INTRODUCTION:

Pyrazoles are five member ring heterocyclic compounds, have some structural features with two nitrogen atoms in adjacent position and are also called as Azoles¹. Recently Pyrazole derivatives have been found in nature, β- [1-pyrazolyl]alanine was isolated from the seeds of water melons [Citurllus lanatus]. The best described property of almost every group of pyrazoles is in the treatment of inflammation and inflammation associated disorders, such as arthritis². Pyrazole derivatives are the subject of many research studies due to their widespread potential biological activities such as antimicrobial³, antiviral⁴, antitumor^5,6, antihistaminic⁷, antidepressant⁸, insecticides⁹ and fungicides⁹.

Several pyrazole derivatives have been found to possess significant activities such as 5-α-red-uctase inhibitor ¹⁰, antiproliferative¹¹, antiparasitic¹², herbicides¹³. A good number of pyrazoles have also been reported to have interesting biological activities like anti-inflammatory¹⁴ and antiprotozoal^15,16 which render them valuable active ingredients of medicine and plant protecting agents. Further, current literature indicates 1, 2-pyrazole derivatives to possess various biological activities¹⁷.

Human and animal diseases caused by helminthes parasites have great impact on public health. Toxocariasis is an infection caused by the nematode Toxocara commonly found in the intestines of puppies and older dogs (Toxocara canis) and cats (Toxocara cati). Humans become infected either by ingesting embryonated eggs accidentally or eating contaminated food with soil containing the eggs (such as unwashed raw vegetables). Hymenolepiasis is caused by (Hymenolepis nana or Hymenolepis diminuta) the dwarf tapeworm which is the most common cause of all intestinal cestode infections. In an infected person the worms can remain encysted in tissue so infection can persist for years.

Treatment with Praziquantel or Albendazole is recommended alternative to these drugs are now being sought¹⁸. The continuous and longterm reliance on a small range of compounds has led to the development of drug resistance in many helminthic strains. In addition, after treatment with Albendazole or Mebendazole several side effects have been reported in hosts such as gastrointestinal symptoms (epigastric pain, diarrhea, nausea, vomiting); nervous system symptoms (headache, dizziness) and allergic phenomena (edema, rashes, urticaria). Some anthelmintic drugs such as Praziquantel and Albendazole arecontraindicated for certain groups of patients like pregnant and lactating woman¹⁹.

The global burden of both, human and domestic animal parasitic diseases coupled with the emergence of drug resistance has made the development of new chemotherapy a critical need²⁰.

Present study was undertaken to synthesize some novel pyrazole clubbed with quinazolinone compounds and investigating their antifungal and anthelmintic activity.

MATERIALS AND METHODS:

Melting points were determined on an electro thermal apparatus using open capillaries and are uncorrected. Thin-layer chromatography was accomplished on 0.2-mm precoated plates of silica gel G60 F254 (Merck). Visualization was made with UV light (254 and 365nm) or with an iodine vapor. IR spectra were recorded on a Shimadzu-fourier transform infra red (FTIR)-8400 Spectrophotometer using KBr disc. 1H NMR spectra were recorded on a Bruker DPX-400 MHz spectrometer. Chemical shifts are expressed in δ ppm downfield from TMS as an internal standard. The mass spectral data were obtained with a SHIMADZU-GCMS-QC-2010.

EXPERIMENTAL PROCEDURE

Synthesis of 3, 5 dibromo Anthranilic Acid (1):

20gm of anthranilic acid dissolved in 25 c.c. bromine in glacial acetic acid(9 cc bromine in 25 cc glacial acetic acid) was added drop by drop from separating funnel till the redish colour of the liquid persist.Then content was converted to a thick mass. So it will form dibromoanthranilic acid. The reaction was monitored by TLC. After completion of reaction, the solvent was removed under vaccuo when the reaction was completed. The solid was crystallized from methanol to give pure product (2). Their melting points, yields and molecular formula are given in Table-1.

Yield: 79%; mp 202-204 ºC; IR (cm-1): 3312 (N-H stretching of primary amine), 3066 (C-H stretching of aromatic ring), 1726, 1675 (C=O stretching of carboxylic acid), 1604 (N-H deformation of NH2 group), 1560 and 1432 (C=C stretching of aromatic ring), 1004 (C-H in plane bending for aromatic ring); 695(C-Br); 1H NMR (CDCl₃) δ ppm: 7.03-7.58 (m, 2H, ArH), 5.38 (s, 1H, NH), 9.94 (S, 1H, COOH); MS: m/z 293,275,213, 189, 174 , 134; Anal.Calcd. For C₇H₅ Br₂NO₂: C, 28.51; H, 1.71; N, 4.75. Found: C, 28.80; H, 1.64; N, 4.77%.

Synthesis of 6, 8-dibromo-2-methyl-4H-3, 1-benzoxazin-4-one (2)

A mixture of 3, 5 di Bromo Anthranilic Acid (1,0.01 mol) and acetic anhydride (10.2 ml(0.1 mol) was refluxed on gentle flame for 1 hr .The excess of acetic anhydride was distilled off under reduce pressure and the residue was dissolve in petroleum ether and kept a side for 1 hr. The reaction was monitored by TLC. After completion of reaction, the solvent was removed under vaccuo when the reaction was completed. The solid was crystallized from ethanol to give pure product (2). Their melting points, yields and molecular formula are given in Table-1.

Yield: 71%; mp 179-181 ºC; IR (cm-1): 3076,3000 (C-H stretching of aromatic ring), 1689 (C=O stretching of benzoxazin-4-one ring), 1639 (C=N stretching of pyridine ring), 1495 (C=C stretching of aromatic ring), 1127 (C0C stretching of benzoxazin-4-one ring),933 (C-H in plane bending for aromatic ring); 684(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.87 (s, 3H, CH₃), 7.39-7.89 (m, Ar-H); MS: m/z 317,289,276,237,213,158,105; Anal.Calcd. for C₉H₅Br₂NO₂: C, 33.89; H, 1.58; N, 4.39. Found: C, 33.91; H, 1.54; N, 4.42%.

Synthesis of 3-amino-6,8-dibromo-2-methylquinazolin-4(3H)-one (3)

A 100mL round-bottom flask equipped with condenser and septum was charged with a solution of 6,8-dibromo-2-methyl-4H-3,1-benzoxazin-4-one (2, 0.01 mmol) in ethyl alcohol (30 mL),followed by the hydrazine hydrate (0.03 mmol) was added and the mixture was reflux for 2 hr. The reaction was monitored by TLC, after complete the reaction mixture was then cooled down to room temperature, poured into crushed ice. When precipitated, the product is filtered, washed with water, and purified by recrystallization from ethanol to give pure product. Their melting points, yields and molecular formula are given in Table-1.

Yield: 69%; mp 138-140 ºC; IR (cm-1):3361(N-H stretching of primary amine), 3063(C-H stretching of aromatic ring),2914(CH₃ Str.),1682 (C=O stretching of ring), 1599(C=N stretching of pyridine ring), 1451 (C=C stretching of aromatic ring), 880(C-H in plane bending for aromatic ring); 688(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.84 (s, 3H, CH₃), 4.73 (s,2H,NH₂), 7.40-8.07(m,Ar-H); MS: m/z 331,314,303,290,251,227,211,172,103; Anal.Calcd. for C9H7Br2N3O: C, 32.46; H, 2.12; N, 12.62. Found: C, 32.49; H, 2.09; N, 12.58%.

Synthesis of N-(6, 8-dibromo-2-methyl-4-oxoquinazolin-3(4H)- yl) acetamide (4)

A 100mL round-bottom flask equipped with condenser and septum was charged with a solution of 3-amino-6, 8-dibromo-2-methylquinazolin-4(3H)-one (3, 0.01 mmol) in Glacial acetic acid (30 mL), followed by the acetic anhydride (0.03 mmol) was added and the mixture was reflux for 1 hr. The reaction was monitored by TLC, after complete the reaction mixture was then cooled down to room temperature, poured into crushed ice. When precipitated, the product is filtered, washed with water, and purified by recrystallization from ethanol to give pure product. Their melting points, yields and molecular formula are given in Table-1.

Yield: 74%; mp 126-128 ºC; IR (cm-1):3366(N-H stretching of primary amine), 3082(C-H stretching of aromatic ring), 2870(CH₃ Str.), 1703(C=O stretching of Acetyl group), 1673(C=O stretching of ring), 1609(C=N stretching of pyridine ring), 1462 (C=C stretching of aromatic ring), 900(C-H in plane bending for aromatic ring); 689(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.83 (s, 3H, CH₃), 2.50 (s, 3H, CH₃),8.94(s,1H,NH),7.40-8.17(m,Ar-H); MS: m/z 373,345,331,314,293,278, 269,190,103; Anal.Calcd. for C118H9Br2N3O2: C35.23; H, 2.42; N, 11.20. Found: C 35.19; H, 2.44; N, 11.2

Synthesis of N- (6, 8-dibromo-4-oxoquinazolin-3(4H)-yl)-3-(2-hydroxyphenyl) prop-2-enamide (5a)

A mixture of N-(6, 8-dibromo-2-methyl-4-oxoquinazolin-3(4H)- yl) acetamide (4,0.01 mol) was dissolved in ethanol. Then sodium hydroxide(5 ml,30%) solution and salisaldehyde(0.01 mol) were added to the resulting solution with continuous stiring for 10 hr then solution so obtained refluxed on gentle flame for 6 hr . The reaction was monitored by TLC. After complete the reaction mixture was then cooled down to room temperature, poured into crushed ice. When precipitated, the product is filtered, washed with water .The solid was crystallized from ethanol to give pure product (5a).5b were prepared similarly. Their melting points, yields and molecular formula are given in Table-1.

Yield: 70%; mp 152-154 ºC; IR (cm-1):3471(O-H stretching of Hydroxy group) ,3356(N-H stretching of primary amine), 3076(C-H stretching of aromatic ring), 2915(CH₃ Str.), 1730(C=O stretching of keto group), 1673 (C=O stretching of ring), 1607(C=N stretching of pyridine ring), 1449 (C=C stretching of aromatic ring), 876(C-H in plane bending for aromatic ring); 687(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.84 (s,3H,CH₃),10.83(s,1H,OH),9.01(s,1H,NH),6.210-6.216(d,1H,CH),7.19-7.25(d,1H,CH),7.41-8.10(m,Ar-H);MS: m/z 477, 459, 449, 397, 331, 316, 314, 163, 161, 103, Anal.Calcd. for C18H13Br2N3O3: C, 45.12; H, 2.73; N, 8.77. Found: C, 45.16; H, 2.69; N, 8.81%.

Synthesis of 6, 8-dibromo-3-{[5-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]amino}quinazolin-4(3H)-one (6a)

A 100mL round-bottom flask equipped with condenser and septum was charged with a solution of N- (6, 8-dibromo-4-oxoquinazolin-3(4H)-yl)-3-(2-hydroxyphenyl) prop-2-enamide (5a, 0.01 mol) in Glacial acetic acid (30 mL), followed by the Hydrazine hydrate (0.01 mol) was added and the mixture was reflux for4 hr. The reaction was monitored by TLC, after complete the reaction mixture was then cooled down to room temperature, poured into crushed ice. When precipitated, the product is filtered, washed with water, and purified by recrystallization from ethanol to give pure product. Their melting points, yields and molecular formula are given in Table-1.

Yield: 69%; mp 234-236 ºC; IR (cm-1):3472(O-H stretching of Hydroxy group) ,3354(N-H stretching of primary amine), 3074(C-H stretching of aromatic ring), 2901(CH₃ Str.), 1733(C=O stretching of keto group), 1671 (C=O stretching of ring), 1608(C=N stretching of pyridine ring), 1449 (C=C stretching of aromatic ring), 875(C-H in plane bending for aromatic ring); 688(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.84 (s, 3H, CH₃), 10.95 (s, 1H, OH),9.19(s,1H,NH),4.50(s,1H,NH)6.21-6.91(t,1H,CH),7.19-7.29(d,1H,CH),7.40-8.27(m,Ar-H);MS: m/z 491,473,463,411,397,331,178,160,135,120,103; Anal.Calcd. for C18H15Br2N5O2: C, 43.84; H, 3.07; N, 14.20. Found: C, 43.88; H, 3.10; N, 14.22%.

Synthesis of 6, 8-dibromo-3-{[5-(phenyl)-4, 5-dihydro-1H-pyrazol-3-yl] amino} quinazolin-4(3H)-one (6b)

A 100mL round-bottom flask equipped with condenser and septum was charged with a solution of N- (6, 8-dibromo-4-oxoquinazolin-3(4H)-yl)-3-(phenyl) prop-2-enamide (5a, 0.01 mol) in Glacial acetic acid (30 mL),followed by the Hydrazine hydrate (0.01 mol) was added and the mixture was reflux for4 hr. The reaction was monitored by TLC, after complete the reaction mixture was then cooled down to room temperature, poured into crushed ice. When precipitated, the product is filtered, washed with water, and purified by recrystallization from ethanol to give pure product. Their melting points, yields and molecular formula are given in Table-1.

Yield: 78%; mp 230-231 ºC; IR (cm-1):3322(N-H stretching of primary amine), 3079(C-H stretching of aromatic ring), 2932(CH₃ Str.), 1727(C=O stretching of keto group),1672 (C=O stretching of ring), 1616(C=N stretching of pyridine ring), 1485,1460 (C=C stretching of aromatic ring), 924(C-H in plane bending for aromatic ring); 650(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.84 (s, 3H, CH₃), 9.00(s,1H,NH),6.20-6.22(d,1H,CH),7.16-7.25(d,1H,CH),7.42-8.07(m,Ar-H); MS: m/z 475,447,395,371,331,316,161,159,144,119,104; Anal.Calcd. for C18H15Br2N5O: C, 45.31; H, 3.17; N, 14.68. Found: C, 45.37; H, 3.20; N, 14.71%.

Synthesis of 6,8-dibromo-3-({5-(2-hydroxyphenyl)-1-[(4-nitro Phenylamino)methyl]-4,5-dihydro-1H-pyrazol-3-yl}amino)quinazolin-4(3H)-one (7a1)

To a mixture of 6,8-dibromo-3-{[5-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]amino}quinazolin-4(3H)-one (6a) (0.005 mol) , 37% formalin (1 mL) and few drop of Acetic acid in ethanol (20 mL) was added drop wise appropriate p-nitro aniline (0.005 mol) with stirring over 15 min. The stirring was continued for 1h at room temperature and the reaction mixture then warmed for 15 min on a water bath. The mixture was poured into ice-cold water and stored in a refrigerator for 24 hr. The solid thus separated was filtered, washed with water, dried and recrystallized from appropriate solvent. Their melting points, yields and molecular formula are given in Table-1.

Yield: 81%; mp 170-172 ºC; IR (cm-1): 3476(O-H stretching of Hydroxy group) ,3350(N-H stretching of primary amine), 3076(C-H stretching of aromatic ring), 2918(CH₃ Str.), 1732(C=O stretching of keto group), 1670 (C=O stretching ring), 1609(C=N stretching of pyridine ring), 1449 (C=C stretching of aromatic ring), 879(C-H in plane bending for aromatic ring); 685(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.84 (s, 3H, CH₃), 9.72(s,1H,NH),4.47(s,1H,NH)6.57-6.91(t,1H,CH),7.16-7.26(d,1H,CH),7.40-8.11(m,Ar-H); ¹³C NMR (400 MHz, CDCl₃), 19.10,32.40,33.29,36.24,37.45, 40.32, 41.43, 43.05, 46.54, 48.15, 50.28, 51.44, 70.12, 101.12-101.15, 104.76, 105.25,123.25,129.23,144.12,154.66;MS:m/z 641, 623,613,561,520,327,285,150,121,104; Anal. Calcd. for C25H21Br2N7O4: C, 46.48; H, 3.29; N, 15.24. Found: C, 46.46; H, 3.32; N, 15.29%.

Synthesis of 6,8-dibromo-3-({5-(2-hydroxyphenyl)-1-[(4-chloro-Phenylamino)methyl ]-4,5-dihydro-1H-pyrazol-3-yl}amino)quinazolin-4(3H)-one (7a₂)

To a mixture of 6,8-dibromo-3-{[5-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]amino}quinazolin-4(3H)-one (6a) (0.005 mol) , 37% formalin (1 mL) and few drop of Acetic acid in ethanol (20 mL) was added drop wise appropriate p-chloro aniline (0.005 mol) with stirring over 15 min. The stirring was continued for 1h at room temperature and the reaction mixture then warmed for 15 min on a water bath. The mixture was poured into ice-cold water and stored in a refrigerator for 24 hr. The solid thus separated was filtered, washed with water, dried and recrystallized from appropriate solvent. Their melting points, yields and molecular formula are given in Table-1.

Yield: 79%; mp 168-169 ºC; IR (cm-1):3475(O-H stretching of Hydroxy group) ,3354(N-H stretching of primary amine), 3078(C-H stretching of aromatic ring), 2815(CH₃ Str.), 1734(C=O stretching of keto group), 1672 (C=O stretching ring), 1617(C=N stretching of pyridine ring), 1449 (C=C stretching of aromatic ring), 877(C-H in plane bending for aromatic ring); 686(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.88 (s, 3H, CH₃), 9.84(s,1H,NH),5.11(s,1H,NH),3.12-3.16(d,1H,CH),3.80-3.91(t,1H,CH),7.40-8.12(m,Ar-H); ¹³C NMR (400 MHz, CDCl₃) :19.40, 32.40, 33.99, 36.24, 37.78, 39.86, 40.41, 41, 43, 42.50, 43.05, 46.51, 47.55, 48.43, 50.28, 51.84, 72.12, 101.11, 101,26,103.20,105.25,123.25,129.23,144.12,156.99 MS:m/z 630, 612, 604, 550, 520, 491, 316, 299, 274, 139,127,104; Anal.Calcd. for C25H21Br2ClN6O2: C, 47.46; H, 3.35; N, 13.28. Found: C, 47.51; H, 3.38; N, 13.31%.

Table - 1: Physical data of synthesized compounds

Comp. No.	R	R¹	Mol. Formula	Colour	MP (⁰C)	Yield (%)
Comp. No.	R	R¹	Mol. Formula	Colour	MP (⁰C)	Yield (%)
1	---------	------	C₇H₅ Br₂NO₂	Reddish brown	202-204	73
2	---------	------	C₉H₅ Br₂NO₂	Orange	179-181	71
3	---------	------	C₉H₇ Br₂N₃O	Dirty Yellow	138-140	69
4	---------	------	C₉H₉ Br₂N₃O₂	Pale Yellow	126-128	74
5a		--	C₁₇H₁₁Br₂N₃O₃	Grey	152-154	70
5b	C₆H₅	--	C₁₇H₁₁Br₂N₃O₂	Pale Yellow	151-153	73
6a		--	C₁₇H₁₃Br₂N₅O₂	Brick red	234-236	69
6b	C₆H₅	--	C₁₇H₁₃Br₂N₅O	Yellowish brown	230-231	78
7a1			C₂₄H₁₉Br₂N₇O₄	Dirty Yellow	170-172	81
7a2			C₂₄H₁₉Br₂N₆O₂Cl	Brick red	168-169	79
7b1	C₆H₅		C₂₄H₁₉Br₂N₇O₃	Reddish Yellow	164-166	76
7b2	C₆H₅		C₂₄H₁₉Br₂N₆OCl	Brownish Yellow	162-163	78

Synthesis of 6,8-dibromo-3-({5-(phenyl)-1-[(4-nitro Phenylamino)methyl]-4,5-dihydro-1H-pyrazol-3-yl}amino)quinazolin-4(3H)-one (7b₁)

To a mixture of 6,8-dibromo-3-{[5-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]amino}quinazolin-4(3H)-one (6a) (0.005 mol), 37% formalin (1 mL) and few drop of Acetic acid in ethanol (20 mL) was added drop wise appropriate p-nitro aniline (0.005 mol) with stirring over 15 min. The stirring was continued for 1h at room temperature and the reaction mixture then warmed for 15 min on a water bath. The mixture was poured into ice-cold water and stored in a refrigerator for 24 hr. The solid thus separated was filtered, washed with water, dried and recrystallized from appropriate solvent. Their melting points, yields and molecular formula are given in Table-1.

Yield: 76%; mp 164-166 ºC; IR (cm-1):3323(N-H stretching of primary amine), 3076(C-H stretching of aromatic ring), 2811(CH₃ Str.), 1724(C=O stretching of keto group),1672 (C=O stretching), 1611(C=N stretching of pyridine ring), 1471(C=C stretching of aromatic ring), 917(C-H in plane bending for aromatic ring); 687(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.89 (s, 3H, CH₃), 9.81(s,1H,NH),5.10(s,1H,NH)3.86-3.96(t,1H,CH),3.11-3.16(d,1H,CH),7.41-8.12(m,Ar-H);¹³CNMR (400MHz, CDCl₃) : 18.18, 32.40, 33.99, 36.04,37.05, 39.66,40.41,41.05, 42.12, 46.51, 47.51, 48.72, 49.51, 72.03, 100.01-100.07, 103.70, 104.76, 105.75, 123.95, 129.23, 144.02, 145.02. MS: m/z 625, 608, 597, 545, 331, 309, 294, 150, 138, 121, 104; Anal.Calcd. For C25H21Br2N7O3: C, 47.87; H, 3.37; N, 15.63. Found: C, 47.84; H, 3.41; N, 15.83%.

Synthesis of 6,8-dibromo-3-({5-(phenyl)-1-[(4-chloro-Phenylamino)methyl]-4,5-dihydro-1H-pyrazol-3-yl}amino)quinazolin-4(3H)-one (7b₂)

To a mixture of 6,8-dibromo-3-{[5-(phenyl)-4,5-dihydro-1H-pyrazol-3-yl]amino}quinazolin-4(3H)-one (6b₁) (0.005 mol), 37% formalin (1 mL) and few drop of Acetic acid in ethanol (20 mL) was added drop wise appropriate p-chloro aniline (0.005 mol) with stirring over 15 min. The stirring was continued for 1h at room temperature and the reaction mixture then warmed for 15 min on a water bath. The mixture was poured into ice-cold water and stored in a refrigerator for 24 hr. The solid thus separated was filtered, washed with water, dried and recrystallized from appropriate solvent. Their melting points, yields and molecular formula are given in Table-1.

Yield: 78%; mp 162-163 ºC; IR (cm-1): 3364(N-H stretching of primary amine), 3028(C-H stretching of aromatic ring), 2928(CH₃ Str.), 1665(C=O stretching of keto group), 1586(C=N stretching of pyridine ring), 1466,1451 (C=C stretching of aromatic ring), 909(C-H in plane bending for aromatic ring); 751(C-Cl stretching of aromatic ring), 654(C-Br stretching of aromatic ring); 1H NMR (CDCI₃) δ ppm: 1.91 (s, 3H, CH₃), 9.89(s,1H,NH),5.09(s,1H,NH)3.81-3.90(t,1H,CH), 3.10-3.14(d,1H,CH),7.43-8.19(m,Ar-H); ¹³C NMR(400 MHz CDCl₃): 18.44,32.40,33.99,36.04, 37.78,39.66,40.41, 41.03, 42.12,46.05, 47.55, 48.43, 49.04, 49.47, 74.44, 100.00-100.17, 101.11, 103.07, 104.76, 105.25, 129.13, 144.12. MS: m/z 614,586,534,283,139,127,104; Anal. Calcd. for C25H21Br2ClN6O: C, 48.69; H, 3.43; N, 13.63. Found: : C, 48.37; H, 3.49; N, 13.59%.

Antifungal activity

All these compounds screened for antifungal activity against Aspergillus flavus,Candida albicans, and Aspergillus niger following agar diffusion method of assay.Solutions of the test compounds were prepared by dissolving 10mg each in dimethyl formamide (10ml-analytical grade). A reference standard (1mg/ml conc.) was prepared by dissolving 10mg of ketoconazole in 10ml. of dimethyl formamide (Analar grade) Further, dilution was made with dimethyl formamide itself to obtain a solution of 100 mg/ml.

The potato-dextrose-agar medium was sterilized by autoclaving at 121⁰ C (15 lb/sq-inch) for 15 minutes. The Petri-plates, tubes and flask with cotton plugs were sterilized in hot-air oven at 150⁰C, for an hour. In each sterilized Petri-plate, about 30ml. of molten potato-dextrose – agar medium inoculated with respective fungus (6ml. of inoculum in 300ml of potato-dextrose medium) was added separately. After solidification of the medium at room temperature, three discs of 6 mm diameter were made in each plate with sterile borer. Accurately 0.1ml (1000 mg/ml conc.) of test solution was transferred to the discs aseptically and labeled, accordingly.

The reference standard, 0.1ml (100 mg/ml conc.) was also added to the cups in each plate. The plates were kept undisturbed at room temperature for 2 hours, at least to allow the solution to diffuse properly into the potato-dextrose – agar medium. Then the plates were incubated at 20⁰C for 48 hours. The diameter of the zone of inhibition was read with the help of an “antibiotic zone reader” The experiment were performed in triplicate in order to minimize the errors²⁹. The results are presented in Tables 2.

Table – 2: Antifungal activity of Pyrazole derivatives ((7a_1-2, 7b_1-2).

Sl. No.	Compd. No.	Diameter of zone of inhibition mm
Sl. No.	Compd. No.	A. flavus	*P. aeruginosa*	*C. albicans*	*A. niger*
1	7a1	25	26	28	25
2	7a₂	18	14	14	11
3	7b1	28	31	26	27
4	7b2	19	18	18	14
5	Solvent (DMF)	-	-	-	-
6	Ketoconazole	-	-	33	31

Table – 3: Anthelmintic Activity of Pyrazole derivatives (7a_1-2, 7b_1-2).

Group	Treatment	Concentration (mg/ml)	Time taken for paralysis (min)	Time taken for Death (min)
I	Control	----	----		-----
II	Albendazole	10	34.66 ± 0.72		63.83 ± 0.79
III	Piperazine citrate	15	21.83 ± 1.42		A
IV	7a1	10	3.15 ± 0.13		14.75 ± 0.33
V	7a2	10	25.37 ± 0.71		45.00 ± 0.41
VI	7b1	10	13.15 ± 0.13		34.75 ± 0.33
VII	7b2	10	50.63 ± 0.42		94.17 ± 0.91

Evaluation of anthelmintic activity

The anthelmintic activity will be evaluated on adult Indian earthworm Pheritima posthuma due to its anatomical and physiological resemblance with the intestinal roundworm parasites of human beings ²¹. The method of Nirmal et al. 2007 ²², Dash et al. 2003²³ and Ghosh et al. 2005²⁴ was followed for the study. The Pyrazole derivatives (7a1-2, 7b1-2) in concentrations of 10 mg/ml were suspended in 1% gum acacia in normal saline (vehicle). Albendazole (10 mg/ml) and Piperazine citrate (15 mg/ml) in 1% gum acacia in normal saline were used as reference standards for comparison of activity. Six groups of approximately equal sized Indian earthworms consisting six earthworms in each group were released into 50 ml of desired formulation. Group I (control) treated with only vehicle, group II treated with 10 mg/ml Albendazole, group III treated with 15 mg/ml Piperazine citrate, group IV, V,VI and VII were treated with 10, mg/ml of Pyrazole derivatives (7a_1-2, 7b_1-2). Observations were made for the time taken to paralyze and/or death of individual worms. Paralysis was said to occur when the worms do not revive even in normal saline. Death was concluded when the worms lose their motility followed with fading away of their body colour. The results are depicted in table 3.

RESULT:

All the newly synthesized compounds (7a₁,7a₂,7b₁ & 7b₂)-series were screened for their antifungal (Aspergillus flavus,Candida albicans, and Aspergillus niger) and anthelmintic (Pheritima posthuma) activity in-vitro at the doses of 100 mg in 0.1ml of DMF. The most promising compound against tested fungi are 7a₁and7b₁while other compounds showed mild to moderate activity except some compounds 7a₂ & 7b₂ which does not produce any zone of inhibition against tested fungi. The difference spectrum of activity against fungi may be due to chemical modification of test compounds which relate to the Structure Activity Relationship (SAR).

In case of anthelmintic activity it was observed that the 6,8-dibromo-3-({5-(2-hydroxyphenyl)-1-[(4-nitro Phenylamino)methyl]-4,5-dihydro-1H-pyrazol-3-yl}amino) quinazolin-4(3H)-one (7a1) was endowed with anthelmintic property. The tested samples were found to be lethal to the worms at the tested level of concentrations. The activity was also found to be concentration dependent for all different samples tested. Potency of the test samples was found to be inversely proportional to the time taken for paralysis/death of the worms. The activities were comparable with the reference drugs Albendazole and Piperazine citrate.

DISCUSSION:

The results in the Table – 2 showed that compound nos. 7a₁and7b₁registered a zone of inhibition of 28mm, 26mm and 7a₂ & 7b₂ shows zone of inhibition 14 mm, 18 mm against Candida albicans while the standard drug ketoconazole possess 33mm. However, compound nos. 7a₁and7b₁exhibited the zone of inhibition between 25mm, 27mm and 7a₂ & 7b₂ shows zone of inhibition 11-14 mm against against A. Niger. At the same time the standard drug ketoconazole showed a zone of inhibition of 31mm against the same microorganism. The most promising compound against tested fungi are 7a₁and7b₁while other compounds showed mild to moderate activity except some compounds 7a₂ & 7b₂ which does not produce any zone of inhibition against tested fungi. The results in the Table – 3 shows that compound 7a1was endowed with anthelmintic property.

CONCLUSION:

The difference spectrum of activity against fungi may be due to chemical modification of test compounds which relate to the Structure Activity Relationship (SAR).

In anthelmintic study it can be concluded that the 6,8-dibromo-3-({5-(2-hydroxyphenyl)-1-[(4-nitroPhenyl amino)methyl]-4,5-dihydro-1H-pyrazol-3-yl}amino) quinazolin-4(3H)-one (7a₁) possesses profound anthelmintic activity against tested worm species. Further studies are needed to establish the mechanism of action for the concerned activity

REFERENCES:

1. Eicher T, Hauptmann S. The Chemistry of Heterocycles: Structure, Reactions, Syntheses, and Applications. 2^nd ed. Wiley-VCH; 2003.

2. John J, Talley, Donald J, Rogier Jr, both of Louis St, Mo. G.D. Searle & Co., Skokie. 1995, Pt No: 5, 434, 178.

3. Pimerova EV, Voronina EV, J. Pharm. Chem 2001; 35: 18-20.

4. Janus SL, Magdif AZ, Erik BP, N.Claus; Chem.1999; 130: 1167-1174.

5. Park HJ, Lee K, Park S, Ahn B, Lee JC, Cho HY, Lee KE. Bio Org. Med. Chem. Lett. 2005; 15: 3307-3312.

6. Bouabdallah I, M’barek LA, Zyad A, Ramadan A, Zidane I, Melhaoui A, Nat.Prod. Res.2006; 20: 1024-1030.

7. Yildirim I, Ozdemir N, Akçamur Y, Dinçer M, Andaç O, Acta Cryst. 2005; E61: 256- 258.

8. Bailey DM, Hansen PE, Hlavac AG, Baizman ER, Pearl J, Defelice AF, Feigenson ME. J. Med. Chem. 1985; 28: 256-260.

9. Chu CK, Cutler J. J. Heterocycl. Chem. 1986; 23: 289-319.

10. Amr AEI, GES, Abdel-Latif NA, Abdlla MM. Acta Pharm, 2006; 56: 1203.

11. Chimichi S, Boccalini M, Hassan MMM, Viola G. Dall Acqua F, Curini M,Tetrahedron. 2006;62: 90.

12. Henry TA.The Plant Alkoloids, Anmol Publicaion Pvt. Ltd. 1999.

13. Kobayashi, Hisafumi, Kato, Motto; Nitani, Fumio. Chem Abstr 1989;106: 297008/g.

14. Nugent Richard A, Marphy Meghan et. al., J. Med. Chem. 1993; 36 (1): 134.

15. Hantoon MA, Minnesota Medicine. 2001:84: 102.

16. Zhang X, Li X., Allan GF, Sbriscia T, J. Med. Chem. 2007; 50 (16): 3857.

17. Abunada NM, H.M. Hasaneen, N.G. Kandile, Miqdad OA, Molecules, 2008; 13: 1501.

18. Meepowpan P, Kittakoop P, Sobhon P, Bartlett A and Whitfield PJ. In vitro screening for anthelmintic and antitumour activity of ethnomedicinal plants from Thailand. J. ethanopharmaology. 2009; 123:479-482.

19. Anthelmintic activity of benzimidazole derivatives against Toxocara canis second-stage larvae and Hymenolepis nana adults. Acta Tropica. 2009; 109:232-235.

20. Navarro AM, Torres BN, Compos AH, Arteche OS, Castillo R, Morales SR, Mulia LY and Luis FH.

21. Thorn GW, Adams RD, Braunwald E., Isselbacher KJ and Petersdorf R.G. Harrisons Principal of Internal Medicine, McGraw Hill Company; 1977,1088.

22. Nirmal SA, Nikalye AG, Jadav RS and Tambe VD, Anthelmintic activity of Martynia annua roots. Indian Drugs. 2007,44(10): 772-773.

23. Dash GK, Mishra S, Panda A, Patro CP and Ganapathy S, Anthelmintic activity of Evolvulus nummularius. Indian Journal of Natural Products. 2003,19(3):24-25.

24. Ghosh T, Maity TK, Bos A and Dash GK, Anthelmintic activity of Bacopa monierri. Indian Journal of Natural Products. 2005,21(2):16-19..

Received on 01.12.2013 Modified on 11.12.2013

Asian J. Research Chem 7(1): January 2014; Page 92-98