25

CHAPTER-III Synthesis and Anticancer Activity of Noval Substituted Pyrazole Derivatives

26

INTRODUCTION PYRAZOLE: Pyrazole is the name given by LUDWIG KNORR to this class of compounds in 1883. The simple doubly unsaturated

compound containing two nitrogen and three carbon atoms in the ring, with the nitrogen atoms neighboring, is known as pyrazole. The reduction products, named as are other rings of five atoms, are pyrazoline and pyrazolidine. Several pyrazoline substitution

products are used in medicine. Many of these are derivatives of 5pyrazolone. Some can be related to 3,5-pyrazolidinedione. For a long time no pyrazole derivative had been found in nature, but in 1959 -(1-pyrazolyl) alanine was isolated from the seeds of water melons (Citurllus lanatus) (L. Fowden). Chemistry: Pyrazole is a colorless solid, m.p. 70C. This

high value (compared with 1-alkyl or aryl substituted pyrazoles) is due to intermolecular hydrogen bonding which results in a dimmer. Pyrazole is a tautomeric substance; the existence of

tautomerism cannot be demonstrated in pyrazole itself, but it can be inferred by the consideration of pyrazole derivatives.

27 Pyrazole exhibits aromatic properties, e.g., it is readily halogenated, nitrated and sulphonated; the group enters at position 4. The following resonating structures are possible for pyrazole. Pyrazole is feebly basic and forms salts with inorganic acids; the imino hydrogen may be replaced by an acyl group. Pyrazole is very resistant to oxidizing and reducing agents, but may be hydrogenated catalytically, first to pyrazoline, and then to pyrazolidine. pyrazole. Oxidation: Pyrazole is resistant to oxidizing agents but the sidechain may be oxidized to carboxylic acid group in presence of potassium permanganate. Reduction: Pyrazole ring system can be reduced with molecular hydrogen and metal catalyst. stronger bases than pyrazole. Alkylation: There is a free N-H group in pyrazole. The free NH group can be alkylated with alkylating agents (alkylhalides, diazomethane or dimethyl sulfate). Electrophilic aromatic substitutions: Pyrazole is an aromatic compound. It readily undergoes electrophilic substitution readily at position 4 through the intermediate formation of arenium ion. Pyrazoline and pyrazolidine are Both of these compounds are stronger bases than

28 The electrophilic substitution is favoured in neutral or basic medium but not in acidic medium. Reaction of pyrazoles with nucleophilies: Halogens of pyrazole derivatives do not undergo substitution reaction. But the presence of electron-withdrawing groups assists nucleophilic substitution of halogens. eg : The halogen of 1-phenylpyrazole readily undergoes nucleophilic substitution. General Synthesis: The synthesis of pyrazoles and their

derivatives generally follow classical methods, the two most important methods for practical purposes being the reaction between hydrazines and -difunctional compounds, and 1,3dipolar cycloadditions. The ring closure is the final step of the reaction of hydrazines with1,3-difunctional compounds. i) Sucrow, studying the chemistry of the enehydrazines, has found several methods for the synthesis of pyrazoles. For example, he has described the cyclization of

monomethylhydrazones of dialkyl oxalacetates to 5-pyrazolones via an enehydrazine. ii) Hart and Brewbaker have described the cyclization of 1,3bis(diazopropane) to pyrazole by a concerted, intramolecular 1,3-dipolar cycloaddition.

29

iii) Pyrazoles have been prepared by the action of hydrazines on heterocyclic derivatives, which react as marked functional groups of a 1,3-difunctional derivative. A carbonyl group can be replaced by a three-membered ring, usually an oxirane or an aziridine or by a -substituted pyrrole or indole derivative.
iv) The addition of aliphatic diazo compounds to acetylenes gives pyrazoles. The same reactions as applied to olefins leads to dihydropyrazoles which are termed pyrazolines (Pechmann) : V) e action of hydrazine or its derivatives on ,-unsaturated aldehydes or ketones yields pyrazolines (the initially formed hydrazones undergo intramolecular addition to the double bond). Pyrazolines can then be oxidized to pyrazoles.

Pharmacological

interest:

Pyrazole

derivatives

constitute

an

interesting class of organic compounds, which are associated with diverse chemical and pharmacological properties. Pyrazolines have received considerable attention in recent years. Pyrazoline

derivatives occupy a unique place in field or medicinal chemistry due to a wide range or biological activities exhibited by them. Several derivatives of these systems find use in medicine described as follows;

30 Phenylbutazone: Chemically, it is 4-butyl-1,2-diphenylpyrazolidine 3,5-dione. It is known for its analgesic anti-inflammatory and

antipyretic actions and finds use for the treatment of rheumatic disorders. Due to its toxicity phenylbutazone should not be

employed routinely as an analgesic and antipyretic. Muzolimine: Muzolimine a 1-substituted 2-pyrazolin-5-one

derivative is a highly active diuretic, differing from the structures of other diuretics since it contains neither a sulfonamide nor a carboxyl group. It has a saluretic effect similar to furosemide and acts in the proximal tubule and in the medullary portion of the ascending limb of the loop of Henle. Phrmacokinetic studies in

dogs, healthy volunteers and in patients with renal insufficiency show that the compound is readily absorbed after oral

administration. Forbisen: Forbisen, 2,2',3,3'-tetramethyl-1,1'-diphenyl-4,4'-bi-3,3'pyrazoline-5,5'-dione a by-product obtained in the manufacture of antipyrine, has been used in bovine anaplasmosis. Oxyphenbutazone: It is a compound having p-hydroxyphenyl group instead of phenyl at position 1 of phenylbutazone. Its uses have been found to be similar to these of phenylbutazone. It also finds use in the treatment of inflammation of the eyes.

Oxyphenbutazone has been one of the active metabolite of phenylbutazone.

31 Sulphinpyrazone: An analogue of phenylbutazone is

sulphinpyrazone which is having a 2-phenylsulphinylethyl group in place of n-butyl group at position 4. It is having a better

therapeutic index as a uricosuric agent; it promotes excretion of uric acid and urate by inhibiting their tubular reabsorption. Feprazone: Structurally, it is similar to phenylbutazone except that the former is having a 3 methylbutenyl substituent at position-4 of pyrazoline-2,5-dione skeleton in place of a butyl substitutent. Feprazone also finds use in the treatment of rheumatic disorders.

Phenazone: It is a pyrazoline derivative which is chemically 2,3dimethyl-1-phenyl 3-pyrazolin-5-one. It forms white crystals or

white crystalline powder. It is freely soluble in water. Phenazone is well known for its analgesic and antipyretic actions. Topically, it is known for its local anaesthetic and styptic actions and solutions having 5% are used locally as ears drops. Phenazone has been

found to affect the metabolism of some other drugs. However, its own metabolism gets affected by drugs that are able to increase or reduce the activity of liver enzymes. Propylphenazone: It is a derivative of phenazone with an isopropyl group attached at position 4. Chemically, it is 4-isopropyl-2,3-

dimethyl-1-phenyl-3-pyrazolin-5-one is having alagesic properties.

32 Analgin: It is a derivative of phenazone which is having N(CH3)CH2SO3Na substituent at position-4. It occurs as an almost white crystalline powder having a scarcely perceptible yellowish tinge. It is freely soluble in water. It is having a bitter taste. The drug is kept in tightly-closed containers. analgesic and antipyretic actions. Pyrazole derivatives have found application in the agrochemical field as insecticides. (a) and O,O-Diethyl 0,0-diethyl O-(3-methyl-5-pyrazolyl) 0-(3-methyl-5-pyrazolyl) It is known for its

phosphate

phosphorothioate (b). 5-Pyrazolone derivatives have found many applications as cotton azo dyes because, even if they were more expensive intermediates, they improved qualities such as brightness and light fastness, Pyrazole Orange. Prompted by the observation that many pyrazoline

derivatives possess anti-inflammatory activity and denaturation of proteins as one of the causes of inflammation is well documented. Production of auto-antigens in certain rheumatic diseases may be due to in-vivo denaturation of proteins. A number of anti-inflammatory drugs are well known to inhibit denaturation of proteins. Almost all non-steroidal anti-

inflammatory drugs (NSAIDs) under current clinical usage are highly acidic in nature and suffer from a common draw back of

33 gastro-intestinal toxicity, thus indicating a clear need to develop a non-acidic non steroidal anti-inflammatory agent. The classical NSAIDs. eg. aspirin like drugs exert their pharmacological action by inhibiting both forms or cycloxygenase isoenzyme. The GIT

ulcerogenic effects are commonly associated with the drugs which inhibit both type of isoenzymes. Classically cox-2 inhibitors are

not acids even then they possess equipotent analgesic, antipyretic and anti-inflammatory activities of same degree as the classic NSAIDs. The Cox-2 inhibitors refecoxib and celecoxib are the

derivative of pyrazole, which in term is derivative of pyrazoline. Several 1,3,5-trisubstituted pyrazolines are reported to possess anti-inflammatory, antiproteolytic, antibacterial, antifungal and insecticidal activities. Also, 1-aryl-2pyrazolines are found to be

useful as antioxidant composition in polymers and in the treatment of cerebral edema. 2-Pyrazolines having aryl

substituents at positions-1,3 and 5 exhibit the phenomenon of fluorescence properties. Pyrazolines exhibiting this phenomenon are extensively used as water soluble optical bleaches. Certain 2pyrazolines attached to pyridine ring have been shown to exhibit fluorescent brightening properties. Pyrazolines attached to indole ring systems have been shown to exhibit monoamine oxidase inhibitor activity. Several halonanilido pyrazolines are used in

textile and cinematographic film industry and they are also found

34 to possess bactericidal, insecticidal, activities. fungicidal, Besides, anaesthetic, fluorinated

analgesic

and

antibacterial

pyrazoles and pyrazolines find their applications as antifertility, antibacterial and antifungal agents. It is also reported that many pyrazoline derivatives have found applications in both pharmaceutical and agrochemical fields as biodegradable agrochemicals. Pyrazoline derivatives are also

found to useful for prevention and treatment or ischemic heart disease, angina, migraine and Parkinsons disease. So pyrazolines have played a crucial part in the development or heterocyclic chemistry and also used a useful synthon in organic synthesis.

REVIEW OF LITERATURE PYRAZOLES: Pyrazoline as antimicrobial and anti bacterial: Sachchar SP and Singh AK1 synthesized several 1-phenyl-3(substituted fluro phenyl)-5-heteroayl-2-pyrazolines (1) and noted their anti microbial as well as anti bacterial actions especially against bacteri-B anthrasis.

Substituted pyrazolines have been reported for their antibacterial activity2-6.

35 Stirrewiberg WE
7

and co-workers reported antibacterial as

well as insecticidal action of some new pyrazoline derivatives (2).

Desai NC et al.,

synthesized several pyrazoline derivatives

of phenothiazines and reported them for moderate to good anti bacterial activity and some compounds exhibited tuberculostatic activity. Ead HA, Hossanen HM, Abdullah MA, Mousaah, have reported the synthesis of two novel series of pyrazoles and 2pyrazolines, and reported their antibacterial activity9. Pyrazolines as antifungal: Sachchar SP
1

extended their studies on several pyrazoline

derivatives and has reported them as antifungal. Aspergillus niger and Helmithosporium sativum were employed as fungi for the testing of fungicidal activity. Ritch S and Horsfall JC
10

synthesized 3, 5 dimethyl -4- nitro

pyrazole and 1, 3, 5 trimethyl-4-nitrosopyrazole compounds and have reported them as anti fungal agents. Their fungicidal activity increased by increasing the size of hydrocarbon side group was also reported by them. Mitra, P and Nayak A11 reported that 5-pyrazolone and its derivatives 4-acetyl-2-pyrazoline-5-one and 2-pyrazoline-5-

36 thione (3) were associated with significant fungicidal activity against the rice blast pathogen Pyricularia oryzae and brown leaf spot pathogen Helminthosporium oryzae12-14. (3) Parvati Mitra and Mitra AG
15

have synthesized 4-N (aryl)

amino methyl-2-pyrazoline-5-one by mannich reaction of 2pyrazoline-5-one derivative. The same authors also reported cobalt II complex with 2-pyrazoline-5-one derivative as having anti fungal activity. Sadasiva Shankar M
16

synthesized several hydroxyl aryl-

pyrazole (4) and tested them for antibacterial as well as antifungal activity where none of the compounds were found to possess anti-bacterial activity. But all compounds were found to possess anti-fungal activity. Antifungal activity has been assessed by employing Drechslera prostrate (Drechs) and Alternatia alternate (Keissler). All compounds tested could inhibit the spore germination at 30-mcg/ml up to a maximum level. Nayak A and Mitra AS
17

synthesized several 4, 4, bis-5-

pyrazoline and 4, 4,unsaturated products and found their fungicidal activity against rice blast pathogen Pyricularia oryazae and brown leaf spot pathogen, Helmithosporium oryzae.

37 Tiwari N, Dwevedi B and Nizamuddin4 synthesized several 1acetyl/aroyl-3-methyl-4-substituted pyrazolines pyrazolines and and anilido -5-aryl

3-methyl-4-substituted 3-methyl-4-substituted

anilido-5-aryl anilido-5-aryl

isoxazolines, and tested against Cephalosporium sacchari, Helmithosporium oryzne, and saprolegina parasitica, acellya Orion all the compounds showned remarkable activity. Mohanthy SK5 reported that 1-phenyl-5-aryl-1-2-pyrazoline 3-4-thiazolidine-2-one derivative exhibited antifungal

properties. The fungicidal activity of these compounds was determined by poisoned food technique at various

concentrations. It was also reported that all compounds inhibit the growth of the fungus Aspergillus flavours. Pyrazoline as antiviral: Sachacha SP (substituted
1

extended their study on several phenyl1-3fluro phenyl)-5-hetero aryl-2-pyrazoline

derivative and found antiviral activity against Sunn hemp rosette Anti-inflammatory activity with pyrazole nucleus: Pyrazolone derivatives as anti inflammatory: Antipyrine was one of the first synthetic Pyrazolyl compound used in medicine and was followed by aminopyrin. They

38 showed anti-inflammatory activity along with analgesic and anti pyretic activities.

R1 Antipyrin Aminopyrin Dipyrone

R2

R3 -CH3 -CH3 -CH3

R4 -H -N-CH3 -N-CH2SO3Na

-C6H5 -CH3 -C6H5 -CH3 -C6H5 -CH3

Another derivative Dipyrone was prepared which showed a strong antiserotonin and anti-oedima activities in rats but found to be clinically disappointing in the treatment of rheumatic arthritis. Other compounds like 4-(N-Nicotinyl amino) derevative was also found to be less toxic and clinically effective in various

inflammation diseases. Frangnly AM, chaaban L, Khalil M and Behkit AA18 have reported anti-inflammatory activity of some new pyrazoles pyrazolines and 4(3H)-quinazolines. Derivative of 3, 5-pyrazolindinedione as anti inflammatory: After the discovery of the 5-pyrazoline derivative as antiinflammatory agents, modifications were made on the basic structure and this resulted in the synthesis of a new compound, phenyl butazone, which was found to be a potent inhibitor of inflammation.

39 A metabolic product of phenyl butazone was found to be less toxic and equally potent as the parent compound. The result was the synthesis of oxyphenbutazone. Modification of the parent compound to increases the acidity of the molecule resulted in the synthesis of a new derivative sulfin pyrazole against gout. (6) R1 Phenyl butazone Oxyphen butazone Sulfin pyrazone -C6H5 -C6H4-OH (P) -C6H5 R2 -C4H9 -C4H9 -CH2-CH2-S-C6H5
12

it was enhanced uricosuric activity and is potent

Pyrazole derivative as anti-inflammatory agent: Coli B

13, 14

reported that pyrazole derivative itself has anti-

inflammatory activity. e.g. Benzylamine (7)(Tantum).

Sarangam S

19

have synthesized number of derivative of

pyrazole (3,4-d) pyrimidine-4-6 diones (8) and screened them for C.N.S. depression properties and anti-inflammatory activity. It was also reported that some derivatives showed anti-inflammatory properties equivalent to aspirin20. Pyrazole derivatives as antidiabetic:

40 Froesch EE21 has reported antidiabetic activity in the 5methyl pyrazole-3-carbaxilic acid (9).

(9) Pyrazole derivative as vasodilator: Burner HR et al.,

22

have reported vasodilator activity in

pyrazole derivative (10).

(10) Pyrazole derivative as hypoglycemic and hypotensive agent: Smith DL23 reported that 3, 5-dimethyl pyrazole and 3methyl pyrazole-5-carboxylic acid exhibited hypoglycemic activity. Arya VP et al.,
24

synthesized several pyrazolidine derivatives

(11-13) and reported the hypotensive activity.

Pyrazolidone derivative as inhibitor: Sweeny MJ antibiotic

25

reported that pyrazopurin which is a natural very effective in inhibiting pyrimidine

was

biosynthesis. Chasin M et al.,

26

discovered a potent new compound

pyrazole pyridine (14 ) that was 60 times more potent as an inhibitor of rat brain PDE than theophyllin.

41 Novinson T
27

reported a series of pyrazole pyrimidine

derivative (15, 16) and reported them to be inhibitor of P.D.E. from rabbit lung and beef heart. In view of the above mentioned important biological

properties of

pyrazoline derivatives. We thought of preparing

some more new pyrazoline derivatives in anticipation that they might possess pronounced biological activity.

42 SCHEME-I

F F O CHO O O CH3 Piperidine CH3COOH O COOCH3

CH3 CH3

H2N S CH3 HN HMPA H2SO4 2 HN S

F O OCH3 N

p-Flouro Benzadehyde

Methy isobutyryl acetate

(SA-1) DDQ Toluene

F O O N S O N NHNH2 N H2-NH2.H2O Ethanol ON S O

F O OCH3 N mCPBA M DC S N

F O OCH3 N

(SNP-1) R O Aceticacid R1 (SNP-2a-j) F O O N S O (SNP-3a-j) N N N R1 R

( SA-3 )

(SA-2)

43 Procedure for the preparation of compound SA-1: p-Fluorobenzaldahyde (5.94g, 0.1 mol), methylisobutyryl acetate (6.33g, 0.1 mol), piperdine (0.42g, 0.01mol) and acetic acid (0.2ml) were placed in a round bottom flask fitted with a condenser and the mixture was heated at 85-90o C for 3hrs. Cooled the reaction mixture and added chloroform (100ml), the separated chloroform layer was washed with 0.1N HCl (50ml), 5% Aq. Sodium bicarbonate (50ml) and finally with water (100ml). The solvent was evaporated completely. The crude mass (12g) was purified by Column chromatography. (Yield- 70%).

Procedure for the preparation of Compound SA-2: Compound SA-1 (10g, 1mol), S-methylthiourea hydrogensulphate (7.93g, 0.8mol), and Hexamethylphosphoramide (15ml) was placed in a round bottom flask and the mixture was heated at 120o-125oC for 24hrs. Cooled the reaction mixture to 80o C to which was and added toluene (50ml) and 5% aq.Sodium bicarbonate (50ml). Stirred the mixture for 30 min at 80-85o C. Cooled the reaction mixture to 25
o

C and separate the toluene layer. It was washed

with water (50ml). The solvent was evaporated under reduced pressure, to get the crude mass. Fresh toluene was added (50ml) to the crude mass in a round bottom flask and added a solution of 2,3-Dichloro-5,6-dicyno-1,4-benzoquinone(DDQ) (4.7g, 0.5mol) in

44 toluene(50ml) slowly for 15min at 25-30o C. Stirred the reaction mixture for 2hrs. Filtered the 2,3-Dichloro-5,6-dicyno-1,4-

hydroquinone (DDHQ) and washed the DDHQ with toluene (10ml) and filtrate was washed with 5%Aq. NaHCO3 (450ml) and water (100ml). The solvent was evaporated to get the solid. The solid was recrystallised from ethanol. (Yield -72 %). Procedure for the preparation of Compound SA-3: In to a clean dry round bottomed flask introduced Compound SA-2 (10g,0.1mol) in dichloromethane(100ml) to which was added a solution of m-choloroperbenzoic acid (13.5g, 0.25mol) in dichloromethane (50ml) for 30min at 0-5oC. Slowly warmed the mixture to 25-30oC and stirred for 2hrs at same temperature. When m-chlorobenzoic acid pricipated out. Filtered the mchlorobenzoic acid and the filtrate was washed with 5% Aq. NaHCO3 (250ml) and with water (100ml). The solvent was evaporated to get the crude mass from which the pure solid is obtained by the addition of hexane (50ml). (Yield - 68%). Preparation of compound SNP-1: The compound SA-3 (10g, 0.1mol) and ethanol (50ml) were placed in a round bottom flask fitted with reflux condenser and added Hydrazine hydrate (99% , 4.26g, 0.3mol) drop by drop with stirring. The reaction mixture was heated under reflux for 18hrs. Cooled the reaction mixture and added to ice cold water with stirring, left

45 overnight at room temperature. The separated solid product was filtered and washed with water. (Yield - 85%).

General Procedure for the Preparation of compound SNP-3a-j: The compound SNP-1 (0.1mol), appropriate chalcone SNP-2a-j (0.1mol) in acetic acid (25 ml) were placed in a round bottom flask fitted with reflux condenser. The reaction mixture was refluxed for 18hrs. Cooled the reaction mixture and added to ice cold water with stirring. The obtained solid product was filtered and washed with water until the filtrate is neutral. (Yields - 61-85%).

46

BIOLOGICAL ACTIVITY ANTICANCER ACTIVITY The anticancer activity of the synthesized compounds was carried out on three cancer cell lines namely HT-29 (Colon cancer), A431 (skin cancer), MCF-7 (Breast cancer), cell lines which were obtained from National center for Cell science (NCCS), Pune, India. The inhibition of the growth of cell lines i.e Cytotoxicity was considered as anticancer activity. Toxicity of test compound in cells was determined by MTT assay based on mitochondrial reduction of yellow MTT tetrazolium dye to a highly colored blue formazan product which was measured as absorbance at 570nm on a

spectrophotometer (spectra max, Molecular devices) and the IC50 values were determined by versus concentration. MATERIALS HT-29 (Colon cancer), A431 (skin cancer), MCF-7 (Breast cancer cell line), cell line was obtained from National center for Cell science (NCCS), Pune, India. DMEM (Dulbeccos Modified Eagles Medium), MTT [3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl plotting % inhibition (from control)

tetrazolium bromide], Trypsin, EDTA were purchased from Sigma Chemicals Co (st.Louis, MO), Fetal bovine serum were purchased

47 from Arrow labs, 96 well flat bottom tissue culture plates were purchased from Tarson. PROCEDURE a) Maintenance of cell lines. The 1 HT-29 (Colon cancer), A431 (Skin cancer),cell lines were grown as adherent in DMEM media, where as MCF-7 (Breast cancer cell line) were grown in MEM media supplemented with 10% fetal bovine serum, 100 g / ml penicillin, 200 g/ml streptomycin, 2mm L-glutamine, and culture was maintained in a humidified atmosphere with 5%CO2. b) Preparation of samples for cytotoxicity Stock solution of 10mg/ml stcok solution in DMSO, from the above stock various dilutions was made with sterile water to get required concentration. CYTOTOXICITY EVALUATION. Toxicity of test compound in cells was determined by MTT assay based on mitochondrial reduction of yellow MTT tetrazolium dye to a highly colored blue formazan product. 1x104 Cells (counted by Trypan blue exclusion dye method)) in 96- well plates were incubated with compounds with series of concentrations tested for 48 hrs at 370C in DMEM/MEM with 10% FBS medium. Then the above media was replaced with 90l of fresh serum free media and 10 l of MTT reagent (5mg/ml) and plates were

48 incubated at 370C for 4h, there after the above media was replaced with 200l of DMSO and incubated at 370C for 10min. The absorbance at 570nm was measured on a spectrophotometer (spectra max, Molecular devices) IC50 values were determined from plot: % inhibition (from control) versus concentration. The results of the Cytotoxicity are tabulated in table 2-4. TABLE 2: Cytotoxicity in MCF cell lines (breast cancer cells). Code SNP-4a Conc. g/ml 200 100 50 10 200 100 50 10 200 100 50 10 200 100 50 10 Triplicates 0.296 0.275 0.244 1.533 1.424 1.21 2.107 2.147 1.928 2.388 2.296 1.964 1.893 1.983 2.028 2.204 2.236 2.01 2.255 2.337 2.288 2.633 2.531 2.303 1.008 0.885 0.877 1.367 1.365 1.342 1.761 1.879 1.7 2.307 2.15 2.187 0.103 0.076 0.084 0.18 0.103 0.084 2.099 2.175 2.381 2.258 2.256 2.236 2.624 2.483 2.426 2.562 2.565 2.341 2.448 2.452 2.266 1.366 1.496 1.522 1.461 1.813 1.967 1.502 1.965 1.823 1.867 2.02 2.298 0.827 0.857 0.932 1.489 1.579 1.47 1.969 1.656 2.118 2.109 2.308 2.217 2.389 2.559 2.659 Average 0.27 1.39 2.06 2.22 1.97 2.15 2.29 2.49 0.92 1.36 1.78 2.21 0.09 0.12 2.22 2.25 2.463 % inhibition IC 50 88.97 113.7 43.61 16.34 10.03 20.10 NA 12.71 6.89 -1.06 62.51 141.2 44.86 27.73 10.08 96.44 52.48 95.03 9.93 8.65

SNP-4b

SNP-4c

SNP-4d

CONTROL

SNP-4e

SNP-4f

200 100 50 10 200 100 50 10

1.46 1.75 1.76 2.06 0.87 1.51 1.91 2.21

44.44 33.57 32.95 21.61 66.84 42.48 27.21 15.92

NA

134.3

CONTROL

49 2.651 2.858 2.857 2.638 2.454 3.018 2.539 2.617 2.278 2.63

TABLE-3: Cytotoxicity in HT cell lines (colon cancer cells). Code SNP-4a Conc. g/ml 200 100 50 10 200 100 50 10 200 100 50 10 200 100 50 10 200 100 50 10 200 100 50 10 % Triplicates Average inhibition IC 50 0.157 0.205 0.175 0.179 64.90 73.2 0.226 0.227 0.207 0.220 56.86 0.291 0.245 0.252 0.263 48.50 0.315 0.31 0.314 0.313 38.63 0.596 0.609 0.63 0.612 -19.93 NA 0.629 0.619 0.657 0.635 -24.51 0.559 0.598 0.608 0.588 -15.36 0.608 0.565 0.583 0.585 -14.77 0.212 0.171 0.148 0.177 65.29 NA 0.331 0.388 0.426 0.382 25.16 0.528 0.545 0.582 0.552 -8.17 0.473 0.462 0.425 0.453 11.11 0.437 0.374 0.512 0.441 13.53 NA 0.464 0.433 0.528 0.475 6.86 0.407 0.41 0.559 0.459 10.07 0.528 0.534 0.494 0.519 -1.70 0.387 0.372 0.383 0.38 47.13 NA 0.349 0.392 0.47 0.40 43.94 0.535 0.376 0.496 0.47 34.86 0.52 0.491 0.449 0.49 32.41 0.498 0.282 0.445 0.41 43.29 NA 0.719 0.455 0.513 0.56 21.90 0.492 0.533 0.49 0.51 29.86 0.46 0.391 0.5 0.45 37.45 1.051 0.477 0.644 0.72

SNP-4b

SNP-4c

SNP-4d

SNP-4e

SNP-4f

CONTROL

50

TABLE-4: Cytotoxicity in A431 Cell Lines (Skin Cancer Cells). % inhibiti on 54.92 37.84 30.07 24.31 17.49 -11.53 -10.67 -15.12 55.50 37.38 33.65 37.47 36.24 2.95 9.61 1.42 66.73 38.06 30.87 -0.77 50.57 18.35 10.20 5.47

Code SNP-4a

SNP-4b

SNP-4c

SNP-4d

SNP-4e

SNP-4f

Conc. g/ml 200 100 50 10 200 100 50 10 200 100 50 10 200 100 50 10 200 100 50 10 200 100 50 10

CONTROL

Triplicates 1.14 1.213 0.92 1.389 1.507 1.617 1.697 1.757 1.623 1.874 1.81 1.811 1.983 1.935 2.072 2.655 2.608 2.834 2.594 2.669 2.772 2.601 2.907 2.85 0.962 1.053 1.216 1.548 1.511 1.487 1.496 1.647 1.674 1.583 1.437 1.52 1.595 1.56 1.474 2.481 2.238 2.327 2.169 2.291 2.102 2.422 2.283 2.452 0.995 0.872 0.588 1.581 1.515 1.475 1.729 1.718 1.655 2.402 2.317 2.718 1.168 1.191 1.289 1.936 2.035 2.055 2.215 2.149 2.263 2.398 2.331 2.247 2.331 2.416 2.496 2.265 2.575 2.668 2.433 2.458 2.667 2.371 2.51 2.359

Average 1.09 1.50 1.69 1.83 2.00 2.70 2.68 2.79 1.08 1.52 1.61 1.51 1.54 2.35 2.19 2.39 0.82 1.52 1.70 2.48 1.22 2.01 2.21 2.33 2.41 2.50 2.52 2.41 2.46

IC 50 171.56

NA

175.16

NA

136.7

208.9

51

Result and Discussions: Certain representative compounds of pyrazoline derivatives were screened for anticancer activity by MTT assay and their IC50 values were determined. Among the compounds screened SNP-4a, SNP-4b, SNP4c, SNP-4d, SNP-4e, and SNP-4f. in MCF cell lines (Breast cancer) the compound SNP-4d showed potent anticancer activity, the rest of the compounds like SNP-4a, SNP-4c and the SNP-4f showed moderate activity, while rest of the compounds did not show any activity worth mentioning. The same compounds were also screened for anticancer against HT cell line (Colon cancer cells). Except SNP-4a which showed much significant activity rest of the compounds showed no activity. Similarly the compounds like SNP4s, SNP-4c, SNP-4e and SNP-4f showed moderate activity against A-431 cell line (skin cancer cells). The rest of the compounds of this series showed no activity. From the above results it can be concluded that substituted pyrazoline moiety still serves as a lead and may give much better results if the molecule is suitably modified and the toxicity studies were carried out. Hence further molecular modification, detailed toxicity studies and elimination of toxic

52 groups is most desirable and may yield better results. Therefore further work in this direction is quite rewarding. REFERENCES 1. Sachchar SP and Singh AK. J Ind Chem Soc 1985; LXII: 142146. 2. 3. 4. Sharma TC and Bokadia MM. J Ind Chem Soc 1980; 19B: 228. Nayak A and Mitra AS. J Ind Chem Soc 1980; LVII: 643-645. Tiwari N, Dwivedi B and Nizamuddin. Bol Chem Farm 1989; 128: 332-335. 5. 6. 7. 8. Mohanty SK and Sridhar R. J Ind Chem Soc 1977; 15: 1146. Mecalcon SE. Agric Chem. 1947; 2,31,45,67. Sirrewiberg WE . Chem Abstr 1978; 89. Desai NC, Trivadi PB, Unnadevi NK, Dave AM and Bhat KN. Indian J Chem 1993; 32B: 760. 9. Ead HA, Hassanun HM and Abdullah MAM. Arch Pharm (Weinheim Ger) 1991; 324: 35-37. 10. Ritch S and Horsfall JC. Chem Abstr 1952; 56,11543. 11. Mitra P and Nayak A. J Ind Chem Soc 1982; 59: 1005. 12. Burns JJ, Yu TF, Arnold Ritterband, James MP, Alexander BG and Bernard BB. J Pharmacol Exp Ther 1957; 119: 418-426. 13. Coli B and Silerstrint. Eup Med Therap. 1957. 14. Rosmer I and Buliard M . Med Pharmacol Exp.1967;16:25. 15. Parvati Mitra AG. J Ind Chem Soc 1981; LVIII: 695-969.

53 16. Sadasiva M and Rajeshwar Rao. J Ind Chem Soc 1982; LIX: 1104. 17. Nayak A and Mitra AS. J Ind Chem Soc 1980; LVII: 643-645. 18. Farghaly AM, Chaaban I, Khalil MA and Behkit AA. Archiv der Pharmazie 1990; 323: 311-315. 19. Sarangam S and Somashekara S. J Ind Chem Soc 1976; 53: 555-559. 20. Reuben G Jone. J Am Chem Soc 1952; 74: 4889-4891. 21. Froesch ER, Waldvogel M, Meyer UA, Jacob A and Labhart A. Mol Pharmacol 1967; 3: 429-441. 22. Brunner H, Eichenberger K, Meier M, Wilhelm M and Schmidt P. Cell Mol Life Sci 1966; 22: 208-209. 23. Smith DL, Forist AA and Dulin WE. J Med Chem 1965; 8: 350353. 24. Arya VP, Grewal RS, David J and Kaul CL. Cell Mol Life Sci 1967; 23: 514-515. 25. Sweeny MJ, Davis FA, Gutowski GE, Hamill RL, Hoffman DH and Poore GA. Cancer Res 1973; 33: 2619-2623. 26. Chasin M and Harris DH. Bio Chem Pharmacol 1972; 21: 2443. 27. Navinson T, Scholten H. Am Chem Soc Abstr Med.1972; 52.