GB2337701A

GB2337701A - Treatment of ischemia with an angiotensin II antagonist

Info

Publication number: GB2337701A
Application number: GB9811312A
Authority: GB
Inventors: Metin Avkran
Original assignee: UNITED MEDICAL AND DENTAL SCHOOLS OF GUYS ST THOMAS HOSPITALS
Current assignee: UNITED MEDICAL AND DENTAL SCHOOLS OF GUYS ST THOMAS HOSPITALS
Priority date: 1998-05-26
Filing date: 1998-05-26
Publication date: 1999-12-01
Also published as: GB2337701A8; GB9811312D0

Abstract

The use of angiotensin II receptor antagonists in modulating intracellular pH and/or cellular ion transport to confer protective effects by administering to a patient with or at risk for tissue ischemia a therapeutically effective amount of said angiotensin II receptor antagonist is disclosed. Use of losartan is exemplified.

Description

2337701

TITLE OF THE INVENTION METH0EfTREATMENT WITH AN ANGIOTENSIN II ANTAGONIST

BACKGROUND OF THE INVENTION

Angiotensin receptors (AT1 and AT2 subtypes) are expressed in ventricular myocardium of many species. Although there is evidence that these receptors may play a role in regulation of myocytes growth,' their roles in other aspects of cardiac physiology and pathophysiology have not been fully defined. (1. Booz GW, Baker KM: Role of type 1 and type 2 angiotensin 10 receptors in angiotensin II-Induced cardiomyocyte hypertrophy. Hypertension 1996; 28:635-640) Angiotensin has been shown to evoke a positive inotropic response in adult mammalian ventricular myocytes, which occurs without augmentation of the intracellular Ca2+ transient and concomitantly with an increase in intracellular pH.2 (2. Ikenouchi H, Barry W11, Bridge JH, Weinberg EO, Apstein CS, Lorell BH: Effects of angiotensin II on intercellular Ca2+ and pH in isolated beating rabbit hearts and myocytes loaded with the indicator indo-1. J. Physiol 1994. 480:203-215) This has led to the hypothesis that an increased myofilament sensitivity to Ca2+ arising from intracellular alkalinization might be largely responsible 2 for the inotropic actions of angiotensin. A possible mechanism underlying the increase in intracelluar pH in response to angiotensin is an increased activity of the sarcolemmal Na+/H+ exchanger, 3.1 although increased activity of other acid extrusion pathways may also be involved. ' (3. Matsul H, Barry W11, Livsey C, Spitzer KW: Angiotensin II stimulates sodium-hydrogen exchange in adult rabbit ventricular myocytes. Cardiovasc. Res. 1995 29:2 15-221. 4. Grace AA, Metcalfe JC, Weissberg PL, Bethell HWL, Vandenberg JT: Angiotensin II stimulates sodium-depen dent proton extrusion in perfused ferret heart, Am. J. Physiol. 1996; 27 O:C 1687 -C 1694. 5. Kohout TA, Rogers TB: Anglotensin II activates the Na+ffiCO symport through a phosphoinsitide-in dep en dent mechanism in cardiac cells. J. Biol. Chem. 1995,270:20432-20438.) 1 Stimulation of sarcolemmal Na+lW- exchanger activity by angiotensin would be expected to have significant adverse consequences in a number of pathological settings. For example, in conditions where removal of cytosolic Ca2+ is impaired, increased myofilament responsiveness to Ca2+ arising from intracellular alkalinization may impair diastolic relaxationIndeed, there is experimental evidence that in pressure overlo ad-in duce d hypertrophy, a condition which is associated with down regulation of the sarcoplasmic reticulum Ca2±ATPase," anglotensin II impairs diastolic relaxation via a mechanism mediated by AT1 receptor.-, (6. Aral M, Matsul H, Perlasamy M: Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ. Res. 1994; 7 4:555-564. 7. Lopez JJ, Lorell BH, Ingelfinger JR, Weinberg EO, Schunkert H, Diamant D, Tang SS: Distribution and function of cardiac anglotensin ATI- and AT2- receptor subtypes in hypertrophied rat hearts. Am. J. Physiol. 1994; 267.- H844H852) Furthermore, there is now substantial evidence that the sarcolemmal Na--IH+ exchanger may be a key mediator of myocardial injury and dysfunction during ischemia and reperfusion,," ' such that novel inhibitors of the exchanger have been developed as therapeutic agents for ischemic heart disease. 111 (8. AvkIran M: Sodium -hydrogen exchange in myocardial ischemia and reperfusion: a critical determinant of injury? in Karmazyn M (ed): Myocardial ischemia: mechanisms, reperfusion, protection. Basel, Birkhauser Verlag, 1996, pp. 299-311. 9. Frbhllch 0, Karmazyn M: The NaH exchanger revisited; an update on Na-H exchange regulation and the role of the exchanger in hypertension and cardiac function in health and disease. Cardiovasc. Res. 1997 -, 36:138-148. 10- Scholz W, Albus U, Counillon L, G6gelein H, Lang HJ, Linz W, Welchert A, Sch61kens BA: Protective effects of HOE642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischemia and reperfusion. Cardiovasc. Res. 1995; 29:260-268) It follows from this that stimulation of sarcolemmal Na+/H4- exchanger activity by intra-cardiac or circulating angiotensin (and other neurohormonal mediators, such as catecholamines,' 1.12 thrombin " and endothelin 1 1) during episodes of myocardial ischemia and reperfusion is likely to exacerbate the 2 unfavorable sequelae of such episodes, such as arrhythmia's, contractile dysfunction and infarction. (11. Wallert MA; Frbhllch 0: al-Ach.energ-ic stimulation of Na-H exchange in cardiac myocytes. Am. J. Physiol. 1992; 263:C1096-CI102. 12. YokoyamaH,YasutakeM,AvkiranM:al-Adrenergic 5 stimulation of sarcolemmal Na+11---1+ exchanger activity in rat ventricular myocytes; evidence for selective mediation by the (xlA- adrenoceptor subtype. Circ. Res. 1998; in press. 13. Yasutake M, Haworth RS, King A, Avkiran M: Thrombin activates the sarcolemmal Na+111+ exchanger: evidence for a receptor-mediated mechanism involving protein kinase C. Circ. Res. 1996; 79:705-715. 14. Kramer BK, Smith TW, Kelly RA. Endothelin and increased contractility in adult rat ventricular myocytes. Role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na+/H+ exchanger.

Cire. Res. 1991; 68:269-279) In light of the above, angiotensin-mediated regulation of sarcolemmal Na+/H+ exchanger activity is likely to be pathophysiological significance. Therefore, identification of the cellular signaling mechanisms involved in such regulation of sarcolemmal Na+11---1+ exchanger activity is not only an important scientific goal but may also reveal a mechanistic basis for some of the reported actions of anglotensin and angiotensin receptor antagonists in cardiovascular disease.

SUMMARY OF THE INVENTION

A method for modulating intracellular pH and/ or cellular ion transport to confer protective effects by administering to a patient with or at risk for tissue ischemia a therapeutically effective amount of an angiotensin 11 antagonist. The method wherein the tissue ischemia is defined as cardiac ischemia, kidney ischemia or brain ischemia. The method wherein the anglotensin 11 antagonist is selected from the group consisting of:

candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, 13MS-184698, 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4y1)methyl - 5, 77 - dimethyl- 2 -ethyl - 311 -imidazo [4,5 -b]p yri dine, BAY106,734, BIBR363, CL329167, E41717, EM1)73495, HN65021, HR7,20, HOE720, 1 LRB08 1, SC52458, SL9 10 102, UP2696, YM358, EM1)66397, ME322 1, TAK536, 13MS184698, CG142112A, CGP49870, CP148130, E4188, EM1)66684, EXP9954, FR 1153332, GA0050, KT3579, LF70156, LRB057 > LY266099, LY301875, PD123177, PD126055, SC51757, SC54629, U96849, UK77778,WAY126227,WK1260,WK1492,YH1498,andYM314'12.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1:

Schematic illustration of the experimental protocols used to study (A) effect of anglotensin II on sarcolemmal Na+111+ exchanger activity, (B) effect of ATI receptor stimulation of Na+lH+ exchanger activity, (C) effect of losartan on the response to AT1 receptor stimulation, and (D) effect of AT2 receptor stimulation on Na+/H+ exchanger activity.

Figure 2:

(A) JH at pHI 6.90 (J 1-16.9) during the first (open bars) and second (solid bars) acid pulses and (B) the change in J 116.9 during the second pulse relative to the first (AJ H6.9), in control cells and in cells which were exposed to 10, 100 or 1000 nM angiotensin 11 throughout the second pulse (n=8 cells per group), ANG: anglotensin 11.

Figure 3: Representative pHi recordings obtained during the first and second () acid pulses in (A) a control cell and in cells exposed to 100 nM angiotensin 11 throughout the second pulse, (B), alone or (C) in conjunction with 100 nM PD123319. ANG: anglotensin 11, PD: PD123319.

Figure 4 (A) JH at pHi 6.90 (J H6.9) during the first (open bars) and second (solid bars) acid pulses and (B) the change in J H6.9 during the second pulse relative to the first, (M H6.9), in control cells and in cells which were 4 exposed to various combinations of angiotensin 11 (100 nM) and PD123319 (10, 30 or 100 nM) during the second pulse (n=8 cells per group, p <0.05). ANG. angiotensin II, PD. PD123319.

Figure 5 JH versus-pHI curves obtained during 2 consecutive acid pulses in (A) control cells and in cells exposed to 100 nM anglotensin II throughout the second pulse, (B) alone or (C) in conjunction with 100 nM PD123319. Open symbols indicate first pulse and solid symbols second pulse JH values (n=8 cells per group, p <0.05 versus first pulse). ANG: anglotensin II, PD:

PD123319.

Figure 6:

Representative pHI recordings obtained dulling the first and second () acid pulses in cells exposed to 100 nM angiotensin II throughout the second pulse (A) alone, (B) in conjunction with 100 nM PD123319 or (C) in conjunction with 100 nM P1) 123319 and 100 nM losartan. ANG: angiotensin 11, PD:PD123319, LOS: losartan.

Figure 71: (A) JH at pHi 6.90 W H6.9) during the first (open bars) and second (solid bars) acid pulses and (B) the change in J H6.9 during the second pulse relative to the first (M H6.9), in control cells and in cells which were exposed to various combinations of angiotensin 11 (100 nM) and P1) 1233 19 25 (100 nn. and losartan (10, 30 or 100 nn during the second pulse (n=10 cells per group, p <0.05). ANG: angiotensin II, PD: PD123319, LOS-losartan.

Figure 8:

JH versus-pHI curves obtained during 2 consecutive acid pulses in cells exposed to 100 nM angiotensin II throughout the second pulse, (A) alone, (B) in conjunction with 100 nM P1) 123319 or (C) in conjunction with 100 nM PD123319 and 100 nM losartan. Open symbols indicate first pulse and solid symbols second pulse JH values (n=10 cells per group, p <0.05 versus first pulse). ANG: angiotensin IT, PD: PT) 123319, LOSAosartan.

Figure 9:

(A) JH at pHI 6.90 W H6.9) during the first (open bars) and second (solid bars) acid pulses and (B) the change in J H6.9 during the second pulse relative to the first (AJ H6.9), in control cells and in cells which were exposed to various combinations of anglotensin 11 (100 nM) and losartan (10, or 100 nn during the second pulse (n=9 cells per group).

ANG:angiotensin 11, LOSAosartan.

DETAILED DESCRIPTION OF THE INVENTION

This invention concerns a method for modulating intracellular pH and/ or cellular ion transport to confer protective effects by administering to a patient with or at risk for tissue ischemia a therapeutically effective amount of an angiotensin IT antagonist.

An embodiment of this invention is the method for modulating intracellular pH and/ or cellular ion transport to confer cardioprotective effects by administering to a patient with or at risk for tissue ischemia a therapeutically effective amount of an angiotensin 11 receptor antagonist selected from the group consisting of: candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, 13MS-184698, 3(T (tetrazol-5-yl)-1, F-biphen -4 -yl)methyl - 5,7- dimethyl -2 -ethyl 3H - 1mi dazo [4,5 - blpyridine, BAY106734, B11311363, CL329167, E4177, EM1)713495, HN65021, HR7 20, HOE 7 20, LRB08 1, SC52458, SL9 10 102, UP2696, YM358, EM1)66397, ME322 1, TAK536, BMS 184698, CG142112A, CGP49870, CP148130, E4188, EM1)66684, EXP9954, F111153332, GA0050, KT3.579, LF-10156, LR13057, LY266099, LY301875, PD123177, PD126055, SC51757, SC54629,1196849, UK77778, WAY126227, WK1260, WK1492,YM498, andYM31471 2. The preferred anglotensin 11 receptor antagonists useful in this method are: candesartan cilexetil, eprosartan, irbesartan, losartan, 6 tasosartan, telmisartan, valsartan, BMS- 184698 and 3-(2'-(tetrazol-5-yl)- 1, Fbiphen - 4-y1)m ethyl - 5,7- dimethyl- 2 ethyl- 31-1-1mi dazo [+5 - bl p yridine.

Another embodiment of the invention is the method for modulating intracellular pH intracellular pH and/ or cellular ion transport to confer protective effects by administering to a patient with or at risk for tissue ischemia a therapeutically effective amount of an imidazole anglotensin II receptor antagonist of formula I N R7 R 8 R6 N 1 (CH2)r R R2 3 R 1 wherein:

RI is:

7 0 4-CO,H; 4-CO,R'- -0-S"-OH. -S03 3)20H.

11 1 H -C(CF OH 0 11 6H 0 11 6H OH 0 N-N 12. 1 -,/ -N; 4-NHSO2CF3 -CONHOR 1 NH '5'-OH -S02 2 -C -F 3 N 27 6H H N-N )/, ' N; 4 N _ H 4-X R13,1 R 13 F F 4-x- 3 4-X F R 2 R 13 F 1 Q-P X \ R 13 8 0 11 HNC 1 HOC 11 0 N-N 1 4-CONH Al N..; H )11 02H 4-CONHNHSO2CF3.4-CONFf--CHCH2C6H 5 (/-isomer) HO,C W' 4-CON 1 - N-N 1 4 z W"'4-J,' N) CF3" (/-isomer) C02H H R 13 0 R 13 4-,"NH 4-X-- 4-N -R or R 4 0 R 2 0 R 20 s -C-NHS02- (CH2) R2 is H Cl; Br; I; F> N02, CN; alkyl of 1 to 4 carbon atoms; acyloxy of 1 to 4 carbon atoms; alkoxy of 1 to 4 carbon atoms; C02H; C02R9; HNS02CH3: NHS02CF3; N-N CONHOR 12 S02NH2- 1 N. aryi- or fury[ N H R3 is H). Cl, Br, I or F; alkyl of 1 to 4 carbon atoms or alkoxy of 1 to 4 carbon atoms; R4 is CN, N02 or C02R 1 R.S is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms alkenyl or alkynyl of 2 to 4 carbon atoms; R6 is alkyl of 2 to 10 carbon atoms, alkenyl or alkynyl of 3 to 10 carbon atoms or the same groups substituted with F Or C02R 14; cycloalkyl of 3 to 8 carbon atoms, cycloalkylalkyl, of 4 to 10 carbon atoms, cycloalkylalkenyl or cycloalkylalkynyl 5 to 10 carbon atoms; (CH2)sZ(CH2), ,R5 optionally substituted with F 9 Or C02R14; benzyl substituted on the phenyl iling with 1 or 2 halogens, alkoxy of 1 to 4 carbon atoms, alkyl of 1 to 4 carbon atoms or nitro; R 1 is H, F, Cl, Br, 1, N02, CLY2t,+ 1, where v=1-6, C05; CN,0 11 16 -C-R straight or branched alkyl of 1 to 6 carbon atoms; phenyl or phenylalkyl, where alkyl is 1 to 3 carbon atoms; or substituted phenyl or substituted phenylalkyl, where alkyl is 1 to 3 carbon atoms, substituted with one or two substituents selected from alkyl of 1 to 4 carbon atoms, F, Cl, Br, OH, OCH3, CF3, and COOR, where R is H, alkyl of 1 to 4 carbon atoms, or phenyl; R8 is H, CN, alkyl of 1 to 10 carbon atoms, alkenyl of 3 to 10 carbon atoms, or the same groups substituted with F; phenylalkenyl wherein the aliphatic portion is 2 to 6 carbon atoms; -(CH2)iiiimidazol-l-yl; - (CH2)iii-1,2,3-triazolyl optionally substituted with one or two group selected from C02CH3 or alkyl of 1 to 4 carbon atoms; -(CH2)s tetrazolyl; / 0 -(CH2)ii-1CH-Rll; -(CH2)nOCR14; -(CH2)nSI115; OR 17 R 14 0 0 -CH=CH(CH2)sCHOR15; -CH=CH(CH2)sCR16; -&16- 0 11 -CH=CH(CH2)sOCR 11 -, (CH2)s-CH-COR 16 \ CH3 0 Y Y -(CH2)nCR16 -(CH2)n0CNHI110; -(C112)nNIZI1COR10 0 v -(CH2)11,NR1ICNHR10; -(CH2)11,NRI1SO2R10; Y U -(CH2)11.NR11CR10; -(CH2)111K -(CH2)111,0NO2-, -CH2N1 -(CH2),11NO2; -CH=N-NRIIRI-I; 0 1 1 (CH2)m-N C) 0 N-N (CH2)S---, N CF3 H 0 1 11 /-\ M12MAL -N \- N CH30 N=N ---(CH2)s NH R 4 - (CHAn-N \- N -,p CH30 -CH--N-NH-S02 --G, or -CH=N-NH-</ 1 N N H R24 0 1 R 21.

R91s: -CH-OCR, RIO is alkyl of 1 to 6 carbon atoms or perfluoroalkyl of 1 to 6 carbon atoms, 1-adamantyl, I-naphthyl, P(Pnaphthyl)ethyl, or (CH2)pC6H5; R 11 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl or benzyl; R 12 is H, methyl or benzyl; R13 is -C02H; -C02R9; -CH2CO2H, -CH2CO2R9,- 11 0 0 to 0 0 0 OH 0 01 1 01 -O-S-OH; -0-P-OH; -SO3R -NHP-OR -C - P - OH; 1 1 1 1 X OH OH OH R27 OH -PO3112; X(CF3)20H; -NHS02CH3- -NHS02CF3- -NHCOCF3; -CONHOR12; S02NH2-, N-N N -N N-N Al N., 2, ' -, A' 1 - N CH2- N CONH N' 31 H H -CONHNHSO2CF3; N-N N CF3 H N=N NH R 4 R 14 is H, alkyl or perfluoroalkyl of 1 to 8 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl or benzyl; R 15 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, benzyl, acyl of 1 to 4 carbon atoms, phenacyl; R16 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, (CH2)pC6H5, OR17, or NR181119; R17 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl or benzyl; R18 and R19 independently are H, alkyl of 1 to 4 carbon atoms, phenyl, benzyl, a-methylbenzyl, or taken together with the nitrogen form a iling of the formula /- (C H2)t -N \-/ Q, Q is NR20, 0 or CH2; R20 is H, alkyl of 1-4 carbon atoms, or phenylR21 is alkyl of 1 to 6 carbon atoms, -NR22R23, or -CHCH2CO2CH3:

NH2 R22 and R23 independently are II, alkyl of 1 to 6 carbon atoms, benzyl, or are taken together as (CH2)u,where u is 3-6.1 12 R24 is H, CH3 or -C6H5-, R25 is NR27R28, OR28, NWONH2, NWSNH2, - NHS02 -C- CH31 or -NHS02 -0 R26 is hydrogen, alkyl with from 1 to 6 carbon atoms, benzyl, or allyl; R27 and R28 are independently hydrogen, alkyl with from 1 to 5 carbon atoms, or phenyl; R29 and R30 are independently alkyl of 1-4 carbon atoms or taken together are -(CH2)q-; R31 is H, alkyl or 1 to 4 carbon atoms, -CH2CH=CH2 or CH2C6H4R32; X is a carbon-carbon single bond, -CO-, -CH2-, -0-, -S-> NH-, -N-) -CON-, -NCO-, -OCH2-, -CH20-, -SCH2-, -CH2S-, 1 1 % R26 R23 R23 -NHC(R270(R28)-, -NR23S02-, -S02NR23-, -CH=CH-, -CF=CF CH=CF-, -CF=CH-, -CH2CH2-, X(R27)(R28)NH-, OR 14 OCOR 17 NR 25 _CF 2CF2 C - or R 29o OR 30 -C- 1 Yis 0 or S; z is 0, NRI 1, or S; m is 1 to 5; n is 1 to 10; p is 0 to 3; q is 2 to 3; r is 0 to 2, 1 1 s is 0 to 5; t is 0 or 1; and pharmaceutically acceptable salts of these compounds; provided that: (1) the R 1 group is not in the ortho position; (2) when R I is R 3 -X-- 1 R 3 -:71 R 2 X is a single bond, and R13 is C02H, or N -N IIA IN N H then R13 must be in the ortho or meta position; or when R1 and X are as above and R 13 is NHS02CF3 or NHS02C113, R 13 must be ortho; (3) when R l is R 3 -X --- ( 1 -:

3 R R4 and X is other than a single bond, then R 13 must be ortho except when X=NR23C0 and R13 is NHS02CF3 Or NHS02CH3, then R13 must be ortho or meta; 14 (4) when R I is 4-CO2H or a salt thereof, R6 cannot be S-alkyl; (5) when R1 is 4-CO2H or a salt thereof, the substituent on the 4position of the imidazole cannot be CH20H, CH20COCH3, Or CH2C0211; (6) when R I is R 13 -X 3 R R 2 X is -OCH2-, and R 13 is 2-CO2H, and R7 is H then R6 is not C2H5S; (7) when R l is CF3SO2HN - CONH b and R6 is n-hexyl then R j and R8 are not both hydrogen; (8) when R I is CF3SO2HN -NHCO b R6 is not methoxybenzyI; (9) the R6 group is not -CHFCH2CH2C113 or C1120H; (10) when r=O, R 1 is R 13 X --I -:-'i R 3 R 2 0 0 X is -NH-C-, R13 is 2-NHSO2CF3, and R6 is n-propyl, then R7 and R8 are not -C02CH3; (11) whenr=O,Rlis R 13 X R 2 0 Y X is NH-C-, R 13 is 2-COOH, and RG is n-propyl, then R' and R8 are not C02CHI (12) when r=l, R 13 Rl=x R 3 R2 1 1 X is a single bond, R' is Cl, and RS is -CHO, then R13 is not 3(tetrazol-5-yl).. (13) when r=1, R 13 RX __( \ _1 R 3 R 2 16 X is a single bond, R7 is Cl, and R8 R13 is not 4-(tetrazol-5-yl).

is -CHO, then A preferred embodiment of the invention is the method as recited above, using the imidazole anglotensin II receptor antagonist, of formula l- R 6,2 N R 8 1 CH2 R' R 7 wherein:

R l is -C02H; -NHS02CF3; N-N R 13 R 2 ---N; -X - \i/ 1 - N or QVP X 13 1 R R6 is alkyl of 3 to 10 carbon atoms, alkenyl of 3 to 10 carbon atoms, alkynyl of 3 to 10 carbon atoms, cycloalkyl of 3 to 8 carbon atoms, benzyl substituted on the phenyl ring with up to two groups selected from alkoxy of 1 to 4 carbon atoms, halogen, alkyl of 1 to 4 carbon atoms, and nitro; R8 is phenylalkenyl wherein the aliphatic portion is 2 to 4 carbon atoms, -(CH2)iti-imidazol- lyl, -(CH2)m 1,2,3-triazolyl optionally substituted with one or two groups selected from C02CH3 or alkyl of 1 to 4 carbon atoms, 0 pp (CH2)171-tetrazolyl, -(CH2)y1OR11; -(CH2)iiOCR14; 0 R14 of -CH=CH(CH2)sCR16, -CH=CH(CH2)sCHOR15; 0 0 to 09 -(CH2)11.CR16; -(CH2)11NHCOR10; -(C112)11NHSO2R10; 0 I/ -(CH2),I.F; -CR1 R13 is -C02H, -C02R9, NHS02CF3, S03H; N-N or IN AN H R16 is H, alkyl of 1 to 5 carbon atoms, OR17, or NRISR19; X is carbon-carbon single bond, -CO-, -CON-, -CH2CH2-, -NCO-, h 1 R23 R23 -OCH2-, -CH20-, -SCH2-, -CH2S-, -NHCH2-, -CH2NH- or - CH=CH-; and pharmaceutically acceptable salts of these compounds.

A further preferred embodiment of the invention is the method as recited above, using the imidazole anglotensin II receptor antagonist of formula L R2 is H, alkyl of 1 to 4 carbon atoms, halogen, or alkoxy of 1 to 4 carbon atoms., R6 is alkyl, alkenyl or alkynyl of 3 to 7 carbon atoms; 0 9A R7 is H, Cl, Br, Ct,F2,+1, where v=1-3, or -CR16; 18 0 R 14 Y' 1 R81s-(CH2)111,0R11--(CH2)inO R14;-CH=CH-CHOR15.

c 0 0 -(CH2)mCRIG; -C112NHdORIO; - (CH2),NHSO2Wo., N -N --CH2 A N " H 16. N 'or -COR 1 RIO is CF3, alkyl of 1 to 6 carbon atoms or phenyl; R 11 is H, or alkyl of 1 to 4 carbon atoms; R13 is C02H; C02CH2OCOC(CH3)3: NHS02CF3; N -N and A /N N H R 14 is H, or alkyl of 1 to 4 carbon atoms; R 15 is H, alkyl of 1 to 4 carbon atoms, or acyl of 1 to 4 carbon atoms; R 16 is H, alkyl of 1 to 5 carbon atoms; OR 17; or r-\ -N \ / 0 m is 1 to 5; 1.5 X is single bond, -0--, -CO-; -NHCO-; or -OCH2-; and pharmaceutically acceptable salts.

A preferred embodiment of this invention is the method as recited above, using an imidazole angiotensin 11 receptor antagonist selected from the group consisting of:

2 -Butyl -4-chloro- P [(T- (1 H -tetrazol -5) -y1)bIphenyl -4 -yl)m ethyl] - 5 - (hydroxymethyl)lmldazole.

2 -Butyl-4-chloro- I- [(2'-carboxybiphenyl-4-yl)methyll-5-(h,,droxymethyl)lmldazole.

2-Butyl-4-chloro- P[(T-c arboxybiphenyl - 4 -yl) methyl] -5-[(methoxy- 1 () carbonyl)aminomethyllimidazole.

2-Butyl-4-chloro- 1 - [(2'-carboxybiph enyl- 4 -yl)m ethyl] -5- [(propoxy carbonyl)aminomethyllimidazole.

2-Butyl-4-chloro- I- [(2'-carboxybiphenyl- 4-yl) methyl] imidazole- 5 carboxaldehyde.

2-Butyl- 1-[(2'-carboxybiphenyl-4-yl)methyll-lmidazole-5-carbox- aldehyde.

2-(1E-Butenyl)-4-chloro- 1- [ (2'-carboxybiphenyl-4-yl) methyl] - 5 (hydi.oxymethyl)imidazole.

2-(1E-Butenyl)-4-chloro- 1-[(2'-carboxybiphenyl-4-yl)methyllimidazole-5-carboxaldehyde.

2-Propyl-4-chloro- 1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyll-5(Iiydi.oxymethyl)lmldazole.

2-Propyl-4-chloro- 1[(2'-(IH-tetrazol-5-yl)blphenyl-4-yl)methyllimidazole-5-carboxaldehyde.

2-Butyl-4-chloro- 1- [T- (IH -tetrazol- 5 -yl)biph enylA -yl)m ethyl] imldzole- 5 -carbox aldehyde.

2-(IE-Butenyl)-4-chloro- 1-[(2'-(1H-tetrazol-5-yl)blphenyl-4yl)methyll-"3-hydroxymethyl)lmldazole.

2(1E-Butenyl)-4-chloro- 1-[(2'-(1H-tetrazol-5-yl)blphenyl-4- yl)m ethyl] imidazole- 5 -carboxaldehyde.

2-Butyl-4-chloro- 1-[(2'-(IH-tetrazol-5-yl)-biphenyl-4-yl)niethylj imidazole-5-carboxylic acid.

2 -Prop yl-4-chloro- 1- [(2 -'(1H-tetrazol-5-yl)-biphenyl-4-yl)methvlj imidazole-5-carboxylic acid.

2 -Propyl - 4 -trifluorom ethyl- I- [(T- (III -tetrazol- 5 - yl)blphen yl -4 yl)methyllimidazole-5-carboxyhe acid.

2-Propyl-4-trifluoromethyl-l-[(2'-(1H-tetrazol5-yl)blphenyl-4- yl) m ethyl] - 5 - (Ii y droxyIm ethyl)imidazol e.

2-Butyl-4-trifluoromethyl-l-[(2'-(1H-tetrazol-5-yl)biphenyl-4yl)meth,,lllmldazole-5-carboxylle acid.

2 -Prop yl- 4 - trifi u orom ethyl - I- [(T- (carboxybiphenyl - 4y1)methyll - imidazole-5-carboxaldehyde.

2-Propyl-4-pentafluoroethyl- 1-[(2'-(IH-tetrazol-5-yl)bphenyl-4yl)methyll-5-(hydroxymethyl)lmldazole.

2-Propyl- 1-[(2-(1H-tetrazol-5-yl)blphenyl-4-yl)methyllimidazole-4,5,dicarboxylic acid.

2 -Prop yl -4-p en taflu oroethyl- P [(T- (1H-tetrazol - 5 -yl)blph enyl 4 yl)tn ethyl] imidazole- 5 -carb oxylic acid.

2-Propyl-4-pentafluoroethyl-[(2'-(IH-tetrazol-5-yl)blphenyl-4yl)meth.ylllmldazole-5-carboxaldehyde, or its ph arm aceutic ally acceptable salt thereof.

A more preferred embodiment of the invention is the method as recited above, using the imidazole angiotensin 11 receptor antagonist of formula 1:

2 -B utyl -4-chloro - I- [(2'-tetrazol- 5 -yl)blph enyl- 4-yll methyl] - 5 - (hych-oxy methyl)imidazole (also known as losartan); and 2-Butyl-4-chloro- 1-[(2'-tetrazol-5-yl)biphenyl-4-vllmethylimidazole-5carboxylic acid (also known as EXP-3 17 4) or its ph arm aceutic ally acceptable salt thereof.

Use of an anglotensin 11 receptor antagonist for the manufacture of a medicament for the modulation of intracellular pH and/or cellular ion transport to confer protective effects in a patient with or at risk for tissue ischemia or in a patient at risk of tissue ischemia.

Dose Ranges The magnitude of therapeutic dose of useful in this method of treatment will, of course, vary with the nature of, and the severity of the condition to be treated and with the particular compound utilized and its route of administration and pharmacokinetic profile and will vary upon the clinician's.ludgment. It will also vary according to the age, weight and response of the individual patient. An effective dosage amount of the acti,,,e component can thus be determined by the clinician after a consideration of all the criteria and using is best Judgment on the patient's behalf. Depending on these considerations, anticipated daily doses would be in the range of 1 ing to about 500 mg administered using an appropriate dosing regimen to maintain an effective plasma concentration.

Pharmaceutical Compositions Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention. For example, oral, intraperitoneal, parenteral, subcutaneuous,Intramuscular, transdermal, subkingual intravenous and topical may be employed. Dosage forms include tablets, troches, dispersions, suspensions, wafers, solutions, capsules, creams, ointments, aerosols, and the like.

The pharmaceutical compositions of a compound useful in the present method as an active ingredient or a ph arm aceutic ally acceptable salt thereof, and may also contain a ph arm aceutic ally acceptable carrier and optionally other therapeutic ingredients. The term "ph arm aceu tic ally acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.

The compositions include compositions suitable for oral, parenteral. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.

In practical use, the compounds exhibiting this activity can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.

Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or gTanules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-inoil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Desirably, each tablet contains from about 1 ing to about 500 mg of the active ingTedient and each cachet. or capsule contains from about 1 to about 500 mg of the active ingredient.

The examples disclosed herein are representative of the instant invention, but should not be construed as limiting the scope of the invention.

23) EXAMPLE

METHODS A. Isolation of Venticular-Mvocytes:

Ventnicular myocytes were isolated from the hearts of adult male Wistar rats (200-250 g body weight) or adult male New Zealand White rabbits (2.0-2.5 kg body weight) using a collagenase-based enzymatic digestion technique. 12, 13. 15. 1G (15. Haworth RS, Yasutake M, Brooks G, Avkiran M. Cardiac Na+111+ exchanger during postnatal development in the rat: changes in mRNA expression and sarcolemmal activity- J. Mol. Cell Cardiol. 1997-, 29:321-332. 16. Shipolini AR, Yokoyama H, Galinanes M, Edmondson SJ, Hearse DJ, Avkiran M. Na+/H+ exchanger activity does not contribute to protection by ischemic preconditioning in the isolated rat heart.

Circulation 1997 -, 96. 3617-362 5.) In brief, animals were anaesthetised by inhalation of diethyl ether (rats) or inravenous injection of sodium pentobarbitone (rabbits) and hearts were excised and perfused (37C) in the Langendorff mode for four sequential periods, as follows: (1) with Tyrode's solution (in mN: NaCI, 135; KCI, 5-4; CaC12, L8; MgC12, 0.5; mHEPES, 10; glucose 10; adjusted to pH 7.4 at 34'C with Na011) for 5 min. (11) with nominally Ca2+ -free Tyrode's solution (in mM: NaCI, 137; KCl, 5.4; NaH2P04, 0.33-, MgC12 L0; HEPES, 10; glucose, 10; adjusted to pH -1.2 at 3CC with NaOH) for 5.5 min, (111) with nominally Ca2±free Tyrode's solution CaC12 L8; MgCL,, 0.5 HEPES, 10; glucose 10 adjusted to pH -4.4 at 340C with NaOH) for 5 min, containing collagenase (Worthington Type 1, 94 U ml) for 10 min, and (1v) with storage buffer (in mM: KOH, 718-, KCI, 30; KH2P0 t, 30-1 MgSO 1, 3; EGTA, 0.5; HEPES, 10; glutamic acid, 50; taurine, 20-, glucose, 10-, adjusted to pH 7.2 at 34"C with KOH) for 5 min. All solutions were gassed with 100% Oz,. After the perfusion procedure, the ventricles were removed and chopped into several pieces in storage buffer. The tissue fragments were then gently agitated to facilitate cell dispersion and the cell suspension was maintained in storage buffer at 25 OC for at least 1 h prior to use in the micro epiflu orescence studies.

24 B. Determination of Sarcolemmal NA+11-1, Exchanger Activity.

The pH-sensitive fluorescent dye carboxy seminaphtorhodafluor- 1 (C-SNARF1) was used to monitor pH in single ventricular myocytes, as we have described previously. Cells loaded with C-SNARF- 1 were placed on a glass coverslip, in a 150 ul chamber and were continuously superfused (3.5 mL/min) with Tyrode's solution (34'C) of the composition described above. Since cells were maintained in bicarbonate free medium throughout the experimental protocol, the rates of acid efflux (JiiS) calculated during recovery from intracellular acidosis (see below) could be used as indicators of sarcolemmal Na+/H+ exchanger activity.

C. Experimental Protocols:

The project encompassed 4 sequential studies, the basic protocols for which are illustrated in Figure 1. In all cases, cells (n=8- 10 per group) were subjected to intracellular acidosis by transient (3 mill) exposure to 20 mM NffiCl (first acid pulse), after 5 to 10 mill of superfusion with normal Tyrode's solution (pH 7.4). Following a 6 min period of NffiCI washout, cells were superfused with normal Tyrode's solution for an additional 7 mill, prior to a second transient exposure to NffiCI (second acid pulse). In control cells, both acid pulses occurred under identical conditions.

When studying the effects of angiotensin 11 (10, 100 or 1000 nM.,), this was present throughout the second pulse (i.e. during exposure to and washout of NHiCI). When studying the effects of angiotensin 11 in the presence of PD123319 (10, 30 or 100 nXl) and/or losartan (10, 30 or 100 nmi), the antagonist(s) were included in all solutions from 3 mill before the second 2.5 pulse to the end of the experiment. The effects of the highest concentration of each antagonist (100 n11), alone or in combination, in the absence of anglotensin were also studied in the appropriate protocols. JH-versus-pHI curves were constructed using data obtained dulling the pH. recovery phases following both acid pulses, to identify and quantify drug induced shifts of the curve that, are indicative of a change in sarcolemmal Na+1111 exchanger activity. I2J:' D. Solutions All buffer chemicals were purchased from Merck (BDH). NH Cl (20 mM) was added directly to Tyrode's solution without osmotic compensation. Losartan (Merck Sharp & Dohme), PD123319 (RBI) and anglotensin 11 (Sigina) were all dissolved in deionised water and added to Tyrode's solution (L 1000 dilution) to obtain the required concentrations shortly before the beginning of experiements on each day.

E. Statistical Analysis Experiments within each study (which comprised 4-6 study groups) were carried out in a randomized manner. Data are expressed as mean SEM. Within individual groups, the paired t test was used to assess changes in Ji at identical pHi (e.g. Jii(;,, at pH,- 6.90) between the first and second acid pulses. Inter-group comparison of the change in Jii(;) (AJilf;.!)) was carried out by ANOVA followed by the Bonferroni t test. A value of p<0.05 was considered significant.

RESULTS A. Effect of Anziotensin on Sarcolemmal Na+11---1+ Exchanger Activity:

This study was carried out to determine the effect of anglotensin on sarcolemnal Na+111+ exchanger activity. Figure 2 shows the effects of exposure to 10- 1000 nM angiotensin II on J i(;.) in rat ventricular myocytes. Over the concentration range studied, anglotensin had no significant effect on J i was sustained throughout the pH range 6.70-71. 10 (data not shown)Similar observations were made in rabbit ventricular myocytes also (data not shown).

These data illustrate that non-selective stimulation of cardiac angiotensin receptors has no effect on sarcolemmal Na+11---1+ exchanger activity. B. Effect of AT i Receptor Stimulation on Sarcolemmal Na+fflExchanger Activity:

Since AT, and AT2 receptors have been shown to mediate opposing actions in some cell systems, 1,17 18 this 26 study was carried out in rat, ventricular myocytes to determine the effect of selective AT, receptor stimulation on sarcolemmal Na+/H- exchanger activity. (17. Stoll M, Steckelings UM, Paul M, Bottarl SP, Metzger R, Unger T: The anglotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J. Clin. Invest. 1995; 95:651-657. 18. Nakailma M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R, Zhang L, 1-1orluchi M, Pratt RE, Dzau VJ: The anglotensin 2 type 2 (AT2) receptor antagonizes the growth effects of the ATI receptor: gain - of-function study using gene transfer. Proc.

Natl. Acad. Scl. USA 1995; 92:10663-1066-1.) Figure 3 shows representative recordings of pH. obtained during the first and second pulses in a control cell and in cells that were exposed to 100 nM angiotensin II during the second pulse, alone or in the presence of 100 nM P1) 123319 (AT, antagonist). As expected, the pH, recovery profiles dulling the first, and second pulses were similar in the control cell (panel A). When the second pulse was in the presence of angiotensin alone, pHi recovery from acidosis was again unaffected (panel B); however, when agiotensin was administered together with P1) 12 3 19 (to selectively stimulate at AT i recep tor) recovery from acidosis was greatly accelerated (panel C).

Figure 4 shows the effects of the various treatments on Jiii;,ij Anglotensin alone again did not significantly change Ji,i.!, confirming the findings of the first study. However, a significant increase in in response to anglotensin was revealed in the presence of 30 and 100 nM of P1) 123319 (panel A). This was reflected by a significant increase in A/ii,;. !j relative to control in the group that received ang-lotensin in the presence of nM PD123319 (panel B). That the increase in Jii,., was induced by ano-lotensin rather than by the AT2 antagonist itself was confirmed b the y lack of effect of PD123319 when given at 100 nM.

Figure 5 shows the Ji-versus-IM; curves obtained during the first and second pulses in the 3 key groups within the study. The second pulse curve was not shifted significantly in the control group (panel A), indicating the absence of a temporal change in sarcolemmal Na+11+ exchanger activity throughout the pH, range 6.75-10. The secondpulsecur- ve 2.5 27 was also not shifted in the presence of anglotensin alone (panel B.) However, when anglotensin was given in the presence of PD 1233 19 (panel C), Ji i was significantly increased throughout the pHi range covered (6. 75-7.00).

These data suggest that selective stimulation of the ATi receptor 5 by angiotensin signifcantly increases sarcolemmal Na+lH+ exchanger activityHowever, this effect is counteracted by simultaneous stimulations of the AT2 receptor, such that angiotensin alone does not alter exchanger activity. C. Effect of Losartan on the Response to ATI Receptor Stimulation:

This study was carried out to determine whether the increase in sarcolemmal Na+/H+ exchanger activity induced in rat ventricular myocytes by the anglotensin II/PD123319 combination is inhibitable by losartan, thereby confirming that the response is mediated selectively via the AT I receptor. Figure 6 shows representative recordings of pHi obtained during the first and second pulses in cells exposed to 100 nM an iotensin 11 during 91:n the second pulse in the absence of any antagonist (panel A), in the presence of 100 nM PD 123319 (panel B) or in the presence of both 100 nM P1) 123319 and 100 nM losartan (panel C). As before, pHi recovery from acidosis was greatly accelerated when anglotensin was administered together with PD123319, to selectively stimulate the AT1 receptor. However, this effect was completely abolished by the coadminstration of losartan.

Fig-tire 7 shows the effects of the various treatments on JH6.9. The significant increase in JH6.9 that occurred in response to the angiotensin II/P1) 123319 combination was abolished by both 30 and 100 nM losartan (panel A). Consequently, the the groups which received these concentrations of losartan., JH6.9 was similar to control and significantly less than that observed in cells exposed to anglotensin IIIPD123319 combination in the absence of losartan (panel B). The combined administration of losartan and PI) 123319 (at 100 nM each) in the absence of anglotensin had no effect on JH6.9 or AJH6.9.

Figure 8 shows the JH-versus-pH; curves obtained during the first and second pulses in the 3 key groups within this study. As illustrated, 28 losartan abolished the increase in J11 in response to the anglotensin IIIPD123319 combination throughout the pHI range covered (6.75-7.05).

These data show that losartan inhibits the increase in sarcolemmal Na+/H+ exchanger activity induced by the anglotensin IIIPD123319 combination, confirming that exchanger stimulation by this intervention is mediated selectively via the AT l receptor subtype.

D. Effect of AT- Receptor Stimulation on Sarcoleminal Na+/H+ Exchancer Activity:

This study was carried out to determine whether the ability of AT2 receptor stimulation to inhibit ATI receptor-mediated activation of the sarcolemmal Na+111+ exchanger in rat ventricular myocytes arises from direct inhibition of exchanger activity. Figure 9 shows the effects on JH6.9 Of exposure to 100 iiM angiotensin II, alone or in the presence of 10, 30 or 100 nM losartan (to selectively stimulate the AT2 receptor). Anglotensin had no significant effect on M6.9 in any of the groups (panel A); consequently, J116.9 was similar to control in all cases (panel B). The lack of effect of anglotensin on JH was sustained throughout the pH range 6.64-7. 10 Losartan alone 100 nM also had no effect.

These data shown that the ability of AT2 receptor stimulation to inhibit AT1 receptor-mediated activation of the sarcolemmal Na+/H+ exchanger does not arise from direct inhibition of the exchanger. It is likely, therefore, that this novel negative-regulatory action is mediated via interruption of the signaling pathway(s) that link AT1 receptor stimulation to exchanger activation.

AnInotensin II increases sarcolemmal Na+fH- exchanger activity in cardiac ventricular myocytes by stimulation of the ATi receptor.

The AT, receptor-mediated increase in sarcolemmal Na+/11+ exchanger activity is counteracted by stimulation of the AT.2 receptor.

The AT2 receptor-mediated negative regulatory effect is likely to arise from inhibition of exchanger-stimulatory signaling pathway(s) rather than from inhibition of the exchanger.

29 a This is the first report of a dual regulatory mechanism in an glotensin - mediated modulation of sarcolemmal Na+/H+ exchanger activity in cardiac ventricular myocytes. Sarcolemmal Na+/H+ exchanger activity is believed to be an important determinant of the severity of cardiac injury and dysfunction, in particular the incidence of ventricular fibrillation 111. 19-21 in myocardial ischemia and repeifusion (for recent reviews, see AvkiraWl, Avkiran and Yasutake 2-"and Frdhheh and Karmazynl). (19. Yasutake M, Ibuki C, Hearse DJ, Avkiran M: Na+/W- exchange and reperfusion arrhythmias: protection by intracoronary infusion of a novel inhibitor. Am. J. Physiol. 1994; 267:112430-H2440. 20. Yasutake M, Avkiran M: Exacerbation of reperfusion arrhythmias by (xl-adrenergic stimulation: a potential role for recep toy -mediated activation of sarcolemmal sodium-hydrogen exchange. Cardiovasc. Res. 1995. 1995; 29:222- 230. 2 1. Xue YX, Aye NN, Hashimoto K.. Antiarrhythmic effects of HOE642, a novel Na+lH+ exchange inhibitor, on ventricular arrhythmias in animal hearts. Eur. J. Pharmacol. 1996; 31,47:309316. 22. Avkiran M, Yasutake M: Role of the sarcolemmal Na+IH-exchanger in arrhythmogenesis during reperfusion of ischemic myocardium, in Fliegel L (ed): The Na/H Exchanger. Austin, RG Landes, 1996, pp. 17 3187) Indeed, the strength of pre- clinical evidence in this area has led to the clinical evaluation of the cardioprotective efficacy of cariporide (a direct inhibitor of the Na+/H+ exchanger) in patients with acute coronary syndromes, in the current GUARDIAN study.

On the basis of the present data, it is reasonable to speculate that selective antagonists of the AT, receptor such as losartan may exhibit cardioprotective actions. This may occur by inhibiting activation of the sarcolemmal Na+/H+ exchanger during ischemic episodes, not only by angiotensin itself (through blockade of positive regulatory ATireceptors) but also by other neurohormonal mediators (through enhanced stimulation of negative regulatory AT2 receptors). Indeed, in view of the evidence that the AT2:ATi receptor ratio is increased in the failing human heart, 2',.2 1 it is possible that such a cardioprotective mechanism may have contributed to the reduction in sudden cardiac death in elderly heart failure patients treated with losartan versus those treated with captopril, in the recent, ELITE study.25 (23. Asano K, Dutcher DL, Port J1), Minobe WA, Tremmel KI), Roden RL, Bohlmeyer TJ, Bush EW, Jenkin MJ, Abraham WT, Raynolds MV, Zisman LS, perryman MB, Bristow MR. Selective downreg-ulation of the 5 anglotensin II AT I receptor subtype in failing human ventricular myocardium Circulation 1997; 95A193-1200.24. Haywood GA, Gullestad L, Katsuya T, Hutchinson HG, Pratt RE, Horiuchi M, Fowler MB: ATI and AT2 angiotensin receptor gene expression in human heart failure. Circulation 1997; 95.1201-1206. 25. Pitt B, Segal R, Martinex FA, Meurers G, Cowley AJ, Thomas 1, Deedwania PC, Ney DE, Snavely DB, Chang PI: Randomised trial of losartan versus captopril in patients over 65 with heart failure (Evaluation of Losartan in the Elderly Study, ELITE). Lancet 1997; 349:7147 -752.) In the light of the above, further investigation of anglotensinmediated reg-ulation of sarcolemmal Na+11+ exchanger activity appears warranted. In particular, it would be of significant value to (i) identify the mechanism(s) underlying the negative regulatory action of AT2 receptor stimulation and (ii) determine the efficacy of this pathway in inhibiting Na+111+ exchanger activation by other neurohormonal mediators that are associated with myocardial ischemia, such as catecholamines., thrombin and endothelin.

31

Claims

WHAT IS CLAIMED IS:

1. A method for modulating intracellular pH and/ or cellular ion transport, to confer protective effects by administering to a patient with or at risk for tissue ischemia a therapeutically effective amount of an 5 anglotensin 11 receptor antagonist.

2. A method for modulating intracellular pH and/ or cellular ion transport to confer protective effects by administering to a patient, with or at risk for tissue ischemia a therapeutically effective amount of an anglotensin II receptor antagonist selected from the group consisting ofcandesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, 13MS- 184698, 3-(2'-(tetrazol-D'-yl)- 1, F-biphen4yl)methyl-5,-i-dimethyl-2-ethyl-3H-Imidazo[4,5-blpyridine, BAY1067134, BIBR363, C1,329167, E4177, EM1)73495, HN65021, H11720, HOE720, LRB08 1, SC52458, SL910102, UP2696, YM358, EM1)66397, ME322 1, TAK536, 13MS184698, CG142112A, CGP49870, CP148130, E4188, EM1)66684, EXP9954, F111153332, GA0050, KT3579, LF70156, LR13057, LY266099, LY301875, PD123177, PD126055, SC51757, SC54629, U96849, UK77-178, WAY12622-1, WK1260, WK1492, Y1-11498, andYM31472.

3. The method as recited in Claim 2, wherein the anglotensin II receptor antagonist is selected from the group consisting of.

candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, BMS-184698 and 3-(2'-(tetrazol-5-yl)-1,1'-biphen4- yl)methyl- 5,7- dimethyl- 2 -ethyl- 31-1-1midazo [4,5 -bl pyridine.

32 4. A method of modulating intracellular pH and/ or cellular ion transport to confer protective effects by administering to a patient with or at, risk for tissue ischemia a therapeutically effective amount of an anglotensin II receptor antagonist of formula N R 7 R 6 A N 11.1 R 8 1 (CH2)r R' 23 3 R R 1 wherein:

RI is:

1 1 0 4-CO,H; 4-CO,R'. -0-SI'-OR -S03 3)2 11 1 H- -C(CF OH.

OH 0 11 6H 0 1 -P03H21 -NH-P-OH-, 4-NHSO2CH3, 6H OH 0 N-N 12.

4-NHSO2CF3-, -CONHOR, NH -S02 2 -C -F 3 N 27 6H H N-N,,41 N _-N; H 4X R 13 R 13 F F 4-X " 4-X- F C\, R 3 ' R 2 R 13 F Q__P X \ R13 34 0 11 -HNC 1 HOC 11 0 N-N 4-CONH 'I, N..; H )11 C02H 4-CONHNHSO2CF3. 4-CONH- CHCH2C6H5 (/-isomer) 11 4-CON 1.

(/-isomer) C02H H02C R 4 J z \ 1 R 11 ' N-N 4 -J,' N CF H R 13 0 R 13 -1 3 4 4-N. or 4 -4yiN H -X--0 -R R 4 0 R 2 0 R 20 s_o -C-NHS02- (CH2) \ / 1.5 3 R2 is H Cl; Br; I; F N02; CN; alkyl of 1 to 4 carbon atoms; acyloxy of 1 to 4 carbon atoms; alkoxy of 1 to 4 carbon atoms; C0211; C02R9; HNS02CH3, NHS02CF3; N-N 12. CONHOR. S02NH2- 1 N. aryi. or furyl,1A N 1 1 1 H R3 is H; Cl, Br, 1 or F; alkyl of 1 to 4 carbon atoms or alkoxy of 1 to 4 carbon atoins; R4 is CN, N02 or C02R 1 R5 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms alkenyl or alkynyl of 2 to 4 carbon atoms; R6 is alkyl of 2 to 10 carbon atoms, alkenyl or alkynyl of 3 to 10 carbon atoms or the same groups substituted with F or C02R 14; cycloalkyl of 3 to 8 carbon atoms, cycloalkylalkyl, of 4 to 10 carbon atoms; cycloalkylalkenyl or cycloalkylalkynyl 5 to 10 carbon atoms; (CH2)sZ(CH2)17,R5 optionally substituted with F or C021114; benzyl substituted on the phenyl ring with 1 or 2 halogens, alkoxy of 1 to 4 carbon atoms, alkyl of 1 to 4 carbon atoms or nitro; R7 is H, F, Cl, Br, 1, N02, Cj2t,+L where v=1-6, C05-1 CN; 0 11 16 -C-R straight or branched alkyl of 1 to 6 carbon atoms; phenyl or phenylalkyl, where alkyl is 1 to 3 carbon atoms; or substituted phenyl or substituted phenylalkyl, where alkyl is 1 to 3 carbon atoms, substituted with one or two substituents selected from alkyl of 1 to 4 carbon atoms, F, Cl, Br, OH, OCHI CF3, and COOR, where R is H, alkyl of 1 to 4 carbon atoms, or phenyl; R8 is H, CN, alkyl of 1 to 10 carbon atoms, alkenyl of 3 to 10 carbon atoms, or the same groups substituted with R. phenylalkenyl wherein the aliphatic portion is 2 to 6 carbon atoms; -(CH2)niimidazol- PyLl -(CH2)m1,2,3-triazolyl optionally substituted with one or two group selected from C02CH3 or alkyl of 1 to 4 carbon atoms; -(CH2)s tetrazolyl; -(CH2)n-ICH-RI I; -(CH2)71,OCR14, -(CH2)iiSR15; 9 OR17 R14 0 0 -CH=CH(CH2)sCHOR15, -CH=CH(CH2)sCR16, -CR16; 0 of -CH=CH(CH2)sOCRI I; (CH2)s-CH-COR16,. 1 CH3 0 Y Y c COR10; (CH2)nCR 16 -(CH2)nO #'NHR 10; -(CH2)nNR 11 " 0 I -(CH2)1INR11CNHR10-, -(C112)11,NR11SO2R10.

)6 Y F1 -(CH2)1INRI1CRIO; -(CH2),?tF; -(CH2)17LON02,- -CH2N1 -(CH2)171NO2; -CH=N-NRI1R17; 0 Z- 1 - (CH2)m-N n 0 N-N (C H2)9- N)CF3 H 0 11 /11 (CHAn-lC-N N CH30 -CH-N-NH-S02 -0 1 R24 0 1 P R9 is: -CH-OCR21, RIO is alkyl of 1 to 6 carbon atoms or perfluoroalkyl of 1 to 6 carbon atoms, 1-adamantyl, I-naplithyl, P(Pnaplithyl)ethyl, or (CH2)pC6H5; R 11 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl or benzyl; R 12 is H, methyl or benzyl-, R13 is -C02H; -C02R9; -CH2CO2H, -CH2CO2R9; 37 N=N --(CH2) NH 4 R (CH2)n-N N CH30 1 N or -CH=N-NH-</ 1 7 N H 0 0 11 99 0 OH to 1 1 1 1 1 OH OH OH R27 OH -P03H2; X(CF3)20H. -NHS02CH3. -NHS02CF3; -NHCOCF3; -CONHOR12; -S02NH2-, N-N N -N N-N N AI IN.'),,' N N CH2 N -CONW N 31 H H CONHNHSO2CF3-, N-N. or 1 X N CF3 H N=N ---,y NH ' R 4 R14 is H, alkyl or perfluoroalkyl of 1 to 8 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl or benzyl; R15 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, benzyl, acyl of 1 to 4 carbon atoms, phenacyl; R 16 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, (CH2)pC6H5, OR17, or NR18RIT, R17 is H, alkyl of 1 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl or benzyl; R 18 and R 19 independently are H, alkyl of 1 to 4 carbon atoms, phenyl, benzyl, (x-methylbenzyl, or taken together with the nitrogen form a iling of the formula /- (C H2)t -N \--i Q., Q is NR20, 0 or CH2; R20 is H, alkyl of 1-4 carbon atoms, or phenyl, R21 is alkyl of 1 to 6 carbon atoms, -NR22R23, or -CHCH2CO2CH3; 9 NH2 R22 and R23 independently are H, alkyl of 1 to 6 carbon atoms., benzyl, or are taken together as (CH2)u where u is 3-6,- 18 R24 is H, CH3 Or -C6H5; R25 is NR27R28, OR28, NHCONH2, NWSNH2, NHS02 -C)- CH3.1 or -NHS02 __0 R26 is hydrogen, alkyl with from 1 to 6 carbon atoms, benzyl, or allyl; R27 and R28 are independently hydrogen, alkyl with from 1 to 5 carbon atoms, or phenyl; R29 and R30 are independently alkyl of 1-4 carbon atoms or taken together are -(CH2)q-; R31 is H, alkyl or 1 to 4 carbon atoms, -CH2CH=CH2 or CH2C6H4R32; X is a carbon-carbon single bond, -CO-, -CH2-, -0-, -S-, -NH-, -N-, -CON-, -NCO-, -OCH2-, -CH20-, -SCH2-, -CH2S-, 1 1 % R26 R23 R23 -NHC(R27)(R28)-, -NR23S02-, -S02NR23-, -CH=CH-, -CF=CF-, - CH=CF-, -CF=CH-, -CH2CH2-, X(R271)(R28)NH-, _CF2CF2 1 /A R 29o OR 30 -C- 1 Yis 0 or S-7 Z is 0, NRI I, or S; m is 1 to 5; n is 1 to 10; p is 0 to 3; q is 2 to 3; r is 0 to 2; OR 14 OCOR'7 NR 25 C - or 1 -H- -H- - 11 39 s is 0 to 5; t is 0 or 1; and pharmaceutically acceptable salts of these compounds; provided that:

(1) the R l group is not in the ortho position; (2) when R I is R l- -X-1 - R' Rz X is a single bond, and R13 is C02H, or N -N ,,k N '.N H then R13 must be in the ortho or meta position., or when R I and X are as above and R13 is NHS02CF3 or NHS02C R 13 must be ortho; (3) when R l is R 3 R 3 R 2 H3, and X is other than a single bond, then R 13 must be ortho except when X=NR23C0 and R13 is NHS02CF3 Or NHS02CH3, then R 13 must be ortho or meta- 1 (4) when RI is 4-CO2H or a salt thereof, R6 cannot be S-alkyl,when RI is 4-CO2H or a salt thereof, the substituent, on the 4position of the imidazole cannot be CH20H, CH20COCH3, Or CH2C0211; (6) when R 1 is -X 1 R 13 R 3 --cl, R 2 X is -OCH2C2115S; (7) when R I is and R 13 is 2-CO2H, and R7 is H then R6 is not CF3SO2HN -CONH b and RG is n-hexyl then R i and R8 are not both hydrogen; (8) when R I is CF3SO2HN -NHCO R6 is not methoxybenzyl; (9) the R6 group is not -ClIKH2CH2CH3 or CH2011; (10) when r=O, R I is 41 R 13 X R3 R 2 0 P' X is -NI-I-C-, R13 is 2-NHSO2CF3, and R6 is n-propyl, then R7 and R8 are not, -C02CHI (11) when r=O, R 1 is R 13 X R 2 0 11 X is NH-C-, R13 is 2-COOH, and R6 is n-propyl, then R' and RS are not C02CHI (12) when r=l, R 13 R '=X _1 R 3 R 2 1 X is a single bond, R7 is Cl, and RS is -CHO, then R 13 is not 3(tetrazol- 5-yl); (13) when r=1, _CR 13 R1=X _1 ', R 3 RI, 42 X is a single bond, R7 is Cl, and RS is -CHO, then R13 is not 4-(tetrazol- 5-yl).

5. The method as recited in Claim 4, using the angiotensin 11 receptor antagonist of formula L R 6" N R 8 1 CH2 R' R 7 wherein:

RI is -C02H,- -NHS02CF3-, N-N R 13 - R2 N---N; -X-\ H or QXY9 1 R 13 R6 is alkyl of 3 to 10 carbon atoms, alkenyl of 3 to 10 carbon atoms, alkynyl of 3 to 10 carbon atoms, cycloalkyl of 3 to 8 carbon atoms, benzyl substituted on the phenyl ring with up to two groups selected from alkoxy of 1 to 4 carbon atoms, halogen, alkyl of 1 to 4 carbon atoms, and nitro; R8 is phenylalkenyl wherein the aliphatic portion is 2 to 4 carbon atoms, -(CH2)tn -imidazol- lyl, -(CH2)i?? 1,2,3-triazolyl optionally substituted with one or two groups selected from C02CH3 Or alkyl of 1 to 4 carbon atoms, 4 3 0 1# (CH2)171-tetrazolyl, -(C112)71,0R1 I; -(CH2biOCR14 0 R14 #1 1 -CH=CH(CH2)sCR16, -CH=CH(CH2)sCHOR15; 0 0 -(CH2)iiCR16; -(CH2)IINH"OR10- -(CH2),,NHSO2R10; c 0 -(CH2)jj; -CR16 1 R13 is -C02H, -C02R9, NHS02CF3; S03H; N-N or "N - H R16 is H, alkyl of 1 to 5 carbon atoms, OR17, or NR18R19; X is carbon-carbon single bond, -CO-, CON-, -CH2CH2-, -NCO-, R23 R23 -OCH2-, -CH20-, -SCH2-, -CH2S-, -NHCH2-, -CH2NHor - CH=CH-, and pharmaceutically acceptable salts of these compounds.

6. The method as recited in Claim 5, using the anglotensin 11 receptor antagonist of formula 1 wherein" R2 is H, alkyl of 1 to 4 carbon atoms, halogen, or alkoxy of 1 to A carbon atoms; R6 is alkyl, alkenyl or alkynyl of 3 to

7 carbon atoms; 0 R7 is H, Cl, Br, C(Y2t.1+1, where v=1-3, or -CR16.

R14 RS is -(CH2)i7i0R1 I; -(CH2)111OCR14. -CH=CH-CHOR15-, 0 p# 0 0 if -(CH2)mCR16.; -CH2NWOR10; 44 N -N - (CH2),NHSO2R'O-, N or -COR 16- CH2 A N H R 10 is CF3, alkyl of 1 to 6 carbon atoms or phenyl; R 11 is H, or alkyl of 1 to 4 carbon atoms; R13 is C02H; C02CH2OCOC(CH3)31 NHS02CF3; N -N and A IN N H R 14 is H, or alkyl of 1 to 4 carbon atoms; R 15 is H, alkyl of 1 to 4 carbon atoms, or acyl of 1 to 4 carbon atoms; R 16 is H, alkyl of 1 to 5 carbon atoms; OR 17; or r-\ -N \,-/ 0.

m is 1 to 5; Xis single bond, -0-; -CO-; -NHCO-; or -OCH2-; and pharmaceutically acceptable salts- 7. The method as recited in Claim 4, wherein the angiotensin 11 receptor antagonist of formula 1 is selected from the group consisting of:

2 -B utyl-4-chloro- P [(T- (1 H-tetrazol - 5 -y1)biphenylA -yl)m ethyl] 5 - (hydroxymethyl)lmidazole.

2 -Bu tyl-4 -chloro - P [(2'-carb oxybiph enyl - 4-yl) methyl] - 5 (hydroxy methyl)lmldazole.

2 -Bu tyl -4 -chloro - 1- [(T-carb oxybiphenyl -4-yl) methyl] - 5 - [(meth oxy carbonyl) aminom ethyl] imi dazole.

2 -Bu tyl -4 -chloro - P [(2'-carb oxybiph enyl -4 -yl) methyl] - 5 [(prop oxy carbonvl)anilnomethyllimidazole.

2-Butyl-4-chloro- 1-[(2'-carboxybiphenyl-4-,,1)methyllimidazole-5carboxaldehyde- 2-Butyl1-[(2'-carboxybiphenyl-4-yl)methyll-lmidazole-5-carboxaldehyde.

2 -(1E-Butenyl)-4-chloro- 1- [(2'-carboxybiphenyl-4-yl)methyl] -5(hydi. oxymethyl)lmidazole.

2-(IE-Butenyl)-4-chloro- P[(2'-carb oxybiphenyl- 4-yl) methyl] imidazole5 -carb ox aldehyde.

2-Propyl-4-chloro- P[(T- (1H -tetrazol - 5 -y1)biphenyl- 4-yl) m ethyl] 5(hydroxymethyl)lmldazole.

2-Propyl-4-chloro- 1 [(T- (1 H-tetrazol- 5 -y1)biphenyl-4-y1) methyl] imidazole-5-carboxaldehyde.

2-Butyl-4-chloro- 1-[2'-(IH-tetrazol-5-yl)blphenyl-4-yl)methyllimldzole-5carboxaldehyde.

2-(1E-Butenyl)-4-chloro- 1-[(2'-(1H-tetrazol-5-yl)biphenyl-4yl)methyll -5 -hydroxymethyl)lmldazole.

2(1E-Butenyl)-4-chloro- 1-[(2'-(IH-tetrazol-5-yl)blphenyl-4yl)methyllimidazole-5-carboxaldehyde.

2-Butyl-4-chloro- 1-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl)methyllimidazole-5-carboxylic acid.

2-Propyl-4-chloro-l-[(2-'(1H-tetrazol-5-yl)-biphenyl-4-yl)methyllimidazole-5-carboxylic acid.

2 -Prop yl- 4-trifluoromethyl- 1- [(T- (1 H-tetr azol- 5 -y1)bIphenyl- 4 yl)methyllimidazole-5-carboxylic acid 2 - Propyl-4-trifluorom ethyl- I- [ (T- (1 H - tetrazol- 5 -y1)bIphenyl - 4 yl)methyll-5-(hydroxylmethyl)lmldazole.

2.5 2-Butyl-4-trifluoromethyl- 1-[(2'-(1H-tetrazol-5-yl)blphenyl-4- yl) methyl] imidazole - 5 -carb oxylic acid.

2 -Prop 571-4-triflu orom ethyl- I- [(T- (carboxybiphei-iyl - 4 y1)methyll imidazole-5-carboxaldehyde.

2 -Prop yl-4-p entafluoroethyl - I- [(T- (1 H- tetrazol- 5 -yl)blph enyl 4yl)methyll-5-(liydroxymethyl)lmldazole.

2-Propyl-l-[(2-(1H-tetrazol-5-yl)blphenyl-4-yl)methyllimidazole-4,5, dicarboxylic acid.

46 2-Propyl-4-pentafluoroethyl- 1-[(2'-(1H-tetrazol-5-yl)blphenyl-4- yl) m ethyl] imi dazole - 5 -carb oxylic acid.

2-Propyl-4-pentafluoroethyl-[(2'-(1H-tetrazol-5-yl)blphenyl4yl)methyllimidazole-5-carboxaldehyde, or a pharmaceutically acceptable salt thereof

8. The method as recited in Claim 7, wherein the anglotensin 11 receptor antagonist of formula I is: 2 -Butyl-4-chloro- 1- [(Ttetrazol- 5 - y1)biphenyl -4-yll methyl] -5-(hydroxy- methyl)lmldazole,. and 2-Butyl-4-chloro-l-[(2'-tetrazol-5-yl)blphenyl-4-vllmethylimidazole5carboxylic acid or a pharmaceuticafly acceptable salt thereof.

9. Use of an anglotensin TI receptor antagonist for the 1,5 manufacture of a medicament for the modulation of intracellular pH and/or cellular ion transport to confer protective effects in a patient with or at risk for tissue ischemia or in a patient at risk of tissue ischemia.

4 7