Friday , 23 August 2019

SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL SCREENING OF SOME NOVEL BENZOIN DERIVATIVES

About author:
P. Deepthi
Dept. of Pharmaceutical Chemistry
JNTUA – OTRI, Anantapur,India
E-mail: deepthi.pharma07@gmail.com

INTRODUCTION :A natural product is a chemical compound  or substance produced by a living organism – found in nature that usually has a pharmacological or biological activity for use in pharmaceutical drug discovery, drug design. A natural product can be considered as such even if it can be prepared by   total synthesis.These small molecules provide the source or inspiration for the majority of FDA-approved agents and continue to be one of the major sources of inspiration for drug discovery. In particular, these compounds are important in the treatment of life-threatening conditions. Natural products,since the very beginning,have served as atemplate for the development of many important classes of drugs.plants are remarkable in their ability to produce a vast array of diverse metabolites varying in structural complexity and biological activity.Plants continue to retain their historical significance  as an importance source of novel compounds, useful directly medicinally as ,as model compounds for synthetic or semi synthetic structure medications and optimization and as bio chemical  and pharmacological probes. Today, natural products and their derivatives stil lrepresent over 50% of all drugs  in clinical use ,with higher plant derived natural products representing 25% of the total. It has been reported that about a  quarter of all the prescription drugs from community pharmacies in the world still contain plant extracts or active principles of plant origin.
Natural products isolated from higher plants arid microorganisms have been providing novel, clinically active drugs. The key to the success of discovering naturally occurring therapeutic agents rests on bioassay-guided fractionation and purification procedures. Screening of both synthetic organic compounds and extracts of natural products has had an impressive history of identifying active agents. For example, there are about 50 commercially available anticancer drugs (excluding endocrines) which have been approved to date by the USFDA, and significantly, the drugs based on natural products represent almost 1/3 of these total approved agents. A most recent addition is taxol (approved in 1992, and the semi-synthetic in 1995), a natural product derived from the Pacific yew tree Taxus brevifolia, which is used for the treatment of ovarian and breast cancer.
DESCRIPTION :
Natural products may be extracted from  tissues    of terrestrial plants, marine organisms or   microorganisms fermentation   broths. A crude (untreated) extract from any one of these sources typically contains novel, structurally diverse chemical compounds, which the natural environment is a rich source of. Chemical diversity in nature is based on biological and geographical diversity, so researchers travel around the world obtaining samples to analyze and evaluate in bio-assays.This effort to search for natural products is known as bio-prospecting. Pharmacognosy provides the tools to identify, select and process natural products destined for medicinal use. Usually, the natural product compound has some form of biological activity and that compound is known as the  active principle  such a structure can act as a lead compound Many of today’s medicines are obtained directly from a natural source.
Isolation and purification :
The lead compound (or active principle) is present in a mixture of other compounds from a natural source, it has to be isolated and purified. The ease with which the active principle can be isolated and purified depends much on the structure, stability, and quantity of the compound. For example,  Alexander Fleming  recognized the antibiotic qualities of  penicillin   and its remarkable non-toxic nature to humans, but he disregarded it as a clinically useful drug because he was unable to purify it. He could isolate it in aqueous solution, but whenever he tried to remove the water, the drug was destroyed. It was not until the development of new experimental procedures such as  freeze drying and  chromatography  that the successful isolation and   purification  of penicillin and other natural products became feasible.
Synthetic drugs :
Synthetic drugs are artificially produced substances for the illicit market which are almost wholly manufactured from chemical compounds in illicit laboratories (amphetamine, benzodiazepines). scientists alter the molecular structure of the natural molecule until they had made a drug that worked like the natural drug, but without the side effects. Aspirin was one of the first nature-inspired synthetic drugs. This class of synthetic drugs is very important because a huge number of drugs that are like morphine in action have been synthesised in attempts to duplicate its’ usefulness and to find an ideal potent analgesic free of habit forming properties Synthetic opioides are totally synthetically produced drugs that have similar effects and the same basic structural elements as morphine (examples are methadone, fentanyl, pethidin).
The synthetic drug trade is multi – dimensional in terms of precursor chemical availability, manufacturing equipment (both sophisticated and improvised), expertise and movement of the finished product. Since clandestine drug laboratories can be located in any part of the world, the trafficking of synthetic drugs can constitute either a domestic or international problem. No single organized crime group or region dominates the synthetic drug trade , as evidenced by the increase in seizures of both laboratories and drugs in every part of the world.Screening of both synthetic organic compounds and extracts of natural products has had an impressive history of identifying active agents. For example, there are about 50 commercially available anticancer drugs (excluding endocrines) which have been approved to date by the USFDA, and significantly, the drugs based on natural products represent almost 1/3 of these total approved agents. A most recent addition is taxol (approved in 1992, and the semi-synthetic in 1995), a natural product derived from the Pacific yew tree Taxus brevifolia, which is used for the treatment of ovarian and breast cancer.
Benzoin is an    organic compound    consisting of an ethylene bridge flanked by phenyl  groups and with hydroxyl  and ketone functional groups.It appears as off-white crystals, with a light  camphor  odor. Benzoin is synthesized from  benzaldehyde in the benzoin condensation. Benzoin is not a constituent of    benzoin resin  obtained from the  benzoin tree  (Styrax). The main component in these natural products is benzoic acid.
MATERIALS & METHODS :
Compound-I
BENZOIN(S.d-finechem.Ltd,Mumbai)Hydrazine sulphate  (IDPC-Hyderabad).Sodium acetate  (S.D-fine chem. Ltd, Mumbai)ethanol(Changshu yang Yuan chemical ltd).
Step-I: Dissolve 1gm of hydrazine and 1.5gm of crystallized sodium acetate in 8-10ml of water and added 0.5gm of the benzoin and shaken and added alcohol until the clear solution was obtained. Shaken the mixture for a few minutes and allowed to stand for some time. Hydrazine crystallized from the cold solution on standing. Filtered off the crystals and washed with cold water and recrystalised from ethanol diluted with water.
Step-II: Suspend about  0.5gm  of  the obtained compound in 10ml of 5%NaoH solution in a small conical flask.Add dropwise 1ml of benzoylchloride,the flask should be well stoppered and shaken frequently .The reaction mixture should be kept cool through out the addition.At last ,shake the reaction mixture frequently for 5-10minutes and filter off the product.Wash the derivative with wate and recrystallise from ethanol.
Compound- II
BENZOIN(S.d-fine chem. Ltd, Mumbai) Isoniazid was presented as a gift sample by Director, JNTU, OTRI, Ananthapur.Sodium acetate (S.D-fine chem. Ltd, Mumbai). ethanol(Changshu yang Yuan chemical ltd).
Step-I: Dissolve 1gm of Isoniazid and 1.5gm of crystallized sodium acetate in 8-10ml of water and added 0.5gm of the benzoin and shaken and added alcohol until the clear solution was obtained. Shaken the mixture for a few minutes and allowed to stand for some time. Isoniazid crystallized from the cold solution on standing. Filtered off the crystals and washed with cold water and recrystalised from ethanol diluted with water.
Step-II: Suspend about  0.5gm  of  the obtained compound in 10ml of 5%NaoH solution in a small conical flask.Add dropwise 1ml of benzoylchloride,the flask should be well stoppered and shaken frequently .The reaction mixture should be kept cool through out the addition.At last ,shake the reaction mixture frequently for 5-10minutes and filter off the product.Wash the derivative with wate and recrystallise from ethanol.
Compound -III
BENZOIN(S.d-fine chem. Ltd, Mumbai)Semicarbazone ( Sisco research laboratoratories, Mumbai).Sodium acetate (S.D-fine chem. Ltd, Mumbai).ethanol(Changshu yang Yuan chemical ltd).
Step-I: Dissolve 1gm of  Semicarbazone and 1.5gm of crystallized sodium acetate in 8-10ml of water and added 0.5gm of the benzoin and shaken and added alcohol until the clear solution was obtained. Shaken the mixture for a few minutes and allowed to stand for some time. Semicarbazone crystallized from the cold solution on standing. Filtered off the crystals and washed with cold water and recrystalised from ethanol diluted with water.
Step-II: Suspend about  0.5gm  of  the obtained compound in 10ml of 5%NaoH solution in a small conical flask.Add dropwise 1ml of benzoylchloride,the flask should be well stoppered and shaken frequently .The reaction mixture should be kept cool through out the addition.At last ,shake the reaction mixture frequently for 5-10minutes and filter off the product.Wash the derivative with wate and recrystallise from ethanol.
Compound- IV
BENZOIN (S.d-fine chem. Ltd, Mumbai)2, 4-DNP (S.d-fine chem. Ltd, Mumbai), Sodium acetate (s.d-fine chem. Ltd,Mumbai) ethanol(Changshu yang Yuan chemical ltd).
Step-I: Dissolve 1gm of 2,4-dinitrophenyl hydrazine and 1.5gm of crystallized sodium acetate in 8-10ml of water and added 0.5gm of the benzoin and shaken and added alcohol until the clear solution was obtained. Shaken the mixture for a few minutes and allowed to stand for some time. 2,4-dinitrophenyl hydrazine crystallized from the cold solution on standing. Filtered off the crystals and washed with cold water and recrystalised from ethanol diluted with water.
Step-II: Suspend about  0.5gm  of  the obtained compound in 10ml of 5%NaoH solution in a small conical flask. Add dropwise 1ml of benzoylchloride, the flask should be well stoppered and shaken frequently .The reaction mixture should be kept cool through out the addition. At last ,shake the reaction mixture frequently for 5-10minutes and filter off the product.Wash the derivative with wate and recrystallise from ethanol.
ANTIMICROBIAL ACTIVITY :
Compounds Minimum inhibitory concentration (MIC) was evaluated by the disc diffusion test using standard inoculums of 10-5 CFU mL-1 .
Sample preparations
Compounds were dissolved and diluted with water and prepared serial dilutions. Compound serial dilutions were performed out of Initial concentrations 1µg/ml greater than once for agar disc diffusion method (i.e.1µg/ml for pure compounds and then prepared serial dilutions 500 µg/ml, 250 µg/ml, 100 µg/ml, 50 µg/ml and 10 µg/ml).
Agar disc diffusion method
Disc diffusion test was carried out according to standard procedure:
4 mm filter paper discs (What man, no. 3) were impregnated with 20 mL of each of the different dilutions. The discs were allowed to remain at room temperature until complete diluents evaporation and kept under refrigeration until ready to be used. Discs loaded with compounds were placed onto the surface of the agar. Streptomycin (25µg/ml)  discs were prepared and paper discs impregnated with 20 mL of diluents used to dilute compounds were used as control.
Test bacteria
The antimicrobial activity of compounds was assessed against five bacteria species: Staphylococcus aureus, pseudomonas varicose, pseudomonas auraginosa, bacillus substalis and Escherichia coli. Overnight cultures were kept for 24 h at 36ºC ± 1ºC and the purity of cultures was checked after 8 h of incubation. After 24 h of incubation, bacterial suspension (inoculums) was diluted with sterile physiological solution, for the diffusion.
RESULT & DISCUSSION :
Compound -I
The compound 1 was synthesized and its purity was determined by using TLC,themelting point of compound 1 was found to be 122oC,the compound 1 exhibited RF of about 0.78using solvent system,Water: ethanol in 3:2 ratio ,Percentage yield was found to be 36.5% .
IR spectrum showed peak of N-H at 3414.03cm-1,Aromatic C-H stretching at 3062.87cm-1 ,C-H stretching at 2934.39cm-1 ,C=O at 1678.56cm-1,Aromatic at 754.17-602.55cm-1.The UV spectrum showed λmax  at 245.5nm.
Compound-II
The compound 2 was synthesized and its purity was determined by using TLC,the melting point of compound 2 was found to be 138 oC,the compound 2 exhibited RF of about 0.26 using solvent system,Ether: Water (3:2).Percentage yield was found to be 23.2%.
IR spectrum showed peak of N-H at 3411.89cm-1,Aromatic C-H stretching at 2929.83cm-1 ,C-H stretching at 2363.57cm-1 ,C=O at 1676.75cm-1,Aromatic at 752.50-608.11cm-1.The UV spectrum showed λmax  at 251.5nm.
Compound-III
The compound 3 was synthesized and its purity was determined by using TLC,the melting point of compound 3 was found to be 820 C,the compound 3 exhibited RF of about 0.46 using solvent system n-hexane:ether (4:1 ).Percentage yield was found to be 56.5%.
IR spectrum showed peak of N-H at 3413.91cm-1,Aromatic C-H stretching at 2835.68cm-1 ,C-H stretching at 2364.9cm-1 ,C=O at 1684.12cmAromatic at 705.52-666.18cm-1.The UV spectrum showed λmax  at 230nm.
Compound-IV
The compound 4 was synthesized and its purity was determined by using TLC,the melting point of compound 4 was found to be 1100 c,the compound 4 exhibited RF of about 0.8 using solvent system. water: ethanol( 3:3).Percentage yield was found to be 61.5%.
IR spectrum showed peak of N-H at 3068.87cm-1,C-H stretching at 2835.36cm-1  ,C=O at 1686.31cm-1,N-O stetching at 1422.56cm-1Aromatic N-H stretching at 706.47-662.48cm-1.The UV spectrum showed λmax  at 275.5nm.
Compound -I has shown very mild  anti bacterial activity against both gram positive and gram negative bacterias.Among them it was quite sensitive towards Escherichia coli.
Compound-II has no significant anti bacterial activity.
Compound-III was found to be effective against Escherichia coli and has inhibited the growth of pseudomonas aureus and bacillus subtilis at its higher concentration.
Compound-IV has shown very mild anti bacterial activity against both gram positive and gram negative bacterias.
CONCLUSION :
The above study concludes that substitution of bulkier groups to the carbonyl carbon and replacement of the hydroxyl(H) hydrogen by alkylation demolishes the anti bacterial activity of benzoin.Thus, this study can be used in the future as a precaution during the design of further   derivatives of benzoin.

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