INTRODUCTION:

Chalcones, analogs of 1,3-diarylprop-2-en-1-one, form a wide class of compounds containing two aromatic rings bound with vinyl ketone fragment. They are useful in synthesis of various heterocyclic compounds. Chalcones of plant origin are known¹.Chalcones present great interest as compounds exhibiting antimalarial², antibactrial³, antifibrogenic⁴, anticancer⁵, antitrichomonal⁶, anti-inflammatory⁷, antileishmanial⁸, cytotoxic and anti-trypanosoma cruzi⁹ activities. While the flavonoid compounds are a group of natural products found in fruits, vegetables, nuts, seeds and flowers as well as in teas and are important constituent of human diet. They have been demonstrated to possess antioxdidant¹⁰, antihypertensive¹¹, antiallergic¹², antinocicepative¹³, trypsin inhibitors¹⁴, plant growth regulator¹⁵, antibacterial and antifungal¹⁶ activities.

In view of these observations and in continuation of our work on biologically active chalcones and their heterocycles¹⁷, we have synthesized some new chalcones and flavones and studied for their antibacterial activity as well as effect on seeds germination of wheat.

MATERIALS AND METHODS:

All melting points are taken in open glass capillaries and were found uncorrected. The purity of compounds has been checked by TLC on silica gel G. The IR spectra in KBr were recorded on Shimadzu spectrophotometer and ¹HNMR spectra were recorded in DMSO on Varian Inova 300 FT MHz spectrophotometer using TMS as internal standard (d ppm). Elemental analyses were performed on a Perkin-Elmer 240 CHN elemental analyzer.

Synthesis of Chalcones (3a-g):-

Equimolar quantities of halo substituted 2-hydroxyacetophenone (0.01mol) and 5-chloro-2-methoxybenzaldehyde (0.01 mol) were dissolved in ethanol (15 ml), under stirring and aqueous KOH (50%, 10 ml) was added dropwise. The reaction mixture was stirred at room temperature and kept for 14-16 hr. The reaction mixture was diluted with water and acidified with 10% HCl. The separated solid was filtered and cryststallised from DMF to give compounds (3a-g).

1-(2’-hydroxy-3’,5’-diiodophenyl)-3-(2-methoxy-5-chlorophenyl)-2-propen-1-one (3a):

IR (KBr) cm^-1: 2995(-OH), 1635(C=O), 1569,

1492(ring C=C), 1051(C-O).

¹HNMR (300 MHz, DMSO): δ 3.93 (s, 3H, OCH₃), 6.93 (d, 1H, H_α), 7.33 (d, 1H, H_β), 7.38- δ8.17 (m, 5H, Ar-H), 12.89 (s, 1H, Ar-OH).

1-(2’-hydroxy-3’-iodo-5’-chlorophenyl)-3-(2-methoxy-5-chlorophenyl)-2-propen- 1-one (3b):

IR (KBr) cm^-1: 3032(-OH), 1630(C=O), 1569,

1496(ring C=C), 1052(C-O).

¹HNMR (300 MHz, DMSO): δ 3.95 (s, 3H, OCH₃), 6.98 (d, 1H, H_α), 7.29 (d, 1H, H_β), 7.35- δ8.17 (m, 5H, Ar-H), 13.05 (s, 1H, Ar-OH).

1-(2’-hydroxy-3’-iodo-5’-methylphenyl)-3-(2-methoxy-5-chlorophenyl)-2-propen- 1-one (3c):

IR (KBr) cm^-1: 3066(-OH), 1632(C=O), 1569,

1487(ring C=C), 1045(C-O).

¹HNMR (300 MHz, DMSO): δ δ2.40 (s, 3H, CH₃), 3.93 (s, 3H, OCH₃), 6.91 (d, 1H, H_α), 7.32 (d, 1H, H_β), 7.34- δ8.17 (m, 5H, Ar-H), 13.23(s, 1H, Ar-OH).

1-(2’-hydroxy-3’-bromo-5’-chlorophenyl)-3-(2-methoxy-5-chlorophenyl)-2-propen- 1-one (3d):

IR (KBr) cm^-1: 3078(-OH), 1637(C=O), 1571,

1490(ring C=C), 1055(C-O).

¹HNMR (300 MHz, DMSO): δ 3.97 (s, 3H, OCH₃), 7.02 (d, 1H, H_α), 7.35 (d, 1H, H_β), 7.39- δ8.21 (m, 5H, Ar-H), 12.95(s, 1H, Ar-OH).

1-(2’4’-dihydroxy-3’,5’-diiodophenyl)-3-(2-methoxy-5-chlorophenyl)-2-propen- 1-one (3e):

IR (KBr) cm^-1: 3393(-OH), 1635(C=O), 1574,

1485 (ring C=C), 1050(C-O).

¹HNMR (300 MHz, DMSO): δ 3.89 (s, 3H, OCH₃), 6.93 (d, 1H, H_α), 7.30 (d, 1H, H_β), 7.39- δ8.21 (m, 4H, Ar-H), 10.83(s, 1H, 4’Ar-OH), 13.32(s, 1H, 2’Ar-OH).

1-(2’4’-dihydroxy-3’,5’-dichlorophenyl)-3-(2-methoxy-5-chlorophenyl)-2-propen- 1-one (3f):

IR (KBr) cm^-1: 3405(-OH), 1635(C=O), 1575,

1485 (ring C=C), 1056(C-O).

¹HNMR (300 MHz, DMSO): δ 3.94 (s, 3H, OCH₃), 6.98 (d, 1H, H_α), 7.32 (d, 1H, H_β), 7.42- δ8.30 (m, 4H, Ar-H), 10.95(s, 1H, 2’Ar-OH), 13.25(s, 1H, 4’Ar-OH).

1-(2’4’-dihydroxy-3’,5’-dibromophenyl)-3-(2-methoxy-5-chlorophenyl)-2-propen- 1-one (3f):

IR (KBr) cm^-1: 3396(-OH), 1637(C=O), 1570,

1480 (ring C=C), 1050(C-O).

¹HNMR (300 MHz, DMSO): δ 3.93 (s, 3H, OCH₃), 6.95 (d, 1H, H_α), 7.30 (d, 1H, H_β), 7.38- δ8.25 (m, 4H, Ar-H), 10.89(s, 1H, 2’Ar-OH), 13.27(s, 1H, 4’Ar-OH).

Synthesis of Flavones (4a-g):-

Chalcone (0.01 mol) was suspended in DMSO (10 ml) and a crystal of iodine was added to it. The mixture was refluxed for 30 min. and diluted with water. The solid obtained was filtered off, washed with 20% sodium thiosulfate and crystallised from aqueous ethyl alcohol to give compounds (4a-g). It gave positive Mg/HCl test (yellow colouration).

2’-Methoxy-5’-chloro-6,8-diiodoflavone (4a):

IR (KBr) cm^-1: 1645(C=O), 1570, 1495 (ring C=C).

¹HNMR (300 MHz, DMSO): δ 3.93 (s, 3H, OCH₃), 7.06 (s, 1H, COCH), 7.43- δ8.20 (m, 5H, Ar-H).

2’-Methoxy-5’,6-dichloro-8-iodoflavone (4b):

IR (KBr) cm^-1: 1643(C=O), 1571, 1493(ring C=C).

¹HNMR (300 MHz, DMSO): δ 3.95 (s, 3H, OCH₃), 6.98 (s, 1H, COCH), 7.30- δ8.15 (m, 5H, Ar-H).

2’-Methoxy-5’-chloro-6-methyl-8-iodoflavone (4c):

IR (KBr) cm^-1: 1632(C=O), 1565, 1490(ring C=C).

¹HNMR (300 MHz, DMSO): δ δ2.45 (s, 3H, CH₃), 3.93 (s, 3H, OCH₃), 6.99 (s, 1H, COCH), 7.37- δ8.20 (m, 5H, Ar-H).

2’-Methoxy-5’,6-dichloro-8-bromoflavone (4d):

IR (KBr) cm^-1: 1645(C=O), 1568, 1495(ring C=C).

¹HNMR (300 MHz, DMSO): δ 3.95 (s, 3H, OCH₃), 7.06 (s, 1H, CHOH), 7.40- δ8.10 (m, 5H, Ar-H).

2’-Methoxy-5’-chloro-6,8-diiodo-7-hydroxyflavone (4e):

IR (KBr) cm^-1: 3405(-OH), 1639(C=O), 1570, 1489 (ring C=C).

¹HNMR (300 MHz, DMSO): δ 3.93 (s, 3H, OCH₃), 7.02 (s, 1H, CHOH), 7.35- δ8.17 (m, 4H, Ar-H), 10.92(s, 1H, Ar-OH).

2’-Methoxy-5’,6,8-trichloro-7-hydroxyflavone (4f):

IR (KBr) cm^-1: 3405(-OH), 1644(C=O), 1579, 1489 (ring C=C).

¹HNMR (300 MHz, DMSO): δ 3.97 (s, 3H, OCH₃), 7.06 (s, 1H, CHOH), 7.45- δ8.25 (m, 4H, Ar-H), 10.97(s, 1H, Ar-OH).

2’-Methoxy-5’-chloro-6,8-dibromo-7-hydroxyflavone (4g):

IR (KBr) cm^-1: 3400(-OH), 1647(C=O), 1567, 1485 (ring C=C).

¹HNMR (300 MHz, DMSO): δ 3.95 (s, 3H, OCH₃), 7.01 (s, 1H, CHOH), 7.43- δ8.33 (m, 4H, Ar-H), 10.95(s, 1H, Ar-OH).

Antibacterial screening:-

The newly synthesized compounds were subjected to antibacterial screening by using cup plate diffusion method^18,19for zone of inhibition. The antibacterial activity was tested against Escherichia coli, Bacillus subtilis, Xanthomanas citri and Ervinia carotovara. Ampicillin was used as standard antibiotic and DMF was used as solvent control.

Table 1: Analytical and Physical data of Chalcones (3a-g) and Flavones (4a-g):-

Compound code	Mol. Formula	Mol. Wt.	Yield (%)	M.P. (⁰C)	Elemental Analysis % Found (Cal.) C H X (Cl,Br,I)
3a	C₁₆H₁₁O₃Cl I₂	540.5	68	167	35.69 (35.52) 1.91 (2.03) 53.45 (53.56)
3b	C₁₆H₁₁O₃Cl₂I	449	58	187	42.88 (42.76) 2.51 (2.40) 44.19 (44.09)
3c	C₁₇H₁₄O₃Cl I	428.5	76	137	47.74 (47.60) 3.37(3.26) 37.95 (37.92)
3d	C₁₆H₁₁O₃Cl₂Br	402	80	161	47.83 (47.76) 2.82 (2.73) 37.59 (37.56)
3e	C₁₆H₁₁O₄Cl I₂	556.5	66	177	34.37 (34.50) 2.06 (1.97) 51.95 (52.02)
3f	C₁₆H₁₁O₄Cl₃	373.5	63	144	51.52 (51.40) 3.05 (2.94) 28.54 (28.54)
3g	C₁₆H₁₁O₄ClBr₂	462.5	71	160	41.60 (41.51) 2.29 (2.37) 42.25 (42.27)
4a	C₁₆H₉O₃Cl I₂	538.5	74	156	35.79 (35.65) 1.59 (1.67) 53.61 (53.76)
4b	C₁₆H₉O₃Cl₂I	447	68	197	42.84 (42.95) 2.15 (2.01) 44.19 (44.29)
4c	C₁₇H₁₂O₃Cl I	426.5	62	165	47.74 (47.83) 2.95 (2.81) 37.98 (38.10)
4d	C₁₆H₉O₃Cl₂Br	400	64	173	48.11 (48.00) 2.38 (2.25) 37.63 (37.76)
4e	C₁₆H₉O₄Cl I₂	554.5	76	149	34.73 (34.62) 1.70 (1.62) 52.11 (52.20)
4f	C₁₆H₉O₄Cl₃	371.5	70	159	51.55 (51.68) 2.34(2.42) 28.54 (28.66)
4g	C₁₆H₉O₄ClBr₂	460.5	59	177	41.78 (41.69) 2.05 (1.95) 42.37 (42.45)

Table 2: Antibacterial activity data of synthesized compounds

Compound code	Zone of inhibition (mm)
Compound code	B. subtilis	E. coli	X. citri	E. carotovara
3a	17	19	15	20
3b	19	20	21	20
3c	20	18	18	21
3d	23	19	21	20
3e	25	25	24	24
3f	28	27	27	29
3g	26	28	25	26
4a	12	17	11	14
4b	22	24	18	20
4c	17	19	11	16
4d	12	14	15	11
4e	26	27	25	26
4f	27	26	21	28
4g	25	28	27	26
Control	----	----	----	----
Ampicillin 100mg/ml	25	26	25	27

Laminar airflow bench was swapped with 70% alcohol and UV lamp was switched on. After 30 min, the UV lamp was switched off. All reagents, media, inoculums and glassware were placed in laminar airflow bench observing all aseptic conditions.

The plates inoculated within minutes of the preparation of suspension, so that the density does not change. A sterile cotton swab over was dipped into the suspension and the media was inoculated by even streaking of the swab over the entire surface of the plate in three directions.

After the inoculums had dried, cups of diameter 6mm were made in agar plate with the sterile cork borer. The drugs solutions were added to these cups with a micropipette and the plate were then incubated at 37⁰C for 24 hours. The zone of inhibition was measured using mm scale. (Table-2)

Germination Assay:-

The newly synthesized compounds were tested for effect on seed germination of wheat (Triticum aestivum) by using moist blotter plate method²⁰. In this method ten seeds of wheat were arranged on the blotter paper (8.5cm) in pre sterilized Petri plates (10cm). The control was treated with only distilled water, then, 2ml (0.01%) of each solution and distilled water were added to the seeds on the blotter paper.

The experiments were carried out under natural light and at room temperature for ten days. The seed germination in the form of root length and shoot length were measured (in cm) at the end of experiment.

Table 3: Effect of synthesized compounds on seed germination of Wheat (Triticum aestivum)

Compound code	Seed germination of wheat (cm)
Compound code	Mean of shoot length	Mean of Root length
3a	5.3	6.2
3b	6.6	10.3
3c	6.4	9.9
3d	11.2	10
3e	5.2	12.2
3f	5.9	5.1
3g	5.4	10.1
4a	8.3	8.4
4b	5.3	6.3
4c	6.6	10.1
4d	5.3	6.4
4e	8.6	5.5
4f	5.1	5.8
4g	7.9	6.6
Control	5.2	5.9

RESULT AND DISCUSSION:

Chalcones (3a-g) and flavones (4a-g) were synthesized. The structures of newly synthesized compounds have been confirmed on the basis of elemental analysis and spectral data. From antibacterial screening, it was found that compound 3e, 3f, 3g, 4e, 4f and 4g exhibited good antibacterial activity against all bacteria at a concentration of 100mg/ml.

Chalcones and flavones have been tested their effect on seed germination of Wheat. All chalcones and flavones showed maximum seed germination than control (distil water) and no any growth of fungi was observed on seeds. The present study reveals that chalcones and flavones which contain iodine, chlorine, bromine, methoxy and hydroxy substituents showed good antibacterial than the standard drug Ampicillin. Chalcones and flavones were acting as a growth promoting agents for seed germination of wheat and further work in this direction is under study.

AKNOWLEDGEMENT:

Authors are also grateful to UGC New Delhi for sanctioning Major Research Grant and the Director, IICT, Hyderabad for providing spectral analysis of newly synthesized compounds.The authors are thankful to Principal, Yeshwant Mahavidyalaya, Nanded for providing laboratory facilities

REFERNCES:

1. Hijova E.Bioavailability of Chalcones. Bratisl Lek Listy 2006; 107(3): 80-84.

2. Liu M, Wilairat P and Mei-Lin G. Antimalarial Alkoxylated and Hydroxylated Chalcones: Structure-Activity Relationship Analysis. J.Med.Chem. 2001; 44(25): 4443-4452.

3. Opletalova V. Chalcones and Their Heterocyclic Analogues as Potential Therapeutic Agents of Bacterial Diseases. Cesk.Slov.Farm.2000; 49: 278-284.

4. Lee SH, Nan JX, Zhao YZ, Woo SW, Park EJ, Kang TH, Seo GS, Kim YC and Sohn DH. The Chalcone Butein from Rhus verniciflua shows antifibrogenic activity. Planta. Med.2003; 69: 990-994.

5. Konieczny MT, Konieczny W, Sabisz M, Skladanowski A, Wakiec R, Augustynowicz-Kopec E and Zwolska Z. Synthesis of Isomeric, Oxathiolone fused Chalcones, and Comparison Their Activity towards various Microorganisms and Human Cancer Cells Line. Chem. Pharm. Bull. 2007; 55: 817-820.

6. Oyedapo AO, Mankanju VO, Adewunmi CO, Iwalewa EO and Adenowo TK. Antitrichomonal Activity of 1,3-Diaryl-2-propen-1-ones on Trichomonas Gallinae. Afr.J.Trad. CAM 2004; 1:55-62.

7. Jin F, Jin XY, Jin YL, Sohn DW, Kim S-A, Sohn DH, Kim YC and Kim HS. Structural Requirements of 2’,4’,6’-Tris (methoxymethoxy) chalcone Derivatives for Anti-inflammatory Activity: The Importance of a 2’-Hydroxy Moiety. Arch.Pharm.Res.2007; 30(11): 1359-1367.

8. Narender T, Khaliq T, Shweta, Nishi, Goyal N and Gupta S. Synthesis of Chromenchalcones and Evaluation of their in vitro Antileishmanial Activity. Bioorg.Med.Chem. 2005; 13: 6543-6550.

9. Aponte JC, Verastegui M, Malaga E, Zimic M, Quiliano M, Vaisberg AJ, Gilman RH and Hammond GB. Synthesis, Cytotoxicity and Anti-Trypanosoma cruzi Activity of New Chalcones. J.Med.Chem. 2008; 51: 6230-6234.

10. Yoo H, Kim SH, Lee J, Kim HJ, Seo SH, Chung BY, Jin C and Lee YS. Synthesis and Antioxidant Activity of 3-Methoxyflavones. Bull.Korean Chem.Soc. 2005; 26(12):2057-2060.

11. Li JX, Xub B, Chai Q, Liu ZX, Zhao AP and Chan LB. Antihypertansive Effect of Total Flavonoid fraction of Astragalus complanatus in Hypertensive Rats. Chin.J.Physiol. 2005; 48: 101-106.

12. Inoue T, Sugimoto Y, Masuda H and Kamei C. Antiallergic Effect of Flavonoid Glycosides obtained from Mentha piperita L. Biol.Pharm.Bull. 2002; 25: 256-259.

13. Umamaheswari S, Viswanathan S, Sathiyasekaran B W C, Parvathavarthini S and Ramaswamy S. Antinociceptive Activity of Certain Dihydroxy Flavones. Indian J.Pharm.Sci. 2006; 68(6): 749-753.

14. Maliar T, Jedinak A, Kadrabova J and Sturdik E. Structural Aspects of Flavonoids as Trypsin Inhibitors. Eur.J.Med.Chem. 2004; 39: 241-248.

15. Keriko J M, Nakajima S, Baba N, Isozaki Y and Iwasa J. Plant Growth Regulators from Kenyan Plant, Psiadia punctulata. Sci.Rep.Agr.OKAYAMA 1995; 84: 7-11.

16. Mostahar S, Katun P and Islam A. Synthesis of Two Vanillin Ring Containing Flavones by Different Methods and Studies of Their Antibacterial and Antifungal Activities. J.Biol.Sci. 2007; 7(3): 514-519.

17. a) Vibhute YB and Basser MA. Synthesis of 6-Hydroxy-3-naphthylidene-5,6’-Benzoflavanone. J.Indian Chem. Soc. 2001; 78: 319. b) Vibhute YB and Basser MA. Synthesis and Activity of a new series of Chalcones as Antibacterial Agents. Indian J.Chem. 2003; 42B: 202-205. c) Mokle SS, Sayyed MA, Kothawar and Chopde. Studies on Synthesis and Antimicrobial Activity of some new Iodochalcones, Flavones and Flavonols. Int. J. Chem. Sci. 2004; 2(1): 96-100. d) Mokle SS, Sayyed MA, Bhusare SR, Pawar RP and Vibhute YB. Synthesis and Antibacterial Activity of new Pyrazoline and Benzothiazepines derivatives. Chemistry: An Indian Journal 2005; 2(9): 302-305.

18. Collins CH. Microbiological Methods. Buterworths, London.1967.

19. Kavangh F. Analytical Microbiology.Acedemic Press, New York.1963.

20. Neergaard and Paul. Seed Pathology. The Mac Millan Press Ltd., Vol.I-III. London.1977.