Thermal solvent-free synthesis of chromonyl chalcones, pyrazolines and their in vitro antibacterial, antifungal activities

Abstract

A facile and ecofriendly synthesis of new chromonyl chalcones 3a-b from 3-formylchromone 1 and active methyl compounds 2a-b is reported under thermal solvent-free heating condition in good yields. The chromonyl chalcones 3a-b were used as intermediates under green condition for the synthesis of new bioactive pyrazoline derivatives 4a-f. The compounds were tested for antimicrobial activity by disk diffusion assay with slight modifications against Gram-positive, Gram-negative strains of bacteria as well as fungal strains. The investigation of antimicrobial screening revealed that compounds 3a-b and 4a-f showed antibacterial and antifungal activities.

Keywords::

• 3-Formylchromone
• green synthesis
• antimicrobial activity

Introduction

Human infections particularly those involving microorganisms like bacteria and fungi cause serious damages in tropical and subtropical countries of the world and these infections are a major threat to public health despite tremendous growth in human chemotherapeutic medicine. These infections can occur in invasive form, and are an increasing problem due to the increase of their incidence in hospitals, especially in patients who are undergoing cancer treatment, transplantation or are immune suppressed for other reasons¹. Methicillin-resistant Staphylococcus aureus (MRSA) is common Gram-positive pathogen of nosocomial infections which account for outbreaks and are increasing in frequency^2,3. Escherichia coli is responsible for the world’s most common and serious infectious diseases like invasive dysentery and diarrhea^4,5. The different parasitic bacteria such as S. aureus, S. pyogenes, S. typhimurium, E. coli have important effect on the human’s mucosal health. The infection with these microorganisms may have significant impact on huge demolition of host tissues and severe diseases^6,7. Thus, antibiotics provide the main basis for the therapy of microbial (bacterial and fungal) infections. However, overuse of antibiotics has become the major factor for the emergence and dissemination of multi-drug resistant strains of several groups of microorganisms⁸. Furthermore, the drugs available are either too expensive or have undesirable side effects or contraindications⁹. The aim of this work is the synthesis of new molecules able to inhibit the growth of Gram-positive, Gram-negative bacteria and fungi.

Chromones comprise a vast array of oxygen containing compounds ubiquitous in plants¹⁰. They form basic nucleus of flavones and have been recognized as the essential component of pharmacophores of a large number of bioactive molecules¹¹. Interest in chromone containing structures stems from their widespread occurrence in molecules that exhibit significant antimicrobial¹², antioxidant¹³, neuroprotective¹⁴, HIV-inhibitory¹⁵ and anticancer¹⁶ activities. In addition, chromone derivatives are also active at benzodiazepine receptors, lipoxygenases and cyclooxygenases¹⁷. Chalcones and pyrazolines are another important class of compounds with wide spread biological applications^18,19.

Growing concern about environmental damage leads to an urgent requirement for the development of efficient and environmentally benign chemical processes for the synthesis of new molecules. Solvent-free reactions are gaining importance in this context as the procedure demonstrates obvious and significant advantages²⁰. However, most of the reported solvent-free accomplishments focus on reactions promoted by microwave irradiation, hand grinding and mechanical milling^21–23. In contrast, reports on direct heating of the reagents under thermal heating conditions without any solvent are relatively less explored. To the best of our knowledge, no reports have been so far made in the synthesis of chromonyl chalcones and derivatives in solvent-free thermal heating condition. Thus, in the present study, we wish to report the novel synthesis of series of chromonyl chalcones and pyrazolines in excellent yields, under thermal solvent-free heating condition and their evaluation for antimicrobial activities.

Materials and methods

Chemistry

Melting points were taken in Riechert Thermover (Austria)instrument and are uncorrected. The IR spectra were recorded on Perkin Elmer (Spectro Lab, United Kingdom) RXI spectrometer in KBr, ¹H NMR on Bruker DRX 300 MHz spectrometer and Bruker Avance (Switzerland) II 400 MHz spectrometer using tetramethyl silane as the internal standard and DMSO-d₆/CDCl₃ as solvent. Mass spectra were obtained on Jeol-SX-102 (FAB) spectrometer. The microanalytical data were collected on Carlo Erba (Carlo Erba Instruments, Germany) analyzer model 1108. 3-Formylchromone 1²⁴, hydrazinobenzothiazole²⁵ and 5-acetylbarbituric acid 2b²⁶ were synthesized by reported methods. Dehydroacetic acid 2a was purchased from E. Merck (Merck, Darmstadt, Germany). Other chemicals were of commercial grade and used without further purification. The purity of the compounds was checked by thin layer chromatography (TLC) on glass plates coated with silica gel G₂₅₄ (E. Merck) using chloroform-methanol (3:1 v/v) mixture as mobile phase and visualized by iodine vapours.

General procedure for the synthesis of chalcones (3a-b) under conventional method

To a solution of 3-formylchromone 1 (1.00 g, 5.7 mmol) and dehydroacetic acid 2a (0.965 g, 5.7 mmol)/5-acetyl barbituric acid 2b (0.970 g, 5.7 mmol) in methanol (12 mL), pyridine (0.5 mL) was added. The reaction mixture was stirred at room temperature/refluxed for a specified time. After completion of the reaction (checked by TLC), the reaction mixture was concentrated and cooled at room temperature. The yellow solid, which precipitated out, was filtered, washed with methanol and dried. The compounds were purified by recrystallization from chloroform-methanol (1:3 v/v) mixture as shining yellow crystals.

General procedure for the synthesis of chalcones (3a-b) under solvent-free condition

A mixture of 3-formylchromone 1 (1.00 g, 5.7 mmol), dehydroacetic acid 2a (0.965 g, 5.7 mmol)/5-acetyl barbituric acid 2b (0.970 g, 5.7 mmol) and pyridine (0.3 mL) were mixed well and air dried. The reaction mixture was then heated at 80 ˚C in a 100 mL beaker. After completion of the reaction (checked by TLC), the reaction mixture was slurred in water (25 mL). The yellow solid obtained was filtered, washed with methanol and dried. The compounds were purified by recrystallization from chloroform-methanol (1:3 v/v) mixture as shining yellow crystals.

(2E)-1-(4-Hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-3-(4-oxo-4H-1-benzopyran-3-yl)-2-propen-1-one (3a)

Pale yellow crystals; mp 235–237°C. ¹H NMR (CDCl₃, 300 MHz) δ: 2.28 (3H, s, CH₃), 5.96 (1H, s, C-5′), 7.36-7.73 (3H, m, Ar-H), 7.87 (1H, d, J = 15.9 Hz, Ha), 8.32 (1H, s, C-2), 8.35 (1H, dd, J = 7.8 Hz, 1.8 Hz, C-5), 8.77 (1H, d, J = 15.9 Hz, Hb). IR (KBr) υ: 3437 (OH), 1721 (C=O), 1659 (C=O). MS-FAB m/z (%): 324 (M⁺, 100), 323 (25), 309 (5), 308 (10), 279 (10), 198 (80), 171 (70), 120 (10), 92 (5). Anal. Cald for C₁₈H₁₂O₆: C, 66.67; H, 3.73. Found: C, 66.53; H, 3.69.

(2E)-3-(4-Oxo-4H-1-benzopyran-3-yl)-1-(2,4,6-pyrimidinetrione-5-yl)-2-propen-1-one (3b)

Light yellow crystals; mp >300°C. ¹H NMR (DMSO-d₆, 300 MHz) δ: 7.40 (1H, d, J = 15.2 Hz, Ha), 7.55-7.91 (3H, m, Ar-H), 8.17 (1H, dd, J = 8.0 Hz, 1.6 Hz, C-5), 8.95 (1H, s, C-2), 9.09 (1H, d, J = 15.9 Hz, Hb), 11.06 (1H, s, NH), 11.76 (1H, s, NH). IR (KBr) υ: 3078 (NH), 1740 (C=O), 1643 (C=O). MS-FAB m/z (%): 326 (M⁺, 60), 206 (10), 198 (70), 171 (65), 145 (50), 120 (40). Anal. Cald for C₁₆H₁₀N₂O₆: C, 58.83; H, 3.06; N, 8.58. Found: C, 58.72; H, 3.14; N, 8.44.

General procedure for the synthesis of pyrazolines (4a-e) and bipyrazole (4f) under conventional method

The compound 3a-b (1.00 g, 3 mmol) was dissolved in acetic acid (8 mL) and hydrazines (hydrazine hydrate (0.15 g, 3 mmol)/hydrazinobenzothiazole (0.49 g, 3 mmol)/phenylhydrazine (0.51 g, 3 mmol) added to it. The reaction mixture was stirred at room temperature or refluxed in a heating mantle for specified period of time. After that, the reaction mixture was cooled and poured into ice-cold water (20 mL). The solid, which precipitated out, was filtered, washed with water and dried. Purification of 4a-f was achieved by recrystallization from chloroform-methanol (1:4 v/v) mixture.

General procedure for the synthesis of pyrazolines (4a-e) and bipyrazole (4f) under solvent-free heating method

The compound 3a-b (1.00 g, 3 mmol) and hydrazines (hydrazine hydrate (0.15 g, 3 mmol)/hydrazinobenzothiazole (0.49 g, 3 mmol)/phenylhydrazine (0.51 g, 3 mmol) were mixed well and heated at 80°C in a 100 mL beaker. After completion of the reaction (checked by TLC), the reaction mixture was slurred in water (20 mL). The solid, obtained was filtered, washed with methanol and dried. The compounds were purified by recrystallization from chloroform-methanol (1:4 v/v) mixture to get 4a-f.

3-(4-Hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-5-(4-oxo-4H-1-benzopyran-3-yl)pyrazoline (4a)

White solid; mp 260–262°C. ¹H NMR (CDCl₃, 300 MHz) δ: 2.27 (3H, s, CH₃), 3.69-3.92 (2H, m, He, Hf), 5.20-5.26 (1H, m, Hd), 6.04 (1H, s, C-5′), 6.68 (1H, s, NH), 7.35-7.68 (3H, m, Ar-H), 8.02 (1H, s, C-2), 8.16 (1H, d, J = 7.9 Hz, C-5), 12.78 (1H, s, OH, D₂O exchangeable). IR (KBr) υ: 1700 (C=O), 1665 (C=O). MS-FAB m/z (%): 338 (M⁺, 60), 337 (70), 309 (5), 281 (5), 217 (5), 193 (15), 189 (5), 185 (5), 163 (10), 120 (15), 93 (45), 92 (10). Anal. Cald for C₁₈H₁₄N₂O₅: C, 63.90; H, 4.17; N, 8.28. Found: C, 63.83; H, 4.24; N, 8.22.

1-Benzothiazolyl-3-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-5-(4-oxo-4H-1-benzopyran-3-yl)pyrazoline (4b)

Pale yellow solid; mp >300°C. ¹H NMR (DMSO-d₆, 300 MHz) δ: 2.48 (3H, s, CH₃), 3.07–4.14 (2H, m, He, Hf), 5.44–5.50 (1H, m, Hd), 6.29 (1H, s, C-5′), 8.02 (1H, d, J = 7.1 Hz, C-5), 7.05-7.81 (7H, m, Ar-H), 8.41 (1H, s, C-2) 12.23 (1H, s, OH, D₂O exchangeable). IR (KBr) υ: 1727 (C=O), 1649 (C=O). MS-FAB m/z (%): 471 (M⁺, 100), 443 (5), 415 (5), 428 (5), 322 (5), 307 (50), 177 (5), 120 (20). Anal. Cald for C₂₅H₁₇N₃O₅S: C, 63.68; H, 3.63; N, 8.91. Found: C, 63.61; H, 3.67; N, 8.87.

5-(4-Oxo-4H-1-benzopyran-3-yl)-3-(2,4,6-pyrimidinetrione-5-yl)pyrazoline (4c)

White solid; mp >300°C. ¹H NMR (DMSO-d₆, 300 MHz) δ: 3.43-3.49 (1H, m, He), 3.82–3.89 (1H, m, Hf), 4.69–4.75 (1H, m, Hd), 7.49–7.84 (3H, m, Ar-H), 6.69 (1H, s, NH), 8.09 (1H, dd, J = 8.0 Hz, 1.2 Hz, C-5), 8.29 (1H, s, C-2), 10.31 (1H, s, NH), 10.45 (1H, s, NH). IR (KBr) υ: 3214 (NH), 3069 (NH), 1721(C=O), 1628 (C=O). MS-FAB m/z (%): 340 (M⁺, 75), 326 (25), 322 (30), 206 (5), 198 (5), 171 (30), 145 (5), 120 (30). Anal. Cald for C₁₆H₁₂ N₄ O₅: C, 56.47; H, 3.52; N, 16.47. Found: C, 56.31; H, 3.46; N, 16.54.

1-Phenyl-5-(4-oxo-4H-1-benzopyran-3-yl)-3-(2,4,6-pyrimidinetrione-5-yl)pyrazoline (4d)

Pale yellow solid; mp >300°C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 3.51–3.56 (1H, m, He), 3.73–3.78 (1H, m, Hf), 4.42–4.44 (1H, m, Hd), 7.25–7.45 (8H, m, Ar-H), 7.55 (1H, s, C-2), 7.79 (1H, dd, J = 7.6 Hz, 1.8 Hz, C-5), 10.60 (1H, s, NH), 10.76 (1H, s, NH). IR (KBr) υ: 3068 (NH), 1737 (C=O), 1649 (C=O). MS-FAB m/z (%): 416 (M⁺, 70), 415(5), 326 (45), 198 (40) 171 (30), 120 (20). Anal. Cald for C₂₂H₁₆N₄O₅: C, 63.46; H, 3.84; N, 13.46. Found: C, 63.50; H, 3.71; N, 13.38.

1-Benzothiazolyl-5-(4-oxo-4H-1-benzopyran-3-yl)-3-(2,4,6-pyrimidinetrione-5-yl)pyrazoline (4e)

Brown crystals; mp >300°C. ¹H NMR (DMSO-d₆, 400 MHz) δ: 3.81–3.87 (1H, m, He), 4.10–4.17 (1H, m, Hf), 5.49–5.53 (1H, m, Hd), 7.12–7.76 (7H, m, Ar-H), 8.15 (1H, dd, J = 8.0 Hz, 1.6 Hz, C-5), 8.27 (1H, s, C-2), 11.17 (1H, s, NH), 11.56 (1H, s, NH). IR (KBr) υ: 3069 (NH), 1701(C=O), 1644 (C=O). MS-FAB m/z (%): 473 (M⁺, 50), 326 (20), 198 (10), 145 (20), 120 (30). Anal. Cald for C₂₃H₁₅N₅O₅S: C, 58.35; H, 3.17; N, 14.79. Found: C, 58.27; H, 3.25; N, 14.71.

1-Phenyl-3-[4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl]-5-[5-(2-hydroxyphenyl)-1-phenyl pyrazol-4-yl]pyrazoline (4f)

Red crystals; mp 250–253°C. ¹H NMR (CDCl₃, 300 MHz) δ: 2.35 (3H, s, CH₃), 3.38–3.50 (1H, m, He), 3.66–4.11 (1H, m, Hf), 4.99–5.34 (1H, m, Hd), 6.01 (1H, s, C-5′), 6.90-7.13 (4H, m, Ar-H), 7.18–7.42 (14H, m, Ar-H), 7.68 (1H, s, Hc), 13.27 (1H, s, OH, D₂O exchangeable). IR (KBr) υ: 3432 (OH), 1701 (C=O). MS-FAB m/z (%): 504 (M⁺, 70), 476 (5), 460 (15), 449 (8), 445 (10), 379 (5), 352 (20), 279 (15). Anal. Cald for C₃₀H₂₄N₄O₄: C, 71.41; H, 4.79; N, 11.10. Found: C, 71.26; H, 4.84; N, 11.06.

Antibacterial studies

The newly synthesized compounds were screened for their antibacterial activity against Streptococcus pyogenes (clinical isolate), MRSA (+ve), Pseudomonas aeruginosa (ATCC-27853), Klebsiella pneumoniae (clinical isolate) and E. coli (ATCC-25922) bacterial strains by disk diffusion method^27,28. A standard inoculums (1–2 × 10⁷ c.f.u./mL 0.5 McFarland standards) was introduced on to the surface of sterile agar plates, and a sterile glass spreader was used for even distribution of the inoculums. The disks measuring 6 mm in diameter were prepared from Whatman no. 1 filter paper and sterilized by dry heat at 140°C for 1 h. The sterile disks previously soaked in a known concentration of the test compounds were placed in nutrient agar medium. Solvent and growth controls were kept. Ciprofloxacin (30 µg) was used as positive control while the disk poured in DMSO was used as negative control. The plates were inverted and incubated for 24 h at 37°C. The susceptibility was assessed on the basis of diameter of zone of inhibition against Gram-positive and Gram-negative strains of bacteria. Inhibition zones were measured and compared with the controls. The bacterial zones of inhibition values are given in Table 1.

Table 1.  Antibacterial activity of compounds 3a-4f.

Compounds	Gram-positive bacteria						Gram-negative bacteria
	Staphylococcus pyogenes			MRSA*			Pseudomonas aeruginosa			Klebsiella pneumoniae			Escherichia coli
	Zone	MIC	MBC	Zone	MIC	MBC	Zone	MIC	MBC	Zone	MIC	MBC	Zone	MIC	MBC
3a	13.5 ± 0.4	25	100	13.4 ± 0.4	25	50	15.1 ± 0.6	50	100	12.6 ± 0.2	25	50	13.3 ± 0.2	25	100
3b	11.4 ± 0.5	25	50	11.1 ± 0.5	50	100	13.2 ± 0.4	25	50	10.7 ± 0.5	50	100	11.1 ± 0.5	25	50
4a	21.5 ± 0.2	25	100	20.6 ± 0.4	50	100	26.3 ± 0.6	50	100	21.5 ± 0.2	50	>100	22.4 ± 0.5	50	>100
4b	20.3 ± 0.4	25	100	19.8 ± 0.3	50	100	22.4 ± 0.2	50	>100	18.4 ± 0.5	50	>100	19.9 ± 0.4	50	100
4c	18.1 ± 0.5	25	100	17.3 ± 0.4	25	>100	20.1 ± 0.4	100	>100	16.1 ± 0.5	50	>100	17.5 ± 0.3	50	>100
4d	15.2 ± 0.4	50	100	14.5 ± 0.2	50	100	12.2 ± 0.3	50	>100	14.5 ± 0.4	100	>100	15.5 ± 0.3	100	>100
4e	10.4 ± 0.4	50	100	10.1 ± 0.3	50	>100	12.2 ± 0.2	50	>100	10.5 ± 0.3	50	>100	11.1 ± 0.4	50	>100
4f	15.2 ± 0.2	50	100	10.7 ± 0.4	50	100	15.2 ± 0.3	50	100	13.3 ± 0.2	50	100	11.8 ± 0.5	50	>100
Standard	23.0 ± 0.2	12.5	12.5	22.0 ± 0.2	6.25	12.5	32.0 ± 0.3	12.5	25	19.0 ± 0.2	6.25	25	27.0 ± 0.2	6.25	12.5
DMSO	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–

Diameter of zone of inhibition (mm), MIC and MFC results of compounds 3a-4f and positive control griseofulvin.Positive control (Standard); ciprofloxacin and negative control (DMSO) measured by the Halo Zone Test (unit, mm). *Methicillin resistant S. aureus (MRSA +ve). MIC (µg/mL), minimum inhibitory concentration, i.e., the lowest concentration of the compound to inhibit the growth of bacteria completely; MBC (µg/mL), minimum bactericidal concentration, i.e., the lowest concentration of the compound for killing the bacteria completely.

Minimum inhibitory concentrations (MICs) were determined by broth dilution technique. The nutrient broth, which contained logarithmic serially two fold diluted amount of test compound and controls were inoculated with approximately 5 × 10⁵ c.f.u/mL of actively dividing bacteria cells. The cultures were incubated for 24 h at 37°C and the growth was monitored visually and spectrophotometrically. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as MIC. To obtain the minimum bactericidal concentration (MBC), 0.1 mL volume was taken from each tube and spread on agar plates. The number of c.f.u was counted after 18–24 h of incubation at 35°C. MBC was defined as the lowest drug concentration at which 99.9% of the inoculums were killed. The minimum inhibitory concentration and minimum bactericidal concentration are given in Table 1.

Antifungal studies

Antifungal activity was also done by disk diffusion method. For assaying antifungal activity Candida albicans, Aspergillus fumigatus, Trichophyton mentagrophytes and Penicillium marneffei were recultured in DMSO by agar diffusion method^29,30. Sabourauds agar media was prepared by dissolving peptone (1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 mL) and adjusting pH to 5.7. Normal saline was used to make a suspension of spore of fungal strain for lawning. A loopful of particular fungal strain was transferred to 3 mL saline to get a suspension of corresponding species. 20 mL of agar media was poured into each Petri dish. Excess of suspension was decanted and the plates were dried by placing in an incubator at 37°C for 1 h. Using an agar punch, wells were made and each well was labeled. A control was also prepared in triplicate and maintained at 37°C for 3–4 days. The fungal activity of each compound was compared with griseofulvin as standard drug. Inhibition zones were measured and compared with the controls. The fungal zones of inhibition values are given in Table 2. The nutrient broth, which contained logarithmic serially two fold diluted amount of test compound and controls was inoculated with approximately 1.6 × 10⁴–6 × 10⁴ c.f.u/mL. The cultures were incubated for 48 h at 35°C and the growth was monitored. The lowest concentration (highest dilution) required to arrest the growth of fungi was regarded as MIC. To obtain the minimum fungicidal concentration (MFC), 0.1 mL volume was taken from each tube and spread on agar plates. The number of c.f.u. was counted after 48 h of incubation at 35°C. MFC was defined as the lowest drug concentration at which 99.9% of the inoculums were killed. The MIC and MFC are given in Table 2.

Table 2.  Antifungal activity of compounds 3a-4f.

Compounds	Candida albicans			Aspergillus fumigatus			Trichophyton mentagrophytes			Penicillium marneffei
	Zone	MIC	MFC	Zone	MIC	MFC	Zone	MIC	MFC	Zone	MIC	MFC
3a	22.2 ± 0.4	12.5	50	19.7 ± 0.3	12.5	50	15.8 ± 0.9	25	100	14.3 ± 0.2	25	50
3b	21.3 ± 0.3	12.5	25	19.6 ± 0.4	25	100	15.4 ± 0.2	12.5	50	12.9 ± 0.3	25	50
4a	20.1 ± 0.3	25	100	18.1 ± 0.2	25	100	14.3 ± 0.3	25	100	11.7 ± 0.2	50	100
4b	20.1 ± 0.3	25	100	17.2 ± 0.5	50	100	13.7 ± 0.4	50	100	10.1 ± 0.4	50	100
4c	19.8 ± 0.5	50	100	17.6 ± 0.6	50	100	12.1 ± 0.3	50	100	10.3 ± 0.6	25	100
4d	18.1 ± 0.3	25	100	16.5 ± 0.4	25	100	11.7 ± 0.5	50	100	9.2 ± 0.4	50	100
4e	17.9 ± 0.3	50	100	15.5 ± 0.5	50	>100	10.4 ± 0.2	50	>100	8.9 ± 0.3	50	100
4f	19.4 ± 0.5	50	100	17.1 ± 0.6	100	>100	12.1 ± 0.4	50	100	10.3 ± 0.6	50	>100
Standard	30.0 ± 0.2	6.25	25	27.0 ± 0.2	12.5	12.5	24.0 ± 0.3	6.25	25	20.0 ± 0.5	12.5	25
DMSO	–	–	–	–	–	–	–	–	–	–	–	–

Diameter of zone of inhibition (mm), MIC and MFC results of compounds 3a-4f and positive control griseofulvin. Positive control (griseofulvin) and negative control (DMSO) measured by the Halo Zone Test (Unit, mm). MIC (µg/mL), minimum inhibitory concentration, i.e., the lowest concentration of the compound to inhibit the growth of fungi completely; MFC (µg/mL), minimum fungicidal concentration, i.e., the lowest concentration of the compound for killing the fungi completely.

Results and discussion

Chemistry

3-Formylchromone has emerged as an important starting material for incorporation of chromone moiety, ever since its convenient synthesis was reported in 1970s²⁴. It has been used as an important synthon due to the presence of three electron deficient centers viz C-2, C-4 carbonyl and C-3 formyl groups. In the present study, we carried out the reaction of 3-formylchromone 1 and active methyl compounds viz dehydroacetic acid 2a and 5-acetyl barbituric acid 2b under different reaction conditions. Initially reactions were conducted at room temperature in the presence of basic catalyst pyridine. The reactions afforded the expected chromonyl chalcones 3a-b in 18–25 hours with 62–70% yields (Scheme 1). The reactions carried using methanol as a solvent under conventional heating condition were found to be completed in 10–16 h, but there was no substantial increase in yields of products. This prompted us to carry out reactions under solvent-free heating technique by simply heating the starting materials with the catalyst. To our pleasant surprise, the reactions were completed in short span of time (12–15 min) with considerable increase in yields (88–92%) and were devoid of any toxic waste products. Thus, the present protocol explains the superiority of thermal solvent-free reactions over the conventional methods. The products obtained were characterized by elemental and spectral (IR, ¹H NMR and mass spectrometry) analysis.

Scheme 1.  Formation of chalcones 3a-b and their reaction with different hydrazines to give compounds 4a-f.

The ¹H NMR spectrum of the compound 3b showed trans olefinic protons Ha and Hb as ortho coupled doublets at δ 7.40 (J = 15.2 Hz) and 9.09 (J = 15.9 Hz), respectively. The value of spin-spin coupling constant J_ab, in the range of 15–16 Hz, is indicative of the E-configuration of chalcone. The three aromatic protons of chromone moiety were discernible in the form of multiplet at δ 7.55–7.91 whereas C-2 and C-5 protons appeared as a singlet and doublet of doublet at δ 8.95 and 8.17 respectively. The NH protons of barbituric acid moiety appeared as two broad singlets at δ 11.06 and 11.76.

Nitrogen bases such as hydrazines react with α,β-unsaturated carbonyl compounds to give pyrazolines³¹. With the objective of synthesizing pyrazolines containing chromone moiety, chromonyl chalcones 3a-b were allowed to react with different hydrazines in conventional method using acetic acid as solvent and under solvent-free heating conditions. As expected, pyrazolines 4a-e were obtained from hydrazine hydrate, hydrazinobenzothiazole and phenylhydrazine (Scheme 1) and the results were comparable with reactions performed under conventional procedures. However, in case of reaction of 3a with phenylhydrazine, the mixture did not afford the desired pyrazoline, instead a bipyrazole 4f was obtained due to the ring opening reaction of pyrone ring both under conventional and thermal solvent-free methods. All the newly synthesized compounds were characterized by elemental and spectral (IR, ¹H NMR and mass spectrometry) analysis.

The ¹H NMR spectrum of compound 4f indicated the absence of resonances attributable to C-2 olefinic and C-5 protons of chromone moiety, whereas compound 4a/4b showed these diagnostic signals of C-2 proton as singlet at δ 8.02, 8.41 and C-5 proton as doublet at δ 8.16, 8.02 respectively. These observations suggest the opening of pyrone ring under the action of phenylhydrazine to lead the formation of bipyrazole 4f. The other prominent peaks as obtained are explained in the experimental part.

Pharmacology

The investigation of antibacterial screening data revealed that all the tested compounds 3a-4f showed moderate to good bacterial inhibition. Among the tested bacterial strains, good inhibitory results were obtained against S. pyogenes and E. coli. The structural activity study showed that chalcones 3a, 3b and their derivatives 4a-f have varying degrees of microbial inhibition. The antimicrobial activity seemed to be dependent on the nature of heterocyclic moieties. A comparative study (Table 1) also revealed that chromonyl chalcones 3a-b, are more potent antibacterial agents than chromonyl pyrazolines 4a-f. Compounds 3a, 3b, 4a, 4b and 4c showed good inhibition (MIC = 25 µg/mL) against S. pyogenes. The maximum inhibition was observed in 3a and 4c against S. aureus (MRSA +ve). Except 3a and 3b all other compounds showed moderate to less activity results against P. aeruginosa, K. pneumoniae and E. coli bacterial strains. The MICs i.e., the lowest concentration of the compounds to inhibit the growth of bacteria completely were in the range of 25–100 µg/mL. The minimum bactericidal concentrations (MBCs) i.e., the lowest concentration of the compounds for killing the bacteria completely were found to be two, three or four folds higher than the corresponding MIC results.

The antifungal screening data of the compounds also revealed good to moderate activity. Among the tested fungal strains, good inhibitory results were obtained against C. albicans, and A. fumigatus. Again the comparative study (Table 2) revealed that chromonyl chalcones 3a-b, were found to have better inhibitory action than pyrazolines 4a-f. The compounds 3a, 3b, 4a, 4b and 4d showed effective inhibition results (MIC = 12.5–25 µg/mL) against C. albicans. The maximum inhibition was observed in 3a, 3b and 4a against A. fumigatus, and T. mentagrophytes fungal cultures. The inhibitory activity against P. marneffei was significantly higher for the compounds 3a, 3b and 4c than the other tested compounds. Compounds 4c-f were moderately active against most of the fungal strains. The MICs, i.e., the lowest concentration of the compounds to inhibit the growth of fungi completely were in the range of 12.5–100 µg/mL. The MFCs, i.e., the lowest concentration of the compounds for killing the fungi completely were found to be two, three or four folds higher than the corresponding MIC results.

Considering the results obtained from antifungal and antibacterial tests together, it is noteworthy to mention that tested compounds are more active towards fungi than bacteria. Thus, the nature of heterocycles and basic skeleton of molecule have significant influence on the extent of antibacterial and antifungal activities. A comparative study of the activity results (Tables 1 and 2) with standard drugs (ciprofloxacin, griseofulvin) revealed that none of the compound exceeds the activity of commercial drugs. However, compounds have produced the marked enhancement in the potency of these analogues as antibacterial and antifungal agents.

Molecular targets of the synthesized compounds on the bacteria and fungi were identified using BioSpec Module of Raasi Suite. Every molecule has multiple targets on bacteria and fungi. The synthesized molecules have the following molecular targets: ATP-binding cassette, sub-family C, member 1 isoform 1 inhibitor, Pyruvate kinase, muscle isoform M2 activator, Eukaryotic translation initiation factor 4γ, 1 isoform 4 inhibitor.

Conclusions

In summary, a clean and convenient synthesis of novel chromone derivatives has been developed. The procedure offers several advantages including mild reaction conditions as well as simple experimental and product isolation procedures, thus, making the current “green protocol” as a useful and attractive methodology for the synthesis of series of novel heterocycles in excellent yields from cheap and readily available starting materials. The antibacterial, antifungal screening data revealed that newly generated compounds are good antimicrobial agents. The newly generated compounds can be used as template for future development through modification and derivatization to design more potent and selective antimicrobial agents.

Acknowledgments

The authors are thankful to University Grants Commission, New Delhi for providing financial assistance in the form of major research project [F.No. 37-15/2009 (SR)], SAIF, CDRI, Lucknow, India for providing analytical, spectral data and Mr Sameer Chaudhary, MD RASA Life Science Informatics, Pune, India for providing drug target identification using BioSpec Module of Raasi Suite.

Declaration of Interest

The authors have declared no conflicts of Interest.