Accepted Manuscript Synthesis and bioactive evaluation of a novel series of coumarinazoles Guri L.V. Damu, Sheng-Feng Cui, Xin-Mei Peng, Qin-Mei Wen, Gui-Xin Cai, Cheng-He Zhou PII: DOI: Reference:

S0960-894X(14)00529-0 http://dx.doi.org/10.1016/j.bmcl.2014.05.029 BMCL 21643

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

24 January 2014 23 April 2014 10 May 2014

Please cite this article as: Damu, G.L.V., Cui, S-F., Peng, X-M., Wen, Q-M., Cai, G-X., Zhou, C-H., Synthesis and bioactive evaluation of a novel series of coumarinazoles, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.029

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 2

Graphical Abstract

Synthesis and bioactive evaluation of a novel series of coumarinazoles

Leave this area blank for abstract info.

Guri L V Damu§†, Sheng-Feng Cui§, Xin-Mei Peng§, Qin-Mei Wen, Gui-Xin Cai* and Cheng-He Zhou*

4

Bioorganic & Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

3 4

Synthesis and bioactive evaluation of a novel series of coumarinazoles

5Guri

L V Damu§†, Sheng-Feng Cui§, Xin-Mei Peng§, Qin-Mei Wen, Gui-Xin Cai* and Cheng-He Zhou ∗

6 7Institute of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China 8 9

A R T IC LE IN F O

A B S TR A C T

Article history: Received Revised Accepted Available online

A series of novel coumarinazoles were designed, synthesized, and characterized by IR, NMR, MS and HRMS spectra. The bioactive assay for the newly prepared compounds against six bacteria and five fungi manifested that most new compounds exhibited good or even stronger antibacterial and antifungal activities in comparison with reference drugs Chloromycin, Norfloxacin and Fluconazole. Bis-azole alcohols 7a and 7d−e showed better anti-C. utilis activity than mono-azole derivatives 4a and 4d−e at the tested concentrations, and they were more potent than the clinical Fluconazole. While triazole alcohol 7a gave comparable anti-C. albicans and anti-C. mycoderma activity to Fluconazole and better anti-MRSA activity than mono-triazole one 4a and clinical Norfloxacin. 1H-Benzoimidazol-2-ylthio c oumarin derivatives 4e and 7e gave the strongest anti-E .coli JM109 efficacy. Oxiran-2-ylmethoxy moiety was found to be a beneficial fragment to improve antibacterial and antifungal activity to some extent.

Keywords: Coumarin Azole Antibacterial Antifungal log p

10 Azole compounds have been attracting increasing interest due 1 11to their large potentiality in medicinal chemistry. Azole 12heterocycles are able to easily bind with the enzymes and 13receptors in organisms through weak interactions such as 14coordination bonds, hydrogen bonds, ion-dipole, cation-π, π-π 15stacking and hydrophobic effect as well as van der Waals force 16etc. A number of azole drugs such as Fluconazole, Itraconazole, 17Voriconazole, Posaconazole and so on have been prevalently 2 18used in the anti-infective therapy. Successful usage of these 19drugs in clinic inspires more research towards azole antimicrobial 20drugs. Although the extensively clinical use of triazole anti21infective agents has ever revolutionized the treatment of many 22infectious diseases, some of them are still limited by poor 23activities towards intractable fungi, high frequency of renal 24toxicity and several adverse effects as well as emergence of 25increasing resistant microbes, which are becoming one of the 26most paramount public health threats. These situations force 3 27researchers to develop structurally new antimicrobial agents. 28Much literature work has shown that the introduction of triazolyl 29ethanol moiety can play an important role in exerting 30antimicrobial action, which not only remarkably enhances the 31antimicrobial activities, but also broadens the antimicrobial 4 32spectrum.

——— §

2013 Elsevier Ltd. All rights reserved.

Important group to bind enzymes and receptors: Azole ring: bind with the prosthetic group iron(II) of cytochrome P450 Hydroxyl group: Form hydrogen bond with H310 in the active site

N

N

N N

N OH

N N F

F

N

N N OH

N

O

OH O

R O

N

Change Different azoles Regulate physicochemical properties and affinity

Target compounds Clinical Fluconazole O 33 34 Fig. 1. Design of coumarin-derived azole alcohols as analogs of Fluconazole

35 Coumarin derivatives exhibit extensive potentiality in 36 medicinal chemistry with the latent ability to exert noncovalent 37 interactions with various active sites in organisms. Related fields 38 have been increasingly attracting special interest due to their 39 potential outstanding contributions in the prevention and 40 treatment of diseases, such as antibacterial, antifungal, 41 anticancer, antiviral, anti-HIV, anti-psoriasis, anti-coagulant, 42 anti-tubercular, anti-malarial, anti-inflammatory, antioxidant 5 43 agents and so on. Numerous efforts have been focusing on the 44 research and development of coumarin derivatives as potential

These authors contributed equally to this work Postdoctoral fellow from Indian Institute of Chemical Technology (IICT), India * Corresponding author: Tel/Fax: +86 23 68254967; e-mail: [email protected] (Cheng-He Zhou), [email protected] (Gui-Xin Cai). †

5

45drugs. So far some coumarin derivatives, for example, Warfarin, 46Acenocoumarol, Armillarisin A, Hymecromone, Cyclocumarol, 47Carbochromen, Dicoumarol, and Ensaculin have been approved 48for therapeutic purposes in clinic. Currently there is a huge 49scientific and commercial interest in the discovery of potent and 50safe coumarin-based agents in the treatment of infective diseases 6 51for their special ability against resistant strains.

55 skeleton and changed different kinds of azole rings including 56 triazole, benzotriazole, benzimidazole and thiol-benzimidazole to 57 generate a novel class of coumarin-derived azole alcohols as 58 novel analogs of Fluconazole (Fig. 1). The newly synthesized 59 coumarinazoles might be expected to show potentiality against 60 bacterial and fungal strains, especially drug-resistant 61 microorganism. Therefore, their antibacterial and antifungal 62 activities were evaluated in vitro against six bacteria and five 63 fungi. The lipophilicity/hydrophilicity and structure-activity 64 relationship (SAR) were also discussed.

52 In continuation of our ongoing interest in the development of 7 53new antimicrobial agents, herein we incorporated triazolyl 54ethanol, an important fragment in Fluconazole, into coumarin

R1

CH3 ii

iii

R1 = H HO

OH

1 i

HO

O

O

2

OH

O

O

O

O 4a− −e

3

R1 = OH

OH

CH3 O

CH3

O

O

CH3

iv O

5

O

4,7 Im, a =

O

O N

N

Im OH

iii HO

Im

iv O

O

CH3

CH3

O

b=

N

N

O

O

O

O

N c=

O

Im

7a− −e

6 N

N

d=

N

N e=

OH N S N H

N

H3C

65

CH3

66Scheme 1. Reagents and conditions: (i) ethyl acetoacetate, oxalic acid, 80−100 °C, 8−10 h; (ii) ethyl acetoacetate, piperidine, -5−30 °C, 8−10 h; (iii) 267(chloromethyl)oxirane, K2CO3, reflux, 4−6 h; (iv) triazole, benzotriazole, benzimidazole or thiol-benzimidazole, K2CO3 , EtOH, 50−70 °C, 6−8 h. 68 69 The target coumarinazoles were prepared from commercially 99 for benzimidazole derivatives 4c and 7c. It seemed that the 70available phenols, and the synthetic route was outlined in 100 combination of benzimidazole and coumarin was not beneficial 71Scheme 1. The cyclization of resorcinol and phloroglucinol 101 to exert their antimicrobial behavior. The fungal C. utilis was 72respectively with ethyl acetoacetate in the presence of oxalic 102 more sensitive to mono-azole coumarins 4a and 4d−e and bis73acid to form the corresponding 7-hydroxy coumarin 2 with 103 azole derivatives 7a and 7d−e at the concentrations of 2–4 74excellent yield of 93% and 5,7-dihydroxy-coumarin 5 in 90% 104 µg/mL than the reference Fluconazole (MIC = 8 µg/mL), while 75yield. The latter was further O-alkylated by commercial 2- 105 coumarin triazole alcohol 7a gave comparable anti-C. albicans 76(chloromethyl)oxirane to yield racemic 5,7- and 7-(oxiran-2- 106 and anti- C. mycoderma activity to reference Fluconazole (MIC 77ylmethoxy)-2H-chromen-2-one derivatives 3 and 6 in high yields 107 = 1 and 4 µg/mL). It was noticeable that mono-substituted 78(80−91%), and subsequently the later were opened ring by 108 oxiran-2-ylmethoxy coumarin 3 (MIC = 1 µg/mL) and its double 79different azoles in ethanol using sodium bicarbonate as base to 109 derivative 6 (MIC = 16 and 8 µg/mL) exhibited much superior 80produce racemates 4a−e and 7a−e with excellent yields. All the 110 activities against A. flavus and B. yeast to reference drug 1 13 81new compounds were confirmed by H NMR, C NMR, IR, MS 111 Fluconazole (MIC = 256 and 16 µg/mL). Furthermore, 82and HRMS spectra. 112 intermediates 3 and 6 showed good antifungal activity against 113 other tested fungal strains. These might suggested this fragment 83 All the newly synthesized coumarinazoles were evaluated in 114 was beneficial to improve antifungal activity to some extent. 84vitro for their antimicrobial activities against two Gram-positive 85bacteria (Micrococcus luteus ATCC 4698 and Methicillin 115 The antibacterial assay showed that most prepared coumarin 86Resistant Staphylococcus aureus N315), four Gram-negative 116 azole alcohols exhibited moderate to weak activities against 87bacteria (Pseudomonas aeruginosa, Bacillus subtilis, Shigella 117 Gram-positive bacteria in comparison with clinical Norfloxacin 88dysenteriae and Escherichia coli JM109), and five fungi 118 and Chloromycin. However, bis-triazole coumarin 7a gave better 89(Candida utilis, Aspergillus flavus, Beer yeast, Candida 119 anti-MRSA activity (MIC = 8 µg/mL) than mono-triazole one 4a 90albicans, and Candida mycoderma) by two folds serial dilution 120 (MIC = 32 µg/mL) even better than Norfloxacin (MIC = 16 91technique recommended by National Committee for Clinical 121 µg/mL), which suggested that triazole alcohol fragment should 8 92Laboratory Standards (NCCLS) with the positive control of 122 be helpful for anti-MASR. The biological activities against 93clinically antimicrobial drugs Fluconazole, Norfloxacin and 123 Gram-negative bacteria showed that bis-1H-benzoimidazol-294Chloromycin. The antimicrobial tests were carried out for three 124 ylthio coumarin 7e displayed similar anti-E .coli JM109 ability 95times. 125 to mono-one 4e (MIC = 0.5 µg/mL), which was much better than 126 references Norfloxacin (MIC = 32 µg/mL) and Chloromycin 96 The obtained results as depicted in Table 1 revealed that most 127 (MIC = 16 µg/mL). It was noticed that intermediates 3 and 6 97of coumarin azole alcohols could effectively inhibit the growth 128 showed comparable or superior antibacterial activities against B. 98of the tested bacterial and fungal strains to some extent, except 129 Subtilis, S. dysenteriae and E .coli JM109 to standard

6

130 Norfloxacin and Chloromycin. These results confirmed 131 incorporation of azole alcohol moiety was helpful

that the 132 antimicrobial activities. Surprisingly, oxiran-2-ylmethoxy for the 133 fragment exerted great effect on the bioactivity to some extent.

134 Table 1. In vitro antimicrobial data as MIC (µg/mL) for compounds 3, 4a−e, 6, 7a−e a Fungi Gram-positive bacteria Gram-negative bacteria Compds C. A. B. C. C. P. B. S. E .coli M. luteus MRSA utilis flavus yeast albicans mycoderma aeruginosa Subtilis dysenteriae JM109 3 32 1 1 16 8 4 128 8 2 4 0.5 4a 4 6 32 4 16 16 32 4 32 32 64 4b 16 256 8 4 16 32 16 32 16 64 16 4c 512 >512 512 128 128 512 512 >512 128 512 128 4d 4 128 32 16 16 128 64 64 128 64 128 4e 0.5 16 64 32 16 16 32 64 32 64 0.5 6 16 16 8 8 8 8 64 8 4 4 0.5 7a 4 32 32 1 4 32 8 32 16 16 1 7b 16 256 32 8 16 16 32 32 64 64 32 7c 512 512 >512 >512 64 128 512 128 >512 >512 >512 7d 4 64 16 16 32 64 32 16 128 16 32 7e 2 64 16 8 16 32 64 16 64 32 0.5 Fluconazole 8 256 16 1 4 Chloromycin 8 16 32 32 32 32 Norfloxacin 2 8 1 16 4 16 135 a C. utilis, Candida utilis; A. flavus, Aspergillus flavus; B. yeast, Beer yeast; C. albicans, Candida albicans; C. mycoderma, Candida mycoderma; M. luteus, 136 Micrococcus luteus ATCC 4698; MRSA, Methicillin-Resistant Staphylococcus aureus N315; P. aeruginosa, Pseudomonas aeruginosa; B. Subtili, Bacillus 137 subtilies; S. dysenteriae, Shigella dysenteriae; E. Coli JM109, Escherichia coli 138 139 Lipophilicity/hydrophilicity governs various biological 173 derivatives 4e and 7e showed the strongest anti-E .coli JM109 140 processes such as the transportation, distribution, metabolism 174 efficacy, which were much better than Norfloxacin and 141 and secretion of biological molecules. A good knowledge of the 175 Chloromycin. It was found that oxiran-2-ylmethoxy moiety 142 lipophilicity/hydrophilicity is essential to predict the 176 seemed to be especially beneficial fragment to improve 143 transportation and activity of drugs. Therefore, the 177 antifungal activity against fungal strains A. flavus and B. yeast to 144 lipophilicity/hydrophilicity expressed as octanol/water partition 178 some extent. Other related work, including the thermodynamic 145 coefficient (log P) was calculated experimentally by traditional 179 properties, the antibacterial and antifungal action mechanism, the 146 saturation shake flask method combining with UV-vis 180 interactions between highly active compounds and HSA, the 147 spectrophotometric approach (Supplementary Information). The 181 effect factors on anti-microbial activities of the target 148 obtained log P values of compounds 4a−e and 7a−e given in 182 compounds such as different kinds of substituted coumarin 149 Table 2 indicated that compounds 4a, 4e and 7b with low log P 183 derivatives, other heterocyclic azoles (imidazole, tetrazole and 150 values showed good antibacterial activities in comparison with 184 their derivatives, etc.), as well as the toxicity investigation along 151 other target compounds with high log P values. These might be 185 with in vivo bioactive evaluation are active in progress in our 152 explained by the possibility that higher lipophilic compounds 186 group. All these will be reported in the future full paper. 153 were unfavorable for being delivered to the binding sites in 154 organism, and indicated the significant role of suitable 187 Acknowledgments 155 lipophilicity in drug design. 188 This work was partially supported by National Natural 156 Table 2. The values of log P for compounds 4a–e and 7a–e 189 Science Foundation of China [No. 21372186, 21172181, 190 81350110338, 81250110089, 81350110523 (The Research Compds 4a 4b 4c 4d 4e 191 Fellowship for International Young Scientists from the 192 International (Regional) Cooperation and Exchange Program)], Log P 0.45 0.60 0.55 0.61 0.49 193 the key program from Natural Science Foundation of Chongqing 194 (CSTC2012jjB10026), the Specialized Research Fund for the Compds 7a 7b 7c 7d 7e 195 Doctoral Program of Higher Education of China (SRFDP 196 20110182110007). Log P 0.65 0.48 0.49 0.61 0.58 157 In summary, a series of coumarinazoles as novel type of 158 antimicrobial agents were successfully prepared through an easy, 159 convenient and economic synthetic procedure. Their structures 160 were characterized by IR, NMR, MS and HRMS spectra. The in 161 vitro antibacterial and antifungal evaluation showed that most 162 synthesized coumarinazoles except for benzimidazole 163 derivatives could effectively inhibit the growth of all tested 164 bacteria and fungi. Bis-azole coumarin derivatives 7a and 7d−e 165 gave comparable anti-C. utilis activity to mono-azole derivatives 166 4a and 4d−e, which were more potent than the clinical 167 Fluconazole. Moreover, triazole alcohol coumarin 7a also 168 exhibited comparable anti-C. albicans and anti- C. mycoderma 169 activity to Fluconazole. In particular, bis-triazole coumarin 7a 170 gave better anti-MRSA activity than mono-triazole one 4a and 171 even better than clinical Norfloxacin. Among all the prepared 172 coumarin azole alcohols, 1H-benzoimidazol-2-ylthio coumarin

197 References and notes 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213

7

1.

2.

3.

4.

(a) Peng, X. M.; Cai, G. X.; Zhou, C. H. Curr. Top. Med. Chem. 2013, 13, 1963; (b) Zhang, L.; Peng, X. M.; Damu, G. L. V.; Geng, R. X.; Zhou, C. H. Med. Res. Rev. 2014, 34, 340; (c) Jin Z. Nat. Prod. Rep. 2011, 28, 1143. (a) Zhou, C. H.; Wang, Y. Curr. Med. Chem. 2012, 19, 239; (b) Bairagi, S. H.; Salaskar, P. P.; Loke, S. D.; Surve, N. N.; Tandel, D. V; Dusara, M. D. Int. J. Pharm. Res. 2012, 4, 16; (c) Damu, G. L. V.; Wen, Q. M.; Cui, S. F.; Peng, X. M.; Zhou, C. H. CN Patent 2012, CN103422813(A). (a) Anand, P.; Singh, B.; Singh, N.; Bioorg. Med. Chem. 2012, 20, 1175; (b) Kostova, I.; Bhatia, S.; Grigorov, P.; Curr. Med. Chem. 2011, 18, 3929; (c) Hopkins, A. L.; Bickerton, G. R.; Carruthers, I. M.; Boyer, S. K.; Rubin, H.; Overington, J. P. Curr. Top. Med. Chem. 2011, 11, 1292. (a) Pasqualotto, A. C.; Thiele, K. O.; Goldani, L. Z. Curr. Opin. Invest. Drugs, 2010, 11, 165; (b) Cui, S. F.; Ren, Y.; Zhang, S. L.;

214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292

5. 6.

7.

8.

Peng, X. M.; Damu, G. L. V.; Geng, R. X.; Zhou, C. H. Bioorg. Med. Chem. Lett. 2013, 23, 3267. Peng, X. M.; Damu, G. L. V.; Zhou, C. H. Curr. Pharm. Des. 2013, 19, 3884. (a) Grazul, M.; Budzisz, E. Coord. Chem. Rev. 2009, 253, 2588; (b) Ma, K.; Thomason, L. A. M.; McLaurin, J. Adv. Pharmacol. 2012, 64, 177; (c) Damu, G. L. V.; Wen, Q. M.; Cui, S. F.; Li, Q. X.; Peng, X. M.; Zhou, C. H. CN Patent 2012, CN103422800 (A). (a) Lv, J. S.; Peng, X. M.; Kishore, B.; Zhou, C. H. Bioorg. Med. Chem. Lett., 2014, 24, 308; (b) Yin, B. T.; Yan, C. Y.; Peng, X. M.; Zhang, S. L.; Rasheed, S.; Geng, R. X.; Zhou, C. H. Eur. J. Med. Chem. 2014, 71, 148; (c) Zhang, H. Z.; Damu, G. L. V.; Cai, G. X.; Zhou, C. H. Eur. J. Med. Chem. 2013, 64, 329; (d) Zhang, S. L.; Chang, J. J.; Damu, G. L.V.; Fang, B.; Zhou, X. D.; Geng, R. X.; Zhou, C. H. Bioorg. Med. Chem. Lett. 2013, 23, 1008; (e) Zhang, F. F.; Gan, L. L. Zhou, C. H. Bioorg. Med. Chem. Lett. 2010, 20, 1881. Experimental: melting points were recorded on X-6 melting point apparatus and uncorrected. TLC analysis was done using precoated silica gel plates. FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer (Bio-Rad, Cambridge, MA, USA) using KBr pellets in the 400–4000 cm-1 range. NMR spectra were recorded on a Bruker AV 300 spectrometer using TMS as an internal standard. The chemical shifts were reported in parts per million (ppm), the coupling constants (J) are expressed in hertz (Hz) and singlet (s), doublet (d) and triplet (t), broad (br) as well as multiplet (m). The mass spectra (MS) were recorded on LCMS–2010A and the high-resolution mass spectra (HRMS) were recorded on an IonSpec FT-ICR mass spectrometer with ESI resource. Synthesis of 4-methyl-7-(oxiran-2-ylmethoxy)-2H-chromen-2-one (3). A mixture of 7-hydroxy-4-methyl coumarin 2 (1.76 g, 10 mmol), potassium carbonate (2.07 g, 15 mmol) and epichlorohydrin (15 mL, 200 mmol) was refluxed for 3−4 h. After the reaction was completed (monitored by TLC, eluent, chloroform/methanol, 200−50/1, V/V), the mixture was cooled to room temperature. The excess 2-(chloromethyl) oxirane was evaporated under reduced pressure, and then water was added. The residue was extracted with chloroform (3 × 20 mL), and the combined organic phase was dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (eluent, chloroform/methanol, 200/1, V/V) to give the desired compound 3 (0.294 g) as white solid. Yield: 68.9%; mp: 124−125 ºC; IR (KBr) cm-1: 3407 (OH), 3129, 2964 (Ar-H), 2931, 2810 (CH2 ), 1722 (C=O), 1629, 1512 (aromatic frame), 1442, 1394, 1373, 1281, 1202, 1155, 1026, 984, 962, 830, 762, 730 cm–1 ; 1 H NMR (300 MHz, DMSO-d6) δ: 8.49 (s, 1H, triazole 3-H), 7.99 (s, 1H, triazole 5-H), 7.72−7.69 (d, 1H, J = 9 Hz, coumarin 5-H), 7.00−6.98 (m, 2H, coumarin 6,8-H), 6.23 (s, 1H, coumarin 3-H), 5.60−5.58 (d, 1H, J = 6 Hz, OH), 4.38−4.31 (m, 2H, phenylOCH2 ), 4.18 (bs, 1H, CHOH), 4.09−4.04 (m, 2H, triazole-CH2), 2.40 (s, 3H, coumarin 4-CH3) ppm; 13C NMR (75 MHz, DMSOd6) δ: 161.9 (coumarin 2-C), 160.5 (coumarin 7-C), 155.1 (coumarin 9-C), 153.8 (coumarin 4-C), 151.8 (triazole 3-C), 145.4 (triazole 5-C), 126.9 (coumarin 5-C), 113.7 (coumarin 3-C), 112.8 (coumarin 10-C), 111.7 (coumarin 6-C), 101.7 (coumarin 8-C), 70.7 (coumarin 7-OCH2), 67.6 (CHOH), 52.2 (triazole-CH2), 18.6 (coumarin-CH3) ppm; ESI-MS (m/z): 301.5 [M]+, 273.1 [M-CO]+; HRMS (TOF) calcd. for C15 H15N3O4 [M+H]+, 302.1096; found, 302.1143. Synthesis of 7-(3-(1H-benzo[d]imidazol-2-ylthio)-2-hydroxy propoxy)-4-methyl-2H-chromen-2-one (4e). To a stirred suspension of potassium carbonate (0.151 g, 1.2 mmol) in ethanol was added 1H-benzo[d]imidazole-2-thiol (0.150 g, 1.0 mmol). The mixture was stirred at 60 o C for 1 h. The reaction was cooled to room temperature, compound 3 (0.302 g, 1.0 mmol) was added at the room temperature and stirred for 10 h under reflux. After the reaction came to end, solvent was evaporated and the residue was extracted with chloroform (3 × 20 mL). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent, chloroform/methanol, 20−10/1, V/V) to afford white solid (0.168 g). Yield: 56%; mp:168−169 °C; IR (KBr) ν: 3419 (OH), 3210 (NH), 2970 (Ar-H), 2658 (CH2), 1698 (C=O), 1630, 1610 (aromatic frame), 1455, 1390, 1349, 1267, 1287, 1204, 1148, 1109, 1071, 980, 744 cm–1; 1 H NMR (300 MHz, DMSO-d6) δ: 12.58 (s, 2H, benzoimidazole-2-thiol N-H), 7.65−7.63 (m, 2 coumarin 8-H), 7.35−7.33 (m, 2H, coumarin 5,6-H), 7.09-7.06 (d,

293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330

8

9.

2H, benzoimidazole-2-thiol 4,8-H), 6.97-6.94 (d, 1H, benzoimidazole-2-thiol 5,6-H), 6.20 (s, 1H, coumarin olefin 3-H), 5.78 (s, 2H, OH), 4.19−4.17 (m, 2H, S-CH2), 4.13 (s, 2H, chiralH), 3.58−3.55 (m, 2H, -OCH2), 2.38 (s, 3H, coumarin 4-CH3) ppm; ESI-MS (m/z): [M+H]+, 383; HRMS (TOF) calcd. for C21H19 N4 O4 S[M+H]+, 383.1066; found, 383.1061. Synthesis of 5,7-bis(2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propoxy)4-methyl-2H-chromen-2-one (7a). To a stirred suspension of potassium carbonate (0.151 g, 1.2 mmol) in ethanol was added 1,2,4-triazole (1.382 g, 2.0 mmol). The mixture was stirred at 60 o C for 1 h. The reaction was cooled to room temperature, compound 6 (0.302 g, 1.0 mmol) was added at the room temperature and stirred for another 10 h under reflux. After the reaction came to end, solvent was evaporated and the residue was extracted with chloroform (3 × 20 mL). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent, chloroform/methanol, 60/5, V/V) to afford white solid 0.365 g. Yield: 82 %; 1H NMR (300 MHz, DMSO-d6) δ: 8.50, 8.49 (s, 2H, triazole 3-H), 8.00, 7.99 ( s, 2H, triazole 5-H), 6.62−6.61 (d, 1H, J = 3 Hz, coumarin 8-H), 6.51−6.50 (d, 1H, J = 3 Hz, coumarin 6H), 6.05 (s, 1H, coumarin 3-H), 5.61−5.57 (bs, 2H, OH), 4.37−4.29 (m, 4H, phenyl-OCH2), 4.27−4.25 (bs, 2H, CHOH), 4.08−4.01 (m, 4H, triazole-CH2 ), 2.59 (s, 3H, coumarin 4-CH3) ppm; 13 C NMR (75 MHz, DMSO-d6) δ: 162.3 (coumarin 2-C), 160.2 (coumarin 7-C), 158.6 (coumarin 5-C), 156.9 (coumarin 9C), 154.9 (coumarin 4-C), 152.0, 151.9 (triazole 3-C), 145.5, 145.4 (triazole 5-C), 111.4 (coumarin 3-C), 104.7 (coumarin 10C), 97.1 (coumarin 6-C), 94.9 (coumarin 8-C), 71.5, 70.8 (coumarin 7-OCH2), 67.8, 67.7 (CHOH), 52.6, 52.4 (triazoleCH2 ), 24.7 (coumarin-CH3) ppm; ESI-MS (m/z): 443.1 [M+H]+, 414.1 [M-CO]+ ; HRMS (TOF) calcd. for C20H22 N6 O6 [M+H]+, 443.1634; found, 443.1680. National Committee for Clinical Laboratory Standards Approved standard Document. M27-A2, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, National Committee for Clinical Laboratory Standards, Wayne, PA, 2002.