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Synthesis and biological evaluation of novel hybrid chalcone derivatives as vasorelaxant agents
European Journal of Medicinal Chemistry 45 (2010) 3986e3992
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel hybrid chalcone derivatives as vasorelaxant agents Xiaowu Dong a, Lilin Du a, Zhichao Pan a, Tao Liu a, Bo Yang b, Yongzhou Hu a, * a b
ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 January 2010 Received in revised form 25 May 2010 Accepted 26 May 2010 Available online 2 June 2010
With the aim to further improve the vasorelaxant activities of chalcones, nine hybrid chalcone derivatives conjugated with nitric oxide (NO) donor or 1,4-dihydropyridyl (1,4-DHP) moiety were designed and synthesized based on molecular hybridization strategy. Their vasorelaxant activities were evaluated in aortic rings with endothelium pre-contracted with phenylephrine (PE). All of these compounds showed preferable vasorelaxant activities which were more potent than their parent compounds. Compounds 8c and 15c, the most potent compounds, would be promising structural templates for the development of novel vasorelaxant agents. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Hybrid chalcones Nitric oxide donor 1,4-Dihydropyridyl moiety Vasorelaxant agent
1. Introduction Flavonoids are a group of naturally occurring phenolic phytochemicals with diverse pharmacological properties, such as antibacterial, anti-viral, anti-allergic, anti-hepatotoxic, anti-ulcer action and anti-cancer [1e3]. Recently, they have been recognized as compounds with potent biological activities that may be active in prevention of chronic diseases such as cardiovascular disease [4]. Flavonoids showed beneficial impact on parameters associated with atherosclerosis, including lipoprotein oxidation, platelet aggregation and vascular reactivity [5e7]. Also, the vasorelaxant activities of flavonoids were extensively studied in isolated vascular preparation and animal models [8,9], suggesting that their vasorelaxant mechanisms were involved with the modulation of nitric oxide (NO) release, Ca2þ entry and inhibition of PKC [10e13]. Superior to traditional vasodilators, the additional antioxidant properties of flavonoids were also demonstrated to be beneficial in treatment of cardiovascular diseases [14]. All these facts prompt the strategies to develop novel and more potent flavonoids as vasorelaxant agents. Previously, we have described the synthesis and biological evaluation of flavonoid derivatives as vasodilators [15e17]. Structure-activities relationship (SAR) studies indicated that chalcones
* Corresponding author. Tel./fax: þ86 571 88208460. E-mail address: [email protected] (Y. Hu). 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.05.054
exhibited favorable vasorelaxant activities and the hydroxyl group at the 2 position of chalcones was an interesting point for further modification. Herein, molecular hybridization strategy which is a common method in drug design [18] was applied to improve the potency of chalcones and retain their multi-cardiovascular protective mechanisms. NO donors [19,20] or 1,4-dihydropyridyl (DHP) moieties [21,22] which are both important pharmacophores for vasodilators were introduced into the hybrid molecules. Different NO donors (nitrate or furoxan) were chosen to modulate the NO release from the final products, while 1,4-DHP moiety was introduced to regulate the Ca2þ entry blockage. Consequently, nine hybrid chalcone derivatives (Fig. 1) were synthesized and tested for their vasorelaxant activities through evaluation in aortic rings. 2. Results and discussion 2.1. Chemistry 2.1.1. Synthesis of the hybrid chalcone derivatives conjugated with NO donors 7b, 8aec and 9a The synthetic routes of hybrid chalcone derivatives conjugated with NO donors 7b, 8aec and 9a are summarized in Scheme 1. 4Formyl-3-methyl-furoxan 4a was obtained using crotonaldehyde 3 as the starting material according to the literature [23]. Compound 4a was transformed to its isomer 4b by heating over 98 h in boiling toluene in moderate yield. Reduction of compounds 4a,b using NaBH4 in dioxane, following by treatment with thionyl chloride
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Fig. 1. Hybrid chalcone derivatives 7b, 8aec, 9a and 15aed.
afforded (chloromethyl)furoxans 5a,b, respectively. Nucleophilic substitution of chalcone 1a (or 1b) with (chloromethyl)furoxans 5a, b (or 1,2-dibromoethane) in the presence of potassium carbonate in acetone resulted in compounds 6aed. Treatment of 6a,b and AgNO3 in acetonitrile yielded nitrates 7a,b. Deprotection of the phenol function of 7a and 6c,d in methanol/3N aq. HCl (5:1, v/v) under reflux afforded compounds 8aec. Etherization of nitrate 8a using 4-
chloromethyl-3-methyl-furoxan in the presence of potassium carbonate provided compound 9a. 2.1.2. Synthesis of the hybrid chalcone derivatives conjugated with 1,4-DHP moiety The synthesis of the target compounds 15aed is outlined in Scheme 2. Treatment of m-nitrobenzaldehyde, methyl acetoacetate
Scheme 1. Synthesis of hybrid chalcone derivatives 7b, 8aec and 9a. Reagents and conditions: (a) acetic acid, NaNO2, ice bath; (b) toluene, reflux; (c) NaBH4, dioxane, r.t.; (d) SOCl2, 0 C to r.t.; (e) K2CO3, acetone, reflux; (f) AgNO3, acetonitrile, r.t.; (g) 3 N HCl, methanol, reflux; (h) K2CO3, acetone, 5a, reflux.
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Scheme 2. Synthesis of hybrid chalcone derivatives 15aed. Reagents and conditions: (a) NH4OAc, microwave, 120 C; (b) NaOH, Methanol/H2O (6:1, v/v), 70 C; (c) K2CO3, 2, reflux; (d) K2CO3, acetone, reflux; (e) 3 N HCl, methanol, reflux.
and ammonium acetate under microwave irradiation afforded 1,4dihydropyridine 10. Selective hydrolysis of 10 in the presence of sodium hydroxide gave key intermediate monoacid 11 [24,25], which was then reacted with dibromoethane to yield compound 12. Condensation of 12 with chalcones 13aed in the presence of potassium carbonate in acetone under reflux afforded the hybrid derivatives 14aed. Deprotection of the phenol function of 14aed was carried out by reaction with methanol/3N aq. HCl (5:1, v/v) under reflux to yield desired compounds 15aed in good yield. 2.2. Vasorelaxation activity and SAR Vasorelaxation activities of hybrid chalcone derivatives 7b, 8aec, 9a and 15aed were evaluated in aortic rings with endothelium pre-contracted with 1 mM phenylephrine (PE). Quercetin, a well known vasodilator, was used as the positive control. The results were summarized in Table 1. All tested compounds promoted relaxation in a dose-dependent manner, and their maximal effects were observed at 300 mM as shown in Fig. 2. SAR of Chalcone hybrid derivatives conjugated with NO donors: Obviously, the vasorelaxant activities of hybrid chalcone derivatives 7b, 8aec and 9a bearing NO donor were increased when compared with that of 2,4,6-trihydroxy-30 ,40 -methylenedioxyl-chalcone 16, a good vasodilator (EC50 ¼ 22.5 mM, Emax ¼ 104.1%) reported in our previous studies [15]. Judged by the activities of compounds 8aec, the 4-methyl-3-methylene-furoxan moiety showed the most favorable effect on enhancing vasorelaxant activities as exemplified
Table 1 Vasorelaxant activities of hybrid chalcone derivatives 7b, 8aec, 9a and 15aed. Compd.
EC50 (mM)
Emax (%)
7b 8a 8b 8c 9a 15a 15b 15c 15d 16a Quercetina
8.4 13.4 12.1 8.9 9.4 9.3 6.4 2.9 9.7 22.5 244.0
104.0 102.9 95.7 100.1 96.0 98.6 101.9 104.0 102.9 104.1 91.3
a
Data available in our previous studies [15].
3.6 5.9 6.2 4.2 6.7 6.9 7.7 12.3 9.1 17.4 13.2
in compounds 8c (EC50 ¼ 8.9 mM, Emax ¼ 100.1%). Besides, increasing the number of nitrates linked to chalcone led to the enhanced vasorelaxant activities. For example, both of compound 7b bearing three nitrates (EC50 ¼ 8.4 mM, Emax ¼ 104.0%) and compound 9a bearing nitrate and furoxane (EC50 ¼ 9.4 mM, Emax ¼ 96.0%) exhibited more potent activities than compound 8a (EC50 ¼ 13.4 mM, Emax ¼ 102.9%). SAR of Chalcone hybrid derivatives conjugated to 1,4-DHP moiety: Comparing the vasorelaxant activities of hybrid compounds 15aed and 16 indicated that the introduction of 1,4-DHP moiety significantly enhanced the biological activities. For instance, the vasorelaxant activity of compound 15c was increased by approximately 6.7-fold comparing with 16. Insight into the observed effects of different substituent on chalcones revealed that the introduction of a para-hydroxyl group to the B-ring of chalcone showed optimal effect (ie. 15c: EC50 ¼ 2.9 mM, Emax ¼ 104.0%), while introduction of hydroxyl groups at the C-5 position of A-ring of chalcone didn’t exhibit enhanced activity (ie.15a: EC50 ¼ 9.3 mM, Emax ¼ 98.6%; 15d: EC50 ¼ 9.7 mM, Emax ¼ 102.9%). 3. Conclusion A series of novel hybrid chalcones derivatives conjugated with NO donors or 1,4-DHP moiety were designed and synthesized based on the hybrid strategy. Biological activity evaluation showed most target compounds exhibit significantly improved vasorelaxant activities. The preliminary structureeactivity relationships studies revealed that 4-methyl-3-methylene-furoxan moiety was the preferable fragment when linked with chalcones, and that the para-hydroxyl group on B-ring of chalcone was the most optimal when conjugated with 1,4-DHP moieties. Consequently, compound 8c and 15c would be promising structural templates for the development of novel vasorelaxant agents. 4. Experiments 4.1. Chemistry Reagents and solvents were purchased from commercial sources. Melting-points were obtained on a B-540 Büchi melting-point apparatus and are uncorrected. Intermediates and target compounds were characterized based on 1H NMR (Brüker AM500)
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Fig. 2. Effects of hybrid chalcone derivatives on relaxation in aortic rings with endothelium pre-contracted with 1 mM phenylephrine (PE). (a) The tested compounds 7b, 8aec and 9a were added cumulatively to achieve the appropriate concentrations; (b) The tested compounds 15aed were added cumulatively to achieve the appropriate concentrations. Results were expressed as means standard error of mean in terms of percentage relaxation of the contraction to PE (n ¼ 3e4).
and 13C NMR (Brüker AM125). Mass spectra were recorded on an Esquire-LC-00075 spectrometer. High resolution mass spectral (HRMS) analyses were recorded on Brüker DALTONICS APEX III mass spectrometer using electron spray ionization. Chalcone 1a, b [15], furoxane 4a,b [23] and 1,4-DHPs 11 [24,25] were synthesized according to the literature. 4.2. General procedure for the synthesis of furoxane 5a,b NaBH4 (300 mg, 7.9 mmol) was added to a cold solution of 4a (or 4b) (500 mg, 3.84 mmol) in dioxane (5 mL). The reaction mixture was stirred for 12 h at room temperature. The residue obtained by concentration of the solution was treated with water and extracted with ethyl acetate (15 mL 3). The organic phase was evaporated in vacuo to give pale yellow oil, to which thionyl chloride (1.67 g, 14.1 mmol) was added dropwise in ice-water bath. The reaction mixture was kept under stirring at room temperature for 12 h. Upon finishing of the reaction, water (50 mL) was added, and the mixture was extracted with ethyl acetate (20 mL 3). Evaporating of the dried combined organic layers afforded the crude product as an oily residue which was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (10:1e6:1). 4.2.1. 3-Methyl-4-chloromethyl-furoxane 5a Pale yellow oil (45%); 1H NMR (CDCl3, 400 MHz, d): 2.22 (s, 3H, CH3); 4.39 (s, 2H, CH2). 4.2.2. 3-Chloromethyl-4-methyl-furoxane 5b Pale yellow oil (38%); 1H NMR (CDCl3, 400 MHz, d): 2.45 (s, 3H, CH3); 4.26 (s, 2H, CH2). 4.2.3. General procedure for the synthesis of compounds 6aed A solution of compounds 1, potassium carbonate and 1,2dibromoethane 2 (or 5a,b) in acetone (20 mL) was refluxed for 2e10 h. After cooled to room temperature, the mixture was filtered and the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (10:1e4:1) to give expected product. 4.2.4. 3-(Benzo[d][1,3]dioxol-5-yl)-1-(2-(2-bromoethoxy)-4,6-bis (methoxymethoxy)phenyl)prop-2-en-1-one (6a) Reagent: compound 1a (300 mg, 1.29 mmol), potassium carbonate (356 mg, 2.58 mmol), 1,2-dibromoethane (4.83 g, 25.7 mmol); yellow syrup (359 mg, 56%). 1H NMR (CDCl3, 500 MHz, d): 3.40 (s, 3H), 3.51 (s, 3H), 3.52 (t, 2H, J ¼ 6.5 Hz), 4.26 (t, 2H,
J ¼ 6.5 Hz), 5.19 (s, 2H), 5.22 (s, 2H), 6.00 (s, 2H), 6.34 (d, 1H, J ¼ 2.0 Hz), 6.55 (d, 1H, J ¼ 2.0 Hz), 6.79 (d, 1H, J ¼ 8.5 Hz), 6.83 (d, 1H, J ¼ 16.5 Hz), 7.00 (dd, 1H, J ¼ 1.5, 8.5 Hz), 7.07 (d, 1H, J ¼ 1.5 Hz), 7.28 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 495. 4.2.5. 3-(Benzo[d][1,3]dioxol-5-yl)-1-(2,4,6-tris(2-bromoethoxy) phenyl)prop-2-en-1-one (6b) Reagent: compound 1b (300 mg, 1.00 mmol), potassium carbonate (276 mg, 2.00 mmol), 1,2-dibromoethane (4.83 g, 25.7 mmol); yellow syrup (416 mg, 67%). 1H NMR (CDCl3, 500 MHz, d): 3.52 (t, 4H, J ¼ 6.5 Hz), 3.66 (t, 2H, J ¼ 6.5 Hz), 4.26 (t, 4H, J ¼ 6.5 Hz), 4.30 (t, 2H, J ¼ 6.5 Hz), 6.00 (s, 2H), 6.18 (s, 2H), 6.79 (d, 1H, J ¼ 7.5 Hz), 6.82 (d, 1H, J ¼ 16.5 Hz), 7.00 (dd, 1H, J ¼ 1.5, 7.5 Hz), 7.06 (d, 1H, J ¼ 1.5 Hz), 7.26 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 619. 4.2.6. 4-((2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-bis (methoxymethoxy)phenoxy)methyl)-3-methyl-1,2,5-oxadiazole 2oxide (6c) Reagent: compound 1a (200 mg, 0.52 mmol), potassium carbonate (142 mg, 1.04 mmol) and 4a (80.8 mg, 0.55 mmol); yellow syrup (206 mg, 80%). 1H NMR (CDCl3, 500 MHz, d): 2.31 (s, 3H), 3.37 (s, 3H), 3.51 (s, 3H), 5.00 (s, 2H), 5.10 (s, 2H), 5.20 (s, 2H), 6.01 (s, 2H), 6.43 (d,1H, J ¼ 2.0 Hz), 6.57 (d,1H, J ¼ 2.0 Hz), 6.75 (d,1H, J ¼ 16.5 Hz), 6.80 (d, 1H, J ¼ 8.0 Hz), 6.96 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.01 (d, 1H, J ¼ 1.5 Hz), 7.21 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 501. 4.2.7. 3-((2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-bis (methoxymethoxy)phenoxy)methyl)-4-methyl-1,2,5-oxadiazole 2oxide (6d) Reagent: compound 1a (200 mg, 0.52 mmol), potassium carbonate (142 mg, 1.04 mmol) and 4b (81 mg, 0.55 mmol); yellow syrup (196 mg, 76%). 1H NMR (CDCl3, 500 MHz, d): 2.12 (s, 3H), 3.38 (s, 3H), 3.51 (s, 3H), 5.10 (s, 2H), 5.11 (s, 2H), 5.20 (s, 2H), 6.01 (s, 2H), 6.48 (d,1H, J ¼ 2.0 Hz), 6.57 (d,1H, J ¼ 2.0 Hz), 6.76 (d,1H, J ¼ 16.5 Hz), 6.80 (d, 1H, J ¼ 8.0 Hz), 6.97 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.03 (d, 1H, J ¼ 1.5 Hz), 7.21 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 501. 4.3. General procedure for the synthesis of nitrate 7a,b To a stirred solution of 6a,b in acetonitrile (5 mL), silver nitrate was added. The reaction mixture was stirred in the dark for 50e70 h. The reaction mixture was then filtered, and the filtrate was evaporated under reduced pressure to afford a dark-yellow solid which was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (5:1e2:1) to give expected product.
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4.3.1. 2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-bis (methoxymethoxy)phenoxy)ethyl nitrate (7a) Reagent: compound 6a (300 mg, 0.61 mmol), silver nitrate (277 mg, 1.633 mmol); yellow syrup (225 mg, 78%). 1H NMR (CDCl3, 500 MHz, d): 3.40 (s, 3H), 3.51 (s, 3H), 4.23 (t, 2H, J ¼ 6.5 Hz), 4.71 (t, 2H, J ¼ 6.5 Hz), 5.20 (s, 2H), 5.23 (s, 2H), 6.00 (s, 2H), 6.34 (d, 1H, J ¼ 2.0 Hz), 6.55 (d, 1H, J ¼ 2.0 Hz), 6.80 (d, 1H, J ¼ 8.5 Hz), 6.83 (d, 1H, J ¼ 16.5 Hz), 7.01 (dd, 1H, J ¼ 1.5, 8.5 Hz), 7.06 (d, 1H, J ¼ 1.5 Hz), 7.29 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 478. 4.3.2. 2,20 ,200 -(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)benzene1,3,5-triyl)tris(oxy)tris(ethane-2,1-diyl) trinitrate (7b) Reagent: compound 6b (400 mg, 0.64 mmol), silver nitrate (277 mg, 1.633 mmol); yellow solid (237 mg, 78%), mp 101e103 C. 1 H NMR (CDCl3, 500 MHz, d): 4.27 (t, 4H, J ¼ 6.5 Hz), 4.33 (t, 2H, J ¼ 6.5 Hz), 4.75 (t, 4H, J ¼ 6.5 Hz), 4.88 (t, 2H, J ¼ 6.5 Hz), 6.05 (s, 2H), 6.23 (s, 2H), 6.82 (d, 1H, J ¼ 16.5 Hz), 6.85 (d, 1H, J ¼ 7.5 Hz), 7.04 (dd, 1H, J ¼ 1.5, 7.5 Hz), 7.11 (d, 1H, J ¼ 1.5 Hz), 7.27 (d, 1H, J ¼ 16.5 Hz). 13C NMR (Acetone-d6, 125 MHz, d): 191.2, 164.0, 162.6, 151.1, 146.3, 144.4, 132.3, 121.8, 119.6, 115.4, 108.2, 107.8, 102.1, 94.3, 66.6, 62.9, 58.2. HRMS(ESI): Found 568.0980 ([M þ H]þ), calcd for C22H21N3O15: 567.0973. 4.4. General procedure for the synthesis of compounds 8aec To a solution of 6c,d (or 7a) in methanol (2 mL), 3 N aq. HCl (0.4 mL) was added. The resulting mixture was refluxed for 45 min, then poured into cold water and extracted with ethyl acetate (5 mL 3 Times). The organic phase was washed with brine and then dried over anhydrous Na2SO4. After removal of the solvent, the residue was purified by chromatography on silica gel using gradient elution with petroleum ethereethyl acetate (8:1e3:1) to give expected product. 4.4.1. 2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5dihydroxyphenoxy)ethyl nitrate (8a) Reagent: compound 7a (100 mg, 0.21 mmol); pale yellow solid (65 mg, 80%), mp 193e194 C. 1H NMR (Acetone-d6, 500 MHz, d): 4.57 (t, 2H, J ¼ 6.5 Hz), 5.12 (t, 2H, J ¼ 6.5 Hz), 6.07 (d, 1H, J ¼ 2.0 Hz), 6.12 (s, 2H), 6.13 (d, 1H, J ¼ 2.0 Hz), 6.93 (d, 1H, J ¼ 8.0 Hz), 7.29 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.33 (d, 1H, J ¼ 1.5 Hz), 7.76 (d, 1H, J ¼ 16.5 Hz), 7.90 (d, 1H, J ¼ 16.5 Hz), 9.54 (s, 1H, OH), 14.17 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 193.4, 165.9, 162.3, 161.1, 150.0, 147.3, 144.4, 130.3, 125.8, 123.6, 109.2, 108.4, 107.6, 102.1, 96.6, 93.3, 70.1, 64.8. HRMS(ESI): Found 390.0755 ([M þ H]þ), calcd for C18H15NO9: 389.0747. 4.4.2. 4-((2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5dihydroxyphenoxy)methyl)-3-methyl-1,2,5-oxadiazole 2-oxide (8b) Reagent: compound 6c (150 mg, 0.3 mmol); pale yellow solid (68 mg, 55%), mp 220e222 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.13 (s, 3H), 5.44 (s, 2H), 6.00 (s, 2H), 6.01 (d, 1H, J ¼ 2.0 Hz), 6.26 (d, 1H, J ¼ 2.0 Hz), 6.88 (d, 1H, J ¼ 8.0 Hz), 7.08 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.09 (d, 1H, J ¼ 1.5 Hz), 7.63 (d, 1H, J ¼ 15.0 Hz), 7.78 (d, 1H, J ¼ 15.0 Hz), 9.52 (brs, 1H, OH), 14.13 (s, 1H, OH). ESI-MS: m/z [M þ H]þ 413. 13C NMR (Acetone-d6, 125 MHz, d): 192.8, 166.1, 163.2, 160.8, 155.4, 151.2, 147.2, 144.3, 130.8, 124.9, 124.1, 112.3, 109.2, 108.8, 107.2, 102.3, 96.5, 93.6, 60.5, 7.8. HRMS(ESI): Found 413.0920 ([M þ H]þ), calcd for C20H16N2O8: 412.0907. 4.4.3. 3-((2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5dihydroxyphenoxy)methyl)-4-methyl-1,2,5-oxadiazole 2-oxide (8c) Reagent: compound 6c (150 mg, 0.3 mmol); pale yellow solid (78 mg, 63%), mp 198e201 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.47 (s, 3H), 5.33 (s, 2H), 6.11 (d, 1H, J ¼ 2.0 Hz), 6.12 (s, 2H), 6.22 (d,
1H, J ¼ 2.0 Hz), 6.92 (d, 1H, J ¼ 8.0 Hz), 7.15 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.17 (d, 1H, J ¼ 1.5 Hz), 7.68 (d, 1H, J ¼ 15.0 Hz), 7.72 (d, 1H, J ¼ 15.0 Hz), 9.50 (brs, 1H, OH), 14.10 (s, 1H, OH). 13C NMR (Acetoned6, 125 MHz, d): 193.0, 165.9, 163.2, 161.2, 154.8, 150.7, 148.3, 144.4, 130.5, 124.8, 123.6, 112.9, 109.1, 108.9, 108.0, 102.1, 96.6, 93.6, 58.6, 8.2. HRMS(ESI): Found 413.0918 ([M þ H]þ), calcd for C20H16N2O8: 412.0907. 4.4.4. 4-((4-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3-hydroxy-5-(2(nitrooxy)ethoxy)phenoxy)methyl)-3-methyl-1,2,5-oxadiazole 2oxide (9a) A solution of compound 8a (50 mg, 0.13 mmol), potassium carbonate (35 mg, 0.25 mmol) and 5a in acetone (3 mL) was refluxed for 10 h. The mixture was filtered and the solution was concentrated in vacuo. The residue was purified by chromatography on silica gel with petroleum ethereethyl acetate (3:1) to give product 9a as pale yellow solid (36 mg, 56%), mp 95e97 C. 1H NMR (CDCl3, 400 MHz, d): 2.14 (s, 3H), 4.55 (t, 2H, J ¼ 6.5 Hz), 5.13 (t, 2H, J ¼ 6.5 Hz), 5.46 (s, 2H), 6.05 (d, 1H, J ¼ 2.0 Hz), 6.10 (s, 2H), 6.21 (d, 1H, J ¼ 2.0 Hz), 6.84 (d, 1H, J ¼ 8.0 Hz), 7.06 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.11 (d, 1H, J ¼ 1.5 Hz), 7.65 (d, 1H, J ¼ 15.6 Hz), 7.81 (d, 1H, J ¼ 15.6 Hz), 13.95 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 193.4, 165.6, 164.3, 161.8, 157.5, 150.1, 148.3, 143.8, 130.3, 124.9, 123.3, 113.5, 109.7, 109.2, 108.7, 102.3, 96.2, 93.5, 71.8, 64.9, 60.6, 8.2. HRMS(ESI): Found 502.1014 ([M þ H]þ), calcd for C22H19N3O11: 501.1020. 4.5. 3-(2-Bromoethyl)-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)1,4-dihydropyridine-3,5-dicarboxylate (12) A solution of compound 11 (1.2 g, 3.61 mmol), potassium carbonate (0.99 g, 7.22 mmol) and 1,2-dibromoethane 2 (13.5 g, 72.2 mmol) in acetone (20 mL) was refluxed for 5 h. The mixture was filtered and the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (4:1e2:1) to give title product as yellow syrup (1.05 g, 66%). 1H NMR (CDCl3, 500 MHz, d): 2.37 (s, 3H), 2.39 (s, 3H), 3.46 (m, 2H), 3.65 (s, 3H), 4.37 (m, 2H), 5.12 (s, 1H), 5.83 (s, 1H), 7.38 (t, 1H, J ¼ 8.0 Hz), 7.67 (dd, 1H, J ¼ 2.0, 8.0 Hz), 8.00 (dd, 1H, J ¼ 2.0, 8.0 Hz), 8.11 (d, 1H, J ¼ 8.0 Hz). ESI-MS: m/z [M þ H]þ 439. 4.6. General procedure for the synthesis of compounds 14a,d A solution of compound 12, potassium carbonate and appropriate chalcone 13a,d in acetone (5 mL) was refluxed for 4e12 h. The mixture was filtered and the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (4:1e1:1) to give expected product. 4.6.1. 3-(2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-bis (methoxymethoxy)phenoxy)ethyl)-5-methyl-2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14a) Reagent: compound 12 (200 mg, 0.45 mmol), potassium carbonate (124 mg, 0.9 mmol) and 13a (152 mg, 0.45 mmol); pale yellow syrup (238 mg, 70%). 1H NMR (CDCl3, 500 MHz, d): 1H NMR (CDCl3, 400 MHz, d): 2.22 (s, 3H), 2.32 (s, 3H), 3.39 (s, 3H), 3.48 (m, 2H), 3.60 (s, 3H), 4.28 (m, 2H), 5.01 (s, 1H), 5.12 (s, 2H), 5.20 (s, 2H), 5.60 (s, 1H), 6.05 (s, 2H), 6.29 (d, 1H, J ¼ 2.0 Hz), 6.52 (d, 1H, J ¼ 2.0 Hz), 6.82 (d, 1H, J ¼ 8.5 Hz), 6.92 (d, 1H, J ¼ 16.0 Hz), 7.05 (dd, 1H, J ¼ 1.5, 8.5 Hz), 7.11 (d, 1H, J ¼ 1.5 Hz), 7.28 (t, 2H, J ¼ 8.0 Hz), 7.58 (d, 1H, J ¼ 16.0 Hz), 7.61 (d, 1H, J ¼ 8.0 Hz), 7.96 (dd, 1H, J ¼ 1.5, 8.0 Hz), 8.01 (d, 1H, J ¼ 1.5 Hz). ESI-MS: m/z [M þ H]þ 746.
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4.6.2. 3-(2-(2-(3-Bromophenyl)acryloyl)-(3,5-bis (methoxymethoxy)phenoxy)ethyl)-5-methyl-2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14b) Reagent: compound 12 (200 mg, 0.45 mmol), potassium carbonate (124 mg, 0.9 mmol)and 13b (193 mg, 0.45 mmol); pale yellow syrup (203 mg, 57%). 1H NMR (CDCl3, 400 MHz, d): 2.24 (s, 3H), 2.34 (s, 3H), 3.40 (s, 3H), 3.51 (m, 2H), 3.62 (s, 3H), 4.26 (m, 2H), 5.00 (s, 1H), 5.11 (s, 2H), 5.18 (s, 2H), 5.64 (s, 1H), 6.30 (d, 1H, J ¼ 2.0 Hz), 6.52 (d, 1H, J ¼ 2.0 Hz), 6.90 (d, 1H, J ¼ 16.0 Hz), 7.22 (t, 2H, J ¼ 7.5 Hz), 7.27 (t, 2H, J ¼ 8.0 Hz), 7.40 (d, 1H, J ¼ 7.5 Hz), 7.46 (d, 1H, J ¼ 7.5 Hz), 7.55 (d, 1H, J ¼ 16.0 Hz), 7.60 (d, 1H, J ¼ 8.0 Hz), 7.61 (s, 1H), 7.92 (dd, 1H, J ¼ 1.5, 8.0 Hz), 8.03 (d, 1H, J ¼ 1.5 Hz). ESI-MS: m/z [M þ H]þ 781. 4.6.3. 3-(2-(3,5-Bis(methoxymethoxy)-2-(3-(4-(methoxymethoxy) phenyl)acryloyl)phenoxy)ethyl) 5-methyl-2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14c) Reagent: compound 12 (200 mg, 0.45 mmol), potassium carbonate (124 mg, 0.9 mmol) and 13c (184 mg, 0.45 mmol); pale yellow syrup (219 mg, 63%). 1H NMR (CDCl3, 400 MHz, d): 2.19 (s, 3H), 2.33 (s, 3H), 3.39 (s, 3H), 3.46 (m, 2H), 3.47 (s, 3H), 3.51 (s, 3H), 3.62 (s, 3H), 4.28 (m, 2H), 5.04 (s, 1H), 5.11 (s, 2H), 5.18 (s, 2H), 5.20 (s, 2H), 5.63 (s, 1H), 6.30 (d, 1H, J ¼ 2.0 Hz), 6.53 (d, 1H, J ¼ 2.0 Hz), 6.81 (d, 1H, J ¼ 16.0 Hz), 7.00 (d, 2H, J ¼ 8.5 Hz), 7.30 (t, 1H, J ¼ 8.0 Hz), 7.42 (d, 2H, J ¼ 8.5 Hz), 7.61 (d, 1H, J ¼ 16.0 Hz), 7.94 (dd, 1H, J ¼ 2.0, 8.0 Hz), 8.04 (d, 1H, J ¼ 2.0 Hz). ESI-MS: m/z [M þ H]þ 763. 4.6.4. 3-(2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-5(methoxymethoxy)phenoxy)ethyl) 5-methyl 2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14d) Reagent: compound 12 (200 mg, 0.45 mmol), potassium carbonate (124 mg, 0.9 mmol)and 13d (149 mg, 0.45 mmol); pale yellow syrup (219 mg, 70%). 1H NMR (CDCl3, 500 MHz, d): 2.21 (s, 3H), 2.34 (s, 3H), 3.44 (s, 3H), 3.44 (m, 2H), 3.63 (s, 3H), 4.23 (m, 2H), 5.01 (s, 1H), 5.12 (s, 2H), 5.62 (s, 1H), 6.02 (s, 2H), 6.65 (dd, 1H, J ¼ 2.0, 8.0 Hz), 6.73 (d, 1H, J ¼ 2.0 Hz), 7.02 (d, 1H, J ¼ 16.0 Hz), 6.85 (d, 1H, J ¼ 8.4 Hz), 7.14 (dd, 1H, J ¼ 2.0, 8.4 Hz), 7.20 (d, 1H, J ¼ 2.0 Hz), 7.35 (t, 2H, J ¼ 8.0 Hz), 7.55 (d, 1H, J ¼ 16.0 Hz), 7.80 (d, 1H, J ¼ 8.0 Hz), 7.97 (dd, 1H, J ¼ 2.0, 8.0 Hz), 8.03 (d, 1H, J ¼ 2.0 Hz). ESI-MS: m/z [M þ H]þ 687. 4.7. General procedure for the synthesis of hybrid compound 15aed To a solution of 14aed in methanol (2 mL), 3 N HCl aqueous (0.4 mL) was added. The resulting mixture was refluxed for 1.5 h, then poured into cold water and extracted with ethyl acetate (5 mL 3 Times). The organic phase was washed with brine and then dried over anhydrous Na2SO4. After removal of the solvent, the residue was purified by chromatography on silica gel using gradient elution with petroleum ethereethyl acetate (2:1e1:2) to give expected product. 4.7.1. 3-(2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5dihydroxyphenoxy)ethyl)-5-methyl 2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (15a) Reagent: compound 14a (100 mg, 0.13 mmol), pale yellow solid (57 mg, 65%); mp 162e165 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.32 (s, 3H), 2.33 (s, 3H), 3.54 (s, 3H), 4.24 (m, 1H), 4.46 (m, 1H), 4.49 (m, 1H), 4.62 (m, 1H), 5.02 (s, 1H), 5.64 (s, 1H), 6.08 (s, 2H), 6.12 (d, 1H, J ¼ 2.0 Hz), 6.13 (s, 1H, J ¼ 2.0 Hz), 6.95 (d, 1H, J ¼ 8.0 Hz), 7.30 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.33 (d, 1H, J ¼ 1.5 Hz), 7.56 (t, 2H, J ¼ 8.0 Hz), 7.74 (d, 1H, J ¼ 16.5 Hz), 7.87 (dd, 1H, J ¼ 2.0, 8.0 Hz), 7.88 (d, 1H, J ¼ 16.5 Hz), 8.00 (d, 1H, J ¼ 2.0 Hz), 8.10 (d, 1H, J ¼ 16.5 Hz), 9.54 (s, 1H, OH), 14.17 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d):
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192.3, 169.6, 169.5, 167.5, 163.93, 162.32, 151.2, 150.4, 149.2, 147.8, 145.6, 144.7, 138.5, 132.3, 130.3, 129.3, 127.9, 124.7, 124.6, 122.6, 109.2, 108.9, 107.2, 103.8, 102.1, 96.6, 94.2, 93.8, 67.9, 61.3, 52.0, 35.6, 16.3. HRMS(ESI): Found 659.1687 ([M þ H]þ), calcd for C34H30N2O12: 658.1799. 4.7.2. 3-(2-(2-(3-(3-Bromophenyl)acryloyl)-3,5dihydroxyphenoxy)ethyl)5-methyl,6-dimethyl -4-(3-nitrophenyl)1,4-dihydropyridine-3,5-dicarboxylate (15b) Reagent: compound 14b (100 mg, 0.13 mmol), pale yellow solid (62 mg, 70%); mp 148e150 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.31 (s, 6H), 3.52 (s, 3H), 4.25 (m, 1H), 4.45 (m, 1H), 4.49 (m, 1H), 4.63 (m, 1H), 5.00 (s, 1H), 5.65 (s, 1H), 6.10 (d, 1H, J ¼ 2.5 Hz), 6.11 (d, 1H, J ¼ 2.5 Hz), 7.26 (t, 1H, J ¼ 8.0 Hz), 7.29 (t, 1H, J ¼ 8.0 Hz), 7.52 (dd, 1H, J ¼ 1.0, 8.0 Hz), 7.56 (t, 2H, J ¼ 8.0 Hz), 7.63 (d, 1H, J ¼ 16.5 Hz), 7.77 (d, J ¼ 1.0 Hz), 7.87 (dd, 1H, J ¼ 1.0, 8.0 Hz), 8.00 (d, 1H, J ¼ 1.0 Hz), 8.13 (d, 1H, J ¼ 16.5 Hz), 9.52 (s, 1H, OH), 14.40 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 193.4, 169.8, 169.5, 165.6, 163.4, 162.1, 149.2, 145.6, 144.9, 144.7, 138.9, 138.1, 136.5, 132.3, 131.1, 130.7, 129.5, 128.1, 127.2, 126.1, 125.1122.2, 109.2, 106.8, 96.6, 93.8, 95.4, 94.2, 67.5, 61.5, 52.1, 34.9, 16.8. HRMS(ESI): Found 693.1017 ([M þ H]þ), calcd for C33H29BrN2O10: 692.1006. 4.7.3. 3-(2-(3,5-Dihydroxy-2-(3-(4-hydroxyphenyl)acryloyl) phenoxy)ethyl)-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)-1,4dihydropyridine-3,5-dicarboxylate (15c) Reagent: compound 14c (100 mg, 0.13 mmol), pale yellow solid (48 mg, 58%); mp 172e174 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.33 (s, 6H), 3.56 (s, 3H), 4.24 (m, 1H), 4.46 (m, 2H), 4.50 (m, 1H), 4.65 (m, 1H), 5.01 (s, 1H), 5.67 (s, 1H), 6.12 (d, 1H, J ¼ 2.0 Hz), 6.13 (d, 1H, J ¼ 2.0 Hz), 6.82 (d, 2H, J ¼ 8.5 Hz), 7.56 (t, 2H, J ¼ 8.5 Hz), 7.59 (d, 2H J ¼ 8.0 Hz), 7.63 (d, 1H, J ¼ 16.5 Hz), 7.87 (dd, 1H, J ¼ 2.0, 8.5 Hz), 8.00 (d, 1H, J ¼ 2.0 Hz), 8.07 (d, 1H, J ¼ 16.5 Hz), 9.05 (s, 1H, OH), 9.55 (s, 1H, OH), 14.33 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 194.2, 169.5, 169.3, 164.3, 163.4, 162.1, 160.1, 150.2, 148.1, 145.6, 144.7, 138.7, 136.6, 129.7, 129.5, 127.4, 126.8, 125.3, 124.6, 116.4, 108.9, 96.3, 94.0, 93.8, 93.5, 67.7, 62.3, 52.0, 35.8, 16.7. HRMS(ESI): Found 631.1859 ([M þ H]þ), calcd for C33H30N2O11: 630.1850. 4.7.4. 3-(2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-5hydroxyphenoxy)ethyl)-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)1,4-dihydropyridine-3,5-dicarboxylate (15d) Reagent: compound 14d (100 mg, 0.15 mmol), pale yellow solid (67 mg, 72%); mp 139e141 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.29 (s, 3H), 2.30 (s, 3H), 3.49 (s, 3H), 4.27 (m, 1H), 4.42 (m, 2H), 4.50 (m, 1H), 5.03 (s, 1H), 5.61 (s, 1H), 6.04 (s, 2H), 6.57 (d, 1H, J ¼ 2.5 Hz), 6.59 (s, 1H), 6.78 (d, 1H, J ¼ 3.0 Hz), 7.06 (d, 1H, J ¼ 3.0 Hz), 7.07 (s, 1H), 7.25 (t, 1H, J ¼ 8.0 Hz), 7.50 (d, 1H, J ¼ 15.5 Hz), 7.58 (d, 1H, J ¼ 8.0 Hz), 7.65 (d, 1H, J ¼ 15.5 Hz), 7.77 (d, 1H, J ¼ 2.5 Hz), 7.86 (d, 1H, J ¼ 3.0 Hz), 8.03 (s, 1H), 9.09 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 191.1, 169.6, 169.4, 162.2, 161.6, 150.8, 148.3, 147.7, 144.9, 144.6, 143.4, 139.3, 136.6, 132.2, 130.7, 129.5, 127.9, 124.5, 123.7, 121.9, 119.8, 109.4, 109.2, 108.8, 102.9, 102.4, 96.6, 94.2, 67.7, 62.3, 52.0, 35.8, 16.5. HRMS(ESI): Found 643.1860 ([M þ H]þ), calcd for C34H30N2O11: 642.1850. 4.8. Vasodilatory effect assay Vascular rings were prepared from the aorta of male Male SpragueeDawley rats (four to six months old and weighing on average 250 g), and contraction studies were performed following the general procedure detailed in the literature [26]. After an equilibration period of at least 1 h, isometric contractions induced by PE (1 mM) were obtained. When contraction of the tissue in
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response to this vasoconstrictor agent had stabilized (after about 20 min), cumulatively increasing concentrations of the tested compounds were added to the bath at 15e20 min intervals (the time needed to obtain steady-state relaxation). Control tissues were simultaneously subjected to the same procedures, but omitting the compounds and adding the vehicle. The flavonoidsinduced maximal relaxation (Emax) in aortic rings was calculated as a percentage of the contraction in response to PE (1 mM). The half maximum effective concentration (EC50) was defined as the concentration of the flavonoids that induced 50% of maximum relaxation from the contraction elicited by PE (1 mM) and was calculated from the concentration response curve by nonlinear regression (curve fit) using GraphPad Prism (Version 4.0). Acknowledgement The authors are grateful to support from the Administration of Traditional Chinese Medicine of Zhejiang Province (NO. 2009CB033), the National Key Tech Project for Major Creation of New drugs (NO. 2009ZX09501-003) and the supports from the School of Medicine, Zhejiang University for carrying out vasorelaxant effect assays. References [1] W. Ren, Z. Qiao, H. Wang, L. Zhu, L. Zhang, Med. Res. Rev. 23 (2003) 519e534. [2] G.D. Carlo, N. Mascolo, A.A. lzzo, F. Capasso, Life Sci. 65 (1999) 337e353. [3] E. Tripoli, M.L. Guardia, S. Giammanco, D.D. Majo, M. Giammanco, Food Chem. 104 (2007) 466e479. [4] M. Gross, Pharmaceut. Biol. 42 (2004) 21e35. [5] A. Koenig, C. Roegler, K. Lange, A. Daiber, E. Glusa, J. Lehmann, Bioorg. Med. Chem. Lett. 17 (2007) 5881e5885.
[6] Z. Nowakowska, Eur. J. Med. Chem. 42 (2007) 125e137. [7] L. Hooper, P. AKroon, E.B. Rimm, J.S. Cohn, I. Harvey, K.A. Cornu, J.J. Ryder, W. L. Hall, A. Cassidy, Am. J. Clin. Nutr. 88 (2008) 38e50. [8] F. Fusi, S. Saponara, F. Pessina, B. Gorelli, G. Sgaragli, Eur. J. Nutr. 42 (2003) 10e17. [9] J. Duarte, F. Perez-vizcaino, J. Jimenez, J. Tamargo, A. Zarzuelo, Stud. Nat. Prod. Chem. 25 (2001) 565e605. [10] Y.H. Zhang, Y.S. Park, T.J. Kim, L.H. Fang, H.Y. Ahn, J.T. Hong, Y. Kim, C.K. Lee, Y. P. Yun, Gen. Pharmacol. 35 (2002) 341e347. [11] S.K. Mishra, S.E. Abbot, A. Choudhury, M. Cheng, N. Khatab, N.J.R. Maycock, A. Zavery, P.I. Aaronson, Cardiovasc. Res. 46 (2000) 539e546. [12] H.D. Jiang, Q. Xia, X.X. Wang, J.F. Song, I.C. Jiang, Pharmazie 60 (2005) 444e447. [13] Z. Pan, T. Feng, L. Shan, B. Cai, W. Chu, H. Niu, Y. Lu, B. Yang, Phytother. Res. 22 (2008) 1428e1433. [14] M. Galisteo, M.F. García-Saura, R. Jiménez, C.I. Villar, A. Zarzuelo, F. Vargas, J. Duarte, Mol. Cell. Biochem. 259 (2004) 91e99. [15] X. Dong, T. Liu, J. Yan, P. Wu, J. Chen, Y. Hu, Bioorg. Med. Chem. 17 (2009) 716e726. [16] X. Dong, L. Qi, C. Jiang, J. Chen, E. Wei, Y. Hu, Bioorg. Med. Chem. Lett. 19 (2009) 3196e3198. [17] X. Dong, J. Chen, C. Jiang, T. Liu, Y. Hu, Arch. Pharm. 342 (2009) 428e432. [18] C. Viegas-Junior, A. Danuello, V. da Silva Bolzani, E.J. Barreiro, C.A.M. Fraga, Curr. Med. Chem. 14 (2007) 1829e1852. [19] A.M. Gasco, A. Di Stilo, G. Sorba, A. Gasco, R. Ferioli, G. Folco, M. Civellic, P. Carusoc, Eur. J. Med. Chem. 28 (1993) 433e438. [20] S.J. Lewis, M.D. Travis, M.P. Hashmi-Hill, K. Sandock, T.P. Robertson, J.N. Bates, Vasc. Pharmacol. 45 (2006) 383e394. [21] D.J. Triggle, D.A. Langs, R.A. Janis, Med. Res. Rev. 9 (1989) 123e180. [22] J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon, A.G. Gilman (Eds.), The Pharmacological Basis of Therapeutics, nineth ed. McGraw-Hill, New York, 1996. [23] R. Fruttero, B. Ferrarotti, A. Serafino, A.D. Stilo, A. Gasco, J. Heterocycl. Chem. 26 (1989) 1345e1347. [24] L. Wang, G. Chen, Chem. Ind. Times 21 (6) (2007) 27e29. [25] T. Ogawa, K. Hatayama, H. Maeda, Y. Kita, Chem. Pharm. Bull. 42 (1994) 1579e1589. [26] L. Qian, H. Wang, W. Qiu, H. Huang, I.C. Bruce, Q. Xia, Vasc. Pharmacol. 45 (2006) 374e382.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel hybrid chalcone derivatives as vasorelaxant agents Xiaowu Dong a, Lilin Du a, Zhichao Pan a, Tao Liu a, Bo Yang b, Yongzhou Hu a, * a b
ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 January 2010 Received in revised form 25 May 2010 Accepted 26 May 2010 Available online 2 June 2010
With the aim to further improve the vasorelaxant activities of chalcones, nine hybrid chalcone derivatives conjugated with nitric oxide (NO) donor or 1,4-dihydropyridyl (1,4-DHP) moiety were designed and synthesized based on molecular hybridization strategy. Their vasorelaxant activities were evaluated in aortic rings with endothelium pre-contracted with phenylephrine (PE). All of these compounds showed preferable vasorelaxant activities which were more potent than their parent compounds. Compounds 8c and 15c, the most potent compounds, would be promising structural templates for the development of novel vasorelaxant agents. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Hybrid chalcones Nitric oxide donor 1,4-Dihydropyridyl moiety Vasorelaxant agent
1. Introduction Flavonoids are a group of naturally occurring phenolic phytochemicals with diverse pharmacological properties, such as antibacterial, anti-viral, anti-allergic, anti-hepatotoxic, anti-ulcer action and anti-cancer [1e3]. Recently, they have been recognized as compounds with potent biological activities that may be active in prevention of chronic diseases such as cardiovascular disease [4]. Flavonoids showed beneficial impact on parameters associated with atherosclerosis, including lipoprotein oxidation, platelet aggregation and vascular reactivity [5e7]. Also, the vasorelaxant activities of flavonoids were extensively studied in isolated vascular preparation and animal models [8,9], suggesting that their vasorelaxant mechanisms were involved with the modulation of nitric oxide (NO) release, Ca2þ entry and inhibition of PKC [10e13]. Superior to traditional vasodilators, the additional antioxidant properties of flavonoids were also demonstrated to be beneficial in treatment of cardiovascular diseases [14]. All these facts prompt the strategies to develop novel and more potent flavonoids as vasorelaxant agents. Previously, we have described the synthesis and biological evaluation of flavonoid derivatives as vasodilators [15e17]. Structure-activities relationship (SAR) studies indicated that chalcones
* Corresponding author. Tel./fax: þ86 571 88208460. E-mail address: [email protected] (Y. Hu). 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.05.054
exhibited favorable vasorelaxant activities and the hydroxyl group at the 2 position of chalcones was an interesting point for further modification. Herein, molecular hybridization strategy which is a common method in drug design [18] was applied to improve the potency of chalcones and retain their multi-cardiovascular protective mechanisms. NO donors [19,20] or 1,4-dihydropyridyl (DHP) moieties [21,22] which are both important pharmacophores for vasodilators were introduced into the hybrid molecules. Different NO donors (nitrate or furoxan) were chosen to modulate the NO release from the final products, while 1,4-DHP moiety was introduced to regulate the Ca2þ entry blockage. Consequently, nine hybrid chalcone derivatives (Fig. 1) were synthesized and tested for their vasorelaxant activities through evaluation in aortic rings. 2. Results and discussion 2.1. Chemistry 2.1.1. Synthesis of the hybrid chalcone derivatives conjugated with NO donors 7b, 8aec and 9a The synthetic routes of hybrid chalcone derivatives conjugated with NO donors 7b, 8aec and 9a are summarized in Scheme 1. 4Formyl-3-methyl-furoxan 4a was obtained using crotonaldehyde 3 as the starting material according to the literature [23]. Compound 4a was transformed to its isomer 4b by heating over 98 h in boiling toluene in moderate yield. Reduction of compounds 4a,b using NaBH4 in dioxane, following by treatment with thionyl chloride
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Fig. 1. Hybrid chalcone derivatives 7b, 8aec, 9a and 15aed.
afforded (chloromethyl)furoxans 5a,b, respectively. Nucleophilic substitution of chalcone 1a (or 1b) with (chloromethyl)furoxans 5a, b (or 1,2-dibromoethane) in the presence of potassium carbonate in acetone resulted in compounds 6aed. Treatment of 6a,b and AgNO3 in acetonitrile yielded nitrates 7a,b. Deprotection of the phenol function of 7a and 6c,d in methanol/3N aq. HCl (5:1, v/v) under reflux afforded compounds 8aec. Etherization of nitrate 8a using 4-
chloromethyl-3-methyl-furoxan in the presence of potassium carbonate provided compound 9a. 2.1.2. Synthesis of the hybrid chalcone derivatives conjugated with 1,4-DHP moiety The synthesis of the target compounds 15aed is outlined in Scheme 2. Treatment of m-nitrobenzaldehyde, methyl acetoacetate
Scheme 1. Synthesis of hybrid chalcone derivatives 7b, 8aec and 9a. Reagents and conditions: (a) acetic acid, NaNO2, ice bath; (b) toluene, reflux; (c) NaBH4, dioxane, r.t.; (d) SOCl2, 0 C to r.t.; (e) K2CO3, acetone, reflux; (f) AgNO3, acetonitrile, r.t.; (g) 3 N HCl, methanol, reflux; (h) K2CO3, acetone, 5a, reflux.
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Scheme 2. Synthesis of hybrid chalcone derivatives 15aed. Reagents and conditions: (a) NH4OAc, microwave, 120 C; (b) NaOH, Methanol/H2O (6:1, v/v), 70 C; (c) K2CO3, 2, reflux; (d) K2CO3, acetone, reflux; (e) 3 N HCl, methanol, reflux.
and ammonium acetate under microwave irradiation afforded 1,4dihydropyridine 10. Selective hydrolysis of 10 in the presence of sodium hydroxide gave key intermediate monoacid 11 [24,25], which was then reacted with dibromoethane to yield compound 12. Condensation of 12 with chalcones 13aed in the presence of potassium carbonate in acetone under reflux afforded the hybrid derivatives 14aed. Deprotection of the phenol function of 14aed was carried out by reaction with methanol/3N aq. HCl (5:1, v/v) under reflux to yield desired compounds 15aed in good yield. 2.2. Vasorelaxation activity and SAR Vasorelaxation activities of hybrid chalcone derivatives 7b, 8aec, 9a and 15aed were evaluated in aortic rings with endothelium pre-contracted with 1 mM phenylephrine (PE). Quercetin, a well known vasodilator, was used as the positive control. The results were summarized in Table 1. All tested compounds promoted relaxation in a dose-dependent manner, and their maximal effects were observed at 300 mM as shown in Fig. 2. SAR of Chalcone hybrid derivatives conjugated with NO donors: Obviously, the vasorelaxant activities of hybrid chalcone derivatives 7b, 8aec and 9a bearing NO donor were increased when compared with that of 2,4,6-trihydroxy-30 ,40 -methylenedioxyl-chalcone 16, a good vasodilator (EC50 ¼ 22.5 mM, Emax ¼ 104.1%) reported in our previous studies [15]. Judged by the activities of compounds 8aec, the 4-methyl-3-methylene-furoxan moiety showed the most favorable effect on enhancing vasorelaxant activities as exemplified
Table 1 Vasorelaxant activities of hybrid chalcone derivatives 7b, 8aec, 9a and 15aed. Compd.
EC50 (mM)
Emax (%)
7b 8a 8b 8c 9a 15a 15b 15c 15d 16a Quercetina
8.4 13.4 12.1 8.9 9.4 9.3 6.4 2.9 9.7 22.5 244.0
104.0 102.9 95.7 100.1 96.0 98.6 101.9 104.0 102.9 104.1 91.3
a
Data available in our previous studies [15].
3.6 5.9 6.2 4.2 6.7 6.9 7.7 12.3 9.1 17.4 13.2
in compounds 8c (EC50 ¼ 8.9 mM, Emax ¼ 100.1%). Besides, increasing the number of nitrates linked to chalcone led to the enhanced vasorelaxant activities. For example, both of compound 7b bearing three nitrates (EC50 ¼ 8.4 mM, Emax ¼ 104.0%) and compound 9a bearing nitrate and furoxane (EC50 ¼ 9.4 mM, Emax ¼ 96.0%) exhibited more potent activities than compound 8a (EC50 ¼ 13.4 mM, Emax ¼ 102.9%). SAR of Chalcone hybrid derivatives conjugated to 1,4-DHP moiety: Comparing the vasorelaxant activities of hybrid compounds 15aed and 16 indicated that the introduction of 1,4-DHP moiety significantly enhanced the biological activities. For instance, the vasorelaxant activity of compound 15c was increased by approximately 6.7-fold comparing with 16. Insight into the observed effects of different substituent on chalcones revealed that the introduction of a para-hydroxyl group to the B-ring of chalcone showed optimal effect (ie. 15c: EC50 ¼ 2.9 mM, Emax ¼ 104.0%), while introduction of hydroxyl groups at the C-5 position of A-ring of chalcone didn’t exhibit enhanced activity (ie.15a: EC50 ¼ 9.3 mM, Emax ¼ 98.6%; 15d: EC50 ¼ 9.7 mM, Emax ¼ 102.9%). 3. Conclusion A series of novel hybrid chalcones derivatives conjugated with NO donors or 1,4-DHP moiety were designed and synthesized based on the hybrid strategy. Biological activity evaluation showed most target compounds exhibit significantly improved vasorelaxant activities. The preliminary structureeactivity relationships studies revealed that 4-methyl-3-methylene-furoxan moiety was the preferable fragment when linked with chalcones, and that the para-hydroxyl group on B-ring of chalcone was the most optimal when conjugated with 1,4-DHP moieties. Consequently, compound 8c and 15c would be promising structural templates for the development of novel vasorelaxant agents. 4. Experiments 4.1. Chemistry Reagents and solvents were purchased from commercial sources. Melting-points were obtained on a B-540 Büchi melting-point apparatus and are uncorrected. Intermediates and target compounds were characterized based on 1H NMR (Brüker AM500)
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Fig. 2. Effects of hybrid chalcone derivatives on relaxation in aortic rings with endothelium pre-contracted with 1 mM phenylephrine (PE). (a) The tested compounds 7b, 8aec and 9a were added cumulatively to achieve the appropriate concentrations; (b) The tested compounds 15aed were added cumulatively to achieve the appropriate concentrations. Results were expressed as means standard error of mean in terms of percentage relaxation of the contraction to PE (n ¼ 3e4).
and 13C NMR (Brüker AM125). Mass spectra were recorded on an Esquire-LC-00075 spectrometer. High resolution mass spectral (HRMS) analyses were recorded on Brüker DALTONICS APEX III mass spectrometer using electron spray ionization. Chalcone 1a, b [15], furoxane 4a,b [23] and 1,4-DHPs 11 [24,25] were synthesized according to the literature. 4.2. General procedure for the synthesis of furoxane 5a,b NaBH4 (300 mg, 7.9 mmol) was added to a cold solution of 4a (or 4b) (500 mg, 3.84 mmol) in dioxane (5 mL). The reaction mixture was stirred for 12 h at room temperature. The residue obtained by concentration of the solution was treated with water and extracted with ethyl acetate (15 mL 3). The organic phase was evaporated in vacuo to give pale yellow oil, to which thionyl chloride (1.67 g, 14.1 mmol) was added dropwise in ice-water bath. The reaction mixture was kept under stirring at room temperature for 12 h. Upon finishing of the reaction, water (50 mL) was added, and the mixture was extracted with ethyl acetate (20 mL 3). Evaporating of the dried combined organic layers afforded the crude product as an oily residue which was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (10:1e6:1). 4.2.1. 3-Methyl-4-chloromethyl-furoxane 5a Pale yellow oil (45%); 1H NMR (CDCl3, 400 MHz, d): 2.22 (s, 3H, CH3); 4.39 (s, 2H, CH2). 4.2.2. 3-Chloromethyl-4-methyl-furoxane 5b Pale yellow oil (38%); 1H NMR (CDCl3, 400 MHz, d): 2.45 (s, 3H, CH3); 4.26 (s, 2H, CH2). 4.2.3. General procedure for the synthesis of compounds 6aed A solution of compounds 1, potassium carbonate and 1,2dibromoethane 2 (or 5a,b) in acetone (20 mL) was refluxed for 2e10 h. After cooled to room temperature, the mixture was filtered and the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (10:1e4:1) to give expected product. 4.2.4. 3-(Benzo[d][1,3]dioxol-5-yl)-1-(2-(2-bromoethoxy)-4,6-bis (methoxymethoxy)phenyl)prop-2-en-1-one (6a) Reagent: compound 1a (300 mg, 1.29 mmol), potassium carbonate (356 mg, 2.58 mmol), 1,2-dibromoethane (4.83 g, 25.7 mmol); yellow syrup (359 mg, 56%). 1H NMR (CDCl3, 500 MHz, d): 3.40 (s, 3H), 3.51 (s, 3H), 3.52 (t, 2H, J ¼ 6.5 Hz), 4.26 (t, 2H,
J ¼ 6.5 Hz), 5.19 (s, 2H), 5.22 (s, 2H), 6.00 (s, 2H), 6.34 (d, 1H, J ¼ 2.0 Hz), 6.55 (d, 1H, J ¼ 2.0 Hz), 6.79 (d, 1H, J ¼ 8.5 Hz), 6.83 (d, 1H, J ¼ 16.5 Hz), 7.00 (dd, 1H, J ¼ 1.5, 8.5 Hz), 7.07 (d, 1H, J ¼ 1.5 Hz), 7.28 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 495. 4.2.5. 3-(Benzo[d][1,3]dioxol-5-yl)-1-(2,4,6-tris(2-bromoethoxy) phenyl)prop-2-en-1-one (6b) Reagent: compound 1b (300 mg, 1.00 mmol), potassium carbonate (276 mg, 2.00 mmol), 1,2-dibromoethane (4.83 g, 25.7 mmol); yellow syrup (416 mg, 67%). 1H NMR (CDCl3, 500 MHz, d): 3.52 (t, 4H, J ¼ 6.5 Hz), 3.66 (t, 2H, J ¼ 6.5 Hz), 4.26 (t, 4H, J ¼ 6.5 Hz), 4.30 (t, 2H, J ¼ 6.5 Hz), 6.00 (s, 2H), 6.18 (s, 2H), 6.79 (d, 1H, J ¼ 7.5 Hz), 6.82 (d, 1H, J ¼ 16.5 Hz), 7.00 (dd, 1H, J ¼ 1.5, 7.5 Hz), 7.06 (d, 1H, J ¼ 1.5 Hz), 7.26 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 619. 4.2.6. 4-((2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-bis (methoxymethoxy)phenoxy)methyl)-3-methyl-1,2,5-oxadiazole 2oxide (6c) Reagent: compound 1a (200 mg, 0.52 mmol), potassium carbonate (142 mg, 1.04 mmol) and 4a (80.8 mg, 0.55 mmol); yellow syrup (206 mg, 80%). 1H NMR (CDCl3, 500 MHz, d): 2.31 (s, 3H), 3.37 (s, 3H), 3.51 (s, 3H), 5.00 (s, 2H), 5.10 (s, 2H), 5.20 (s, 2H), 6.01 (s, 2H), 6.43 (d,1H, J ¼ 2.0 Hz), 6.57 (d,1H, J ¼ 2.0 Hz), 6.75 (d,1H, J ¼ 16.5 Hz), 6.80 (d, 1H, J ¼ 8.0 Hz), 6.96 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.01 (d, 1H, J ¼ 1.5 Hz), 7.21 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 501. 4.2.7. 3-((2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-bis (methoxymethoxy)phenoxy)methyl)-4-methyl-1,2,5-oxadiazole 2oxide (6d) Reagent: compound 1a (200 mg, 0.52 mmol), potassium carbonate (142 mg, 1.04 mmol) and 4b (81 mg, 0.55 mmol); yellow syrup (196 mg, 76%). 1H NMR (CDCl3, 500 MHz, d): 2.12 (s, 3H), 3.38 (s, 3H), 3.51 (s, 3H), 5.10 (s, 2H), 5.11 (s, 2H), 5.20 (s, 2H), 6.01 (s, 2H), 6.48 (d,1H, J ¼ 2.0 Hz), 6.57 (d,1H, J ¼ 2.0 Hz), 6.76 (d,1H, J ¼ 16.5 Hz), 6.80 (d, 1H, J ¼ 8.0 Hz), 6.97 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.03 (d, 1H, J ¼ 1.5 Hz), 7.21 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 501. 4.3. General procedure for the synthesis of nitrate 7a,b To a stirred solution of 6a,b in acetonitrile (5 mL), silver nitrate was added. The reaction mixture was stirred in the dark for 50e70 h. The reaction mixture was then filtered, and the filtrate was evaporated under reduced pressure to afford a dark-yellow solid which was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (5:1e2:1) to give expected product.
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4.3.1. 2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-bis (methoxymethoxy)phenoxy)ethyl nitrate (7a) Reagent: compound 6a (300 mg, 0.61 mmol), silver nitrate (277 mg, 1.633 mmol); yellow syrup (225 mg, 78%). 1H NMR (CDCl3, 500 MHz, d): 3.40 (s, 3H), 3.51 (s, 3H), 4.23 (t, 2H, J ¼ 6.5 Hz), 4.71 (t, 2H, J ¼ 6.5 Hz), 5.20 (s, 2H), 5.23 (s, 2H), 6.00 (s, 2H), 6.34 (d, 1H, J ¼ 2.0 Hz), 6.55 (d, 1H, J ¼ 2.0 Hz), 6.80 (d, 1H, J ¼ 8.5 Hz), 6.83 (d, 1H, J ¼ 16.5 Hz), 7.01 (dd, 1H, J ¼ 1.5, 8.5 Hz), 7.06 (d, 1H, J ¼ 1.5 Hz), 7.29 (d, 1H, J ¼ 16.5 Hz). ESI-MS: m/z [M þ H]þ 478. 4.3.2. 2,20 ,200 -(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)benzene1,3,5-triyl)tris(oxy)tris(ethane-2,1-diyl) trinitrate (7b) Reagent: compound 6b (400 mg, 0.64 mmol), silver nitrate (277 mg, 1.633 mmol); yellow solid (237 mg, 78%), mp 101e103 C. 1 H NMR (CDCl3, 500 MHz, d): 4.27 (t, 4H, J ¼ 6.5 Hz), 4.33 (t, 2H, J ¼ 6.5 Hz), 4.75 (t, 4H, J ¼ 6.5 Hz), 4.88 (t, 2H, J ¼ 6.5 Hz), 6.05 (s, 2H), 6.23 (s, 2H), 6.82 (d, 1H, J ¼ 16.5 Hz), 6.85 (d, 1H, J ¼ 7.5 Hz), 7.04 (dd, 1H, J ¼ 1.5, 7.5 Hz), 7.11 (d, 1H, J ¼ 1.5 Hz), 7.27 (d, 1H, J ¼ 16.5 Hz). 13C NMR (Acetone-d6, 125 MHz, d): 191.2, 164.0, 162.6, 151.1, 146.3, 144.4, 132.3, 121.8, 119.6, 115.4, 108.2, 107.8, 102.1, 94.3, 66.6, 62.9, 58.2. HRMS(ESI): Found 568.0980 ([M þ H]þ), calcd for C22H21N3O15: 567.0973. 4.4. General procedure for the synthesis of compounds 8aec To a solution of 6c,d (or 7a) in methanol (2 mL), 3 N aq. HCl (0.4 mL) was added. The resulting mixture was refluxed for 45 min, then poured into cold water and extracted with ethyl acetate (5 mL 3 Times). The organic phase was washed with brine and then dried over anhydrous Na2SO4. After removal of the solvent, the residue was purified by chromatography on silica gel using gradient elution with petroleum ethereethyl acetate (8:1e3:1) to give expected product. 4.4.1. 2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5dihydroxyphenoxy)ethyl nitrate (8a) Reagent: compound 7a (100 mg, 0.21 mmol); pale yellow solid (65 mg, 80%), mp 193e194 C. 1H NMR (Acetone-d6, 500 MHz, d): 4.57 (t, 2H, J ¼ 6.5 Hz), 5.12 (t, 2H, J ¼ 6.5 Hz), 6.07 (d, 1H, J ¼ 2.0 Hz), 6.12 (s, 2H), 6.13 (d, 1H, J ¼ 2.0 Hz), 6.93 (d, 1H, J ¼ 8.0 Hz), 7.29 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.33 (d, 1H, J ¼ 1.5 Hz), 7.76 (d, 1H, J ¼ 16.5 Hz), 7.90 (d, 1H, J ¼ 16.5 Hz), 9.54 (s, 1H, OH), 14.17 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 193.4, 165.9, 162.3, 161.1, 150.0, 147.3, 144.4, 130.3, 125.8, 123.6, 109.2, 108.4, 107.6, 102.1, 96.6, 93.3, 70.1, 64.8. HRMS(ESI): Found 390.0755 ([M þ H]þ), calcd for C18H15NO9: 389.0747. 4.4.2. 4-((2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5dihydroxyphenoxy)methyl)-3-methyl-1,2,5-oxadiazole 2-oxide (8b) Reagent: compound 6c (150 mg, 0.3 mmol); pale yellow solid (68 mg, 55%), mp 220e222 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.13 (s, 3H), 5.44 (s, 2H), 6.00 (s, 2H), 6.01 (d, 1H, J ¼ 2.0 Hz), 6.26 (d, 1H, J ¼ 2.0 Hz), 6.88 (d, 1H, J ¼ 8.0 Hz), 7.08 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.09 (d, 1H, J ¼ 1.5 Hz), 7.63 (d, 1H, J ¼ 15.0 Hz), 7.78 (d, 1H, J ¼ 15.0 Hz), 9.52 (brs, 1H, OH), 14.13 (s, 1H, OH). ESI-MS: m/z [M þ H]þ 413. 13C NMR (Acetone-d6, 125 MHz, d): 192.8, 166.1, 163.2, 160.8, 155.4, 151.2, 147.2, 144.3, 130.8, 124.9, 124.1, 112.3, 109.2, 108.8, 107.2, 102.3, 96.5, 93.6, 60.5, 7.8. HRMS(ESI): Found 413.0920 ([M þ H]þ), calcd for C20H16N2O8: 412.0907. 4.4.3. 3-((2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5dihydroxyphenoxy)methyl)-4-methyl-1,2,5-oxadiazole 2-oxide (8c) Reagent: compound 6c (150 mg, 0.3 mmol); pale yellow solid (78 mg, 63%), mp 198e201 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.47 (s, 3H), 5.33 (s, 2H), 6.11 (d, 1H, J ¼ 2.0 Hz), 6.12 (s, 2H), 6.22 (d,
1H, J ¼ 2.0 Hz), 6.92 (d, 1H, J ¼ 8.0 Hz), 7.15 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.17 (d, 1H, J ¼ 1.5 Hz), 7.68 (d, 1H, J ¼ 15.0 Hz), 7.72 (d, 1H, J ¼ 15.0 Hz), 9.50 (brs, 1H, OH), 14.10 (s, 1H, OH). 13C NMR (Acetoned6, 125 MHz, d): 193.0, 165.9, 163.2, 161.2, 154.8, 150.7, 148.3, 144.4, 130.5, 124.8, 123.6, 112.9, 109.1, 108.9, 108.0, 102.1, 96.6, 93.6, 58.6, 8.2. HRMS(ESI): Found 413.0918 ([M þ H]þ), calcd for C20H16N2O8: 412.0907. 4.4.4. 4-((4-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3-hydroxy-5-(2(nitrooxy)ethoxy)phenoxy)methyl)-3-methyl-1,2,5-oxadiazole 2oxide (9a) A solution of compound 8a (50 mg, 0.13 mmol), potassium carbonate (35 mg, 0.25 mmol) and 5a in acetone (3 mL) was refluxed for 10 h. The mixture was filtered and the solution was concentrated in vacuo. The residue was purified by chromatography on silica gel with petroleum ethereethyl acetate (3:1) to give product 9a as pale yellow solid (36 mg, 56%), mp 95e97 C. 1H NMR (CDCl3, 400 MHz, d): 2.14 (s, 3H), 4.55 (t, 2H, J ¼ 6.5 Hz), 5.13 (t, 2H, J ¼ 6.5 Hz), 5.46 (s, 2H), 6.05 (d, 1H, J ¼ 2.0 Hz), 6.10 (s, 2H), 6.21 (d, 1H, J ¼ 2.0 Hz), 6.84 (d, 1H, J ¼ 8.0 Hz), 7.06 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.11 (d, 1H, J ¼ 1.5 Hz), 7.65 (d, 1H, J ¼ 15.6 Hz), 7.81 (d, 1H, J ¼ 15.6 Hz), 13.95 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 193.4, 165.6, 164.3, 161.8, 157.5, 150.1, 148.3, 143.8, 130.3, 124.9, 123.3, 113.5, 109.7, 109.2, 108.7, 102.3, 96.2, 93.5, 71.8, 64.9, 60.6, 8.2. HRMS(ESI): Found 502.1014 ([M þ H]þ), calcd for C22H19N3O11: 501.1020. 4.5. 3-(2-Bromoethyl)-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)1,4-dihydropyridine-3,5-dicarboxylate (12) A solution of compound 11 (1.2 g, 3.61 mmol), potassium carbonate (0.99 g, 7.22 mmol) and 1,2-dibromoethane 2 (13.5 g, 72.2 mmol) in acetone (20 mL) was refluxed for 5 h. The mixture was filtered and the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (4:1e2:1) to give title product as yellow syrup (1.05 g, 66%). 1H NMR (CDCl3, 500 MHz, d): 2.37 (s, 3H), 2.39 (s, 3H), 3.46 (m, 2H), 3.65 (s, 3H), 4.37 (m, 2H), 5.12 (s, 1H), 5.83 (s, 1H), 7.38 (t, 1H, J ¼ 8.0 Hz), 7.67 (dd, 1H, J ¼ 2.0, 8.0 Hz), 8.00 (dd, 1H, J ¼ 2.0, 8.0 Hz), 8.11 (d, 1H, J ¼ 8.0 Hz). ESI-MS: m/z [M þ H]þ 439. 4.6. General procedure for the synthesis of compounds 14a,d A solution of compound 12, potassium carbonate and appropriate chalcone 13a,d in acetone (5 mL) was refluxed for 4e12 h. The mixture was filtered and the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography using gradient elution with petroleum ethereethyl acetate (4:1e1:1) to give expected product. 4.6.1. 3-(2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-bis (methoxymethoxy)phenoxy)ethyl)-5-methyl-2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14a) Reagent: compound 12 (200 mg, 0.45 mmol), potassium carbonate (124 mg, 0.9 mmol) and 13a (152 mg, 0.45 mmol); pale yellow syrup (238 mg, 70%). 1H NMR (CDCl3, 500 MHz, d): 1H NMR (CDCl3, 400 MHz, d): 2.22 (s, 3H), 2.32 (s, 3H), 3.39 (s, 3H), 3.48 (m, 2H), 3.60 (s, 3H), 4.28 (m, 2H), 5.01 (s, 1H), 5.12 (s, 2H), 5.20 (s, 2H), 5.60 (s, 1H), 6.05 (s, 2H), 6.29 (d, 1H, J ¼ 2.0 Hz), 6.52 (d, 1H, J ¼ 2.0 Hz), 6.82 (d, 1H, J ¼ 8.5 Hz), 6.92 (d, 1H, J ¼ 16.0 Hz), 7.05 (dd, 1H, J ¼ 1.5, 8.5 Hz), 7.11 (d, 1H, J ¼ 1.5 Hz), 7.28 (t, 2H, J ¼ 8.0 Hz), 7.58 (d, 1H, J ¼ 16.0 Hz), 7.61 (d, 1H, J ¼ 8.0 Hz), 7.96 (dd, 1H, J ¼ 1.5, 8.0 Hz), 8.01 (d, 1H, J ¼ 1.5 Hz). ESI-MS: m/z [M þ H]þ 746.
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4.6.2. 3-(2-(2-(3-Bromophenyl)acryloyl)-(3,5-bis (methoxymethoxy)phenoxy)ethyl)-5-methyl-2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14b) Reagent: compound 12 (200 mg, 0.45 mmol), potassium carbonate (124 mg, 0.9 mmol)and 13b (193 mg, 0.45 mmol); pale yellow syrup (203 mg, 57%). 1H NMR (CDCl3, 400 MHz, d): 2.24 (s, 3H), 2.34 (s, 3H), 3.40 (s, 3H), 3.51 (m, 2H), 3.62 (s, 3H), 4.26 (m, 2H), 5.00 (s, 1H), 5.11 (s, 2H), 5.18 (s, 2H), 5.64 (s, 1H), 6.30 (d, 1H, J ¼ 2.0 Hz), 6.52 (d, 1H, J ¼ 2.0 Hz), 6.90 (d, 1H, J ¼ 16.0 Hz), 7.22 (t, 2H, J ¼ 7.5 Hz), 7.27 (t, 2H, J ¼ 8.0 Hz), 7.40 (d, 1H, J ¼ 7.5 Hz), 7.46 (d, 1H, J ¼ 7.5 Hz), 7.55 (d, 1H, J ¼ 16.0 Hz), 7.60 (d, 1H, J ¼ 8.0 Hz), 7.61 (s, 1H), 7.92 (dd, 1H, J ¼ 1.5, 8.0 Hz), 8.03 (d, 1H, J ¼ 1.5 Hz). ESI-MS: m/z [M þ H]þ 781. 4.6.3. 3-(2-(3,5-Bis(methoxymethoxy)-2-(3-(4-(methoxymethoxy) phenyl)acryloyl)phenoxy)ethyl) 5-methyl-2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14c) Reagent: compound 12 (200 mg, 0.45 mmol), potassium carbonate (124 mg, 0.9 mmol) and 13c (184 mg, 0.45 mmol); pale yellow syrup (219 mg, 63%). 1H NMR (CDCl3, 400 MHz, d): 2.19 (s, 3H), 2.33 (s, 3H), 3.39 (s, 3H), 3.46 (m, 2H), 3.47 (s, 3H), 3.51 (s, 3H), 3.62 (s, 3H), 4.28 (m, 2H), 5.04 (s, 1H), 5.11 (s, 2H), 5.18 (s, 2H), 5.20 (s, 2H), 5.63 (s, 1H), 6.30 (d, 1H, J ¼ 2.0 Hz), 6.53 (d, 1H, J ¼ 2.0 Hz), 6.81 (d, 1H, J ¼ 16.0 Hz), 7.00 (d, 2H, J ¼ 8.5 Hz), 7.30 (t, 1H, J ¼ 8.0 Hz), 7.42 (d, 2H, J ¼ 8.5 Hz), 7.61 (d, 1H, J ¼ 16.0 Hz), 7.94 (dd, 1H, J ¼ 2.0, 8.0 Hz), 8.04 (d, 1H, J ¼ 2.0 Hz). ESI-MS: m/z [M þ H]þ 763. 4.6.4. 3-(2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-5(methoxymethoxy)phenoxy)ethyl) 5-methyl 2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (14d) Reagent: compound 12 (200 mg, 0.45 mmol), potassium carbonate (124 mg, 0.9 mmol)and 13d (149 mg, 0.45 mmol); pale yellow syrup (219 mg, 70%). 1H NMR (CDCl3, 500 MHz, d): 2.21 (s, 3H), 2.34 (s, 3H), 3.44 (s, 3H), 3.44 (m, 2H), 3.63 (s, 3H), 4.23 (m, 2H), 5.01 (s, 1H), 5.12 (s, 2H), 5.62 (s, 1H), 6.02 (s, 2H), 6.65 (dd, 1H, J ¼ 2.0, 8.0 Hz), 6.73 (d, 1H, J ¼ 2.0 Hz), 7.02 (d, 1H, J ¼ 16.0 Hz), 6.85 (d, 1H, J ¼ 8.4 Hz), 7.14 (dd, 1H, J ¼ 2.0, 8.4 Hz), 7.20 (d, 1H, J ¼ 2.0 Hz), 7.35 (t, 2H, J ¼ 8.0 Hz), 7.55 (d, 1H, J ¼ 16.0 Hz), 7.80 (d, 1H, J ¼ 8.0 Hz), 7.97 (dd, 1H, J ¼ 2.0, 8.0 Hz), 8.03 (d, 1H, J ¼ 2.0 Hz). ESI-MS: m/z [M þ H]þ 687. 4.7. General procedure for the synthesis of hybrid compound 15aed To a solution of 14aed in methanol (2 mL), 3 N HCl aqueous (0.4 mL) was added. The resulting mixture was refluxed for 1.5 h, then poured into cold water and extracted with ethyl acetate (5 mL 3 Times). The organic phase was washed with brine and then dried over anhydrous Na2SO4. After removal of the solvent, the residue was purified by chromatography on silica gel using gradient elution with petroleum ethereethyl acetate (2:1e1:2) to give expected product. 4.7.1. 3-(2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5dihydroxyphenoxy)ethyl)-5-methyl 2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (15a) Reagent: compound 14a (100 mg, 0.13 mmol), pale yellow solid (57 mg, 65%); mp 162e165 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.32 (s, 3H), 2.33 (s, 3H), 3.54 (s, 3H), 4.24 (m, 1H), 4.46 (m, 1H), 4.49 (m, 1H), 4.62 (m, 1H), 5.02 (s, 1H), 5.64 (s, 1H), 6.08 (s, 2H), 6.12 (d, 1H, J ¼ 2.0 Hz), 6.13 (s, 1H, J ¼ 2.0 Hz), 6.95 (d, 1H, J ¼ 8.0 Hz), 7.30 (dd, 1H, J ¼ 1.5, 8.0 Hz), 7.33 (d, 1H, J ¼ 1.5 Hz), 7.56 (t, 2H, J ¼ 8.0 Hz), 7.74 (d, 1H, J ¼ 16.5 Hz), 7.87 (dd, 1H, J ¼ 2.0, 8.0 Hz), 7.88 (d, 1H, J ¼ 16.5 Hz), 8.00 (d, 1H, J ¼ 2.0 Hz), 8.10 (d, 1H, J ¼ 16.5 Hz), 9.54 (s, 1H, OH), 14.17 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d):
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192.3, 169.6, 169.5, 167.5, 163.93, 162.32, 151.2, 150.4, 149.2, 147.8, 145.6, 144.7, 138.5, 132.3, 130.3, 129.3, 127.9, 124.7, 124.6, 122.6, 109.2, 108.9, 107.2, 103.8, 102.1, 96.6, 94.2, 93.8, 67.9, 61.3, 52.0, 35.6, 16.3. HRMS(ESI): Found 659.1687 ([M þ H]þ), calcd for C34H30N2O12: 658.1799. 4.7.2. 3-(2-(2-(3-(3-Bromophenyl)acryloyl)-3,5dihydroxyphenoxy)ethyl)5-methyl,6-dimethyl -4-(3-nitrophenyl)1,4-dihydropyridine-3,5-dicarboxylate (15b) Reagent: compound 14b (100 mg, 0.13 mmol), pale yellow solid (62 mg, 70%); mp 148e150 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.31 (s, 6H), 3.52 (s, 3H), 4.25 (m, 1H), 4.45 (m, 1H), 4.49 (m, 1H), 4.63 (m, 1H), 5.00 (s, 1H), 5.65 (s, 1H), 6.10 (d, 1H, J ¼ 2.5 Hz), 6.11 (d, 1H, J ¼ 2.5 Hz), 7.26 (t, 1H, J ¼ 8.0 Hz), 7.29 (t, 1H, J ¼ 8.0 Hz), 7.52 (dd, 1H, J ¼ 1.0, 8.0 Hz), 7.56 (t, 2H, J ¼ 8.0 Hz), 7.63 (d, 1H, J ¼ 16.5 Hz), 7.77 (d, J ¼ 1.0 Hz), 7.87 (dd, 1H, J ¼ 1.0, 8.0 Hz), 8.00 (d, 1H, J ¼ 1.0 Hz), 8.13 (d, 1H, J ¼ 16.5 Hz), 9.52 (s, 1H, OH), 14.40 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 193.4, 169.8, 169.5, 165.6, 163.4, 162.1, 149.2, 145.6, 144.9, 144.7, 138.9, 138.1, 136.5, 132.3, 131.1, 130.7, 129.5, 128.1, 127.2, 126.1, 125.1122.2, 109.2, 106.8, 96.6, 93.8, 95.4, 94.2, 67.5, 61.5, 52.1, 34.9, 16.8. HRMS(ESI): Found 693.1017 ([M þ H]þ), calcd for C33H29BrN2O10: 692.1006. 4.7.3. 3-(2-(3,5-Dihydroxy-2-(3-(4-hydroxyphenyl)acryloyl) phenoxy)ethyl)-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)-1,4dihydropyridine-3,5-dicarboxylate (15c) Reagent: compound 14c (100 mg, 0.13 mmol), pale yellow solid (48 mg, 58%); mp 172e174 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.33 (s, 6H), 3.56 (s, 3H), 4.24 (m, 1H), 4.46 (m, 2H), 4.50 (m, 1H), 4.65 (m, 1H), 5.01 (s, 1H), 5.67 (s, 1H), 6.12 (d, 1H, J ¼ 2.0 Hz), 6.13 (d, 1H, J ¼ 2.0 Hz), 6.82 (d, 2H, J ¼ 8.5 Hz), 7.56 (t, 2H, J ¼ 8.5 Hz), 7.59 (d, 2H J ¼ 8.0 Hz), 7.63 (d, 1H, J ¼ 16.5 Hz), 7.87 (dd, 1H, J ¼ 2.0, 8.5 Hz), 8.00 (d, 1H, J ¼ 2.0 Hz), 8.07 (d, 1H, J ¼ 16.5 Hz), 9.05 (s, 1H, OH), 9.55 (s, 1H, OH), 14.33 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 194.2, 169.5, 169.3, 164.3, 163.4, 162.1, 160.1, 150.2, 148.1, 145.6, 144.7, 138.7, 136.6, 129.7, 129.5, 127.4, 126.8, 125.3, 124.6, 116.4, 108.9, 96.3, 94.0, 93.8, 93.5, 67.7, 62.3, 52.0, 35.8, 16.7. HRMS(ESI): Found 631.1859 ([M þ H]þ), calcd for C33H30N2O11: 630.1850. 4.7.4. 3-(2-(2-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-5hydroxyphenoxy)ethyl)-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)1,4-dihydropyridine-3,5-dicarboxylate (15d) Reagent: compound 14d (100 mg, 0.15 mmol), pale yellow solid (67 mg, 72%); mp 139e141 C. 1H NMR (Acetone-d6, 500 MHz, d): 2.29 (s, 3H), 2.30 (s, 3H), 3.49 (s, 3H), 4.27 (m, 1H), 4.42 (m, 2H), 4.50 (m, 1H), 5.03 (s, 1H), 5.61 (s, 1H), 6.04 (s, 2H), 6.57 (d, 1H, J ¼ 2.5 Hz), 6.59 (s, 1H), 6.78 (d, 1H, J ¼ 3.0 Hz), 7.06 (d, 1H, J ¼ 3.0 Hz), 7.07 (s, 1H), 7.25 (t, 1H, J ¼ 8.0 Hz), 7.50 (d, 1H, J ¼ 15.5 Hz), 7.58 (d, 1H, J ¼ 8.0 Hz), 7.65 (d, 1H, J ¼ 15.5 Hz), 7.77 (d, 1H, J ¼ 2.5 Hz), 7.86 (d, 1H, J ¼ 3.0 Hz), 8.03 (s, 1H), 9.09 (s, 1H, OH). 13C NMR (Acetone-d6, 125 MHz, d): 191.1, 169.6, 169.4, 162.2, 161.6, 150.8, 148.3, 147.7, 144.9, 144.6, 143.4, 139.3, 136.6, 132.2, 130.7, 129.5, 127.9, 124.5, 123.7, 121.9, 119.8, 109.4, 109.2, 108.8, 102.9, 102.4, 96.6, 94.2, 67.7, 62.3, 52.0, 35.8, 16.5. HRMS(ESI): Found 643.1860 ([M þ H]þ), calcd for C34H30N2O11: 642.1850. 4.8. Vasodilatory effect assay Vascular rings were prepared from the aorta of male Male SpragueeDawley rats (four to six months old and weighing on average 250 g), and contraction studies were performed following the general procedure detailed in the literature [26]. After an equilibration period of at least 1 h, isometric contractions induced by PE (1 mM) were obtained. When contraction of the tissue in
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response to this vasoconstrictor agent had stabilized (after about 20 min), cumulatively increasing concentrations of the tested compounds were added to the bath at 15e20 min intervals (the time needed to obtain steady-state relaxation). Control tissues were simultaneously subjected to the same procedures, but omitting the compounds and adding the vehicle. The flavonoidsinduced maximal relaxation (Emax) in aortic rings was calculated as a percentage of the contraction in response to PE (1 mM). The half maximum effective concentration (EC50) was defined as the concentration of the flavonoids that induced 50% of maximum relaxation from the contraction elicited by PE (1 mM) and was calculated from the concentration response curve by nonlinear regression (curve fit) using GraphPad Prism (Version 4.0). Acknowledgement The authors are grateful to support from the Administration of Traditional Chinese Medicine of Zhejiang Province (NO. 2009CB033), the National Key Tech Project for Major Creation of New drugs (NO. 2009ZX09501-003) and the supports from the School of Medicine, Zhejiang University for carrying out vasorelaxant effect assays. References [1] W. Ren, Z. Qiao, H. Wang, L. Zhu, L. Zhang, Med. Res. Rev. 23 (2003) 519e534. [2] G.D. Carlo, N. Mascolo, A.A. lzzo, F. Capasso, Life Sci. 65 (1999) 337e353. [3] E. Tripoli, M.L. Guardia, S. Giammanco, D.D. Majo, M. Giammanco, Food Chem. 104 (2007) 466e479. [4] M. Gross, Pharmaceut. Biol. 42 (2004) 21e35. [5] A. Koenig, C. Roegler, K. Lange, A. Daiber, E. Glusa, J. Lehmann, Bioorg. Med. Chem. Lett. 17 (2007) 5881e5885.
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