 mesh mesh sieve and mixed with the mixture from step 3.

Crospovidone and croscarmellose (25 grams each) were sieved through a 30 mesh mesh screen and mixed with the mixture from step 3. 5. NOTE: For the tablet only of 5 mg, the povidone was sieved through a 30 mesh mesh sieve and mixed with the mixture from step 4. 6. The powder mixture of step 4 (25 mg and 125 mg tablet) or from step 5 (tablets of 5 mg) was blended for 2 or 4 minutes in a Hobart mixer or equivalent planetary mixer. 7. The mixture from step 6 was granulated with distilled water (approximately 30 to 40 ml per 100 grams of granulate). The water was added for 5 to 10 minutes and the mixture was continued for another 2 to 4 minutes. 8. The granulate was transferred to paper-coated trays and dried in an oven at 60øC until the moisture content was less than 2.5%. 9. The granules were sifted by hand through a 20 mesh mesh sieve. 10. Citric acid (45 grams), crospovidone (25 grams) and croscarmellose (25 grams) were sieved by hand through a 30 mesh mesh sieve. The granules from step 9 and the sieved materials from step 10 were transferred to a V-blender or equivalent and mixed for about 10 minutes. 12. Magnesium stearate (12 grams) was mixed with one portion (approximately 50 to 100 grams) of the mixture from step 11 and then sieved through a 20 mesh mesh screen. 13. The mixture from step 12 was blended with the mixture from step (a) in a V-blender or equivalent apparatus, approximately 4 minutes, for 74 357 4805.PG.03 * 11. 14. The blend of step 13 is compressed into a rotary press. The tablet weights were set according to the formula (e.g., 150 mg total weight for a 25 mg tablet).

Example 12

Comparisons of bioavailability

The following compositions were prepared comprising compound I and the bioavailability of each composition in fasted dogs was determined.

Composition SI

A composition comprising the dihydrochloride salt of compound I (115.9 mg), Avicel (300.0 mg), crospovidone (47.6 mg), sodium starch glycolate (47.6 mg) and magnesium stearate (5.0 mg) was made into a tablet.

Composition S2

A composition comprising the dihydrochloride salt of compound I (115.9 mg), Avicel (500.0 mg), crospovidone (195.2 mg), sodium starch glycolate (195.2 mg) and magnesium stearate (10.0 mg) was made into a tablet.

Composition S3

A composition comprising the dihydrochloride salt of compound I (110.4 mg), PEG 1450 (100 mg), Avicel (100 mg) and lactose (100 mg) was encapsulated (hard gelatin capsules, 000, for human use). 74 357 4805.PG.03 -40-

Composition S4

A composition comprising the monomethanesulfonate salt of compound I (120.3 mg), Aviceil (250 mg), crospovidone (150 mg) and magnesium stearate (2.5 mg) was made into a tablet.

Composition S5

A composition containing the dimethanesulfonate salt of compound I (120.5 mg), Avice ® (150 mg) and lactose (150 mg), was encapsulated (capsules with hard, gray gelatin shell, # 000, for human use).

Protocol for oral bioavailability studies

Dogs (beagle dogs of both sexes, weighing about 10 kg) were left unattended for at least 12 hours prior to measurement. They resumed feeding after the last blood sample was collected (24 hours). The water was always at his disposal.

A dose equivalent to 50 or 100 milligrams of compound I, followed by either 20 or 50 ml of water, was administered through the mouth to each dog through a feed tube.

Blood samples were collected at the following times: 0, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 hours. Blood samples of 3 ml volume were anti-coagulated with EDTA. The plasma was separated by centrifugation and frozen until analyzed. Concentrations of compound X in blood plasma were determined by HPLC. The bioavailability was calculated as follows:% Bioavailability = oral (AUC) x D / (intravenous AUC) x 100. (AUC) and intravenous (AUC) are the areas below the curves of the change in plasma concentration in function after oral and intravenous administration, respectively. D is defined as the ratio between intravenous and oral doses. In particular, oral D = (DOSE) iv / (DOSE). The intravenous dose was 3.0 mg / kg and at C 74 357 4805.PG.03

The oral dose was about 5-20 mg / kg or about 10-20 mg / kg. The value of intravenous (AUC) used corresponds to the mean value of a separate group of six dogs. The data on bioavailability and Cmax (maximal concentration) for compositions 1-3 and S1-S5 are shown in Table 1.

Table 1

Comnosicão Ns. Ns. of dogs Average biodegradability C max ua / ml 1 (EX IA) 8 69.8 2.45 2 (Ex. 1B) 8 44.0 1.76 3 (Ex. 1C) 12 71.1 4.17 4 (Ex. 1D) 8 72.4 2.6 SI 6 44.0 2.7 S2 6 31.0 1.2 S3 8 18.0 1.3 S4 8 9.2 0.8 S5 8 35.0 1.5

These data indicate that compound I is up to 7.8 times more bioavailable when administered orally in the form of a tablet comprising a pharmaceutically acceptable organic polycarboxylic acid, compared to an encapsulated tablet or powder without the organic polycarboxylic acid.

The compounds of formula II are renin inhibitors and have an excellent degree of activity and specificity in the treatment of hypertension in humans or other mammals. The novel compounds of the present invention are also useful in the treatment of congestive heart failure in humans or other mammals. The compounds of formula II are also useful in the treatment of vascular problems in humans or other mammals, especially in vascular diseases associated with diabetes, such as diabetic nephropathy, diabetic neuropathy and diabetic retinopathy. The compounds of formula II are also useful in the treatment of renal diseases in humans or other mammals, in particular in chronic and acute renal insufficiencies. Compounds of formula II are also useful in the treatment of psoriasis in humans or other mammals. The total daily dose of a compound II, administered to a human or other mammal in a single or divided dose, may be in amounts, for example, 0.001 to 10 mg / kg body weight, daily, and more usually 0.01 to 10 mg / kg. The dosage unit compositions may contain such quantities or submultiples thereof to complete the daily dose. The amount of the active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the patient treated and the particular mode of administration.

It is to be understood, however, that the specific dosage level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, age, weight, general health, sex, , the time of administration, the route of administration, the rate of excretion, the drug combination, and the severity of the particular disease in which the therapy is being performed. The foregoing is merely illustrative of the invention and is not intended to limit the invention to the disclosed compounds, methods and compositions. Variations and changes which are obvious to those skilled in the art are considered to fall within the scope and nature of the invention which is defined in the appended claims.

Claims (10)

74 357 4805.PG.03 A pharmaceutical composition comprising a compound of the formula: in which R 1 is 4-piperazinyl, 1-methyl-4-piperazinyl, 1-methyl-1-oxo-4-piperazinyl, 2-oxo-4-piperazinyl, 4-morpholinyl, 4-thiomorpholinyl or 1-methyl- 4-homopiperazinyl; R2 is benzyl, p-methoxybenzyl, 2-phenylethyl, 1-naphthylmethyl or 2-naphthylmethyl; R 3 is 4-thiazolyl, 2-amino-4-thiazolyl, 2-thiazolyl, 5-thiazolyl, 1-pyrazolyl, 3-pyrazolyl, 1-imidazolyl, n-propyl, isopropyl, CH 3 S- or CH 3 SCH 2 -; R4 is lower alkyl or cyclopropyl; R 5 is hydrogen or lower alkyl; and X is CH2 or NH; or a pharmaceutically acceptable salt, ester or prodrug thereof, and a pharmaceutically acceptable organic polycarboxylic acid. Composition according to Claim 1, characterized in that the organic polycarboxylic acid is citric acid. A pharmaceutical composition comprising a compound of the formula: 74 357 4805.PG.03 44 in which R 1 is 4-piperazinyl, 1-methyl-4-piperazinyl, 1-methyl-1-oxo-4-piperazinyl, 2-oxo-4-piperazinyl, 4-morpholinyl, 4-thiomorpholinyl or 1-methyl- 4-homopiperazinyl; R2 is benzyl, p-methoxybenzyl, 2-phenylethyl, 1-naphthylmethyl or 2-naphthylmethyl; R 3 is 4-thiazolyl, 2-amino-4-thiazolyl, 2-thiazolyl, 5-thiazolyl, 1-pyrazolyl, 3-pyrazolyl, 1-imidazolyl, n-propyl, isopropyl, CH 3 S- or CH 3 SCH 2 -; R4 is lower alkyl or cyclopropyl; R 5 is hydrogen or lower alkyl; and X is CH2 or NH; or a pharmaceutically acceptable salt, ester or prodrug thereof, from about 1% by weight to about 20% by weight of a pharmaceutically acceptable organic polycarboxylic acid and from about 30% by weight to about 90% by weight of weight of a load. A composition according to claim 3, characterized in that the organic polycarboxylic acid is citric acid and the filler is microcrystalline cellulose. A pharmaceutical composition comprising a compound of the formula: or a pharmaceutically acceptable salt, ester or prodrug thereof, from about 1% by weight to about 20% by weight of a pharmaceutically acceptable organic polycarboxylic acid, and from about 30% by weight to about 90% by weight of weight of a load. Composition according to Claim 5, characterized in that the organic polycarboxylic acid is citric acid and the filler is microcrystalline cellulose. 74 357 4805.PG.03 -45- A pharmaceutical composition comprising from about 4.0 wt.% To about 30 wt.% Of a compound of the formula: or a pharmaceutically acceptable salt, ester or prodrug thereof, from about 1% by weight to about 20% by weight of a pharmaceutically acceptable organic polycarboxylic acid, and from about 30% by weight to about 90% by weight of a filler. Composition according to Claim 7, characterized in that the organic polycarboxylic acid is citric acid and the filler is microcrystalline cellulose. A pharmaceutical composition comprising from about 4.0 wt.% To about 30 wt.% Of a compound of the formula: or a pharmaceutically acceptable salt, ester or prodrug thereof, from about 3% by weight to about 5% by weight of citric acid, from about 40% by weight to about 80% by weight of microcrystalline cellulose, from about 2% by weight to about 4% by weight of crospovidone, from about 2% by weight to about 4% by weight of croscarmellose sodium and from about 0.5% by weight to about 74 357 4805.PG.03 -46- 1.5% by weight of magnesium stearate. A pharmaceutical tablet comprising (A) about 5.3 mg of the hydrochloride salt of the compound of the formula: about 99.5 mg of microcrystalline cellulose, about 4.0 mg of citric acid, about 4.6 mg of crospovidone, about 4.6 mg of croscarmellose sodium, about 6.0 mg of povidone and about of 1.0 mg of magnesium stearate; or (B) about 26.3 mg of the hydrochloride salt of the compound of the formula: about 105 mg of microcrystalline cellulose, about 7.5 mg of citric acid, about 5.0 mg of crospovidone, about 5.0 mg of croscarmellose sodium and about 1.2 mg of magnesium stearate; or (C) about 131.4 mg of the hydrochloride salt of the compound of the formula: (following formula) 74 357 4805.PG.03 about 314 mg of microcrystalline cellulose, about 22.8 mg of citric acid, about 14 mg of crospovidone, about 14 mg of croscarmellose sodium and about 3.8 mg of magnesium stearate. Lisbon, 'IdOUT. 1992 By ABBOTT LABORATORIES = 0 0FICIAL AGENT =