Indian Journal of Experimental Biology Vol., November 00, pp. - Efficacy of -methyl--hydroxy coumarin derivatives against vectors Aedes aegypti and Culex quinquefasciatus Madhavi Deshmukh a, Pushpa Pawar b, Mary Joseph b, Usha Phalgune c, Rajashree Kashalkar a & Nirmala R Deshpande d a Department of Chemistry, Sir Parshurambhau College, Pune 00, India b Department of Entomology, National Chemical Laboratory, Pune 00, India c Central NMR Facility, National Chemical Laboratory, Pune 00, India d T R Ingle Research Laboratory, Department of Chemistry, Sir Parshurambhau College, Pune 00, India Received April 00, revised 0 August 00 -Methyl--hydroxy coumarin is considered as a lead molecule as a biopesticide. Its mono bromo and tribromo derivatives were synthesized. Two more derivatives were synthesized by acylation. Compound (,,-tribromo--hydroxy- -methyl-chromen--one) was found to be the most potent against IV th instar larvae of C. quinquefasciatus and A. aegypti the LC 0 being. and. ppm respectively. It showed 00% larval mortality at ppm against A. aegypti and at 0 ppm against C. quinquefasciatus. Compounds and (,,-tribromo--hydroxy--methyl-chromen- -oxo-h-chromen--yl acetate) showed remarkable ovicidal activity. Significant reduction of 0-% hatching of eggs of both mosquito species was observed at the highest dose of 00 ppm. The hatched larvae showed 00% mortality in the successive instars. Compounds and (-bromo--hydroxy--methyl-chromen--one and -bromo--methyl- -oxo-h-chromen--yl acetate) showed moderate activity against both mosquito species. Keywords: Aedes aegypti, Culex quinquefasciatus, Larval mortality, -Methyl--hydroxy coumarin, vicidal activity The major disease burden in India is contributed by vector borne diseases like Malaria, Filaria and Dengue fever. Management of the disease vector for Dengue fever using synthetic chemicals has failed due to vector resurgence and environmental pollution. Consequently an intensive effort has been made to find alternative control measures. ne of the control measures is the use of environment friendly and easily biodegradable natural insecticide of plant origin. Coumarins are one of the major class of phenolic compounds occur in green plants, fungi and bacteria. Coumarins, which are primarily benzo-α-pyrone derivatives, comprise a large class of naturally occurring, low molecular weight, polyphenolic compounds. These compounds display a remarkable array of pharmacological and biochemical activities. Coumarin derivatives are also used as drug and pesticide intermediates. Several natural products with a coumarin moiety have been reported to have multiple biological activities. The compounds derived from natural coumarin, scopoletin, showed insect antifeedants activity. Due to the unquestionable Telephone : +-0-0 E-mail: madhaviarya@rediffmail.com, anurag.deshpande@rediffmail.com importance of coumarins considerable efforts have been made to prepare compounds bearing single substitution, or more complicated systems, including heterocyclic rings mainly at -, - and/or -positions. Keeping in view the pesticidal potential of this class of the compounds, the toxic effects of the coumarin derivatives have been evaluated on larvae of the Culex quinquefasciatus and Aedes aegypti. Materials and Methods Synthesis of compounds Compound : To the cold solution of H (0% aq.; ml; 0 M) in methanol (0 ml), drop wise addition of H (0% aq.; ml; 0. 00 M) was carried out. The mixture was stirred for min at room temperature. To this cold solution, -methyl--hydroxy coumarin (0. g; 0 M) was added. The reaction mixture was stirred for min. White solid separated out within min. Compound (0. g; %) was crystallized from hot methanol. Purified compound showed a sharp melting nature at o C. UV spectrum showed λ max at. and nm. ( H, C=mg/ml, Ab= 0.0,0.). IR spectrum showed characteristic bands at. (hydroxy), 00 (-C-H stretching), 0 (unsaturated
DESHMUKH et al.: EFFICACY F CUMARIN DERIVATIVES AGAINST AEDES & CULEX lactone carbonyl), 0. unsaturated ester carbonyl),. (conjugated double bond),.,. (aromatic ring) and., 0. (C- stretching),.(substituted ) cm -. Compound : Compound (0. g; 0. M) was treated with acetic anhydride to get the clear solution and a drop of pyridine was added. The white product compound (0. g; %) separated out immediately. Compound, white solid, purified by using ethanol as a solvent. Purified compound showed a sharp melting nature at 0 o C. UV spectrum showed λ max at. and nm. (( H, C=mg/ml, Ab= 0.0, 0.). IR spectrum of compound showed characteristic bands at 00 (olefinic -CH stretching),. cm - for unsaturated lactone carbonyl,..0 cm - for ester carbonyl. Compound : -methyl--hydroxy coumarin (0. g;. mm) was added in acetic acid (0 ml) and stirred for min. to ensure dissolution. K ( g; 0. mm) was added in the reaction mixture and it was stirred for further 0 min to dissolve completely. H (0%; 0. ml;. mm) was charged and reaction mixture was further stirred for hr. The reaction mixture was poured in cold water and stirred for half an hour. Yellowish white solid compound was obtained. It was filtered and washed with water till free from acid. Compound, white solid, purified by crystallisation using ethanol as a solvent. It showed a sharp melting nature at o C. UV spectrum showed λ max at, nm (CHCl, C= mg/ml, Ab=0., 0.). IR spectrum showed characteristic bands at 0 (hydroxy), 00 (aromatic -CH stretching), 0.(unsaturated lactone carbonyl), 0. and Table H NMR spectral data of compounds - 0.(aromatic ring), 00. (-C- stretching),.,,, (aromatic CH bending region) and a sharp band at 0.(-) cm -. Compound : Compound (0. g; 0. M) was treated with acetic anhydride to get the clear solution and a drop of pyridine was added. The white product compound (0. g; %) separated out immediately. Compound, white solid, purified by crystallization using ethanol as solvent. It showed a sharp melting nature at 0 o C. UV spectrum showed λ max at and nm (CHCl, C=mg/ml, Ab=.,.). IR spectrum showed characteristic bands at 00 (olefinic -CH stretching), 0 (unsaturated lactone carbonyl),.0 (ester carbonyl), (double bond) 0.,. (aromatic ring),. (substituted ) cm -. H NMR and C NMR data of the compounds - are exhibited in Tables and respectively. Biological assay Larvicidal assay: A. aegypti and C. quiquefasciatus mosquitoes were reared at ± o C and 0 ± % RH. Early IV th instar larvae and hr old eggs were taken for the experiment from a routine culture being maintained at National Chemical Laboratory. IV th instar larvae were fed with yeast and dog biscuits powder in : proportion. Standard methods for testing the susceptibility of mosquito larvae to the synthetic compounds, as suggested by WH were followed in all the experiments. Compounds were tested to determine the larvicidal activity by making serial dilutions like 0,, 0 and 00 mg/ml solutions and used in the larvicidal bioassay against two mosquito species. The bioassays were conducted at room temperature ( ο ± ο C) by R R R Compd Functional groups R R H NMR (δ ppm) a+b ** R = R = R =, R =H.0 (s, H, -H),.(s,H, ),.00 (s, H, Ar-H) b ** R = R =R =,.(s,h, =C- ),.(s,h,,. (s, H, Ar-H) R =- -C= * a R = R = R = H, R =.(s,h, ),. (dd,j=hz and Hz, H,Ar-H),. (d,j= Hz,H,Ar-H),. (d, J= Hz, H, Ar-H), a *** R =, R = R = H R =- -C= *00 MHz, **00 MHz, ***00 MHz; a CDCl b, DMS-d. (s, H,-H).(s, H, =C- ),.(s, H, ),. (m, H, Ar-H),. (d, J= Hz, H, Ar-H)
0 INDIAN J EXP BIL, NVEMBER 00 exposing 0 larvae in each concentration of the compound in a volume of 0 ml of water taken in 00 ml beaker. Five replicates for each concentration and the control (with solvent only) were tested.the larval mortality in each concentration and control was recorded after every hr of continuous exposure. The corrected mortality was determined using Abbott s formula whenever required. The dose mortality data were analyzed by log probit method of Finney, lethal concentration for 0% mortality was calculated. Atom No. Table C NMR spectral data of compounds - a+b ** b ** * a a *** C -. (s).0(s)..(s) C 0.(s) 0.(s) 0..(s) C.(s).(s). 0.(s) C. (d).(d).0.(d) C.0(d).(s)..(d) C. (s).0(s)..0(s) C 0.(d).(s) 0. (d) C -.0 (s).0(s)..0 (s) C - 0.(s).(s) 0..(s) C-.(q) (q)..(q) C- 0.(q) 0.(q) C-.(s).(s) *00 MHz, **00 MHz, ***00 MHz; a CDCl, b DMS-d vicidal assay A small strip of whatman filter paper containing 0- hr old eggs of A.aegypti were cut. This strip contained 0-0 eggs. These were counted and immersed in 0 ml of water. In C. quinquefasciatus the egg raft was lightly tapped by the brush on a filter paper and around 0-0 eggs were collected and immersed in 0 ml water. Desired concentrations of the compound were released in water medium containing eggs. Percent hatching was observed after hr of treatment. Development in successive instars was also observed. Hatched larvae were provided with food (powdered yeast tablets and dog buiscuits in : ratio). Any mortality occurring in any instar was taken into account and total % mortality in larval instars was calculated. Five replicates were taken for each concentration and the experiment was repeated three times in both the bioassay. Results The results obtained against A. aegypti larvae (Table ) revealed that 00% mortality was achieved only in Compound treated larvae. There was 00% kill even at the lowest concentration of ppm (Table ). The LC 0 was lowest for Compound (. ppm). Compound exhibited 00% larval kill at the highest dose of 0 ppm. Compound and showed comparatively less activity. In C. quinquefasciatus (Table ) the least LC 0 (. ppm) was shown by Table Larvicidal activity of -methyl- hydroxy coumarin derivatives against A. aegypti (A) and C. quinquefasciatus (B) IV th instar larvae [Values are mean ±SE] Sample Mortality (%) at ppm LC 0 Fiducial limit Chi square 0 00 0 0. lower- upper χ Compound A 00± 00± B 00± 00± Compound 00± 0.± A. B 00±.±. Compound.± ± A.. 00± 00±.±..±..± 0. 00± 00±.±. 0.±. ± 0..±..±..±...-. 00±. ±.... ±...±.0 0.- 0...0± 0 0..- 0....-... 0...0 B.0±. Compound. A ±..±. 0.±..±..± 0. 0± 0. 0 0.0 0.-.0.0 0.-... B.0±. ±..± 0.0 0± 0. 0..- 0..
DESHMUKH et al.: EFFICACY F CUMARIN DERIVATIVES AGAINST AEDES & CULEX Compound. The 00% larval kill was achieved at the lowest concentration of 0 ppm. Compound also exhibited 00% kill at 0 ppm and 0-0% kill at lower concentration of 00 and 0 ppm respectively. H 0 Compound :,, -Tribromo--hydroxy--methylchromen--one Compound and did not show 00% kill even at the highest concentration. Treatments with the coumarin derivatives on the eggs of A. aegypti (Table ) showed that the percentage hatching was remarkably H 0 Compound : -omo--hydroxy--methyl-chromen- -one ' H C ' Compound :,, -Tribromo--methyl- - oxo- Hchromen--yl acetate 0 ' H C ' Compound : -omo--methyl- -oxo-hchromen--yl acetate Table vicidal effect of -methyl--hydroxy coumarin derivatives on A. aegypti and C. quinquefasciatus eggs [Values are mean ±SE] 0 Compound Dose Hatching (%) of treated A. aegypti eggs Post embryonic mortality (%) of (I-II)*A. aegypti Hatching (%) of treated C. quinquefasciatus Eggs Post embryonic mortality (%) of (I-III)* C. quinquefasciatus Compound 00 0 0.±0..±0..0±.0.±0. 00± 00± 00±.0±..±0..±0. 0.±0..±0. 00± 00± 00±.±.0 Compound 00 0 0.±0..±0..±.0.±.0 00± 0.±0..0±0..±0..±.0.0±..±.0 0.±.0 00±.±..±0..±0. Compound 00 0 0.±..±..±.0.0±..±..±0..±0..±0. 0.±..±.0.±.0 0.±..±.0.±0..±0. 0.±0. Compound 00 0 0.±..±.0 0.±..0±..±0..0±0.0.±0..0±0..±..±..±..±..±.0 0.±..±0..±0. Control 00± 0.0±.0.±0. * I - III instar larval mortality
INDIAN J EXP BIL, NVEMBER 00 reduced in case of Compound, the results indicated that Compound has highest ovicidal activity than the other compounds. It showed -% hatching at the concentration from 00-0 ppm (Table ). In C. quinquefasciatus eggs only - % hatching was observed on various concentrations taken. Compound was found to be active at 00 ppm. At lower concentrations (0 ppm) the hatching declined by 0%. Compound was found to be active at 00 and 0 ppm by giving 0-% of hatching in case of C. quinquefasciatus. Compounds and were less potent and the percentage hatching was more than 0% in case of both the mosquitoes. Compound showed 00% post embryonic mortality in both the mosquitoes at all the concentrations except 0 ppm. Larvae died in very st instar stage. In case of both the mosquito treated compound there was 00% kill at 00 ppm in very first instar stage. However in case of remaining concentrations the hatched larvae were killed in nd instar stage. Compound showed the PEM in nd instar at 00 ppm but in lower concentration ( 0-0 ppm) the mortality was carried till rd instar. Compound exhibited the PEM in rd instar at all the concentration and it was observed that the mortality was insignificant in case of both the mosquitoes. Discussion From the experiment it revealed that the larvae and eggs of C. quiquefasciatus were more susceptible than the larvae of A.aegypti. The LC 0 being only. ppm in C. quiquefasciatus against IV th instar larvae where as treatment with eggs showed % hatching less than the Aedes aegypti. However there was 00% PEM in case of both the mosquitoes at higher concentration taken (00 and 0 ppm). The larval death occurred in very st instar stage in all the concentrations except at 0 ppm. Mortality occurring in successive instars was observed mostly during moulting from st instar to nd instar stage which reflects that the embryonic effect was carried over upto the nd instar when treated at higher concentration. At lower concentration (0-0 ppm) the effect was carried till rd instar which ultimately affected total percentage of normal adults emerging out of the treatments. n the basis of the larvicidal and ovicidal activity, the potency of compounds was graded as Compound > Compound > Compound > Compound. A greater potency of Compound is due to bromine atoms present at C- and C- positions. These results are in conformity with the results of Nofal et al. The results showed that such compounds can be utilized in vector control programme. Acknowledgement Thanks are due to the Principal and Head of the Department of Chemistry, Sir Parshurambhau College, Pune, and Dr A Sen Entomology Department NCL, Pune, India, for co-operation. References Sukumar K, Perich M J & Boobar I R, Botanical derivatives in mosquito Control: A review, J Am Mosq Control Assoc, () 0 Murray R D, Mendez H J & own S A, The natural coumarins, ccurrence chemistry and biochemistry (John Wiley and Sons, New York). Shinde D B, Shingare M S, Gupta P P & Srimal R C, Synthesis of heteroaryloxyhydroxypropanes and their PCA activities, Indian J Chem, B () (),0. Nagaiah K, Krupadanam G L & David S G, Synthesis of angular furano - or pyrano-fused coumarins from natural scopoletin, Indian J Chem, B () (00),. Fylaktakidou, Konstantina C, Hadjipavlou-Litina, Dimitra J, Litinas K E & Nicolaides Demetrios N., Natural and synthetic coumarin derivatives with anti-inflammatory/ antioxidant activities, Curr Pharmaceut Design, 0 (0) (00), Abbot W S, A method of computing the effectiveness of insecticides, J Econ Entomol, () Nofal Z M, El-Zahar M I & Abd El-Karim S S, New Coumarin detrivatives with expected biological activity, Molecules, ( 000).