AU748469B2

AU748469B2 - Endothelin antagonists

Info

Publication number: AU748469B2
Application number: AU85921/98A
Authority: AU
Inventors: Steven A. Boyd; Kenneth J. Henry; Charles W Hutchins; Hwan-Soo Jae; Jeffrey A. Kester; Steven A. King; Gang Liu; Bryan K Sorensen; Bruce G. Szczepankiewicz; Andrew S. Tasker; Thomas W. Von Geldern; Martin Winn; Steven J. Wittenberger
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1996-02-13
Filing date: 1998-07-27
Publication date: 2002-06-06
Anticipated expiration: 2017-02-12
Also published as: TR200501137T2; BG104216A; TR200101233T2; EP1003740A2; NO20000542D0; TW552260B; PL342500A1; CA2297894A1; IL134175A0; NO20000542L; WO1999006397A3; WO1999006397A2; TR200101234T2; HUP0003484A2; BR9815296A; HUP0003484A3; JP2001512119A; TR200000993T2; AU8592198A; SK1452000A3

Description

ENDOTHELIN ANTAGONIST TECHNICAL FIELD The present invention relates to a compound which is an endothelin antagonist, and to compositions for antagonizing endothelin.

BACKGROUND OF THE INVENTION Endothelin (ET) is a 21 amino acid peptide that is produced by endothelial cells.

ET is produced by enzymatic cleavage of a Trp-Val bond in the precursor peptide big endothelin (Big ET). This cleavage is caused by an endothelin converting enzyme (ECE).

Endothelin has been shown to constrict arteries and veins, increase mean arterial blood 0t pressure, decrease cardiac output, increase cardiac contractility in vitro, stimulate mitogenesis in vascular smooth muscle cells in vitro, contract non-vascular smooth muscle including guinea pig trachea, human urinary bladder strips and rat uterus in vitro, increase airway resistance in vivo, induce formation of gastric ulcers, stimulate release of atrial natriuretic factor in vitro and in vivo, increase plasma levels of vasopressin, aldosterone and catecholamines, inhibit release of renin in vitro and stimulate release of gonadotropins in vitro.

It has been shown that vasoconstriction is caused by binding of endothelin to its receptors on vascular smooth muscle (Nature 332 411 (1988), FEBS Letters 231 440 (1988) and Biochem. Biophys. Res. Commun. 154 868 (1988)). An agent which 20 suppresses endothelin production or an agent which binds to endothelin or which inhibits the binding of endothelin to an endothelin receptor will produce beneficial effects in a variety of therapeutic areas. In fact, an anti-endothelin antibody has been shown, upon intrarenal infusion, to ameliorate the adverse effects of renal ischemia on renal vascular resistance and glomerular filtration rate (Kon, et al., J. Clin. Invest. 83 1762 (1989)). In 25 addition, an anti-endothelin antibody attenuated the nephrotoxic effects of intravenously administered cyclosporin (Kon, et al., Kidney Int. 37 1487 (1990)) and attenuated infarct size in a coronary artery ligation-induced myocardial infarction model (Watanabe, et al., Nature 344 114(1990)).

Clozel et al. (Nature 365 759-761 (1993)) report that Ro 46-2005, a nonpeptide ET-A/B antagonist, prevents post-ischaemic renal vasoconstriction in rats, prevents the decrease in cerebral blood flow due to subarachnoid hemorrhage (SAH) in rats, and decreases MAP in sodium-depleted squirrel monkeys when dosed orally. A similar effect of a linear tripeptide-like ET-A antagonist, BQ-485, on arterial caliber after SAH has also been recently reported (S.Itoh, T. Sasaki, K. Ide, K. Ishikawa, M. Nishikibe, and M.

AL Yano, Biochem. Biophys. Res. Comm., 195 969-75 (1993)). These results indicate that agents which antagonize ET/ET receptor binding will provide therapeutic benefit in the indicated disease states.

Agents with the ability to antagonize ET/ET receptor binding have been shown to be active in a number of animal models of human disease. For example, Hogaboam et al (EUR. J. Pharmacol. 1996, 309, 261-269), have shown that an endothelin receptor antagonist reduced injury in a rat model of colitis. Aktan et al (Transplant Int 1996, 9, 201-207) have demonstrated that a similar agent prevents ischemia-reperfusion injury in kidney transplantation. Similar studies have suggested the use of endothelin antagonists in the treatment of angina, pulmonary hypertension, Raynaud's disease, and migraine. (Ferro and Webb, Drugs 1996, 51,12-27).

Abnormal levels of endothelin or endothelin receptors have also been associated with a number of disease states, including prostate cancer (Nelson et al, Nature Medicine 1995, 1, 944-949), suggesting a role of endothelin in the pathophysiology of these diseases.

Wu-Wong et al (Lfe Sciences 1996, 58, 1839-1847) have shown that both endothelin and endothelin antagonists bind tightly to plasma proteins, serum albumin. This plasma protein binding can decrease the effectiveness with which the antagonists inhibit endothelin's action. Thus, endothelin antagonists with reduced plasma protein binding may be more effective than highly bound congeners.

20 DISCLOSURE OF THE INVENTION :In accordance with the present invention, there is provided the compound 2 S,3R,4S]-2-(4-Methoxyphenyl)-4-(1,3-benzodioxol-5-yl) [(N-butyl-N-(4-dimethylaminobutyl)amino)carbonylmethyl]-pyrrolidine-3-carboxylic acid or a pharmaceutically acceptable salt thereof.

S 25 The invention also provides a pharmaceutical composition comprising 2 S,3R,4S]-2-(4-Methoxyphenyl)-4-(1,3-benzodioxol-5-yl)- 1 -[(N-butyl-N-(4-dimethyl- Saminobutyl)amino)carbonylmethyl]-pyrrolidine-3-carboxylic acid together with one or more pharmaceutically acceptable carriers, excipients, diluents and/or adjuvants.

Scheme I illustrates the general procedure for preparing the compound of the invention and related compounds. A p-ketoester 1, where E is loweralkyl or a carboxy protecting group is reacted with a nitro vinyl compound 2, in the presence of a base (for example, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or sodium ethoxide or sodium hydride and the like) in an inert solvent such as toluene, benzene, tetrahydrofuran or ethanol and the like. The condensation product 3 is reduced (for example, hydrogenation ing a Raney nickel or platinum catalyst). The resulting amine cyclizes to give the dihydro pyrrole 4. Reduction of 4 (for example, sodium cyanoborohydride or catalytic hydrogenation and the like) in a protic solvent such as ethanol or methanol and the like gives the pyrrolidine compound 5 as a mixture of cis-cis, trans,trans and cis,trans products. Chromatographic separation removes the cis-cis isomer leaving a mixture of the trans,trans and cis,trans isomers which is further elaborated. The cis-cis isomer can be epimerized (for example, using sodium ethoxide in ethanol) to give the trans,trans isomer and then carried on as described below. The pyrrolidine nitrogen is acylated or sulfonylated with R 3 -X (R 3 is R 4 or R 6

-S(O)

2 and X is a leaving group such as a halide (Cl is preferred) or X taken together with R 4 or R 6 -S(0) 2 forms an activated to ester including esters or anhydrides derived from formic acid, acetic acid and the like, alkoxycarbonyl halides, N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxybenzotriazole, N-hydroxy-5-norbomene-2,3-dicarboxamide, 2,4,5-trichlorophenol and the like) or alkylated with R 3 -X where X is a leaving group (for example, X is a halide (for example, Cl, Br or I) or X is a leaving group such as a sulfonate (for example, mesylate, tosylate, triflate and the like)) in the presence of a base such as diisopropyl ethylamine or triethylamine and the like to give the N-derivatized pyrrolidine 6 which is still a mixture of trans,trans and cis,trans isomers. Hydrolysis of the ester 6 (for example, using a base such a sodium hydroxide in EtOH/H 2 0) selectively hydrolyzes the trans,trans ester to give a mixture of 7 and 8, which are readily separated.

0** o* *g Co e Scheme I

CO

2

E

R kklNO0 2 R, C0 2

E

N0 2

N

A

2 C0 2

E

H

C0 2

E

Mixture of cis-cis Twas-Trans Cis-Tranls 4, 4**e .4 4O S

S

*4

S*

S *94* *4 *5

S

.SS*

0 5**4 5* S S S S 5* 5* *0

S

jX-R 3 /R 3 E C0 2

E

Mixture of Trans-Trans Cis-Trans I H 2 01 C0 2

H

Trans-Trarls N .R3 C0 2

E

Cis-Trans For acylation of the pyrrolidine nitrogen with N-(Q-hydroxyalkyl)-N-alkyl haloacetamnides, further transformations of the alkylation product are possible (Scheme 11). Activation (for example using miethanesulfonyl chloride) of the alcohol, followed by displacement with halogen (for example, using lithim bromide) provides the corresponding halide. Displacement of halide with an amine, for example dimethylamine, provides the corresponding amino ester, which may be hydrolyzed as previously described to provide product.

Scheme 11 0 R c k 0HR H N CVt M s C I N l C O~ 2. Li~r 0I COE 0 'R 0 *R

(CH

3 2 NH 'R u' NaO OS S R N~~Ny~~N

"COOH

0 0 S R The foregoing may be better understood by reference to the following examples which are provided for illustration and not intended to limit the scope of the inventive concept. The following abbreviations are used: DBU for 1,8-diazabicyclo[5.4.0]undec-7ene, EtOAc for ethyl acetate, EtOH for ethanol, TFA for trifluoroacetic acid and THF for tetrahydrofuran.

EXAMPLE 1 trans, trans-2-(4-Methoxyphenyl)-4-(1,3-benzodioxol-5-yl)- -(propylaminocarbonylmethyl) -pyrrolidine-3-carboxylic acid EXAMPLE 1A 0o Ethyl 2 -(4-methoxybenzoyl)-4-nitromethyl-3-(1,3-benzodioxole-5-yl)butyrate To ethyl 4 -methoxybenzoyl)acetate (23.0 g, 0.104 mol), prepared by the method of Krapcho et al., Org. Syn. 47, 20 (1967), and 5-( 2 -nitrovinyl)-1,3-benzodioxole (17.0 g, 0.088 mol) dissolved in 180 mL of toluene and heated to 800 C was added 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU, 0.65 g) with stirring. The mixture was heated until all the nitro starting material dissolved. The solution was stirred without heating for minutes (min) and then an additional 0.65 g of DBU was added. After stirring an additional 45 minutes, thin layer chromatography ethyl acetate in methylene chloride) indicated the absence of nitro starting material. Toluene (200 mL) was added, and the organic phase was washed with dilute hydrochloric acid and NaCl solution. The 20 organic phase was dried over sodium sulfate and then concentrated under reduced pressure. The residue obtained was chromatographed on silica gel eluting with 3:1 hexane-ethyl acetate to give 21.22 g of the desired product as a mixture of isomers and 9.98 g. of recovered ethyl 4 -methoxybenzoyl)acetate.

S.i EXAMPLE 1B 25 Ethyl 2 -(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-4,5-dihydro-3H-pyrrole-3carboxylate The compound resulting from Example 1A (21 g) in 500 mL of ethanol was hydrogenated under 4 atmospheres of hydrogen pressure using a Raney nickel 2800 .catalyst (51 (The Raney nickel was washed with ethanol three times before use.) The catalyst was removed by filtration, and the solution was concentrated under reduced pressure. The residue obtained was chromatographed on silica gel eluting with 8.5% ethyl acetate in methylene chloride to give 12.34 g of the desired product.

.doc:clb EXAMPLE 1C Ethyl 2-(4-methoxyphenyl-4-( 1,3-benzodioxol-5-yl)-pyrrolidine-3-carboxylate) as a mixture ofcis-cis; trans,trans; and cis,trans-isomers The compound resulting from Example 1B (11.89 g, 0.324 mol) was dissolved in 27 mL of tetrahydrofuran and 54 mL of ethanol. Sodium cyanoborohydride (2.35 g, 0.374 mol) and 5 mg bromocresol green were added. To this blue solution was added dropwise a solution of 1:2 concentrated HCI in ethanol at such a rate that the color was kept at light yellow-green. After the yellow color persisted without additional HC1, the solution was stirred an additional 20 minutes. The solution was concentrated in vacuo and then partitioned between chloroform and an aqueous potassium bicarbonate solution. The organic phase was separated, dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on silica gel eluting with 85:15 ethyl acetatehexane to give 5.96 g. of a mixture of 64% trans,trans-compound and 34% cis,transcompound. Further elution with pure ethyl acetate gave 0.505 g of an unknown solid followed by 3.044 g of pure cis,cis-compound.

EXAMPLE 1D trans, trans-2-(4-Methoxyphenyl)-4-(1,3-benzodioxol-5-yl)- 1-(propyaminocarbonylmethyl)-pyrrolidine-3-carboxylic acid The mixture of 64% trans,trans- and 34% cis,trans-pyrrolidines (the mixture 20 resulting from Example 1C) (5.72 g, 15.50 mmol), ethyldiisopropylamine (4.20 g, 32.56 and N-propyl bromoacetamide (3.42 g, 19.0 mmol), prepared by the method of Weaver, W. E. and Whaley, W. J. Amer. Chem. Soc., 69 515 (1947), in 30 mL of acetonitrile was heated at 500 C for 1 hour. The solution was concentrated in vacuo. The residue was dissolved in toluene, shaken with potassium bicarbonate solution, dried over S 25 sodium sulfate and concentrated in vacuo to give 7.16 g of product as a mixture of trans,trans- and cis,trans-ethyl esters.

This mixture was dissolved in a solution of 50 mL of ethanol and 15 mL of water containing 5.00 g of sodium hydroxide and stirred for 3 hours at room temperature. The solution was concentrated in vacuo and 60 mL of water added. The mixture was extracted with ether to remove the unreacted cis,trans- ethyl ester. The aqueous phase was treated with hydrochloric acid until slightly cloudy. It was then further neutralized with acetic acid to give the crude acid product. The crude product was filtered and purified by dissolving it in tetrahydrofuran, drying over sodium sulfate, concentrating in vacuo, and crystallizing from ether to give 3.230 g of the title compound. m.p. 151-1530 C. 'H NMR

A

(CD

3 OD, 300 MHz) 5 0.87 J=7 Hz, 3H), 1.49 (sextet, J=7 Hz, 2H), 2.84 J=16 Hz, [R:\LIBZ]05685.doc:clb 8 1H), 2.95-3.20 (in, 4H), 3.20 J=16 Hz, 1H), 3.34-3.42 (in, 1H), 3.58-3.66 (in, 1H), 3.78 3H), 3.88 J=10 Hz, 1H), 5.92 2H), 6.75 J=8 Hz, 1H), 6.86 (dd, J=8 Hz, J=1 Hz, 1H), 6.90 J=9 Hz, 2H), 7.02 J=1 Hz, 1H), 7.40 J=9 Hz, 2H).

EXAMPLE 2 trans, trans-2-(4-Methoxyphenyl)-4-(J, 4-benzodioxan-6-yl) -1-(propylaminocarbonylmethyl)-pyrrolidine-3-carboxylic acid Using the procedures described in Example 1 and substituting 6-(2-nitrovinyl)- 1,4-benzodioxane for 5-(2-nitrovinyl)-1,3-benzodioxole afforded the title compound. m.p.

80-8 1 C. 'H NMR (CDC1 3 300 MHz) 6 0.89 J=7Hz, 3H), 1.49 (sextet, J=7Hz, 2H), 2.78 J=16Hz, lH), 2.92 J=IOHz, 1H), 3.05-3.43 (in, 5H), 3.24 J=l6Hz, 1H), 3.52-3.62 (in, 1H), 3.80 3H), 3.80 J=lOHz, IH), 4.27 4H), 6.74-6.93 (in, 7.29 J=9Hz, 2H). MS (DCIINH 3 m/e 455 EXAMPLE 3 trans, trans-2-(4-Methoxyphenyl)-4-(], 3-benzodioxol-S-yl)-J-(NN-di(n-butyl)aminocarbonylmethyl)-pyrrolidine-3-carboxylic Acid The title compound was prepared using the procedures described in Example 1.

m.p. 123-125' C. 'H NMR (CDCl 3 300 MHz) 8 0.79 J=7 Hz, 3H), 0.85 J=7 Hz, 3H), 1.00-1.50 (in, 8H), 2.74 J=13 Hz, 1H), 2.90-3.09 (in, 4H), 3.23-3.50 (in, 3H), 3.38 J=13 Hz, 1H), 3.52-3.62 (in, 1H), 3.75 J=10 Hz, 1H), 3.78 3H), 5.93 (dd, eg 20 J=2 Hz, 4 Hz), 6.71 J=8 Hz, 1H), 6.81-6.89 (in, 3H), 7.03 J=2 Hz, 1H), 7.30 (d, J=9 Hz, 2H). MS (DCI/Nil 3 m/e 511 Anal calcd for C 2 9

H

3 8

N

2 0 6 C, 68.21; H, Pg...7.50; N, 5.49. Found: C, 68.07; H, 7.47; N, 5.40.

EXAMPLE 4 trans, trans-I -(NN-Di(n-butyl)aminocarbonylmethyl)-2-(4-methoxyphenyl)-4-(l, 3-benzo- 25 dioxol-S-yl)pyrrolidine-3-carboxylic Acid Using the procedures described in Examples 2 and 3, the title compound was prepared as a white solid. m.p. 74-760 C. 'H NMR (CDC1 3 300 MHz) 6 0.80 J=6 Hz, 3H), 0.88 J=8 Hz, 3H), 1.08 (sextet, J=8 Hz, 2H), 1.21-1.48 (in, 6H), 2.75 J=12 Hz, C. 1H), 2.95-3.09 (in, 4H), 3.26-3.59 (mn, 5H), 3.75 J=9 Hz, 1H), 3.79 3H), 4.28 (s, 4H), 6.78 J=9 Hz, 1H), 6.85 J=9 Hz, 2H), 6.91 J=3 Hz, 9 Hz, 1H), 6.98 (d, J=3 Hz, IH), 7.32 J=9 Hz, 2H). MS (DCIN} 3 m/e 525 EXAMPLE Alternate Preparation of (+)-trans, trans-i -(NN-Di(n-butyl)aminocarbonylmethyl)-2-(4methoxyphenyl)-4-(1, 3-benzodioxol-5-yl)pyrrolidine-3-carboxylic acid Hydrochloride 7 Salt [R:ULBZ]05685.doc:cb EXAMPLE N,N-Dibutyl bromoacetamide To a solution ofbromoacetyl bromide (72.3 mL, 830 mmol) in toluene (500 mL) cooled to 00 C was added a solution of dibutylamine (280.0 mL, 1.66 mol) in toluene (220 s mL) via an addition funnel maintaining the reaction temperature below 100 C. Upon completion of the-addition, the reaction mixture was stirred at 00 C for 15 minutes. A solution of 2.5% aqueous H 3 P0 4 (500 mL) was slowly introduced, and the reaction mixture was allowed to warm to room temperature with vigorous stirring. The solution is phosphoric acid by weight. The layers were separated and the organic phase washed 0o with water (500 mL) and concentrated to provide the bromoacetamide as a solution in toluene.

EXAMPLE 5-(2-Nitrovinyl)-1, 3-benzodioxole To piperonal (15.55 kg, 103.5 mol) under mechanical stirring and under nitrogen was added ammonium acetate (13.4 kg, 173.8 mol), acetic acid (45.2 kg), and nitromethane (18.4 kg, 301.4 mol) sequentially. The mixture was warmed to 700 C. After about 30 minutes, the yellow product began to crystallize. The reaction temperature was raised to 800 C and stirred for about 10 hours until minimal piperonal remains. The somewhat thick reaction mixture was cooled to 100 C and filtered. The precipitate was 20 washed with acetic acid (2 x 8 kg) and then water (2 x 90 kg). The product was dried under a nitrogen purge and then in a vacuum oven at 500 C for 2 days to afford 15.94 kg of the title compound as a bright yellow solid.

EXAMPLE 4-Methoxybenzoyl acetate a* 25 To potassium t-amylate (25 wt 50.8 kg, 99.26 mol) in toluene (15.2 kg) cooled to 50 C under mechanical stirring and under nitrogen was added a mixture of 4- C: methoxyacetophenone (6.755 kg, 44.98 mol) and diethyl carbonate (6.40 kg, 54.18 mol) in toluene over 1 hour maintaining the temperature below 100 C. The reaction mixture o was heated to 600 C for 8 hours until no 4 -methoxyacetophenone was detected by HPLC.

The mixture was cooled to 200 C and quenched by adding to a mixture of acetic acid (8 kg) and water (90 kg) over 30 minutes while maintaining the temperature at <200 C. The layers were separated, and the organic layer was washed with 5% sodium bicarbonate solution (41 kg) and concentrated to 14.65 kg. The temperature is maintained below 500 C during the distillation. The yellow product concentrate was assayed by HPLC against an external standard and the yield was found to be 9.40 kg [R:\LIBZ]05685.doc:clb EXAMPLE Ethyl 2-(4-methoxybenzoyl)-4-nitromethyl-3-(1,3-benzodioxol-5-yl) butyrate To the compound resulting from Example 5B (7.5 kg, 37.9 mol) suspended in THF (56 kg) with mechanical stirring under nitrogen was added the compound resulting from Example 5C (8.4 kg, 37.9 mol). The mixture was cooled to 170 C, sodium ethoxide (6.4 g, 0.095 mol) was added, and the reaction was stirred for 30 minutes. After about minutes, the nitrostyrene was completely dissolved. Sodium ethoxide (6.4 g, 0.095 mol) was added, and the mixture was stirred at 250 C until HPLC shows less than 1 area ketoester remaining. The reaction was concentrated to 32.2 kg which was determined by HPLC assay to be 14.9 kg EXAMPLE Ethyl cis, cis-2-(4-methoxyphenyl)-4-(1, 3-benzodioxol-5-yl) pyrrolidine-3-carboxylate Raney nickel (20.0 from which the water had been decanted, was charged to a :.i stirred hydrogenator equipped with a thermocouple. THF (20 mL), the crude compound 15 resulting from Example 5D (40.82 g, 0.0482 mol), and acetic acid (2.75 mL, 0.0482 mol)

I*

were added sequentially. The mixture was put under a hydrogen atmosphere at 60 psi until the hydrogen uptake slowed dramatically. TFA was added, and the mixture was hydrogenated at 200 psi until HPLC shows no residual imine and <2 area nitrone. The .catalyst was filtered away and washed with 100 mL of methanol. The filtrate was assayed 20 by HPLC and found to contain 13.3 g (75% yield) of the cis, cis-pyrrolidine compound.

The filtrate was concentrated and chased with additional THF (200 mL) to give a final volume of 100 mL. The mixture was neutralized with 2 N NaOH solution (50 mL), diluted with water (200 mL), and extracted with ethyl acetate (2 x 100 mL). The combined nearly colorless ethyl acetate layers were assayed against an external standard S 25 by HPLC to be 13.0 g of the title compound.

EXAMPLE Ethyl trans, trans-2-(4-methoxyphenyl)-4-(1, 3-benzodioxol-5-yl)pyrrolidine-3-carboxylate The solution of the compound resulting from Example 5E (38.1 g, 0.103 mol) was chased with ethanol (200 mL) to a final volume of 100 mL and sodium ethoxide (3.40 g, 0.050 mol) was added. The mixture was heated to 750 C. When HPLC shows of the cis,cis isomer remaining, the mixture was cooled to room temperature. The product was assayed by HPLC against an external standard and found to contain 34.4 g yield) of the title compound. The crude compound solution was concentrated and the residue taken up in isopropyl acetate (400 mL). The organic layer was washed with ALi water (2 x 150 mL) and then extracted with 0.25 M phosphoric acid solution (2 x 400 [R:\LIBZ]05685.doc:clb 11 mL). The combined phosphate layers were stirred with ethyl acetate (200 mL) and neutralized to pH 7 with solid sodium bicarbonate (21 The organic layer was separated and found to contain 32.9 g of the title compound.

EXAMPLE Ethyl (2R, 3R, 4S)-(+)-2-(4-methoxyphenyl)-4-(1, 3-benzodioxol-5-yl)pyrrolidine-3carboxylate, mandelate salt The solution resulting from Example 5F was chased with acetonitrile (100 mL) to give a final volume of 50 mL. (S)-(+)-Mandelic acid (2.06 g, 0.0136 mmol) was added and allowed to dissolve. The mixture was seeded with the product and allowed to stir at room temperature for 16 hours. The reaction mixture was cooled to 0° C and stirred for hours. The product was filtered and dried in a vacuum oven with a nitrogen purge for 1 day at 500 C to give 5.65 g of the title compound. The purity of the product can be determined by chiral HPLC using Chiralpak AS, isocratic elution with 95:5:0.05 hexaneethanol-diethylamine; flow 1 mL/min.; UV detection at 227 nm. Retention times: 15 enantiomer: 15.5 min.; (-)-enantiomer: 21.0 min.

:o :EXAMPLE (2R, 3R, 4S)-(+)-2-(4-methoxyphenyl)-4-(1, 3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonylmethyl)-pyrrolidine-3-carboxylic acid The compound resulting from Example 5G (20.0 g, 0.0383 mol) was suspended 20 in ethyl acetate (150 mL) and 5% sodium bicarbonate solution (150 mL). The mixture was stirred at room temperature until the salt dissolved and carbon dioxide evolution had ceased. The organic layer was separated and concentrated. The residue was chased with acetonitrile (200 mL) to a final volune of 100 mL and cooled to 100 C.

o. Diisopropylethylamine (11.8 mL, 0.0574 mol) and the compound resulting from Example 25 5A (10.5 g, 0.0421 mol) were added, and the mixture was stirred for 12 hours at room temperature. The reaction mixture was concentrated and chased with ethanol (200 mL) to a final volume of 100 mL. Sodium hydroxide solution 20 mL, 0.200 mol) was added, and the mixture was heated at 600 C for 4 hours until HPLC showed no starting material remaining. The reaction mixture was poured into water (400 mL) and washed with hexanes (2 x 50 mL). The aqueous layer was washed with hexane (2 x 20 mL). A stirred mixture of the aqueous layer and ethyl acetate (400 mL) was neutralized to pH with concentrated HC1 (12 mL). The organic layer was separated and found to contain 18.3 g (94% yield) of the title compound.

[R:\LIBZ]05685.doc:clb 12 EXAMPLE 51 (2R,3R,4S)-(+)-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-l-(N,N-di(n-butyl)aminocarbonylmethyl)-pyrrolidine-3-carboxylic acid hydrochloride salt To a solution of the compound of Example 5H in ethyl acetate at room temperature in a mechanically stirred vessel equipped with a thermocouple, was added 39.4 mL of 1 N HCI in ethanol (0.0394 mol) The resultant solution was filtered to remove foreign matter, concentrated in vacuo, and chased with ethyl acetate (400 mL). The solution was seeded repeatedly, as the solvent was removed, until crystallization was 0o initiated. The mixture was concentrated to a volume of 100 mL, and the product was filtered and washed with ethyl acetate (25 mL). The resultant white solid was dried in a vacuum oven under a nitrogen purge at 500 C to afford 17.6 g of the title compound.

EXAMPLE 6 [2S, 3R, 4S]-2-(4-Methoxyphenyl)-4-(1, 3-benzodioxol-5-yl)- -[(N-butyl-N-(4-dimethylaminobutyl)amino)carbonylmethyl]-pyrrolidine-3-carboxylic Acid EXAMPLE 6A N-butyl-4-hydroxybutyramide To 30 mL (390 mmol) of g-butyrolactone was added 45 ml (455 mmol) of n- 20 butylamine. The solution was heated at 850 C for 1.5 hr, then the excess n-butylamine was removed in vacuo. The product crystallized on standing to give about 62 g of a colorless, low melting solid.

EXAMPLE 6B N-butyl-4-hydroxybutyl Chloroacetamide 25 To an ice cooled solution of 3.40 g (91.9 mmol) of LiAlH 4 in 90 mL of THF was added 2.4 mL of 98% H 2

SO

4 dropwise, with stirring. After bubbling had ceased, a solution of 4.7 g of the compound of Example 6A in 10 mL of THF was added. The mixture was stirred at reflux for 24 hr, then cooled with an ice bath and quenched by sequential dropwise addition of 1.7 mL H 2 0, and 17 mL of 25% w/v aqueous NaOH. The white precipitate was filtered, and washed with about 50 mL of THF. The combined filtrate and washings were concentrated to 3.85 g of an oil. To an ice cooled solution of this material in 35 mL of ethyl acetate was added a solution of 5.0 g (29.2 mmol) of chloroacetic anhydride in 10 mL of ethyl acetate. The solution was stirred at 00 C for min, then extracted with saturated aqueous NaHCO 3 solution (1 x 25 mL), 2M NaOH (1 x s 25 mL), 5% NH 4 OH (1 x 25 mL), 1 M HC1 (1 x 25 mL), and brine (1 x 25 mL), dried (R:\LIBZ]05685.doc:clb 13 over MgSO 4 filtered, and concentrated in vacuo to an oil. The product was purified via silica gel chromatography, eluting with 98:2 diethyl ether: methanol, to give 1.52 g (31%) of a colorless oil.

EXAMPLE 6C Ethyl [2S,3R,4S]-2-(4-Methoxyphenyl)-4-(1, 3-benzodioxol-5-yl)-]-[(N-butyl-N-(4hydroxybutyl)amino)carbonylmethyl]-pyrrolidine-3-carboxylate To 1.52 g (6.85 mmol) of the compound of Example 6B was added 2.75 g (7.44 mmol) of the ethyl [2S,3R,4S]-2-(4-Methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrrolidine-3-carboxylate (prepared by neutralization of the compound of Example 10 mL of DMSO, and 2 mL of N,N-diisopropylethylamine. The solution was stirred at ambient temperature for 22 h, then poured into 100 mL of water and extracted with diethyl ether (3 x 25 mL). The combined ether layers were washed with water (1 x mL), 4% H 3 P0 4 (1 x 25 mL), saturated aqueous NaHCO 3 solution (1 x 25 mL), and brine (1 x 25 mL), dried over MgSO 4 filtered, and concentrated to an oil. This was 15s purified via silica gel chromatography, eluting with 98:2 diethyl ether: methanol to give 3.0 g of a colorless oil.

.i EXAMPLE 6D Ethyl [2S,3R, 4S]-2-(4-Methoxyphenyl)-4-(1,3-benzodioxol-5-yl) [(N-butyl-N-(4-bromobutyl)amino)carbonylmethyl]-pyrrolidine-3-carboxylate TO an ice cooled solution of 2.80 g (5.05 mmol) of the compound of Example 6C in 27 mL of diethyl ether was added 1.4 mL (10 mmol) of triethylamine, then 0.58 mL of methanesulfonyl chloride. A white precipitate formed, and the suspension was stirred at 00 C for 20 min. The reaction was diluted with 75 mL of diethyl ether, then extracted with saturated aqueous NaHCO 3 solution (2 x 25 mL), 5% NH4OH (2 x 25 mL), and brine (1 x 25 25 mL), dried over MgSO 4 filtered, and concentrated to 3.0 g of a colorless oil. To this material in 45 mL of DMF was added 6.0 g (69 mmol) of LiBr. The reaction warmed to about 500 C, then gradually cooled. The solution was stirred at ambient temperature for 4h, then poured into 450 mL of water, and extracted with diethyl ether (3 x 100 mL). The combined ether layers were back extracted with water (1 x 100 mL), and brine (1 x 100 mL), dried over MgSO 4 filtered, and concentrated in vacuo to an oil. The product was purified via silica gel chromatography, eluting with 3:1 diethyl ether: petroleum ether, to give 2.65 g of a colorless oil.

[R:\LIBZ]05685.doc:clb EXAMPLE 6E [2S,3R,4S]-2-(4-Methoxyphenyl)-4-(1,3-benzodioxol-5-yl)- -[(N-butyl-N-(4-dimethylaminobutyl)amino)carbonylmethyl]-pyrrolidine-3-carboxylic Acid To a solution of the compound of Example 6D (0.825 g, 1.34 mmol) in 3 mL of ethanol was added 5 mL of 4.07M dimethylamine in ethanol; the resultant solution was heated at reflux for 75 min. Solvents were removed in vacuo. The residue was purified by flash chromatography on silica gel, eluting with 9:1 dichloromethane/methanol. The resultant material was taken up in 5 mL of 1.4N NaOH in 5:1 ethanol/water and stirred at ambient temperature for 14 hrs. Solvents were removed in vacuo; the residue was taken up in water, then adjusted to pH 6-7 with IM HCI (about 7 mL required). The mixture was extracted with EtOAc (3 the aqueous layer was concentrated in vacuo. The residue was washed 3 x with acetonitrile; the combined washes were filtered through Celite and concentrated to give 596 mg of a white foam.

o o The ability of the compound of the invention to lower blood pressure can be demonstrated according to the methods described in Matsumura, et al., Eur. J. Pharmacol.

185 103 (1990) and Takata, et al., Clin. Exp. Pharmacol. Physiol. 10 131 (1983).

The ability of the compound of the invention to treat congestive heart failure can 20 be demonstrated according to the method described in Margulies, et al., Circulation 82 2226 (1990).

The ability of the compound of the invention to treat myocardial ischemia can be demonstrated according to the method described in Watanabe, et al., Nature 344 114 (1990).

25 The ability of the compound of the invention to treat coronary angina can be demonstrated according to the method described in Heistad, et al., Circ. Res. 54 711 (1984).

The ability of the compound of the invention to treat cerebral vasospasm can be demonstrated according to the methods described in Nakagomi, et al., J. Neurosurg. 66 915 (1987) or Matsumura, et al., Life Sci. 49 841-848 (1991).

The ability of the compound of the invention to treat cerebral ischemia can be demonstrated according to the method described in Hara et al., European. J. Pharmacol.

197 75-82, (1991).

R:\LIBZ]05685.doc:clb The ability of the compound of the invention to treat acute renal failure can be demonstrated according to the method described in Kon, et al., J. Clin. Invest. 83 1762 (1989).

The ability of the compound of the invention to treat chronic renal failure can be demonstrated according to the method described in Benigni, et al., Kidney Int. 44 440- 444(1993).

The ability of the compound of the invention to treat gastric ulceration can be demonstrated according to the method described in Wallace, et al., Am. J. Physiol. 256 G661 (1989).

The ability of the compound of the invention to treat cyclosporin-induced nephrotoxicity can be demonstrated according to the method described in Kon, et al., Kidney Int. 37 1487 (1990).

The ability of the compound of the invention to treat endotoxin-induced toxicity (shock) can be demonstrated according to the method described in Takahashi, et al., 15 Clinical Sci. 79 619 (1990).

The ability of the compound of the invention to treat asthma can be demonstrated according to the method described in Potvin and Varma, Can. J. Physiol. and Pharmacol.

67 1213 (1989).

The ability of the compound of the invention to treat transplant-induced 20 atherosclerosis can be demonstrated according to the method described in Foegh, et al., Atherosclerosis 78 229-236 (1989).

The ability of the compound of the invention to treat atherosclerosis can be demonstrated according to the methods described in Bobik, et al., Am. J. Physiol. 258 C408 (1990) and Chobanian, et al., Hypertension 15 327 (1990).

25 The ability of the compound of the invention to treat LPL-related lipoprotein disorders can be demonstrated according to the method described in Ishida, et al., Biochem. Pharmacol. 44 1431-1436 (1992).

The ability of the compound of the invention to treat proliferative diseases can be demonstrated according to the methods described in Bunchman ET and CA Brookshire, Transplantation Proceed. 23 967-968 (1991); Yamagishi, et al., Biochem. Biophys. Res.

Comm. 191 840-846 (1993); and Shichiri, et al., J. Clin. Invest. 87 1867-1871 (1991).

Proliferative diseases include smooth muscle proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus ,_..erythematosus, diabetic retinopathy or other retinopathies, psoriasis, scleroderma, prostatic hyperplasia, cardiac hyperplasia, restenosis following arterial injury or other pathologic stenosis of blood vessels.

The ability of the compound of the invention to treat acute or chronic pulmonary hypertension can be demonstrated according to the method described in Bonvallet et al., Am. J. Physiol. 266 H1327 (1994). Pulmonary hypertension can be associated with congestive heart .failure, mitral valve stenosis, emphysema, lung fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), altitude sickness, chemical exposure, or may be idiopathic.

The ability of the compound of the invention to treat platelet aggregation, and thrombosis, can be demonstrated according to the method described in McMurdo et al.

Eu. J. Pharmacol. 259 51 (1994).

The ability of the compound of the invention to treat cancers can be demonstrated according to the method described in Shichiri, et al., J. Clin. Invest. 87 1867 (1991).

15 is The ability of the compound of the invention to treat IL-2 (and other cytokine) mediated cardiotoxicity and vascular permeability disorders can be demonstrated according to the method described in Klemm et al., Proc. Nat. Acad. Sci. 92 2691 (1995).

The ability of the compound of the invention to treat nociception can be demonstrated according to the method described in Yamamoto et al., J. Pharmacol. Exp.

20 Therap. 271 156 (1994).

The ability of the compound of the invention to treat colitis can be demonstrated according to the method described in Hogaboam et al (EUR. J. Pharmacol. 1996, 309, o 261-269).

The ability of the compound of the invention to treat ischemia-repurfusion injury 0. 25 in kidney transplantation can be demonstrated according to the method described in Aktan et al (Transplant Int 1996, 9, 201-207).

The ability of the compound of the invention to treat angina, pulmonary hypertension, raynaud's disease, and migraine can be demonstrated according to the method described in Ferro and Webb (Drugs 1996, 51, 12-27).

The compound of the present invention can be used in the form of salts derived from inorganic or organic acids. These salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quatemized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil-soluble or dispersible products are thereby obtained.

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

Basic addition salts can be prepared in situ during the final isolation and 15 purification of the compound of the invention, or separately by reacting the carboxylic acid function with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia, or an organic primary, secondary or tertiary amine. Such pharmaceutically acceptable salts include, but are not limited to, cations based on the alkali and alkaline earth metals, such as sodium, lithium, 20 potassium, calcium, magnesium, aluminum salts and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines useful for the formation of base addition salts include diethylamine, 25 ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

The compound of the invention is useful for antagonizing endothelin in a human or other mammal. In addition, the compound of the present invention is useful (in a human or other mammal) for the treatment of hypertension, acute or chronic pulmonary hypertension, Raynaud's disease, congestive heart failure, myocardial ischemia, reperfusion injury, coronary angina, cerebral ischemia, cerebral vasospasm, chronic or acute renal failure, non-steroidal antiinflammatory drug induced gastric ulceration, cyclosporin induced nephrotoxicity, endotoxin-induced toxicity, asthma, fibrotic or proliferative diseases, including smooth muscle proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus Serythematosus, diabetic retinopathy or other retinopathies, psoriasis, scieroderma, :\LIBZ05685.doc:clb prostatic hyperplasia, cardiac hyperplasia, restenosis following arterial injury or other pathologic stenosis of blood vessels, LPL-related lipoprotein disorders, transplantationinduced atherosclerosis or atherosclerosis in general, platelet aggregation, thrombosis, cancers, prostate cancer, IL-2 and other cytokine mediated cardiotoxicity and permeability disorders, and nociception, especially treatment of bone pain associated with bone cancer.

Total daily dose administered to a host in single or divided doses may be in amounts, for example, from 0.001 to 1000 mg/kg body weight daily and more usually 0.1 to 100 mg/kg for oral administration or 0.01 to 10 mg/kg for parenteral administration.

Dosage unit compositions may contain such amounts of submultiples thereof to make up the daily dose.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

15 It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, tine of .0 9 administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.

o 20 The compound of the present invention may be administered orally, parenterally, sublingually, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral 25 as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-propanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including .do:cib 19 synthetic monb- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may 0o also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

i Liquid dosage forms for oral administration may include pharmaceutically 15 acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise S.i adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

The compound of the present invention can also be administered in the form of 20 liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically aceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present 25 invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic.

Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.

A representative solid dosage form, for example, a tablet or a capsule, comprises: Compound of the invention: 35% w/w Starch, Pregelatinized, NF 50% w/w Microcrystalline Cellulose, NF 10% w/w Talc, Powder, USP 5% w/w L:\LBZ]05685.doc:clb While the compound of the invention can be administered as the sole active pharmaceutical agent, it can also be used in combination with one or more cardiovascular agents independently selected from diuretics, adrenergic blocking agents, vasodilators, calcium channel blockers, renin inhibitors, angiotensin converting enzyme (ACE) inhibitors, angiotensin II antagonists, potassium channel activators and other cardiovascular agents.

Representative diuretics include hydrochlorothiazide, chlorothiazide, acetazolamide, amiloride, bumetanide, benzthiazide, ethacrynic acid, furosemide, indacrinone, metolazone, spironolactone, triamterene, chlorthalidone and the like or a pharmaceutically acceptable salt thereof.

Representative adrenergic blocking agents include phentolamine, phenoxybenzamine, prazosin, terazosin, tolazine, atenolol, metoprolol, nadolol, propranolol, timolol, carteolol and the like or a pharmaceutically acceptable salt thereof.

Representative vasodilators include hydralazine, minoxidil, diazoxide, is nitroprusside and the like or a pharmaceutically acceptable salt thereof.

Representative calcium channel blockers include amrinone, bencyclane, diltiazem, fendiline, flunarizine, nicardipine, nimodipine, perhexilene, verapamil, gallopamil, nifedipine and the like or a pharmaceutically acceptable salt thereof.

Representative renin inhibitors include enalkiren, zankiren, RO 42-5892, PD- 20 134672 and the like or a pharmaceutically acceptable salt thereof.

Representative angiotensin II antagonists include DUP 753, A-81988 and the S. like.

Representative ACE inhibitors include captopril, enalapril, lisinopril and the like or a pharmaceutically acceptable salt thereof.

25 Representative potassium channel activators include pinacidil and the like or a pharmaceutically acceptable salt thereof.

Other representative cardiovascular agents include sympatholytic agents such as methyldopa, clonidine, guanabenz, reserpine and the like or a pharmaceutically acceptable salt thereof.

The compound of the invention and the cardiovascular agent can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compound in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as separate 3-7 ompositions or as a single dosage form containing both agents.

0.0 .r IBZ105685.doc:cIb I When administered as a combination, the therapeutic agents can be formulated as separate compositions which are given at the same time or different times, or the therapeutic agents can be given as a single composition.

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Claims

1. [2S,3R,4S]-2-(4-Methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-[(N-butyl- N-( 4 -dimethylaminobutyl)amino)carbonylmethyl]-pyrrolidine-3-carboxylic acid, or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising [2S,3R,4S]-2-(4-Methoxy- phenyl)-4-(1,3-benzodioxol-5-yl)-1-[(N-butyl-N-(4-dimethylaminobutyl)amino)carbonyl- methyl]-pyrrolidine-3-carboxylic acid together with one or more pharmaceutically acceptable carriers, excipients, diluents and/or adjuvants.

3. A pharmaceutical composition according to claim 2 further comprising 0t one or more cardiovascular agents.

4. A pharmaceutical composition according to claim 3 wherein the cardiovascular agents are selected from diuretics, adrenergic blocking agents, vasodilators, calcium channel blockers, renin inhibitors, angiotensin converting enzyme (ACE) inhibitors, angiotensin II antagonists and potassium channel activators. Dated 27 March, 2002 Abbott Laboratories Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 0 5 0 So [R:\LIBZ]05685.doc:clb