Synthesis and Cytotoxic Studies of Newer 3-(1-Benzofuran-2-Yl)-5-(Substituted Aryl) Isoxazole

 

Chetan M. Bhalgat¹*, Sachin L. Patil², Sandeep K. Chitale¹, Krantisinha Randive¹, Kumar G. Patil¹ and Santosh Patil¹

¹Department of Pharmaceutical Chemistry, S.A.C. College of Pharmacy, B.G.Nagara- 571448, Mandya              

²Principal, Mayani College of Pharmacy, Mayani- 415102, Tal: Khatav, Dist: Satara

*Corresponding Author E-mail: chetanbhalgat2004@yahoo.co.in

ABSTRACT:

The 3-(1-benzofuran-2-yl)-5-(substituted phenyl) isoxazole have been synthesized by the reaction of Benzofuran chalcone with hydroxylamine hydrochloride in presence of sodium acetate in ethanol. All the compounds were synthesized by conventional method and characterized by IR, ¹H NMR and mass spectral data. The new synthesized derivatives I1-I7 was evaluated for in vitro cytotoxic activity on HeLa cell lines. Compounds I1-I7 was scheduled for evaluation against full panel of human cervical cancer cell lines at the minimum seven concentrations at two fold dilutions.

 

 

 

INTRODUCTION:

Heterocyclic synthesis has emerged as powerful technique for generating new molecules useful for drug discovery¹. Heterocyclic compounds provide scaffolds on which pharmacophores can arrange to yield potent and selective drugs².

Benzofuran nucleus may be combined with nitrogen heterocycles in different ways. Several benzofuran compounds are reported to posses, antibacterial³, antifungal⁴, anti-inflammatory⁵, antidepressant⁶, analgesic⁷ and hypoglycemic⁸ activities. It has already been pointed out that; benzofuran nucleus is very rarely associated with a nitrogen heterocycle. Several isoxazole derivatives are found to possess antitubercular⁹, antibacterial⁹ and antifungal⁹ activities.

In our present research work we are synthesizing new compound i.e. benzofuran fused with isoxazole ring. Cytotoxic drugs remain the mainstay of cancer chemotherapy and are being administered with novel ways of therapy such as inhibitor of signals¹⁰. It is therefore important to discover novel cytotoxic agents with spectra of activity and toxicity that differ from those current agents^10,11. On the other hand, benzofuran ring system incorporated with different heterocyclic moieties has wide spectrum of anticancer against different types of carcinoma^12-17.

 

MATERIALS AND METHODS:

I] Experimental section:

Reactions and purity of compounds were monitored by TLC (silica gel G60) using ethylacetate: benzene (1:1) solvent system and the spots were identified by iodine vapor chamber. Melting points were determined in open capillary using paraffin bath and are uncorrected. The IR spectra of the compounds were recorded on NICOLET380 FT-IR spectrophotometer using KBr pellets. ¹H NMR spectra were recorded in DMSO on a 300 MHz Shimadzu FT-NMR (δ in ppm) relative to TMS as internal standard. The mass spectra were recorded on Triple Quadruple LC-MS with ESI source. Mfg. SCIEX at 70eV.

 

Synthesis of benzofuran chalcones:

Benzofuran chalcones were synthesized according to reported methods from 2-acetyl benzofuran and various aromatic aldehydes^{9, 18-19}.

 

Synthesis of 33-(1-benzofuran-2-yl)-5-(phenyl) isoxazole (I1):

An equimolar mixture of chalcone, C1 (0.02 mol) and hydroxylamine hydrochloride (0.02 mol) were dissolved in ethanol (25ml) and refluxed for 6 h in presence of catalytic amount of sodium acetate. 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. Finally the compound synthesized namely 3-(1-benzofuran-2-yl)-5-phenyl-1,2-oxazole (I1). The completion of the reaction was monitored by TLC. Similarly various isoxazole derivatives I 2-7 were prepared (Figure 1).

Figure 1: Scheme of synthesis

II] In-vitro Evaluation of Cytotoxicity activity:²⁰

The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 10⁵ cells/ml using DMEM medium containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 µl of different test concentrations of test drugs were added on to the partial monolayer in microtitre plates. The plates were then incubated at 37°C for 3 days in 5% CO₂ atmosphere and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the sample solutions in the wells were discarded and 50 µl of

 

MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37°C in 5% CO₂ atmosphere. The supernatant was removed and 100 µl of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (CTC₅₀) values is generated from the dose-response curves for each cell line.

% Growth inhibition =    100 – (OD_T/OD_C) X 100

Where, OD_T =Mean OD of individual test group and OD_C =Mean OD of control group

 

RESULTS AND DISCUSSION:

All the synthesized compounds physical data and spectral data have been shown in table1 and 2, respectively. All compounds were tested for cytotoxic assay in seven different concentrations and repeated twice. The compound which having average CTC₅₀ (mg/ml) value greater than 1000 is considered as nontoxic to cells and below this value the compound confirmed cytotoxic property. The cytotoxic and/or growth inhibitory effect of the compound, I1-7, were tested against HeLa cell lines of two fold dilution of seven concentrations ranging from 1000-15.5 mg/ml. The percentage growth was evaluated spectrophotometrically vs control not treated with test agents. A 24 h continuous drug exposure protocol was followed and cell viability by MTT assay was used to estimate cell viability or growth. For the compound, the 50% cytotoxic concentration (CTC₅₀) and total growth inhibition percentages (TGI) were obtained for each cell lines.  The concentration of test drug needed to inhibit cell growth by 50% (CTC₅₀) values is generated from the dose-response curves for each cell line. The percentage growth concentration which is greater than CTC₅₀ value is considered as cytotoxic active concentration. The percentage growth concentration which is minimum than the CTC₅₀ value was considered as nontoxic concentration.

 

Among the compounds I1-I7 tested for cytotoxic activity in HeLa, the compound I4 shows potent cytotoxic effect, while compound I5 shows moderate cytotoxic effect on HeLa cells with CTC₅₀ value of 376.25 and 762.25 mg/ml respectively (Fig.-2). All the other compounds proved to be nontoxic with CTC₅₀ values above 1000 µg/ml (table-3 and 4)

Table 1: Physical data of synthesized compound

Compound Code	Chemical Name	R	Melting Point (0C)	Yield (%)	Rf Value
I1	3-(1-benzofuran-2-yl)-5-(phenyl)isoxazole	-H	120-121	89	5.8
I2	3-(1-benzofuran-2-yl)-5-(4-nitrophenyl)isoxazole	-NO2 (p)	172-174	54	5.4
I3	3-(1-benzofuran-2-yl)-5-(3-nitrophenyl)isoxazole	-NO2 (m)	Semisolid	78	4.9
I4	4-[3-(1-benzofuran-2-yl)isoxazol-5-yl]phenol	-OH (p)	157-159	60	5.7
I5	4-[3-(1-benzofuran-2-yl)isoxazol-5-yl]-2-ethoxyphenol	-C2H5O (m),-OH (p)	117-119	58	6.1
I6	3-(1-benzofuran-2-yl)-5-(3-methoxyphenyl)isoxazole	CH3O (m)	125-126	90	5.8
I7	3-(1-benzofuran-2-yl)-5-(3,4,5-trimethoxyphenyl)isoxazole	-CH3O (o, m, p)	semisolid	74	5.3

 

Table 2: Spectral data of synthesized compound:

Compound Code	IR (KBr cm-1)	1H NMR (DMSO δ ppm)	Mass M+1 m/z
I1	2926 (-CH str), 1453 (CH=CH), 1378 (C-O-C), 753 (Ar-H)	7.3-6.7(m, 5H, Benzofuran), 6.8(s, 1H, Isoxazole), 7.8-7.5(m, 5H, Ar-H)	262.3
I2	1454 (CH=CH), 1345 (NO str), 753 (Ar-H), 2957 (-CH str)	7.5-7.1(m, 5H, Benzofuran), 6.9(s, 1H, Isoxazole), 8.2-7.8(m, 4H, Ar-H)	307.3
I3	1459 (CH=CH), 1357 (NO str), 753 (Ar-H), 2963 (-CH str)	7.5-7.0(m, 5H, Benzofuran), 6.3(s, 1H, Isoxazole), 8.3-7.7(m, 4H, Ar-H)	307.4
I4	3356 (OH), 1456 (CH=CH), 2926 (-CH str), 1157 (C-O-C)	7.9-7.3(m, 5H, Benzofuran), 6.6(s, 1H, Isoxazole), 7.4-6.9(m, 4H, Ar-H), 4.2(s, 1H, OH)	278.3
I5	3337 (OH), 2956 (-CH str), 1248 (C-O-C), 752 (Ar-H), 1454 (CH=CH)	7.5-7.1(m, 5H, Benzofuran), 6.6(s, 1H, Isoxazole), 6.6-6.8(m, 3H, Ar-H), 4.6(s, 1H, OH), 3.9(m, 2H, CH2) , 1.6(t, 3H, CH3)	322.4
I6	3372 (NO), 2955 (CH str), 1455 (CH=CH), 1601 (C=C)	3.8 (m, 3H of OCH3), 8.0-6.9 (m, 8H, Ar-H), 5.4 (s, 1H of Isoxazole), 3.8 (m, 3H of OCH3)	292.3
I7	1125 (C-O-C), 1455 (CH=CH), 1590 (C=C), 2955 (-CH str)	7.4-7.1 (m, 7H, Aryl-H), 6.8 (s, 1H of isoxazole), 3.8-3.2 (m, 9H of OCH3)	352.3

Table3: Cytotoxicity properties of test compounds of I1-I7 on HeLa cell line

Compound Code	Test Concentration	% Cytotoxicity		CTC50 (µg/ml)		Average CTC50 (µg/ml)
	(µg/ml)	n1	n2	n1	n2
I1	1000	36	33.85	<1000	<1000	<1000
	500	19.56	13.65
	250	1.42	0
	125	0	0
	62.5	0	0
	31.2	0	0
	15.6	0	0
I2	1000	23	24.75	<1000	<1000	<1000
	500	11.85	9.06
	250	0	1.05
	125	0	0
	62.5	0	0
	31.2	0	0
	15.6	0	0
I3	1000	41.09	38.65	<1000	<1000	<1000
	500	12.35	9.87
	250	0	0
	125	0	0
	62.5	0	0
	31.2	0	0
	15.6	0	0
I-4	1000	81.18	82.28	372.5	380.0	376.25
	500	70.88	72.08
	250	27.78	28.32
	125	0	0
	62.5	0	0
	31.2	0	0
	15.6	0	0
I5	1000	71.35	74.35	775.5	750	762.25
	500	24.56	26.58
	250	0	0
	125	0	0
	62.5	0	0
	31.2	0	0
	15.6	0	0
I6	1000	18.56	16.5	<1000	<1000	<1000
	500	1.54	0.8
	250	0	0
	125	0	0
	62.5	0	0
	31.2	0	0
	15.6	0	0
I7	1000	11.05	8.56	<1000	<1000	<1000
	500	0	0
	250	0	0
	125	0	0
	62.5	0	0
	31.2	0	0
	15.6	0	0

Figure 2: Cytotoxicity effect of various compounds

        

Table 4: Cytotoxicity property (CTC₅₀) of test compounds of I1-I7 on HeLa cell line

Concentration (µg/ml)	CTC50 (µg/ml)
	I1	I2	I3	I4	I5	I6	I7
1000	>1000	>1000	>1000	376.25	762.25	>1000	>1000
500
250
125
62.5
31.2
15.6

 

 

CONCLUSION:

Benzofuran with fused isoxazole compounds are reported to posses, antibacterial, antifungal, anti-inflammatory and antitubercular activities. Here these moieties are fused and screened for cytotoxic activity. Among these derivatives 4-Hydroxy benzaldehyde, 3-ethoxy,4-Hydroxy benzaldehyde substituted derivative (I4 and I5) showed good cytotoxic activity. Above results establish the fact that benzofuran with fused isoxazole can be a rich source for exploitation.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Dr B. Ramesh, Principal SAC College of pharmacy, B.G. Nagara for providing research facilities and encouragement. The authors are also thankful to Dept. of USIC Karnataka University, Dharwad for providing Spectra.

 

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Received on 28.08.2010          Modified on 15.09.2010

Accepted on 30.09.2010         © RJPT All right reserved