Accepted Article
Received Date : 19-Jun-2014 Revised Date : 22-Sep-2014 Accepted Date : 24-Sep-2014 Article type : Research Article
Design, Synthesis and Pharmacological Evaluation of Novel NO-Releasing Benzimidazole Hybrids as Potential Antihypertensive Candidate Yanchun Zhanga, Jinyi Xub,, Yunman Li c, Yaohe Quan b, Xiaoming Wu b, a. Department of Medicinal Chemistry, Anhui University of Chinese Medicine, 103 Meishan Road, Hefei 230031, PR China b. State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China c. Department of Physiology, China Pharmaceutical University, 24 TongJia Xiang, Nanjing 210009, PR China
Abstract: Two series of novel NO-releasing benzimidazole derivatives (8a–e, 9a-g) were designed and synthesized by coupling nitro ester and furoxan NO-donor moieties with benzimidaozole biphenyl skeleton. The NO releasing assay indicated that all the target compounds had different level of NO releasing ability. Furthermore, the isolated organ assay (rat aortic strips) was used to evaluate the antagonism of Ang II induced vasoconstriction ability. It was observed that the pA2 values of compounds 8e and 9e were better than that of lead compound 6. Moreover, the pharmacological investigation showed that the antagonism of Ang II-induced pressure response by oral administration of compound 8e was obviously superior to that of lead compound 6, and comparable to that of the positive control Losartan. These results suggested that NO-releasing hybrids may provide a promising approach for the discovery of novel antihypertensive agents.
Keywords: Nitric oxide (NO); Benzimidazole derivatives; Ang II antagonism; Antihypertension
Corresponding author:
E-mail address: [email protected], [email protected]
1. Introduction Nitric oxide (NO), multifunctional messenger molecule with diverse physiological functions, plays an important part in cardiovascular system, such as maintenance of vascular tone, regulation of arterial pressure (SAP), inhibition of platelet aggregation and neutrophils on the endothelium and suppression of smooth muscle cell proliferation, which also protect against ischemia injury [1-3].
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12442 This article is protected by copyright. All rights reserved.
Accepted Article
“Sartans”, the first-choice class of antihypertensive drugs, however, lack the enhancement of the NO-mediated vasorelaxing and protecting effect due to bradykinin preservation [4-5]. In recent years, on the basis of knowledge about NO properties, a lot of hybrid drugs have been witnessed. For example, NO-releasing aspirin, NO-releasing β-blocking agents [6-7], molecules possess improved pharmacological profile or reduced adverse effects after integrated with NO-donor groups were researched. WB1106, NO-Losartan have been synthesized, with improved antihypertensive effects of the “native” drugs, such as anti-ischemic, cardio-protective properties and antiplatelet effects [8-9].
Figure 1. The structure of lead compound 6 Inspired by the obtained interesting results from our previous research, in which an NO-donor moiety connected to a ‘native’ molecule could markedly enhance its therapeutic impact [10, 12],
we designed two series
of NO-releasing benzimidazole hybrids by coupling lead compound 6 (Figure 1) which had moderate Ang II antagonism activity, with NO-donors via different linkers (Table 1). Herein, we would like to report the synthesis and biological evaluation of these novel NO releasing benzimidazole derivatives. Table 1 NO-Releasing Benzimidazole Hybrids
Compd.
Linker
8a
-CH2CH2-
8b
-CH2CH2CH2-
8c
-CH2CH2CH2CH2-
8d
-(CH2)5-
8e
NO donor
-ONO2
-CH2 PhCH2-
Compd.
Linker
9a
-CH2CH2-
9b
-CH2CH2CH2-
9c
-CH2CH2CH2CH2-
9d
-CH2CH2CHCH3-
9e
-CH2 PhCH2-
9f
-CH2CH2OCH2CH2-
9g
-CH2C
NO donor
CCH2-
2. Chemistry Target compounds 8a-e, 9a-g were prepared via the synthetic route outlined in Scheme 1, 2, 3. The furoxans 1a-g were synthesized with thiophenol as described in Scheme 1. In Scheme 2, intermediate 2 was obtained according to the reported methods
[10, 11]
. Key compound 6 was delivered as follows. Abenzene-1,
2-diamine was cyclized with PPA at 100°C to obtain compound 4, which was converted to compound 5 with
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Accepted Article
methyl 4'-(bromomethyl)biphenyl-2-carboxylate (3) and NaH in DMF. Then by saponification of compound 5 with NaOH, key compound 6 was obtained. Then compound 6 was esterized with alkyl dibromide to give compounds 7a-d which were further converted into their corresponding target nitrooxy derivatives 8a-d by reaction with silver nitrate in acetonitrile under refluxing and at darkness. Compound 6 was condensed with compound 2, 1a-g respectively in dry CH2Cl2 in the presence of N, N-dicyclohexylcarbodiimide (DCC) and catalytic N, N-(dimethylamino)pyridine (DMAP) to afford the corresponding target compounds 8e, 9a-g (Scheme 3).
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3. Results and discussion 3.1. NO-releasing test of target compounds in vitro As shown in Figure 2 and 3, all the target compounds produced different levels of NO in vitro determined by Griess assay [13]. It was found that NO-donating derivatives 8e released the maximum amount of NO and 9e did secondly. The maximum amount of NO released by compound 8e was up to 0.77 μmol/L (Figure 4). Generally, the series of nitrooxy NO donor hybrids (8a-e) displayed better NO releasing ability than furoxans (9a-g).
Figure 2. NO release assay in vitro of target compounds with nitrooxy
Figure 3. NO release assay in vitro of target compounds with furoxan
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Figure 4. NO release assay in vitro between 8e and 9e 3.2. Ang II Antagonist Activity Assay All the titled compounds were evaluated for antagonism of Ang II induced vascular contraction in vitro on rat aortic rings. As shown in Table 2, lead compound 6 had moderate pA2 value, whereas the introducing with NO donor increased the ability of inhibition remarkably. The results showed that the Ang II antagonist activity of compounds 8e and 9e were obviously superior to Losartan (Table 2).
Table 2 Inhibition of Ang II induced vascular contraction in vitro on rat aortic rings
Compd.
pA2（ x ）
Compd.
pA2（ x ）
Losartan
7.904
9f
7.913
6
7.904
9b
7.915
8a
7.947
9c
7.957
8b
7.958
9a
7.881
8c
7.956
9e
8.099
8d
7.947
9d
7.873
8e
8.033
9g
7.821
3.3 Effects on Ang II Induced Pressor Response in Conscious Normotensive Rats Compounds 8b, 8e and 9e, which shown better antagonism of Ang II induced vascular contraction, were chosen for further antihypertensive evaluation. Tested compounds exhibited the similar mode as losartan did. The hybrid derivative 8b, 8e and 9e, showed maximum values longer than losartan for 60 min (Figure 5, 6). It was speculated that these may be due to the synergy effect of ‘native’ molecule with NO donor.
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Accepted Article
Figure. 5 Inhibition of the Ang II (100 ng/kg iv)-induced pressor response in conscious normotensive rata by 3 mg/kg of 8b, 8e, 9e and Losartan
Figure 6. Inhibition of the Ang II (100 ng/kg iv)-induced pressor response in conscious normotensive rata by 6 mg/kg of 8e, 9e and Losartan. We also noticed that even the active part was covered with NO donor, the tested compounds still showed the inhibition of the Ang II induced pressor response almost immediately after the compounds were administered. It was supposed that the inhibition may be induced by the initial releasing of NO. As showed in Figure 7, the inhibition of tested compounds was dose related.
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Accepted Article
Figure 7. Inhibition of the Ang II (100 ng/kg iv)-induced pressor response by 8e (n=3) and Losartan. Significance levels p
Received Date : 19-Jun-2014 Revised Date : 22-Sep-2014 Accepted Date : 24-Sep-2014 Article type : Research Article
Design, Synthesis and Pharmacological Evaluation of Novel NO-Releasing Benzimidazole Hybrids as Potential Antihypertensive Candidate Yanchun Zhanga, Jinyi Xub,, Yunman Li c, Yaohe Quan b, Xiaoming Wu b, a. Department of Medicinal Chemistry, Anhui University of Chinese Medicine, 103 Meishan Road, Hefei 230031, PR China b. State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China c. Department of Physiology, China Pharmaceutical University, 24 TongJia Xiang, Nanjing 210009, PR China
Abstract: Two series of novel NO-releasing benzimidazole derivatives (8a–e, 9a-g) were designed and synthesized by coupling nitro ester and furoxan NO-donor moieties with benzimidaozole biphenyl skeleton. The NO releasing assay indicated that all the target compounds had different level of NO releasing ability. Furthermore, the isolated organ assay (rat aortic strips) was used to evaluate the antagonism of Ang II induced vasoconstriction ability. It was observed that the pA2 values of compounds 8e and 9e were better than that of lead compound 6. Moreover, the pharmacological investigation showed that the antagonism of Ang II-induced pressure response by oral administration of compound 8e was obviously superior to that of lead compound 6, and comparable to that of the positive control Losartan. These results suggested that NO-releasing hybrids may provide a promising approach for the discovery of novel antihypertensive agents.
Keywords: Nitric oxide (NO); Benzimidazole derivatives; Ang II antagonism; Antihypertension
Corresponding author:
E-mail address: [email protected], [email protected]
1. Introduction Nitric oxide (NO), multifunctional messenger molecule with diverse physiological functions, plays an important part in cardiovascular system, such as maintenance of vascular tone, regulation of arterial pressure (SAP), inhibition of platelet aggregation and neutrophils on the endothelium and suppression of smooth muscle cell proliferation, which also protect against ischemia injury [1-3].
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12442 This article is protected by copyright. All rights reserved.
Accepted Article
“Sartans”, the first-choice class of antihypertensive drugs, however, lack the enhancement of the NO-mediated vasorelaxing and protecting effect due to bradykinin preservation [4-5]. In recent years, on the basis of knowledge about NO properties, a lot of hybrid drugs have been witnessed. For example, NO-releasing aspirin, NO-releasing β-blocking agents [6-7], molecules possess improved pharmacological profile or reduced adverse effects after integrated with NO-donor groups were researched. WB1106, NO-Losartan have been synthesized, with improved antihypertensive effects of the “native” drugs, such as anti-ischemic, cardio-protective properties and antiplatelet effects [8-9].
Figure 1. The structure of lead compound 6 Inspired by the obtained interesting results from our previous research, in which an NO-donor moiety connected to a ‘native’ molecule could markedly enhance its therapeutic impact [10, 12],
we designed two series
of NO-releasing benzimidazole hybrids by coupling lead compound 6 (Figure 1) which had moderate Ang II antagonism activity, with NO-donors via different linkers (Table 1). Herein, we would like to report the synthesis and biological evaluation of these novel NO releasing benzimidazole derivatives. Table 1 NO-Releasing Benzimidazole Hybrids
Compd.
Linker
8a
-CH2CH2-
8b
-CH2CH2CH2-
8c
-CH2CH2CH2CH2-
8d
-(CH2)5-
8e
NO donor
-ONO2
-CH2 PhCH2-
Compd.
Linker
9a
-CH2CH2-
9b
-CH2CH2CH2-
9c
-CH2CH2CH2CH2-
9d
-CH2CH2CHCH3-
9e
-CH2 PhCH2-
9f
-CH2CH2OCH2CH2-
9g
-CH2C
NO donor
CCH2-
2. Chemistry Target compounds 8a-e, 9a-g were prepared via the synthetic route outlined in Scheme 1, 2, 3. The furoxans 1a-g were synthesized with thiophenol as described in Scheme 1. In Scheme 2, intermediate 2 was obtained according to the reported methods
[10, 11]
. Key compound 6 was delivered as follows. Abenzene-1,
2-diamine was cyclized with PPA at 100°C to obtain compound 4, which was converted to compound 5 with
This article is protected by copyright. All rights reserved.
Accepted Article
methyl 4'-(bromomethyl)biphenyl-2-carboxylate (3) and NaH in DMF. Then by saponification of compound 5 with NaOH, key compound 6 was obtained. Then compound 6 was esterized with alkyl dibromide to give compounds 7a-d which were further converted into their corresponding target nitrooxy derivatives 8a-d by reaction with silver nitrate in acetonitrile under refluxing and at darkness. Compound 6 was condensed with compound 2, 1a-g respectively in dry CH2Cl2 in the presence of N, N-dicyclohexylcarbodiimide (DCC) and catalytic N, N-(dimethylamino)pyridine (DMAP) to afford the corresponding target compounds 8e, 9a-g (Scheme 3).
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3. Results and discussion 3.1. NO-releasing test of target compounds in vitro As shown in Figure 2 and 3, all the target compounds produced different levels of NO in vitro determined by Griess assay [13]. It was found that NO-donating derivatives 8e released the maximum amount of NO and 9e did secondly. The maximum amount of NO released by compound 8e was up to 0.77 μmol/L (Figure 4). Generally, the series of nitrooxy NO donor hybrids (8a-e) displayed better NO releasing ability than furoxans (9a-g).
Figure 2. NO release assay in vitro of target compounds with nitrooxy
Figure 3. NO release assay in vitro of target compounds with furoxan
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Figure 4. NO release assay in vitro between 8e and 9e 3.2. Ang II Antagonist Activity Assay All the titled compounds were evaluated for antagonism of Ang II induced vascular contraction in vitro on rat aortic rings. As shown in Table 2, lead compound 6 had moderate pA2 value, whereas the introducing with NO donor increased the ability of inhibition remarkably. The results showed that the Ang II antagonist activity of compounds 8e and 9e were obviously superior to Losartan (Table 2).
Table 2 Inhibition of Ang II induced vascular contraction in vitro on rat aortic rings
Compd.
pA2（ x ）
Compd.
pA2（ x ）
Losartan
7.904
9f
7.913
6
7.904
9b
7.915
8a
7.947
9c
7.957
8b
7.958
9a
7.881
8c
7.956
9e
8.099
8d
7.947
9d
7.873
8e
8.033
9g
7.821
3.3 Effects on Ang II Induced Pressor Response in Conscious Normotensive Rats Compounds 8b, 8e and 9e, which shown better antagonism of Ang II induced vascular contraction, were chosen for further antihypertensive evaluation. Tested compounds exhibited the similar mode as losartan did. The hybrid derivative 8b, 8e and 9e, showed maximum values longer than losartan for 60 min (Figure 5, 6). It was speculated that these may be due to the synergy effect of ‘native’ molecule with NO donor.
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Accepted Article
Figure. 5 Inhibition of the Ang II (100 ng/kg iv)-induced pressor response in conscious normotensive rata by 3 mg/kg of 8b, 8e, 9e and Losartan
Figure 6. Inhibition of the Ang II (100 ng/kg iv)-induced pressor response in conscious normotensive rata by 6 mg/kg of 8e, 9e and Losartan. We also noticed that even the active part was covered with NO donor, the tested compounds still showed the inhibition of the Ang II induced pressor response almost immediately after the compounds were administered. It was supposed that the inhibition may be induced by the initial releasing of NO. As showed in Figure 7, the inhibition of tested compounds was dose related.
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Figure 7. Inhibition of the Ang II (100 ng/kg iv)-induced pressor response by 8e (n=3) and Losartan. Significance levels p
