Sage Journals: Discover world-class research

Abstract

A series of 1,3,4-oxadiazole-fused and piperazine-fused quinazoline derivatives is synthesized and evaluated as antibacterial agents. The synthetic protocol involves an efficient Suzuki C–C cross-coupling reaction on the quinazoline ring followed by formation of 1,3,4-oxadiazole intermediates. These are further treated with piperazine bases in the presence of formalin in methanol to furnish the final N-Mannich products. The majority of these compounds show potent antibacterial activities against several different strains of Gram-positive bacteria including multidrug-resistant clinical isolates. The cytotoxic activity assay demonstrates that the synthesized compounds do not affect cell viability on Human cervical (HeLa) cells at their minimum inhibitory concentrations. The newly synthesized compounds are characterized by infrared spectroscopy, ¹H NMR, ¹³C NMR, mass spectrometry, and elemental analysis.

Keywords

1,3,4-oxadiazoles antimicrobial activity piperazines quinazolines Suzuki coupling

Introduction

The investigation of new heterocycles that demonstrate potency toward multiple biological targets remains an intriguing scientific endeavor. There has been a growing demand for the development of more sustainable antibiotics in recent decades, particularly those targeting the expansion of multidrug resistance in pathogenic microbes. In view of this, we have combined different moieties, such as quinazoline, piperazine, and oxadiazole, in a single molecule. Compounds bearing a quinazoline moiety have been reported to possess a wide spectrum of biological and therapeutic properties, such as anti-inflammatory,^1–3 antiplasmodial,⁴ antibacterial,⁵ antitumor,⁶ antimicrobial,^7,8 anthelmintic,⁹ and anticonvulsant properties.¹⁰ Expecting an enhancement of biological activity, we have incorporated a 1,3,4-oxadiazole moiety as a potential bioactive site in our systems. The large number of synthetic compounds with a 1,3,4-oxadiazole nucleus plays a vital role in medicinal chemistry. They are known to possess various biological activities such as antimicrobial,^11–13 antituberculosis,^14,15 anti-inflammatory,¹⁶ antimalarial,¹⁷ and anticancer activities.¹⁸ Moreover, the literature survey revealed that analogs with inserted N-substituted phenyl piperazine bases have been reported to demonstrate a wide range of pharmacological activities.^19–23

In continuation of our efforts to find new promising pharmacologically active molecules, we report here the synthesis and in vitro antimicrobial activity of quinazoline analogs by applying an efficient palladium-catalyzed C–C Suzuki coupling. The synthesized compounds were tested in vitro with various antibacterial strains, including multidrug-resistant clinical isolates such as methicillin-resistant Staphylococcus aureus (MRSA CCARM 3167 and MRSA CCARM 3506) and quinolone-resistant Staphylococcus aureus (QRSA CCARM 3505 and QRSA CCARM 3519) by recording the minimum inhibitory concentrations (MICs).

Results and discussion

Chemistry

The synthetic strategy to prepare the target compounds is illustrated in Scheme 1. The initial analog, 4-[(2-chloroquinazolin-4-yl)oxy]benzonitrile (2), was prepared by the reaction of 2,4-dichloroquinazoline (1) with 4-hydroxybenzonitrile under basic conditions. The next step was consolidation of the Suzuki cross-coupling reaction to facilitate C–C bond formation between the carbon atom of the quinazoline ring and the phenyl ring of 4-ethoxycarbonylphenyl boronic acid pinacol ester to form ethyl 4-[4-(4-cyanophenoxy)quinazoline-2-yl]benzoate 3. The resulting ester derivative was then treated with 90% hydrazine hydrate in ethanol to yield the corresponding hydrazide derivative 4. The cyclization of this analog with carbon disulfide in ethanolic potassium hydroxide furnished the hydrazinecarbodithioate salt 5, which was further treated with sulfuric acid and hydrochloric acid at cooled temperature to furnish the corresponding 1,3,4-oxadiazole derivative 6. This was treated with different 1-(4-substituted phenyl)piperazines in the presence of formalin in methanol to furnish the final Mannich bases 7a–i. All the IR, ¹H NMR, ¹³C NMR, and MS spectral data of synthesized compounds were in accordance with the assumed structures (Please see the supplement material). The purity of the synthesized compounds was monitored by thin-layer chromatography (TLC) and ascertained by elemental analysis.

Scheme 1.

Synthetic protocol for the preparation of analogs 7a-i.

Antibacterial activity

The in vitro antimicrobial activities (Table 1) of the synthesized analogs were evaluated against three Gram-positive bacteria (Staphylococcus aureus RN4220, KCTC 503, and KCTC 209) and one Gram-negative bacterium (Escherichia coli CCARM 1356). It was observed that many of the synthesized analogs displayed excellent efficacy against all the three Gram-positive bacteria, specifically S. aureus RN4220 and S. aureus KCTC503 (MIC values of ⩽64 μg mL⁻¹) as compared to the control drugs. The introduction of fluoro, trifluoromethyl, hydroxy, and methoxy functional groups on the phenyl ring attached to the piperazine moiety significantly enhanced the antibacterial activity. The trifluoromethyl-piperazine-fused quinazoline derivative 7f showed more promising activity against S. aureus RN4220 at a 2 μg mL⁻¹ MIC level. Moreover, fluoro-substituted (7d), hydroxy-substituted (7h), and methoxy-substituted (7i) derivatives were half-fold less active than 7f. The methoxy-piperazine-fused derivative 7i was also found to be active against S. aureus KCTC 503 with the highest MIC level of 2 μg mL⁻¹. Moreover, a similar analog was found to be active against S. aureus KCTC 209 with half-fold less activity. However, most of the synthesized analogs were found to be inactive (MIC values of >64 μg mL⁻¹) against the Gram-negative bacterium E. coli CCARM 1356, except for the hydroxy-substituted 7h and methoxy-substituted 7i (MIC values of 32 μg mL⁻¹).

Table 1.

Inhibitory activities (MIC, µg mL⁻¹) of compounds 7a–i against bacteria.

Compound	R	S. aureus			E. coli
Compound	R	4220^a	503^b	209^c	1356^d
7a	H	32	64	>64	>64
7b	Cl	16	16	16	>64
7c	Br	16	8	16	>64
7d	F	4	16	8	>64
7e	CH₃	32	32	32	>64
7f	CF₃	2	4	16	>64
7g	NO₂	32	32	32	>64
7h	OH	4	4	16	32
7i	OCH₃	4	2	8	32
Oxacillin		1	1	1	>64
Norfloxacin		2	2	4	16

Staphylococcus aureus RN4220.

Staphylococcus aureus KCTC503.

Staphylococcus aureus KCTC209.

Escherichia coli CCARM 1356.

The synthesized analogs were further evaluated for their inhibitory activity against selected methicillin-resistant S. aureus (MRSA) and quinolone-resistant S. aureus (QRSA) (Table 2). It is clear from the data that all of the compounds exhibited higher levels of inhibitory activity than oxacillin against MRSA 3167 (methicillin-resistant S. aureus CCARM 3167) and MRSA 3506 (methicillin-resistant S. aureus CCARM 3506). The synthesized analogs, 7f and 7i, showed the most potent levels of antibacterial activity (1–2 μg mL⁻¹) against multidrug-resistant clinical isolates MRSA 3167 and 3506. Moreover, analogs 7f, 7h, and 7i were found to be potent against quinolone-resistant S. aureus CCARM 3505 and S. aureus CCARM 3519 with MIC levels of 4 μg mL⁻¹.

Table 2.

Inhibitory activities (MIC, µg mL⁻¹) of compounds 7a–i against clinical isolates of multidrug-resistant Gram-positive bacterial strains.

Compound	R	Multidrug-resistant Gram-positive strains
		MRSA		QRSA
		3167^a	3506^b	3505^c	3519^d
7a	H	32	32	32	64
7b	Cl	4	8	8	16
7c	Br	4	8	8	16
7d	F	4	4	8	8
7e	CH₃	16	8	16	16
7f	CF₃	2	2	4	4
7g	NO₂	16	16	16	32
7h	OH	4	4	4	4
7i	OCH₃	1	2	4	4
Oxacillin		>64	>64	1	1
Norfloxacin		4	4	>64	>64

Methicillin-resistant S. aureus CCARM 3167.

Methicillin-resistant S. aureus CCARM 3506.

Quinolone-resistant S. aureus CCARM 3505.

Quinolone-resistant S. aureus CCARM 3519.

The cytotoxic properties of compounds 7f, 7h, and 7i were also investigated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay to determine whether their observed antibacterial activity was caused by selective toxicity toward the bacterial cells and the results are shown in Table 3.

Table 3.

Cytotoxic activity of compounds 7f, 7h, and 7i against HeLa cells.

Compound	R	IC₅₀ (µg mL⁻¹)^a,b
7f	CF₃	19.39
7h	OH	23.56
7i	OCH₃	21.78

HeLa cell (Human cervical) monolayers were used as an in vitro model of cervicovaginal epithelium for testing the cytotoxicity of the new compounds.

IC₅₀ is defined as the concentration at which 50% growth inhibition is observed.

These compounds did not affect cell viability of Human cervical (HeLa) cells at their MICs, but showed cytotoxicity at much higher concentrations. Hence, the inconsistency between the antibacterial activity and cytotoxicity of compounds 7f, 7h, and 7i suggests that there may be an antibacterial mechanism operating different from that of cytotoxicity.

Conclusion

In conclusion, a series of oxadiazole- and piperazine-integrated quinazoline derivatives has been synthesized through an efficient Suzuki C–C cross-coupling reaction and the product tested for their antibacterial efficacy. The results demonstrated that most of the synthesized analogs demonstrate good levels of antibacterial activity against Gram-positive bacteria, including multidrug-resistant strains of clinical isolates. The presence of trifluoromethyl, hydroxy, and methoxy groups on the piperazine ring is essential for the compound to show high antibacterial activity between 2 and 8 μg mL⁻¹ (7f, 7h, and 7i) MIC levels against Staphylococcus aureus. Moreover, methoxy-piperazine-substituted quinazoline analog 7i shows highest antibacterial activity (1 μg mL⁻¹) against methicillin-resistant S. aureus CCARM 3167. This analog also shows high antibacterial activity at 4 μg mL⁻¹ MIC level against quinolone-resistant S. aureus CCARM 3505 and CCARM 3519. Potent analogs were evaluated for their cytotoxicity and exhibited no significant influence on the cell viability of HeLa cells at their MIC (1 µg mL⁻¹).

Materials and methods

General

All reactions were carried out under a nitrogen atmosphere. Air- and moisture-sensitive solvents and solutions were transferred via syringe or stainless steel cannula. All chemicals including 1-(4-substituted phenyl)piperazine bases were purchased from Sigma-Aldrich, Merck, and Fluka. Solvents used were of analytical grade. Anhydrous potassium carbonate was stored in a nitrogen-filled glovebox, ground, and was taken out in small quantities and stored in a desiccator. All reactions were routinely checked by TLC. TLC was performed on aluminum-backed silica gel plates (silica gel 60 F254 grade, Merck DC) with spots visualized by UV light. Column chromatography was performed on silica gel LC 60A (70–200 µm). Melting points were determined in open capillaries on a Veego electronic apparatus VMP-D (Veego Instrument Corporation, Mumbai, India) and are uncorrected. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker 400 MHz model spectrometer using DMSO-d₆ as the solvent and tetramethylsilane (TMS) as the internal standard with a ¹H resonance frequency of 400 MHz and a ¹³C resonance frequency of 100 MHz. ¹H NMR and ¹³C NMR chemical shifts are reported as parts per million (ppm) downfield from TMS (Me₄Si). The splitting patterns are designated as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet. The mass spectra were measured using a Waters Micromass Q-Tof Micro instrument (time of flight (TOF) mass spectrometer). Column chromatography was performed on a 1½ foot (2.5 cm diameter) glass column using silica gel LC 60A (70–200 µm). Elemental analysis was carried out using a Heraeus Carlo Erba 1180 C,H,N,S analyzer (Hanau, Germany).

4-[(2-Chloroquinazolin-4-yl)oxy]benzonitrile (2)

An oven-dried Schlenk flask was charged with a magnetic stir bar, 2,4-dichloroquinazoline (1) (10 mmol), 4-hydroxybenzonitrile (11 mmol), potassium carbonate (15 mmol), and anhydrous acetone (35 mL). The reaction slurry was stirred vigorously at 30°C for 12 h. After the completion of the reaction, the slurry was treated with ice cold water (200 mL), and the resulting precipitates were filtered off.²⁴ The crude product was recrystallized from acetonitrile.

White solid, yield: 2.14 g, 76%; m.p. 167–171°C; IR (KBr, cm⁻¹): 2850 (C–H), 2220 (C≡N), 1238 (C–O–C), 779 (C–Cl); ¹H NMR (400 MHz, DMSO-d₆): δ 8.15 (dd, J = 6.8, 1.8 Hz, 1H, quinazoline), 7.95–7.78 (m, 3H, quinazoline), 7.77–7.46 (m, 4H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 167.3, 164.9, 146.4, 139.4, 135.0, 132.5, 129.4, 127.4, 123.6, 122.4, 121.2 (Ar-C), 114.1 (C≡N), 93.9 (Ar-C-C≡N).

Ethyl 4-[4-(4-cyanophenoxy)quinazolin-2-yl]benzoate (3)

In an oven-dried round-bottomed flask, a mixture of 4-[(2-chloroquinazolin-4-yl)oxy]benzonitrile (2) (10 mmol), tetrakis (triphenylphosphine)palladium(0) (0.13 mmol), 2M sodium carbonate aqueous solution (5 mL), and 4-ethoxycarbonylphenylboronic acid pinacol ester (15 mmol) in dimethoxyethane (DME) (50 mL) was stirred under a nitrogen atmosphere for 15 min and then heated to reflux temperature for 24 h. The progress of the reaction was monitored by TLC (60% EtOAc in hexane). At the end of the reaction, the solvent was evaporated. The residue was dissolved in CH₂Cl₂ and filtered through a pad of Celite. The filtrate was washed with water (2 × 250 mL), and the organic layer was dried over sodium sulfate. Purification of the residue was achieved by silica gel column chromatography using EtOAc/hexane, 6:4 to give the desired product.²⁵

White solid, yield: 3.20 g, 81%; m.p. 219–221°C; IR (KBr, cm⁻¹): 2949 (C–H), 2220 (C≡N), 1236 (C–O–C); ¹H NMR (400 MHz, DMSO-d₆): δ 8.45 (dd, J = 6.8, 1.8 Hz, 1H, quinazoline), 8.27–7.87 (m, 3H, quinazoline), 7.72–7.21 (m, 8H, ArH); 4.07 (q, J = 5.9 Hz, 2H, OCH₂CH₃), 1.35 (t, J = 6.0 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ 179.8, 166.9 (COO), 155.9, 154.1, 153.4, 150.6, 134.3, 133.2, 131.8, 129.6, 127.9, 126.3, 121.7, 121.5, 121.1, 115.8 (Ar-C), 105.8 (C≡N), 100.0 (Ar-C-C≡N), 64.0 (CH₂CH₃), 15.1 (CH₂ CH₃).

4-[4-(4-Cyanophenoxy)quinazolin-2-yl]benzohydrazide (4)

An oven-dried Schlenk tube was charged with a magnetic stir bar, ethyl 4-[4-(4-cyanophenoxy)quinazolin-2-yl]benzoate (3) (10 mmol) and hydrazine hydrate (10 mmol) in ethanol (60 mL) and heated under reflux for 6 h. The reaction mixture was cooled to room temperature and the separated solid was collected by filtration, washed with ethanol, and recrystallized from methanol. The product was obtained as pale yellow solid. The progress of the reaction was monitored by TLC (60% EtOAc in hexane).²⁶

Pale yellow solid, yield: 2.82 g, 74%, m.p. 263–265°C; IR (KBr, cm⁻¹): 3319 (N–H), 2993 (C–H), 2220 (C≡N), 1176 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 9.88–9.71 (m, 1H, CONHNH₂), 8.45 (dd, J = 7.6, 1.4 Hz, 1H, quinazoline), 8.24 (m, 3H, quinazoline), 7.84–7.22 (m, 8H, ArH), 4.21 (s, 2H, –NH₂). ¹³C NMR (100 MHz, DMSO-d₆): 171.6, 165.3 (C=O), 162.6, 153.2, 150.6, 138.5, 134.4, 134.0, 131.7, 129.6, 129.3, 129.2, 128.6, 127.9, 126.4, 125.7 (Ar-C), 105.4 (C≡N), 96.8 (Ar-C-C≡N).

Potassium 2-{4-[4-(4-cyanophenoxy)quinazolin-2-yl]benzoyl}hydrazinecarbodithioate (5)

An oven-dried Schlenk tube was charged with a magnetic stir bar, 4-[4-(4-cyanophenoxy)quinazolin-2-yl]benzohydrazide (4) (10 mmol), potassium hydroxide (10 mmol), and ethanol (60 mL). The reaction mixture was stirred for 1 h in an ice bath. Carbon disulfide (15 mmol) was added dropwise to the above pre-cooled reaction mixture, which resulted in the formation of a pale yellow precipitate. The pale yellow precipitate was filtered, washed with cold acetone (2 × 10 mL), and dried in a vacuum oven to obtain 5.²⁶

4-({2-[4-(5-Thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]quinazolin-4-yl}oxy)benzonitrile (6)

Using an oven-dried Schlenk tube, charged with a magnetic stir bar, potassium 2-{4-[4-(4-cyanophenoxy)quinazolin-2-yl]benzoyl}hydrazinecarbodithioate (5) (10 mmol) was added in very small portions to a hydrochloric acid (10 mL, 0–5°C) until the solution became homogeneous. The homogeneous mixture was then poured onto crushed ice. The separated precipitate was filtered under vacuum, washed with water, and dried in a vacuum oven to obtain 6. The progress of the reaction was monitored by TLC (60% EtOAc in hexane).²⁶

White solid, yield: 2.87 g, 68%, m.p. 211–215°C; IR (KBr, cm⁻¹): 3319 (N–H), 2993 (C–H), 2220 (C≡N), 1612 (C=N of oxadiazole), 1247 (C=S), 1176 (N–N, oxadiazole), 1141 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 8.44 (s, 1H, NH), 8.18 (dd, J = 6.2 Hz, 1H, quinazoline), 7.84–7.54 (m, 3H, quinazoline), 7.44–6.36 (m, 8H, ArH). ¹³C NMR (100 MHz, DMSO-d₆): δ 182.1 (C=S), 170.7, 169.1, 159.6, 150.2, 142.4, 140.2, 134.4, 131.0, 129.7, 129.0, 127.5, 126.3, 121.4, 117.2, 115.4 (Ar-C), 108.4 (C≡N), 100.0 (Ar-C-C≡N).

General synthetic procedure for preparation of analogs 7a–i

An oven-dried Schlenk tube was charged with a magnetic stir bar, compound 6 (10 mmol), 40% formalin (12 mmol), and ethanol (60 mL). The reaction mixture was stirred for 30 min at room temperature. Next, the appropriate 1-(4-substituted phenyl)piperazine (10 mmol) was dissolved in methanol and added dropwise to the above stirred mixture and allowed to reflux for 3–8 h. The progress of the reaction was monitored by TLC (60% EtOAc in hexane). After completion of the reaction, the mixture was allowed to cool and poured onto crushed ice. The precipitate formed was collected by filtration, washed with water, and dried in a vacuum oven to furnish 7a–i. Purification of the residue was achieved by silica gel column chromatography using EtOAc/hexane, 6:4 to give the desired product.²⁶

4-{[2-(4-{4-[(4-Phenylpiperazin-1-yl)methyl]-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl}phenyl)quinazolin-4-yl]oxy}benzonitrile (7a)

White solid, yield: 4.30 g, 72%, m.p. 194–195°C; IR (KBr, cm⁻¹): 3209 (C–H), 2218 (C≡N), 1608 (C=N of oxadiazole), 1257 (C=S), 1174 (N–N), 1112 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 8.51 (dd, J = 6.4 Hz, 1H, quinazoline), 8.21–7.79 (m, 3H, quinazoline), 7.57–7.25 (m, 13H, ArH), 4.92 (s, 2H, N–CH₂–N), 3.89 (br s, 4H, piperazine), 3.51 (br s, 4H, piperazine). ¹³C NMR (100 MHz, DMSO-d₆): δ 172.2 (C=S), 168.4, 167.2, 157.3, 156.7, 150.2, 147.3, 143.3, 139.7, 137.0, 135.0, 133.4, 131.0, 129.4, 128.2, 126.2, 125.3, 123.6, 120.4, 119.2, 116.2 (Ar-C), 105.2 (C≡N), 97.8 (Ar-C-C≡N), 62.2 (CH₂), 50.4, 45.6. MS (ESI): m/z 598.3 [M+1]⁺. Anal. Calcd. For C₃₄H₂₇N₇O₂S: C, 68.32; H, 4.55; N, 16.40; S, 5.36. Found: C, 68.25; H, 4.68; N, 16.27; S, 5.28.

4-({2-[4-(4-{[4-(4-Chlorophenyl)piperazin-1-yl]methyl}-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]quinazolin-4-yl}oxy)benzonitrile (7b)

White solid, yield: 5.31 g, 84%, m.p. 283–286°C; IR (KBr, cm⁻¹): 3134 (C–H), 2230 (C≡N), 1612 (C=N of oxadiazole), 1237 (C=S), 1198 (N–N), 1103 (C–O–C), 747 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆): δ 8.47 (dd, J = 6.8 Hz, 1H, quinazoline), 7.92–7.78 (m, 3H, quinazoline), 7.63–7.04 (m, 12H, ArH), 4.83 (s, 2H, N–CH₂–N), 3.75 (br s, 4H, piperazine), 3.63 (br s, 4H, piperazine). ¹³C NMR (100 MHz, DMSO-d₆): δ 178.0 (C=S), 167.5, 163.8, 156.5, 154.2, 149.3, 149.1, 147.4, 143.1, 141.4, 140.9, 139.8, 132.5, 131.0, 130.7, 129.1, 128.3, 127.6, 126.9, 123.2, 118.9 (Ar-C), 104.7 (C≡N), 96.5 (Ar-C-C≡N), 63.0 (CH₂), 48.8, 44.5. MS (ESI): m/z 632.5 [M+1]⁺. Anal. Calcd. for C₃₄H₂₆ClN₇O₂S: C, 64.60; H, 4.15; N, 15.51; S, 5.07. Found: C, C, 64.71; H, 4.26; N, 15.49; S, 4.92.

4-({2-[4-(4-{[4-(4-Bromophenyl)piperazin-1-yl]methyl}-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]quinazolin-4-yl}oxy)benzonitrile (7c)

Light brown solid, yield: 4.73 g, 70%, m.p. 240–241°C; IR (KBr, cm⁻¹): 3089 (C–-H), 2225 (C≡N), 1617 (C=N of oxadiazole), 1240 (C=S), 1150 (N–N), 1123 (C–O–C), 938 (C-Br). ¹H NMR (400 MHz, DMSO-d₆): δ 8.21 (dd, J = 7.0 Hz, 1H, quinazoline), 8.04–7.93 (m, 3H, quinazoline), 7.79–7.30 (m, 12H, ArH), 4.85 (s, 2H, N–CH₂–N), 3.78 (br s, 4H, piperazine), 3.41 (br s, 4H, piperazine). ¹³C NMR (100 MHz, DMSO-d₆): δ 179.3 (C=S), 168.5, 167.0, 159.3, 157.8, 155.4, 153.1, 149.2, 148.6, 147.1, 145.5, 143.0, 141.7, 138.6, 135.2, 130.7, 128.4, 128.0, 125.1, 123.9, 120.4 (Ar-C), 107.7 (C≡N), 95.4 (Ar-C-C≡N), 60.4 (CH₂), 50.3, 46.7. MS (ESI): m/z 676.1 [M+1]⁺. Anal. Calcd. for C₃₄H₂₆BrN₇O₂S: C, 60.36; H, 3.87; N, 14.49; S, 4.74. Found: C, 60.44; H, 3.76; N, 14.39; S, 4.77.

4-({2-[4-(4-{[4-(4-Fluorophenyl)piperazin-1-yl]methyl}-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]quinazolin-4-yl}oxy)benzonitrile (7d)

White solid, yield: 4.80 g, 78%, m.p. 267–269°C; IR (KBr, cm⁻¹): 3125 (C–H), 2220 (C≡N), 1597 (C=N of oxadiazole), 1345 (C–F), 1232 (C=S), 1186 (N–N), 1120 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 8.41 (dd, J = 7.0 Hz, 1H, quinazoline), 8.17–8.02 (m, 3H, quinazoline), 7.95–7.47 (m, 12H, ArH), 4.78 (s, 2H, N–CH₂–N), 3.88 (br s, 4H, piperazine), 3.53 (br s, 4H, piperazine). ¹³C NMR (100 MHz, DMSO-d₆): δ 180.4 (C=S), 168.4, 166.9, 154.8, 154.1, 152.3, 150.4 (C-F), 149.7, 147.5, 146.2, 144.1, 141.7, 139.2, 136.4, 134.8, 131.3, 129.4, 128.9, 127.6, 125.3, 119.3 (Ar-C), 106.0 (C≡N), 97.1 (Ar-C-C≡N), 62.6 (CH₂), 51.3, 46.1. MS (ESI): m/z 616.4 [M+1]⁺. Anal. Calcd. for C₃₄H₂₆FN₇O₂S: C, 66.33; H, 4.26; N, 15.92; S, 5.21. Found: C, 66.19; H, 4.15; N, 16.01; S, 5.27.

4-({2-[4-(5-Thioxo-4-{[4-(p-tolyl)piperazin-1-yl]methyl}-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]quinazolin-4-yl}oxy)benzonitrile (7e)

Pale White solid, yield: 4.22 g, 69%, m.p. 255–256°C; IR (KBr, cm⁻¹): 3089 (C–H), 2218 (C≡N), 1617 (C=N of oxadiazole), 1244 (C=S), 1159 (N–N), 1132 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 8.50 (dd, J = 6.2 Hz, 1H, quinazoline), 8.09–7.93 (m, 3H, quinazoline), 7.66–7.25 (m, 12H, ArH), 5.02 (s, 2H, N–CH₂–N), 3.90 (br s, 4H, piperazine), 3.55 (br s, 4H, piperazine), 2.29 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 176.3 (C=S), 169.0, 167.5, 159.4, 157.3, 155.3, 152.7, 151.0, 149.6, 147.3, 145.4, 140.3, 138.9, 133.5, 130.2, 129.5, 128.7, 124.1, 123.6, 121.4, 118.8 (Ar-C), 105.0 (C≡N), 97.2 (Ar-C-C≡N), 60.6 (CH₂), 51.5, 46.2, 14.3 (CH₃). MS (ESI): m/z 612.3 [M+1]⁺. Anal. Calcd. for C₃₅H₂₉N₇O₂S: C, 68.72; H, 4.78; N, 16.03; S, 5.24. Found: C, 68.66; H, 4.86; N, 16.09; S, 5.14.

4-[(2-{4-[5-Thioxo-4-({4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}methyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl]phenyl}quinazolin-4-yl)oxy]benzonitrile (7f)

White solid, yield: 4.19 g, 63%, m.p. 289–293°C; IR (KBr, cm⁻¹): 3111 (C–H), 2222 (C≡N), 1589 (C=N of oxadiazole), 1239 (C=S), 1185 (N–N), 1119 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 8.68 (dd, J = 7.2 Hz, 1H, quinazoline), 8.23–8.01 (m, 3H, quinazoline), 7.82–7.45 (m, 12H, ArH), 4.90 (s, 2H, N–CH₂–N), 3.87 (br s, 4H, piperazine), 3.46 (br s, 4H, piperazine). ¹³C NMR (100 MHz, DMSO-d₆): δ 180.7 (C=S), 167.5, 167.0, 157.8, 156.4, 155.2, 153.7, 149.6 (C-F), 147.5, 144.9, 142.6, 139.8, 135.2, 131.5, 129.1, 128.9, 128.0, 126.4, 125.0, 123.5, 122.8, 118.9 (Ar-C), 106.4 (C≡N), 95.1 (Ar-C-C≡N), 63.0 (CH₂), 50.7, 45.2. MS (ESI): m/z 666.2 [M+1]⁺. Anal. Calcd. for C₃₅H₂₆F₃N₇O₂S: C, 63.15; H, 3.94; N, 14.73; S, 4.82. Found: C, 63.10; H, 3.97; N, 14.70; S, 4.79.

4-({2-[4-(4-{[4-(4-Nitrophenyl)piperazin-1-yl]methyl}-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]quinazolin-4-yl}oxy)benzonitrile (7g)

Light yellow solid, yield: 4.76 g, 74%, m.p. 247–249°C; IR (KBr, cm⁻¹): 3044 (C–H), 2230 (C≡N), 1602 (C=N of oxadiazole), 1255 (C=S), 1180 (N–N), 1134 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 8.32 (dd, J = 6.8 Hz, 1H, quinazoline), 8.05–7.88 (m, 3H, quinazoline), 7.66–7.29 (m, 12H, ArH), 4.88 (s, 2H, N–CH₂–N), 3.72 (br s, 4H, piperazine), 3.46 (br s, 4H, piperazine). ¹³C NMR (100 MHz, DMSO-d₆): δ 177.3 (C=S), 168.9, 167.4, 156.2, 155.9, 153.0, 151.3, 149.7, 146.2, 144.5, 141.9, 138.2, 134.6, 131.8, 129.4, 128.8, 128.0, 126.9, 125.4, 123.5, 120.3 (Ar-C), 105.7 (C≡N), 97.2 (Ar-C-C≡N), 60.8 (CH₂), 49.7, 43.4. MS (ESI): m/z 643.1 [M+1]⁺. Anal. Calcd. for C₃₄H₂₆N₈O₄S: C, 63.54; H, 4.08; N, 17.44; S, 4.99. Found: C, 63.48; H, 4.12; N, 17.36; S, 5.04.

4-({2-[4-(4-{[4-(4-Hydroxyphenyl)piperazin-1-yl]methyl}-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]quinazolin-4-yl}oxy)benzonitrile (7h)

White solid, yield: 4.59 g, 75%, m.p. 189–192°C; IR (KBr, cm⁻¹): 3057 (C–H), 2220 (C≡N), 1615 (C=N of oxadiazole), 1231 (C=S), 1189 (N–N), 1129 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 8.72 (dd, J = 7.2 Hz, 1H, quinazoline), 8.44–8.17 (m, 3H, quinazoline), 7.94–7.36 (m, 12H, ArH), 5.27 (s, 1H, OH), 4.03 (s, 2H, N–CH₂–N), 3.68 (br s, 4H, piperazine), 3.24 (br s, 4H, piperazine). ¹³C NMR (100 MHz, DMSO-d₆): δ 176.9 (C=S), 168.1, 166.5, 155.4, 155.0, 151.1, 149.8, 149.0, 146.0, 142.3, 139.5, 136.0, 130.2, 129.7, 128.5, 127.3, 126.8, 126.0, 125.4, 122.9, 120.3 (Ar-C), 105.5 (C≡N), 96.4 (Ar-C-C≡N), 62.8 (CH₂), 49.8, 44.1. MS (ESI): m/z 614.5 [M+1]⁺. Anal. Calcd. for C₃₄H₂₇N₇O₃S: C, 66.54; H, 4.43; N, 15.98; S, 5.22. Found: C, 66.57; H, 4.45; N, 15.91; S, 5.27.

4-({2-[4-(4-{[4-(4-Methoxyphenyl)piperazin-1-yl]methyl}-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl]quinazolin-4-yl}oxy)benzonitrile (7i)

White solid, yield: 4.46 g, 71%, m.p. 199–203°C; IR (KBr, cm⁻¹): 3089 (C–H), 2224 (C≡N), 1590 (C=N of oxadiazole), 1224 (C=S), 1168 (N–N), 1123 (C–O–C). ¹H NMR (400 MHz, DMSO-d₆): δ 8.45 (dd, J = 6.8 Hz, 1H, quinazoline), 8.25–7.91 (m, 3H, quinazoline), 7.77–7.31 (m, 12H, ArH), 4.26 (s, 3H, OCH₃), 3.98 (s, 2H, N–CH₂–N), 3.61 (br s, 4H, piperazine), 3.34 (br s, 4H, piperazine). ¹³C NMR (100 MHz, DMSO-d₆): δ 173.2 (C=S), 169.5, 167.0, 155.9, 154.5, 153.2, 150.7, 149.3, 147.8, 143.5, 140.6, 138.2, 135.0, 131.7, 128.9, 127.5, 127.0, 126.4, 125.7, 122.1, 116.3 (Ar-C), 104.2 (C≡N), 97.5 (Ar-C-C≡N), 62.0 (CH₂), 57.6 (OCH₃), 50.9, 46.3. MS (ESI): m/z 628.7 [M+1]⁺. Anal. Calcd. for C₃₅H₂₉N₇O₃S: C, 66.97; H, 4.66; N, 15.62; S, 5.11. Found: C, 67.03; H, 4.70; N, 15.58; S, 5.07.

Pharmacology

The in vitro antimicrobial activity was performed using the broth microdilution method and the MIC with different strains, including multidrug-resistant clinical isolates. The newly prepared compounds were screened for their antibacterial activity against Gram-positive bacteria, Staphylococcus aureus (RN4220, KCTC 503 and KCTC 209) and a Gram-negative bacterium, Escherichia coli CCARM 1356. The strains of multidrug-resistant clinical isolates were multidrug-resistant Staphylococcus aureus (MRSA CCARM 3167 and MRSA CCARM 3506) and quinolone-resistant Staphylococcus aureus (QRSA CCARM 3505 and QRSA CCARM 3519). Clinical isolates were collected from various patients hospitalized in several clinics. Test susceptible microbacteria were grown to mid-log phase in Mueller-Hinton broth (MHB) and diluted 1000-fold in the same medium. The stock solutions of the synthesized compounds in DMSO were poured into 96-well plates and used to obtain final concentrations of 64–0.5 µg mL⁻¹ by the twofold serial dilution technique. Oxacillin and Norfloxacin were used as positive controls for bacteria. Suspensions of microbacteria were prepared to contain approximate 10⁵ CFU mL⁻¹ and applied to 96-well plates with serially diluted compounds to be tested and incubated at 37°C for 24 h. The MIC (expressed in µg mL⁻¹) was the lowest concentration of the test substance that completely inhibited growth of the microbacteria. The microbacteria growth was measured by the absorption at 630 nm using a microtiter enzyme-linked immunosorbent assay (ELISA) reader. All experiments were carried out three times.²⁷

In vitro cytotoxicity

Cytotoxicity activity assay: Human cervical (HeLa) cell monolayers were used as an in vitro model of cervicovaginal epithelium for testing the cytotoxicity of the new compounds. HeLa cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with fetal bovine serum (FBS; 10%) and antibiotics (penicillin–streptomycin mixture (100 U mL⁻¹)). Cells at 80%–90% confluence were split by trypsin (0.25% in phosphate-buffered saline (PBS); pH 7.4), and the medium was changed at 24 h intervals. The cells were cultured at 37°C in a 5% CO₂ incubator. The cells were grown to three passages and approximately 1 × 10⁴ cells were seeded into each well of a 96-well plate and allowed to incubate overnight to allow cells to attach to the substrate. After 24 h, the medium was replaced with DMEM supplemented with 10% FBS containing various concentrations of test compounds and incubated for 48 h. Then, 10 µL of MTT solution (5 mg mL⁻¹ in PBS) was added to each well. After incubation for 4 h, the medium was removed and the resulting formazan crystals were dissolved with 100 µL of DMSO. After shaking for 10 min, the optical density was measured at 570 nm using a microtiter ELISA reader. The assay was conducted four times. The IC₅₀ values were defined as the concentrations inhibiting 50% of cell growth.²⁸

Supplemental Material

Supplementary_Infomation – Supplemental material for Investigation of the antibacterial activity of new quinazoline derivatives against methicillin and quinolone resistant Staphylococcus aureus

Supplemental material, Supplementary_Infomation for Investigation of the antibacterial activity of new quinazoline derivatives against methicillin and quinolone resistant Staphylococcus aureus by Amit B Patel in Journal of Chemical Research

Footnotes

Acknowledgements

The author is thankful to the Administration of Daman & Diu, Principal and Vice Principal, Government College, Daman for encouragement and facilities. He is also thankful to SAIF, Panjab University and Centre of Excellence, Vapi, India, for spectral analyses.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Amit B Patel

Supplemental material

Supplemental material for this article is available online.

References

Baraka

. Boll Chim Farm 2001; 140: 90.

Balakumar

Lamba

Kishore

, et al. Eur J Med Chem 2010; 45: 4904.

Alagarsamy

Raja Solomon

Dhanabal

. Bioorg Med Chem 2007; 15: 235.

Wright

. Nat Prod Rep 2010; 27: 961.

Bedi

Kumar

Mahajan

. Bioorg Med Chem Lett 2004; 14: 5211.

Patel

Kumari

Chikhalia

. Res Chem Intermed 2015; 41: 4439.

Aly

. Chin J Chem 2003; 21: 339.

Patel

Kumari

Chikhalia

. Indian J Chem 2015; 54: 260.

Rad-Moghadam

Samavi

. J Heterocycl Chem 2006; 43: 913.

10.

Jatav

Mishra

Kashaw

, et al. Eur J Med Chem 2008; 43: 1945.

11.

Adelstein

. J Med Chem 1973; 16: 309.

12.

Modi

. J Saudi Chem Soc 2012; 16: 327.

13.

Saha

Tanwar

Marella

, et al. Mini-Rev Med Chem 2013; 13: 1027.

14.

Macaev

Ribkovskaia

Pogrebnoi

, et al. Bioorg Med Chem 2011; 19: 6792.

15.

Dhumal

Deshmukh

Bhosle

, et al. Bioorg Med Chem Lett 2016; 26: 3646.

16.

Omar

Mahfouz

Rahman

. Eur J Med Chem 1996; 31: 819.

17.

Ladani

Patel

. New J Chem 2015; 39: 9848.

18.

Luo

Liu

Chen

, et al. MedChemComm 2016; 7: 263.

19.

Letavic

Keith

, et al. Bioorg Med Chem Lett 2008; 18: 39.

20.

Baraldi

del Carmen Nuñez

Morelli

, et al. J Med Chem 2003; 46: 1318.

21.

Wang

Shi

Zhan

, et al. Chin J Chem 2015; 33: 1124.

22.

Papadopoulou

Geronikaki

Hadjipavlou-Litina

. Farmaco 2014; 60: 969.

23.

Chhatriwala

Patel

, et al. J Chem Res 2014; 38: 611.

24.

Wang

Wei

Deng

, et al. Arch Pharm 2009; 342: 671.

25.

Patel

Kumari

, et al. Med Chem Res 2013; 22: 367.

26.

Patel

Kumari

, et al. Lett Org Chem 2013; 9: 478.

27.

Sagar

Das

Koratkar

, et al. Cancer Lett 1992; 63: 189.

28.

Wayne

. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 6th ed. Wayne, PA: NCCLS, 2003, pp. M06-A07.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

1.48 MB