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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.51 no.2 Concepción June 2006

http://dx.doi.org/10.4067/S0717-97072006000200018 

 

J. Chil. Chem. Soc., 51, Nº 2 (2006) , pags: 927-932

 

SYNTHESIS, CHARACTERIZATION AND IN VITRO ANTIFUNGAL EVALUATION OF NOVEL BENZIMIDAZO[1,2-c]QUINAZOLINES

 

B. A. INSUASTY1, H. TORRES2, J. QUIROGA1, R. ABONÍA1, R. RODRÍGUEZ3, M. NOGERAS4, A. SÁNCHEZ4, C. SAITZ5, S. L. ALVAREZ6 , S. A. ZACCHINO7*

1Grupo de Investigación de Compuestos Heterocíclicos, Departamento de Química, Universidad del Valle, Cali, Colombia; 2Departamento de Ciencias Básicas, Universidad Nacional de Colombia Sede Palmira, Palmira, Colombia; 3Departamento de Química, Universidad Nacional de Colombia, Bogotá, Colombia, 4Departamento de Química Inorgánica y Orgánica, Universidad de Jaén, Jaén, España; 5Departamento de Química Orgánica y Fisico-Química, Universidad de Chile, Santiago de Chile, Chile, 6Morfología, 7Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.


SUMMARY

The synthesis of a series of new benzimidazo[1,2-c]quinazolines starting from 2-nitrobenzaldehyde and o-phenylendiamine is described. The structure elucidation of the products is based on detailed NMR analysis of experiments such as 1H-COSY, NOESY, DEPT, HSQC and HMBC. These compounds showed antifungal properties only against dermatophytes. Among them, those with electron-donor substituents on the 6-phenyl ring inhibited mainly T. rubrum and E. floccosum with MICs between 25-250 µg/mL and M. canis, M. gypseum and T. mentagrophytes with MICs between 50-250 µg/mL. Structures with electron-withdrawing substituents on the phenyl ring did not show any activities up to 250 µg/ml. Methyl substituents on the benzimidazole ring seem negatively affect the antifungal behaviour of this series.

KEYWORDS: benzimidazoles, quinazolines, benzimidazo[1,2-c]quinazolines, antifungal evaluation, dermatophytes


INTRODUCTION

Due to the increasing number of immunocompromised individuals, fungal infections have increased steadily in the last two decades, affecting millions of people worldwide. Opportunistic systemic mycoses are associated to high rates of deaths and skin fungal infections, although not life threatening, debilitate patients' quality of life, with the additional danger that they can spread to other areas of the body and to other individuals.

Although several drugs have been developed for the treatment of systemic an superficial mycoses, there are in fact a limited number of efficacious antifungal drugs [1,2]. Many of the currently available drugs have undesirable side-effects or are very toxic, produce recurrence, or lead to the development of resistance [3]. As a consequence, there is a real need for a next generation of antifungal agents [4].

Considering that compounds containing the quinazoline skeleton have showed varied interesting antifungal properties [5-7] and on the basis of our current project aimed at synthetic strategies for preparing heterocyclic compounds containing the quinazoline ring, we have previously synthesized and in vitro evaluated tetrahydropyrazolo[1,5-c]quinazolines for antifungal properties, showing that they do not possess any antifungal activity up to 250 µg/mL [8].

As a continuation of our previous studies, we report here the synthesis, antifungal properties and structure-activity relationships of a series of benzimidazo[1,5-c]quinazolines, not reported up to date in the literature, prepared by using 2-nitrobenzaldehyde and o-phenylendiamines as starting materials.

The use of 2-nitrobenzaldehyde for the design and synthesis of new quinazoline compounds is being subject of a growing interest [9,10].

In turn, substituted benzimidazoles have showed to be interesting structures from the point of view of their potential antimicrobial activity. So, interesting antifungal properties have been described for ciclopentyl and ciclohexyl-imidazoles [11], benzimidazolecarboxamides [12, 13] and sulfinyl benzimidazoles [14].

Particularly benzimidaquinazolines have showed virucide properties [15], making these compounds interesting candidates for being submitted to antifungal evaluation. It is known that one of the strategies for finding new antifungal agents is the Comparative Design. In this approach, compounds known to be active against any microorganism different from fungi, should be tested for antifungal properties since they or their analogues could become effective against known opportunistic infections [16].

EXPERIMENTAL PROCEDURES

GENERAL

All starting material were commercially available research-grade chemicals obtained from Aldrich (Steinheim, Germany), Merck (Darmstadt, Germany) and Sigma (St Louis, MO, USA). Melting points were determined in a Büchi Melting Point Apparatus (New Castle, Delaware, England) and are uncorrected. The 1H- and 13C nmr spectra were run on a Bruker DPX 300 spectrometer (Bruker Karslruhe, Germany) operating at 300 MHz and 75 MHz respectively, using dimethyl sulfoxide-d6 as solvent and tetramethylsilane as internal standard. The mass spectra were recorded on a Fisons-Platform interface APCI in MeOH and recorded on a Hewlett Packard HP Engine-5989 spectrometer (Palo Alto, California, USA) (equipped with a direct inlet probe) a operating at 70 eV. The elemental analysis have been obtained using a LECO CHNS-900 equipment (St. Joseph, Michigan, USA). The compound 3 was prepared according to reported methods.12

General procedure for the synthesis of 6-aryl-5,6-dihydrobenzimidazo[1,2-c]quinazolines (4a-l)

A solution of equimolar amounts (16 mmol) of the corresponding diamine 3 and benzaldehyde in ethanol (10 mL) was treated with acetic acid (1 mL). The mixture was stirred at room temperature for 10 minutes and then allowed in the refrigerator overnight. The resulting solid was collected and recrystallized from ethanol.

6-Phenyl-5,6-dihydrobenzimidazo[1,2- c]quinazoline (4a).

This compound was obtained according to general procedure as white crystals, mp 220 ºC, yield 70%; 1H NMR: d = 6.84 (t, 1H, H-2); 6.88 (d, 1H, H-4, 3J=7.7 Hz); 7.09 (d, 1H, H-6, 3J=1.3 Hz); 7.10 (t, 1H, H-9); 7.15 (d, 1H, H-8, 3J=6.7 Hz); 7.18 (t, 1H, H-10); 7.26 (t, 1H, H-3); 7.32 (m, 5H, H-o, m, p); 7.62 (d, 1H, NH, 3J=1.3 Hz); 7.67 (d, 1H, H-11, 3J=7.9 Hz); 7.98 (dd, 1H, H-1, 3J=7.7 Hz, 4J=1.3 Hz). 13C NMR: d = 68.2 (C-6); 110.9 (C-8); 112.3 (C-12b); 115.2 (C-4); 118.6 (C-2); 119.0 (C-11); 122.4 (C-9); 122.6 (C-10); 125.0 (C-1); 126.4 (2C, C-m); 129.1 (2C, C- o); 129.3 (C-p); 132.0 (C-3); 133.2 (C-7a); 140.8 (C-i); 143.5 (C-4a); 144.2 (C-11a); 147.3 (C-12a). MS (FAB): m/z (%) = 299 (M++2)

Anal. Calcd. for C20H15N3: C, 80.78; H, 5.08; N, 14.13. Found: C, 80.73; H, 5.12; N, 14.06.

6-(4-Chlorophenyl)-5,6-dihydrobenzimidazo[1,2 -c]quinazoline (4b).

This compound was obtained according to general procedure as white crystals, mp 207 ºC, yield 82%; 1H NMR: d = 6.85 (t, 1H, H-2); 6.86 (d, 1H, H-4, 3J=7.4 Hz); 7.13 (s, 1H, H-6); 7.14 (t, 1H, H-9); 7.20 (t, 1H, H-10); 7.23 (d, 1H, H-8, 3J=7.9 Hz); 7.26 (t, 1H, H-3); 7.44 (d, 2H, H-o, 3J=8.5 Hz); 7.40 (d, 2H, H-m, 3J=8.5 Hz); 7.65 (s, 1H, NH); 7.67 (d, 1H, H-11, 3J=7.8 Hz); 7.96 (d, 1H, H-1, 3J=7.5 Hz). 13C NMR: d = 67.3 (C-6); 110.8 (C-8); 112.3 (C-12b); 115.3 (C-4); 118.8 (C-2); 119.1 (C-11); 122.6 (C-9); 122.7 (C-10); 125.1 (C-1); 128.2 (2C, C-o); 129.2 (2C, C- m); 132.1 (C-3); 133.1 (C-7a); 133.9 (C-i); 139.8 (C-p); 143.2 (C-4a); 144.2 (C-11a); 147.1 (C-12a). MS (FAB): m/z (%) = 333 (M++2, 100).

Anal. Calcd. for C20H14ClN3: C, 72.40; H, 4.25; N, 12.66. Found: C, 72.38; H, 4.19; N, 12.61.

6-(4-Bromophenyl)-5,6-dihydrobenzimidazo[1,2 -c]quinazoline (4c).

This compound was obtained according to general procedure as white crystals, mp 209 ºC, yield 87%; 1H NMR: d = 6.85 (t, 1H, H-2); 6.86 (d, 1H, H-4, 3J=7.8 Hz); 7.14 (s, 1H, H-6); 7.16 (m, 2H, H-9, H-10); 7.19 (d, 2H, H-o, 3J=8.4 Hz); 7.28 (d, 1H, H-8, 3J=7.3 Hz); 7.29 (t, 1H, H-3); 7.50 (d, 2H, H-m, 3J=8.4 Hz); 7.68 (d, 1H, H-11, 3J=7.9 Hz); 7.69 (s, 1H, NH); 7.95 (d, 1H, H-1, 3J=7.3 Hz). 13C NMR: d = 67.3 (C-6); 110.8 (C-8); 112.2 (C-12b); 115.3 (C-4); 118.7 (C-2); 119.1 (C-11); 122.5 (C-i); 122.6 (C-9); 122.7 (C-10); 125.1 (C-1); 128.4 (2C, C-o); 132.1 (3C, C- m, C-3); 133.1 (C-7a); 140.2 (C-p); 143.2 (C-4a); 144.2 (C-11a); 147.1 (C-12a). MS: m/z (%) = 377 (M+ +1, 100), 379 (74).

Anal. Calcd. for C20H14BrN3: C, 63.84; H, 3.75; N, 11.17. Found: C, 63.77; H, 3.72; N, 11.22.

6-(4-Fluorophenyl)-5,6-dihydrobenzimidazo[1,2 -c]quinazoline 4n.

This compound was obtained according to general procedure as yellow crystals, mp 204 ºC, yield 85%; 1H NMR: d = 6.82 (s, 1H, NH); 6.86 (t, 1H, H-2); 7.10-7.40 (m, 9H, H-arom.); 7.62 (s, 1H, H-6); 7.67 (d, 1H, H-11, 3J=9.0 Hz); 7.97 (d, 1H, H-1, 3J=9.0 Hz). 13C NMR: d = 67.0 (C-6); 110.4 (C-8); 111.8 (C-12b); 114.8 (C-4); 118.2 (C-2); 118.7 (C-11); 122.0 (C-9); 122.2 (C-10); 124.6 (C-1); 128.0 (2C, C-o); 131.6 (2C, C- m); 132.2 (C-3); 132.7 (C-7a); 136.6 (C-i); 142.9 (C-4a); 143.8 (C-11a); 146.7 (C-12a); 163.8 (C-p). MS (EI): m/z (%) = 315 (M+, 60); 220 (27); 114 (100).

Anal. Calcd. for C20H14FN3: C, 76.18; H, 4.47; N, 13.33. Found: C, 76.31; H, 4.41; N, 13.21.

6-(4-Nitrophenyl)-5,6-dihydrobenzimidazo[1,2- c]quinazoline (4e).

This compound was obtained according to general procedure as white crystals, mp 252 ºC, yield 91%; 1H NMR: d = 6.86 (t, 1H, H-2); 6.87 (d, 1H, H-4, 3J=7.5 Hz); 7.16 (t, 1H, H-9); 7.22 (t, 1H, H-10); 7.27 (t, 1H, H-3); 7.34 (s, 1H, H-6); 7.35 (d, 1H, H-8, 3J=7.6 Hz); 7.44 (d, 2H, H-o, 3J=8.4 Hz); 7.69 (d, 1H, H-11, 3J=7.8 Hz); 7.81 (s, 1H, NH); 7.98 (d, 1H, H-1, 3J=7.8 Hz); 8.19 (d, 2H, H-m, 3J=7.8 Hz). 13C NMR: d = 66.3 (C-6); 110.2 (C-8); 111.8 (C-12b); 114.9 (C-4); 118.5 (C-2); 118.8 (C-11); 122.4 (C-9); 122.4 (C-10); 124.1 (2C, C-m); 124.7 (C-1); 126.9 (2C, C- o); 131.8 (C-3); 132.6 (C-7a); 142.3 (C-4a); 143.7 (C-11a); 146.5 (C-12a); 147.2 (C-i); 147.6 (C-p). MS (EI): m/z (%) = 342 (M+, 50), 220(50), 194(100).

Anal. Calcd. for C20H14N4O2 : C, 70.17; H, 4.12; N, 16.37. Found: C, 70.21; H, 4.20; N, 16.31.

6-(4-Methoxyphenyl)-5,6-dihydrobenzimidazo[1,2 -c]quinazoline (4f).

This compound was obtained according to general procedure as white crystals, mp 187 ºC, yield 65%; 1H NMR: d = 3.7 (s, 3H, OCH3); 6.83 (t, 1H, H-2); 6.87 (d, 1H, H-4, 3J=8.3 Hz); 6.90 (d, 2H, H-m, 3J=8.7 Hz). 6.98 (d, 1H, H-6, 3J=1.7 Hz ); 7.06 (t, 1H, H-9); 7.07 (d, 1H, H-8, 3J=7.6 Hz); 7.15 (m, 1H, H-10); 7.25 (d, 2H, H-o, 3J=8.7 Hz); 7.28 (t, 1H, H-3); 7.50 (d, 1H, NH, 3J=1.7 Hz); 7.65 (d, 1H, H-11, 3J=7.9 Hz); 7.95 (d, 1H, H-1, 3J=7.7 Hz, 4J=1.2 Hz). 13C NMR: d = 55.5 (OCH3); 68.1 (C-6); 111.0 (C-8); 112.3 (C-12b); 114.5 (2C, C- m); 115.2 (C-4); 118.5 (C-2); 119.0 (C-11); 122.3 (C-9); 122.5 (C-10); 125.0 (C-1); 127.9 (2C, C-o); 132.0 (C-3); 132.4 (C-i); 133.2 (C-7a); 143.7 (C-4a); 144.3 (C-11a); 147.4 (C-12a); 160.1 (C-p). MS (FAB): m/z (%) = 329 (M++2).

Anal. Calcd. for C21H17N3O: C, 77.04; H, 5.23; N, 12.84. Found: C, 77.11; H, 5.18; N, 12.88.

6-(3,4,5-Trimethoxyphenyl)-5,6-dihydrobenzimidazo[1,2 -c]quinazoline (4g).

This compound was obtained according to general procedure as white crystals, mp 217 ºC, yield 65%; IH NMR: d = 3.61 (s, 6H, OCH3); 3.62 (s, 3H, OCH3); 6.70 (s, 2H, H-o); 6.85 (t, 1H, H-2); 6.90 (d, 1H, H-4, 3J=7.8 Hz); 6.93 (d, 1H, H-6, 3J=1.4 Hz); 7.10 (m, 2H, H-9, H-8); 7.17 (m, 1H, H-10); 7.27 (t, 1H, H-3); 7.53 (d, 1H, NH, 3J=1.4 Hz); 7.65 (d, 1H, H-11, 3J=7.8 Hz); 7.97 (dd, 1H, H-1, 3J=7.7 Hz, 4J=1.4 Hz). 13C NMR: d = 55.76 (2C, OCH3); 59.93 (OCH3); 68.28 (C-6); 103.76 (2C, C-o); 111.94 (C-12b); 114.73 (C-4); 118.24 (C-2); 118.59 (C-11); 121.98 (C-10); 122,07 (2C, C-8, C-9); 124.52 (C-1); 131.52 (C-3); 132.86 (C-11a); 135.53 (C-i); 137.95 (C-p); 143.34 (C-4a); 143.77 (C-7a); 146.99 (C-12a); 152.93 (2C, C- m). MS (EI): m/z (%) = 387 (M+, 34), 220 (25), 194 (100).

Anal. Calcd. for C23H21N3O3 : C, 71.30; H, 5.46; N, 10.85. Found: C, 71.37; H, 5.38; N, 10.80.

9,10-Dimethyl-6-phenyl-5,6-dihydrobenzimidazo[1,2 -c]quinazoline (4h).

This compound was obtained according to general procedure as white crystals, mp 230 ºC (desc.), yield 63%; 1H NMR: d = 2.21 (s, 3H, CH3); 2.28 (s, 3H, CH3); 6.81 (t, 1H, H-2); 6.83 (d, 1H, H-4, 3J=8.0 Hz); 6.97 (s, 1H, H-8); 7.00 (d, 2H, H-6, 3J=1.7 Hz); 7.21 (m, 3H, 2H-m, H-p); 7.31 (m, 3H, 2H-o, H-3); 7.43 (s, 1H, H-11); 7.55 (d, 1H, NH, 3J=1.7 Hz); 7.9 (d, 1H, H-1, 3J=6.8 Hz). 13C NMR: d = 20.3 (CH3); 20.5 (CH3); 67.4 (C-6); 110.9 (C-8); 112.7 (C-12b); 115.1 (C-4); 118.5 (C-2); 119.2 (C-11); 124.7 (C-1); 126.2 (2C, C- m); 129.1 (3C, 2C-o, C-p); 130.9 (C-9); 131.1 (C-10, C-p); 131.6 (C-3); 131.8 (C-7a); 141.1 (C-i); 142.8 (C-4a); 143.1 (C-11a); 146.4 (C-12a). MS (FAB): m/z (%) = 327 (M+ +2).

Anal. Calcd. for C22H19N3: C, 81.20; H, 5.89; N, 12.91. Found: C, 81.23; H, 5.81; N, 12.86.

9,10-Dimethyl-6-(4-chlorophenyl)-5,6 -dihydrobenzimidazo[1,2-c]quinazoline (4i).

This compound was obtained according to general procedure as white crystals, mp 277 ºC, yield 78%; 1H NMR: d = 2.23 (s, 3H, CH3); 2.29 (s, 3H, CH3); 6.83 (t, 2H, H-2 y H-4); 7.07 (s, 2H, H-6, H-8); 7.20 (d, 3H, H-o y H-3, 3J=7.9 Hz); 7.39 (d, 2H, H-m, 3J=7.9 Hz); 7.45 (s, 1H, H-11); 7.60 (s, 1H, NH); 7.93 (d, 1H, H-1, 3J=7.3 Hz). 13C NMRM: d = 20.3 (CH3); 20.5 (CH3); 67.0 (C-6); 110.8 (C-8); 112.7 (C-12b); 115.3 (C-4); 118.7 (C-2); 119.3 (C-11); 124.8 (C-1); 127.9 (2C, C-o); 129.1 (2C, C- m);, 131.1 (C-9); 131.2 (C-10); 131.7 (2C, C-3, C-7a); 133.7 (C-p); 140.1 (C-i); 142.8 (C-4a); 142.8 (C-11a); 146.3 (C-12a). MS: m/z (%) = 361 (M++2, 100), 363 (34)

Anal. Calcd. for C22H18N3Cl: C, 73.43; H, 5.04; N, 11.68. Found: C, 73.51; H, 5.11; N, 11.61.

9,10-Dimethyl-6-(4-bromophenyl)-5,6 -dihydrobenzimidazo[1,2-c]quinazoline (4j).

This compound was obtained according to general procedure as white crystals, mp 278 ºC, yield 79%; 1H NMR: d = 2.23 (s, 3H, CH3); 2.28 (s, 3H, CH3); 6.81 (t, 1H, H-2); 6.82 (d, 1H, H-4, 3J=7.6 Hz); 7.04 (d, 1H, H-6, 3J=2.0 Hz); 7.06 (s, 1H, H-8); 7.11 (d, 2H, H-o, 3J=8.5 Hz); 7.21 (t, 1H, H-3); 7.44 (s, 1H, H-11); 7.51 (d, 2H, H-m, 3J=8.5 Hz); 7.59 (d, 1H, NH, 3J=2.0 Hz); 7.91 (d, 1H, H-1, 3J=7.2 Hz). 13C NMR: d = 20.3 (CH3); 20.5 (CH3); 67.0 (C-6); 110.8 (C-8); 112.6 (C-12b); 115.2 (C-4); 118.7 (C-2); 119.3 (C-11); 122.3 (C-i); 124.8 (C-1); 128.2 (2C, C-o); 131.1 (C-9); 131.3 (C-10); 131.6 (C-7a); 131.7 (C-3); 132.1 (2C, C- m);, 140.5 (C-p); 142.7 (C-4a); 142.8 (C-11a); 146.2 (C-12a). MS: m/z (%) = 405 (M+ +1, 100), 407 (68)

Anal. Calcd. for C22H18N3Br: C, 65.36; H, 4.49; N, 10.39. Found: C, 65.42; H, 4.43; N, 10.47.

9,10-Dimethyl-6-(4-nitrophenyl)-5,6 -dihydrobenzimidazo[1,2-c]quinazoline (4k).

This compound was obtained according to general procedure as white crystals, mp 316 ºC, yield 88%; 1H NMR: d = 2.24 (s, 3H, CH3); 2.29 (s, 3H, CH3); 6.83 (t, 1H, H-2); 6.84 (d, 1H, H-4, 3J=8.1 Hz); 7.18 (s, 1H, H-8); 7.23 (t, 1H, H-3); 7.27 (d, 1H, H-6, 3J=2.2 Hz); 7.37 (d, 2H, H-o, 3J=8.7 Hz); 7.47 (s, 1H, H-11); 7.78 (d, 1H, NH, 3J=2.2 Hz); 7.95 (d, 1H, H-1, 3J=8.1 Hz); 8.18 (d, 2H, H-m, 3J=8.7 Hz). 13C NMR: d = 20.3 (CH3); 20.5 (CH3); 66.5 (C-6); 110.7 (C-8); 112.7 (C-12b); 115.4 (C-4); 118.9 (C-2); 119.4 (C-11); 124.5 (2C, C-m); 124.8 (C-1); 127.2 (2C, C- o);, 131.3 (C-7a); 131.5 (C-9); 131.6 (C-10); 131.8 (C-3); 142.3 (C-4a); 142.8 (C-11a ); 146.1 (C-12a); 147.9 (2C, C-i, C-p). MS (FAB): m/z (%) = 372 (M++2).

Anal. Calcd. for C22H18N4O2 : C, 71.34; H, 4.90; N, 15.13. Found: C, 71.39; H, 4.84; N, 15.17.

9,10-Dimethyl-6-(4-methoxyphenyl)-5,6 -dihydrobenzimidazo[1,2-c]quinazoline (4l).

This compound was obtained according to general procedure as white crystals, mp 229 ºC, yield 55%; 1H NMR: d = 2.21 (s, 3H, CH3); 2.27 (s, 3H, CH3); 3.68 (s, 3H, OCH3); 6.82 (t, 2H, H-2); 6.84 (d, 1H, H-4, 3J=7.5 Hz); 6.87 (d, 2H, H-m, 3J=8.7 Hz). 6.95 (s, 2H, H-6, H-8); 7.15 (d, 2H, H-o, 3J=8.7 Hz); 7.21 (t, 1H, H-3); 7.43 (s, 1H, H-11); 7.51 (s, 1H, NH); 7.91 (d, 1H, H-1, 3J=7.7 Hz). 13C NMR: d = 20.3 (CH3); 20.6 (CH3); 55.5 (OCH3); 67.6 (C-6); 111.0 (C-8); 112.6 (C-12b); 114.4 (2C, C- m); 115.1 (C-4); 118.4 (C-2); 119.1 (C-11); 124.7 (C-1); 127.5 (2C, C-o); 130.8 (C-9); 131.0 (C-10); 131.6 (C-3); 131.7 (C-7a); 133.2 (C-i); 142.8 (C-11a); 143.3 (C-4a); 146.5 (C-12a); 159.9 (C-p). MS (FAB): m/z (%) = 357 (M+ +2)

Anal. Calcd. for C23H21N3: C, 77.72; H, 5.96; N, 11.82. Found: C, 77.67; H, 5.89; N, 11.77.

BIOLOGICAL EVALUATION

Microorganisms and media

The microorganisms used for the fungistatic evaluation were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) or were clinical isolates kindly provided by Centro de Referencia Micológica (C, CEREMIC), Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531-(2000)-Rosario, Argentina. Candida albicans ATCC 10231, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 32264, Aspergillus flavus ATCC 9170, Aspergillus fumigatus ATCC 26934, Aspergillus niger ATCC 9029, Trichophyton mentagrophytes ATCC 9972. Microsporum canis C 112, Trichophyton rubrum C 113, Epidermophyton floccosum C 114, Microsporum gypseum C 115, and Candida tropicalis C131. The strains were maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and subcultured every 15 days to prevent pleomorphic transformations. Spore suspensions were obtained according to reported procedures [17] and adjusted to 106 spores/cells with colony forming ability/mL.

Antifungal assays

The antifungal activity of benzimidazo[1,2-c]quinazolines was evaluated with the agar dilution method by using Sabouraud-chloramphenicol agar for both yeast and dermatophyte species as previously described [18,19]. Stock solutions of compounds (10 mg/mL in DMSO) were diluted to give serial two-fold dilutions that were added to each medium resulting in concentrations ranging from 0.10 to 250 µg/mL. The minimum inhibitory concentration (MIC) for each compound was defined as the lowest concentration that produces no visible fungal growth after the incubation time. Amphotericin B (Sigma Chem. Co.), Ketoconazole (Janssen Pharmaceutica) and Terbinafine (Novartis) were used as positive controls

RESULTS AND DISCUSSION

CHEMISTRY

The synthesis of benzimidazo[1,2-c]quinazolines 4 was performed from 2-(2-aminophenyl)benzimidazole 3, following Scheme 1. The starting compound was readily prepared in high yield by reduction of 2-(2-nitrophenyl)benzimidazole 2. The benzimidazolo[1,2-c]quinazolines were obtained by reaction with aldehydes in ethanol/acetic acid mixture.

i) 2-O2NC6H4CHO, EtOH/HCl; ii) Ni-Raney/MeOH, N2H4.H2O;
iii) R'CHO/EtOH,AcOH
 
Scheme 1. Synthesis of benzimidazo[1,2-c]quinazolines using o-phenylendiamines as starting materials

The formation of 4 as the unique product of reaction was confirmed by spectroscopic analyses. The IR spectra of compounds 4 measured in KBr pellets showed a band of the elongation vibrations corresponding to the C=N group at 1620-1640 cm-1 and another band at 3420-3450 cm-1 attributed to the NH-group.

Together with the aromatic proton signals at 7.16-7.57 ppm, a broad singlet at 7.5-7.8 ppm corresponding to the NH group and a hardly resolved doublet at 6.9-7.4 of the proton in the 6-position of the quinazoline ring (3J = 1.4-1,7 Hz) were observed in the 1H-NMR spectra of compounds 4a-l.

DEPT experiments in the 13C-NMR spectra allowed to assign the signals belonging to quaternary, tertiary and primary carbon atoms for compounds 4a-l and corroborated the proposed structures. The full assignment of the signals is supported by 2D COSY, HMQC and HMBC NMR spectra.

The presence of a molecular ion at m/z 299 (M++2) in the mass spectrum was consistent with the structure 4a. All spectra described in this paper are available from authors on request.

NTIFUNGAL ASSAYS

Compounds 4 were assayed for antifungal properties against a panel of human opportunistic pathogenic yeasts, filamentous fungi as well as dermatophytes with the agar dilution method. To carry out the antifungal evaluation, concentrations up to 250 µg/mL were incorporated to growth media according to reported procedures [18, 19].

Three type of antifungal drugs in clinical use were incorporated to the assays as positive controls. They have been chosen considering that each one acts through a different mechanism of action and in addition, are commonly used for treating infections provoked for fungal species included in the test panel. Amphotericin B (which acts by binding to ergosterol forming pores in the fungal membrane) [20, 21] is also the drug of choice for systemic mycoses produced by Candida and other yeasts many times resistant to azoles [22, 23, 24]. Terbinafine acts by inhibiting the enzyme squalene epoxidase, which catalyzes the epoxidation of squalene in the biosynthesis of ergosterol. It is the drug of choice for the treatment of dermatophytoses [25, 26]

Retocoazole, which Ketoconazole, which possesses the typical azole ring in its structure (ring that is also present in benzimidazoles tested here) acts by inhibiting the biosynthesis of ergosterol trough the inhibition of 14-demethylase. Azoles are intensely used for the treatment of all mycoses and many fungi have developed resistance to them [27].

Results showed than none of the compounds tested was active neither against the yeasts C. albicans, S. cerevisiae or C. neoformans nor against the filamentous fungi A. niger, A. fumigatus or A. flavus (results not shown). In contrast, some compounds of the series showed antifungal effects against dermatophytes (Table 1).


Table 1: MICs values (mg/mL) of benzimidazole[1,2-c]quinazolines acting against human opportunistic pathogenic fungi.
M.c.: Microsporum canis C 112. M.g.: Microsporum gypseum C 115, T.r.: Trichophyton rubrum C 113, T.m.: Trichophyton mentagrophytes ATCC 9972, E.f.: Epidermophyton floccosum C 114. Amp= Amphotericin B. Ket=Ketoconazole.

Terb=Terbinafine.

Since dermatophytes are a group of fungi which characteristically infect the keratinized areas of the body and dermatomycoses are very difficult to eradicate, it is very interesting to note that these benzimidazo[1,2-c]quinazolines showed activity against dermatophytes and not against another type of fungi.

To evaluate the structure-activity relationships, the effect of electron-donor and electron-withdrawing substituents on the 6-phenyl ring of structures possessing A) a non-substituted benzimidazole (compounds 4a-g); B) a 9,10-dimethyl substituted benzimidazole (compounds 4h-l) were analyzed.

Among compounds with non-substituted benzimidazole (type A), only structures 4a, 4f, 4g showed significant activity against all the dermatophytes tested. Non-substituted compound 4a showed a moderate activity with MICs between 100-125 µg/mL. The introduction of electron-withdrawing substituents such as Cl, Br, F and NO2 on the 6-phenyl ring rendered compounds 4b, 4c, 4d and 4e respectively, all of them devoid of antifungal activity. In contrast, the replacement of the para-H atom in structure 4a by a donor substituent such as OCH3 group (®4f), enhanced the activity 2.5-5 times in T. mentagrophytes, E. floccosum and T. rubrum. The addition of two extra OCH3 groups (4f ®4g) does not improve the activity, both compounds showing the same MICs against all dermatophytes tested.

Regarding the influence of methyl substituents on both 9 and 10-positions of the benzimidazole ring (compounds type B, 4h-4l), results showed that this structural change produced a decrease in the antifungal activity in compounds without substituent or with donor groups (compare activities of 4h/4a; 4l/4f), and do not modify the lack of activity of structures with electron-withdrawing substituents (compare activities of 4b/4i; 4c/4j or 4e/4k) particularly against T. rubrum, T. mentagrophytes and E. floccosum. Figure 1 illustrates this effect for T. rubrum.


 
Figure 1: Comparative MICs in mg/ml of (9,10-Me-) vs. (9,10-H) -6-(4-R-phenyl)benzimidazo[1,2-c]quinazolines against Trichophyton rubrum. R= H, Cl, Br, NO2, OCH3

It is interesting to note that antifungal benzimidazole quinazolines showed activities only against dermatophytes and among them particularly against T. rubrum and E. floccosum. T. rubrum is the cause of approximately 80-93% of chronic and recurrent dermatophyte infections in human beings [28] and E. floccosum along with T. rubrum are the most frequent ethiological agents of tinea cruris, infection that affect the perianal and perineal areas of adult men producing bilateral erythematous lesions which are highly contagious. In addition E. floccosum produces arthroconidia which survive for a longer time than those of other dermatophytes, therefore constituting an environmental source of contagion, which could lead to recurrent outbreaks of dermatophytosis in individuals and in institutions [28].

Although benzimidazole compounds presented here showed lower activities than the available antifungal drugs, it is important to take in account that resistance to all antifungal agents in clinical use is growing at an alarming rate due in part to the treatment of the chronic different tineas with the same broad spectrum antifungal agent. So, narrow spectrum compound are intensely needed for treating dermatomycoses where the ethiological agent is known [29]. Therefore, we hope that the study of the antifungal properties of new benzimidazole-quinazoline compounds open new avenues for the development of another antifungals which, in the future, could be useful for treating the obstinate dermal fungal infections.

ACKNOWLEDGEMENTS

Authors are grateful to COLCIENCIAS, to Universidad del Valle and to the Spanish "Ministerio de Educación, Cultura y Deporte (Programa de Cooperación con Iberoamérica, AECI)" for financial support. S.A.Z. thanks to the Agencia de Promoción Científica y Tecnológica de la Argentina, PICTR #00260. This research is part of Project X.7-PIBEAFUN (Iberian-American Project on Search and Development of Antifungal Agents) of the Iberian-American Program for the Development of Science and Technology (CYTED).

 

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e-mail: szaabgil@citynet.net.ar

 

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