SEMICARBAZONE: A VERSATILE THERAPEUTIC ANALOGUE AND IT`S PHARMACOLOGICAL EVALUATION

ABOUT AUTHORS:
Tripti Verma*, Chandra Shekhar Sharma, HemendraPratap Singh
Bhupal Nobles’ College of Pharmacy,
Udaipur, Rajasthan, India
triptiphsoni22@gmail.com

ABSTRACT:
Semicarbazones are compounds which are synthesized by a condensation reaction between a ketone or aldehyde and semicarbazide. Semicarbazone have biological activity against many of the most common species of bacteria. The biological properties of semicarbazonesare often related to metal ion coordination. The literature survey revealed that semicarbazones had been emerged as a compound with broad range of activities including anti-fungal, anti-bacterial, anticonvulsant, anticancer, antimicrobial activity and anti-inflammatory activity. Semicarbazone also used as ligand as the cytotoxic agent, spectrophotometric agents as well for the analysis of metal ions and in qualitative organic analysis of carbonyl compounds. In the present study we have focused on the Pharmacological evaluation of semicarbazone analogues. As per activity there are different Pharmacological evaluation methods of synthesized compound.

REFERENCE ID: PHARMATUTOR-ART-1934

INTRODUCTION:
A semicarbazone is a derivative of imines formed by a condensation reaction between a ketone or aldehyde and semicarbazide. They are classified as imine derivatives because they are formed from the reaction of an aldehyde or ketone with the terminal -NH2 group of semicarbazide, which behaves very similarly to primary amines.The semicarbazide which are the raw material of semicarbazone have been known to have biological activity against many of the most common species of bacteria (Dorgan et al., 1999)1.Semicarbazone, themselves are of much intrest due to a wide spectrum of anti-fungal and anti-bacterial activities (Singhal and Paul 2011a)2. Recently some researcher had reviewed of bioactivity of semicarbazones and they have exhibited anti-convulsant (Pandeyaet al., 2000)3, anti-tubercular (Sriramet al., 2004)4, anti-oxidant (Singhal and Paul 2011b)5, anti-microbial, analgesic, anti-pyretic (Singhal and Paul 2011c)6 and anti-inflammatory activity (Singh et al., 2010)7.

Semicarbazone:
In organic chemistry, semicarbazone is a derivative of semicarbazide which contains an additional ketone functional group.

Figure 1: General Structure of semicarbazone analogues

Dimmocket al., reported an extensive series of semicarbazone⁸. The lead compound amongthe (aryloxyl) arylsemicarbazones was 4-(4-fluorophenoxy) benzaldehydesemicarbazone.Preclinical evaluations have been completed and an (IND) has been filed. The compound is apotent sodium channel blocker (Na+) and it is planned to be developed for the treatment ofneuropathic pain. Phase I Clinical trials are scheduled in the near future.

The semicarbazone is formed when ammonia related a compound (nucleophiles) such as semicarbazide is added to the carbonyl group (=CO), they form imine like derivatives (Fig.2).

Figure 2: Synthesis of semicarbazone analogues

The conversion of aldehydes and ketones into imine like derivatives is an exothermic and pH dependent reaction.

General method for the synthesis of semicarbazone analogues
The general method for the synthesis of semicarbazone analogues is presented in Figure 3.

Figure 3: General method for the synthesis of semicarbazone analogues

Applications of semicarbazones and their complexes:
The metal complexes of semicarbazones play an essential role in agriculture, pharmaceutical and industrial chemistry and they are used as catalysts, in various biological systems, polymers and dyes, besides some uses antifertility and enzymatic agents. The biological properties of semicarbazonesare often related to metal ion coordination. Firstly, lipophilicity, which controls the rate of entry in to the cell, is modified by coordination⁸. Also, the metal complex can be more active than the free ligand. The mechanism of action can involve binding to a metal in vivo or the metal complex may be a vehicle for activation of the ligand as the cytotoxic agent. Moreover, coordination may lead to significant reduction of drug-resistance⁹. They are also used as spectrophotometric agents as well for the analysis of metal ions¹⁰ and are frequently used in the qualitative organic analysis of carbonyl compounds¹¹.

Recently, thiosemicarbazones have been synthesized and screened against the three parasitic cysteine proteases cruzain, falcipain-2, and rhodesain and against their respective parasite sources, Trypanosomacruzi, Plasmodiumfalciparium, and Trypanosoma brucei¹². The results obtained suggested that thiosemicarbazones represent validated leads that kill several species of protozoan parasites through the inhibition of cysteine proteases as well as through action against other targets. Furthermore, semicarbazones, which can also be regarded as urea derivatives, have gained considerable importance¹³in recent years in the design of enzyme inhibitors¹⁴, as replacement for the amide (–CO–NH–) bond in peptidomimetics¹⁵.

Anti-Fungal and anti-bacterial activity:
Micrbial resistance towards drug creates a very serious problem since last 3 decades because of this development of resistance many drug are now useless which were very effective (Cohen 1992; Cunha 1998)^16,17. Moreover, the toxic effect produced by these anti-biotic is also reducing their significance. So, for the need for new anti-microbial is always being there. Anti-fungal screening of synthesised chalconesemicarbazone derivative was conducted using a filter paper disc method.

The synthesised compound were screened for their anti-fungal activity against four fungi, Candida albicans, Aspergillus niger, Asperillusorzane, Penicilliumcitrinum at 100 ug ml^-1involving disc diffusion method with saburoud dextrose agar (Hi-media).

The synthesized compound were screened for their anti-bacterial activity against two gram positive (Bacillus subtilis, Micrococusluteus) and two gram negative (Klebsiellaaerogenes, Salmonella paratyphi) bacteria at 100 ug/ml involving disc diffusion method with Mueller- Hinter agar media (Hi-media)^18,19. Anti-bacterial activity determined by measuring zone inhibition of in millimetre around each of the disc and compared with standard Ciprofloxacin (100 ug/ml). Anti-bacterial activity was classified as Standard (>27 mm) Highly active (21-27 mm), moderately active (15-21 mm), least active (12-15 mm) and less than 12 mm was taken as inactive²⁰.

Anti-microbial activity:
Ahsanet al., reported the antimicrobial activity of a series of sixteen N1-(3-chloro-4-fluorophenyl)-N4-substituted semicarbazone derivatives were synthesized and subjected to computational pharmacokinetic studies to predict molecular properties. Among them the compound 2-(4-hydroxybenzylidene)-N-(3-chloro-4-fluorolphenyl)hydrazinecarboxamide (a) was found to be the most active compound showed good antibacterial inhibition while the compound 2-(4-methoxybenzylidene)-N-(3-chloro-4-fluorolphenyl) hydrazinecarboxamide(b) was moderately active against fungal strains. They have noticed the compounds bearing OH and NO₂ group at the phenyl ring at position 4 exhibited good antibacterial activity while compound bearing OCH₃ at the phenyl ring at position 4 exhibited moderate antifungal activity²¹.

During the course of Sriramet al. work on the synthesis and screening of new drugs fortuberculosis, they have identified N1-(4-acetamido phenyl)-N4-(2-nitrobenzylidene) semicarbazone, which inhibited in vitro M. tuberculosis H₃₇Rv; 100% inhibition at 1.56

/ml. This paper was first of its kind in which aryl semicarbazones are reported to possessantimycobacterials potency greaterthanp-aminosalicylic acid, ethionamide, ethambutol,ciprofloxacin and kanamycin.

The thioureido derivatives of 4-aminoacetophenone aryl semicarbazone have been prepared by Mishra et al. These derivatives have been characterized on the basis of different physicochemical evidences. The anti-HIV-1 (HTLV-IIIB) and HIV-2 (ROD) activity and cytotoxicity of the compounds were tested. The compound (a) and (b) showed maximum protection among the series⁴.

Several novel semicarbazone derivatives were prepared by Cerecettoet al. from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde, and tested in vitro as potential antitrypanosomal agents. The compounds were prepared in good to excellent yields in 2-3 steps from readily available starting materials. Some derivatives were found to be active against T.cruzi with an activity similar to that of niturtimox²².

Several novel semicarbazone derivatives were prepared from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde and semicarbazides bearing a spermidine mimetic moiety by Cerecetto et al. These compounds were tested in vitro as potential antitrypanosomal agents and some of them, together with the parent compounds, 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives, were also evaluated in vivo using infected mice. Two of the compounds (a-b) displayed the highest in vivo activity. A correlation was found between lipophilic hydrophilic properties and trypanocidal activity, high RM values being associated with low in vivo effects²³.

To investigate the relationship between antimicrobial activities and the molecular structures of nickel (II) complexes with thiosemicarbazone and semicarbazone ligands, nickel (II) complexes with ligands Hmtsc(a), Hatsc(b), and H2 dmtsc(c), were prepared and characterized by Kasuaga et al. Their antimicrobial activities were evaluated by the MIC against four bacteria (B.subtilis, S. aureus, E. coli and P. aeruginosa), two yeasts (C. albicans and S. cerevisiae) and two molds (A. niger and P. citrinum). The 4-coordinate, diamagnetic nickel (II) complexes showed antimicrobial activities which were different from those of free ligands or the starting nickel (II) compounds. [Ni(mtsc)(Oac)] showed selective and effective antimicrobial activites against two gram positive bacteria (B. subtili sand S. aureus) and modest activities against a yeast (S. cerevisiae), [Ni(mtsc)Cl] exhibited moderate activities against a gram positive bacterium (S. aureus) and [Ni(atsc)(Oac)] showed modest activities against gram positive bacteria (B. subtilis and S. aureus)²⁴.

Pharmacological Evaluation:

Antifungal activity
The fungicidal activity of all the compounds was studied at 1000 ppm concentration in vitro. Plant pathogenic organisms used were C. albicansand A. clavatus. The antifungal activity of all the compounds was measured on each of these plant pathogenic strains on a potato dextrose agar (PDA) medium such a PDA medium contained potato 200 gm., dextrose 20 gm., agar 20 gm., and water 1 liter. Five days old cultures were employed. The compounds to be tested were suspended (1000 ppm) in a PDA medium and autoclaved at 120 ⁰C for 15 min and at 15 atm. pressure. These media were poured into sterile Petri plates and the organisms were inoculated after cooling the Petri plates²³. The percentage inhibition for fungi was calculated after five days using the formula given below,

100 (X - Y)
% of Inhibition = -------------
X

Where,
X = Area of colony in control plate.
Y = Area of colony in test plate

Anti-bacterial activity
The study has been conducted according to the method adopted by Cruickshank et al. Nutrient agar broth was melted in a water bath and cooked to 45 °C with gentle shaking to bring about uniform cooling. It was inoculated with 0.5-0.6 ml of 24 hour old culture especially and mixed well by gentle shaking before pouring on the sterilized Petri dish (25 ml each). The poured material was allowed to set (1.5 hour) and there after the “cups” was made bypunching into the agar surface with a sterile cork borer and soaping out the punched part of agar. Into this “cups” 0.1 ml of test solution (prepared by dissolving 100 ml of sample in 10 ml DMF) was added by sterile micropipette. The plates were noted. The antibacterial activities of all compounds are compared against Ampicilin as a standard drug²⁴.

Anticonvulsant activity:
Epilepsy, one of the most frequent neurological disorders,is a major public health issue, affecting about 4% of individuals over their lifetime²⁵. There is currently a need forimproved agents for the treatment of seizure disorders, since available drugs are effective in only 60–80% of epileptic patients²⁹.

We have recently been investigating various aryl-substituted semicarbazones as potential anticonvulsant agents (Pandeyaet al., 2000; 2004a; 2004b; 2005a; Thirumuruganetal.,2006)^3,27-30 and compound N1-(2,6-dimethylphenyl)-N4-(4-N,N-dimethyl aminobenzaldehyde) semicarbazones showed an ED₅₀(effective dose at 50% of the total population) of 18.33 mg/kg and TD₅₀(toxic dose at 50% of the total population) greater than 250 mg/kg, resulting in a high protection index of >13.64 when compared to phenytoin (Yogeeswariet al., 2005b). The rationale behind the development of semicarbazonesis their structural dissimilarity to exciting antiepileptic drugs, so it was hoped that such novel compounds would not have the side effects seen with many of the currently available medications (Kadaba, 1984)²⁶. Aryl semicarbazones have also been shown to possess excellent anticonvulsant activity in the maximal electroshock seizure (MES) in both mice and rats and also against clonic seizures induced by pentylenetetrazole (PTZ) in mice, being more activethan some conventional antiepileptic drugs, beside their low neurotoxicity. While fitting semicarbazones to the binding site hypothesis, it is likely that two-electron donor atoms in semicarbazones group and aryl ring align at the complimentary area on a macromolecular complex with the receptor in vivo (Yogeeswariet al., 2005b)³³. The analysis of the distance relationship showed that aryl semicarbazones fulfilled the essential demands of the pharmacophore when compared with the phenytoin, carbamazepine, denzinamide, and remacemide(Yogeeswari et al., 2005a; 2005b)^31,32. The presence of electron-rich atom/group attached at the paraposition of the aryl ring showed increased potency in the MES screen. Substitution in the aryl ring by halogens led to a number of semicarbazones with low ED₅₀ values in the rat oral MES screen accompanied by high protective index values (Dimmocket al., 1995)³⁴. Hence, in our earlier study, 4-bromophenyl group was considered important and led to compounds with better activity profile with very low neurotoxicity and no sedative-hypnotic activity (Pandeyaet al., 2000)³. Recently we reported on variously substituted aryl

semicarbazones with the order of activity being found to be 4-F>2-Br=3-Br=4-Cl>4-CH₃>4-Br>3-Cl>3- CH₃ with respect to the primary aryl group (Yogeeswariet al., 2006)³⁵. We also reported some 3-chloro-2-methylphenyl semicarbazones as potential anticonvulsant agents (Yogeeswariet al., 2004a)³⁰. In continuation of our work on disubstituted aryl semicarbazones,The synthesis and anticonvulsant evaluation of newer 4-bromo-3-methylphenyl semicarbazones for exploring the structure-activity relationships of aryl semicarbazones.

Recently, Unverferthet al.,³⁶ suggested a pharmacophore model for structurally different anticonvulsants containing aryl rings and electron donor and hydrogen bond donor/acceptor functions. During the last five years, semicarbazonesemerged as novel anticonvulsant entities in the laboratories of Dimmock³⁴ and Pandeya³. The structural requirementsin the semicarbazone series are: a lipophilic aryl ring, a distal aryl ring and a hydrogen-bonding domain (HBD). The lipophilic aryl ring with chloro, bromo or nitro groups has been found to be essential for anticonvulsant activity. The distal aryl ring is also implicated at the binding site. The HBD in semicarbazone series has been suggested by Dimmock to be the terminal –NHCONH₂. Pandeyaet al.³⁷ suggested this HBD to be adjacent to the lipophilic aryl ring, but it was not confirmed as some of the compounds with –CH₂– in place of –NH– were showing activity.

The synthesized a series of N-(4,6-substituted diphenylpyrimidin-2-yl) semicarbazones and tested for their anticonvulsant activity against the two seizure models, maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ). Most of the compounds displayed good anticonvulsant activity with lesser neurotoxicity. To assess the unwanted effects of the compounds on liver, estimation of enzymes and proteins was carried out. The most active compound of the series was N1-[4-(4-Chlorophenyl)-6-(3,4-dimethoxyphenyl)-pyrimidin-2-yl]- N4-(4-nitrobenzaldehyde) semicarbazone (2) devoid of any neurotoxicity³⁸.

Many new pharmacophores have been developed over the last twenty years. Functionalizedamino acids and alpha-amino amides are two classes of antiepileptic drugs that exhibit pronounced anticonvulsant activity.Lacosamide and safinamide are two recently launched antiepileptic drugs resulting from this approach. Enaminoneshave been extensively investigated as anticonvulsants.

Pharmacological Evaluation:
The maximal electro-shock (MES)-induced convulsions
The maximal electro-shock (MES)-induced convulsions in animals represent grand mal type of epilepsy. In MES-convulsion electroshock is applied through the corneal electrodes. Through optic stimulation cortical excitation is produced. The MES-convulsions are divided into five phases such as (a) tonic flexion, (b) tonic extensor, (c) clonic convulsions, (d) stupor and (e) recovery or death.

Requirements:Rats (150-200g), Drug

Equipment: Electro-convulsiometer, corneal electrode (apply 50 mA current for 0.2 sec), stop-watch.

Procedure:

Animal were weighed and numbered and divided into two groups each consisting of 4-5 mice. One group were used as control and the other for sample compound treatment.
The corneal electrodes were placed on the cornea of the animal and the prescribed current were applied. The readings of different stages of convulsions i.e. (a) tonic flexion, (b) tonic extensor phase, (c) clonic convulsions, (d) stupor and (e) recovery or death were noted. The time (sec) spend by the animal in each phase of the convulsions were noted. The whole procedure were repeated with other animals of control group also.
The sample compound were injected intraperitoneally to a group of 4-5 mice. After 30 min, the animals were subjected to electroconvulsions as described in step2.
The reduction in time or abolition of tonic extensor phase of MES- convulsions were noted³⁸.

PTZ -Induced Convulsions in Rats
Chemo-convulsions due to PTZ(Pentylenetetrazole)which produce clonic–type of convulsions resemble petit mal type of convulsions in man.Albino rats of 150-200 g body weight were divided into five groups of six animals each. The firstgroup, receiving saline orally, served as control whereas the second group received 4mg/kg of diazepam intraperitoneally (i.p); the third group received a sub protective dose of diazepam, i.e. 0.5 mg/kg i.p.; the fourth received 50 mg/kg of BM (p.o); and the fifth group received a sub protective dose of diazepam, i.e. 0.5 mg/kg and 50 mg/kg of BM. After an hour, all the animals were injected with 80 mg/kg pentylenetetrazolintraperitoneally and presence or absence of clonic convulsions was noted for each animal. The % latencies of clonic convulsions were noted, and numerically transformed to a seizure score (S) calculated from the formula: S = 1- (control Latency/ drug seizure Latency) In the case of control animal; S = 0, whereas for animals that did not experience seizures latencies of infinity, S =1. This numerical transformation enabled inclusion of all animals in the statistical analysis, irrespective of whether they had a seizure or not. The mortality in the 24h following PTZ, in the different pretreatment groups was also recorded³⁸.

Anticancer activity:
The anticancer activities of vanillinsemicarbazone³⁹against Ehrlich Ascites Carcinoma have been reported. EAC cells are experimental tumormodels used worldwide in cancer research. In 1907, Paul Ehrlich discovered this tumor in the mammary gland of a white mouse, and thetumor was named after him. The present form of EAC cells has been developed by Loewenthaland Jahn⁴⁰ from one of the several carcinoma lines⁴¹. Khanamet al.⁴² showed that acetone semicarbazone exhibits anticancer activities against EAC cells in Swiss albino mice. Other than Schiff bases, Schiff base complexes with transition metals have also been investigated for their anticancer activities against EAC cells in Swiss albino mice^43,44. In the present investigation, we selected benzophenonesemicarbazone (BSC) as the test compound and studied its anticancer activities against EAC cells in vivo. Cancer chemotherapy causes myelosuppression and anemia⁴⁵because of the reduction of both red blood cell (RBC) content and hemoglobin percentage. In support of this anticancer study, hematological parameters have also been studied accordingly.

Pharmacological Evaluation:
Hematological parameters in normal and tumorbearing mice
The effects of the test compound on hematological parameters were studied in both normal and tumor-bearing mice. For the tumor-bearing mice, the treatment was initiated after 24 h of tumor transplantation and continued for 10 days. The hematological parameters in the normal mice were studied using a similar method used for the tumor-bearing mice. Blood was drawn out from the tail vein of the mice from each group on days 5, 10, 15, and 25 for such studies⁴³.

Effects of the test compound on normal peritoneal cells
The effects of test compound on the normal peritoneal cells were determined by counting the total peritoneal cells and the number of macrophages. Four groups of mice (4 in each group) were used in the experiment. Groups 1 to 3 were treated with BSC at doses of 5, 15, and 25 mg/kg (i.p.), respectively, for three consecutive days. Group 4 was used as the untreated control. After 24 h of the last treatment, each animal was injected with 5 mL normal saline (0.98%) into the peritoneal cavity and was sacrificed. The intraperitoneallyexuded cells and the number of macrophages were countedwith 1% neutral red by using a hemocytometer⁴⁴.

Anti-oxidant activity:
Free radicals are an atom or molecule that bears an unpaired electron and is extremely reactive, capable of engaging in rapid change reaction that destabilize other molecules andgenerate many more free radicals. In plants and animals these free radicals are deactivated by antioxidants. These antioxidants act as an inhibitor of the process of oxidation, even at relatively small concentration and thus have diverse physiological role in the body. The body is constantly exposed to the negative and sometimes lethal effects of oxidants during normal physiological processes. The harmful free radicals such as hydroxyl, peroxyl and the superoxideanion are constantly being produced as a result of metabolicreactions in living systems. On a daily basis, up to 5% of inhaled oxygen may be converted to reactive oxygen species (ROS). These ROS have the ability to bind to cellular structures, and have been implicated in number of pathological processes such as aging, inflammation, reoxygenationof ischemic tissues, atherosclerosis, cancer and even Parkinson’s disease in men⁶⁰.Two processes, which produce free radicals in vivo, have been identified and named the Fenton reaction and the Haber-weiss reaction⁴⁶. Antioxidants play an important role in animal health.Antioxidants play an important role in animal health. Conventional antioxidants have been shown to improve animal performance during conditions characterized by increased tissue oxidant levels such as stress, injury and infections⁴⁷. The semicarbazone is an electron withdrawing group and exhibited antioxidant activity. Favourablesubstitution may increase their free radical scavenging effect⁴⁸.

Pharmacological Evaluation:

DPPH Free Radical Scavenging Assay
The antioxidant activity of the synthesized semicarbazonederivatives was evaluated using the DPPH free radical scavenging assay. 200μl of test sample solution (100μg/ml) was added to 4 ml of 100μM methanolic DPPH. After vortexing, the mixture was incubated for 20 minutes at room temperature and the absorbance at 517 nm was measured. Ascorbic acid (100μg/ml) was used as standard. A blank was prepared without adding standard or test compound. Lower the absorbance of the reaction mixture indicates higher free radical scavenging activity⁴⁸. The capability to scavenge the DPPH radical was calculated using the following equation,

A control - A test
DPPH scavenged (%)= ------------------ × 100
A control

Where,
A control is the absorbance of the control reaction and

A test is the absorbance in the presence of the test compounds. The antioxidant activity of the chalcone semicarbazones is expressed comparing withstandard ascorbic acid.

Anti-inflammatory activity:
Non-steroidal anti-inflammatory drugs (NSAID's) are widely used in the treatment of pain and inflammation. NSAID's reduce the pain and swelling associated with arthritis by blocking the metabolism of arachidonic acid (AA) through the enzyme cyclooxygenase (COX) and thereby the production of prostaglandins, e.g. PGE2, which sensitizes nociceptors at nerve fiber terminals^49,50. Additionally, the 5-lipoxygenase (5-LO) products such as leukotriene B₄ (LTB₄) also contributes to the hyperalgesia seen during inflammation by decreasing the mechanical and thermal thresholds of C fibers⁵¹. Leukotrienes, especially LTB4 together with prostaglandins are implicated in the acute ulceration induced by NSAID's⁵². For these reasons, compounds that achieve dual inhibition of the enzymes COX and 5-LO reduce side effects and improved the efficacy in the combat of pain in inflammatory diseases⁵³. Some previously synthesized hydrazone analogues were also described as a dual COX/5-LO inhibitor⁵⁴. Furthermore, there are several reports about the synthesis and pharmacological evaluation of new bioactive N-arylhydrazones acting at the AA cascade enzyme level⁵⁵. As a part of research program⁵⁶Shafieeet al also find analgesic and anti-inflammatory activity of 4-(2- phenoxyphenyl) semicarbazones⁵⁷. Chalcones belong to the flavonoid family from plant origin and some of them possess anti- inflammatory activity. Recently, several natural and synthetic chalcone derivatives werereported to inhibit inducible nitric oxide synthase (iNOS)-catalyzed NO production in cell cultures⁵⁸. Butein, a natural chalcone, has anti-inflammatory activity. One synthetic derivative of butein, 2',4',6'-tris(methoxymethoxy)chalcone (TMMC), has potent anti-inflammatory activity via an HO-1 (haeme-oxygenase) dependent pathway⁵⁹

From the literature it have also been concluded that the semicarbazone⁵⁷ and chalcone, both the moieties have good anti-inflammatory and analgesic activities. In the present studywe have used pharmacophore hybridization technique of drug design and designed a pharmacophoremodel ‘chalconesemicarbazone’, which is having hydrogen acceptor site, hydrogen donor site, lipophilic site etc, which may help in binding with receptors and plays an important role in pharmacological activities. Antiphlogistic agents are also called anti-inflammatory agents which minimize the vasodilation/vascular permeability/inflammation induced by phlogistic agents.

Pharmacological Evaluation:

Anti-inflammatory
Animals were divided into control, standard and different test groups comprising of six animals in each group. They were fasted overnight with free access to water before experiment. The anti-inflammatory activity was determined in vivousing the carrageenan-induced rat paw edema test. In all groups, acute inflammation was produced by sub-planter injection of 0.1ml of freshly prepared 1% suspension of carrageenan. in the right hind paw of the rats 1 h after ip. Administration of the compounds and paw volume was measured plethysmometrically at 0, 1, 2 and 3hr. The test compounds (30mg/kg) was administered i.p. in DMSO, standard group was treated with diclofenac (50mg/kg) i.p. 1 hrs before by the injection and control group received only DMSO. Anti-inflammatory activity was expressed as percent of inhibition of the edema when compared with the control group. Mean difference in paw volume was measured statically by student t test⁵⁵. Mean defference in paw volume was measured and percentage inhibition was calculated by using formula,

% inhibition of edema = (Vc-Vt / Vc) x 100,

Where,
Vt and Vc are the mean paw volume of test group and control group, respectively.

Analgesic activity

Tail immersion method
Adult wistar rats weighing 75-100 gm were used for tail immersion method. The animals were placed into individual restraining cages leaving the tail hanging out freely. The animals were allowed to adapt to the cages for 30 min. before testing. The lower 5cm portion of the tail was marked and this part of tail was immersed in the water bath thermostatically maintained at 55°C. The withdrawal time of the maximum cut off time immersion was 15sec. to avoid the injury of the tissue of the tail.

The animal were divided into 13 groups consisting of 3 animal in each group.

Grouping was done as follows.

Group 1- served as control (DMSO)
Group 2 –served as standard (Diclofenac Sodium)
Title – served as test compounds

The initial reading was taken immediately before administration of the test and standard drugs and than 1, 2 , 3 and 4 hrs after the administration .the tail flick latency difference or mean increase in latency after drug administration was used to indicate the analgesia produced by test and standard drugs.

CONCLUSION:
Semicarbazone moiety and its various derivatives studied frequently in the past time and foundpotent in various pharmacological activity.Semicarbazones have been developed as versatile therapeutic analogue. This article mainly focused on the various derivatives of semicarbazones and their Pharmacological Evaluation method. Thus by studying all the derivatives showing different activities like Anti-fungal, Anti-bacterial, Anti-microbial, Anti-Cancer, Anti-oxidant and Anti-inflammatory.Semicarbazone also used as ligand as the cytotoxic agent, spectrophotometric agents as well for the analysis of metal ions and in qualitative organic analysis of carbonyl compounds. Also main role in design of enzyme inhibitors and kill several species of protozoan parasites through the inhibition of cysteine proteases.We can say that semicarbazone analogues have been explored in past years and is still be used for future development of new drugs.

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