ISRN Organic Chemistry

Volume 2013 (2013), Article ID 635384, 9 pages

http://dx.doi.org/10.1155/2013/635384

Research Article

Design, Synthesis, and In Vitro Antimicrobial Evaluation of Fused Pyrano[3,2-e]tetrazolo[1,5-c]pyrimidines and Diazepines

Sankari Kanakaraju, P. Sagar Vijay Kumar, Bethanamudi Prasanna, and G. V. P. Chandramouli

Department of Chemistry, National Institute of Technology, Warangal, Andhra Pradesh 506 004, India

Received 16 June 2013; Accepted 18 July 2013

Academic Editors: M. D'Auria, J. C. Menéndez, D. Sémeril, and J. Wu

Copyright © 2013 Sankari Kanakaraju et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

A series of novel pyranochromene-containing tetrazoles fused with pyrimidinethiones, pyrimidines, and diazepines 3a–f, 4a–f, and 5a–f were synthesized by condensation of the corresponding tetrazoles 2a–f with carbon disulfide, benzaldehyde, and 4-methoxy phenacyl bromide, respectively. The compound 2a–f was obtained by reaction of pyrano[3,2-c]chromenes 1a–f with sodium azide. The structures of the newly synthesized compounds 2a–f to 5a–f were established on the basis of their elemental analyses, IR, ¹H NMR, ¹³C NMR, and mass spectral data. All of the title compounds were subjected to in vitro antibacterial testing against four pathogenic strains and antifungal screening against two fungi. Preliminary results indicate that some of them exhibited promising activities and that they deserve more consideration as potential antimicrobials.

1. Introduction

Pyrano[3,2-c]chromene derivatives are a class of important heterocycles with a wide range of biological properties [1] such as spasmolytic, diuretic, anticoagulant, anticancer, and antianaphylactic activities [2–5].

Tetrazoles represent an important class of heterocyclic compounds with wide ranging applications [6]. The synthesis of novel tetrazole derivatives and the investigation of their chemical and biological behavior has gained more importance in the recent decades for biological and pharmaceutical reasons. They have found use in pharmaceuticals as lipophilic spacers and carboxylic acid surrogates, which improves oral absorption [7]. Their derivatives were reported to possess broad spectrum of biological activity in both medicinal and pharmaceutical areas such as antinociceptive [8], antibacterial [9], antifungal [10], anti-HIV, anticancer, immunosuppressive [11], anti-inflammatory [12] and antiulcer [13], antiproliferative [14], antiallergic [15], and analgesic [16] activities.

On the other hand, pyrimidine scaffold was the base of many bioactive molecules such as antitubercular, antimicrobial [17], antiviral [18], antitumor [19, 20], anti-inflammatory, analgesic [21], antioxidant [22], antiproliferative [23], and antileishmanial agents [24]. Consequently, synthetic methodologies for synthesis of novel pyrimidines or pyrimidine fused compounds are of particular interests to organic and medicinal chemists.

The diazepine family represents one of the most prominent classes of privileged scaffolds in the field of drugs and pharmaceuticals. These compounds are widely used as anticonvulsant, antianxiety, analgesic, sedative, antidepressive, and hypnotic agents [25–27].

In view of the abovementioned facts, it was envisaged that these active pharmacophores, if linked together, would generate novel molecular templates which are likely to exhibit interesting biological properties. Hence, in continuation of our interest in the synthesis of biologically active heterocycles [28, 29], we report herein the synthesis and antimicrobial evaluation of some new heterocyclic compounds like pyranochromenes-containing tetrazoles. These tetrazoles are fused to six and seven-membered heterocyclic rings like pyrimidines, pyrimidinethiones, and diazepines. This combination was suggested in an attempt to investigate the influence of such structure variation on the anticipated biological activities, hoping to add some synergistic biological significance to the target molecules.

2. Results and Discussion

The synthetic strategies adopted for the synthesis of the target compounds are depicted in Scheme 1. The starting materials used in the present scheme, that is, pyrano[3,2-c]chromenes 1a–f, were prepared according to the previously reported procedure [30]. Pyrano[3,2-c]chromenes 1a–f were allowed to react with sodium azide in presence of NH₄Cl in DMF to give the corresponding tetrazoles 2a–f, which on reaction with CS₂ in presence of pyridine yielded the corresponding pyrimidine-5-thiones 3a–f. Condensation of tetrazoles 2a–f with benzaldehyde furnished the respective pyrimidines 4a–f. The compounds 5a–f were obtained by condensing tetrazoles 2a–f with 4-methoxyphenacyl bromide under reflux in ethanol. The structures of all of the newly synthesized compounds were elucidated on the basis of their spectral (IR, ¹H NMR, ¹³C NMR, and mass) and elemental analyses data. The synthesized compounds 2a–f, 3a–f, 4a–f, and 5a–f were also assayed for their antimicrobial activities.

Scheme 1: Schematic representation for the synthesis of fused tetrazole derivatives. Reagents and conditions: (i) NaN₃, DMF, NH₄Cl, 120°C, 7 h; (ii) CS₂, pyridine, reflux, 10 h; (iii) conc. HCl, benzaldehyde, MeOH, reflux, 16 h; (iv) 4-methoxyphenacyl bromide, EtOH, AcONa, reflux, 16 h.

The formation of tetrazoles 2a–f from pyrano[3,2-c]chromenes 1a–f was confirmed by their IR, ¹H NMR, and ¹³C NMR. In IR spectrum the disappearance of sharp absorption band (–CN) around 2200 cm⁻¹ and the appearance of band (–NH) around 3300 cm⁻¹ showed the formation of tetrazole, while in ¹H NMR the tetrazole NH proton was observed as a singlet at 10.01–10.21 ppm and in ¹³C NMR the tetrazole carbon was observed around 159.18–159.82 ppm. Furthermore, the condensation product 3a–f was confirmed by its IR spectrum which showed absence of the characteristic absorption band due to the –NH₂ group, and according to the IR spectroscopic data of these 3a–f compounds which have tetrazolopyrimidin-5-thione structure, the observation of the C=S stretching band at 1165–1181 cm⁻¹, and the absence of an absorption band at about 2550–2600 cm⁻¹ region cited for SH group have proved that these compounds were in the thionic form. In their ¹³C NMR, peak due to C=S appeared at 189.72–190.16 ppm. The formation of 4a–f from 2a–f was confirmed by their IR spectra in which no –NH₂ stretching vibrations were observed, and were well supported by their ¹H NMR and ¹³C NMR. In ¹H NMR, peaks shown at 6.22–6.34 ppm were assigned to pyrimidine CH proton, and in ¹³C NMR the signal was observed at 63.51–64.38 ppm due to pyrimidine carbon. Other signals were found in accordance to the established structures. Furthermore, the conversion of tetrazoles 2a–f to fused tetrazolodiazepines 5a–f was also well confirmed by their IR, ¹H NMR, and ¹³C NMR spectra. The disappearance of characteristic peaks due to NH₂ and NH groups of 2a–f clearly indicated the smooth cyclization. Their ¹H NMR spectra showed a singlet at 6.13–6.32 ppm for two protons, which is attributed to the methylene protons of diazepine ring, while in ¹³C NMR the signal that appeared at 48.43–49.06 ppm was assigned to methylene carbon of diazepine ring. For all of the compounds, the band displayed at 1700–1719 cm⁻¹ was due to the lactone carbonyl group, which was observed around 160.22–161.93 ppm in ¹³C NMR. The signal due to pyran proton, present in all of the compounds, appeared as a singlet at 4.56–5.12 ppm. All of the other aromatic and aliphatic protons were observed at the expected regions. Mass spectra of almost all of the compounds showed peaks, in agreement with their molecular weights. However, in some cases, peaks were also observed. For all of the compounds, the elemental analyses values were in good agreement with the theoretical data. Full characterization details were provided in the Experimental section.

2.1. Antimicrobial Activity

The antibacterial activity of the synthesized compounds 2a–f, 3a–f, 4a–f, and 5a–f was screened against the Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus and the Gram-negative bacteria, that is, Pseudomonas aeruginosa and Escherichia coli using nutrient agar medium. The antifungal activity of the compounds was tested against Candia albicans and Aspergillus niger using Potato dextrose agar (PDA) medium. The minimum inhibitory concentration (MIC) was carried out using microdilution susceptibility method [31]. Ciprofloxacin was used as a standard antibacterial drug, and fluconazole was used as a standard antifungal drug. The observed data on the antimicrobial activity of compounds and control drugs are given in Table 1.

Table 1: Minimum inhibitory concentration (MIC, g/mL) of the synthesized compounds 2a–f, 3a–f, 4a–f, and 5a–f.

Compound
Gram-positive bacteria
Gram-negative bacteria
Fungi

B. subtilis
S. aureus
P. aeuroginosa
E. coli
C. albicans
A. niger

2a
400
400
—
—
400
400

2b
400
200
200
400
200
50

2c
400
400
400
200
50
400

2d
400
400
200
400
400
200

2e
200
400
400
200
200
400

2f
—
—
—
—
—
—

3a
400
400
200
200
400
200

3b
25
100
100
50
100
50

3c
200
400
200
400
200
200

3d
400
200
200
400
200
400

3e
25
100
100
25
50
100

3f
400
400
400
400
400
400

4a
400
400
400
400
200
400

4b
100
50
200
100
50
200

4c
400
200
400
400
400
400

4d
200
400
400
200
400
200

4e
200
100
50
100
200
50

4f
400
200
400
400
400
200

5a
—
—
—
—
—
—

5b
200
400
—
400
—
200

5c
—
400
400
—
400
400

5d
400
200
200
—
200
—

5e
200
400
200
400
200
400

5f
—
—
—
—
—
—

Ciprofloxacin
6.25
6.25
6.25
6.25

Fluconazole

6.25
6.25

The MIC values were determined as the lowest concentration that completely inhibited visible growth of the microorganisms. The investigation of antibacterial screening (Table 1) revealed that some of the newly synthesized compounds showed moderate-to-good inhibition at 25–100 μg/mL in DMSO. Amongst all of the compounds, compounds 3b and 3e exhibited good activities against B. subtilis (MIC: 25 μg/mL) and E. coli (MIC: 50 and 25 μg/mL) and moderate activities against S. aureus and P. aeruginosa. Compound 4b displayed good activity against S. aureus (MIC: 50 μg/mL), whereas compound 4e exhibited good activity against P. aeuroginosa (MIC: 50 μg/mL).

The investigation of antifungal screening (Table 1) revealed that some of the newly synthesized compounds showed moderate-to-good inhibition at 25–100 μg/mL in DMSO. Among the tested compounds, compounds 2b, 3b, and 4e were found to be more active than other compounds against A. niger (MIC: 50 μg/mL). Compounds 2c, 3e, and 4b possess good activities against C. albicans (MIC: 50 μg/mL). Compounds 2f, 5a, and 5f showed no activity against the tested species. Remaining compounds showed moderate-to-least activity against both bacteria and fungi. The structure activity relationship of the synthesized compounds revealed that the compounds having diazepine ring showed the least activity compared with other compounds. The presence of chloro and methoxy group enhances the activities of the compounds. The maximum antimicrobial activity was observed with compounds 3b, 3e, 4b, and 4e.

3. Conclusion

In summary, a series of novel pyranochromene-containing fused tetrazole derivatives have been synthesized and characterized by spectral and elemental analyses. All of the newly synthesized compounds were screened for their in vitro antimicrobial activities. Among the screened samples, 3b, 3e, 4b, and 4e showed significant antibacterial and antifungal activities compared with other tested samples.

4. Experimental

Melting points were recorded in open capillary and were uncorrected. Column chromatography was performed using silica-gel (100–200 mesh size) purchased from Thomas Baker, and thin-layer chromatography (TLC) was carried out using aluminium sheets precoated with silica-gel 60F₂₅₄ purchased from Merck. IR spectra (KBr) were obtained using a Bruker WM-4(X) spectrometer (577 model). ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a Bruker WM-400 spectrometer in DMSO- with TMS as an internal standard. Mass spectra (ESI) were carried out on a JEOL SX-102 spectrometer. CHN analysis was done by the Carlo Erba EA 1108 automatic elemental analyzer. The chemicals and solvents used were of commercial grade and were used without further purification unless otherwise stated.

4.1. General Procedure for the Synthesis of 2-Amino-4-aryl-3-(1H-tetrazol-5-yl)pyrano[3,2-c]chromen-5(4H)-ones (2a–f)

To a mixture of compound 1a–f (10 mmol) in DMF (25 mL), sodium azide (0.78 g, 12 mmol) and NH₄Cl (0.64 g, 12 mmol) were added, and the reaction mixture was stirred at 120°C for 7 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature and poured into ice cold water (100 mL). The solid separated was filtered, washed with water, dried, and purified by column chromatography on silica-gel using hexane/ethyl acetate (7 : 3) as eluent to afford compound 2a–f.

4.2. 2-Amino-4-phenyl-3-(1H-tetrazol-5-yl)pyrano[3,2-c]chromen-5(4H)-one (2a)

White solid, yield 2.58 g (72%), m.p. 213–216°C; IR (KBr, cm⁻¹): 3386, 3278, 3196, 2992, 1706, 1660, 1567, 1497, 1310, 1279, 1110, 1051; ¹H NMR (400 MHz, DMSO-): 4.74 (s, 1H, H-4), 7.24–7.34 (m, 5H, ArH), 7.41 (brs, 2H, NH₂), 7.45–7.52 (m, 2H, ArH), 7.69–7.89 (m, 1H, ArH), 7.91 (d, 1H, ArH), 10.01 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 36.96, 89.03, 103.97, 112.93, 119.21, 122.43, 124.62, 127.09, 127.61, 128.49, 132.87, 143.31, 152.10, 153.38, 157.97, 159.50, 161.10; MS m/z: 360 . Anal. Calcd for C₁₉H₁₃N₅O₃: C, 63.51; H, 3.65; N, 19.49. Found: C, 63.62; H, 3.71; N, 19.38 %.

4.3. 2-Amino-4-(4-chlorophenyl)-3-(1H-tetrazol-5-yl)pyrano[3,2-c]chromen-5(4H)-one (2b)

White solid, yield 2.67 g (68%), m.p. 192–194°C; IR (KBr, cm⁻¹): 3397, 3284, 3193, 2990, 1713, 1664, 1551, 1493, 1321, 1281, 1121, 1063; ¹H NMR (400 MHz, DMSO-): 4.66 (s, 1H, H-4), 7.30 (d, 2H, ArH), 7.36–7.44 (m, 4H, ArH + NH₂), 7.48 (d, 1H, ArH), 7.54 (t, 1H, ArH), 7.78 (t, 1H, ArH), 7.92 (d, 1H, ArH), 10.14 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 37.12, 88.12, 103.89, 113.88, 119.73, 123.34, 125.48, 129.32, 130.51, 132.66, 133.71, 143.15, 153.14, 154.41, 158.13, 159.64, 160.25; MS m/z: 394 . Anal. Calcd for C₁₉H₁₂ClN₅O₃: C, 57.95; H, 3.07; N, 17.78. Found: C, 57.83; H, 3.14; N, 17.86%.

4.4. 2-Amino-4-(4-fluorophenyl)-3-(1H-tetrazol-5-yl)pyrano[3,2-c]chromen-5(4H)-one (2c)

Brown solid, yield 2.26 g (60%), m.p. 161–164°C; IR (KBr, cm⁻¹): 3416, 3378, 3296, 2998, 1719, 1666, 1566, 1491, 1322, 1284, 1114, 1053; ¹H NMR (400 MHz, DMSO-): 4.62 (s, 1H, H-4), 7.28 (d, 2H, ArH), 7.34–7.46 (m, 4H, ArH + NH₂), 7.50 (d, 1H, ArH), 7.58 (t, 1H, ArH), 7.75 (t, 1H, ArH), 7.94 (d, 1H, ArH), 10.06 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 38.17, 88.43, 103.72, 112.67, 120.14, 123.42, 125.36, 129.12, 130.64, 132.48, 133.52, 143.76, 153.96, 154.71, 158.25, 159.73, 160.41; MS m/z: 378 . Anal. Calcd for C₁₉H₁₂FN₅O₃: C, 60.48; H, 3.21; N, 18.56. Found: C, 60.59; H, 3.26; N, 18.43%.

4.5. 2-Amino-3-(1H-tetrazol-5-yl)-4-p-tolylpyrano[3,2-c]chromen-5(4H)-one (2d)

White solid, yield 2.34 g (63%); m.p. 208–210°C; IR (KBr, cm⁻¹): 3392, 3287, 3206, 2982, 1708, 1661, 1555, 1494, 1319, 1273, 1125, 1057; ¹H NMR (400 MHz, DMSO-): 2.38 (s, 3H, CH₃), 4.63 (s, 1H, H-4), 7.24 (d, 2H, ArH), 7.32–7.40 (m, 4H, ArH + NH₂), 7.48 (d, 1H, ArH), 7.54 (t, 1H, ArH), 7.71 (t, 1H, ArH), 7.84 (d, 1H, ArH), 10.02 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 20.94, 37.62, 88.26, 103.21, 118.83, 119.76, 122.48, 125.28, 129.32, 130.61, 131.56, 133.68, 142.64, 153.06, 154.24, 158.41, 159.18, 160.22; MS m/z: 374 . Anal. Calcd for C₂₀H₁₅N₅O₃: C, 64.34; H, 4.05; N, 18.76. Found: C, 64.46; H, 4.11; N, 18.62%.

4.6. 2-Amino-4-(4-methoxyphenyl)-3-(1H-tetrazol-5-yl)pyrano[3,2-c]chromen-5(4H)-one (2e)

Orange solid, yield 2.95 g (76%); m.p. 231–233°C; IR (KBr, cm⁻¹): 3419, 3376, 3284, 2992, 1716, 1668, 1566, 1492, 1320, 1283, 1117, 1061; ¹H NMR (400 MHz, DMSO-): 3.84 (s, 3H, OCH₃), 4.71 (s, 1H, H-4), 6.92 (d, 2H, ArH), 7.18–7.34 (m, 4H, ArH + NH₂), 7.45 (d, 1H, ArH), 7.51 (t, 1H, ArH), 7.70 (t, 1H, ArH), 7.89 (d, 1H, ArH), 10.16 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 38.84, 55.90, 89.24, 105.13, 113.72, 114.81, 120.12, 123.26, 125.43, 129.52, 133.55, 136.17, 152.94, 153.82, 157.56, 158.24, 159.72, 160.36; MS m/z: 390 . Anal. Calcd for C₂₀H₁₅N₅O₄: C, 61.69; H, 3.88; N, 17.99. Found: C, 61.74; H, 3.77; N, 17.87%.

4.7. 2-Amino-4-(3-nitrophenyl)-3-(1H-tetrazol-5-yl)pyrano[3,2-c]chromen-5(4H)-one (2f)

White solid, yield 2.26 g (56%); m.p. 198–200°C; IR (KBr, cm⁻¹): 3408, 3388, 3294, 2990, 1718, 1663, 1561, 1495, 1318, 1264, 1122, 1051; ¹H NMR (400 MHz, DMSO-): 4.92 (s, 1H, H-4), 7.42–7.52 (m, 4H, ArH + NH₂), 7.64 (t, 1H, ArH), 7.73 (t, 1H, ArH), 7.84 (d, 1H, ArH), 7.92 (d, 1H, ArH), 8.12 (d, 1H, ArH), 8.16 (s, 1H, ArH), 10.21 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 38.92, 89.43, 103.82, 113.71, 119.62, 123.11, 123.23, 123.64, 125.46, 130.29, 133.69, 135.36, 146.61, 148.27, 153.13, 154.56, 158.81, 159.82, 160.74; MS m/z: 405 . Anal. Calcd for C₁₉H₁₂N₆O₅: C, 56.44; H, 2.99; N, 20.78. Found: C, 56.50; H, 3.08; N, 20.67%.

4.8. General Procedure for the Synthesis of Fused Pyrano[3,2-e]tetrazolo[1,5-c]pyrimidin-5-thiones (3a–f)

A mixture of compound 2a–f (2 mmol) and carbon disulfide (0.15 g, 0.12 mL, 2 mmol) in pyridine (10 mL) was refluxed on a water bath for 10 h. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature, then poured into ice cold water (20 mL), and neutralized with hydrochloric acid (1 : 1). The solid obtained was filtered, washed with water, dried, and recrystallized from ethanol to afford compound 3a–f.

4.9. 14-Phenyl-5,6-dihydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidin-5-thione (3a)

White solid, yield 0.54 g (68%); m.p. 226–228°C; IR (KBr, cm⁻¹): 3399, 2990, 1710, 1664, 1598, 1505, 1310, 1261, 1181, 1089; ¹H NMR (400 MHz, DMSO-): 5.03 (s, 1H, H-4), 7.09–7.57 (m, 7H, ArH), 7.87–7.95 (m, 2H, ArH), 10.09 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 32.45, 86.53, 104.19, 118.14, 123.63, 124.25, 126.65, 127.99, 128.09, 132.02, 139.88, 144.59, 152.11, 155.59, 156.08, 160.12, 161.93, 190.02; MS m/z: 403 . Anal. Calcd for C₂₀H₁₁N₅O₃S: C, 59.84; H, 2.76; N, 17.45. Found: C, 59.88; H, 2.85; N, 17.34%.

4.10. 14-(4-Chlorophenyl)-5,6-dihydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidin-5-thione (3b)

White solid, yield 0.57 g (66%); m.p. 185–188°C; IR (KBr, cm⁻¹): 3406, 2998, 1718, 1668, 1583, 1508, 1323, 1267, 1177, 1082; ¹H NMR (400 MHz, DMSO-): 4.76 (s, 1H, H-4), 7.28 (d, 2H, ArH), 7.37 (d, 2H, ArH), 7.51 (d, 1H, ArH), 7.56 (t, 1H, ArH), 7.77 (t, 1H, ArH), 7.95 (d, 1H, ArH), 10.21 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 33.44, 86.17, 103.74, 113.42, 119.76, 123.29, 124.82, 129.46, 130.32, 132.71, 133.54, 142.88, 152.96, 153.88, 157.12, 160.47, 161.12, 189.82; MS m/z: 436 . Anal. Calcd for C₂₀H₁₀ClN₅O₃S: C, 55.11; H, 2.31; N, 16.07. Found: C, 55.16; H, 2.23; N, 16.15%.

4.11. 14-(4-Fluorophenyl)-5,6-dihydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidin-5-thione (3c)

White solid, yield 0.52 (63%); m.p. 173–176°C; IR (KBr, cm⁻¹): 3386, 2985, 1712, 1666, 1577, 1512, 1321, 1256, 1167, 1076 cm⁻¹; ¹H NMR (400 MHz, DMSO-): 4.84 (s, 1H, H-4), 7.36 (d, 2H, ArH), 7.48 (d, 2H, ArH), 7.54 (d, 1H, ArH), 7.62–7.74 (m, 2H, ArH), 7.94 (d, 1H, ArH), 10.34 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 34.21, 87.34, 103.48, 112.56, 120.71, 123.56, 124.22, 128.96, 130.82, 132.45, 133.68, 143.18, 153.27, 154.40, 157.57, 160.19, 161.56, 189.93; MS m/z: 421 . Anal. Calcd for C₂₀H₁₀FN₅O₃S: C, 57.28; H, 2.40; N, 16.70. Found: C, 57.39; H, 2.47; N, 16.62%.

4.12. 14-(4-Methylphenyl)-5,6-dihydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]-pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidin-5-thione (3d)

White solid, yield 0.49 g (60%); m.p. 190–192°C; IR (KBr, cm⁻¹): 3411, 2982, 1700, 1661, 1592, 1507, 1313, 1266, 1172, 1083; ¹H NMR (400 MHz, DMSO-): 2.30 (s, 3H, CH₃), 4.91 (s, 1H, H-4), 7.12 (d, 2H, ArH), 7.38 (d, 2H, ArH), 7.45 (d, 1H, ArH), 7.52–7.79 (m, 2H, ArH), 7.91 (d, 1H, ArH), 10.28 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 20.96, 34.52, 87.64, 103.18, 118.36, 119.82, 122.57, 125.39, 125.43, 130.72, 131.44, 133.84, 142.72, 153.14, 154.36, 158.74, 159.72, 161.78, 189.72; MS m/z: 416 . Anal. Calcd for C₂₁H₁₃N₅O₃S: C, 60.71; H, 3.15; N, 16.86. Found: C, 60.84; H, 3.20; N, 16.75%.

4.13. 14-(4-Methoxyphenyl)-5,6-dihydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidin-5-thione (3e)

White solid, yield 0.50 g (59%); m.p. 200–203°C; IR (KBr, cm⁻¹): 3393, 2994, 1711, 1653, 1588, 1503, 1322, 1268, 1165, 1081; ¹H NMR (400 MHz, DMSO-): 3.86 (s, 3H, OCH₃), 4.93 (s, 1H, H-4), 6.98 (d, 2H, ArH), 7.22 (d, 2H, ArH), 7.48 (d, 1H, ArH), 7.54 (t, 1H, ArH), 7.76 (t, 1H, ArH), 7.89 (d, 1H, ArH), 10.33 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 34.64, 55.96, 88.71, 105.24, 113.64, 114.78, 120.22, 123.64, 125.49, 129.31, 133.57, 136.23, 152.88, 153.91, 158.71, 159.76, 159.88, 161.28, 189.83; MS m/z: 432 . Anal. Calcd for C₂₁H₁₃N₅O₄S: C, 58.46; H, 3.04; N, 16.23. Found: C, 58.39; H, 3.14; N, 16.32%.

4.14. 14-(3-Nitrophenyl)-5,6-dihydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidin-5-thione (3f)

White solid, yield 0.45 g (51%); m.p. 211–213°C; IR (KBr, cm⁻¹): 3396, 2997, 1716, 1666, 1591, 1511, 1314, 1266, 1180, 1079; ¹H NMR (400 MHz, DMSO-): 5.10 (s, 1H, H-4), 7.44 (d, 1H, ArH), 7.51 (t, 1H, ArH), 7.62 (t, 1H, ArH), 7.75 (t, 1H, ArH), 7.82 (d, 1H, ArH), 7.94 (d, 1H, ArH), 8.10 (d, 1H, ArH), 8.18 (s, 1H, ArH), 10.48 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 34.79, 88.76, 103.93, 113.62, 119.84, 123.32, 123.43, 123.79, 125.62, 130.71, 133.82, 135.43, 146.67, 148.54, 153.12, 155.93, 159.72, 160.74, 161.87, 190.16; MS m/z: 447 . Anal. Calcd for C₂₀H₁₀N₆O₅S: C, 53.81; H, 2.26; N, 18.83. Found: C, 53.92; H, 2.35; N, 18.78%.

4.15. General Procedure for the Synthesis of Fused Pyrano[3,2-e]tetrazolo[1,5-c]pyrimidines (4a–f)

To a mixture of compound 2a–f (2 mmol) and benzaldehyde (0.212 g, 0.20 mL, 2 mmol) in methanol (10 mL), conc. HCl (0.5 mL) was added, and the reaction mixture was refluxed for 16 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature and neutralized with saturated sodium bicarbonate solution; the solid separated was filtered, washed with water, dried, and recrystallized from ethanol to afford compound 4a–f.

4.16. 5,14-Diphenyl-6-hydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidine (4a)

White solid, yield 0.54 g (61%); m.p. 168–170°C; IR (KBr, cm⁻¹): 3384, 2997, 1714, 1635, 1593, 1368, 1261, 1123; ¹H NMR (400 MHz, DMSO-): 4.58 (s, 1H, H-4), 6.32 (s, 1H, CH), 7.14–7.58 (m, 11H, ArH), 7.82–7.86 (m, 2H, ArH), 7.94 (d, 1H, ArH), 9.60 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 35.96, 63.51, 88.32, 103.86, 113.74, 118.44, 123.30, 123.41, 123.90, 124.00, 125.27, 126.63, 127.87, 131.53, 131.85, 140.57, 143.77, 152.24, 153.69, 156.82, 159.68, 160.36; MS m/z: 448 . Anal. Calcd for C₂₆H₁₇N₅O₃: C, 69.79; H, 3.83; N, 15.65. Found: C, 69.72; H, 3.92; N, 15.57%.

4.17. 14-(4-Chlorophenyl)-5-phenyl-6-hydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidine (4b)

White solid, yield 0.51 g (53%); m.p. 155–158°C; IR (KBr, cm⁻¹): 3391, 2982, 1717, 1665, 1571, 1372, 1258, 1132; ¹H NMR (400 MHz, DMSO-): 4.78 (s, 1H, H-4), 6.24 (s, 1H, CH), 7.10–7.46 (m, 9H, ArH), 7.52 (d, 1H, ArH), 7.58 (t, 1H, ArH), 7.79 (t, 1H, ArH), 7.94 (d, 1H, ArH), 9.93 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 36.24, 63.72, 88.41, 104.76, 114.54, 118.73, 124.44, 125.59, 126.68, 127.82, 129.46, 130.28, 131.47, 132.61, 133.48, 138.12, 142.23, 153.17, 154.53, 156.22, 159.83, 160.48; MS m/z: 482 . Anal. Calcd for C₂₆H₁₆ClN₅O₃: C, 64.80; H, 3.35; N, 14.53. Found: C, 64.86; H, 3.46; N, 14.44%.

4.18. 14-(4-Fluorophenyl)-5-phenyl-6-hydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidine (4c)

Brown solid, yield 0.49 g (53%); m.p. 187–189°C; IR (KBr, cm⁻¹): 3387, 2987, 1711, 1658, 1589, 1366, 1267, 1129; ¹H NMR (400 MHz, DMSO-): 4.83 (s, 1H, H-4), 6.22 (s, 1H, CH), 7.12–7.45 (m, 9H, ArH), 7.56 (d, 1H, ArH), 7.63 (t, 1H, ArH), 7.74 (t, 1H, ArH), 7.96 (d, 1H, ArH), 10.10 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 36.48, 63.76, 88.53, 103.74, 113.78, 119.94, 122.53, 124.64, 126.72, 127.39, 129.65, 130.48, 131.62, 132.73, 133.66, 139.12, 143.13, 153.70, 154.49, 157.11, 159.32, 160.53; MS m/z: 466 . Anal. Calcd for C₂₆H₁₆FN₅O₃: C, 67.09; H, 3.46; N, 15.05. Found: C, 67.18; H, 3.37; N, 15.10%.

4.19. 14-(4-Methylphenyl)-5-phenyl-6-hydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidine (4d)

White solid, yield 0.53 g (58%); m.p. 174–176°C; IR (KBr, cm⁻¹): 3371, 2982, 1706, 1669, 1584, 1371, 1256, 1125; ¹H NMR (400 MHz, DMSO-): 2.32 (s, 3H, CH₃), 4.96 (s, 1H, H-4), 6.28 (s, 1H, CH), 7.08–7.40 (m, 9H, ArH), 7.46 (d, 1H, ArH), 7.56 (t, 1H, ArH), 7.71 (t, 1H, ArH), 7.93 (d, 1H, ArH), 10.18 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 20.96, 37.08, 64.16, 88.72, 103.32, 118.78, 119.72, 122.53, 125.36, 126.57, 128.71, 129.43, 130.53, 132.19, 133.58, 138.23, 139.21, 143.13, 153.70, 154.49, 157.11, 159.32, 160.57; MS m/z: 462 . Anal. Calcd for C₂₇H₁₉N₅O₃: C, 70.27; H, 4.15; N, 15.18. Found: C, 70.33; H, 4.24; N, 15.10%.

4.20. 14-(4-Methoxyphenyl)-5-phenyl-6-hydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidine (4e)

White solid, yield 0.57 g (60%); m.p. 165–168°C; IR (KBr, cm⁻¹): 3388, 2997, 1701, 1665, 1591, 1360, 1267, 1129; ¹H NMR (400 MHz, DMSO-): 3.85 (s, 3H, OCH₃), 4.98 (s, 1H, H-4), 6.30 (s, 1H, CH), 7.01–7.44 (m, 9H, ArH), 7.50 (d, 1H, ArH), 7.54 (t, 1H, ArH), 7.73 (t, 1H, ArH), 7.84 (d, 1H, ArH), 10.14 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): δ 37.43, 55.48, 64.34, 88.76, 104.07, 112.39, 114.73, 120.32, 123.59, 125.14, 126.37, 127.68, 129.38, 131.73, 133.81, 137.70, 138.32, 152.72, 154.68, 156.36, 158.42, 159.58, 160.53; MS m/z: 478 . Anal. Calcd for C₂₇H₁₉N₅O₄: C, 67.92; H, 4.01; N, 14.67. Found: C, 67.81; H, 4.06; N, 14.58%.

4.21. 14-(3-Nitrophenyl)-5-phenyl-6-hydro-13-oxo-5H,13H,14H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidine (4f)

Yellow solid, yield 0.50 g (51%); m.p. 191–194°C; IR (KBr, cm⁻¹): 3399, 2982, 1718, 1659, 1582, 1368, 1251, 112; ¹H NMR (400 MHz, DMSO-): 5.12 (s, 1H, H-4), 6.34 (s, 1H, CH), 7.11–7.38 (m, 5H, ArH), 7.46 (d, 1H, ArH), 7.53 (t, 1H, ArH), 7.60 (t, 1H, ArH), 7.76 (t, 1H, ArH), 7.88 (d, 1H, ArH), 7.94 (d, 1H, ArH), 8.12 (d, 1H, ArH), 8.20 (s, 1H, ArH) 10.24 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-): 37.82, 64.38, 88.83, 104.29, 113.68, 120.07, 123.33, 124.12, 125.57, 126.37, 127.41, 128.03, 131.32, 132.12, 133.73, 136.51, 138.56, 145.87, 148.42, 153.32, 154.66, 158.27, 159.87, 160.67; MS m/z: 493 . Anal. Calcd for C₂₆H₁₆N₆O₅: C, 63.41; H, 3.27; N, 17.07. Found: C, 63.48; H, 3.16; N, 17.16%.

4.22. General Procedure for the Synthesis of Fused Pyrano[3,2-e]tetrazolo[1,5-c][1,4]diazepines (5a–f)

A mixture of compound 2a–f (2 mmol), 4-methoxyphenacyl bromide (0.45 g, 2 mmol), and sodium acetate (0.19 g, 2.4 mmol) in ethanol (15 mL) was refluxed for 16 h. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature and then poured into water (30 mL). The solid separated was filtered, washed with water, dried, and purified by column chromatography on silica-gel using hexane/ethyl acetate (6 : 4) as eluent to obtain compound 5a–f.

4.23. 15-Phenyl-6-(4-methoxyphenyl)-13-oxo-14H,15H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c][1,4]diazepine (5a)

White solid, yield 0.56 g (58%); m.p. 138–140°C; IR (KBr, cm⁻¹): 2954, 1700, 1634, 1608, 1509, 1364, 1288, 1138, 1108; ¹H NMR (400 MHz, DMSO-): 3.85 (s, 3H, OCH₃), 4.58 (s, 1H, H-4), 6.13 (s, 2H, CH₂), 7.10–7.15 (m, 2H, ArH), 7.28 (d, 2H, ArH), 7.37 (d, 2H, ArH), 7.43–7.53 (m, 3H, ArH), 7.70–7.77 (m, 2H, ArH), 7.82 (d, 1H, ArH), 7.93 (d, 1H, ArH); ¹³C NMR (100 MHz, DMSO-): 37.02, 48.43, 55.13, 103.90, 105.73, 113.01, 118.67, 119.21, 122.48, 124.61, 126.02, 126.25, 127.08, 128.22, 129.23, 132.85, 142.14, 152.14, 153.35, 157.22, 157.80, 159.53, 160.22, 161.87; MS m/z: 490 . Anal. Calcd for C₂₈H₁₉N₅O₄: C, 68.71; H, 3.91; N, 14.31. Found: C, 68.80; H, 3.83; N, 14.36%.

4.24. 15-(4-Chlorophenyll-6-(4-methoxyphenyl)-13-oxo-14H,15H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c][1,4]diazepine (5b)

White solid, yield 0.54 g (52%); m.p. 154–156°C; IR (KBr, cm⁻¹): 2973, 1707, 1651, 1591, 1511, 1371, 1272, 1129, 1112; ¹H NMR (400 MHz, DMSO-): 3.86 (s, 3H, OCH₃), 4.56 (s, 1H, H-4), 6.17 (s, 2H, CH₂), 6.98 (d, 2H, ArH), 7.29 (d, 2H, ArH), 7.44 (d, 1H, ArH), 7.56 (d, 2H, ArH), 7.61 (t, 1H, ArH), 7.72 (t, 1H, ArH), 7.84 (d, 2H, ArH) 7.98 (d, 1H, ArH); ¹³C NMR (100 MHz, DMSO-): 37.12, 48.51, 55.76, 104.23, 105.84, 113.14, 114.32, 119.71, 123.81, 124.93, 126.42, 128.72, 130.02, 130.86, 132.54, 133.67, 142.24, 152.28, 153.76, 156.92, 157.38, 159.63, 160.32, 161.78; MS m/z: 524 . Anal. Calcd for C₂₈H₁₈ClN₅O₄: C, 64.19; H, 3.46; N, 13.37. Found: C, 64.24; H, 3.57; N, 13.31%.

4.25. 15-(4-Fluorophenyl)-6-(4-methoxyphenyl)-13-oxo-14H,15H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c][1,4]diazepine (5c)

White solid, yield 0.51 g (51%); m.p. 173–176°C; IR (KBr, cm⁻¹): 2989, 1704, 1648, 1606, 1512, 1371, 1266, 1134, 1104; ¹H NMR (400 MHz, DMSO-): 3.86 (s, 3H, OCH₃), 4.64 (s, 1H, H-4), 6.19 (s, 2H, CH₂), 6.96 (d, 2H, ArH), 7.32 (d, 2H, ArH), 7.49 (d, 1H, ArH), 7.54 (d, 2H, ArH), 7.65 (t, 1H, ArH), 7.71 (t, 1H, ArH), 7.87 (d, 2H, ArH) 7.94 (d, 1H, ArH); ¹³C NMR (100 MHz, DMSO-): 37.43, 48.62, 55.81, 103.92, 105.74, 112.86, 114.36, 119.93, 123.25, 125.61, 126.32, 129.23, 130.13, 130.87, 132.85, 133.26, 143.17, 152.28, 153.70, 156.87, 157.47, 159.44, 160.53, 161.49; MS m/z: 508 . Anal. Calcd for C₂₈H₁₈FN₅O₄: C, 66.27; H, 3.58; N, 13.80. Found: C, 66.21; H, 3.67; N, 13.88%.

4.26. 15-(4-Methylphenyl)-6-(4-Methoxyphenyl)-13-oxo-14H,15H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c][1,4]diazepine (5d)

White solid, yield 0.55 g (55%); m.p. 165–168°C; IR (KBr, cm⁻¹): 2983, 1709, 1641, 1614, 1511, 1360, 1273, 1131, 1109; ¹H NMR (400 MHz, DMSO-): 2.32 (s, 3H, CH₃), 3.89 (s, 3H, OCH₃), 4.68 (s, 1H, H-4), 6.22 (s, 2H, CH₂), 6.98 (d, 2H, ArH), 7.29–7.35 (m, 4H, ArH), 7.48 (d, 1H, ArH), 7.56 (t, 1H, ArH), 7.72 (t, 1H, ArH), 7.89 (d, 2H, ArH) 7.98 (d, 1H, ArH); ¹³C NMR (100 MHz, DMSO-): 20.94, 38.23, 48.71, 55.90, 103.53, 105.76, 114.47, 118.39, 119.68, 122.85, 125.83, 126.41, 129.26, 130.31, 130.72, 131.66, 133.53, 142.87, 153.12, 154.43, 156.93, 157.59, 159.52, 160.12, 161.23; MS m/z: 504 . Anal. Calcd for C₂₉H₂₁N₅O₄: C, 69.18; H, 4.20; N, 13.91. Found: C, 69.27; H, 4.25; N, 13.81%.

4.27. 6,15-Di-(4-methoxyphenyl)-13-oxo-14H,15H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c][1,4]diazepine (5e)

White solid, yield 0.59 g (57%); m.p. 201–203°C; IR (KBr, cm⁻¹): 2991, 1711, 1667, 1600, 1521, 1371, 1265, 1142, 1101; ¹H NMR (400 MHz, DMSO-): 3.84 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 4.77 (s, 1H, H-4), 6.28 (s, 2H, CH₂), 6.98–7.10 (m, 4H, ArH), 7.39 (d, 2H, ArH), 7.48 (d, 1H, ArH), 7.54 (t, 1H, ArH), 7.70 (t, 1H, ArH), 7.81 (d, 2H, ArH) 7.93 (d, 1H, ArH); ¹³C NMR (100 MHz, DMSO-): 38.41, 48.83, 55.89, 55.96, 105.21, 105.93, 113.78, 114.82, 120.23, 123.71, 125.38, 127.08, 129.37, 130.43, 133.62, 136.81, 152.73, 153.88, 155.13, 156.22, 157.83, 158.34, 159.77, 160.43, 161.76; MS m/z 520 . Anal. Calcd for C₂₉H₂₁N₅O₅: C, 67.05; H, 4.07; N, 13.48. Found: C, 67.11; H, 4.14; N, 13.39%.

4.28. 15-(3-Nitrophenyl)-6-(4-methoxyphenyl)-13-oxo-14H,15H-[1]benzopyrano[3′,4′:5,6]pyrano[3,2-e][1,2,3,4]tetrazolo[1,5-c][1,4]diazepine (5f)

White solid, yield 0.51 g (48%); m.p. 228–230°C; IR (KBr, cm⁻¹): 2987, 1708, 1658, 1603, 1501, 1376, 1263, 1127, 1118; ¹H NMR (400 MHz, DMSO-): 3.89 (s, 3H, OCH₃), 4.93 (s, 1H, H-4), 6.32 (s, 2H, CH₂), 7.00 (d, 2H, ArH), 7.41 (d, 1H, ArH), 7.51 (t, 1H, ArH), 7.63 (t, 1H, ArH), 7.75 (t, 1H, ArH), 7.80 (d, 1H, ArH), 7.89 (d, 2H, ArH), 7.98 (d, 1H, ArH), 8.10 (d, 1H, ArH), 8.16 (s, 1H, ArH); ¹³C NMR (100 MHz, DMSO-): 38.71, 49.06, 55.98, 104.74, 105.91, 113.82, 114.67, 119.58, 123.62, 123.94, 124.21, 125.64, 126.91, 130.39, 131.13, 133.64, 135.67, 146.72, 148.31, 153.32, 154.66, 157.38, 158.32, 159.84, 160.77, 161.83; MS m/z: 535 . Anal. Calcd for C₂₈H₁₈N₆O₆: C, 62.92; H, 3.39; N, 15.72. Found: C, 62.81; H, 3.45; N, 15.62%.

5. Biological Protocol

5.1. Antimicrobial Activity

All of the newly synthesized compounds 2a–f, 3a–f, 4a–f, and 5a–f were screened for their antibacterial activities against the Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and the Gram-negative bacteria (Pseudomonas aeuroginosa and Escherichia coli). The antifungal activity of the compounds was assayed against Candia albicans and Aspergillus niger. The MICs of the compound assays were carried out using the microdilution susceptibility method. Ciprofloxacin was used as a reference antibacterial agent. Fluconazole was used as a reference antifungal agent. The test compounds, ciprofloxacin and fluconazole, were dissolved in DMSO at concentration of 800 μg/mL, and they were then diluted in culture medium (nutrient agar for bacteria and Potato dextrose agar for fungi ), and two-fold serial dilution of the solution was prepared (400, 200, 100, 50, 12.5, and 6.25 μg/mL). The tubes were incubated at 36°C for 24 h and 48 h for bacteria and fungi, respectively. The minimum inhibitory concentrations (MICs, μg/mL) of the compounds were recorded as the lowest concentration of each chemical compound in the tubes with no turbidity (i.e., no growth) of inoculated bacteria/fungi.

Acknowledgment

The authors are thankful to the Director of the National Institute of Technology, Warangal, India, for providing research facilities and financial support.

References

G. R. Green, J. M. Evans, A. K. Vong, A. R. Katritzky, C. W. Rees, and E. F. Scriven, Comprehensive Heterocyclic Chemistry, vol. 5, Pergamon Press, Oxford, UK, 1995.

W. O. Foye, Principi Di Chemico Frmaceutica, Piccin, Padova, Italy, 1991.

L. Bonsignore, G. Loy, D. Secci, and A. Calignano, “Synthesis and pharmacological activity of 2-oxo-(2H) 1-benzopyran-3-carboxamide derivatives,” European Journal of Medicinal Chemistry, vol. 28, no. 6, pp. 517–520, 1993.

L. L. Andreani and E. Lapi, “On some new esters of coumarin-3-carboxylic acid wit balsamic and bronchodilator action,” Bollettino Chimico Farmaceutico, vol. 99, pp. 583–586, 1960.

E. C. Witte, P. Neubert, and A. Roesch, “7-(piperazinyl propoxy)-2H-1-benzopyran-2-ones,” Ger. Offen DE, 3427985, Chemical Abstracts 104, 224915f, 1986

R. N. Butler, A. R. Katritzky, C. W. Rees, and E. F. V. Scriven, Comprehensive Heterocyclic Chemistry, vol. 4, Pergamon Press, Oxford, UK, 1996.

R. J. Herr, “5-Substituted-1H-tetrazoles as carboxylic acid isosteres: medicinal chemistry and synthetic methods,” Bioorganic and Medicinal Chemistry, vol. 10, no. 11, pp. 3379–3393, 2002.

A. Rajasekaran and P. P. Thampi, “Synthesis and antinociceptive activity of some substituted-{5-[2-(1,2,3,4- tetrahydrocarbazol-9-yl)ethyl]tetrazol-1-yl}alkanones,” European Journal of Medicinal Chemistry, vol. 40, no. 12, pp. 1359–1364, 2005.

R. M. Demarinis, J. R. E. Hoover, and G. L. Dunn, “A new parenteral cephalosporin. SK&F 59962: 7 trifluoromethylthioacetamido 3 (1 methyl 1H tetrazol 5 ylthiomethyl) 3 cephem 4 carboxylic acid. Chemistry and structure activity relationships,” Journal of Antibiotics, vol. 28, no. 6, pp. 463–470, 1975.

R. S. Upadhayaya, S. Jain, N. Sinha, N. Kishore, R. Chandra, and S. K. Arora, “Synthesis of novel substituted tetrazoles having antifungal activity,” European Journal of Medicinal Chemistry, vol. 39, no. 7, pp. 579–592, 2004.

A. Dlugosz, “Synthesis and biological activity of pyrimidobenzodiazepine derivatives. New ring systems: triazolo- and tetrazolo-pyrimidobenzodiazepines,” Pharmazie, vol. 50, no. 3, pp. 180–182, 1995.

E. E. Knaus and P. Kumar, “Synthesis and antiinflammatory activity of 5-(1,2-dihydropyridyl)-tetrazol-2-acetic acids, esters and amides,” European Journal of Medicinal Chemistry, vol. 28, no. 11, pp. 881–885, 1993.

K. Terashima, T. Tanimura, H. Shimamura et al., “Studies on antiulcer agents. II. Antiulcer properties of N-(1H-tetrazol-5-yl)-2-anilino-5-pyrimidinecarboxamides inhibiting release of histamine from passively sensitized rat peritoneal mast cells,” Chemical and Pharmaceutical Bulletin, vol. 43, no. 6, pp. 1042–1044, 1995.

A. S. Gundugola, K. L. Chandra, E. M. Perchellet, A. M. Waters, J.-P. H. Perchellet, and S. Rayat, “Synthesis and antiproliferative evaluation of 5-oxo and 5-thio derivatives of 1,4-diaryl tetrazoles,” Bioorganic and Medicinal Chemistry Letters, vol. 20, no. 13, pp. 3920–3924, 2010.

A. Nohara, H. Kuriki, T. Saijo, H. Sugihara, M. Kanno, and Y. Sanno, “Studies on antianaphylactic agents. 5. Synthesis of 3-(1H-tetrazol-5-yl)chromones, a new series of antiallergic substances,” Journal of Medicinal Chemistry, vol. 20, no. 1, pp. 141–145, 1977.

A. Rajasekaran and P. P. Thampi, “Synthesis and analgesic evaluation of some 5-[β-(10-phenothiazinyl) ethyl]-1-(acyl)-1,2,3,4-tetrazoles,” European Journal of Medicinal Chemistry, vol. 39, no. 3, pp. 273–279, 2004.

N. R. Kamdar, D. D. Haveliwala, P. T. Mistry, and S. K. Patel, “Design, synthesis and in vitro evaluation of antitubercular and antimicrobial activity of some novel pyranopyrimidines,” European Journal of Medicinal Chemistry, vol. 45, no. 11, pp. 5056–5063, 2010.

D. Hocková, A. Holý, M. Masojídková et al., “Synthesis and antiviral activity of 2,4-diamino-5-cyano-6-[2- (phosphonomethoxy)ethoxy]pyrimidine and related compounds,” Bioorganic and Medicinal Chemistry, vol. 12, no. 12, pp. 3197–3202, 2004.

R. Lin, S. G. Johnson, P. J. Connolly et al., “Synthesis and evaluation of 2,7-diamino-thiazolo[4,5-d] pyrimidine analogues as anti-tumor epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors,” Bioorganic and Medicinal Chemistry Letters, vol. 19, no. 8, pp. 2333–2337, 2009.

A. A. Aly, M. Ramadan, A. M. Mohamed, and E. A. Ishak, “Thieno[2, 3-d]pyrimidines in the synthesis of new fused heterocyclic compounds of prospective antitumor and antioxidant agents,” Journal of Heterocyclic Chemistry, vol. 49, no. 5, pp. 1009–1018, 2012.

M. T. Chhabria, H. G. Bhatt, H. G. Raval, and P. M. Oza, “Synthesis and biological evaluation of some 5-ethoxycarbonyl-6-isopropylamino-4-(substitutedphenyl)aminopyrimidines as potent analgesic and anti-inflammatory agents,” Bioorganic and Medicinal Chemistry Letters, vol. 17, no. 4, pp. 1022–1024, 2007.

Y. Kotaiah, N. Harikrishna, K. Nagaraju, and C. V. Rao, “Synthesis and antioxidant activity of 1, 3, 4-oxadiazole tagged thieno[2, 3-d]pyrimidine derivatives,” European Journal of Medicinal Chemistry, vol. 58, pp. 340–345, 2012.

A. M. Marini, F. da Settimo, S. Salerno et al., “Synthesis and in vitro antiproliferative activity of new substituted benzo[3′,2′:5,6]thiopyrano[4,3-d]pyrimidines,” Journal of Heterocyclic Chemistry, vol. 45, no. 3, pp. 745–749, 2008.

A. Agarwal, R. Ramesh, A. Ashutosh, N. Goyal, P. M. S. Chauhan, and S. Gupta, “Dihydropyrido[2,3-d]pyrimidines as a new class of antileishmanial agents,” Bioorganic and Medicinal Chemistry, vol. 13, no. 24, pp. 6678–6684, 2005.

J. K. Landquist, A. R. Katritzky, and C. W. Rees, Comprehensive Heterocyclic Chemistry, vol. 1, Pergamon Press, Oxford, UK, 1984.

U. Rudolph and H. Möhler, “GABA-based therapeutic approaches: GABAA receptor subtype functions,” Current Opinion in Pharmacology, vol. 6, no. 1, pp. 18–23, 2006.

L. Costantino and D. Barlocco, “Privileged structures as leads in medicinal chemistry,” Current Medicinal Chemistry, vol. 13, no. 1, pp. 65–85, 2006.

S. Kanakaraju, B. Prasanna, S. Basavoju, and G. V. P. Chandramouli, “Ionic liquid catalyzed one-pot multi-component synthesis, characterization and antibacterial activity of novel chromeno[2, 3-d]pyrimidine-8-amine derivatives,” Journal of Molecular Structure, vol. 1017, pp. 60–64, 2012.

S. Kanakaraju, B. Prasanna, and G. V. P. Chandramouli, “An efficient one-pot three-component synthesis of novel sulfanyl tetrazoles using ionic liquids,” Journal of Chemistry, vol. 2013, Article ID 104690, 6 pages, 2013.

S. Abdolmohammadi and S. Balalaie, “Novel and efficient catalysts for the one-pot synthesis of 3,4-dihydropyrano[c]chromene derivatives in aqueous media,” Tetrahedron Letters, vol. 48, no. 18, pp. 3299–3303, 2007.

P. R. Murray, E. J. Baron, M. A. Pfaller et al., Manual of Clinical Microbiology, American Society of Microbiology, Washington, DC, USA, 1995.