Synthesis, in Vivo Anti-inflammatory, and in Vitro Antimicrobial Activity of New 5-Benzofuranyl Fused Pyrimidines

Abstract

Chalcone (3) has been synthesized as a new chalcone derivative bearing benzofuran moiety at 1 position. Such chalcone was used as a model dielectrophile applied to react with some nucleophiles such as 5-amino pyrazoles, 5-amino-1,2,4-triazole, 2-aminobenzimidazole, and 6-uraciles under Michael reaction conditions and resulted in a new series of fused pyrimidines such as pyrazolo[1,5-a]pyrimidines 7a–e, [1,2,4]-triazolo[1,5-a]pyrimidine 9, pyrimido[1,2-a]benzimidazole 11, and synthesis of pyrido[2,3-d]pyrimidinones 13a and b. The structures of the synthesized target heterocyclic compounds were confirmed by microanalytical and spectral data such as Fourier transform (FT)-IR, ¹H-NMR, and MS spectra. The newly synthesized compounds were evaluated for their anti-inflammatory and antimicrobial activities; most showed significant activities.

The development of an effective therapeutic agent for the management of inflammation has undergone continual evolution leading to the emergence of more efficacious classes of drugs. Chalcones have been synthesized in order to develop drugs active against cancer,^1,2) malaria,^3,4) tuberculosis,⁵⁾ and cardiovascular diseases⁶⁾ or for their properties to modulate the regulation of biochemical pathways like nitric oxide (NO)⁷⁾ or tyrosine kinase.⁸⁾

Combination of pharmacophores on the same scaffold is a well established approach to the synthesis of more potent drugs.⁹⁾ Heterocycles containing benzofuran nucleus are known to have a wide range of potent pharmacological activities. Brazilian propolis naturally occurring benzofuran derivatives were found to have mild cytotoxic activity against highly liver-metastatic murine colon 26-L5 carcinoma and human HT-1080 fibrosarcoma cells.¹⁰⁾ Ailanthoidol has been reported to have antiviral, antioxidant and antifungal activities.¹¹⁾ Furthermore, most of compounds prepared from 2-acetylbenzofurans have antimicrobial, antitumor, anti-inflammatory activity, and they are used in treatment of cardiac arrhythmias.^12–15)

Motivated by these findings coupled with our ongoing program in the field of chalcones and other heterocyclic compounds conducted with them as anti-inflammatory agents.^16,17) In the present work, we decided to synthesize some new chalcones containing benzofuran moiety; in addition, a novel series of fused pyrimidines will be synthesized with a potential to act as dual anti-inflammatory–antibacterial agents with minimal gastrointestinal (GI) side effects and high safety margin.

Results and Discussion

Chemistry

It was considered worthwhile to explore the reaction for chalcone formation, which usually synthesized using Claisen–Schmidt reaction in basic medium and polar solvent. They are prepared by condensation reactions of acetophenone or substituted acetophenone with benzaldehyde or substituted benzaldehyde in ethanolic NaOH solution.¹⁸⁾ Herein, the condensation reaction of 2-acetylbenzofuran (1) with 4-piperidinylbenzaldehyde (2) in EtOH in the presence of NaOH afforded the propenone (chalcone) 3 (Chart 1). The structure of chalcone 3 was established through elemental and spectral analysis data. Its IR spectrum showed a strong peak at 1648 cm⁻¹ characteristic for unsaturated ketone and the ¹H-NMR spectrum exhibited signals of each of the olefinic double bond protons as two doublets at δ=6.70 and 7.52 ppm. Configuration of the title chalcone across such double bond is proposed as E which is in agreement with the basis single crystal X-ray crystallography founded by Hussein et al.¹⁹⁾

Chart 1. Chalcone Derivative 3 Formation

In an effort to synthesize new compounds with dual anti-inflammatory-antibacterial potential; the chalcone 3 was reacted with substituted 5-amino pyrazoles 4a–e^20,21) in EtOH in the presence of catalytic amount of piperidine; the fused pyrazolo-pyrimidine derivatives 7a–e were furnished as sole product. Such results are in agreement with recent findings founded by Pinku et al.,²²⁾ where, the dihydropyrazolo[1,5-a]pyrimidine derivative was not detected in the reaction. IR, ¹H-NMR and mass spectra for compounds 7a–e were found to be consistent with the proposed structures, where IR spectra exhibited the absorption bands for two C=N functions in each of the four condensed compounds and substantiated the absorption bands of the NH₂ groups in compounds 7d and e at ν_max=3278 and 3285 cm⁻¹, respectively, ¹H-NMR for these two compounds displayed two singlets at 6.0 and 6.15 for the two (D₂O-exchangeable, NH₂) groups, mass spectra gave the expected molecular weights for all of the four synthesized compounds (cf. Chart 2 and Experimental).

Chart 2. Formation of Pyrazolo-Pyrimidine Derivatives 7a–e

The stereo-controlled construction of carbon–nitrogen (C–N) bonds is an important topic in modern organic synthesis, driven by the significance of the biologically and synthetically interesting products formed.^23–26) Among a number of methods for this stereoselective transformation, the Michael addition of nitrogen nucleophiles, also known as aza-Michael addition, offers a straightforward C–N bond formation and has been successfully employed in the synthesis of diversified bioactive natural products.^27–32) Until now, various types of nitrogen nucleophiles have been explored in asymmetric aza-Michael addition such as hydrazines, amines, amides, pyrazoles.^16,17)

This reaction involves the reaction of aminopyrazoles 4a–e with such chalcone 3 (α,β-unsaturated ketone) under basic conditions. Although in all reactions between a dinucleophile (hydrazines, hydroxylamine, aminopyrazoles, etc.) with a dielectrophile of the type above mentioned, two compounds can be formed.³³⁾ Since only pyrazolo[1,5-a]pyrimidines 7a–e were isolated it was assumed that the reaction starts by the 1,4-Michael addition of NH on the CC double bond followed by the condensation of the NH₂ on the carbonyl group. So such reaction has been proceeded via the not isolated intermediates 5a–e and 6a–e.

In the same fashion, chalcone 3 was reacted with 5-amino-1,2,4-triazole 8 under the same reaction conditions mentioned for 7a–e formation and the fused triazolo-pyrimidine 9 was obtained. Pyrimido[1,2-a]benzimidazole derivative 11 was formed via interaction of chalcone 3 with 2-aminobenzimidazole 10 in absolute EtOH. The disappearance of the absorption bands characteristic for the two NH₂ groups which are present in the starting compounds 8 and 10 from the IR and ¹H-NMR spectra of compounds 9 and 11 in addition to their exact molecular weights given by mass spectra and elemental analysis are confirmatory for their structures (cf. Chart 3 and Experimental).

Chart 3. Formation of Fused Pyrimidines

Finally, refluxing chalcone 3 with 6-uraciles 12a and b in absolute EtOH afforded the pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione 13a and the thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one 13b derivatives, respectively. The IR spectrum of 13a showed an absorption band at ν_max=3338 and 3115 cm⁻¹ assignable for two NH groups; in addition, it showed absorption bands at 1678 and 1645 cm⁻¹ characteristic for two C=O amide groups. On the other hand, ¹H-NMR spectrum of compound 12a confirmed the disappearance of the NH₂ signal, while compound 13a displayed two singlets for NH’s (D₂O-exchangeable) (cf. Chart 3 and Experimental).

Biological Evaluation

In Vivo Anti-inflammatory Activity

The anti-inflammatory activity of the synthesized compounds (7a–e, 9, 11, 13a, b) and Ibuprofen as a reference drug were screened on Carrageenan-induced hind paw edema model^33,34) at different time intervals (Table 1). All screened compounds showed significant reduction in rat paw edema reflecting their anti-inflammatory activity. Notably, the percent inhibition reached its maximum value at the 4th hour, compounds 7d, e, and 13b showed the maximum potency at the 4th hour.

Table 1. Inhibitory Effect of the Synthesized Compounds and Ibuprofen on Carrageenan-Induced Edema

Compound	Inhibition % of edema
	1 h	2 h	3 h	4 h
Control	1.4±0.08b,c)	2.2±0.09a)	2.9±0.036b)	2.4±0.07a,b)
Ibuprofen	4.33c)±0.12	12.91d,e)±0.45	26.50d)±0.96	36.06e)±2.21
7a	1.20d)±0.09	23.27e)±1.03	20.01e)±0.72	37.26e)±2.23
7b	4.29c)±0.24	16.86c)±0.41	25.29d)±0.84	34.06c)±1.32
7c	9.26e)±2.42	14.11d)±2.85	27.87c)±0.89	36.24e)±2.54
7d	51.47a)±3.25	53.63a)±3.57	58.26a)±4.52	63.05a)±3.67
7e	36.15b)±2.42	57.15a)±4.25	52.78b)±2.65	76.24a)±4.22
9	23.08c)±2.16	39.63b)±3.56	36.07d)±1.03	52.24d)±0.39
11	18.07e)±1.20	29.16a)±4.21	27.78b)±1.56	41.72c)±2.13
13a	0.32e)±0.01	8.58e)±0.22	21.87e)±0.88	35.71e)±1.35
13b	0.37e)±0.01	14.01d)±0.85	26.50d)±1.24	56.32b)±2.52
LSD at 5%	5.50	4.12	4.53	3.58

a) Dose levels: test compounds (50 mg/kg body wt), ibuprofen (10 mg/kg body wt). b) Values are expressed as the mean±S.E.M. and analyzed by ANOVA. c) Values in parentheses (percentage anti-inflammatory activity, AI%). d) Significantly different compared to respective control values, p<0.05. e) Significantly different compared to respective control values, p<0.01.

In Vitro Antimicrobial Activity

Some of the synthesized compounds were screened for their antibacterial and antifungal activities at (5 µg/mL in N,N-dimethylformamide (DMF)) concentration against one Gram-positive bacteria (Staphylococcus aureus ATC C 29213), two Gram-negative bacteria (Escherichia coli ATC C 25922, Pseudomonas aeruginosa ATC C 27953), and three yeast (Fusarium oxysporium, Aspergillus niger, Candida albicans). Ciprofloxacin and nystatin were respectively used as standard antibacterial and antifungal references. Most of the tested compounds showed excellent antimicrobial activities with respect to the control drugs. In addition, the obtained data revealed significant antifungal activities especially compound 7a compared to that of the reference standard drug nystatin. All tested compounds except 7a showed good bactericidal activities against Staphylococcus aureus. Escherichia coli showed high sensitivity to 7d and 13a whereas 7b and d showed high potency against Pseudomonas aeruginosa (Table 2).

Table 2. Bactericidal and Fungicidal Activity of Newly Synthesized Compounds and Ciprofloxacin and Nystatin^a)

Compd. No.	Staphylococcus aureus	Escherichia coli	Pseudomonas aeruginosa	Candida albicans	Aspergillus niger	Fusarium oxysporium
7a	0	0	0	10	15	21
7b	11	0	10	8	17	0
7d	9	18	11	0	19	12
7e	8	0	0	6	0	19
13a	13	18	0	0	19	0
Cf	28	25	26	—	—	—
Nystin	—	—	—	24	22	24

a) The activities are based on the diameters of zones of inhibition in mm. One milliliter of stock solution (5 µg/mL in DMF) was applied in each hole of each paper disk. Cf=Ciprofloxacin (antibacterial agent), zone of inhibition: 0–13 mm (low); 14–16 mm (moderate); >16 mm (high); 0=no inhibition.

Results of antimicrobial activities were shown in Table 2. Data in Table 2 revealed that most of tested compounds have superior significant antifungal potency to antibacterial potency. Compound 7d was found to have the most potent activity similar to the antifungal drug nystatin against Fusarium oxysporium. All tested compounds except 7a showed good bactericidial activity against Staphylococcus aureus. Escherichia coli showed high sensitivity to 7a and 13a, whereas 7b and d showed high potency against Pseudomonas aeruginosa.

Conclusion

We successfully obtained a novel series of fused pyrimidine derivatives with high functionality based on chalcone derivative bearing benzofuran moiety at 1 position. Also the plethora of research described in this manuscript indicates a wide spectrum of pharmacological activities exhibited by the prepared compounds produced here as anti-inflammatory and antimicrobial agents.

Experimental

Reagents and Instruments

Reagents and solvents were used as obtained from the supplier without further purification. All melting points reported are uncorrected and were determined on a Stuart electric melting point apparatus. Elemental analysis were carried out in the Micro Analytical Center, Cairo University, Giza, Egypt. TLC was performed on Merk TLC aluminium sheets silica gel 60 F₂₅₄ with detection by UV quenching at 254 nm. IR spectra (in KBr, cm⁻¹) were recorded on Fourier transform (FT)-IR 1430 Perkin-Elmer spectrophotometer. ¹H-NMR spectra were recorded on a Varian 300 MHz (Germany, 1999) with residual proton signal of the deuterated solvent as the internal reference (δ_H=7.26 ppm for CDCl₃ and δ_H=2.51 ppm for DMSO-d₆). Tetramethylsilane (TMS) was used as an internal standard with chemical shifts δ in ppm from downfield to upfield. Electron ionization (EI)-MS were recorded on a GC-MS-HP model MS5988.

Synthesis of (E)-1-(Benzofuran-2-yl)-3-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one (3)¹⁹⁾

To a stirred solution of 2-acetylbenzofuran (1) (1.6 g, 10 mmol) and 4-piperidinylbenzaldehyde (2) (1.89 g, 10 mmol) in ethanol (30 mL), 10% aqueous sodium hydroxide (10 mL) was added portion-wise at room temperature for 10 min, the reaction mixture was further stirred for 3 h. The resulting solid was filtered, washed with water, dried and crystallized from EtOH/DMF to afford chalcone 3. orange powder; 85% yield; mp 178–179°C; ¹H-NMR (DMSO-d₆) δ: 1.40–1.56 (6H, m, piperidine H), 2.98–3.26 (4H, m, piperidine H), 6.70–6.72 (1H, d, J=14.0 Hz, –CO–CH=), 6.88–6.92 (2H, d, J=14.0 Hz, Ar-H), 7.52–7.54 (1H, d, J=14 Hz,=CH-Ar), 7.61–7.82 (6H, m, Ar-H), 8.16 (1H, s, benzofuran H). IR (KBr) cm⁻¹: 3099 (CH-Ar), 1648 (C=O), 1578 (CH olefinic). MS m/z: 331 (M⁺, 100), 330 (68), 274 (26), 247 (28), 214 (56), 186 (27), 145 (87), 117 (7). Anal. Calcd for C₂₂H₂₁NO₂: C, 79.73; H, 6.39; N, 4.23. Found: C, 79.56; H, 6.21; N, 4.17.

Synthesis of Pyrazolo[1,5-a]pyrimidines (7a–e), [1,2,4]-Triazolo[1,5-a]pyrimidine (9) and Pyrimido[1,2-a]benzimidazole (11)

To a suspension of chalcone 3 (0.33 g, 1.0 mmol) in absolute ethanol (30 mL), the appropriate amines 4a–e, 10 or 8 (1.0 mmol) and piperydine (0.3 mL) were added. The reaction mixture was refluxed for 15 h, the mixture was left to cool. The crystals separating on cooling were filtered off and crystallized from EtOH/DMF.

5-(Benzofuran-2-yl)-2-phenyl-7-(4-(piperidin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine (7a)

Yellow powder; yield 72%; mp 258–259°C; ¹H-NMR (DMSO-d₆) δ: 1.44–1.52 (6H, m, piperidine), 2.96–3.02 (4H, m, piperidine), 6.03 (1H, s, pyrazole), 6.80–6.88 (2H, d, J=8.6 Hz, Ar-H), 7.00–7.78 (11H, m, Ar-H), 7.80 (1H, s, pyrimidine), 8.14 (1H, s, benzofuran). IR (KBr) cm⁻¹: 3055 (CH, Ar), 1611 (C=N), 1514 (C=N). MS m/z: 472 (M⁺+2, 100), 471 (M⁺+1, 94), 470 (M⁺, 13), 443 (46), 369 (10), 331 (26), 186 (33), 117 (9). Anal. Calcd for C₃₁H₂₆N₄O: C, 79.12; H, 5.57; N, 11.91. Found: C, 79.39; H, 5.64; N, 11.68.

5-(Benzofuran-2-yl)-2-phenyl-3-(phenyldiazenyl)-7-(4-(piperidin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine (7b)

Orange powder; yield 65%; mp 165–166°C; ¹H-NMR (DMSO-d₆) δ: 1.47–1.56 (6H, m, piperidine) 3.19–3.31 (4H, m, piperidine), 6.85–6.92 (2H, d, J=8.6 Hz, Ar-H), 7.35–7.82 (16H, m, Ar-H), 8.10 (1H, s, pyrimidine), 8.15 (1H, s, benzofuran). IR (KBr) cm⁻¹: 3061 (CH, Ar), 1642 (C=N), 1579 (N=N). MS m/z: 576 (M⁺+1, 2.30), 575 (M⁺, 1.00), 381 (3), 330 (100), 274 (9), 246 (5), 129 (9), 117 (14), 84 (15). Anal. Calcd for C₃₇H₃₀N₆O: C, 77.33; H, 5.26; N, 14.62. Found: C, 77.48; H, 5.44; N, 14.50.

5-(Benzofuran-2-yl)-3-((4-chlorophenyl)diazenyl)-2-phenyl-7-(4-(piperidin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine (7c)

Orange powder; yield 68%; mp 188–190°C; ¹H-NMR (DMSO-d₆) δ: 1.49–1.56 (6H, m, piperidine) 3.25–3.31 (4H, m, piperidine), 6.93–6.95 (2H, d, J=8.6 Hz, Ar-H), 7.33–7.72 (15H, m, Ar-H), 8.07 (1H, s, pyrimidine), 8.13 (1H, s, benzofurane). IR (KBr) cm⁻¹: 3100 (CH, Ar), 1641 (C=N), 1579 (N=N). MS m/z: 609 (M⁺, 2.30), 491 (3), 381 (3), 330 (18), 219 (9), 132 (100), 117 (20), 84 (26). Anal. Calcd for C₃₇H₂₉ClN₆O: C, 72.96; H, 4.80; N, 13.80. Found: C, 73.12; H, 5.08; N, 13.65.

5-(Benzofuran-2-yl)-3-(phenyldiazenyl)-7-(4-(piperidin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-2-amine (7d)

Orange red powder; yield 72%; mp 209–210°C; ¹H-NMR (DMSO-d₆) δ: 1.48–1.56 (6H, m, piperidine), 3.21–3.30 (4H, m, piperidine), 6.00 (2H, s, D₂O-exchangeable, NH₂), 6.86–6.88 (2H, d, J=8.6 Hz, Ar-H), 7.10–7.50 (11H, m, Ar-H), 7.79 (1H, s, pyrimidine), 7.81 (1H, s, benzofurane). IR (KBr) cm⁻¹: 3278 (NH₂), 2926 (CH, Ar), 1645 (C=N), 1566 (N=N). MS m/z: 513 (M⁺, 0.5), 303 (23), 381 (3), 210 (20), 129 (87), 117 (47), 84 (34), 82 (100); Anal. Calcd for C₃₁H₂₇N₇O: C, 72.50; H, 5.30; N, 19.09. Found: C, 72.76; H, 5.18; N, 19.34.

5-(Benzofuran-2-yl)-3-((4-chlorophenyl)diazenyl)-7-(4-(piperidin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-2-amine (7e)

Orange powder; yield 70%; mp 267–268°C; ¹H-NMR (DMSO-d₆) δ: 1.48–1.56 (6H, m, piperidine), 3.27–3.31 (4H, m, piperidine), 6.15 (2H, s, D₂O-exchangeable, NH₂), 6.86–6.88 (2H, d, J=8.6 Hz, Ar-H), 7.00–7.60 (10H, m, Ar-H), 7.82 (1H, s, pyrimidine), 7.86 (1H, s, benzofurane H). IR (KBr) cm⁻¹: 3285 (NH₂), 2932 (CH, Ar), 1591 (C=N), 1563 (N=N). MS m/z: 548 (M⁺, 1.1), 464 (2), 310 (6), 236 (8), 116 (7), 84 (8), 82 (100); Anal. Calcd for C₃₁H₂₆ClN₇O: C, 67.94; H, 4.78; N, 17.89. Found: C, 67.69; H, 4.66; N, 17.72.

5-(Benzofuran-2-yl)-7-(4-(piperidin-1-yl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (9)

Yellow powder; yield 70%; mp 182–183°C; ¹H-NMR (DMSO-d₆) δ: 1.44–1.59 (6H, m, piperidine), 2.95–3.10 (4H, m, piperidine) 6.93–6.95 (2H, d, J=8.6 Hz, Ar-H), 7.33–7.71 (6H, m, Ar-H), 7.82 (1H, s, pyrimidine), 8.14 (1H, s, benzofurane), 8.69 (1H, s, triazole). IR (KBr) cm⁻¹: 3061 (CH, Ar), 1641 (C=N), 1581 (C=N). MS m/z: 395 (M⁺, 1.2), 278 (12), 222 (5), 167 (11), 117 (18), 84 (83), 83 (100). Anal. Calcd for C₂₄H₂₁N₅O: C, 72.89; H, 5.35; N, 17.71. Found: C, 73.21; H, 5.54; N, 18.03.

2-(Benzofuran-2-yl)-4-(4-(piperidin-1-yl)phenyl)pyrimido[1,2-a]benzimidazole (11)

Pale yellow powder; yield 75%; mp 268–270°C; ¹H-NMR (DMSO-d₆) δ: 1.46–1.52 (6H, m, piperidine), 3.04–3.20 (4H, m, piperidine), 6.83–6.85 (2H, d, J=8.6 Hz, Ar-H), 7.00–7.49 (10H, m, Ar-H), 7.65 (1H, s, pyrimidine), 7.75 (1H, s, benzofuran). IR (KBr) cm⁻¹: 2931 (CH, Ar), 1626 (C=N), 1573 (C=N). MS m/z: 444 (M⁺, 0.12), 387 (2.0), 327 (10.0), 228 (3.0), 117 (5), 84 (10), 83 (100); Anal. Calcd for C₂₉H₂₄N₄O: C, 78.36; H, 5.44; N, 12.60. Found: C, 78.64; H, 5.58; N, 12.37.

Synthesis of Pyrido[2,3-d]pyrimidines 13a and b

To a solution of chalcone 3 (0.33 g, 1.0 mmol) in DMF (30 mL), 6-aminouracile or 6-aminothiouraciles 12a and b (1.0 mmol) was added. The reaction mixture was heated under reflux for 35 h. After cooling, the precipitate was collected by filtration and recrystallized from EtOH/DMF to afford 13a and b, respectively.

7-(Benzofuran-2-yl)-5-(4-(piperidin-1-yl)phenyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (13a)

Yellow powder; yield 72%; mp 285–286°C; ¹H-NMR (DMSO-d₆) δ: 1.45–1.52 (6H, m, piperidine), 3.04–3.15 (4H, m, piperidine), 6.60 (1H, s, D₂O-exchangeable, NH), 6.82–6.86 (2H, d, J=8.6 Hz, Ar-H), 7.00–7.56 (6H, m, Ar-H), 7.89 (1H, s, pyridine), 8.15 (1H, s, benzofuran), 9.68 (1H, s, D₂O-exchangeable, NH). IR (KBr) cm⁻¹: 3338 (NH), 3115 (NH), 2968 (CH, Ar), 1678 (C=O amide), 1645 (C=O amide), 1595 (C=N). MS m/z: 438 (M⁺, 3.0), 354 (6), 326 (8), 268 (14), 192 (11), 117 (36), 82 (100); Anal. Calcd for C₂₆H₂₂N₄O₃: C, 71.22; H, 5.06; N, 12.78. Found: C, 71.53; H, 4.83; N, 12.97.

7-(Benzofuran-2-yl)-5-(4-(piperidin-1-yl)phenyl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (13b)

Yellow powder; yield 75%; mp 298–300°C; ¹H-NMR (DMSO-d₆) δ: 1.48–1.61 (6H, m, piperidine), 3.02–3.14 (4H, m, piperidine), 6.30 (1H, s, D₂O-exchangeable, NH), 6.72–6.84 (2H, d, J=8.6 Hz, Ar-H), 6.95–7.69 (6H, m, Ar-H), 7.82 (1H, s, pyridine), 8.13 (1H, s, benzofuran), 8.33 (1H, s, D₂O-exchangeable, NH). IR (KBr) cm⁻¹: 3325 (NH), 3093 (NH), 2929 (CH, Ar), 1642 (C=O amide), 1576 (C=N), 1173 (C=S). MS m/z: 455 (M⁺+1, 6), 454 (M⁺, 2.0), 453 (23), 332 (27), 272 (25), 243 (64), 158 (61), 122 (100), 117 (24), 82 (61); Anal. Calcd for C₂₆H₂₂N₄O₂S: C, 68.70; H, 4.88; N, 12.33; S, 7.05. Found: C, 68.99; H, 4.61; N, 12.65;S, 6.74.

Pharmacology

Anti-inflammatory Screening

Carrageenan-induced rat paw edema.

To evaluate the possible anti-inflammatory properties of the synthesized compounds, either vehicle (10 mL/kg), ibuprofen (10 mg/kg), or the synthetic compounds at the same dose level. The compounds were given orally 30 min before injection of 1% carrageenan into the sub plantar area of the rats, right hind paw. Paw volume was measured at 0, 1, 2, 3, and 4 h after carrageenan injection by using a plethysmometer (UgoBasile, Milan, Italy).

The institutional and national guide for the care and use of laboratory animals was followed where the study was approved by Alexandria University Animal Ethics Committee, Egypt.

Statistical Analysis

Results were expressed as the mean±standard error of the mean (S.E.M.). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. p<0.05 was considered statistically significant.

Antimicrobial Screening

Applying the agar plate diffusion technique³⁵⁾ some of the newly synthesized compounds were screened in vitro for antimicrobial activity against representative Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), yeast (Fusarium oxysporium, Aspergillus niger, Candida albicans). In this method a standard 5-mm diameter sterilized filter paper disc impregnated with the compound (5 µg/mL in DMF) was placed on an agar plate seeded with the tested organism. The plates were incubated for 24 h at 37°C for bacteria and 28°C for fungi. The zone of inhibition of bacterial and fungal growth around the disc was observed.

Conflict of Interest

The authors declare no conflict of interest.

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