New chalcones and thiopyrimidine analogues derived from mefenamic acid: microwave-assisted synthesis, anti-HIV activity and cytotoxicity as antileukemic agents

2.1. Chemistry

The synthetic strategy followed toward the synthesis of different chalcone derivatives of mefenamic acid is outlined in Scheme 1. Thus, the acid chloride 5, prepared by the treatment of mefenamic acid 4 with PCl₅ under microwave irradiation (MWI), was treated with p-amino-acetophenone 6 to give the amide analogue 7 (60%). Alternatively, the amide 7 was prepared, in 83% after chromatography, by the coupling of mefenamic acid 4 with 6 in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) at 0°C and then at room temperature. The intermediate 7 was shared by all chalcone molecules synthesized. Treatment of 7 with the appropriate 4-substituted benzaldehydes (e.g. benzaldehyde, 4-chloro-, 4-bromo-, 4-dimethylamino-, 4-nitro and 4-tolualdehydes) in the presence of 20% NaOH for 6–8 min under MWI afforded, after chromatographic, the desired chalcone derivatives 8–13, respectively, in 70–80% yield. Alternatively, the analogues 8–13 were prepared in 75–85% yield by refluxing of 7 with substituted benzaldehydes in the presence of ethanolic NaOH for 4–5 h.

Scheme 1:

Reagents and conditions: (i) PCl₅, EtOH, room temperature, 3 h; (ii) method a: 5, MWI, 5 min; (iii) method b: 4, DCC, DMAP, CH₂Cl₂, room temperature, 16 h; (iv) method a: X–C₆H₄CHO, MWI, EtOH, 20% NaOH, 6–8 min; (v) method b: X–C₆H₄CHO, 20% NaOH, EtOH, room temperature, 4–5 h.

The structures of 7 and 8–13 were assigned on the basis of their NMR (¹H, ¹³C and 2D), which showed rather similar patterns for the aromatic scaffold.

The ¹H NMR spectra of 8–13 were characterized by the presence of additional aromatic and olefinic protons and carbon atoms, indicative for the formation of new chalcone analogues. The ¹H NMR spectra of 8–13 showed doublets at the regions δ=8.40–8.31 ppm (J~7.9 Hz) assigned for H-3, while the doublets at the regions δ=7.80–7.56 and 8.40–8.06 ppm (J=15.0–16.2 Hz) were assigned for the olefin protons Hα and Hβ of the chalcone moiety, respectively. The other aromatic and aliphatic protons have been fully analyzed (cf Experimental section). In the ¹³C NMR spectra of 7 and 8–13, the carbonyl carbon atoms (C=O and C_amide=O) appeared at the regions δ=190.2–189.7 and 166.2–165.8 ppm, respectively. Carbon atoms of the chalcone residue (Cα and Cβ) resonated at δ=122.1–119.0 and 149.2–139.3 ppm, respectively, while the resonances at δ=121.2–120.1, 150.3–144.9 and 119.5–117.8 ppm were assigned to the aromatic carbon atoms 1, 2 and 3, respectively. The signals at the regions δ=135.3–131.2, 118.4–116.3 and 128.4–128.0 ppm were attributed to carbon atoms 4, 5 and 6, respectively. Other aromatic carbon atoms as well as the substituents were fully analyzed (cf Experimental section).

Compound 10 was selected for further NMR experiments. The gradient heteronuclear multiple-bond correlation [35] NMR spectrum of 10 showed that six ³J_H,C couplings were observed: H-6 at δ_H=7.64 ppm with the C=O carbon atom at δ_C=189.7 ppm, while the carbonyl carbon atom of the NHCO group at δ_C=165.8 ppm coupled with both aromatic protons 3′′-H+5′′-H at δ_H=7.85 ppm, as well as with Hβ at δ_H=8.09 ppm. Further, two ³J_H,C couplings between Cβ at δ_C=145.1 ppm and both aromatic carbon atoms 2′′′-H+6′′-H at δ_H=7.60 ppm were observed. Additionally, a ³J_H,C coupling between Hα at δ=7.58 ppm and the amide carbon atom at δ_C=165.8 ppm was witnessed (Fig. 2).

Fig. 2:

J_H,C correlations in the HMBC NMR spectrum of 10.

We selected the chalcones 8 and 11 as precursors for the synthesis of new thiopyrimidine derivatives, aiming at examining their anti-HIV activity as well as cytotoxicity against human lymphocyte MT-4 cells in comparison to those of the chalcone analogues 8–13. Thus, the treatment of 8 and 11 with thiourea in 20% NaOH and EtOH under microwave irradiation (400 W, 180°C) for 3–6 min afforded, after neutralization with HCl and purification, the thiopyrimidine derivatives 14 and 15 in 65% and 60% yield, respectively (Scheme 2).

Scheme 2:

Synthesis of 2-((2,3-dimethylphenyl)amino)-N-(4-(6-aryl-2-thioxo-1,2-dihydropyrimidine-4-yl)phenyl)benzamides.

The structures of 14 and 15 were determined from their IR, ¹H and ¹³C NMR spectra. In the ¹H NMR spectra, H-5 of the thiopyrimidine ring appeared as singlets at δ=8.05 and 8.06 ppm, respectively, while the downfield doublets at δ=8.49 and 8.48 ppm were assigned to 2-H of the aromatic ring. The other aromatic and substituent protons were analyzed (cf Experimental section). The ¹³C NMR spectra showed signals at δ=181.2 and 181.8 ppm, which were attributed to C=S of the thiopyrimidine ring, respectively. The resonances at δ=175.0, 175.2 and 166.8, 166.9 ppm were assigned to C-4 of the thiopyrimidine moiety and to the carbonyl carbon atom of the amide group, respectively. C-5 and C-6 of the thiopyrimidine moiety resonated at δ=163.9, 163.4 and δ=109.6 and 110.1 ppm, respectively. The aromatic carbon atom C-1′′′ was appeared at δ=130.2 and 125.4 ppm, respectively, while C_4′′′-NMe₂ of 15 resonated at δ=153.9 ppm. The other aromatic and substituent carbon atoms were fully assigned (cf Experimental section).

2.2. In vitro anti-HIV activity

Compounds 8–15 were evaluated for their in vitro anti-HIV-1 (strain III_B) and anti-HIV-2 (strain ROD) activity, which were monitored by the inhibition of the virus-induced cytopathic effect in the human T-lymphocyte (MT-4) cells by MTT assay [36]. The results are listed in Table 1, in which the data for nevirapine (BOE/BIRG587) [37] and azidothymidine (AZT) [38] are included for comparison purposes. The cytotoxicity of the compounds was determined in parallel. All the compounds showed no activity against HIV-1 and HIV-2, except 9 and 11, which showed IC₅₀ >2.17 and >2.06 μm, respectively, with a selectivity index (SI) of <1, meaning no selectivity could be witnessed. The cytotoxicity of 9 and 11 against the human CD4⁺ lymphocytes MT-4 (C type adult T leukemia cells) (CC₅₀=2.17 and 2.06 μm, respectively) is remarkable and encourages to investigate the compounds further as lead structures.

4.1 General

Melting points are uncorrected and were measured on a Büchi melting point apparatus B-545 (Büchi Labortechnik AG, Switzerland). Microanalytical data were obtained with a Vario EL (Shimadzu, Japan). NMR spectra were recorded at 400 and 600 MHz (¹H) and at 150.91 MHz (¹³C) (Bruker, Germany) with tetramethylsilane as the internal standard. Heteronuclear assignments were verified by ¹H-¹³C HMBC experiments. Heteronuclear assignments were verified by ¹H-¹³C HMBC experiments. Microwave-supported reactions were performed in a SmithSynthesizer (Personal Chemistry AB), monomode microwave cavity at 2.45 GHz, temperature control by automated adjustment of irradiation power in a range from 0 to 300 W in Biotage microwave reaction vials (2–5 mL) with Teflone septum and an aluminum crimp top.

4.2 2-((2,3-Diethylphenyl)amino)benzoyl chloride (mefenamyl chloride) (5)

To a solution of mefenamic acid (4) (241 mg, 1.00 mmol) in abs. EtOH (10 mL) was added slowly PCl₅ (208 mg, 1.00 mmol) and the mixture was heated under reflux for 2 h. The mixture was kept overnight at 0–4°C and the solid was filtered and directly used for the next step without further purification.

4.3 4-(Acetylphenyl)-2-((2,3-dimethylphenyl)amino)benzamide (7)

Method a: A mixture of 5 (78 mg, 1.00 mmol) and p-aminoacetophenone (41 mg, 3.00 mmol) was irradiated under microwave for 5 min. The mixture was cooled to room temperature and rinsed with CHCl₃ (2×10 mL) and the combined extract was washed successively with a solution of 2 m HCl (10 mL) and a solution of 5% NaHCO₃ (10 mL). The organic layer was dried (MgSO₄) and evaporated to dryness. The crude product was purified by washing with pentane, dried and recrystallized from EtOH to give 7 (215 mg, 60%); m.p. 122–124°C. – ¹H NMR ([D₆]DMSO): δ=10.25 (s, 1H, NH_amid), 8.40 (d, 1H, J=7.9 Hz, 3-H), 8.06 (d, 1H, J=15.0 Hz, Hβ), 7.87 (d, 2H, J=7.8 Hz, 3′′-H+5′′-H), 7.85 (d, 2H, J=7.8 Hz, 2′′-H+6′′-H), 7.64 (d, 1H, J=7.7 Hz, 6-H), 7.59 (d, 1H, J=15.0 Hz, Hα), 7.45 (m, 1H, 4-H), 6.99 (m, 1H, 5-H), 6.89 (m, 1H, 5′-H), 6.70 (d, 1H, J=8.0 Hz, 4′-H), 6.32 (d, 1H, J=8.0 Hz, 6′-H), 5.20 (s, 1H, NHPh), 3.34 (s, 3H, C_3′-Me), 2.62 (COMe), 2.12 (s, 3H, C_2′-Me) ppm. – ¹³C NMR ([D₆]DMSO): δ=190.1 (COMe), 166.2 (C_amide=O), 149.3 (C-2), 145.1 (C-1′′), 139.1 (C-1′), 137.2 (C-3′), 135.5 (C-4′′), 135.3 (C-4), 133.2 (C-2′), 130.4 (C-3′′+C-5′′), 128.3 (C-6), 125.1 (C-6′), 123.1 (C-5′), 122.7 (C-2′′+C-6′′), 120.1 (C-1), 117.8 (C-3+C-4′), 116.3 (C-5), 21.3, 20.7 (2×MePh) ppm. – C₂₃H₂₂N₂O₂ (348.44): calcd. C 77.07, H 6.19, N 7.82; found C 76.91, H 6.02, N 7.59.

Method b: To a solution of appropriate carboxylic acid derivative (1.00 mmol) and phenol or amine derivative (1.00 mmol) in CH₂Cl₂ (10 mL), DMAP (0.20 mmol) and DCC (1.10 mmol) were added and the resulting solution was stirred for 16 h at room temperature. The reaction mixture was quenched with 0.5 n HCl and extracted with CH₂Cl₂. The organic phase was washed with a 1% NaHCO₃ solution and brine, dried over Na₂SO₄ and evaporated under vacuum. The residue was purified by flash column chromatography. Elution with CH₂Cl₂–MeOH (0–5%) as eluents afforded 7 (290 mg, 81%); m.p. and mixed m.p. with other physical properties were identical for those of the authentic sample prepared in method a.

4.4 General procedure for the preparation of chalcone derivatives (8–13)

Method a: To a mixture of 7 (358 mg, 1.00 mmol) in EtOH and 20% NaOH (40 mg, 1.00 mmol) was added substituted benzaldehyde (1.00 mmol) and the mixture was stirred under microwave irradiation for 6–8 min. The reaction progress was monitored by thin-layer chromatography (TLC). After completion of the reaction, the mixture was poured onto ice-cold water and neutralized with dil. HCl. The resulting solid was filtered, dried and recrystallized from EtOH.

4.5 General procedure for the synthesis of 2-((2,3-dimethylphenyl)amino)-N-(4-(6-aryl-2-thioxo-1,2-dihydropyrimidin-4-yl)phenyl)benzamides (14 and 15)

A mixture of chalcone (1.00 mmol), thiourea (76 mg, 1.0 mmol) and 20% NaOH (112 mg, 2.0 mmol) in EtOH (10 mL) was heated under MWI for 3–6 min, and the reaction was monitored by TLC. After cooling, the mixture was poured onto cold ice-water and acidified with dil. HCl, filtered, washed with water and dried. The product was recrystallized from EtOH to give the desired thiopyrimidine analogue.

4.6 In vitro anti-HIV assay

Evaluation of the antiviral activity of the test compounds against HIV-1 strain III_B and HIV-2 strain (ROD) in MT-4 cells was performed using the MTT assay as previously described. Stock solution (10×final concentrations) of the test compound was added in 25 μL volumes to two series of triplicate wells to allow simultaneous evaluation of their effects on mock- and HIV-infected cells at the beginning of each experiment. Serial five-fold dilutions of test compounds were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman Instruments, Fullerton, CA, USA). Untreated control HIV- and mock-infected cell samples were included for each sample. HIV-1 (III_B) or HIV-2 (ROD) stock (50 μL) at 100–300 CCID50 (50% cell culture infectious dose) or culture medium was added to the microtiter tray. Mock-infected cells were used to evaluate the effect of test compounds on uninfected cells in order to assess the cytotoxicity of the test compound. Exponentially growing MT-4 cells were centrifuged for 5 min at 1000 rpm, and the supernatant was discarded. The MT-4 cells were resuspended at 6×105 cells/mL, and an amount of 50 μL volume was transferred to the microtiter tray wells. Five days after infection, the viability of mock- and HIV-infected cells were examined spectrophotometrically by the MTT assay. The MTT assay is based on the reduction of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Across Organics, Geel, Belgium) by mitochondrial dehydrogenase of metabolically active cells to a blue purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Multiscan Ascent Reader, Labsystem, Helsinki, Finland) at two wavelengths (540 and 690 nm). All data were calculated using the median optical density value of three wells.