Vol. 63, No. 275 Chem. Pharm. Bull. 63, 75–80 (2015)
Regular Article
Synthesis and Antimicrobial Activity of Amino Linked Heterocycles Lingaladinne Mallikarjuna Reddy, Guda Dinneswara Reddy, Adivireddy Padmaja, and Venkatapuram Padmavathi* Department of Chemistry, Sri Venkateswara University; Tirupati 517 502, Andhra Pradesh, India. Received July 16, 2014; accepted November 18, 2014 Amino-linked benzoxazolyl/benzothiazolyl/benzimidazolyl quinazolines were prepared and their antimicrobial activity studied. The nitro-substituted benzothiazolyl quinazoline (8f) may be a potential antibacterial agent against Staphylococcus aureus and nitro-substituted benzimidazolyl quinazoline (9f) may be a potential antifungal agent against Aspergillus niger. Key words
benzoxazole; benzothiazole; benzimidazole; 2,4-dichloroquinazoline; antimicrobial activity
Heterocyclic compounds have attracted substantial attention due to their broad biological and therapeutic applications. The exploration of new heterocycles that can accommodate potency to multiple biological targets remains an intriguing scientific endeavor. As an important pharmacophore, quinazoline has a variety of biological activities such as anticancer,1) antibacterial2) and anti-inflammatory.3) Different azoles, benzoxazoles, benzothiazoles and benzimidazoles are associated with diverse pharmacological activities viz., analgesic, antituberculosis,4) antimicrobial,5,6) anti-inflammatory, antioxidant and antiviral.7) Synthetic analogues possessing these core structures have considerable medicinal interest and are identified as the important heterocyclic motifs for the development of novel therapeutics. In continuation to extend our search for the development of a variety of heteroaromatics with different pharmacophoric units8) the present work synthesis of amino linked benzoxazolyl/benzothiazolyl/benzimidazolyl quinazolines as antimicrobial precursors has been taken up.
Chemistry
The synthetic intermediate 2,4-dichloroquinazoline (3) was obtained by the reaction of substituted anthranilic acid with urea followed by chlorination with POCl3 in the presence of N,N-dimethylaniline.9) The benzo[d]oxazol-2-amine (4), benzo[d]thiazol-2-amine (5) and 1H-benzo[d]imidazol-2-
Chart 1.
amine (6) were produced from the respective o-disubstituted benzenes with cyanogen bromide in ethanol.10–12) The amino linked tris heterocyclic compound, N2,N4-di(benzo[d]oxazol-2-yl)quinazoline-2,4-diamine (7) was prepared by the reaction of 3 with 4 in the presence of a catalytic amount of conc. HCl. Similarly, N2,N4-di(benzo[d]thiazol-2-yl)quinazoline-2,4-diamine (8) and N2,N4-di(1H-benzo[d]imidazol-2-yl)quinazoline-2,4-diamine (9) were obtained by treating 3 with 5 and 6, respectively (Chart 1). The 1H-NMR spectra of 7a, 8a and 9a displayed a broad singlet at δ 9.32, 9.49 and at 9.12 ppm due to NH protons in addition to the signals of aromatic protons. Besides, compound 9a exhibited another broad singlet at δ 11.98 ppm for NH of benzimidazole. The signals of NH disappeared when D2O was added. The structures of 7–9 were further established by IR, 13C-NMR and elemental analyses.
Biological Results
Antimicrobial Activity Compounds 7–9 were tested for antimicrobial activity at three different concentrations 50, 75 and 100 µg/well. The results of antibacterial activity shown in Table 1 and Fig. 1 revealed that Gram-positive bacteria were more susceptible towards the tested compounds than Gramnegative bacteria. The bis benzothiazolyl quinazolines (8) exhibited higher activity than bis benzoxazolyl quinazolines (7)
Synthesis of Amino Linked Heterocycles
To whom correspondence should be addressed. e-mail: [email protected] * © 2015 The Pharmaceutical Society of Japan
Chem. Pharm. Bull.
76 Table 1.
Vol. 63, No. 2 (2015)
The in Vitro Antibacterial Activity of Compounds 7–9 Diameter of zone of inhibition (mm) Gram positive
Compound
7a 7b 7c 7d 7e 7f 8a 8b 8c 8d 8e 8f 9a 9b 9c 9d 9e 9f Chloramphenicol Control (DMSO)
S. aureus
Gram negative B. subtilis
P. aeruginosa
K. pneumoniae
50 (µg/ well)
75 (µg/ well)
100 (µg/ well)
50 (µg/ well)
75 (µg/ well)
100 (µg/ well)
50 (µg/ well)
75 (µg/ well)
100 (µg/ well)
50 (µg/ well)
75 (µg/ well)
100 (µg/ well)
— — 8±2 — — 10±2 12±2 22±2 25±1 8±2 — 30±3 18±1 20±2 — — 23±2 30±3 —
— 8±1 10±2 — — 13±2 14±1 24±1 28±2 10±1 9±2 33±2 10±2 19±2 23±1 8±1 — 25±1 33±1 —
— 10±2 13±1 — — 14±2 17±1 27±2 30±2 13±2 12±1 35±2 11±3 22±3 25±2 10±2 9±2 27±1 35±2 —
— — — — — — 8±1 14±1 16±1 — — 20±1 — 10±1 12±1 — — 14±2 32±2 —
— — 8±1 — — — 10±2 15±2 17±2 8±2 — 22±2 10±2 13±1 14±2 — — 17±1 34±2 —
— 8±2 11±2 — — — 11±2 18±1 20±2 12±1 10±2 25±1 13±2 15±2 16±1 — — 19±2 38±1 —
— — — — — — — — 11±1 — — 14±1 — — — — — 8±2 25±3 —
— — — — — — — 10±2 14±2 — — 16±2 — — 9±2 — — 11±1 27±2 —
— — — — — — — 12±1 15±2 — — 19±1 — 11±2 13±1 — — 14±2 30±2 —
— — 8±1 — — 9±2 11±1 16±2 18±1 — — 19±1 10±1 13±2 15±2 — — 17±1 38±1 —
— 10±1 11±2 — — 12±1 12±2 18±1 20±2 11±3 — 21±2 12±2 15±1 16±2 — — 19±2 40±2 —
— 12±2 14±1 — — 14±2 14±2 21±2 22±3 15±2 13±2 23±1 14±1 17±2 19±1 10±2 8±2 22±1 42±3 —
—: No activity. ±: Standard deviation.
Fig. 1.
The in Vitro Antibacterial Activity of Compounds 7–9
and bis benzimidazolyl quinazolines (9). It was noticed that 8c and 8f having chloro and nitro substituent on the aromatic ring displayed greater activity particularly on Staphylococcus aureus. In fact the compound 8f exhibited similar activity when compared with the standard drug chloramphenicol at all tested concentrations. However, the compounds 9c and 9f showed good activity on Staphylococcus aureus. Amongst all the tested compounds, 7b, 7c and 7f exhibited least activity whereas the other derivatives of 7 were not active. It was further observed that chloro, bromo and nitro substituted compounds showed higher activity than those having methyl and
methoxy substituents. This indicated that electron withdrawing substituents enhanced the activity than electron donating ones. In fact, the compounds having methoxy substituent displayed least activity which may be due to mesomeric effect. The results of antifungal activity shown in Table 2 and Fig. 2 indicated that all the tested compounds displayed higher activity on Aspergillus niger than on Penicillium chrysogenum. The compounds 8 and 9 exhibited higher activity than 7. The results further revealed that bis benzimidazolyl quinazolines (9) displayed greater activity than bis benzothiazolyl quinazolines (8). The activity exhibited by 9f on A. niger was
Chem. Pharm. Bull. Vol. 63, No. 2 (2015)77 Table 2.
The in Vitro Antifungal Activity of Compounds 7–9 Diameter of zone of inhibition (mm) A. niger
Compound 50 µg/well 7a 7b 7c 7d 7e 7f 8a 8b 8c 8d 8e 8f 9a 9b 9c 9d 9e 9f Ketoconazole Control (DMSO)
— — — — — 8±1 10±2 14±2 18±2 — — 22±1 — 16±2 22±2 — — 29±1 31±2 —
75 µg/well
P. chrysogenum 100 µg/well
— — 8±2 — — 10±1 12±1 17±1 20±1 — — 25±2 8±1 19±1 25±2 — — 32±2 33±3 —
— 9±3 11±1 — — 12±2 15±2 19±2 23±2 — — 28±1 10±2 21±2 27±1 — — 36±1 36±2 —
50 µg/well — — — — — — — 8±1 10±2 — — 11±2 — 12±1 15±1 — — 17±2 35±1 —
75 µg/well — — — — — — — 9±2 12±1 — — 13±2 — 14±2 17±2 — — 19±1 36±2 —
100 µg/well — — — — — 10±1 12±2 15±2 — — 17±2 — 17±1 19±2 — — 21±2 38±3 —
—: No activity. ±: Standard deviation.
Fig. 2.
The in Vitro Antifungal Activity of Compounds 7–9
similar to the standard ketoconazole at 100 µg/well. The structure–antimicrobial activity of the tested compounds revealed that those having benzimidazolyl and benzothiazolyl moieties showed greater activity than the compounds with benzoxazolyl unit. The presence of chloro, bromo and nitro substituents on the quinazoline ring enhanced the activity. The compounds which showed greater antibacterial and antifungal activities are further assayed for the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) and the values are listed in Table 3. MIC is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism. (But it is not sure that the microorganisms are completely killed.) The MBC/MFC is the lowest concen-
tration of antibiotic required to kill a particular bacterium/ fungi. The MBC/MFC involves an additional set of steps performed once the MIC is determined. The antimicrobials are usually regarded as bactericidal/fungicidal if the MBC/ MFC is not greater than four times the MIC.13) The compound 8f exhibited low MIC values against Staphylococcus aureus when compared with 8c, 9c and 9f. In fact, the MIC value of 8f against Staphylococcus aureus is equal to the standard drug chloramphenicol and the MBC value is 2×MIC. On the other hand, the compound 9f displayed low MIC value on Aspergillus niger than 8c, 8f and 9c equal to the standard ketoconazole and the MFC value is 2×MIC. This suggested that nitro substituted benzothiazolyl quinazoline 8f is the potential antibacterial agent against Staphylococcus aureus and nitro
Chem. Pharm. Bull.
78 Table 3.
Vol. 63, No. 2 (2015)
MIC, MBC and MFC of Compounds 8c, 8f, 9c and 9f
Compound 8c 8f 9c 9f Chloramphenicol Ketoconazole
Minimum inhibitory concentration MIC (MBC/MFC) µg/well S. aureus
B. subtilis
P. aeruginosa
K. pneumoniae
A. niger
P. chrysogenum
25 (100) 6.25 (12.5) 50 (200) 50 (200) 6.25 —
200 (—) 100 (>200) 200 (—) 200 (—) 6.25 —
200 (—) 200 (—) — 200 (—) 6.25 —
200 (—) 200 (—) 200 (—) 200 (—) 12.5 —
50 (200) 25 (100) 25 (100) 6.25 (12.5) — 6.25
200 (—) 200 (—) 200 (—) 200 (—) — 12.5
S=Staphylococcus; B=Bacillus; P=Pseudomonas; K=Klebsiella; A=Aspergillus; P=Penicillium.
substituted benzimidazolyl quinazoline 9f is the potential antifungal agent against Aspergillus niger.
Conclusion
The amino linked benzoxazolyl/benzothiazolyl/benzimidazolyl quinazolines were prepared adopting simple and versatile synthetic methodologies and tested for their antimicrobial activity. The nitro substituted benzothiazolyl quinazoline (8f) is the potential antibacterial agent against Staphylococcus aureus (35 mm, MIC 6.25 µg/mL) and nitro substituted benzimidazolyl quinazoline (9f) is the potential antifungal agent against Aspergillus niger (36 mm, MIC 6.25 µg/mL).
Experimental
General Melting points (mp) were determined in open capillaries on a melting temperature apparatus and are uncorrected. The purity of the compounds was checked by TLC (silica gel H, BDH, ethyl acetate–hexane, 1 : 3). The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and the wave numbers were given in cm−1. The 1 H- and 13C-NMR spectra were recorded in dimethylsulfoxide (DMSO)-d6 on a Bruker-400 spectrometer operating at 400 and 100 MHz, respectively. All chemical shifts are reported in δ (ppm) using tetramethylsilane (TMS) as an internal standard. The microanalyses were performed on a Perkin-Elmer 240C elemental analyzer. The compounds 2,4-dichloroquinazoline9) (3), benzo[d]oxazol-2-amine10) (4), benzo[d]thiazol-2-amine11) (5) and 1H-benzo[d]imidazol-2-amine12) (6) were prepared by adopting the literature precedents. General Procedure for the Synthesis of N2,N4Di(benzo[d]oxazol-2-yl)quinazoline-2,4-diamine (7a–d)/ N2,N4-Di(benzo[d]thiazol-2-yl)quinazoline-2,4-diamine (8a– d)/N2, N4-Di(1H-benzo[d ]imidazol-2-yl)quinazo line-2,4-diamine (9a–d) A solution of 2,4-dichloroquinazoline (3) (1.00 mmol), benzo[d]oxazol-2-amine (4)/ benzo[d]thiazol-2-amine (5)/1H-benzo[d]imidazol-2-amine (6) (2.50 mmol) and a catalytic amount of conc. HCl (0.2 mL) in 2-propanol (5 mL) was stirred at room temperature for 6–8 h. After completion of the reaction, the separated solid was filtered, dried and recrystallized from 2-propanol. N2,N4-Di(benzo[d]oxazol-2-yl)quinazoline-2,4-diamine (7a) White needles, yield 75%, mp 136–138°C. IR (KBr) cm−1: 1547 (C=N), 3284 (NH). 1H-NMR (DMSO-d6) δ: 7.03–8.22 (m, 12H, Ar-H), 9.32 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 160.2 (C-2′), 171.6 (C-4), 179.3 (C-2), 111.8, 112.4, 116.3, 122.1, 123.6, 125.7, 127.3, 128.6, 133.2, 144.5, 146.9, 151.7 (aromatic carbons). Anal. Calcd for C22H14N6O2: C, 67.00; H, 3.58; N, 21.31. Found: C, 67.10; H, 3.60; N, 21.46.
N2,N4-Di(benzo[d]oxazol-2-yl)-6-bromoquinazoline-2,4diamine (7b) White needles, yield 69%, mp 171–173°C. IR (KBr) cm−1: 1550 (C=N), 3288 (NH). 1H-NMR (DMSO-d6) δ: 7.00–8.26 (m, 11H, Ar-H), 9.36 ( br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 161.4 (C-2′), 172.3 (C-4), 180.4 (C-2), 108.3, 117.3, 121.4, 137.4, 127.3, 151.8, 143.5, 115.4, 124.1, 122.5, 110.6, 146.5 (aromatic carbons). Anal. Calcd for C22H13BrN6O2: C, 55.83; H, 2.77; N, 17.76. Found: C, 55.96; H, 2.80; N, 17.96. N2,N4-Di(benzo[d]oxazol-2-yl)-6-chloroquinazoline-2,4diamine (7c) White needles, yield 77%, mp 163–165°C. IR (KBr) cm−1: 1553 (C=N), 3296 (NH). 1H-NMR (DMSO-d6) δ: 7.06-8.18 (m, 11H, Ar-H), 9.45 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 162.1 (C-2′), 173.4 (C-4), 181.6 (C-2), 109.1, 110.3, 116.3, 117.8, 122.1, 125.3, 127.2, 129.1, 138.2, 146.1, 148.1, 152.3 (aromatic carbons). Anal. Calcd for C22H13Cl N6O2: C, 61.62; H, 3.06; N, 19.60. Found: C, 61.70; H, 3.05; N, 19.78. N2,N4-Di(benzo[d]oxazol-2-yl)-6-methylquinazoline-2,4diamine (7d) White needles, yield 72%, mp 152–154°C. IR (KBr) cm−1: 1549 (C=N), 3291 (NH). 1H-NMR (DMSO-d6) δ: 2.51 (s, 3H, Ar-CH3), 7.07–8.14 (m, 11H, Ar-H), 9.24 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 23.4 (Ar-CH3), 159.6 (C-2′), 169.6 (C-4), 176.8 (C-2), 111.3, 112.6, 116.1, 133.2, 136.5, 123.6, 124.4, 125.3, 126.4, 145.2, 147.5, 148.6 (aromatic carbons). Anal. Calcd for C23H16N6O2: C, 67.64; H, 3.95; N, 20.58. Found: C, 67.76; H, 3.99; N, 20.79. N2,N4-Di(benzo[d]oxazol-2-yl)-6-methoxyquinazoline-2,4diamine (7e) White needles, yield 74%, mp 159–161°C. IR (KBr) cm−1: 1538 (C=N), 3278 (NH). 1H-NMR (DMSO-d6) δ: 3.92 (s, 3H, Ar-OCH3), 6.98–8.24 (m, 11H, Ar-H), 9.29 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 53.2 (Ar-OCH3), 160.8 (C-2′), 172.7 (C-4), 180.3 (C-2), 98.2, 110.4, 111.6, 115.6, 124.8, 126.1, 127.4, 128.6, 144.8, 146.2, 146.7, 155.5 (aromatic carbons). Anal. Calcd for C23H16N6O3: C, 65.09; H, 3.80; N, 19.80. Found: C, 65.15; H, 3.78; N, 19.92. N2,N4-Di(benzo[d]oxazol-2-yl)-6-nitroquinazoline-2,4-diamine (7f) White needles, yield 78%, mp 168–170°C. IR (KBr) cm−1: 1559 (C=N), 3302 (NH). 1H-NMR (DMSO-d6) δ: 7.12–8.15 (m, 11H, Ar-H), 9.52 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 163.5 (C-2′), 174.3 (C-4), 182.4 (C-2), 107.2, 116.5, 142.6, 127.6, 125.8, 151.6, 145.2, 117.3, 126.6, 124.5, 112.2, 148.3 (aromatic carbons). Anal. Calcd for C22H13N7O4: C, 60.14; H, 2.98; N, 22.31. Found: C, 60.25; H, 2.99; N, 22.49. N2,N4-Di(benzo[d]thiazol-2-yl)quinazoline-2,4-diamine (8a) White needles, yield 76%, mp 162–164°C. IR (KBr) cm−1: 1552 (C=N), 3292 (NH). 1H-NMR (DMSO-d6) δ: 6.92–8.23 (m, 12H, Ar-H), 9.49 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 166.1 (C-2′), 172.3 (C-4), 180.6 (C-2), 110.9, 111.8, 118.1, 119.6,
Chem. Pharm. Bull. Vol. 63, No. 2 (2015)79
120.1, 123.7, 125.4, 127.5, 128.1, 132.4, 151.7, 152.4 (aromatic carbons). Anal. Calcd for C22H14N6S2: C, 61.95; H, 3.31; N, 19.70. Found: C, 62.08; H, 3.34; N, 19.87. N2,N4-Di(benzo[d]thiazol-2-yl)-6-bromoquinazoline-2,4diamine (8b) White needles, yield 80%, mp 193–195°C. IR (KBr) cm−1: 1558 (C=N), 3298 (NH). 1H-NMR (DMSO-d6) δ: 6.86–8.20 (m, 11H, Ar-H), 9.52 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 167.2 (C-2′), 173.6 (C-4), 181.2 (C-2), 109.2, 118.5, 122.1, 138.7, 128.4, 152.1, 152.4, 119.4, 125.4, 120.4, 117.6, 130.3 (aromatic carbons). Anal. Calcd for C22H13BrN6S2: C, 52.28; H, 2.59; N, 16.63. Found: C, 52.35; H, 2.58; N, 16.77. N2,N4-Di(benzo[d]thiazol-2-yl)-6-chloroquinazoline-2,4diamine (8c) White needles, yield 78%, mp 182–184°C. IR (KBr) cm−1: 1564 (C=N), 3315 (NH). 1H-NMR (DMSO-d6) δ: 6.96–8.25 (m, 11H, Ar-H), 9.56 (br s, 2H, NH).13C-NMR (DMSO-d6) δ: 168.3 (C-2′), 174.3 (C-4), 182.7 (C-2), 111.2, 113.1, 119.2, 120.2, 121.3, 122.4, 127.2, 128.2, 129.1, 132.1, 149.1, 154.2 (aromatic carbons). Anal. Calcd for C22H13Cl N6S2: C, 57.32; H, 2.84; N, 18.23. Found: C, 57.28; H, 2.86; N, 18.33. N2,N4-Di(benzo[d]thiazol-2-yl)-6-methylquinazoline-2,4diamine (8d) White needles, yield 73%, mp 168–170°C. IR (KBr) cm−1: 1558 (C=N), 3307 (NH). 1H-NMR (DMSO-d6) δ: 2.58 (s, 3H, Ar-CH3), 6.88–7.92 (m, 11H, Ar-H), 9.38 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 24.2 (Ar-CH3), 165.6 (C-2′), 170.5 (C-4), 177.3 (C-2), 111.8, 112.7, 119.9, 120.8, 121.5, 124.1, 125.8, 126.6, 133.8, 137.2, 149.2, 153.9 (aromatic carbons). Anal. Calcd for C23H16N6S2: C, 62.71; H, 3.66; N, 19.08. Found: C, 62.83; H, 3.69; N, 19.21. N2,N4-Di(benzo[d]thiazol-2-yl)-6-methoxyquinazoline-2,4diamine (8e) White needles, yield 75%, mp 171–173°C. IR (KBr) cm−1: 1545 (C=N), 3286 (NH). 1H-NMR (DMSO-d6) δ: 3.97 (s, 3H, Ar-OCH3), 7.07–8.26 (m, 11H, Ar-H), 9.48 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 54.4 (Ar-OCH3), 167.3 (C-2′), 173.5 (C-4), 182.6 (C-2), 98.6, 111.8, 112.8, 118.6, 119.1, 120.6, 125.9, 127.2, 128.1, 146.8, 152.8, 155.7 (aromatic carbons). Anal. Calcd for C23H16N6S2O: C, 60.51; H, 3.53; N, 18.41. Found: C, 60.58; H, 3.55; N, 18.56. N2,N4-Di(benzo[d]thiazol-2-yl)-6-nitroquinazoline-2,4-diamine (8f) White needles, yield 81%, mp 187–189°C. IR (KBr) cm−1: 1568 (C=N), 3319 (NH). 1H-NMR (DMSO-d6) δ: 7.02–8.18 (m, 11H, Ar-H), 9.59 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 169.4 (C-2′), 175.1 (C-4), 184.2 (C-2), 108.2, 117.6, 143.2, 128.4, 126.5, 152.1, 154.3, 122.1, 127.2, 122.4, 119.6, 132.6 (aromatic carbons). Anal. Calcd for C22H13N7O2S2: C, 56.04; H, 2.78; N, 20.79. Found: C, 56.09; H, 2.79; N, 20.90. N2,N4-Di(1H-benzo[d]imidazol-2-yl)quinazoline-2,4-diamine (9a) White needles, yield 65%, mp 150–152°C. IR (KBr) cm−1: 1531 (C=N), 3273 (NH). 1H-NMR (DMSO-d6) δ: 6.85–8.26 (m, 12H, Ar-H), 9.12 (br s, 2H, NH), 11.98 (br s, 2H, imidazole-NH). 13C-NMR (DMSO-d6) δ: 155.3 (C-2′), 169.5 (C-4), 177.8 (C-2), 110.5, 111.4, 119.6, 123.2, 128.6, 129.6, 132.4, 137.2, 151.3 (aromatic carbons). Anal. Calcd for C22H16N8: C, 67.34; H, 4.11; N, 28.55. Found: C, 67.45; H, 4.09; N, 28.71. N2,N4-Di(benzo[d]thiazol-2-yl)-6-bromoquinazoline-2,4diamine (9b) White needles, yield 63%, mp 188–190°C. IR (KBr) cm−1: 1538 (C=N), 3279 (NH). 1H-NMR (DMSO-d6) δ: 6.81–8.29 (m, 11H, Ar-H), 9.16 (br s, 2H, NH), 12.04 (br s, 2H, imidazole-NH). 13C-NMR (DMSO-d6) δ: 156.1 (C-2′), 170.6 (C-4), 178.1 (C-2), 107.1, 115.4, 120.2, 136.3, 126.4, 150.1, 137.3, 111.2, 119.4 (aromatic carbons). Anal. Calcd for
C22H15BrN8: C, 56.06; H, 3.21; N, 23.77. Found: C, 56.15; H, 3.23; N, 23.92. N2,N4-Di(1H-benzo[d]imidazol-2-yl)-6-chloroquinazoline-2,4-diamine (9c) White needles, yield 70%, mp 181–183°C. IR (KBr) cm−1: 1543 (C=N), 3286 (NH). 1H-NMR (400 MHz, DMSO-d6) δ: 6.91–8.31 (m, 11H, Ar-H), 9.24 (br s, 2H, NH), 12.14 (br s, 2H, imidazole-NH). 13C-NMR (DMSOd6) δ: 157.1 (C-2′), 172.4 (C-4), 179.4 (C-2), 110.4, 113.3, 119.3, 121.2, 128.4, 130.7, 131.4, 139.2, 149.9 (aromatic carbons). Anal. Calcd for C22H15ClN8: C, 61.90; H, 3.54; N, 26.25. Found: C, 61.95; H, 3.55; N, 26.36. N2,N4-Di(1H-benzo[d]imidazol-2-yl)-6-methylquinazoline-2,4-diamine (9d) White needles, yield 66%, mp 172–174°C. IR (KBr) cm−1: 1538 (C=N), 3279 (NH). 1H-NMR (DMSO-d6) δ: 2.42 (s, 3H, Ar-CH3), 6.82–8.16 (m, 11H, Ar-H), 9.05 (br s, 2H, NH), 11.81 (br s, 2H, imidazole-NH). 13C-NMR (DMSO-d6) δ: 22.5 (Ar-CH3), 154.3 (C-2′), 167.1 (C-4), 174.6 (C-2), 111.8, 112.1, 120.4, 124.1, 125.8, 133.8, 137.2, 138.4, 149.2 (aromatic carbons). Anal. Calcd for C23H18N8: C, 67.97; H, 4.46; N, 27.57. Found: C, 68.10; H, 4.49; N, 27.79. N2,N4-Di(1H-benzo[d]imidazol-2-yl)-6-methoxyquinazoline-2,4-diamine (9e) White needles, yield 68%, mp 177–179°C. IR (KBr) cm−1: 1525 (C=N), 3264 (NH). 1H-NMR (DMSO-d6) δ: 3.85 (s, 3H, Ar-OCH3), 6.93–8.12 (m, 11H, Ar-H), 9.18 (br s, 2H, NH), 12.06 (br s, 2H, imidazole-NH). 13 C-NMR (DMSO-d6) δ: 52.8 (Ar-OCH3), 156.8 (C-2′), 168.5 (C-4), 177.8 (C-2), 98.4, 110.2, 111.8, 119.2, 127.6, 129.3, 137.8, 146.7, 155.7 (aromatic carbons). Anal. Calcd for C23H18N8O: C, 65.39; H, 4.29; N, 26.53. Found: C, 65.47; H, 4.31; N, 26.70. N2,N4-Di(benzo[d]thiazol-2-yl)-6-nitroquinazoline-2,4-diamine (9f) White needles, yield 64%, mp 185–187°C. IR (KBr) cm−1: 1548 (C=N), 3292 (NH). 1H-NMR (DMSO-d6) δ: 6.95–8.36 (m, 11H, Ar-H), 9.28 (br s, 2H, NH), 12.18 (br s, 2H, imidazole-NH). 13C-NMR (DMSO-d6) δ: 158.3 (C-2′), 173.2 (C-4), 180.5 (C-2), 106.2, 115.6, 143.2, 128.7, 126.5, 152.1, 139.4, 113.5, 121.2 (aromatic carbons). Anal. Calcd for C22H15N9O2: C, 60.41; H, 3.46; N, 28.82. Found: C, 60.52; H, 3.45; N, 28.98. Biological Assays. Compounds The compounds 7–9 were evaluated for antimicrobial activity by agar well diffusion and broth dilution methods. Cells Bacterial strains Staphylococcus aureus, Bacillus subtilis, Pseudomonus aeruginosa, Klebsiella pneumoniae and fungi Aspergillus niger, Penicillium chrysogenum were obtained from the Department of Microbiology, S.V. University, Tirupati. Antibacterial and Antifungal Assays The in vitro antimicrobial studies were carried out by agar well diffusion method against test organisms.14,15) Nutrient broth (NB) plates were swabbed with 24 h old broth culture (100 µL) of test bacteria. Using the sterile cork borer, wells (6 mm) were made into each petriplate. The compounds were dissolved in DMSO of 5 mg/mL and from this 10 µL, 15 µL and 20 µL (50, 75 and 100 µg/well) were added into the wells by using sterile pipettes. The standard antibiotics, chloramphenicol for antibacterial activity and ketoconazole for antifungal activity (as positive control) were simultaneously tested against the pathogens. The samples were dissolved in DMSO which showed no zone of inhibition acts as a negative control. The plates were incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. After appropriate incubation, the diameter of zone of
80
Chem. Pharm. Bull.
inhibition of each well was measured. Duplicates were maintained and the average values were calculated for eventual antibacterial activity. Minimum Inhibitory Concentration Assay Broth dilution test was used to determine MIC of the above mentioned samples.16,17) Freshly prepared nutrient broth was used as diluents. The 24 h old culture of the test bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonus aeruginosa and Klebsilla pneumoniae and the test fungi Aspergillus niger and Penicillium chrysogenum were diluted 100 fold in nutrient broth (100 µL bacterial cultures in 10 mL NB). The stock solution of the synthesized compounds was prepared in DMSO by dissolving 5 mg of the compound in 1 mL of DMSO. Increasing concentrations of the test samples (1.25, 2.5, 5, 10, 15, 20, 40 µL of stock solution contains 6.25, 12.5, 25, 50, 75, 100, 200 µg of the compounds) were added to the test tubes containing the bacterial and fungal cultures. All the tubes were incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. The tubes were examined for visible turbidity using NB as control. Control without test samples and with solvent was assayed simultaneously. The lowest concentration that inhibited visible growth of the tested organisms was recorded as MIC. Minimum Bactericidal/Fungicidal Concentration To determine the MBC18) and MFC19) for each set of test tubes in the MIC determination, a loopful of broth was collected from those tubes which did not show any growth and inoculated on sterile nutrient broth (for bacteria) and potato dextrose agar (PDA) (for fungi) by streaking. Plates inoculated with bacteria and fungi were incubated at 37°C for 24 h and at 28°C for 48 h, respectively. After incubation, the lowest concentration was noted as MBC (for bacteria) or MFC (for fungi) at which no visible growth was observed. Acknowledgments The authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India for the sanction of Major Research Project. One of the authors Mr. L. Mallikarjuna Reddy is thankful to CSIR, New Delhi, India for the sanction of Junior Research Fellowship. Conflict of Interest interest.
The authors declare no conflict of
References
Vol. 63, No. 2 (2015)
1) Mendelsohn J., Baselga J., Oncogene, 19, 6550–6565 (2000). 2) Nesterova I. N., Radkevich T. P., Granik V. G., Pharm. Chem. J., 25, 786–789 (1991). 3) Chao Q., Deng L., Shih H., Leoni L. M., Genini D., Carson D. A., Cottam H. B., J. Med. Chem., 42, 3860–3873 (1999). 4) Klimešová V., Koci J., Waisser K., Kaustova J., Mollmann U., Eur. J. Med. Chem., 44, 2286–2293 (2009). 5) Sheng C., Xu H., Wang W., Cao Y., Dong G., Wang S., Che X., Ji H., Miao Z., Yao J., Zhang W., Eur. J. Med. Chem., 45, 3531–3540 (2010). 6) Alper-Hayta S., Arisoy M., Temiz-Arpaci O., Yildiz I., Aki E., Ozkan S., Kaynak F., Eur. J. Med. Chem., 43, 2568–2578 (2008). 7) Satyendra R. V., Vishnumurthy K. A., Vagdevi H. M., Rajesh K. P., Manjunatha H., Shruthi A., Eur. J. Med. Chem., 46, 3078–3084 (2011). 8) Patel R. V., Kumari P., Rajani D. P., Chikhalia K. H., Med. Chem. Res., 22, 195–210 (2013). 9) Smits R. A., Adami M., Istyastono E. P., Zuiderveld O. P., van Dam C. M. E., de Kanter F. J. J., Jongejan A., Coruzzi G., Leurs R., de Esch I. J. P., J. Med. Chem., 53, 2390–2400 (2010). 10) Saritha G., Sarangapani M., Prasad G., Swathi C., Der Pharm. Lett., 3, 427–432 (2011). 11) Wu. Y.-Q., Limburg D. C., Wilkinson D. E., Hamilton G. S., J. Het. Chem., 40, 191–193 (2003). 12) Huigens R. W. III, Reyes S., Reed C. S., Bunders C., Rogers S. A., Steinhauer A. T., Melander C., Bioorg. Med. Chem., 18, 663–674 (2010). 13) French G. L., J. Antimicrob. Chemother., 58, 1107–1117 (2006). 14) Chung K. T., Thomasson W. R., Wu-Yuan C. D., J. Appl. Bacteriol., 69, 498–503 (1990). 15) Azoro C., World J. Biotechnol., 3, 347–351 (2002). 16) Janovska D., Kubikova K., Kokoska L., Czech J. Food Sci., 21, 107–110 (2003). 17) Joshi B., Lekhak S., Sharma A., J. Sci. Eng. Technol., 5, 143–150 (2009). 18) Clinical and Laboratory Standards Institute, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, CLSI document M7-Az,” 7th ed., Wayne, Pennsylvania, U.S.A., 2006. 19) “Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard, NCCLS document M27-A2,” 2nd ed., Wayne, Pennsylvania, U.S.A., 2002.
Regular Article
Synthesis and Antimicrobial Activity of Amino Linked Heterocycles Lingaladinne Mallikarjuna Reddy, Guda Dinneswara Reddy, Adivireddy Padmaja, and Venkatapuram Padmavathi* Department of Chemistry, Sri Venkateswara University; Tirupati 517 502, Andhra Pradesh, India. Received July 16, 2014; accepted November 18, 2014 Amino-linked benzoxazolyl/benzothiazolyl/benzimidazolyl quinazolines were prepared and their antimicrobial activity studied. The nitro-substituted benzothiazolyl quinazoline (8f) may be a potential antibacterial agent against Staphylococcus aureus and nitro-substituted benzimidazolyl quinazoline (9f) may be a potential antifungal agent against Aspergillus niger. Key words
benzoxazole; benzothiazole; benzimidazole; 2,4-dichloroquinazoline; antimicrobial activity
Heterocyclic compounds have attracted substantial attention due to their broad biological and therapeutic applications. The exploration of new heterocycles that can accommodate potency to multiple biological targets remains an intriguing scientific endeavor. As an important pharmacophore, quinazoline has a variety of biological activities such as anticancer,1) antibacterial2) and anti-inflammatory.3) Different azoles, benzoxazoles, benzothiazoles and benzimidazoles are associated with diverse pharmacological activities viz., analgesic, antituberculosis,4) antimicrobial,5,6) anti-inflammatory, antioxidant and antiviral.7) Synthetic analogues possessing these core structures have considerable medicinal interest and are identified as the important heterocyclic motifs for the development of novel therapeutics. In continuation to extend our search for the development of a variety of heteroaromatics with different pharmacophoric units8) the present work synthesis of amino linked benzoxazolyl/benzothiazolyl/benzimidazolyl quinazolines as antimicrobial precursors has been taken up.
Chemistry
The synthetic intermediate 2,4-dichloroquinazoline (3) was obtained by the reaction of substituted anthranilic acid with urea followed by chlorination with POCl3 in the presence of N,N-dimethylaniline.9) The benzo[d]oxazol-2-amine (4), benzo[d]thiazol-2-amine (5) and 1H-benzo[d]imidazol-2-
Chart 1.
amine (6) were produced from the respective o-disubstituted benzenes with cyanogen bromide in ethanol.10–12) The amino linked tris heterocyclic compound, N2,N4-di(benzo[d]oxazol-2-yl)quinazoline-2,4-diamine (7) was prepared by the reaction of 3 with 4 in the presence of a catalytic amount of conc. HCl. Similarly, N2,N4-di(benzo[d]thiazol-2-yl)quinazoline-2,4-diamine (8) and N2,N4-di(1H-benzo[d]imidazol-2-yl)quinazoline-2,4-diamine (9) were obtained by treating 3 with 5 and 6, respectively (Chart 1). The 1H-NMR spectra of 7a, 8a and 9a displayed a broad singlet at δ 9.32, 9.49 and at 9.12 ppm due to NH protons in addition to the signals of aromatic protons. Besides, compound 9a exhibited another broad singlet at δ 11.98 ppm for NH of benzimidazole. The signals of NH disappeared when D2O was added. The structures of 7–9 were further established by IR, 13C-NMR and elemental analyses.
Biological Results
Antimicrobial Activity Compounds 7–9 were tested for antimicrobial activity at three different concentrations 50, 75 and 100 µg/well. The results of antibacterial activity shown in Table 1 and Fig. 1 revealed that Gram-positive bacteria were more susceptible towards the tested compounds than Gramnegative bacteria. The bis benzothiazolyl quinazolines (8) exhibited higher activity than bis benzoxazolyl quinazolines (7)
Synthesis of Amino Linked Heterocycles
To whom correspondence should be addressed. e-mail: [email protected] * © 2015 The Pharmaceutical Society of Japan
Chem. Pharm. Bull.
76 Table 1.
Vol. 63, No. 2 (2015)
The in Vitro Antibacterial Activity of Compounds 7–9 Diameter of zone of inhibition (mm) Gram positive
Compound
7a 7b 7c 7d 7e 7f 8a 8b 8c 8d 8e 8f 9a 9b 9c 9d 9e 9f Chloramphenicol Control (DMSO)
S. aureus
Gram negative B. subtilis
P. aeruginosa
K. pneumoniae
50 (µg/ well)
75 (µg/ well)
100 (µg/ well)
50 (µg/ well)
75 (µg/ well)
100 (µg/ well)
50 (µg/ well)
75 (µg/ well)
100 (µg/ well)
50 (µg/ well)
75 (µg/ well)
100 (µg/ well)
— — 8±2 — — 10±2 12±2 22±2 25±1 8±2 — 30±3 18±1 20±2 — — 23±2 30±3 —
— 8±1 10±2 — — 13±2 14±1 24±1 28±2 10±1 9±2 33±2 10±2 19±2 23±1 8±1 — 25±1 33±1 —
— 10±2 13±1 — — 14±2 17±1 27±2 30±2 13±2 12±1 35±2 11±3 22±3 25±2 10±2 9±2 27±1 35±2 —
— — — — — — 8±1 14±1 16±1 — — 20±1 — 10±1 12±1 — — 14±2 32±2 —
— — 8±1 — — — 10±2 15±2 17±2 8±2 — 22±2 10±2 13±1 14±2 — — 17±1 34±2 —
— 8±2 11±2 — — — 11±2 18±1 20±2 12±1 10±2 25±1 13±2 15±2 16±1 — — 19±2 38±1 —
— — — — — — — — 11±1 — — 14±1 — — — — — 8±2 25±3 —
— — — — — — — 10±2 14±2 — — 16±2 — — 9±2 — — 11±1 27±2 —
— — — — — — — 12±1 15±2 — — 19±1 — 11±2 13±1 — — 14±2 30±2 —
— — 8±1 — — 9±2 11±1 16±2 18±1 — — 19±1 10±1 13±2 15±2 — — 17±1 38±1 —
— 10±1 11±2 — — 12±1 12±2 18±1 20±2 11±3 — 21±2 12±2 15±1 16±2 — — 19±2 40±2 —
— 12±2 14±1 — — 14±2 14±2 21±2 22±3 15±2 13±2 23±1 14±1 17±2 19±1 10±2 8±2 22±1 42±3 —
—: No activity. ±: Standard deviation.
Fig. 1.
The in Vitro Antibacterial Activity of Compounds 7–9
and bis benzimidazolyl quinazolines (9). It was noticed that 8c and 8f having chloro and nitro substituent on the aromatic ring displayed greater activity particularly on Staphylococcus aureus. In fact the compound 8f exhibited similar activity when compared with the standard drug chloramphenicol at all tested concentrations. However, the compounds 9c and 9f showed good activity on Staphylococcus aureus. Amongst all the tested compounds, 7b, 7c and 7f exhibited least activity whereas the other derivatives of 7 were not active. It was further observed that chloro, bromo and nitro substituted compounds showed higher activity than those having methyl and
methoxy substituents. This indicated that electron withdrawing substituents enhanced the activity than electron donating ones. In fact, the compounds having methoxy substituent displayed least activity which may be due to mesomeric effect. The results of antifungal activity shown in Table 2 and Fig. 2 indicated that all the tested compounds displayed higher activity on Aspergillus niger than on Penicillium chrysogenum. The compounds 8 and 9 exhibited higher activity than 7. The results further revealed that bis benzimidazolyl quinazolines (9) displayed greater activity than bis benzothiazolyl quinazolines (8). The activity exhibited by 9f on A. niger was
Chem. Pharm. Bull. Vol. 63, No. 2 (2015)77 Table 2.
The in Vitro Antifungal Activity of Compounds 7–9 Diameter of zone of inhibition (mm) A. niger
Compound 50 µg/well 7a 7b 7c 7d 7e 7f 8a 8b 8c 8d 8e 8f 9a 9b 9c 9d 9e 9f Ketoconazole Control (DMSO)
— — — — — 8±1 10±2 14±2 18±2 — — 22±1 — 16±2 22±2 — — 29±1 31±2 —
75 µg/well
P. chrysogenum 100 µg/well
— — 8±2 — — 10±1 12±1 17±1 20±1 — — 25±2 8±1 19±1 25±2 — — 32±2 33±3 —
— 9±3 11±1 — — 12±2 15±2 19±2 23±2 — — 28±1 10±2 21±2 27±1 — — 36±1 36±2 —
50 µg/well — — — — — — — 8±1 10±2 — — 11±2 — 12±1 15±1 — — 17±2 35±1 —
75 µg/well — — — — — — — 9±2 12±1 — — 13±2 — 14±2 17±2 — — 19±1 36±2 —
100 µg/well — — — — — 10±1 12±2 15±2 — — 17±2 — 17±1 19±2 — — 21±2 38±3 —
—: No activity. ±: Standard deviation.
Fig. 2.
The in Vitro Antifungal Activity of Compounds 7–9
similar to the standard ketoconazole at 100 µg/well. The structure–antimicrobial activity of the tested compounds revealed that those having benzimidazolyl and benzothiazolyl moieties showed greater activity than the compounds with benzoxazolyl unit. The presence of chloro, bromo and nitro substituents on the quinazoline ring enhanced the activity. The compounds which showed greater antibacterial and antifungal activities are further assayed for the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) and the values are listed in Table 3. MIC is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism. (But it is not sure that the microorganisms are completely killed.) The MBC/MFC is the lowest concen-
tration of antibiotic required to kill a particular bacterium/ fungi. The MBC/MFC involves an additional set of steps performed once the MIC is determined. The antimicrobials are usually regarded as bactericidal/fungicidal if the MBC/ MFC is not greater than four times the MIC.13) The compound 8f exhibited low MIC values against Staphylococcus aureus when compared with 8c, 9c and 9f. In fact, the MIC value of 8f against Staphylococcus aureus is equal to the standard drug chloramphenicol and the MBC value is 2×MIC. On the other hand, the compound 9f displayed low MIC value on Aspergillus niger than 8c, 8f and 9c equal to the standard ketoconazole and the MFC value is 2×MIC. This suggested that nitro substituted benzothiazolyl quinazoline 8f is the potential antibacterial agent against Staphylococcus aureus and nitro
Chem. Pharm. Bull.
78 Table 3.
Vol. 63, No. 2 (2015)
MIC, MBC and MFC of Compounds 8c, 8f, 9c and 9f
Compound 8c 8f 9c 9f Chloramphenicol Ketoconazole
Minimum inhibitory concentration MIC (MBC/MFC) µg/well S. aureus
B. subtilis
P. aeruginosa
K. pneumoniae
A. niger
P. chrysogenum
25 (100) 6.25 (12.5) 50 (200) 50 (200) 6.25 —
200 (—) 100 (>200) 200 (—) 200 (—) 6.25 —
200 (—) 200 (—) — 200 (—) 6.25 —
200 (—) 200 (—) 200 (—) 200 (—) 12.5 —
50 (200) 25 (100) 25 (100) 6.25 (12.5) — 6.25
200 (—) 200 (—) 200 (—) 200 (—) — 12.5
S=Staphylococcus; B=Bacillus; P=Pseudomonas; K=Klebsiella; A=Aspergillus; P=Penicillium.
substituted benzimidazolyl quinazoline 9f is the potential antifungal agent against Aspergillus niger.
Conclusion
The amino linked benzoxazolyl/benzothiazolyl/benzimidazolyl quinazolines were prepared adopting simple and versatile synthetic methodologies and tested for their antimicrobial activity. The nitro substituted benzothiazolyl quinazoline (8f) is the potential antibacterial agent against Staphylococcus aureus (35 mm, MIC 6.25 µg/mL) and nitro substituted benzimidazolyl quinazoline (9f) is the potential antifungal agent against Aspergillus niger (36 mm, MIC 6.25 µg/mL).
Experimental
General Melting points (mp) were determined in open capillaries on a melting temperature apparatus and are uncorrected. The purity of the compounds was checked by TLC (silica gel H, BDH, ethyl acetate–hexane, 1 : 3). The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and the wave numbers were given in cm−1. The 1 H- and 13C-NMR spectra were recorded in dimethylsulfoxide (DMSO)-d6 on a Bruker-400 spectrometer operating at 400 and 100 MHz, respectively. All chemical shifts are reported in δ (ppm) using tetramethylsilane (TMS) as an internal standard. The microanalyses were performed on a Perkin-Elmer 240C elemental analyzer. The compounds 2,4-dichloroquinazoline9) (3), benzo[d]oxazol-2-amine10) (4), benzo[d]thiazol-2-amine11) (5) and 1H-benzo[d]imidazol-2-amine12) (6) were prepared by adopting the literature precedents. General Procedure for the Synthesis of N2,N4Di(benzo[d]oxazol-2-yl)quinazoline-2,4-diamine (7a–d)/ N2,N4-Di(benzo[d]thiazol-2-yl)quinazoline-2,4-diamine (8a– d)/N2, N4-Di(1H-benzo[d ]imidazol-2-yl)quinazo line-2,4-diamine (9a–d) A solution of 2,4-dichloroquinazoline (3) (1.00 mmol), benzo[d]oxazol-2-amine (4)/ benzo[d]thiazol-2-amine (5)/1H-benzo[d]imidazol-2-amine (6) (2.50 mmol) and a catalytic amount of conc. HCl (0.2 mL) in 2-propanol (5 mL) was stirred at room temperature for 6–8 h. After completion of the reaction, the separated solid was filtered, dried and recrystallized from 2-propanol. N2,N4-Di(benzo[d]oxazol-2-yl)quinazoline-2,4-diamine (7a) White needles, yield 75%, mp 136–138°C. IR (KBr) cm−1: 1547 (C=N), 3284 (NH). 1H-NMR (DMSO-d6) δ: 7.03–8.22 (m, 12H, Ar-H), 9.32 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 160.2 (C-2′), 171.6 (C-4), 179.3 (C-2), 111.8, 112.4, 116.3, 122.1, 123.6, 125.7, 127.3, 128.6, 133.2, 144.5, 146.9, 151.7 (aromatic carbons). Anal. Calcd for C22H14N6O2: C, 67.00; H, 3.58; N, 21.31. Found: C, 67.10; H, 3.60; N, 21.46.
N2,N4-Di(benzo[d]oxazol-2-yl)-6-bromoquinazoline-2,4diamine (7b) White needles, yield 69%, mp 171–173°C. IR (KBr) cm−1: 1550 (C=N), 3288 (NH). 1H-NMR (DMSO-d6) δ: 7.00–8.26 (m, 11H, Ar-H), 9.36 ( br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 161.4 (C-2′), 172.3 (C-4), 180.4 (C-2), 108.3, 117.3, 121.4, 137.4, 127.3, 151.8, 143.5, 115.4, 124.1, 122.5, 110.6, 146.5 (aromatic carbons). Anal. Calcd for C22H13BrN6O2: C, 55.83; H, 2.77; N, 17.76. Found: C, 55.96; H, 2.80; N, 17.96. N2,N4-Di(benzo[d]oxazol-2-yl)-6-chloroquinazoline-2,4diamine (7c) White needles, yield 77%, mp 163–165°C. IR (KBr) cm−1: 1553 (C=N), 3296 (NH). 1H-NMR (DMSO-d6) δ: 7.06-8.18 (m, 11H, Ar-H), 9.45 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 162.1 (C-2′), 173.4 (C-4), 181.6 (C-2), 109.1, 110.3, 116.3, 117.8, 122.1, 125.3, 127.2, 129.1, 138.2, 146.1, 148.1, 152.3 (aromatic carbons). Anal. Calcd for C22H13Cl N6O2: C, 61.62; H, 3.06; N, 19.60. Found: C, 61.70; H, 3.05; N, 19.78. N2,N4-Di(benzo[d]oxazol-2-yl)-6-methylquinazoline-2,4diamine (7d) White needles, yield 72%, mp 152–154°C. IR (KBr) cm−1: 1549 (C=N), 3291 (NH). 1H-NMR (DMSO-d6) δ: 2.51 (s, 3H, Ar-CH3), 7.07–8.14 (m, 11H, Ar-H), 9.24 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 23.4 (Ar-CH3), 159.6 (C-2′), 169.6 (C-4), 176.8 (C-2), 111.3, 112.6, 116.1, 133.2, 136.5, 123.6, 124.4, 125.3, 126.4, 145.2, 147.5, 148.6 (aromatic carbons). Anal. Calcd for C23H16N6O2: C, 67.64; H, 3.95; N, 20.58. Found: C, 67.76; H, 3.99; N, 20.79. N2,N4-Di(benzo[d]oxazol-2-yl)-6-methoxyquinazoline-2,4diamine (7e) White needles, yield 74%, mp 159–161°C. IR (KBr) cm−1: 1538 (C=N), 3278 (NH). 1H-NMR (DMSO-d6) δ: 3.92 (s, 3H, Ar-OCH3), 6.98–8.24 (m, 11H, Ar-H), 9.29 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 53.2 (Ar-OCH3), 160.8 (C-2′), 172.7 (C-4), 180.3 (C-2), 98.2, 110.4, 111.6, 115.6, 124.8, 126.1, 127.4, 128.6, 144.8, 146.2, 146.7, 155.5 (aromatic carbons). Anal. Calcd for C23H16N6O3: C, 65.09; H, 3.80; N, 19.80. Found: C, 65.15; H, 3.78; N, 19.92. N2,N4-Di(benzo[d]oxazol-2-yl)-6-nitroquinazoline-2,4-diamine (7f) White needles, yield 78%, mp 168–170°C. IR (KBr) cm−1: 1559 (C=N), 3302 (NH). 1H-NMR (DMSO-d6) δ: 7.12–8.15 (m, 11H, Ar-H), 9.52 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 163.5 (C-2′), 174.3 (C-4), 182.4 (C-2), 107.2, 116.5, 142.6, 127.6, 125.8, 151.6, 145.2, 117.3, 126.6, 124.5, 112.2, 148.3 (aromatic carbons). Anal. Calcd for C22H13N7O4: C, 60.14; H, 2.98; N, 22.31. Found: C, 60.25; H, 2.99; N, 22.49. N2,N4-Di(benzo[d]thiazol-2-yl)quinazoline-2,4-diamine (8a) White needles, yield 76%, mp 162–164°C. IR (KBr) cm−1: 1552 (C=N), 3292 (NH). 1H-NMR (DMSO-d6) δ: 6.92–8.23 (m, 12H, Ar-H), 9.49 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 166.1 (C-2′), 172.3 (C-4), 180.6 (C-2), 110.9, 111.8, 118.1, 119.6,
Chem. Pharm. Bull. Vol. 63, No. 2 (2015)79
120.1, 123.7, 125.4, 127.5, 128.1, 132.4, 151.7, 152.4 (aromatic carbons). Anal. Calcd for C22H14N6S2: C, 61.95; H, 3.31; N, 19.70. Found: C, 62.08; H, 3.34; N, 19.87. N2,N4-Di(benzo[d]thiazol-2-yl)-6-bromoquinazoline-2,4diamine (8b) White needles, yield 80%, mp 193–195°C. IR (KBr) cm−1: 1558 (C=N), 3298 (NH). 1H-NMR (DMSO-d6) δ: 6.86–8.20 (m, 11H, Ar-H), 9.52 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 167.2 (C-2′), 173.6 (C-4), 181.2 (C-2), 109.2, 118.5, 122.1, 138.7, 128.4, 152.1, 152.4, 119.4, 125.4, 120.4, 117.6, 130.3 (aromatic carbons). Anal. Calcd for C22H13BrN6S2: C, 52.28; H, 2.59; N, 16.63. Found: C, 52.35; H, 2.58; N, 16.77. N2,N4-Di(benzo[d]thiazol-2-yl)-6-chloroquinazoline-2,4diamine (8c) White needles, yield 78%, mp 182–184°C. IR (KBr) cm−1: 1564 (C=N), 3315 (NH). 1H-NMR (DMSO-d6) δ: 6.96–8.25 (m, 11H, Ar-H), 9.56 (br s, 2H, NH).13C-NMR (DMSO-d6) δ: 168.3 (C-2′), 174.3 (C-4), 182.7 (C-2), 111.2, 113.1, 119.2, 120.2, 121.3, 122.4, 127.2, 128.2, 129.1, 132.1, 149.1, 154.2 (aromatic carbons). Anal. Calcd for C22H13Cl N6S2: C, 57.32; H, 2.84; N, 18.23. Found: C, 57.28; H, 2.86; N, 18.33. N2,N4-Di(benzo[d]thiazol-2-yl)-6-methylquinazoline-2,4diamine (8d) White needles, yield 73%, mp 168–170°C. IR (KBr) cm−1: 1558 (C=N), 3307 (NH). 1H-NMR (DMSO-d6) δ: 2.58 (s, 3H, Ar-CH3), 6.88–7.92 (m, 11H, Ar-H), 9.38 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 24.2 (Ar-CH3), 165.6 (C-2′), 170.5 (C-4), 177.3 (C-2), 111.8, 112.7, 119.9, 120.8, 121.5, 124.1, 125.8, 126.6, 133.8, 137.2, 149.2, 153.9 (aromatic carbons). Anal. Calcd for C23H16N6S2: C, 62.71; H, 3.66; N, 19.08. Found: C, 62.83; H, 3.69; N, 19.21. N2,N4-Di(benzo[d]thiazol-2-yl)-6-methoxyquinazoline-2,4diamine (8e) White needles, yield 75%, mp 171–173°C. IR (KBr) cm−1: 1545 (C=N), 3286 (NH). 1H-NMR (DMSO-d6) δ: 3.97 (s, 3H, Ar-OCH3), 7.07–8.26 (m, 11H, Ar-H), 9.48 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 54.4 (Ar-OCH3), 167.3 (C-2′), 173.5 (C-4), 182.6 (C-2), 98.6, 111.8, 112.8, 118.6, 119.1, 120.6, 125.9, 127.2, 128.1, 146.8, 152.8, 155.7 (aromatic carbons). Anal. Calcd for C23H16N6S2O: C, 60.51; H, 3.53; N, 18.41. Found: C, 60.58; H, 3.55; N, 18.56. N2,N4-Di(benzo[d]thiazol-2-yl)-6-nitroquinazoline-2,4-diamine (8f) White needles, yield 81%, mp 187–189°C. IR (KBr) cm−1: 1568 (C=N), 3319 (NH). 1H-NMR (DMSO-d6) δ: 7.02–8.18 (m, 11H, Ar-H), 9.59 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 169.4 (C-2′), 175.1 (C-4), 184.2 (C-2), 108.2, 117.6, 143.2, 128.4, 126.5, 152.1, 154.3, 122.1, 127.2, 122.4, 119.6, 132.6 (aromatic carbons). Anal. Calcd for C22H13N7O2S2: C, 56.04; H, 2.78; N, 20.79. Found: C, 56.09; H, 2.79; N, 20.90. N2,N4-Di(1H-benzo[d]imidazol-2-yl)quinazoline-2,4-diamine (9a) White needles, yield 65%, mp 150–152°C. IR (KBr) cm−1: 1531 (C=N), 3273 (NH). 1H-NMR (DMSO-d6) δ: 6.85–8.26 (m, 12H, Ar-H), 9.12 (br s, 2H, NH), 11.98 (br s, 2H, imidazole-NH). 13C-NMR (DMSO-d6) δ: 155.3 (C-2′), 169.5 (C-4), 177.8 (C-2), 110.5, 111.4, 119.6, 123.2, 128.6, 129.6, 132.4, 137.2, 151.3 (aromatic carbons). Anal. Calcd for C22H16N8: C, 67.34; H, 4.11; N, 28.55. Found: C, 67.45; H, 4.09; N, 28.71. N2,N4-Di(benzo[d]thiazol-2-yl)-6-bromoquinazoline-2,4diamine (9b) White needles, yield 63%, mp 188–190°C. IR (KBr) cm−1: 1538 (C=N), 3279 (NH). 1H-NMR (DMSO-d6) δ: 6.81–8.29 (m, 11H, Ar-H), 9.16 (br s, 2H, NH), 12.04 (br s, 2H, imidazole-NH). 13C-NMR (DMSO-d6) δ: 156.1 (C-2′), 170.6 (C-4), 178.1 (C-2), 107.1, 115.4, 120.2, 136.3, 126.4, 150.1, 137.3, 111.2, 119.4 (aromatic carbons). Anal. Calcd for
C22H15BrN8: C, 56.06; H, 3.21; N, 23.77. Found: C, 56.15; H, 3.23; N, 23.92. N2,N4-Di(1H-benzo[d]imidazol-2-yl)-6-chloroquinazoline-2,4-diamine (9c) White needles, yield 70%, mp 181–183°C. IR (KBr) cm−1: 1543 (C=N), 3286 (NH). 1H-NMR (400 MHz, DMSO-d6) δ: 6.91–8.31 (m, 11H, Ar-H), 9.24 (br s, 2H, NH), 12.14 (br s, 2H, imidazole-NH). 13C-NMR (DMSOd6) δ: 157.1 (C-2′), 172.4 (C-4), 179.4 (C-2), 110.4, 113.3, 119.3, 121.2, 128.4, 130.7, 131.4, 139.2, 149.9 (aromatic carbons). Anal. Calcd for C22H15ClN8: C, 61.90; H, 3.54; N, 26.25. Found: C, 61.95; H, 3.55; N, 26.36. N2,N4-Di(1H-benzo[d]imidazol-2-yl)-6-methylquinazoline-2,4-diamine (9d) White needles, yield 66%, mp 172–174°C. IR (KBr) cm−1: 1538 (C=N), 3279 (NH). 1H-NMR (DMSO-d6) δ: 2.42 (s, 3H, Ar-CH3), 6.82–8.16 (m, 11H, Ar-H), 9.05 (br s, 2H, NH), 11.81 (br s, 2H, imidazole-NH). 13C-NMR (DMSO-d6) δ: 22.5 (Ar-CH3), 154.3 (C-2′), 167.1 (C-4), 174.6 (C-2), 111.8, 112.1, 120.4, 124.1, 125.8, 133.8, 137.2, 138.4, 149.2 (aromatic carbons). Anal. Calcd for C23H18N8: C, 67.97; H, 4.46; N, 27.57. Found: C, 68.10; H, 4.49; N, 27.79. N2,N4-Di(1H-benzo[d]imidazol-2-yl)-6-methoxyquinazoline-2,4-diamine (9e) White needles, yield 68%, mp 177–179°C. IR (KBr) cm−1: 1525 (C=N), 3264 (NH). 1H-NMR (DMSO-d6) δ: 3.85 (s, 3H, Ar-OCH3), 6.93–8.12 (m, 11H, Ar-H), 9.18 (br s, 2H, NH), 12.06 (br s, 2H, imidazole-NH). 13 C-NMR (DMSO-d6) δ: 52.8 (Ar-OCH3), 156.8 (C-2′), 168.5 (C-4), 177.8 (C-2), 98.4, 110.2, 111.8, 119.2, 127.6, 129.3, 137.8, 146.7, 155.7 (aromatic carbons). Anal. Calcd for C23H18N8O: C, 65.39; H, 4.29; N, 26.53. Found: C, 65.47; H, 4.31; N, 26.70. N2,N4-Di(benzo[d]thiazol-2-yl)-6-nitroquinazoline-2,4-diamine (9f) White needles, yield 64%, mp 185–187°C. IR (KBr) cm−1: 1548 (C=N), 3292 (NH). 1H-NMR (DMSO-d6) δ: 6.95–8.36 (m, 11H, Ar-H), 9.28 (br s, 2H, NH), 12.18 (br s, 2H, imidazole-NH). 13C-NMR (DMSO-d6) δ: 158.3 (C-2′), 173.2 (C-4), 180.5 (C-2), 106.2, 115.6, 143.2, 128.7, 126.5, 152.1, 139.4, 113.5, 121.2 (aromatic carbons). Anal. Calcd for C22H15N9O2: C, 60.41; H, 3.46; N, 28.82. Found: C, 60.52; H, 3.45; N, 28.98. Biological Assays. Compounds The compounds 7–9 were evaluated for antimicrobial activity by agar well diffusion and broth dilution methods. Cells Bacterial strains Staphylococcus aureus, Bacillus subtilis, Pseudomonus aeruginosa, Klebsiella pneumoniae and fungi Aspergillus niger, Penicillium chrysogenum were obtained from the Department of Microbiology, S.V. University, Tirupati. Antibacterial and Antifungal Assays The in vitro antimicrobial studies were carried out by agar well diffusion method against test organisms.14,15) Nutrient broth (NB) plates were swabbed with 24 h old broth culture (100 µL) of test bacteria. Using the sterile cork borer, wells (6 mm) were made into each petriplate. The compounds were dissolved in DMSO of 5 mg/mL and from this 10 µL, 15 µL and 20 µL (50, 75 and 100 µg/well) were added into the wells by using sterile pipettes. The standard antibiotics, chloramphenicol for antibacterial activity and ketoconazole for antifungal activity (as positive control) were simultaneously tested against the pathogens. The samples were dissolved in DMSO which showed no zone of inhibition acts as a negative control. The plates were incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. After appropriate incubation, the diameter of zone of
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inhibition of each well was measured. Duplicates were maintained and the average values were calculated for eventual antibacterial activity. Minimum Inhibitory Concentration Assay Broth dilution test was used to determine MIC of the above mentioned samples.16,17) Freshly prepared nutrient broth was used as diluents. The 24 h old culture of the test bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonus aeruginosa and Klebsilla pneumoniae and the test fungi Aspergillus niger and Penicillium chrysogenum were diluted 100 fold in nutrient broth (100 µL bacterial cultures in 10 mL NB). The stock solution of the synthesized compounds was prepared in DMSO by dissolving 5 mg of the compound in 1 mL of DMSO. Increasing concentrations of the test samples (1.25, 2.5, 5, 10, 15, 20, 40 µL of stock solution contains 6.25, 12.5, 25, 50, 75, 100, 200 µg of the compounds) were added to the test tubes containing the bacterial and fungal cultures. All the tubes were incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. The tubes were examined for visible turbidity using NB as control. Control without test samples and with solvent was assayed simultaneously. The lowest concentration that inhibited visible growth of the tested organisms was recorded as MIC. Minimum Bactericidal/Fungicidal Concentration To determine the MBC18) and MFC19) for each set of test tubes in the MIC determination, a loopful of broth was collected from those tubes which did not show any growth and inoculated on sterile nutrient broth (for bacteria) and potato dextrose agar (PDA) (for fungi) by streaking. Plates inoculated with bacteria and fungi were incubated at 37°C for 24 h and at 28°C for 48 h, respectively. After incubation, the lowest concentration was noted as MBC (for bacteria) or MFC (for fungi) at which no visible growth was observed. Acknowledgments The authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India for the sanction of Major Research Project. One of the authors Mr. L. Mallikarjuna Reddy is thankful to CSIR, New Delhi, India for the sanction of Junior Research Fellowship. Conflict of Interest interest.
The authors declare no conflict of
References
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