1. INTRODUCTION:

Coumarins constitute one of the most important place in the world of natural products and synthetic organic chemistry^[1-2^]. Coumarins are present in a group of natural compounds either in the free or combined state, particularly found in a variety of plants of the Orchidaceae, Leguminoceae, Rutacea and Umbelliferae species in the form of benzopyrene derivatives etc. Coumarins have important effects in plant biochemistry and physiology as they act as antioxidants, enzyme inhibitors and precursors of toxic substances. These compounds are involved in the actions of plant growth hormones, growth regulators, the control of respiration, photosynthesis and defense against infection^[3]. Coumarins have long been recognized to possess anti-inflammatory, anti-oxidant, anti-allergic, hepatoprotective, anti-thrombotic, anti-viral and anti-carcinogenic activities ^[4]. In addition to biological activities they are used as additives to food and cosmetics and optical brightening agents ^[5-6].

Hydroxycoumarins are distinctive phenolic compounds. Therefore they act as potent metal chelators and also free radical scavengers ^[7]. They are powerful chain-breaking antioxidants. The very long association of plant coumarins with various animal species and other organisms throughout evolution may account for the extraordinary range of biochemical and pharmacological activities of these chemicals in mammalian and other biological systems^[8]. The coumarins are extremely variable in structure due to the various types of substitutions in their basic structure which can influence their biological activity. The interesting biological activities of the coumarins have made them attractive targets in organic synthesis.

Mosquitoes can spread more diseases than any other group of arthropods and affect millions of people throughout the world. WHO has declared the mosquitoes as “public enemy number one” ^[9]. They act as a vector for most of the life threatening diseases like malaria, yellow fever, dengue fever, chikungunya ferver, filariasis, encephalitis, West Nile virus infection, etc., in almost all tropical and subtropical countries and many other parts of the world.

Due to industrialization people migrate towards industrial areas for job opportunities which resulted in the development of ‘slums’ with improper disposal of water. This gives the viable environment for culex mosquitoes which are the vectors of filariasis and encephalitis to breed in this stagnated polluted water. To prevent production of mosquito borne diseases and to improve quality of environment and public health mosquito manage is essential. The major tool in mosquito control operation is the application of synthetic insecticides such as organochlorine and organophosphate compounds. But this has not been successful process due to human, technical, operational, ecological and economic factors. The main factors are lack of novel insecticides, high cost of synthetic insecticides, concern for environmental sustainability, harmful effect on human health, other non-target populations, their non biodegradable nature, higher rate of biological magnification through ecosystem and increasing insecticide resistance on a global scale ^[10-11].

Therefore if alternative non-chemical methods of suppressing pests and vector mosquitoes can be evolved, they may be accomplished by desired modification of the environment without destroying it. The method should be environment friendly, cost-effective, biodegradable and target specific insecticides against mosquito species. Considering these, the application of eco-friendly alternatives such as biological control of vectors has become the central focus of the control programmme in lieu of the chemical insecticides ^[12].

Also there is necessary to control micro-organism in all the domestic places by medical fields to inhibit the spoilage of foods and industrial products. Therefore the control of micro-organism often depends on establishing conditions which cannot be tolerated by microbes. Anti-microbial conditions can be created by microbicidal agents that specifically kill or inhibit bacteria. Knowledge of condition of microbial growth is important and it can be facilitated by culturing micro-organism and controlling unwanted organism. The present work is designed to synthesis the simple coumarins such as 7-hydroxy-4- methylcoumarin, 7-acetoxy-4-methylcoumarin, 8-acetyl-7-hydroxy-4-methylcoumarin, resacetophenone, 8-acetyl-5-hydroxy-4-methylcoumarin, 4, 7-dimethylcoumarin and evaluate the anti-larvicidal, anti-bacterial properties of the coumarins. Also to identify the whether such compounds are can be utilized for developing newer anti-larvicidal, anti- bacterial drugs.

2 MATERIALS AND METHODS:

2.1 Materials:

Resorcinol, ethylacetoacetate, acetic anhydride, pyridine, anhydrous aluminium chloride, zinc chloride, glacial acetic acid, phosphorous oxychloride, dry benzene, p-cresol and all the organic reagents were analytical grade purchased from Merck, SRL, Qualigens, India. Solutions have been prepared using double distilled water for all the measurements.

2.2 Synthesis of coumarins

Synthesis of six simple coumarins such as 7-hydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 8-acetyl-7-hydroxy-4-methylcoumarin, resacetophenone, 8-acetyl-5-hydroxy-4-methylcoumarin, 4,7-dimethylcoumarin was prepared by pechmann condensation of ethyl acetoacetate with a few phenols.

2.2.1 Synthesis of 7-hydroxy-4-methylcoumarin

The synthetic procedure of 7-hydroxy-4-methylcoumarin is as follows. 50 ml of concentrated H₂SO₄ was cooled in a 250 ml beaker to 10°C in an ice bath. When the temperature was 0°C, a solution of 5 gms of resorcinol was dissolved in 6.75 ml of ethylacetoacetate. While this solution was cooled, conc. H₂SO₄ was added drop by drop over a period of ½ an hour and was brought to room temperature. The reaction mixture was left in a refrigerator overnight. Next day the coumarin product was poured into ice cold water with constant stirring. The separated coumarin solid was filtered, washed with double distilled water. And it was recrystallised from ethyl alcohol.

2.2.2 Synthesis of 7-acetoxy-4-methylcoumarin

The pechmann condensation procedure of 7-acetoxy-4-methylcoumarin is given below. A mixture of 3 ml of acetic anhydride and 5 ml of pyridine were added drop by drop to dissolve the 2.5 gm weighed 7-hydroxy-4-methylcoumarin and it was kept overnight. After 12 hrs the reaction product was poured into crushed ice. The coumarin solid that separated was filtered thoroughly and washed with double distilled water. It was recrystallized from ethyl alcohol.

2.2.3 Synthesis of 8-acetyl-7-hydroxy-4-methylcoumarin

A mixture of 2.5 gms of 7-acetoxy-4-methylcoumarin and 4.5 gms of anhydrous aluminium chloride was heated on an oil bath at 160°C for three hours. It was cooled at room temperature. The mixture was acidified with conc. hydrochloric acid by adding drop by drop and kept at 10°C for 2 hours. The product was filtered and washed with ice cold water. The separated product was filtered, washed with water and recrystallized from ethyl acetoacetate.

Table.1 comparison on the literature melting point, experimental melting point, molecular weight and molecular formula of the coumarins

S.No	Coumarins	Melting point (°C)	Experimental melting point (°C)	Molecular weight	Molecular formula
1.	7-hydroxy-4-methylcoumarin	184	184	176.17	C₁₀H₈O₃
2.	7-acetoxy-4-methylcoumarin	151	150	218.205	C₁₂H₁₀O₄
3.	8-acetyl-7-hydroxy-4-methylcoumarin	148	148	337.3	C₁₉H₁₅NO₅
4.	Resacetophenone	145	144	152.15	C₈H₈O₃
5.	8-acetyl-5-hydroxy-4-methylcoumarin	211	210	218.205	C₁₂H₁₀O₄
6.	4,7-dimethyl coumarin	170	170	174.196	C₁₁H₁₀O₂

2.2.4 Synthesis of resacetophenone

The synthetic procedure of resacetophenone is outlined here briefly. 15 gms of ZnCl₂ was dissolved in 10 ml of hot glacial acetic acid. 10 gms of resorcinol was added slowly to this hot solution with constant stirring. Heating was continued for 20 minutes by maintaining the temperature at 110-120°C. After heating was over, the hot mixture was poured to a mixture of 50 ml of conc. hydrochloric acid and 50 ml of cold distilled water (1:1 ratio). The solid separated was filtered and washed with dil.HCl and then with distilled water. A portion of the sample was recrystallised from alcohol.

2.2.5 Synthesis of 8-acetyl-5-hydroxy-4-methylcoumarin

A mixture of 4 gms of resacetophenone, 3 gms of ethylacetoacetate, 2 ml of phosphorous oxychloride and 10 ml of dry benzene was heated on a steam bath for 5 hrs. When the evolution of hydrochloric acid was stopped, benzene solution was poured and the residue was extracted with benzene twice and the solvent was removed by distillation. The residue was crystallized from alcohol.

2.2.6 Synthesis of 4, 7-dimethylcoumarin

50 ml of conc.sulphuric acid was cooled to 10°C in an ice bath. When the temperature was 0°C, a solution of 5 mg of p-cresol in 6.7 gm of ethylacetoacetate (6.75 ml) was added dropwise with constant stirring. The reaction mixture was left in a refrigerator overnight. The product was poured into ice water. The separated solid was filtered, washed with distilled water and recrystallized from alcohol. Table.1 shows the melting point from literature, experimental melting point, molecular weight and molecular formula of the simple coumarins.

2.3 Characterization of coumarins

Fourier Transform Infrared spectra of the 7-hydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 8-acetyl-7-hydroxy-4-methylcoumarin, resacetophenone, 8-acetyl-5-hydroxy-4-methylcoumarin and 4, 7-dimethylcoumarin were studied. The sample was dispersed in KBr were recorded on a BRUKER, TENSOR-27 FT-IR spectrometer. The sample was grounded with dried potassium bromide (KBr) powder and compressed into a disc, and was then subjected to analysis. Measurements were carried out in the wave number range of 400-4000 cm^-1.

2.4 Anti-larvicidal study of coumarins

24 hours bio-assay study was carried out with six coumarins synthesized such as 7-hydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 8-acetyl-7-hydroxy-4-methylcoumarin, resacetophenone, 8-acetyl-5-hydroxy-4-methylcoumarin and 4, 7-dimethylcoumarin against early second instar and fourth instar larvae of Culex quinquefasciatus [both laboratory colonized larvae and field collected larvae].

2.4.1 Materials and Methods for anti-larvicidal activity

Egg rafts of Culex quinquefasciatus was obtained from centre for research in Medical Entomology at Madurai [ICMR] and was kept in the basin containing water inside after two days. The larvae were fed with powdered mixture of dog biscuits and yeast in the ratio of 3:1. The early fourth star larvae [LCL] were used for the bio assay studies. Field larvae of culex species were collected from Melakottai near Gandhigram. They were reared in the laboratory for two days and bio assay studies were conducted using these larvae [FCL]. The food for the larvae was prepared by taking dog biscuits and yeast powder in the ratio of 3:1 and was dissolved in water. And two or three drops from this solution were poured in to the parval contains as feed.

2.4.2 Preparation of stock and control solution

Stock solution was prepared by 100 mgs of the coumarin compound was weighed, dissolved in distilled water and made up to the mark in the 100 ml standard flask. It was vigorously shaken and this forms stock solution of 1000 ppm. 500 ppm stock solution was prepared by taking 50 ml of the stock solution with 1 mg of coumarin with 1ml of water. Similarly the other concentrations such as 250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm and 15.625 ppm were prepared accordingly. A control solution was taken with only 50 ml of the water. Solutions of 500 ppm, 250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm and 15.625 ppm were prepared for all the six coumarins just before starting the bio assay experiments.

Ten number of earlier second instar and fourth instar larvae of Culex quinquefasciatus which were reared in the laboratory were introduced in the beaker containing test solution. The larvae were fed with one or two drops of larval food. The following parameter was observed for 24 hrs.

2.5 Anti-microbial study of coumarins

2.5.1 Materials and methods

The stock was prepared by weighing 1 mg of the compound was dissolved in hot ethanol and made up to 100 ml with distilled water. The three different cconcentrations such as 10 ppm, 1 ppm and 0.1 ppm were prepared as the test solutions. 10 ppm solution was prepared by 1 ml of stock solution for the bioassay test. Similarly the concentrations such as 1 ppm and 0.1 ppm were prepared. A variety of test organism bacteria capable of human pathogenity were chosen. The characteristic property of the selected bacteria is given in Table.2.

The colonies were maintained by the sub-culture method. The slant was prepared by using nutrient agar and with the help of inoculation loop, the strain was streaked. This slant was incubated for 24 hrs at 37°C. And the loop was touched to the isolated colonies of the pathogen growing on the slant and then inoculated into a take of culture broth. This was also incubated for 24 hrs at 37°C.

2.5.2 Anti-bacterial screening

For complete screening of anti-bacterial activity, the plate dilution technique is used. A test nutrient agar medium was used and the composition of nutrient agar medium is given in Table.3.

Table.3 Composition of nutrient agar medium

S.No	Composition	Quantity (gms)
1.	Beef	3
2.	Peptone	5
3.	Agar	20
4.	Distilled water	1 litre

2.5.3 Plant dilution technique

Plant dilution method was used to measure the minimum inhibitory concentration (MIC). In this method, the solution of the compound (in different concentrations of 10 ppm, 1 ppm and 0.1 ppm) was mixed with the nutrient agar in petriplate. One ml of the compound was used in the each plate to which the nutrient agar was poured (pour plate method) and mixed. A microorganism was streaked onto the plate. After 24 hrs of incubation at 37°C, the change in growth pattern, if any was noted. A control plate was done by a pouring only nutrient agar.

3 RESULTS AND DISCUSSION:

3.1 Mechanism of the Pechmann Condensation for coumarin

The pechmann condensation reaction for the synthesis of simple coumarin is conducted with a strong Bronsted acid such as methane sulfonic acid or a Lewis acid such as AlCl₃. The acid catalyses transesterification as well as keto-enol tautomerisation ^[13-14]:

A Michael Addition leads to the formation of the coumarin skeleton. This addition is followed by rearomatisation:

Subsequent acid-induced elimination of water gives the product:

Table.4 Spectral values of coumarins

S.No	Coumarins	FT-IR values (cm^-1)
1.	7-hydroxy-4-methylcoumarin	3042, 2987, 2970, 2951, 2938, 2919, 2810, 2756, 2605, 2343, 2310, 1612, 1457, 1262, 1067, 839, 691, 525
2.	7-acetoxy-4-methylcoumarin	3491, 3056, 2935, 1744, 1616, 1373, 1265, 1227, 1152, 1131, 1070, 1019, 985, 982
3.	8-acetyl-7-hydroxy-4-methylcoumarin	3646, 3614, 3586, 3547, 3423, 3361, 3216, 1614, 1453, 1370, 1274, 1159, 1136, 1073
4.	Resacetophenone	3193, 3152, 3047, 2928, 2363, 1712, 1638, 1612, 1560, 1473, 1375
5.	8-acetyl-5-hydroxy-4-methylcoumarin	3750, 3504, 3173, 1678, 1603, 1453, 1393, 1276, 1213, 1159, 1134, 1068, 982, 846, 806, 806, 749, 584, 478
6.	4, 7-dimethylcoumarin	3440, 3416, 3394, 3345, 3298, 3184, 3006, 2964, 2859, 1715, 1379, 1250, 1197, 933, 854, 824, 597, 573, 523

3.2 FT-IR Analysis of coumarins

Table.4 shows the spectral values of the synthesized simple coumarins. In 7-acetoxy-4-methylcoumarin the C-H stretching vibrations of the benzene derivatives generally appear in the region of 3000-3100 cm^-1. In this region, the bands are not affected appreciably by the nature of the substituents. The C-H vibration have been found at 3056, 2935 cm^-1 ^[15]. The bands between 1350 and 1650 cm^-1 in the aromatic and hetero aromatic compounds are assigned to C-C stretching vibrations. The presence of carbonyl group in the molecule often given rise to the appearance of the medium intensity band in the single bond region 1373-1200 cm^-1. The IR spectra of 7-hydroxy-4-methylcoumarin showed distinctly strong absorptions at 1265 and 1070 cm^-1 for C-O and at 1616 cm^-1 for C=O. The absorptions are within the range of comparison spectra B₅ at 1070, 1616, and 3056 cm^-1 respectively ^[16]. In 8-acetyl-7-hydroxy-4-methylcoumarin shows the C-C stretching vibrations generally appear at 1614 cm^-1. The C-O-C stretching vibration is found at 1274 cm^-1 ^[17]. In resacetophenone the phenyl vibrations appears at 1473 cm^-1 and 1375 cm^-1.In 8-acetyl-5-hydroxy-4-methylcoumarin C=O- stretching vibration appears at 1712cm^-1, O-H at 3154 cm^-1 and C-H at 2928 cm^-1 ^[18]. FT-IR spectrum of 4,7-dimethyl coumarin shows the band at 1612 cm⁻¹ observed due to C = C stretching ^[19]. Table.4 shows the spectral values of the coumarins is listed below.

3.3 Effect of 7-hydroxy-4-methylcoumarin on second Instar and fourth Instar larvae

In 24 hours, for test solutions of 40 ppm, 20 ppm, 10 ppm, 5 ppm and 1ppm percentage of mortality of Laboratory Colonies Larvae (LCL) were 100%, 92%, 63%, 26% and 4% respectively on second instar. For test solutions of 500 ppm, 125 ppm, 62.5 ppm, 31.25 ppm and 15.625 ppm the percentage mortality of Field Colonies Larvae (FCL) were 100%, 60%, 39%, 28%, 20% and 17% respectively. The LC 50 and LC 90 values were 7.22 and 22.00 for LCL. The LC 50 and LC 90 values were 141.32 and 1518.4 for FCL respectively. For fourth instar larvae, the for test solutions of 40 ppm, 20 ppm, 10 ppm and 5 ppm, percentage of mortality of LCL were 100%, 92%, 51% and 15% respectively. The percentage mortality of FCL were 97%, 51%, 41%, 19%, 11% and 1% respectively for the test solutions of 500 ppm, 125 ppm, 62.5 ppm, 31.25 ppm and 15.625 ppm. For LCL, the LC 50 and LC 90 values were 9.38 and 19.48 and 157.56 and 627.03 for FCL respectively. All other synthesized coumarins, other than 7-hydroxy-4-methylcoumarin did not show any anti-larvicidal effect and the results were not effective. Therefore the best results were observed for 7-hydroxy-4-methylcoumarin. The larvae of culex quinquefasciatus was used in the present study because of their availability and ability to multiply under laboratory conditions. In all the other remaining five coumarins, the percentage of mortality rates was gradually reduced at lower concentrations. Figure.1 shows the Escherchia Coli Entero bactericeae at A-1 ppm, B- 10 ppm and C- Control.

Table.5. Effect of the 7-hydroxy-4-methylcoumarin against larvae of culex quinquefasciatus (II instar) (24 hours observation)

S.No

Larvae tested

Concentration

% of Larval mortality

LOG

LC 50

[LOG LC 90]

LC 50

LC 90

Fiducidal limit for LC [50]

LC [90]

Regression equation

Chi square

LCL

40.0000

20.0000

10.0000

5.0000

1.0000

100

0.85

1.34

7.22

22.00

UL : 11.53

LL: 4.52

UL: 48.85

LL : 9.90

Y=2.72+2.65x

13.08

FCL

500.0000

250.0000

125.0000

62.5000

31.2500

15.0000

100

2.15

3.18

141.32

1518.41

UL : 660.23

LL: 30.25

UL: 107507.2

LL : 21.44

Y=2.32+1.24x

67.74

Table.6. Effect of the 7-hydroxy-4-methylcoumarin against larvae of culex quinquefasciatus (IV instar) (24 hours observation)

S.No

Larvae tested

Concentration

% of Larval mortality

LOG

LC 50

[LOG LC 90]

LC 50

LC 90

Fiducidal limit for LC [50]

LC [90]

Regression equation

Chi square

LCL

40.0000

20.0000

10.0000

5.0000

100

0.9726

1.54

9.39

19.48

UL : 10.37

LL: 8.50

UL: 23.12

LL : 16.41

Y=1.06+4.04x

1.29

Not signifi-cant

FCL

500.0000

250.0000

125.0000

62.5000

31.2500

15.0000

2.19

2.79

157.56

627.03

UL : 247.01

LL: 90.60

UL: 1867.44

LL : 210.54

Y=0.30+2.13x

29.76

Highly signifi-cant

Result of the anti-microbial activity:

As indicated in the methodology the 24 hours bio assay studies were carried out using plate dilution technique. In this technique the bio assay study of the few prepared coumarin compounds after the 24 hrs are given in Table.7.

Table.7. The effect of coumarins against certain pathogenic bacteria (Plate dilution technique) (24 hours)

S.No	Compounds tested	E.Coli
1.	Coumarin III	Susceptible
2.	Coumarin IV	Resistance
3.	Coumarin V	Resistance
4.	Coumarin VII	Resistance
5.	Coumarin IX	Resistance
6.	Coumarin XI	Resistance

In Table.7 susceptible defined as the growth of the organism is inhibited and resistance means the growth of the organism is not inhibited. Growth of the test bacteria in the petriplates was graded as high growth, medium growth, less growth and no growth in comparison with the control. In the control plate, only agar solution was taken and the agar solution had no effect on the bacteria. It was considered as “No Growth”. The effect of 7-hydroxy-4-methylcoumarin against the one pathogenic bacteria i.e., Escherchia Coli Entero bactericeae, showed susceptibility for this organism. This coumarin was to be more susceptible than the remaining five coumarins. Minimal Inhibitory Concentration (MIC) in ppm has been defined as the lowest concentration of drug that will prevent the growth of particular micro-organism. As indicated in above, E-Coli was susceptible at a concentration greater than or equal to 0.1 ppm of 7-hydroxy-4-methylcoumarin. Many substances show anti-microbial property, but only a few of them form potential chemotherapeutic drugs. In the present study, it was found that few of the coumarins selected for study are having anti-microbial activity with difference in their magnitude.

4 CONCLUSIONS:

The six coumarins such as 7-hydroxy-4-methylcoumarin,7-acetoxy-4-methylcoumarin, 8-acetyl-7-hydroxy-4- methylcoumarin, resacetophenone, 8-acetyl-5-hydroxy-4-methylcoumarin and 4, 7-dimethylcoumarin were synthesized by pechmann method by the reaction of ethyleacetoacetate with phenols. The effect of six synthesized coumarins on their anti-larvicidal properties against second and fourth instar larvae of culex quinquefasciatus was investigated. Also the effect of coumarins on their anti-microbial activity was studied. Out of the six coumarins for which anti-larvicidal effects were studied, only 7-hydroxy-4-methylcoumarin was found to show significant effect. Similarly 7-hydroxy-4-methylcoumarin was found to inhibit the growth of the organism Escherchia Coli. Therefore out of six synthesized coumarins 7-hydroxy-4-methylcoumarin showed high anti-larvicidal and anti-microbial activity.

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Received on 18.03.2016 Modified on 18.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 423-429

DOI: 10.5958/0974-360X.2016.00078.0

Selected bacteria/family	Optimum temperature of growth	Morphology	Disease caused
Escherchia coli Entero bactericeae	37° C	Gram-negative rods motile, non-sporing organism measuring 1-3 arranged singly or pairs.	Acute gastroenteritis infection of the urinary track wound infection, pycogenic infection and septicaemia.