CARBOXYLIC ACID AS ANTIHYPERTENSIVE AGENTS

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SYNTHESIS AND BIOLOGICAL ACTIVITY OF 4'-(5-AMINO-6-CHLORO-2- SUBSTITUTED-BENZOIMIDAZOL-1-YLMETHYL)]-BIPHENYL-2-

CARBOXYLIC ACID AS ANTIHYPERTENSIVE AGENTS

M. C. SHARMA^a*, D. V. KOHLI^a, SMITA SHARMA^b

aDepartment of Pharmaceutical Sciences Dr.Hari Singh Gour University Sagar (M.P) 470003 India

bDepartment of Chemistry Yadhunath Mahavidyalya Bhind (M.P) 477001 India

A series of 4`-(5-Amino-6-chloro-2-substituted-benzoimidazol-1-ylmethyl)]-biphenyl-2- carboxylic acid side chain in the 2 position have been synthesized from substituted compounds [1-14] compounds aromatic aldhyde,ayl,alkyl groups and tested for antihypertensive activity in induced hypertensive rats. All the compounds have been found to be less active than Losartan, Telmisartan their structures were assigned with elemental analysis, melting point and spectral analysis like ^IR, 1H NMR,¹³C NMR and EI MS (Received May 23, 2010; accepted June 15, 2010)

Keywords: 5-Amino, angiotensin II, antihypertensive agents

1. Introduction

The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure through the actions of angiotensin II (AII) (vasoconstriction, aldosterone secretion, renal sodium reabsorption, and nor epinephrine release) and thus is an appropriate target for therapeutic intervention in hypertension.¹The renin-angiotensin system (RAS) is known to play an important role in the regulation of blood pressure and electrolyte balance.² Inhibitors of the RAS would be effective for the treatment of hypertension and congestive heart failure. Although angiotensin- converting enzyme (ACE) inhibitors are highly effective and their use has become well- established for the treatment of hypertension and congestive heart failure, they suffer from some side effects such as dry cough and angioedema caused by the nonspecific action of ACE.³ On the other hand, angiotensin II (AII) (the primary effector component of the RAS) receptor antagonists block the RAS at the AII receptor level and are expected to be more specific and effective agents than ACE inhibitors.Angiotensin II (Ang II)a type 1 (AT1) receptor belongs to the G protein- coupled receptor superfamily and mediates virtually all the known physiological actions of Ang II through interaction with heterotrimeric G-protein and subsequent activation of several effector systems (phospholipases C, D, A2, adenyl cyclase, etc.). AT1 receptor shows the seven hydrophobic transmembrane domains forming R-helices in the lipid bilayer of the cell membrane and plays a key role in the reninangiotensin system involved in the regulation of cardiovascular functions and pathophysiology of hypertension.^4,5 The discovery of potent and orally active nonpeptide Ang II antagonists such as losartan and eprosartan has encouraged the development of a large number of similar compounds.⁶ Amongthem, irbesartan, candesartan, valsartan, telmisartan, tasosartan,and olmesartan are on the market. Most of the developed AT1 receptor antagonists are characterized by the presence in their structure of the biphenyl fragment bearing an acidic moiety and differ in the nature of the pendent heterocyclic system (valsartan lacks the heterocyclic moiety) connected to the para position of the proximal phenyl.Substantial effort has been made to find renin inhibitors, although orally active agents have only recently been reported.⁷

*Corresponding author: [email protected]

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No less effort has been devoted to finding AII antagonists, which besides being the most direct way of controlling the RAS could have the additional advantage of lacking the side effects, such as cough and angioedema, observed with ACE inhibitors, as these are probably caused by partial inhibition of the cleavage of bradykinin and substance P. Starting from the initial leads reported by Takeda,⁹ researchers at DuPont discovered losartan, the first orally active AT1 selective nonpeptide AII antagonist that reached the market for the treatment of hypertension (1994, Cozar).

Whereas reports on effectivereplacements of the biphenyl tetrazole “tail” of losratan are scarce, the imidazolic “head” of the molecule, postulated to act mainly to link the required functionalities, has been successfully replaced by a wide variety of cyclic and acyclic structures, leading to a number of compounds currently in clinical trials.¹⁰AngII receptor antagonists are expected to have similar therapeutic effects and indications as the ACE inhibitors without unwanted side effects associated inhibition of other ACE mediated pathways, such as bradykinin metabolism.Intial research in this area led to the discovery of peptide analog such as saralasin ([sar1-Ala8]-AngII) which displayed potent and selective AngII receptor antagonist activity both in vivo and in vitro.However, these peptides had limited therapeutic utility due to partial agonist activity short duration of action and lack of appreciable oral bioavailability¹¹.Only in recent years a number of non peptides AngII antagonists that show promise as inhibitors of the RAS been reported¹².All these antagonists possess a central aromatic nucleus bearing the pharmacophores indispensable for activity and notably a polar function adjustant to biphenyl substituents while a polar function in this area of molecule seems to be necessary to maintain activity¹³. Sartans are appropriately substituted heterocyclic head coupled through a methylene linker to pendent biphenyl system bearing an acidic function; viz. candesartan is an effective competitive Ang II antagonist with benzimidazole nucleus as the heterocyclic head ¹⁴. The substituent at 6-position on the nucleus increases the activity whereas small substituent at 5-position decreases the activity¹⁵. Compounds containing tetrazole nucleus are also reported as AT₁ receptor antagonists and their protypical derivative 3 exhibits non-competitive antagonism¹⁶ and amino group attach with carboxylic group given good biological activity ^{17 ,18}

2. Experimental

Melting points were determined in open capillary tubes and are uncorrected. The time required for completion of the reaction was monitored by TLC using Silica gel-G plates and spots were exposed in iodine chamber. IR spectra were recorded on a Perkin Elmer 1800 (FTIR) spectrometer 1H NMR spectra (DMSO) were taken on a DRX-300 spectrometer (300 MHz) using TMS as internal standard and chemical shifts are expressed in δ ppm.

MCS-6-chloro-2-substitued -1H-benzoimidazole

4-chloro-benzene-1,2-diamine(5.0 gm) was dissolved in a mixture of methanol/water (200 ml, v/v 1:1).To this, different aromatic aldehyde,alkyl and aryl compound (1.1gm) in absolute ethanol (25ml) and Cu (OAc)₂ H₂O (3.1 gm in water (100ml) were added sequentially while stirring the solution. The reaction mixture was then heated to reflux under vigorous stirring for three hours after this a white precipitate was formed. The mixture was filtered hot and then washed with water to afford a yellow solid. The precipitate was again dissolved in ethanol (150ml) and to this, HCl (15 ml) and solution of Na2SxH2O was added in water (100 ml).At this stage, the reaction mixture was heated at reflux for 2.5 hour, resulting in the formation of black slurry.

Reaction mixture was allowed to cool to room temperature and filtered through a pad of celite to remove the precipitated CuS.The filtrate was treated with ammonia solution to pH 8-9 and then concentrated to yield a reddish pale precipitate. After filtration and vacuum evaporation, compound obtained as reddish solid.

MCS-02: 6-chloro-5-nitro-2-substitued -1H-benzoimidazole

65.0 ml of concentrated nitric acid was placed in three necked flask and equal quantity of concentrated sulphuric acid (1:1) was added slowly. The mixture was kept in the ice cold water then compound MCS-01(different R-methyl, ethyl, butyl) (15.10 gm) was mixed in portions during 2 hour under room temperature. After stirred continuously for 14 hours minutes and then

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the reaction mixture was poured slowly over crushed ice with stirring. The precipitated product was filtered out and washes with cold water. The final product recrystillzed from absolute ethanol.

MCS 03: (Biphenyl Carboxylic acid)

35 gm of potassium hydroxide was heated at 170°-192 °C in a three necked flask until fusion. 12.5gm of finely powdered of 9H-Fluorenone was added in five portions over one and half hour with vigorous stirring and the temperature was maintained at 170°-192°C for further one half hour. The fusion mixture was then poured in ice cold water with stirring. The obtained suspension was filtered at vacuum pump and then filtrate was acidify with HCl to pH-5.3 resulting in precipitation of by product which was filtered under suction wash with distilled water and the filtrate was again acidify with Con.HCl. The precipitated product was filtered under suction and dried in air. The product was recrystallized from Absolute ethanol. Product MCS-03 was formed.

Yield:72%.m.p.=152-155°C.IR(KBr):3573-3112(O-Hstr),1711.6(C=OCarboxylic,str), 1331, 1303(C-O-H in-plane bend).¹H-NMR(CDCl3):9.59(1H,s,COOH),7.33- 8.16(1H,m,9H),¹³CNMR(CDCl3)δ:111.2,112.1,115.8,127.5,139.8,FABMS,198.08(100%),199.06(

14.5%),200.12(1.%).Anal. Calcd for C13H10O2: Found: C, 78.77; H, 5.07%.

MCS-04: (4’ Acetylamino methyl biphenyl-2-caboxylic acid)

5 gm of MCS 03 was dissolved in 25 ml of concentrated H₂SO₄.After that acetamide (2.15 gm) and Paraform aldehyde (0.560) gm were added subsequently. The solution was heated at 70°C along with stirring for 4.5 hours. The hot mixture was poured over ice and cold water. The precipitated product was filtered under suction and dried in air. The product was recrystallized from Absolute ethanol. The resulting solid was filtered out.

Yield: 58% m.p.-165^o-169^oC IR (KBr) (cm^-1): 3397.4 (N-H str.), 3262.7(O-H, str), 2986 (C-H str), 2945 (aliphatic C-H str), 1675.2 (C=O str of), 1587.5 (N-H bend of amide), 1495.9(C-N str) 784.6(Benz. Ring); ¹H NMR (300 MHz, CDCl₃) δ:2.03(s,3H,CH3), 9.76(1H,s,COOH),4.32(2H,s,CH₂),7.98(s,1H,-NH);7.09

8.24(m,8H,ArH).¹³CNMR(CDCl3)δ:19.5(CH3),53.7(CH2)112.4,116.1,122.1,125.7,133.5,139.2,144 .1,155.7,170.2,FAB-MS, 269.12(100%), 270.03(18.6),271.07 (2.2%).Anal. Calcd for C16H15NO3: C, 71.36; H, 5.61; N, 5.20%; Found: C, 71.27; H, 5.54; N, 5.12

MCS 05: (4’bromomethylbiphenyl-2-carboxylic acid)

1.4gm of MCS-04 was taken in a RBF. 1.598 gm of phosphorus oxy chloride was added to 4ml of DMF and further addition of xylene (4ml). The reaction mixture was refluxed for 7 ½ hours. The cold solution was washed with water and evaporated to give a light yellow crystalline product. Yield: 52 % m.p.-133^o-136^oC IR (KBr) (cm-1): IR (KBr): 3354 (O-H str.), 2902(C-H str., CH₂), 1679.4 (Carboxylic, C=O str.), 1676-1413 (C=N, C=C str.), 1189 (C-O str), 854.2 (.benz.

ring), 598.7(C-Cl str.) ¹H NMR (300 MHz,

CDCl3)10.07(s,1H,OH),7.118.05(m,8H,ArH),4.64(s,2H,CH2)..¹³CNMR(CDCl3)δ:33.8(CH2)115.9, 117.2,123.4,128.2,136.1,139.2,142.4,151.2,,FABMS,289.12(100%)291.14(97.11%),270.03(18.6), 271.07 (2.2%).Anal.Calcd for C14H11BrO2: C, 57.76; H, 3.81; %; Found: C, 57.71; H, 3.80; %.

MCS06:4-(6-chloro-2-substitued-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2- carboxylic acid

0.4612gm of MCS-02 was dissolved in 20ml of DMF (dimethyl formamide) and stirred vigorously with 1.5gm of potassium carbonate at 27⁰C for one hour. To the resulting mixture 0.482gm of MCS-05 first dissolved in 20 ml of DMF and then was added drop wise with dropping funnel in 1 hour the reaction was allowed to proceed for further 11 hours at room temperature and solvent removed under vacuum. Residue was treated with 20ml of dilute HCl and extracted with ethyl acetate. The organic layer was washed with brine solution, distilled water and dried over anhydrous sodium sulphate. (MCS-06) was obtained.

(1)4-(6-chloro-2-methyl-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2-carboxylic acid

Yield: 72%, m.p. = 179⁰-182⁰C. Anal.Calcd for C₂₂H₁₆ClN₃O₄: Found: C,62.64;H, 4.82;N,9.96 %;IR (KBr): 3398(Broad O-H str.), 2912 (C-H str., CH₃), 2812.0 (C-H str.,

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CH2),1706.2 (carboxylic, C=O str.),1602,1549(C=N and C=Cstr.), 1532-1313 (N-O str., NO2), 1141 (C-N str.), 795.29(ben.Ring),654.4(C-Cl str).1^H NMR (300 MHz, CDCl3) 10.23(s,1H,COOH),7.26-

8.42(m,10H,ArH),2.87(s,3H,CH3),5.12(s,2H,CH2).¹³CNMR(CDCl3)δ:24.4,58.3,111.3,112.1,116.2, 127.1,131.4,133.1,139.1,142.2,147.2,150.4,FAB-MS, 421.082

(2)4-(6-chloro-2-ethyl-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2-carboxylic acid Yield:59%,m.p.=158-163⁰C.Mol.weight435.09,Anal.Calcd for C23H18ClN3O4:C,63.38;H, 4.16;N,9.64 %. IR (KBr): 3294.1(Broad O-H str.), 2994 (C-H str., CH3), 2902 (C-H str., CH2), 2853.6 (C-H str,CH2),1703(carboxylic,C=O str.),1642.3,1532.1 (C=N and C=Cstr.), 1512-1327 (N-O str., NO2), 1123 (C-N str.), 782 (1,4 disub. Benz.Ring) 647.1(C-Cl str). ¹HNMR (300 MHz,

CDCl3) 9.72(s, 1H, COOH), 7.12-8.19(m, 10H,

ArH),2.42(s,3H,CH3),4.99(s,2H,CH2),3.26(s,2H,CH2),.¹³CNMR(CDCl3)δ:21,53.1,60.3,111.4,113.

1,115.3,118.2,127.2,136.2,142.2,144.1,FAB-MS, 435.03(100% )

(3)4-(6-chloro-5-nitro-2-propyl-benzimidazole-1-ylmethyl)-biphenyl-2-carboxylic acid

Yield:59%,m.p.=158-163⁰C.Mol.weight449.11,Anal.Calcd for C24H20ClN3O4:C,64.08;H, 4.46;N,9.34 %. IR (KBr): 3311(Broad O-H str.), 2942 (C-H str., CH3), 2956 (C-H str.,CH2), 2821 (C-H str,CH2), 2123.1 (C-H str,CH2),1711(carboxylic,C=O str.),1622,1532 (C=N and C=Cstr.), 1521-1317 (N-O str., NO2), 1125 (C-N str.), 793 (1,4 disub. Benz.Ring) 657(C-Cl str). ¹HNMR

(300 MHz, CDCl3) 10.12(s, 1H, COOH), 7.02-

8.23(m,10H,ArH),2.41(s,3H,CH3),4.90(s,2H,CH2),3.26(s,2H,CH2),1.69(s,2H,CH2).¹³CNMR(CDCl

3)δ:20,50.1,53.3,65.1,73.7,112.4,114.1,116.1,117.1,122.2,130.2,141.1,145.1,FAB-MS, 449.12(100% )

(4)4-(2-butyl-6-chloro-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2-carboxylic acid Yield:50%,m.p.=163-166⁰C.Mol.weight463.12,Anal.Calcd for C25H22ClN3O4:C,64.78;H, 4.76;N,9.06 %. IR (KBr): 3349.5(Broad O-H str.), 2923 (C-H str., CH3), 2921 (C-H str.,

CH2), 2801 (C-H str,CH2), 2103.0 (C-H str,CH2),1698(carboxylic,C=O str.),1627-1540 (C=N and C=Cstr.), 1503-1311 (N-O str., NO2), 1117 (C-N str.), 801 (1,4 disub. Benz.Ring) 654(C-Cl str). ¹HNMR (300 MHz, CDCl3) 10.22(s, 1H, COOH), 7.09- 8.13(m,10H,ArH),2.53(s,3H,CH3),5.04(s,2H,CH2),3.22(s,2H,CH2),1.53(s,2H,CH2),1.31(s,2H,CH2) .¹³CNMR(CDCl3)δ:22,46.4,53.2,55.3,67.1,71.3,112.2,113.5,117.1,118.1,124.1,132.2,134.1,140.0, FAB-MS, 464.02.

(5)4-(6-chloro-5-nitro-2-pentyl-benzimidazole-1-ylmethyl)-biphenyl-2-carboxylic acid Yield:50%,m.p.=188-194⁰C.Mol.weight477.15,Anal.Calcd for C26H24ClN3O4:C,65.38;H, 5.06;N,8.76 %. IR (KBr): 3357(Broad O-H str.), 2913.5 (C-H str.,CH3), 2874.3 (C-H str.,

CH2), 2846.5 (C-H str,CH2), 2103.0-2153.6 (C-H str,CH2),1718(carboxylic,C=O str.),1627-1517 (C=N and C=Cstr.), 1511-1303 (N-O str., NO2), 1123(C-N str.), 812 (1,4 disub.

Benz.Ring) 648.3(C-Cl str). ¹HNMR (300 MHz, CDCl3) 9.48(s,1H,COOH), 7.31- 8.29(m,10H,ArH),2.74(s,3H,CH3),5.13(s,2H,CH2),3.31(s,2H,CH2),1.65(s,2H,CH2),1.38(s,4H,CH2) .¹³CNMR(CDCl3)δ:21,43.4,51.2,53.3,62.1,73.8,112.8,113.4,116.2,117.2,126.5,130.1,132.1,142.4, FAB-MS, 476.02.

(6)4-(6-chloro-2-(2-chloro-phenyl)-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2- carboxylic acid

Yield:57%,m.p.=223-226^oC. Anal.Calcd for C27H17Cl2N3O4:C,62.51;H,3.30;N,8.11 %; IR (KBr): 3491.2(Broad O-H str.), 2947 (C-H str., CH2), 1704 (carboxylic, C=O str.),1640,1581 (C=N and C=Cstr.),1530-1302 (N-O str., NO2), 1184(C-N str.), 797.8(Benz.Ring)628.0(C- Clstr),651.3(C-Clstr), ¹HNMR(300MHz,CDCl3)9.56(s,1H,COOH),7.18 -

8.23(13H,ArH),5.16(s,2H,CHCl),3.51(s,1H,CHCl)¹³CNMR(CDCl3)δ:42.2,112.3,113.5,117.2,119.

2,125.5,127.1,136.1,139.4,FAB-MS, 517

(7)4-(6-chloro-2-(3-chloro-phenyl)-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2- carboxylic acid

Yield:64%,m.p.=233-236^oC. Anal.Calcd for C27H17Cl2N3O4:C,62.56;H,3.32;N,8.13 %; IR (KBr): 3499.2(Broad O-H str.), 2944 (C-H str., CH2), 1700 (carboxylic, C=O str.),1643,1587 (C=N and C=Cstr.),1532-1312(N-O str., NO2), 1198(C-N str.), 790.8(Benz.Ring)621.1(C-

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Clstr),656.8(C-Clstr), ¹HNMR(300MHz,CDCl3)9.51(s,1H,COOH),7.16-

8.09(13H,ArH),5.18(s,2H,CHCl),3.63(s,1H,CHCl)¹³CNMR(CDCl3)δ:42.2,112.3,113.5,117.2,119.

2,125.5,127.1,136.1,139.4,FAB-MS, 518

(8) 4-(6-chloro-2-(4-chloro-phenyl)-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2- carboxylic acid

Yield:57%,m.p.=248-250^oC. Anal.Calcd for C27H17Cl2N3O4:C,62.56;H,3.32;N,8.13 %; IR (KBr): 3503( O-H str.), 2936 (C-H str., CH2), 1712 (carboxylic, C=O str.),1649,1582 (C=N and C=Cstr.),1524-1302(N-O str., NO2), 1184(C-N str.), 798(Benz.Ring)626.2(C-Clstr),657.8(C- Clstr), ¹HNMR(300MHz,CDCl3)9.51(s,1H,COOH),7.31- 7.98

(13H,ArH),5.11(s,2H,CHCl),3.74(s,1H,CHCl)¹³CNMR(CDCl3)δ:42.2,112.3,113.5,117.2,119.2,12 5.5,127.1,136.1,139.4,FAB-MS, 518

(9) 4-[6-chloro-2-(2-fluoro-phenyl)-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2- carboxylic acid

Yield:53%,m.p.=256-259^oC. Anal.Calcd for C27H17ClFN3O4:C,64.62;H,3.42;N,7.06 %; IR (KBr): 3515( O-H str.), 2914 (C-H str., CH2), 1702 (carboxylic, C=O str.),1665,1532 (C=N and C=Cstr.),1549-1324(N-O str., NO2), 1156(C-N str.), 791(Benz.Ring)626.2(C-Clstr).

1HNMR(300MHz,CDCl3)10.16 (s,1H,COOH),7.14- 8.22

(14H,ArH),5.11(s,2H,CH2),3.51(s,1H,CHCl).¹³CNMR(CDCl3)δ:58.2,111.0,115.1,119.2,121.1,124 .1,133.4,134.0,138.5,FAB-MS, 501.08

(10) 4-[6-chloro-2-(2-hydroxy-phenyl)-5-nitro-benzimidazole-1-ylmethyl)-biphenyl-2- carboxylic acid

Yield:53%,m.p.=236-239^oC. Anal.Calcd for C27H18ClN3O5:C,64.87;H,3.63;N,8.41 %; IR (KBr): 3475( O-H str.), 2947 (C-H str., CH2), 1698 (carboxylic, C=O str.),1613,1554 (C=N and C=Cstr.),1519-1335(N-O str., NO2), 1130(C-N str.), 797(Benz.Ring) 642.4(C-Clstr).

1HNMR(300MHz,CDCl3)10.02 (s,1H,COOH),7.36- 8.51

(10H,ArH),5.11(s,2H,CH₂),3.51(s,1H,CHCl),3.18(s,3H,CH₃),5.07(s,1H,OH),.¹³CNMR(CDCl₃)δ:2 3.2,48.2,112.3,114.1,117.2,120.1,123.1,128,144.2,147.5,FAB-MS, 499.90

MCS-4`-(5-Amino-6-chloro-2-substituted-benzoimidazol-1-ylmethyl)]-biphenyl-2- carboxylic acid

2.5 gm of compound was placed in three necked RBF and dissolved in absolute ethanol and heated to 60⁰C under reflux. To this, 1.2 gm stannous chloride dihydrate was added with slow stirring during 1.5hours and reaction conditions were maintained for further 12 hours. The mixture was cooled to room temperature and pH adjusted to 7.6 with 5% sodium hydroxide solution. The organic layer was washed with brine, distilled water then dried over anhydrous sodium sulphate.

Solvent removed under vacuum and product was obtained.

MCS[1]:4`-(5-Amino-6-chloro-2-methyl-benzoimidazol-1-ylmethyl)]-biphenyl-2- carboxylic acid

Yield: 64%, m.p. = 211⁰-214⁰C. Anal.Calcd for C22H18ClN3O2: Found: C, 67.44; H, 4.62;

N, 10.76 %; IR (KBr): 3412.3(Broad O-H str.), 2943 (C-H str., CH3), 2854 (C-H str., CH2), 1718 (carboxylic, C=O str.), 1615, 1519(C=N and C=Cstr.), 812 (Ben. Ring),632.4(C- Clstr).1^HNMR(300MHz,CDCl3)9.54(s,1H,COOH),7.06-8.22

(m,10H,ArH),2.42(s,3H,CH3),5.00(s,2H,CH2),4.08(s,2H,NH),.¹³CNMR(CDCl3)δ:13,52.3,112.3,11 3.1,115.2,123.1,130.4,135.1,138.1,141.2,143.2,145.1,FAB-MS, 391.10

MCS [2]:4`-(5-Amino-6-chloro-2-ethyl -benzoimidazol-1-ylmethyl)]-biphenyl-2- carboxylic acid

Yield:61%,m.p.=219⁰-223⁰C. Anal.Calcd for C22H20ClN3O2:Found:C,68.04;H, 4.95;N,10.36 %;IR (KBr): 3453(Broad O-H str.), 2903 (C-H str., CH3), 2825 (C-H str., CH2),1710 (carboxylic, C=O str.),1602,1532(C=N and C=Cstr.), 802 (ben.Ring),645(C- Clstr).1^HNMR(300MHz,CDCl3)9.72(s,1H,COOH),7.11-8.02

(m,10H,ArH),2.34(s,3H,CH3),5.04(s,2H,CH2),4.82(s,2H,NH),2.64(s,2H,CH2),.¹³CNMR(CDCl3)δ:

14,58.0,112.9,113.4,116.2,121.1,128.4,135.5,137.2,140.1,142.5,145.9,FAB-MS, 405.21

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MCS [3]:4`-(5-Amino-6-chloro-2-propyl -benzoimidazol-1-ylmethyl)]-biphenyl-2- carboxylic acid

Yield:52%,m.p.=231-235⁰C.Mol.weight449.11,Anal.Calcd for C23H22ClN3O2:C,68.68;H, 5.26;N,10.04 %. IR (KBr): 3392(Broad O-H str.), 2931 (C-H str., CH3), 2909 (C-H str.,

CH2), 2865 (C-H str,CH2), 2154 (C-H str,CH2),1701(carboxylic,C=O str.),1654,1513 (C=N and C=Cstr.), 1114 (C-N str.), 799.5 (1,4 disub. Benz.Ring) 651(C-Cl str). ¹HNMR (300

MHz, CDCl3) 10.00(s, 1H, COOH), 7.12-

8.20(m,10H,ArH),2.48(s,3H,CH3),4.95(s,2H,CH2),4.89(s,2H,NH),2.96(s,2H,CH2),1.69(s,2H,CH2).

13CNMR(CDCl3)δ:17,53.1,55.3,61.1,70.7,111.4,113.1,113.5,114.1,121.2,132.2,145.1,148.1,FAB- MS, 419.14

MCS[4]:4`-(5-Amino-2-butyl-6-chloro-benzoimidazol-1-ylmethyl)]-biphenyl-2 carboxylic acid

Yield:47%,m.p.=163-166⁰C.Mol.weight463.12,Anal.Calcd for C23H24ClN3O4:C,69.21;H, 5.57;N,9.66 %. IR (KBr): 3352.3(Broad O-H str.), 2934.5(C-H str., CH3), 2902 (C-H str.,

CH2), 2862 (C-H str,CH2), 2143.2 (C-H str,CH2),1692(carboxylic,C=O str.),1620-1518 (C=N and C=Cstr.), 1154 (C-N str.), 805 (1,4 disub. Benz.Ring) 650(C-Cl str). ¹HNMR (300 MHz, CDCl₃) 9.52(s, 1H, COOH), 4.49(s,2H,NH), 7.19- 8.10(m,10H,ArH),2.59(s,3H,CH₃),5.00(s,2H,CH₂),3.58(s,2H,CH₂),1.66(s,2H,CH₂),1.38(s,2H,CH₂) .¹³CNMR(CDCl3)δ:20,42.4,50.2,58.3,60.1,75.3,112.7,114.5,116.1,119.1,123.1,136.2,139.1,145.0, FAB-MS, 434.11.

MCS[5]:4`-(5-Amino-6-chloro-2-pentyl-benzoimidazol-1-ylmethyl)]-biphenyl-2 carboxylic acid

Yield:54%,m.p.=237-240⁰C. Anal.Calcd for C26H26ClN3O2:C,69.71;H, 5.86;N,9.38 %. IR (KBr): 3376(Broad O-H str.), 2919(C-H str.,CH₃), 2846 (C-H str.CH₂), 2898 (C-H str,CH₂), 2132 (C-H str,CH₂),1714(carboxylic,C=O str.),1607-1500 (C=N and C=Cstr.), 1129(C-N str.), 810 (1,4 disub. Benz.Ring) 658 (C-Cl str). ¹HNMR (300 MHz, CDCl₃) 9.87(s,1H,COOH),7.35-

8.58(m,10H,ArH), 4.69 (s,2H,NH), 2.56(s,3H,CH₃),5.06(s,2H,CH₂),3.54(s,2H,CH₂),1.72(s,2H,CH₂),1.62(s,4H,CH₂).¹³CNMR(CDCl₃)

δ:20,40.4,57.2,59.3,68.1,74.8,113.8,115.4,118.2,119.2,127.5,132.1,134.1,145.4,FAB-MS, 446.16.

MCS[6]:4`-[5-Amino-6-chloro-2-(2-chloro-phenyl)benzoimidazol-1-ylmethyl)]- biphenyl-2 carboxylic acid

Yield:64%,m.p.=245-247^oC. Anal.Calcd for C₂₇H₁₉Cl₂N₃O₂:C,66.41;H,3.92;N,8.61 %; IR (KBr): 3416.1(Broad O-H str.), 2922 (C-H str., CH₂), 1713 (carboxylic, C=O str.),1623,1512 (C=N and C=Cstr.), 1184(C-N str.), 802.8(Benz.Ring)649.3(C-Clstr),

1HNMR(300MHz,CDCl₃)9.93(s,1H,COOH),7.18-8.23(14H,ArH),5.16(s,2H,CH

Cl),4.24(s,2H,NH)¹³CNMR(CDCl3)δ:47,111.1,115.3,119.5,121.2,127.5,129.3,133.2,142.0,FAB- MS, 488.02

Yield:67%,m.p.=247-250^oC. Anal.Calcd for C27H19Cl2N3O2:C,66.41;H,3.92;N,8.61 %; IR (KBr): 3421(Broad O-H str.), 2925 (C-H str., CH₂), 1710 (carboxylic, C=O str.),1620,1518 (C=N

and C=Cstr.), 1181(C-N str.), 799.2(Benz.Ring)652.1(C-Clstr),

1HNMR(300MHz,CDCl₃)9.98(s,1H,COOH),7.20-8.13(14H,ArH),5.06(s,2H,CH

Yield:55%,m.p.=241-244^oC. Anal.Calcd for C27H19Cl2N3O2:C,66.41;H,3.92;N,8.61 %; IR (KBr): 3414.2(Broad O-H str.), 2925.6 (C-H str., CH2), 1711 (carboxylic, C=O str.),1620,1516 (C=N and C=Cstr.), 1180(C-N str.), 811(Benz.Ring)647.2(C-Clstr),

1HNMR(300MHz,CDCl₃)10.04(s,1H,COOH),7.25-

8.11(14H,ArH),4.96(s,2H,CHCl),4.33(s,2H,NH)¹³CNMR(CDCl3)δ:47,112.1,113.3,115.5,122.2,12 4.5,127.3,131.2,140.2,FAB-MS, 488.02

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MCS[9]:4`-[5-Amino-6-chloro-2-(2-fluoro-phenyl)benzoimidazol-1-ylmethyl)]- biphenyl-2 carboxylic acid

Yield:55%,m.p.=261-264^oC. Anal.Calcd for C27H19ClFN3O2:C,68.71;H,4.06;N,8.91 %; IR (KBr): 3427.4(Broad O-H str.), 2915.3 (C-H str., CH2), 1700.6 (carboxylic, C=O str.),1629,1510

(C=N and C=Cstr.), 1174(C-N str.), 801(Benz.Ring).

1HNMR(300MHz,CDCl3)10.19(s,1H,COOH),7.15-8.26(14H,ArH),4.99(s,2H,CH

Yield:67%,m.p.=266-269^oC. Anal.Calcd for C27H19ClFN3O2:C,68.71;H,4.06;N,8.91 %; IR (KBr): 3422.1(Broad O-H str.), 2918.6 (C-H str., CH2), 1702.4 (carboxylic, C=O str.),1622,1516

Cl),4.92(s,2H,NH)¹³CNMR(CDCl3)δ:42,113.0,114.1,116.2,120.1,122.5,124.3,133.5,142.5 ,FAB-MS, 469.125

Yield:55%,m.p.=262-2647^oC. Anal.Calcd for C27H19ClFN3O2:C,68.71;H,4.06;N,8.91 %;

IR (KBr): 3416.1(Broad O-H str.), 2922.6(C-H str., CH2), 1698 (carboxylic, C=O str.),1654,1521

(C=N and C=Cstr.), 1154(C-N str.), 798.1(Benz.Ring).

MCS[12]:4`-[5-Amino-6-chloro-2-(2-hydroxy-phenyl)benzoimidazol-1-ylmethyl)]- biphenyl-2 carboxylic acid

Yield:55%,m.p.=281-284^oC. Anal.Calcd for C27H20ClN3O3:C,69.01;H,4.29;N,7.54 %; IR (KBr): 3356.8(Broad O-H str.), 2986(C-H str., CH2), 1699.6 (carboxylic, C=O str.),1614,1501

Cl),4.75(s,2H,NH),4.08(s,1H,OH),¹³CNMR(CDCl3)δ:56.8,111.3,115.1,117.2,122.1,127.5, 125.1,131.8,140.9,FAB-MS, 469.13

Cl),4.82(s,2H,NH),4.16(s,1H,OH),¹³CNMR(CDCl3)δ:56.8,111.3,115.1,117.2,122.1,127.5,125.1,13 1.8,140.9,FAB-MS, 470.10

Cl),4.79(s,2H,NH),4.32(s,1H,OH),¹³CNMR(CDCl3)δ:56.8,111.3,115.1,117.2,122.1,127.5,125.1,13 1.8,140.9,FAB-MS, 469.13

3. Pharmacological activity

Procedure for development of hypertention for normotensive rats ²²

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Albino normotensive rats (Wistar Strain) were taken and they were hypertensized by cholinomimetic agents for screening of all the synthesized benzimidazole derivatives for there anti-hypertensive activity. Suspension of test compounds was prepared in 1% w/v sodium carboxy methyl cellulose (sodium CMC) and was administered at dose level of 50 and 100 microgram/kg animal body weight to different groups of five rats each. After administration of dose to animal blood pressure was measured by normotensive tail and cuff method using pressure meter.

Measurements were done after one hour and three hours interval in step-wise manner as follows:

Screening Methods for Anti-hypertensive Activity:

(a) Angiotensin II induced Hypertension:²³(i) Invasive method (Direct method).

(ii) Non-invasive Tail cuff method (Indirect method).

(b) In-vitro determination of vasodilator activity by aortic rings.

4. Experimental techniques

(i) Invasive Method (Direct Method):^23-25 Male albino wistar (150-250 gm) rats were used and housed at 24±1⁰C room temperature. The rats were anaesthetized with sodium chloride 0.9%

solution, Drug solution 10-μg/100ml, and Heparin 500 I.U.solution urethane hydrochloride 50%

w/v solution 80 mg/kg i.p. To set up the instrument firstly the level of mercury in the left arm of manometer was adjusted to 90-100 mm of Hg (normal blood pressure of rat).this was done in steps of 10mm at a time and the physiogram so obtained was used as calibration graph for calculations.

The Jugular vein and carotid artery were surgically cannulated for drug administration for recording the blood pressure respectively. The trachea was cannulated in order to provide artificial respiration to rat during the experiment. By means of three way stop cock and a stainless steel needle at the end of P.E. tubing was attached to arterial cannula for B.P., Transducers and the venus cannula to a syringe. Then both the cannulas were filled by heparinized saline before the administration. Arterial cannula was connected via transducer to physiograph recorder. Several baseline readings of systolic and diastolic pressures were recorded. The physiograph shows the reduction of the blood pressure with compare to losratan. Synthesized compounds were screened in presence of Angiotensin II induced hypertension (0.5 μg/kg i.v.). Observations are given in the table 1, 2.

(ii) Non-invasive Tail cuff Method (Indirect Method):^24-25 Albino rats weighing 200-250 gm were used to screening for all the synthesizes benzimidazole derivatives for antihypertensive activity. Suspension of test compound was prepared in 1% w/v sodium carboxy methyl cellulose and administered at dose level of 50 mg/kg animal body weight to different of six rats each group.Contorl group received an equal quantity of 1% w/v sodium carboxy methyl cellulose suspension. After administration of dose to animal, blood pressure was measured by Non-invasive Tail cuff Method using pressure meter.Measurment were done after 1 hour and 3 hour time interval intensive stepwise. One hour after administration of drug sample, animal was shifted to the restrainer, which restricts the movement of animal. The tail was cleaned with moist cotton to remove the dirty matter and talcum powder was sprayed on tail to make its surface smooth. A tail cuff and pulse transducer was fixed around the tail. Initially animal shows particular pulse level, when the pulse rate is within the normal range. ‘STRAT’ switch is put on and the recorder records the blood pressure as SBP (systolic blood pressure). DBP (Diastolic blood pressure) and MABP (mean arterial blood pressure), which is displayed on monitor. The pressure can be easily read from the pre-calibrated monitor. Once all the values are displayed the recorder is switched off and for next measurement. Some procedures are allowed once when sufficient pulse level is attained.

Observations are given in the table1, 2.

5. Results and discussion

First we synthesized 2-phenyl Benzimidazole. In this method the reactants are condensed in the presence of an oxidant such as cupric acetate. An improvement on the conventional method is the use of sodium bisulfite addition adduct of the aldehyde12.The reactions are carried out in boiling ethanol, yields are good [e.g. 2-phenyl (90%), 2-(3- pyridyl) (97%)] and there is little risk

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of decomposition of liable substituents. Synthetic scheme (Fig. 2) for target compounds was divided into two steps. Step I involved synthesis of 2- aryl, alkyl, aldehyde benzimidazoles by condensation reaction of 4-chlor-o-phenylenediamine with the respective. The corresponding 5- nitro derivatives were prepared by nitration under controlled temperature conditions.¹⁹Step II include the novel sequential combination of three routine reactions to synthesize 2’- carboxybiphenyl methylene chloride. Biphenyl –2-carboxylic acid was prepared by potash fusion of 9H flourenone²⁰ which was then subjected to aromatic substitution reaction using paraformaldehyde and acetamide in conc. sulphuric acid²⁰ to affect intermediate,4- acetamidomethyl biphenyl-2’-carboxylic acid. The required component was identified as third fraction which was subjected to substitution reaction with phosphorus oxychloride in xylene and dimethyl formamide²¹ to produce the pendant moiety 4-(bromomethyl) biphenyl-2’-carboxylic acid and synthesis biphenyl with carboxylic compound²⁶. The synthesized compounds were characterized on the basis of chemical and spectral data. The present work was mainly intended to establish the moieties which are responsible for Angiotension-II inhibition. Ang II antagonism by compounds with same functional group at 2 position has been found to be a function of substituent at 5-position. Presence of amino group has increased the activity substantially over the substituted one ([1] to [14]). The maximum activity has been observed with nitro group (Compound 2, 4, 6, 7, 9, 10, 12, 13, and 14). This suggests that there are some sites in the receptor pocket, which can interact with the functional groups at position 5. Substituted benzimidazole nucleus coupled to carboxylbipheny methyl group has been designed, synthesized and evaluated for angiotensin II antagonism. Compound with amino group at 5-position and aromatic, aryl, alkyl compounds at 2- position have been found to be more potent than losratan and Telmisartan. Higher activity of Compound 2, 4, 6, 7, 9, 10, 12, 13, and 14suggests that this group at 2-position should be either H- bond acceptor and hydrophobic. The higher activity of 5-amino derivatives may be ascribed to the ability of group to act as H-bond acceptor, hydrophobic with respect to the receptor site. The noncompetitive mode of antagonism of further suggests that such receptor pocket may not be accessible to the natural ligand, Ang II.

Acknowledgement

The authors are thankful to Prof.Pratibha Sharma School of chemical sciences DAVV Indore, to given valuable suggestion and help to synthesis experimental work, authors also thankful to Central Drugs Research Institute (CDRI), Lucknow (India) for providing spectro- analytical facilities, respectively.

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Table 1. Hypertension induced in normotensive rat.

After 1hour After 3 hour

Comp. Exp. Animal Albino

(Wistar) Rat SBP DBP MABP SBP DBP MABP

1 145 106 126 141 112 122

2 141 112 127 144 114 126

3 142 111 124 140 106 123

4 147 108 129 142 102 122

5 142 112 127 143 106 123

[1]

6 141 116 128 142 112 122

1 145 111 128 140 107 123

2 143 115 129 138 106 122

3 147 107 127 141 102 123

4 152 110 131 142 106 124

5 148 106 127 141 104 122

[2]

6 144 110 127 138 104 121

1 144 108 126 142 104 123

2 148 106 127 142 106 124

3 151 109 130 146 104 125

4 146 104 125 142 104 123

5 144 106 125 140 102 121

[3]

6 148 104 126 142 106 124

1 140 116 128 139 104 122

2 148 106 126 144 104 124

3 145 113 129 144 100 122

4 141 111 126 140 112 126

5 146 114 130 142 104 123

[4]

6 144 116 130 140 106 123

1 146 108 127 142 106 124

2 143 106 125 139 104 121

3 146 110 128 140 104 122

4 149 111 130 143 106 124

5 152 112 133 145 103 124

[5]

6 150 111 131 146 104 125

1 148 112 130 144 102 123

2 144 114 129 146 106 126

3 142 108 125 146 104 125

4 146 106 126 142 104 123

5 142 110 126 140 116 128

[6]

6 148 102 125 144 106 125

1 140 106 123 142 106 124

2 141 114 128 142 104 123

3 146 108 127 144 104 124

4 148 114 130 144 102 123

5 144 112 132 142 104 123

[7]

6 146 114 12 143 101 122

(11)

1 142 112 127 140 102 121

2 144 116 130 141 101 122

3 142 110 126 139 104 123

4 146 106 126 144 104 124

5 148 106 127 146 102 124

[8]

6 144 104 124 140 100 120

1 140 106 123 138 102 120

2 144 112 127 142 104 123

3 142 114 127 140 101 122

4 148 104 126 144 104 124

5 154 108 132 144 102 123

[9]

6 148 104 126 142 100 121

1 144 112 127 141 102 121

2 142 114 128 144 101 122

3 146 110 126 142 100 120

4 140 108 124 138 102 120

5 144 106 125 142 101 123

[10]

6 142 108 125 138 100 119

1 140 104 122 144 106 125

2 142 106 124 140 106 121

3 146 104 125 144 104 120

4 140 114 127 136 100 121

5 148 108 126 144 102 123

[11]

6 151 112 133 146 101 124

1 144 114 129 142 102 121

2 139 114 127 135 103 119

3 142 106 124 140 102 123

4 144 108 126 142 100 121

5 148 104 126 145 104 124

[12]

6 143 106 124 141 101 122

1 141 110 129 142 108 125

2 138 105 125 139 107 123

3 132 104 128 142 102 122

4 142 103 123 140 102 121

5 141 110 124 143 105 123

1 140 105 128 138 104 121

[13]

6 139 108 124 141 103 122

1 142 113 128 142 104 123

2 141 109 125 144 103 124

3 144 114 128 141 102 121

4 146 104 125 142 102 122

5 144 112 127 141 106 123

[14]

6 148 104 126 145 105 125

Losartan 116 - - - - -

Control

Telmisartan 114 - - - - -

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Table 2. Reduction in blood pressure (mm Hg) at a dose of 50 μgm/kg animal body weight.

After 1hour After 3 hour

Comp. Exp. Animal Albino

(Wistar) Rat SBP DBP MABP SBP DBP MABP

1 123 101 112 122 101 113

2 127 105 112 121 102 115

3 124 104 114 122 102 112

4 121 104 113 123 104 115

5 123 101 112 121 104 112

[1]

6 122 103 113 120 100 111

1 120 100 110 122 102 112

2 122 102 112 121 101 111

3 121 100 111 124 103 113

4 122 103 114 121 104 112

5 120 101 111 120 102 111

[2]

6 118 104 111 123 101 112

1 120 102 111 125 102 113

2 122 106 114 122 100 111

3 123 101 113 126 102 113

4 121 107 114 127 103 115

5 122 102 112 123 107 115

[3]

6 121 106 113 126 102 114

1 126 106 112 128 102 115

2 124 106 115 123 103 113

3 126 104 115 124 104 114

4 124 108 116 122 102 112

5 123 100 113 125 105 115

[4]

6 122 114 114 123 103 114

1 124 111 115 122 102 112

2 127 109 123 124 102 113

3 125 110 122 127 101 114

4 124 112 118 121 102 112

5 126 105 116 127 101 114

[5]

6 126 109 117 122 106 114

1 124 103 115 125 101 113

2 128 105 114 127 102 114

3 133 107 120 122 102 112

4 131 108 123 124 104 114

5 126 102 114 127 102 115

[6]

6 128 104 115 122 102 111

1 126 105 116 121 104 113

2 129 104 117 124 104 114

3 127 102 115 127 102 115

4 126 108 118 122 107 113

5 124 106 115 126 106 116

[7]

6 130 114 122 128 104 117

[8] 1 123 103 113 124 102 113

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2 125 104 115 122 103 112

3 124 105 116 126 104 115

4 125 101 113 123 104 116

5 132 104 118 127 107 117

6 135 105 120 129 102 116

1 123 103 113 124 103 114

2 122 106 114 123 107 115

3 127 101 114 126 106 116

4 125 105 130 123 105 114

5 128 109 119 124 102 113

[9]

6 131 103 117 128 103 115

1 134 102 118 125 106 116

2 132 105 119 129 105 117

3 127 103 115 125 104 114

4 124 101 113 123 104 112

5 128 105 116 125 101 113

[10]

6 124 103 117 122 100 111

1 124 102 113 126 104 115

2 121 101 111 129 103 116

3 124 105 115 123 106 115

4 135 102 119 124 101 112

5 136 101 118 122 104 113

[11]

6 134 100 117 126 104 115

1 122 102 112 122 100 111

2 123 103 116 124 110 117

3 125 104 115 125 106 116

4 122 105 116 131 106 118

5 134 105 119 120 105 113

[12]

6 125 103 114 126 102 114

1 127 104 116 124 105 114

2 125 108 117 122 108 115

3 124 105 115 125 106 116

4 122 109 116 126 106 116

5 125 104 114 122 106 114

[13]

6 123 111 118 128 104 116

1 127 105 116 126 105 115

2 129 108 119 124 104 114

3 122 112 117 122 103 112

4 126 114 120 128 107 117

5 124 111 118 123 104 113

[14]

6 126 104 115 127 107 117

Losartan 116 - - - - -

Control

Telmisartan 114 - - - - -

(14)

HO Cl

N N

N N N HN

O

H₃C CH₃

COOH n-Bu

BPT

N

BPT CH3

CH₃

OH

Losartan Valsartan Tasosartan

N

BPT n-Bu OH

COCH3

Ô Ô Ô ÔH

OH N

N

O

N

N COOH

Cl

n-Bu

O F3COOSHN

Br

Candesartan Milfasartan Saprisartan

N

N N n-Bu

BPT CH3

CH3

N N

HCO2C

n-Pr

BPT O

O

OH C(CH3)2OH CH3

N

O

BPT n-Bu

Telmisartan Olmesartan Irbesartan

N

N COOH

Cl

O Br

N N

N NH

NH N

OH S

BPT CH3

n-Bu

COOCH₃

Zolzsartan Eprosartan Fig. Angiotensin II AT1 selective antagonists

(15)

O

9H-Flourenone

KOH

^COOH

H

₂

SO

₄

,HCHO

CH

₃

C0NH

_{2 ,}

6hrs

COOH H₃COCHNH₂C

POCl

₃

COOH

DMF,K

₂

CO

₃

NH N

R O₂N

NH₂ NH₂

+

R-

R=Alkyl,Aryl R

^!

=Aldehyde,Acid

NH N O R

R^!

NH N

R O₂N

Con.Nitric acid Con.sulphuric

acid

Stirring 14hrs

MCS-01

MCS-03 MCS-04

MCS-05 N

N N⁺

O

-O R O

HO

Br Cl

Cl

Cl Cl

6-Chloro-2-substiued -5-nitro-1H-benzoimidazole

4'-(6-Chloro-2-substiued -5-nitro-benzoimidazol-1- ylmethyl)-biphenyl-2-carboxylic acid

SnCl

₂

.2H

₂

O

N H₂N N

COOH

EtOH

Cl

4'-(5-Amino-6-chloro-2-substitued -benzoimidazol-1-ylmethyl)-biphenyl-2-carboxylic acid

R

SCHEM-

(16)

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[13] D.A.Scheur, M.H. Perrone. Angiotensin type 2 receptors mediate depressor phase of biphasic pressure response. Am J Physiol Regul Integr Comp Physiol. 264: 917-923(1993)

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Naka, J. Med. Chem. 36 (1993) 2182e2195(1993)

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