Acta Chim. Slov. 2004, 51, 447-452.
447
Scientific Paper
SYNTHESIS AND STUDIES OF ANTIMICROBIAL ACTIVITY OF
LANCEOLATIN B
Sayed Alam
Department of Chemistry, Rajshahi University of Engineering & Technology, Rajshahi-6204,
Bangladesh. Tel.: +88-0721-750349; Fax: +88-0721-750105; e-mail: alam_sms@yahoo.com
Received 17-10-2003
Abstract
Phytochemical studies of Pongamia pinnata showed an abundance of [2", 3": 7, 8]furanoflavone (lanceolatin B). Now, it has been synthesized from p-resacetophenone via chalcone precursor followed by the treatment of DMSO/I2, diphenyl sulphide and DDQ, respectively. The antibacterial activity of [2", 3": 7, 8]furanoflavone and its corresponding chalcone were tested by the disc diffusion method for antibacterial effects against Shigella dysenteriae, Salmonella typhi, Streptococcus-P-haemolyticus and Staphylococcus aureus.
Key words: Pongamia pinnata, lanceolatin B, antibacterial activity
Introduction
Pongamia pinnata (Leguminosae) is one of the most useful medicinal plants of India1 as well as Bangladesh.2 Ali its parts have been extensively studied and are known to contain [2", 3": 7, 8]furanoflavone named as lanceolatin B.3"7 Ali parts of the plant have been used as a crude drug for the treatment of tumours, piles, skin diseases, painful rheumatic joints wounds, ulcers, etc.8 Pirrung, M. C. et al9 reported the synthesis of lanceolatin B through dipolar cycloaddition of diazocyclohexene-l, 3-diones, leading to benzofuran derivatives. This paper now reports herein a synthesis of [2", 3": 7, 8]furanoflavone (lanceolatin B) and study the antibacterial activity of this furanoflavone along with its chalcone precursor. The synthetic method of [2", 3":7, 8]furanoflavone has been accomplished in five steps starting from p-resacetophenone (1).
Results and discussion
The synthesis of [2", 3":7, 8]furanoflavone (lanceolatin B) was accomplished starting from p-resacetophenone (1) as shown in Scheme 1 and Scheme 2.
p-Resacetophenone (1) when refluxed with allyl bromide in presence of K2C03 and acetone yielded 4-0-allyresacetophenone10 (2), which on Claisen-rearrangement
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Acta Chim. Slov. 2004, 51, 447-452.
gave 3-C-allylresacetophenoneu (3). The compound 3 was subjected to OSO4/KIO4 oxidative double bond cleavage followed by orthophosphoric acid cyclization to 2-hydroxyfurano(2', 3' : 4, 3)acetophenone12 (4).
HO
OH
CH2=CH-CH2Br ------------------------------)
K2C03
Os04, KIO4
Ortophosphori c aci d
Claisen
rearrangement
HO.
Scheme 1
Claisen condensation of compound 4 with benzaldehyde yielded the corresponding chalcone 5 in good yield (53.70%) and it was obtained as yellow needles, mp 112-114 °C. The UV spectrum of compound 5 showed characteristics absorption band at 254, 276 and 328 nm. The IR spectrum of compound 5 showed absorption frequencies at 3452 and 1645 cm"1 indicating the presence of a hydroxyl, a conjugated carbonyl group and the absorption peaks at 1600 and 1592 cm"1 indicated the presence of unsymmetric ethylenic double bond and aromatic rings, respectively. In its 'H NMR spectrum, a set of trans-oMmic proton at 5 7.58 and 8.04 (each d, J = 16.0 Hz) and a chelated hydroxyl group at 5 12.68 assigned to C2'-OH proton. The lH NMR also exhibited the presence of two olefmic protons of a fused furan ring at 5 7.12 and 7.79 (d, J = 2.1 Hz). In the aromatic region, a set of two ortho-coupled protons of the A-ring at 5 7.05 and 7.91 (J = 8.9 Hz) and five protons of the B-ring at 5 7.21-7.30 (m) were observed.
The compound 5, which on separately treatment with DDQ, DMSO/I2 and diphenyl disulphide gave the desired compound 6 (lanceolatin B) in excellent yield. The spectral data (UV, IR and lH NMR) and melting point of compound 6 were very much similar with the natural sample of [2", 3":7, 8]furanoflavone (lanceolatin B).
Antibacterial activities. The antibacterial activities of compounds 5 and 6 have been assayed using a filter paper disc diffusion method13'14 at the concentration of 100 ug/disc and 200 ug/disc against four human pathogenic bacteria. Among them, two were Gram-positive and the rest two were Gram-negative. The inhibitory effects of compounds 5 and 6 against these organisms are given in Table 1.
O
4
S. Alam: Synthesis and Studies of Antimicrobial Activity of Lanceolatin B
Acta Chim. Slov. 2004, 51, 447-452.
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O
OH                CHO
O               ^^
O
KOH
EtOH/H20
DMSO/I;
O
12
O
or DDQ or PhSSPh
O
Scheme 2. Synthesis of lancheolatin B.
Table 1. Antibacterial screening for the compound 5 and 6"
Compound	Concentration	S. dysenteriae	S. typhi	S-p-haemolyticus 9	S. aureus
5	100 |ag/disc	9	-		10
	200 |ag/disc	15	16	15	14
6	100 |ag/disc	10	9	10	9
	200 |ag/disc	14	15	16	13
K-30*	30 |ag/disc	24	22	23	24
" Inhibitor^ activity is expressed as the diameter (in mm) of the observed inhibition zone. *Kanamycin-30.
The screening result indicate that compounds 5 and 6 showed moderate antibacterial activities to ali tested bacteria, except that compound 5 showed no effect against S. typhi at the concentration of 100 ug/disc.
Minimum lnhibitory Activity. The minimum inhibitory concentration of the compounds 5 and 6 were determined against S. dysenteriae and S-(3-haemolyticus by serial dilution method.15 The MIC level of both the compound 5 and 6 was found 64 ug/mL against L dysenteriae and S-(3-haemolyticus, respectively.
Experimental
Melting points were recorded on Gallenkamp apparatus and are uncorrected. IR spectra (KBr) were measured using a Shimadzu, DR-8001 spectrophotometer, lH NMR spectra (CDC13) were recorded on a Bruker WH 200 MHz instrument with TMS as an internal standard and UV spectra (MeOH) on a LKB 4053 spectrophotometer. Purity of the compounds was checked by tlc. CAUTION: benzene is flammable, may cause cancer, and should be handled with čare.
Synthesis of [2", 3" : 4', 3'] furanochalcone (5). A mixture of 2-hydroxyfurano(2', 3' : 4, 3)acetophenone (4, 10 mmol, 1.76 g) and benzaldehyde (1.1 eqiv., 1.16 g) in ethanolic solution of KOH (5%, 15 mL) was kept at room temperature for about 75 hr. The reaction mixture was diluted with ice-cold water, acidified with cold diluted HC1 and extracted with ether. The ether layer was washed with water, dried over anhydrous
4
5
6
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Na2S04 and evaporated to dryness. It was purified by preparative tlc (petroleum ether : acetone; 10:1) and crystallized from benzene-petroleum ether as yellow needles (yield 1.55 g, 53.70%), mp. 112-114 °C, Rf 0.64 (benzene: acetone; 9:1). Anal. Calcd for Ci7Hi203: C 77.26, H 4.58. Found: C 77.41, H 4.42. UV XMe0H : 254, 276, 328 nm. IR v
L             LZ,        J                                                                                                                                                                                                                          max
3452, 1645, 1600, 1592, 1470, 1420, 1375, 1325 cm"1. TTNMRtCDCls) S7.05 (d, 1H, J 8.9 Hz, C5'-H), 7.12 (d, 1H, J 2.1 Hz, C4"-H), 7.21-7.30 (m, 5H, C2-H, C3-H, C4-H, C5-H and C6-H), 7.58 (d, 1H, J 16.0 Hz, Ca-H), 7.79 (d, 1H, J 2.1 Hz, C5"-H), 7.91 (d, 1H, J 8.9 Hz, C6'-H), 8.04 (d, 1H, J 16.0 Hz, Cp-H), 12.68 (s, 1H, C2'-OH, chelated).
Synthesis of [2", 3" : 7, 8] furanoflavone (6) using DDQ. The chalcone (5, 0 75 mmol, 200 mg) in dry dioxane (50 mL) was added DDQ (0.68 mmol, 155 mg) and the solution refluxed for 3 hr. The product was purified by preparative tlc over silica gel using petroleum ether - benzene (1:2) as developing solvent. It crystallized from chloroform-petroleum ether as colorless needles (yield 120 mg, 60%), mp 137-138 °C (Lit.6 mp 137 °C), Rf 0.46 (benzene - ethyl acetate; 4:1). It gave positive Mg/HCl (yellow colouration) and Labat tests and a blue fluorescence in UV light. Anal. Calcd for C17H10O3: C 77.85, H 3.84. Found: C 77.66, H 3.68. UV XEt0H : 250, 330 nm. IR v 1644, 1580, 1405, 1360, 1345 cm"1. 'H NMR (CDCI3) L6.86 (s, 1H, C3-H), 7.22 (br d, 1H, J 2.1 Hz, C4"-H), 7.55-7.61 (m, 4H, C3'-H, C4'-H, C5'-H, and C6-H), 7.78 (d, 1H, J 2.1 Hz, C5"-H), 7.95 (m, 2H, C2'-H and C6'-H), 8.15 (d, 1H, J 9.9 Hz, C5-H).
Synthesis of [2", 3" : 7, 8] furanoflavone (6) using DMSO/I2. The chalcone (5, 0 75 mmol, 200 mg) was suspended in dimethyl sulfoxide (DMSO, 6 mL) and a crystal of iodine16 was added to it. The mixture was refluxed for 15 min in an oil bath and diluted with water. The solid obtained was filtered off, washed with 20% aq. sodium thiosulfate. It was purified by preparative tlc over silica gel GF254 using hexane-acetone (10:1) as eluting solvent and crystallized from ether as pale yellow needles (yield 135 mg, 67.50%), mp 137-138 °C, R/0.46 (benzene - ethyl acetate; 4:1). It gave positive Mg/HCl and Labat tests and a blue fluorescence in UV light. Spectral data of this flavone (6) was identical to that prepared by DDQ method.
Synthesis of [2", 3" : 7, 8] furanoflavone (6) using Ph-S-S-Ph. The chalcone (5, 0 75 mmol, 200 mg) was pasted with diphenyl sulphide17 (0.68 mmol, 125 mg) in a mortar and the mixture was transferred to a 100 mL three necked round bottom flask equipped with nitrogen inlet and outlet tubes. The central neck was closed by a glass stopper. The
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flask was then dipped into a silicon oil bath and heated at 265 °C under nitrogen atmosphere until the distillation of the thiols formed through the other outlet tube ceased (2.5 hr). The reaction mixture was then cooled at room temperature and 20 mL chloroform was added. The organic layer was washed with water several times. It was dried over anhydrous sodium sulfate and the solvent was removed by distillation. The product crystallized from ether as pale yellow needles (yield 130 mg, 65%), mp 137-138 °C, R/0.46 (benzene - ethyl acetate; 4: 1). It gave positive Mg/HCl and Labat tests and a blue fluorescence in UV light. Spectral data for this flavone (6) were also identical to that prepared by DDQ and DMSO/I2 method.
Antibacterial screening. The antibacterial activities of synthesized compounds 5 and 6 were studied against four human pathogenic bacteria, viz., S. dysenteriae (G), S. typhi (G ), S-ß-haemolyticus (G+) and S. aureus (G+). For the detection of antibacterial activities the filter paper disc diffusion method13'14 was performed. Each sample was dissolved in dimethyl sulfoxide (DMSO), an aliquot (10 µL) of the solution was dropped on to a paper disk (5mm diameter, Whatman No. 2 filterpaper). Kanamycin was used as standard antibiotics for the antibacterial activities. Nutrient Agar (NA) was used as basal medium for test bacteria. These agar media were inoculated with 0.5 mL of the 24 hr liquid cultures containing 107 microorganisms/mL. The diffusion tirne was 24 hr at 5 °C for bacteria. The sample containing air-dried paper disk was then placed on the agar. The incubation tirne was 12 hr at 37 °C for bacteria. Disks with only DMSO were used as control. Inhibitory activity was measured with a transparent ruler (in mm) as the diameter of the observed inhibition zones.
Determination of the Minimum Inhibitory Concentration (MIC). Minimal inhibitory concentration is defined as the lowest concentration that inhibits bacterial growth. For determination of the minimum inhibitory concentration (MIC) the serial dilution technique15 were followed using nutrient broth medium. The MIC value of the compound 5 and 6 were determined against S. dysenteriae (G) and S-ß-haemolyticus (G+).
Acknowledgement
The author is thankful to Mr. Shamim Hossain, Department of Pharmacy, University of Rajshahi, Bangladesh for kind help and co-operation to determine antibacterial activity.
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References
1.  The Wealth of India; Raw materials, CSIR, New Delhi, 1969, 8, 206.
2.  Medicinal Plants of Bangladesh; Chemical constituents and ušes, Asiatic Society of Bangladesh, Dhaka, 1998, 270.
3.  T. Tanaka, M. Iinuma, K. Yuki, Y. Fujii, M. Mizuno, Phytochemistry 1992, 31, 993-998.
4.  P. Lakshmi, G. Srimannarayana, N. V. Subba Rao, Indian J. Chem. 1974, 12, 8-11.
5.   S. B. Malik, P. Sharma, T. R. Seshadri, Indian J. Chem. 1977, 15B, 536-539.
6.   S. K. Talapatra, A. K. Malik, B. Talapatra, Phytochemistry 1982, 19, 1199-1202.
7.   S. Talapatra, A. K. Malik, B. Talapatra, Phytochemistry 1980, 21, 761-766.
8.  B. S. Parmar, K. K. Sahrawat, S. K. Mukerrjee, J. Sci. Indian Res. 1976, 35, 608-611.
9.  M. C. Pirrung, Y. R. Lee, Tetrahedron Lett. 1994, 35, 6231-6234.
10.   S. Rangswami, S. Narayanaswmi, T. R. Seshadri, J. Chem. Soc. 1954, 1871-1873.
11.   W. Baker, O. M. Lothin, J. Chem. Soc. 1935, 628-631.
12.  M. A. Hossain, A. Islam, J. Ban. Acad. Sci. 1999, 23, 9-12.
13.   S. Rollas, N. Kalyoncuoglu, D. Siir-Altiner, Y. Yegenoglu, Pharmazie 1993, 48, 308-311.
14.   A. W. Bauer, W. W. M. Kirby, J. C. Sherris, M. Turek, Am. J. CM. Pathol. 1966, 45, 493-496.
15.   C. Nishina, N. Enoki, S. Tawata, A. Mori, K. Kobayashi, M. Fukushima, Agric. Biol. Chem. 1987, 51, 139-143.
16.   A. G. Doshi, P. A. Soni, B. J. Ghiya, Indian J. Chem. 1986, 258, 759.
17.  Y. Hoshino, T. Ohinata, N. Taken, Buli. Chem. Soc. Jpn. 1986, 59, 2351-2352.
Povzetek
Fitokemične študije Pongamia pinnata so pokazale prisotnost [2", 3": 7, 8]furanoflavona (lanceolatin B). Tega smo pripravili iz 2,4-dihidroksiacetofenona preko halkonskega prekurzorja, s sledečo obdelavo z DMSO/I2, difenil sulfidom in nato DDQ. Antibakterijsko aktivnost pripravljenih spojin smo testirali na Shigella dysenteriae, Salmonella typhi, Streptococcus-P-haemolyticus in Staphylococcus aureus.
S. Alam: Synthesis and Studies of Antimicrobial Activity of Lanceolatin B