Synthesis and Antimicrobial Evaluation of Some Heterocyclic Chalcone Derivatives

Abstract

Some new heterocyclic compounds containing isoxazole, pyrazole and oxadiazole ring systems were prepared from various chalcones. The synthesized compounds have been characterized by elemental analysis and spectral methods. These compounds were screened for their antimicrobial activities.

1. Introduction

Chalcones are synthesized by condensing ketones with aromatic aldehydes in the presence of suitable bases. They are very useful intermediates for the synthesis of five- [1,2], six- [1,3] and seven-membered [4] heterocyclic compounds. Chalcone derivatives exhibit diverse pharmacological activities [5,6,7,8,9,10,11,12,13,14]. It is therefore, not surprising that many synthetic methods have been developed for the preparation of heterocycles starting from chalcone precursors that have been tested for their antimicrobial activities.

2. Results and Discussion

All of our results are shown in Scheme 1. The starting chalcones 1a–c were obtained in good yields by a base catalyzed condensation [15-16] of appropriately substituted benzaldehydes and cyclopropylmethyl ketone [17]. The method is attractive since it specifically generates the (E) isomer [18].

Scheme 1.

Synthesis of 2a–f, 3a–f, 4a–f and 5a–f.

The hydrazones 2a–f were prepared by the reaction of chalcones 1a–c with benzoyl hydrazine derivatives and were subsequently used for the syntheses of various pyrazoles 3a–f and oxadiazoles 4a–f. The IR spectra of these hydrazones revealed the characteristic bands for vinyl CH=CH at 1582–1617, C=N at 1616–1647, C=O at 1664–1698 and NH at 3330–3420 cm⁻¹. The ¹H-NMR spectra showed the presence of a singlet at δ = 9.97–10.82 ppm for the NH proton, a multiplet at δ = 7.17–8.45 ppm characteristic for the aromatic protons and the olefinic =C–CH=CH, a doublet at δ = 6.60–6.95 ppm characteristic for the olefinic =C–CH=CH proton. The cyclopropyl ring protons appeared as two multiplets in the range δ = 1.56–2.64 ppm (CH) and δ = 0.70–1.46 ppm (2CH₂), respectively.

The pyrazole derivatives 3a–f were obtained by treatment of hydrazones 2a–f with 30% hydrochloric acid. The IR of 3a–f showed the characteristic bands for C=C–Ar at 1519–1596, C=N at 1623–1644 and amide carbonyl band at 1660–1686 cm⁻¹, while the ¹H-NMR spectra showed a singlet at δ = 6.64–7.12 ppm for the pyrazole–C₄–H. On the other hand, refluxing of hydrazones 2a–f with acetic anhydride gave the corresponding dihydro-1,3,4-oxadiazole derivatives 4a–f. The mechanism of cyclization reaction has been well studied [19-20]. The IR spectra of the dihydro-oxadiazoles 4a–f lacked the NH, but showed a carbonyl absorption at 1667–1677 cm⁻¹ for the acetyl group. Their structures were further confirmed from the ¹H-NMR spectra which does not reveal the presence of NH signal present in the starting hydrazone 2, moreover, the ¹H-NMR of 4 exhibited a singlet of three protons intensity at δ = 2.10–2.16 ppm for the COCH₃. Finally, treatment of chalcones 1a–c with hydroxylamine hydrochloride in presence of sodium acetate produced isoxazoles 5a–c in moderate yield. The structure of 5 was fully confirmed by spectral method. For example, the IR of 5 does not show the presence of carbonyl band characteristic for the starting chalcone 1. The ¹H-NMR of 5 exhibited a singlet of one proton intensity at δ = 5.42–5.69 ppm characteristic for the isoxazole–C₄–H. Melting points, elemental analysis and spectral methods are outlined in Table 1 and Table 2.

Table 1.

Physical and analytical data of compounds, 2a–f, 3a–f, 4a–f and 5a–c.

Compound	X	Y	Yield (%)	Mp (ºC)	Molecular Formula	Calculated %			Found %
						C	H	N	C	H	N
2a	NO2	Me	72	181	C20 H19 N3 O3	68.77	5.44	12.03	68.71	5.39	12.09
2b	NO2	NO2	79	201	C19 H16 N4 O5	60.00	4.21	14.74	60.06	4.19	14.77
2c	H	H	66	166	C19 H18 N2 O	78.62	6.21	9.66	78.59	6.19	9.62
2d	H	Me	65	180	C20 H20 O N2	78.95	6.58	9.21	79.01	6.49	9.28
2e	OMe	Me	69	190	C21 H22 O2 N2	75.45	6.59	8.38	75.49	6.62	8.44
2f	OMe	H	74	160	C20 H20 O2 N2	75.00	6.25	8.75	74.97	6.26	8.77
3a	NO2	Me	81	183	C20 H17 O3 N3	69.16	4.90	12.10	69.22	4.87	12.08
3b	NO2	NO2	92	197	C19 H14 O5 N4	60.32	3.70	14.81	60.33	3.69	14.77
3c	H	H	62	159	C19 H16 O N2	79.17	5.56	9.72	79.21	5.53	9.71
3d	H	Me	66	164	C20 H18 O N2	79.47	5.96	9.27	79.51	5.91	9.27
3e	OMe	Me	64	171	C21 H20 O2 N2	75.90	6.02	8.43	75.93	6.06	8.51
3f	OMe	H	59	180	C20 H18 O2 N2	75.47	5.66	8.81	75.52	5.60	8.86
4a	NO2	Me	83	201	C22 H21 O4 N3	67.52	5.37	10.74	67.52	5.43	10.80
4b	NO2	NO2	97	210	C21 H18 O6 N4	59.72	4.27	13.27	59.69	4.26	13.22
4c	H	H	59	177	C21 H20 O2 N2	57.90	6.02	8.43	57.84	5.99	8.44
4d	H	Me	58	179	C22 H22 O2 N2	76.30	6.36	8.09	76.28	6.31	8.12
4e	OMe	Me	67	189	C23 H24 O3 N2	73.40	6.38	7.45	73.44	6.39	7.49
4f	OMe	H	80	162	C22 H22 O3 N2	72.93	6.08	7.73	72.91	6.01	7.76
5a	NO2	―	54	165	C12 H10 O3 N2	62.61	4.35	12.17	62.66	4.29	12.21
5b	H	―	49	159	C12 H11 O N	77.84	5.95	7.57	77.90	5.99	7.62
5c	OMe	―	74	161	C13 H13 O2 N	72.56	6.05	6.51	72.60	6.01	6.48

Table 2.

IR and ¹H-NMR spectral data of compounds 2a–f, 3a–f, 4a–f and 5a–c.

Compound	IR cm−1 (KBr)				1H NMR (δ / ppm)a
	C=C	C=N	C=O	NH	Ar–H’s and = C–CH=CH (m)	=C–CH=CH (d), J =12 Hz	Pyrazole-C4–H (s) or isoxazole–C4–H (s)	NH (s)	Cyclopropyl ring H’s		Ar–CH3 (s) Ar–OCH3 and CH3CO–
									CH (m)	2 (CH2) (m)
2a	1592	1629	1678	3420	7.36–7.91	6.72	―	10.66	1.91–2.55	0.75–1.37	2.21
2b	1617	1647	1698	3390	7.23–8.45	6.93	―	10.45	1.80–2.64	0.71–1.40	―
2c	1582	1616	1664	3330	7.19–7.63	6.60	―	9.97	1.82–2.49	0.78–1.29	―
2d	1587	1636	1669	3336	7.29–7.82	6.71	―	10.82	1.71–2.36	0.70–1.40	2.10
2e	1601	1628	1687	3411	7.20–7.71	6.75	―	10.73	1.63–2.40	0.77–1.46	2.19, 3.49
2f	1617	1644	1681	3332	7.17–7.49	6.95	―	9.99	1.56–2.59	0.78–1.16	3.42
3a	1519	1629	1670	―	7.26–7.92 b	―	6.85	―	1.90–2.48	0.69–1.21	2.13
3b	1586	1633	1684	―	7.24–8.33 b	―	7.12	―	1.76–2.66	0.80–1.36	―
3c	1556	1641	1660	―	7.22–7.71 b	―	6.78	―	1.73–2.40	0.71–1.28	―
3d	1590	1644	1678	―	7.24–7.77 b	―	6.64	―	1.75–2.41	0.77–1.33	2.11
3e	1571	1623	1686	―	7.21–7.68 b	―	6.69	―	1.66–2.39	0.71–1.33	2.10,3.44
3f	1596	1633	1664	―	7.11–7.62 b	―	6.74	―	1.49–2.55	0.77–1.19	3.39
4a	1587	1645	1669	―	7.18–7.79 b	―	―	―	1.92–2.41	0.67–1.32	2.01, 2.16
4b	1610	1646	1667	―	7.22–8.22 b	―	―	―	1.70–2.66	0.71–1.40	2.13
4c	1602	1626	1669	―	7.16–7.75 b	―	―	―	1.68–2.39	0.75–1.26	2.11
4d	1594	1630	1670	―	7.33–7.76 b	―	―	―	1.73–2.44	0.71–1.38	1.99, 2.13
4e	1600	1633	1677	―	7.26–7.72 b	―	―	―	1.60–2.47	0.71–1.26	2.13,3.32,2.15
4f	1615	1646	1671	―	7.25–7.51 b	―	―	―	1.47–2.61	0.76–1.33	3.34,2.10
5a	1580	1627	―	―	7.19–7.99 b	―	5.69	―	1.82–2.51	0.71–1.36	―
5b	1571	1629	―	―	7.22–7.62 b	―	5.42	―	1.43–2.45	0.69–1.27	―
5c	1569	1633	―	―	7.26–7.70 b	―	5.46	―	1.57–2.41	0.67–1.29	3.30

^a Solution in DMSO-d₆; ^b The chemical shift only indicates Ar–H’s.

2.1. Antimicrobial Activity

All the synthesized heterocyclic derivatives, pyrazoles 3a–f oxadiazoles 4a–f and isoxazoles 5a–c were assayed for their antimicrobial activity against four test organisms: Staphylococcus aureus ATCC6538P, Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC9027 and Candida albicans ATCC2091 using rifampicin (5 μg/disc) and ampicillin (10 μg/disc) as standard drugs following agar well-diffusion method [21].

The tested heterocyclic compounds showed no significant effect against Pseudomonas aeruginosa and Candida albicans, whereas they showed a potent activity against Staphylococcus aureus and Escherichia coli. The maximum activity (+ + +) (MIC = 25 μg/mL) was indicated for compounds 3a, 3b, 4a, 4b and 5a. These results suggest that the electron-withdrawing nitro group plays a crucial role in enhancing the observed activity.

Compounds 3e, 4c, 4e and 4f showed a moderate activity (+ +) (MIC = 50 μg /mL) against Staphylococcus, while these compounds exhibited slight activity (+) (MIC = 75 μg/mL) against Escherichia coli. All other compounds were inactive towards the different strains of bacteria. The results are summarized in Table 3.

Table 3.

Antibacterial activities of newly synthesized compounds 3–5.

Compound	X	Y	Staphylococcus aureus	Escherichia coli
3a	NO2	Me	+ + +	+ + +
3b	NO2	NO2	+ + +	+ + +
4a	NO2	Me	+ + +	+ + +
4b	NO2	NO2	+ + +	+ + +
5a	NO2	―	+ + +	+ + +
3e	OMe	Me	+ +	+
4c	H	H	+ +	+
4e	OMe	Me	+ +	+
4f 3c 3d 3f 4d 5b 5c	OMe H H OMe H H OMe	H H Me H Me ― ―	+ + ― ― ― ― ― ―	+ ― ― ― ― ― ―

+ + + for maximum activity, MIC = 25 μg/mL; + + for moderate activity, MIC = 50 μg/mL; +for slight activity, MIC = 75 μg/mL and – for inactive.

3. Experimental

3.1. General

Melting points were taken in open capillary tubes using Electrothermal apparatus 9100 ( UK ) and are uncorrected. Microanalyses were performed at Faculty of Science, Cairo University, Cairo, Egypt, using a Elementary Vario el III C, H, N, S Analyzer (Germany). IR spectra were recorded using potassium bromide disks on a Perkin-Elmer 1650 spectrophotometer (Faculty of Science, Alexandria University, Alex, Egypt). ¹H-NMR spectra were determined on a Varian EM-390 MHz spectrophotometer, using TMS as internal standard.

3.2. General Procedure for Preparation of E-l-Cyclopropyl-3-(p-substituted-phenyl)-2-propenenones 1a–c

To a cold solution of sodium hydroxide (3 g) in aqueous ethanol (50 mL, 60%), cyclopropylmethyl ketone (10 mmol), was added dropwise (30 min), while rapidly stirring and the temperature kept below 20 °C, then the desired p-substituted benzaldehyde (10 mmol) was added dropwise (30 min). After five hours, the mixture was left overnight in refrigerator. The separated solid was filtered, washed with water and dried, then recrystallized from ethanol as colorless needles. The physical properties and all the spectral data were as reported in the literature [17].

3.3. General Procedure for Preparation of 1-Cyclopropyl-3-(p-substituted-phenyl)-2-propene-l-aroyl hydrazones 2a–e

A solution of chalcones 1a–c (10 mmol) in ethanol (10 mL) was refluxed with the appropriate aroyl hydrazines (10 mmol) in glacial acetic acid (2 mL) for about six hours, then the reaction mixture was poured onto crushed ice and was kept overnight at room temperature, the separated solid was filtered off, washed successively with water and dried, then recrystallized from methanol. IR and NMR data: see Table 1 and Table 2.

3.4. General Procedure for Preparation of 1-Aroyl-3-cyclopropyl-5-(p-substituted-phenyl)-pyrazoles 3a–e

A solution of the appropriate hydrazone 3a–e (10 mmol) in 30% hydrochloric acid (15 mL) was refluxed for about two hours, the reaction mixture was concentrated, separated solid was filtered off, washed with water, dried and recrystallized from methanol. IR and NMR data: see Table 1 and Table 2.

3.5. General Procedure for Preparation of 3-Acetyl-2-cyclopropyl-2-(p-substituted styryl)-5-(p-substituted phenyl)-1,3,4-oxadiazoles 4a–e

A mixture of the appropriate hydrazone 2a–e (10 mmol) and acetic anhydride (15 mL) was heated under reflux for three hours. After the reaction mixture attained room temperature, it was poured into crushed ice and the oily product deposited was decanted from water and extracted with ether. The ether layer was washed with sodium bicarbonate, followed by water, dried over anhydrous sodium sulphate and evaporated to give the corresponding oxadiazoles 4a–e as needles. IR and NMR data: see Table 1 and Table 2.

3.6. General Procedure for Preparation of 3-Cyclopropyl-5-(p-substituted Phenyl)isoxazole 5a–c

A mixture of chalcone 1a–c (20 mmol), hydroxylamine hydrochloride (20 mmol) and sodium acetate (20 mmol) in ethanol (25 mL) was refluxed for six hours. The mixture was concentrated by distilling out the solvent under reduced pressure and poured into ice-water. The precipitate obtained was filtered, washed and recrystallized from ethanol to give isoxazole 5 as needles. IR and NMR data: see Table 1 and Table 2.

3.7. Determination of Antimicrobial Activity

All the synthesized heterocyclic compounds 3a–f, 4a–f and 5a–f were tested against four different microorganisms: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The agar well-diffusion method was applied for the determination of inhibition zone and minimum inhibitory concentration (MIC). Briefly, 0.75 mL of broth culture containing ca. 106 colon-forming units (CFU) per mL of the test strain was added to 75 mL of nutrient agar medium at 45 °C, mixed well, and then poured into a 15 cm sterile metallic Petri plate. The medium was allowed to solidify, and 8 mm wells were dug with a sterile metallic borer. Then, a DMSO solution of the test sample (1 mL) at 1 mg/mL was added to the respective wells. DMSO served as negative control, and the standard antimicrobial drugs rifampicin (5 μg/disc) and ampicillin (10 μg/disc) were used as positive controls. Triplicate plates of each microorganism strain were prepared and were incubated aerobically at 37 °C for 24 h. The activity was determined by measuring the diameter of zone showing complete inhibition (mm), thereby, the zones were precisely measured with the aid of a Vernier Caliper (precision 0.1 mm).The growth inhibition was calculated with reference to the positive control.

4. Conclusions

In summary, this work demonstrates a rapid, efficient method for synthesis of new heterocyclic compounds of pharmacological interest.