INTRODUCTION:

Considerable attention has been focused on Pyrazolines and substituted Pyrazolines due to their interesting biological activities. They have found to possess anti-fungal¹, anti-depressant^2-7, anti-convulsant⁸, anti-inflammatory^9-12, anti-bacterial^13-14, anti-cancer^15-16, anti-oxidant^17-18, anti-pyretic¹⁹, anti-neoplastic activities^20-21, anti-viral²², anti-amoebic^23-24, Acaricidal agro chemical fungicides or insecticides²⁵, anti-cholinergic^26-27, anti-diabetic²⁸, anti-HIV^29-32, anti-malarial³³, Anesthetic³⁴, Anaxiolytic³⁵, anti-parasitic³⁶, anti-allergic³⁷, anti-microbial^38-40, anti-tuberculosis^41-44,Tyrosinase inhibitor⁴⁵, Blue photo luminescence and electro luminescence⁴⁶, Food and chemical toxicology⁴⁷, Herbicidal^48-50, Hypoglycemic⁵¹, Hypotensive⁵², immuno suppressive⁵³, anti-tumor^54-55.

Moreover, many selectively chloro-substituted organic compounds show peculiar pharmacological and agrochemical properties. The work reported herein was aimed at the preparation of some new pyrazoline derivatives with anticipated biological activities.

EXPERIMENTAL:

General:

All chemicals were used of A.R. grade (either of B.D.H. or Excel-R or Extra pure E. Merck quality). The structures of the compounds were determined by elemental analysis, IR and NMR spectral data. All melting points were measured on an electro thermal melting point apparatus and are uncorrected. The infrared spectra were recorded in potassium bromide disks on a Pye Unicam SP 3-300 or a Shimadzu FT-IR 8101 PC infrared spectrophotometer. The ¹H –NMR (200 MHz) and ¹³C-NMR (50 MHz) spectra were recorded in DMSO-d₆ on a Varian Mercury VX 200 NMR using TMS as the internal reference. Mass spectra were measured on a GCMS-QP 1000 EX spectrophotometer at 70 eV. Purity of the compounds is checked on T.L.C. using Silica Gel-G. Elemental analysis is performed on Carlo-Erba1108 analyzer

Synthesis of Ethyl-2-[2, 3-dichloroanilido] Ethanoate [1]:

A mixture of 2, 3-dichloroaniline (10ml) and diethylmalonate (20ml) was refluxed for forty five minutes in a round bottomed flask fitted with an air condenser of such a length (14") that ethanol formed escaped and diethylmalonate flowed back into the flask. Contents were cooled, ethanol (30 ml) was added, when malon-2,3dichlorodianilide separated out. It was filtered under suction. The filtrate was poured on to crushed ice (Ca160g) and stirred when ethyl-2-(2, 3-dichloroanilido) ethanoate precipitated as green mass. On recrystallization from aqueous ethanol (50%), ester was obtained as white crystals. Yield: 81%, M. P.: 88⁰C, M. W.: 276. Anal. Calculation for C₁₁H₁₁ N₁O₃ Cl₂: Found: C 39.20, H: 03.24, O: 14.25, N: 4.14, Cl: 21.09, Calcd. C: 39.21, H: 03.26, O: 14.26, N: 04.15, Cl: 21.16. IR [KBr] V_max Cm^-1 : 1665-1660 [C=O diketone], 1290 [-C-O- Ester], 760-755 [2,3 disubstituted benzene], 1250 [C-Cl Stretching], 1590, 1520 , 1440 [C=C Ring stretching], 3150 [N-H Stretching], 3040[C-H aromatic], 1330-1322 [C-H Stretching]. PMR (DMSO): δ 4.42 (2H, s, CO-CH₂-CO), 4.0 (2H, s, NH₂), 7.4-8.6 (3H, m, Ar-H), 9.2 (1H, s, CO-NH D₂O exchangeable), 10.6 [1H, s, Ar-NH D₂O exchangeable].

Synthesis of Ethyl-2-[(N-acetyl) 2, 3- dichloroanilido] ethanoate [2]:

Acetyl chloride (4.74 gm; 0.06 mol), dioxane (6 ml), Ethyl-2-(2,3-dichloroanilido) ethanoate (16.56 gm; 0.06 mol) and Triethylamine (5.7 gm; 0.06 mol) were placed in a round bottomed flask carrying reflux condensor having calcium chloride guard tube. The contents were heated on a boiling water bath for two hours and kept over night when triethylamine hydrochloride separated. It was filtered under suction and the filtrate was poured on to crushed ice (Ca180 g) and stirred when ethyl-2-[(N-acetyl) 2, 3-dichloroanilido] ethanoate separated or solid. It was filtered under suction, dried and purified by recrystallisation from aqueous methanol (1:1) in white crystals. Yield = 75.4 %, MP = 90°C Anal. calculation for C₁₃H₁₃ O₄ N₁ Cl₂ : [FW = 318 ] , Calculated: N 02.95 , C 45.64, H 03.38 , O 13.50 , Cl 15.00 , Found : N 02.94, C 45.62 , H 03.37 , O 13.52 , Cl 15.02. IR [KBr] V_max cm^-1 : 1720 [ C=O diketone ], 1300 [ -C-O- Ester], 762[ 2,5- disubstituted benzene ], 1090 [ C-Cl Stretching ], 1590, 1520 , 1440 [C=C Ring stretching ], 3160 [N-H Stretching], 3040[C-H aromatic], 1330-1322 [C-H Stretching ]. PMR (DMSO): δ 4.44 [2H, s, CO-CH₂-CO], 4.1 [2H, s, NH₂], 7.2-8.5 [3H, m, Ar-H], 9.4 [1H, s, CO-NH D₂O exchangeable], 10.8 [1H, s, Ar-NH D₂O exchangeable].

Synthesis of Ethyl-2-[(N-acetyl) 2, 3-dichloroanilido] acetohydrazide [3]:

Ethyl-2-[(N-acetyl) 2, 3-dichloroanilido] ethanoate (9.54 gm; 0.03 mol), ethanol (10 ml) and hydrazine hydrate (15 ml; 80%) were mixed together and stirred for thirty five minutes. Ethyl-2-[(N-acetyl) 2, 3-dichloroanilido] acetohydrazide was filtered under suction and recrystallised from ethanol in white crystals. Yield; 79%, MP = 177°C, MW 304: Anal. calculation for C₁₁ H₁₁N₃ O₃ Cl₂ : Calculated ; N 09.04 ,C 41.32 ,H 03.01 ,O 10.33, Cl 15.28, Found; N 09.01, C 41.30, H 03.00, O 10.31, Cl 15.27 . IR [KBr] V_max cm^-1: 3160 [N-H Stretching], 3048 [C-H aromatic], 1660 [C=O diketone], 1432 [C-Cl aromatic], 1595, 1520, 1445 [ C=C ring stretching]. PMR (DMSO): δ 4.44 (2H, s, CO-CH₂-CO), 4.1 (2H, s, NH₂), 7.2-8.5 (3H, m, Ar-H), 9.4 (1H, s, CO-NH D₂O exchangeable), 10.7 (1H, s, Ar-NH D₂O exchangeable).

Mono cyanoethylation of 2, 3-dichloroaniline [4]:

A 250 ml three necked flask equipped with a stirrer, reflux condenser and thermometer was charged with 2, 3-dichloro aniline (0.1mol, 16.2g), acrylonitrile (0.1mol, 10.6 g) and Cupric acetate monohydrate (1.02g, 4% by weight of the amine). The mixture was stirred and refluxed on boiling water bath for three hours. The dark mixture was then transferred to a 250 ml distilling flask fitted with a 15.2 cm modified vigorous column and the unchanged acrylonitrile was first collect at 100 mm (water pump). The distillation was continued and the unchanged 2, 3-dichloro aniline B.P. 252⁰C/0.5mm was recovered. The N-Cyanoethyl-2, 3-dichloroaniline was obtained as light yellow colored viscous liquid at 175-176⁰C/mm which solidified after keeping overnight. Yield: 15.7g (97%), M.P. 82⁰C

Preparation of Cinnamoyl Chloride [5]:

Cinnamic acid (10 g, 0.067mol) and Thionyl Chloride (12.0 ml) were taken in a round bottomed flask fitted with a reflux condenser carrying a calcium chloride guard tube. The contents were refluxed on a water bath for two and half hours in a fume cupboard until the evolution of HCl gas ceased from the guard tube. After cooling liquid was carefully transferred to a claisen flask and distilled under reduced pressure when unreacted thionyl chloride distilled over first. Cinnamoyl chloride was collected at 165-166⁰C/ 18-20mm pressure.

Synthesis of N-Cinnamoyl –N-2’-Cyanoethyl -2, 3-dichloroaniline [6]:

Solution of cinnamoyl chloride (3.5 g, 0.02 mol), dioxane (2ml), N-2’-cyanoethyl -2, 3-dichloro aniline (7.90g, 0.02 mol) and triethylamine (2.1 g) were placed in a round bottomed flask having a Liebig condenser carrying calcium chloride guard tube. The contents were heated for two hours on a boiling water bath. On keeping overnight triethylamine hydrochloride separated as solid. It was filtered and contents concentrated when crystals separated out. Two crystallizations from ethanol gave shining white needles. Yield: 55 %, M.P.: 156⁰C, Anal. Calculated for C₁₈ H₁₄ Cl₂ N₂O; M.W. 345; N: 4.5, Cl: 11.3 ; found N: 4.3 , Cl : 11.2 % , IR[KBr] V_max Cm^-1 : 3280-3050 (C-H stretching , aromatic ), 2955 and 2890 (C-H Stretching, aliphatic (asymmetric ) and C-H stretching , aliphatic (symmetric), 2215( C-N stretching), 1655(C=C stretching , benzene ring), 1645 C=O (stretching, tertiary amide ), 1615, 1575, 1455, (C=C ring stretching), 1050, 750, (2, 3-disubstituted benzene).

Synthesis of 1-[(2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7]:

A mixture of N-cinnamoyl-N-2’-cyanoethyl -2, 3-dichloroaniline (0.345 g; 0.001 mol), Ethyl-2-[(N-acetyl) 2, 3-dichloroanilido] acetohydrazide (0.304g; 0.001 mol), dioxane (3 ml), and glacial acetic acid (2 drops) was

refluxed for five hours. The solid which separated during the course of heating was filtered under suction and purified by washing thrice with hot ethanol, when the pyrazoline was obtained as yellow needles. Yield: 61%, M.P.: 258⁰C, M.W.: 631, Anal. Calculated for C₂₉H₂₃Cl₄N₅O₃Cl: 13.7; N: 6.8, found Cl: 13.6, N: 6.6%. U.V. [(λ ^{Et OH}_{M ax}nm), log ε]: 214.3 (4.94), 318.9 (4.78). IR[KBr] V_max Cm^-1 : 3300-2860 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching, aromatic , (iii) C-H stretching , aliphatic ], 2240 (C N stretching ), 1660 [ C=O and N-H (amide)] , 1590 ( C=N stretching ), 1580, 1470, 1420 (C=C ring stretching , aromatic ), 1040, 820, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.22-2.46 (2H, s, CH₂), 3.4-3.9 (3H, s, CH₃), 4.12-4.40(1H, s, NH), 6.95-7.40 (13H, m, ArH). 3.17 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.65 H_Z, C₄- H_A of pyrazoline ring). 3.92 (1H, dd J_MA= 17.80 Hz, J_MX = 13.60 Hz, C₄-H_Mof pyrazoline ring) , 4.70 (1H, d, J = 16.13 Hz COCH geminal proton ), 5.58 (1H, dd J_MX12.80 H_{Z ,} J_{AX =}4.60 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 179.58 (C=O), 159.78 (C=N), 141.05, 137.65, 134.45, 131.85 (4C, ArC’s), 132.48, 130.55, 128.66, 125.77, 112.28 (5C, Ar CH’s), 62.67 (CH_2,ester), 61.83 (C-5, pyrazoline), 46.95 (C-4, pyrazoline), 18.86 (CH₃). –MS-FAB⁺: m/z: 631 [M]. Synthetic sequence for new pyrazolines has been outlined in scheme-I.

1-[(2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7a]:

Yield: 61%, M.P.: 258⁰C, M.W.: 631, Anal. Calculated for C₂₉H₂₃Cl₄N₅O₃Cl: 13.7; N: 6.8, found Cl: 13.6, N: 6.6%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 214.3 (4.94), 318.9 (4.78). IR[KBr] V_max Cm^-1 : 3300-2860 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2240 (C N stretching ), 1660 [ C=O and N-H (amide)] , 1590 ( C=N stretching ), 1580, 1470, 1420 (C=C ring stretching , aromatic ), 1040, 820, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.22-2.46 (2H, s, CH₂), 3.4-3.9 (3H, s, CH₃), 4.12-4.40(1H, s, NH), 6.95-7.40 (13H, m, ArH). 3.17 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.65 H_Z, C₄- H_A of pyrazoline ring). 3.92 (1H, dd J_MA= 17.80 Hz, J_MX = 13.60 Hz, C₄-H_Mof pyrazoline ring) , 4.70 (1H, d, J = 16.13 Hz COCH geminal proton ), 5.58 (1H, dd J_MX12.80 H_{Z ,} J_{AX =}4.60 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 179.58 (C=O), 159.78 (C=N), 141.05, 137.65, 134.45, 131.85 (4C, ArC’s), 132.48, 130.55, 128.66, 125.77, 112.28 (5C, Ar CH’s), 62.67 (CH_2,ester), 61.83 (C-5, pyrazoline), 46.95 (C-4, pyrazoline), 18.86 (CH₃). –MS-FAB⁺: m/z: 631 [M].

1- [(o-methyl) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7b]:

Yield: 41%, M.P.: 270⁰C, M.W.: 646, Anal. Calculated for C₃₀ H₂₅Cl₄ N₅O_3,N: 4.4; found N: 4.1, Cl: 9.0; found Cl: 9.1 %. U.V. [(λ ^Et
OH_{M ax}nm), log ε]: 214.6(4.90), 319.4 (4.82). IR[KBr] V_max Cm^-1 : 3300-2890 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond )(II) C-H stretching , aromatic , (iii)C-H stretching,aliphatic], 2242(C N stretching ), 1650 [ C=O and N-H (amide)] , 1580 ( C=N stretching ), 1585, 1478, 1430 (C=C ring stretching , aromatic ), 1045, 822, ( C-Cl stretching , 2,3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.23-2.48 (2H, s, CH₂), 4.16-4.30(1H, s, NH), 6.90-7.45 (13H, m, ArH). 3.10 (1H, dd, J_{AM =}16 H_Z, J_AX = 4.60H_Z, C₄- H_A of pyrazoline ring). 3.98 (1H, dd J_MA= 17.90 Hz, J_MX = 13.80 Hz, C₄-H_Mof pyrazoline ring) , 4.60 (1H, d, J = 16.43 Hz COCH geminal proton ), 5.70 (1H, dd J_MX12.40 H_{Z ,} J_{AX =}4.50 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 181.58 (C=O), 158.74 (C=N), 143.07, 136.54, 133.40, 130.74 (4C, ArC’s), 131.47, 130.36, 126.62, 124.70, 114.31 (5C, Ar CH’s), 63.66 (CH_2,ester), 60.81 (C-5, pyrazoline), 46.91 (C-4, pyrazoline), 18.82 (CH₃). –MS-FAB⁺: m/z: 646 [M].

1- [(m-methyl) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7c]:

Yield: 53%, M.P.: 262⁰C, M.W.: 646, Anal. Calculated for C₃₀H₂₅Cl₄N₅O_3,Cl: 11.7; N: 5.7, found Cl: 11.5, N: 5.3%. U.V. [(λ ^Et
OH_{M ax}nm), log ε]: 212.2 (4.92), 318.6 (4.78). IR[KBr] V_max Cm¹ : 3300-2950 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2240(C N stretching ), 1670 [ C=O and N-H (amide)] , 1575 ( C=N stretching ), 1560, 1430, 1410 (C=C ring stretching , aromatic ), 1050, 815, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.32-2.56 (2H, s, CH₂), 4.35-4.55(1H, s, NH), 6.40-7.20 (13H, m, ArH). 3.10 (1H, dd, J_{AM =}17 H_Z, J_AX = 4.55 H_Z, C₄- H_A of pyrazoline ring). 3.88 (1H, dd J_MA= 17.70 Hz, J_MX = 13.55 Hz, C₄-H_Mof pyrazoline ring) , 4.68 (1H, d, J = 16.16 Hz COCH geminal proton ), 5.66 (1H, dd J_MX12.60 H_{Z ,} J_{AX =}4.40 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 167.56 (C=O), 154.61 (C=N), 143.01, 136.62, 133.43, 130.85 (4C, ArC’s), 132.48, 130.55, 128.66, 125.77, 112.28 (5C, Ar CH’s), 62.67 (CH_2,ester), 61.83 (C-5, pyrazoline), 45.92 (C-4, pyrazoline), 18.84 (CH₃). –MS-FAB⁺: m/z: 646 [M].

1- [(p-methyl) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7d]:

Yield: 58%, M.P.: 248⁰C, M.W.: 666.5, Anal. Calculated for C₃₀ H₂₅Cl₄ N₅O_3,Cl: 12.4; N: 6.1, found Cl: 12.1, N: 5.9%. U.V. [(λ ^{Et OH}_Maxnm), log ε]: 227.3 (4.96), 319.6 (4.70). IR[KBr] V_max Cm^-1 : 3300-3040 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2250(C N stretching ), 1620 [ C=O and N-H (amide)] , 1570 ( C=N stretching ), 1550, 1460, 1430 (C=C ring stretching , aromatic ), 1040, 825, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.14-2.41 (2H, s, CH₂), 4.28-4.35(1H, s, NH), 6.80-7.60(13H, m, ArH). 3.28 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.61 H_Z, C₄- H_A of pyrazoline ring). 3.87 (1H, dd J_MA= 17.79 Hz, J_MX = 13.58Hz, C₄-H_Mof pyrazoline ring) , 4.68 (1H, d, J = 16.45 Hz COCH

Table-I: (Unsubstituted / Substituted) 1-[(2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline

CS. No.	R	Color	M.P. (˚C)	Yield (%)	M.W.	Molecular Formula
7a.	H	Yellow	258	61	631	C₂₉H₂₃Cl₄ N₅O₃
7b.	CH₃(o)	Cream	270	41	646	C₃₀H₂₅Cl₄ N₅O₃
7c.	CH₃(m)	Light Yellow	262	53	646	C₃₀H₂₅Cl₄ N₅O₃
7d.	CH₃(p)	Light Yellow	248	58	666.5	C₃₀H₂₅Cl₄ N₅O₃
7e.	Cl(o)	white	262	49	666.5	C₂₉H₂₂Cl₅ N₅O₃
7f.	Cl(m)	Light Yellow	266	60	666.5	C₂₉H₂₂Cl₅ N₅O₃
7g.	Cl(p)	Cream	261	64	666.5	C₂₉H₂₂Cl₅ N₅O₃
7h.	O-CH₃(o)	Yellow	249	67	662	C₃₀H₂₅Cl₄ N₅O₄
7i.	O-CH₃(m)	White	251	71	662	C₃₀H₂₅Cl₄ N₅O₄
7j.	O-CH₃(p)	Cream	264	73	662	C₃₀H₂₅Cl₄ N₅O₄
7k.	F(p)	Yellow	246	52	650	C₂₉H₂₂Cl₄ N₅O₃F₁
7l.	Br(o)	Dark brown	259	59	711	C₂₉H₂₂Cl₄N₅O₃Br
7m.	O-C₂H₅ (o)	L. Brown	269	63	676	C₃₁H₂₇Cl₄ N₅O₄
7n.	O-C₂H₅ (m)	Brown	255	65	676	C₃₁H₂₇Cl₄ N₅O₄
7o.	O-C₂H₅ (p)	Brown	245	61	676	C₃₁H₂₇Cl₄ N₅O₄
7p.	CO₂H (o)	Brown	258	69	676	C₃₀H₂₃Cl₄ N₅O₅
7q.	CO₂H (m)	Brown	250	64	676	C₃₀H₂₃Cl₄ N₅O₅
7r.	CO₂H (p)	L. brown	254	52	676	C₃₀H₂₃Cl₄ N₅O₅
7s.	Br(m)	Brown	247	57	711	C₂₉H₂₂Cl₄N₅O₃Br
7t.	Br(p)	Brown	252	49	711	C₂₉H₂₂Cl₄N₅O₃Br

All compounds gave satisfactory elemental analysis.

geminal proton ), 6.11 (1H, dd J_MX13.30 H_{Z ,} J_{AX =}4.65 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 174.55 (C=O), 157.77 (C=N), 139.15, 135.65, 133.44, 131.80 (4C, ArC’s), 131.42, 129.85, 126.62, 124.64, 111.17(5C, Ar CH’s), 64.61 (CH_2,ester), 62.81 (C-5, pyrazoline), 45.92 (C-4, pyrazoline), 17.93 (CH₃). –MS-FAB⁺: m/z: 666 [M].

1-[(o-chloro) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7e]:

Yield: 49%, M.P.: 262⁰C, M.W.: 666.5, Anal. Calculated for C₂₉H₂₂Cl₅N₅O_3,Cl: 13.0; N: 5.1, found Cl: 13.1, N: 4.9%. U.V. [(λ ^Et
OH_{M ax}nm), log ε]: 215.5 (5.10), 319.2 (5.16). IR[KBr] V_max Cm^-1 : 3300-3110[broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2290(C N stretching ), 1680 [ C=O and N-H (amide)] , 1540 ( C=N stretching ), 1530, 1490, 1440 (C=C ring stretching , aromatic ), 1080, 890, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 3.10-3.18 (2H, s, CH₂), 4.19-4.55(1H, s, NH), 6.87-7.20 (13H, m, ArH). 3.10 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.62 H_Z, C₄- H_A of pyrazoline ring). 4.05 (1H, dd J_MA= 18.10 Hz, J_MX = 13.90 Hz, C₄-H_Mof pyrazoline ring) , 4.60 (1H, d, J = 16.19 Hz COCH geminal proton ), 5.45 (1H, dd J_MX13.15 H_{Z ,} J_{AX =}5.10 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 164.79 (C=O), 154.72 (C=N), 147.22, 143.60, 138.44, 132.83 (4C, ArC’s), 130.79, 128.85, 123.63, 121.72, 115.26(5C, Ar CH’s), 64.60 (CH_2,ester), 60.92 (C-5, pyrazoline), 47.15 (C-4, pyrazoline), 19.10(CH₃). –MS-FAB⁺: m/z: 666 [M], 667[M+1].

1- [(m-chloro) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7f]:

Yield: 60%, M.P.: 266⁰C, M.W.: 666.5, Anal. Calculated for C₂₉H₂₂Cl₅N₅O_3, Cl: 16.0; N: 6.3, found Cl: 16.2, N: 6.1%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 214.6 (4.97), 322.4 (4.81). IR[KBr] V_max Cm^-1 : 3300-3120 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2240(C N stretching ), 1658 [ C=O and N-H (amide)] , 1605 ( C=N stretching ), 1570, 1460, 1430 (C=C ring stretching , aromatic ), 1070, 830, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.58-2.87 (2H, s, CH₂), 4.35-4.62(1H, s, NH), 7.10-7.55 (13H, m, ArH). 3.34 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.70 H_Z, C₄- H_A of pyrazoline ring). 4.15 (1H, dd J_MA= 17.90Hz, J_MX = 13.20 Hz, C₄-H_Mof pyrazoline ring) , 4.60 (1H, d, J = 16.44 Hz COCH geminal proton ), 5.55 (1H, dd J_MX13.30 H_{Z ,} J_{AX =}4.70 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 178.57 (C=O), 155.65 (C=N), 144.11, 138.64, 135.44, 132.82 (4C, ArC’s), 131.88, 130.15, 126.60, 123.80, 116.26(5C, Ar CH’s), 61.66 (CH_2,ester), 59.95(C-5, pyrazoline), 47.93 (C-4, pyrazoline), 18.95(CH₃). –MS-FAB⁺: m/z: 666 [M], 667 [M+1].

1- [(p-chloro) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7g]:

Yield: 64%, M.P.: 261⁰C, M.W.: 666.5, Anal. Calculated for C₂₉H₂₂Cl₅N₅O_3,Cl: 17.0; N: 6.7, found Cl: 17.2, N: 6.5%. U.V. [(λ ^{Et OH}_{M ax}nm), log ε]: 216.3 (5.20), 340.6 (4.88). IR[KBr] V_max Cm^-1 : 3300-2960 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2290(C N stretching ), 1680 [ C=O and N-H (amide)] , 1620 ( C=N stretching ), 1575, 1465, 1415 (C=C ring stretching , aromatic ), 1035, 825, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.86-3.10 (2H, s, CH₂), 4.19-4.45(1H, s, NH), 6.90-7.42 (13H, m, ArH). 3.28 (1H, dd, J_{AM =}17H_Z, J_AX = 4.68 H_Z, C₄- H_A of pyrazoline ring). 3.70 (1H, dd J_MA= 17.81 Hz, J_MX = 13.30 Hz, C₄-H_Mof pyrazoline ring) , 4.20 (1H, d, J = 16.48 Hz COCH geminal proton ), 5.22(1H, dd J_MX12.89 H_{Z ,} J_{AX =}4.57 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 169.52 (C=O), 157.78 (C=N), 152.20, 148.65, 142.44, 138.85 (4C, ArC’s), 134.48, 132.53, 129.68, 123.77, 126.27 (5C, Ar CH’s), 64.67 (CH_2,ester), 62.60 (C-5, pyrazoline), 47.25 (C-4, pyrazoline), 18.35 (CH₃). –MS-FAB⁺: m/z: 666 [M], 667[M+1].

1- [(o-methoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7h]:

Yield: 67%, M.P.: 249⁰C, M.W.: 662, Anal. Calculated for C₃₀H₂₅Cl₄ N₅O_4,Cl: 14.4; N: 7.1, found Cl: 14.2, N: 7.0%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 215.3 (5.04), 318.4(4.79). IR[KBr] V_max Cm^-1 : 3300-2880 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2270(C N stretching ), 1640 [ C=O and N-H (amide)] , 1575 ( C=N stretching ), 1570, 1455, 1440 (C=C ring stretching , aromatic ), 1050, 810, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.38-2.51 (2H, s, CH₂), 4.29-4.50(1H, s, NH), 6.90-7.20 (13H, m, ArH). 3.27 (1H, dd, J_{AM =}17 H_Z, J_AX = 4.55 H_Z, C₄- H_A of pyrazoline ring). 3.98 (1H, dd J_MA= 17.90 Hz, J_MX = 13.80 Hz, C₄-H_Mof pyrazoline ring) , 4.82 (1H, d, J = 16.23 Hz COCH geminal proton ), 5.51 (1H, dd J_MX11.90 H_{Z ,} J_{AX =}4.40 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 173.52 (C=O), 158.70 (C=N), 144.10, 138.62, 135.65, 130.85 (4C, ArC’s), 133.38, 131.40, 129.46, 123.80, 116.18 (5C, Ar CH’s), 63.66 (CH_2,ester), 63.68(C-5, pyrazoline), 45.92(C-4, pyrazoline), 19.15(CH₃). –MS-FAB⁺: m/z: 662 [M].

1- [(m-methoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7i]:

Yield: 71%, M.P.: 251⁰C, M.W.: 662, Anal. Calculated for C₃₀H₂₅Cl₄ N₅O_4,Cl: 15.2; N: 7.5, found Cl: 15.3, N: 7.2%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 218.1 (4.95), 317.9 (4.68). IR[KBr] V_max Cm^-1 : 3300-2910 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2240(C N stretching ), 1660 [ C=O and N-H (amide)] , 1590 ( C=N stretching ), 1585, 1480, 1410 (C=C ring stretching , aromatic ), 1060, 825, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.12-2.49 (2H, s, CH₂), 4.14-4.45(1H, s, NH), 7.10 -7.40 (13H, m, ArH). 3.22 (1H, dd, J_{AM =}19H_Z, J_AX = 4.59 H_Z, C₄- H_A of pyrazoline ring). 4.10(1H, dd J_MA= 17.80 Hz, J_MX = 13.65 Hz, C₄-H_Mof pyrazoline ring) , 4.74 (1H, d, J = 16.10 Hz COCH geminal proton ), 5.70 (1H, dd J_MX12.40 H_{Z ,} J_{AX =}4.70 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 178.56 (C=O), 153.77 (C=N), 142.05, 139.40, 132.45, 130.80(4C, ArC’s), 131.45, 129.80, 127.84, 125.70, 113.18 (5C, Ar CH’s), 61.67 (CH_2,ester), 62.82 (C-5, pyrazoline), 46.65 (C-4, pyrazoline), 18.42 (CH₃). –MS-FAB⁺: m/z: 662 [M].

1- [(p-methoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7j]:

Yield: 73%, M.P.: 264⁰C, M.W.: 662, Anal. Calculated for C₃₀H₂₅Cl₄ N₅O_4, Cl: 15.7; N: 7.7, found Cl: 15.3, N: 7.7%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 216.4 (4.93), 318.7 (4.76). IR[KBr] V_max Cm^-1 : 3300-2890 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2230(C N stretching ), 1680 [ C=O and N-H (amide)] , 1610 ( C=N stretching ), 1590, 1520, 1460 (C=C ring stretching , aromatic ), 1030, 840, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.20-2.56 (2H, s, CH₂), 4.10-4.80(1H, s, NH), 6.85-7.10 (13H, m, ArH). 3.18 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.62 H_Z, C₄- H_A of pyrazoline ring). 3.97 (1H, dd J_MA= 18.20 Hz, J_MX = 13.50Hz, C₄-H_Mof pyrazoline ring) , 4.80 (1H, d, J = 16.18 Hz COCH geminal proton ), 5.60 (1H, dd J_MX12.70 H_{Z ,} J_{AX =}4.65 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 174.55 (C=O), 158.71 (C=N), 143.10, 138.60, 137.45, 133.85 (4C, ArC’s), 132.48, 130.55, 128.66, 125.75, 114.68 (5C, Ar CH’s), 62.80 (CH_2,ester), 63.20 (C-5, pyrazoline), 46.80 (C-4, pyrazoline), 18.86 (CH₃). –MS-FAB⁺: m/z: 662 [M].

1- [(p-floro) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7k]:

Yield: 52%, M.P.: 246⁰C, M.W.: 650, Anal. Calculated for C₂₉H₂₂Cl₄F₁N₅O_3,Cl: 11.4; N: 5.6, found Cl: 11.2, N: 5.4%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 222.5 (4.98), 317.9 (4.73). IR[KBr] V_max Cm^-1 : 3300-2860 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2250(C N stretching ), 1660 [ C=O and N-H (amide)] , 1575 ( C=N stretching ), 1570, 1460, 1430 (C=C ring stretching , aromatic ), 1070, 860, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.18-2.34 (2H, s, CH₂), 4.16-4.70(1H, s, NH), 6.70-7.10 (13H, m, ArH). 3.16 (1H, dd, J_{AM =}17 H_Z, J_AX = 4.60 H_Z, C₄- H_A of pyrazoline ring). 3.93 (1H, dd J_MA= 17.90 Hz, J_MX = 13.70 Hz, C₄-H_Mof pyrazoline ring) , 4.90 (1H, d, J = 16.40 Hz COCH geminal proton ), 5.55 (1H, dd J_MX12.90 H_{Z ,} J_{AX =}4.55 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 176.47 (C=O), 156.78 (C=N), 142.05, 137.62, 135.45, 132.84 (4C, ArC’s), 130.28, 129.50, 126.60, 122.70, 111.88 (5C, Ar CH’s), 63.10 (CH_2,ester), 62.40 (C-5, pyrazoline), 47.10 (C-4, pyrazoline), 18.95 (CH₃). –MS-FAB⁺: m/z: 650 [M].

1- [(o-bromo) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7l]:

Yield: 59%, M.P.: 259⁰C, M.W.: 711, Anal. Calculated for C₂₉H₂₂Cl₄N₅O₃BrCl: 11.8; N: 5.8, found Cl: 11.5, N: 5.4%. U.V. [(λ ^{Et OH}_Maxnm), log ε]: 210.2 (4.93), 318.7 (4.85). IR[KBr] V_max Cm^-1 : 3300-2880 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2230(C N stretching ), 1620 [ C=O and N-H (amide)] , 1555 ( C=N stretching ), 1605, 1510, 1490 (C=C ring stretching , aromatic ), 1060, 840, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.20-2.54 (2H, s, CH₂), 4.25-4.45(1H, s, NH), 6.80-7.30 (13H, m, ArH). 3.25 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.55 H_Z, C₄- H_A of pyrazoline ring). 4.04 (1H, dd J_MA= 17.70 Hz, J_MX = 13.50 Hz, C₄-H_Mof pyrazoline ring) , 4.80 (1H, d, J = 16.66 Hz COCH geminal proton ), 5.68 (1H, dd J_MX13.10 H_{Z ,} J_{AX =}4.70 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 178.70 (C=O), 158.72 (C=N), 141.10, 138.40, 136.49, 130.85 (4C, ArC’s), 131.48, 130.32, 127.66, 124.77, 113.38 (5C, Ar CH’s), 62.60 (CH_2,ester), 61.84 (C-5, pyrazoline), 45.92 (C-4, pyrazoline), 19.06 (CH₃). –MS-FAB⁺: m/z: 711 [M].

1- [(o-ethoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7m]:

Yield: 63%, M.P.: 269⁰C, M.W.: 676, Anal. Calculated for C₃₁H₂₇Cl₄ N₅O_4, Cl: 13.2; N: 6.5, found Cl: 13.2, N: 6.3%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 212.5 (4.98), 318.4 (4.88). IR[KBr] V_max Cm^-1 : 3300-2920 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2260(C N stretching ), 1640 [ C=O and N-H (amide)] , 1580 ( C=N stretching ), 1590, 1480, 1460 (C=C ring stretching , aromatic ), 1050, 860, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.30-2.44 (2H, s, CH₂), 4.14-4.40(1H, s, NH), 6.80-7.20 (13H, m, ArH). 3.17 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.60 H_Z, C₄- H_A of pyrazoline ring). 3.95 (1H, dd J_MA= 17.80 Hz, J_MX = 13.65 Hz, C₄-H_Mof pyrazoline ring) , 4.55 (1H, d, J = 16.35 Hz COCH geminal proton ), 5.50(1H, dd J_MX12.90 H_{Z ,} J_{AX =}4.65 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 176.58 (C=O), 156.74 (C=N), 140.05, 136.65, 135.45, 132.90 (4C, ArC’s), 131.46, 130.52, 129.66, 126.72, 112.44 (5C, Ar CH’s), 62.90 (CH_2,ester), 61.88 (C-5, pyrazoline), 46.35 (C-4, pyrazoline), 18.80 (CH₃). –MS-FAB⁺: m/z: 676 [M].

1- [(m-ethoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7n]:

Yield: 65%, M.P.: 255⁰C (d), M.W.: 676, Anal. Calculated forC₃₁H₂₇Cl₄ N₅O₄Cl: 13.7; N: 6.7, found Cl: 13.3, N: 6.2%. U.V. [(λ ^{Et OH}_{M ax}nm), log ε]: 210.2 (4.89), 318.5 (4.72). IR[KBr] V_max Cm^-1 : 3300-2890 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2240(C N stretching ), 1670 [ C=O and N-H (amide)] , 1570 ( C=N stretching ), 1580, 1460, 1430 (C=C ring stretching , aromatic ), 1055, 830, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.14-2.26 (2H, s, CH₂), 4.18-4.30(1H, s, NH), 7.0-7.30 (13H, m, ArH). 3.15(1H, dd, J_{AM =}18 H_Z, J_AX = 4.60 H_Z, C₄- H_A of pyrazoline ring). 3.90 (1H, dd J_MA= 17.90 Hz, J_MX = 13.55 Hz, C₄-H_Mof pyrazoline ring) , 4.75(1H, d, J = 16.12 Hz COCH geminal proton ), 5.55(1H, dd J_MX12.70 H_{Z ,} J_AX
=4.50 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 174.54 (C=O), 153.78 (C=N), 143.10, 140.64, 137.45, 136.85 (4C, ArC’s), 133.48, 131.55, 127.66, 124.57, 112.28 (5C, Ar CH’s), 64.65 (CH_2,ester), 62.85 (C-5, pyrazoline), 46.45 (C-4, pyrazoline), 18.95 (CH₃). –MS-FAB⁺: m/z: 676 [M].

1- [(p-ethoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7o]:

Yield: 61%, M.P.: 245⁰C, M.W.: 676, Anal. Calculated forC₃₁H₂₇Cl₄N₅O₄Cl: 12.8; N: 6.3, found Cl: 12.4, N: 6.1%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 218.2 (4.88), 318.6 (4.72). IR[KBr] V_max Cm^-1 : 3300-2930 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2250(C N stretching ), 1640 [ C=O and N-H (amide)] , 1555 ( C=N stretching ), 1590, 1450, 1430 (C=C ring stretching , aromatic ), 1045, 840, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.20-2.46 (2H, s, CH₂), 4.10-4.45(1H, s, NH), 6.90-7.30 (13H, m, ArH). 3.20 (1H, dd, J_{AM =}19 H_Z, J_AX = 4.80 H_Z, C₄- H_A of pyrazoline ring). 3.90 (1H, dd J_MA= 17.60 Hz, J_MX = 13.65Hz, C₄-H_Mof pyrazoline ring) , 4.70 (1H, d, J = 16.20 Hz COCH geminal proton ), 5.65(1H, dd J_MX12.60 H_{Z ,} J_{AX =}4.70 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 181.52 (C=O), 162.78 (C=N), 142.20, 138.65, 137.42, 133.84(4C, ArC’s), 129.88, 128.50, 127.60, 126.75, 110.38 (5C, Ar CH’s), 63.67 (CH_2,ester), 61.83 (C-5, pyrazoline), 46.65 (C-4, pyrazoline), 18.99 (CH₃). –MS-FAB⁺: m/z: 676 [M].

1- [(m-bromo) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7s]:

Yield: 57%, M.P.: 247⁰C, M.W.: 711, Anal. Calculated for C₂₉H₂₂Cl₄N₅O₃Br Cl: 11.4; N: 5.6, found Cl: 11.2, N: 5.2%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 214.3 (4.90), 318.4 (4.70). IR[KBr] V_max Cm^-1 : 3300-2890 [broad band due to (I) N-H

Table-II: Tuberculostatic Activity of new pyrazolines:

S. No.	Compounds	Growth at conc. [mg/mL]
S. No.	Compounds	10	100
7a.	1-[(2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	0
7b.	1- [(o-methyl) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	0
7c.	1- [(m-methyl) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	0
7d.	1- [(p-methyl) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7e.	1- [(o-chloro) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7f.	1- [(m-chloro) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	0
7g.	1- [(p-chloro) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	0
7h.	1- [(o-methoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7i.	1- [(m-methoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	0
7j.	1- [(p-methoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7k.	1- [(p-floro) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7l.	1- [(o-bromo) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7m.	1- [(o-ethoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	0
7n.	1- [(m-ethoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7o.	1- [(p-ethoxy) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7s.	1- [(m-bromo) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+
7t.	1- [(p-bromo) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline	+	+

‘+’ and ‘0’ indicate presence and inhibition of growth respectively.

stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2240 (C N stretching ), 1660 [ C=O and N-H (amide)] , 1570 ( C=N stretching ), 1570, 1490, 1470 (C=C ring stretching , aromatic ), 1050, 830, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.28-2.52 (2H, s, CH₂), 4.13-4.30(1H, s, NH), 6.90-7.55 (13H, m, ArH). 3.15 (1H, dd, J_{AM =}18 H_Z, J_AX = 4.70 H_Z, C₄- H_A of pyrazoline ring). 3.95 (1H, dd J_MA= 17.70 Hz, J_MX = 13.50 Hz, C₄-H_Mof pyrazoline ring) , 4.60 (1H, d, J = 16.10 Hz COCH geminal proton ), 5.80 (1H, dd J_MX12.90 H_{Z ,} J_AX
=4.70 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 178.57 (C=O), 157.77 (C=N), 140.15, 136.64, 134.40, 130.80 (4C, ArC’s), 130.18, 128.75, 127.66, 125.78, 113.19(5C, Ar CH’s), 61.62(CH_2,ester), 61.70 (C-5, pyrazoline), 46.90 (C-4, pyrazoline), 18.75 (CH₃). –MS-FAB⁺: m/z: 711 [M].

1- [(p-bromo) 2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline [7t]:

Yield: 49%, M.P.: 252⁰C, M.W.: 711, Anal. Calculated for C₂₉H₂₂Cl₄N₅O₃Br_,Cl: 9.8; N: 4.8, found Cl: 9.4, N: 4.3%. U.V. [(λ ^{Et OH}_M
axnm), log ε]: 210.2 (4.94), 318.7 (4.76). IR[KBr] V_max Cm^-1 : 3300-2850 [broad band due to (I) N-H stretching, secondary amide (Intra molecular hydrogen bond ), (II) C-H stretching , aromatic , (iii) C-H stretching , aliphatic ], 2250(C N stretching ), 1650 [ C=O and N-H (amide)] , 1580 ( C=N stretching ), 1560, 1480, 1440 (C=C ring stretching , aromatic ), 1040, 840, ( C-Cl stretching , 2, 3-disubstituted aromatic ring ).¹H-NMR (250 MHz, δ ppm, DMSO-d_6,): 2.20-2.44 (2H, s, CH₂), 4.15-4.45(1H, s, NH), 6.90-7.45 (13H, m, ArH). 3.20(1H, dd, J_{AM =}17 H_Z, J_AX = 4.60 H_Z , C₄- H_A of pyrazoline ring). 3.90 (1H, dd J_MA= 17.85 Hz, J_MX = 13.65Hz, C₄-H_Mof pyrazoline ring) , 4.75 (1H, d, J = 16.15 Hz COCH geminal proton ), 5.55 (1H, dd J_MX12.85H_{Z ,} J_{AX =}4.64 Hz, C₅-H_X of pyrazoline ring). ¹³C-NMR: ∂/ppm 180.55 (C=O), 161.78 (C=N), 142.15, 138.65, 136.45, 133.80 (4C, ArC’s), 131.46, 128.50, 127.65, 125.70, 114.27 (5C, Ar CH’s), 62.68 (CH_2,ester), 60.88(C-5, pyrazoline), 47.20 (C-4, pyrazoline), 18.95 (CH₃). –MS-FAB⁺: m/z: 711 [M]. Most of the pyrazolines are high melting point and light yellow or cream colored solids. The data of new products are furnished in table- I.

BIOLOGICAL EVALUATION:

Anti-bacterial activity:

Newly synthesized compounds (7a-t) have been tested for their antibacterial activity against gram positive bacteria S. albus, S. aureus and gram negative bacteria E .Coli and Pseudomonas poisonous by agar plate disc diffusion method at 30 μg/mL concentration. Ampicillin and Tetracycline used as a reference compound. The compound (7a, 7b, 7c, 7f, 7g, 7j, 7m, and 7r ) shown significant activity and the compound (7i, 7k, 7l, 7p, 7t, ) have shown moderate activity.

Anti-fungal activity:

The same compounds were tested for their antifungal activity against Candida albicans, Aspergillus Niger and Alternaria alternata at concentration of 30 mg/mL using sabouraud dextrose agar media. The compound (7c, 7j, 7m, and 7r) shown significant activities and compound (7a, 7b, 7f, and 7g) were found to be moderately active against Candida albicans and Aspergillus Niger. All the other compounds did not show significant activity against the fungi at the concentration used.

Anti-Tubercular Activity:

Some new compounds have been tested for anti- tubercular activity in-vitro using Mycobacterium tuberculosis. The compounds were incorporated into Lowenstein Jensen egg medium having concentrations of 10 and 100 mg/mL and were inoculated with Mycobacterium tuberculosis, H_27,R_Vstrains, incubated at 37⁰C and observed, weekly for the growth of organism for eight weeks. The compound (7a, 7b, 7c, 7f, 7g, 7j, and 7m) inhibited the growth of Mycobacterium tuberculosis at 100mg/mL concentration other compounds were found to be inactive. Results are assembled in table-II.

RESULTS AND DISCUSSION:

Newly synthesized 1-[(2, 3-dichloroanilinomalonyl)-3-(N-2’-cyanoethyl)-2-(N-acetyl) 2, 3-dichloroanilino)]-5- phenyl pyrazoline have been synthesized by the reaction of N-cinnamoyl-N-2’-cyanoethyl-2,3-dichloroaniline with Ethyl-2-[(N-acetyl) 2,3-dichloroanilido] acetohydrazide. Pyrazolines are yellow, cream and brown color solids, having high melting points. Identity of these products has been established by elemental analysis and spectral data. Newly synthesized compounds (7a-t) have been tested for their antibacterial activity against gram positive bacteria S. albus , S. aureus and gram negative bacteria E. Coli and Pseudomonas poisonous .The compound (7a, 7b, 7c, 7f, 7g, 7j, 7m, and 7r ) shown significant activity and the compound (7i, 7k, 7l, 7p, 7t, ) have shown moderate activity. The same compounds were tested for their antifungal activity against Candida albicans, Aspergillus Niger and Alternaria alternata at concentration of 30 mg/mL using sabouraud dextrose agar media. The compound (7c, 7j, 7m, and 7r) shown significant activities and compound (7a, 7b, 7f, and 7g) were found to be moderately active against Candida albicans and Aspergillus Niger. All the other compounds did not show significant activity against the fungi at the concentration used. Some new compounds have been tested for antitubercular activity in-vitro using Mycobacterium tuberculosis. The compounds were incorporated into Lowenstein Jensen egg medium having concentrations of 10 and 100 mg/mL and were inoculated with Mycobacterium tuberculosis, H_27,Rv strains, incubated at 37⁰C and observed, the compound (7a, 7b, 7c, 7f, 7g, 7j, and 7m) inhibited the growth of Mycobacterium tuberculosis at 100mg/mL concentration other compounds were found to be inactive.

CONCLUSION:

Newly synthesized compounds (7a-t) have been tested for their antibacterial activity against gram positive bacteria S. albus , S. aureus and gram negative bacteria E. Coli and Pseudomonas poisonous .The compound (7a, 7b, 7c, 7f, 7g, 7j, 7m, and 7r ) shown significant activity and the compound (7i, 7k, 7l, 7p, 7t) have shown moderate activity. The same compounds were tested for their antifungal activity against Candida albicans, Aspergillus Niger and Alternaria alternata at concentration of 30 mg/mL using sabouraud dextrose agar media. The compound (7c, 7j, 7m, and 7r) shown significant activities and compound (7a, 7b, 7f, and 7g) were found to be moderately active against Candida albicans and Aspergillus Niger. All the other compounds did not show significant activity against the fungi at the concentration used. Some new compounds have been tested for antitubercular activity in-vitro using Mycobacterium tuberculosis. The compounds were incorporated into Lowenstein Jensen egg medium having concentrations of 10 and 100 mg/mL and were inoculated with Mycobacterium tuberculosis, H_27,Rv strains, incubated at 37⁰C and observed, the compound (7a, 7b, 7c, 7f, 7g, 7j, and 7m) inhibited the growth of Mycobacterium tuberculosis at 100mg/mL concentration other compounds were found to be inactive.

ACKNOWLEDGEMENT:

The authors are thankful to Director, C.D.R.I. Lucknow (U.P.), for elemental analysis, Director, Tuberculosis Research Centre, Amargadh (Gujrat), for testing tuberculostatic activity and Director, D.R.D.E. Gwalior (M.P.), for spectral studies, and Director, Cancer Hospital and Research Institute, G.R. Medical College and Birla Institute of Medical Research, Gwalior (M.P.), for Biological activities. We are also grateful to Principal, SMS Government Model Science College, Gwalior (M.P), for providing research facilities.

REFERENCES:

1. Korgaokar, S. S.; Patil, P. H.; Shah, M. J; Parekh, H. H. Indian J. Pharm. Sci. 58, 222-225 (1996).

2. J. C. Jung, E. B. Watkins and M. A. Avery, Heterocycles 65, 77–94,(2005).

3. E. Palaska, M. Aytemir, T. Uzbay and D. Erol, Eur. J. Med. Chem. 36, 539–543, (2001).

4. Julian, L. Med. Hypotheses 69, 684-689, (2007).

5. Rajendra, P. Y.; Lakshmana, R. A.; Prasoona, L.; Murali, K.; Ravi, K. P. Bioorg. Med. Chem. Lett. 15, 5030-5034, (2005).

6. Ruhogluo, O.; Ozdemir, Z.; Calis, U.; Gumusel, B.; Bilgin, AA. Arzneimittelforschung 55, 431-436,(2005).

7. Ozdemir, Z.; Kandilici, HB; Gumusel, B.; Calis, U.; Bilgin, AA. Eur. J. Med. Chem. 42, 373-379,(2007).

8. Ashok Kumar, Sharma S, Bajaj K, Bansal D, Sharma S, Saxena KK, Lata S, Gupta B and Srivastava VK, Ind. J. Chem., 44B, 1979-1984, (2003).

9. Udupi, R. H., Narayanrao, S. and Bhat, A. R. Indian J. Heterocyclic Chemistry, 7, 217- 220,(1998).

10. Amir M, Kumar S. Indian J. Chem 44B: 2532-2537, (2005).

11. Udupi, R. H.; Kushnoor, A.S.; Bhat, A. R. Indian J.Heterocycl. Chem. 8, 63-66, (1998).

12. Amir, M., Kumar, H., Khan, S. A. Bioorg. Med. Chem. Lett. 18, 918-922, (2008).

13. Munawar A. Munawar, Muhammad Azad , Makshoof Athar and Paul W. Groundwater; Chemical Papers, Vol. 62, 3, 288-293, (2008).

14. Sadaf Sadiq Khan and Aurangzeb Hasan; Heterocycl. Commun. 13, 131-138, (2007).

15. Islam MR, Muhsin M. Bangladesh J. Pharmacol. 2, 7-12, (2007).

16. Hull, M.A.; Ko, S.C.W.; Hawcroft, G. Mol. Canc. Ther. 3, 1031-1039, (2004).

17. T. S. Jeong, K. S. Kim, J. R. Kim, K. H. Cho, S. Lee and W. S. Lee, Bioorg. Med. Chem. Lett. 14 , 2719–2723, (2004), DOI: 10.1016/j.bmcl.2004.03.072.

18. T. Saibara, K. Toda, A. Wakatsuki, Y. Ogawa, M. Ono and S. Onishi, Toxicol. Lett. , 51–54, (2003). DOI: 10.1016/S0378- 4274(03)00113-9.

19. El-Zohry MF, Younes MI, Metwally SA. Synthesis 972, (1984).

20. R. Lin, G. Chiu, Y. Yu, P. J. Connolly, S. Li, Y. Lu, M. Adams, A. R. Fuentes- Pesquera, S. L. Emanuel and L. M. Greenberger, Bioorg. Med. Chem. Lett. 17, 4557–4561; (2007) DOI: 10.1016/j. bmcl.2007.05.092.

21. S. Rollas, N. Gulerman and H. Erdeniz, Farmaco 57, 171–174, (2002).

22. Olsen, D. B., A. B. Eldrup, L. Bartholomew, B. Bhat, M. R. Bosserman, A. Ceccacci, L. F. Colwell, J. F. Fay, O. A. Flores, K. L. Getty, J. A. Grobler, R. L. LaFemina, E. J. Markel, G. Migliaccio, M. Prhavc, M. W. Stahlhut, J. E. Tomassini, M. MacCoss, D. J. Hazuda and S. S. Carroll. Antimicrob. Agents Chemother. 48:3944–3953, (2004).

23. Abid M, Azam A. Bioorg Med Chem Lett 16, 2812-6, (2006).

24. Asha Budakoti, Abdul Roouf Bhat; Amir Azam; Eur. J. Med. Chem. Vol. 44, Issue- 3, 1317-1325, (2009).

25. Inoue Y, Kobayashi T, Masu A, Asahina K. Jpn Kokai Tokkyo Koho.1991; JP03197467 [Chem Abstr. 115, 280054p, (1991).

26. A.A. Bekhit, H.M.A. Ashour, A.A. Guemei, Arch. Pharm. 338, 167, (2005).

27. M. Bagheri, M. Shekarchi, M. Jorjani, M.H. Ghahremani, M. A. Shafiee, Arch. Pharm. 337, 25,(2004).

28. J. H. Ahn, H. M. Kim, S. H. Jung, S. K. Kang, K. R. Kim, S. D. Rhee, S. D. Yong, H. G. Cheon and S. S. Kim, Bioorg. Med. Chem. Lett. 14 , 4461–4465, (2004).

29. Joel O, Jean-Yves P, Patricia M, Pascal C, Fretier P, Philippe J, Dereuddre-Bosquet N, Dominique D, and Jean-Louis I, J. Med. Chem. 42, 4733-4740, (1999).

30. Maria L, Barreca, Jan B, Alba C, Erik DC, Laura DL, Hans DH, Monforte AM, Monfort P, Christophe P, RaoA and Maria Z, Design, J. Med. Chem 45, 5410- 5413, (2002).

31. S. D. Bhardwaj, V. S. Jolly, Orient. J. Chem. 12 (1996) 185; Chem. Abstr. 126, 1442174, (1997).

32. Genin MJ, Biles C, Keiser BJ et al, J Med Chem, 43, 1034-40, (2000b).

33. G. V. Subbraju, A. Ranga Nayakulu, D. Parameshwara, Indian J. Heterocycl. Chem. 4 87, (1994).

34. Krishna R B, Panade R, Bhaithwal S P and Parmar S S, Eur Med J Chem., 15567, (1980).

35. Wagner E., Becan L. and Nowakowska E.; Bio. Org. Med. Chem.; 12, 265, (2004).

36. Troeberg, L.; Chen, X.; Flaherty, T. M.; Morty, R. E.; Cheng, M.; Hua, H.; Springer, C.; Mc Kerrow, J. H.; Kenyon, G. L.; Lonsdale-Eccles, J. D.; Coetzer, T. H. T.; Cohen, F. E. Chalcone, Mol. Med. (N.Y.) 6, 660-669, (2000), [Chem. Abstr. 2001, 134, 246896x].

37. B. Roman, Pharmazie 45, 214, (1990).

38. Azarifar, D.; Shaebanzadeh, M.; Molecules 7, 885-895, (2002).

39. Shekarchia M, Pirali-Hamedania B L, Navidpourb N, Adiba and Shafieeb A, J Iranian Chem Soc., 5, 150-158, (2008).

40. Francesc Puig-Basagoiti; Mark Tilgner, Brett M. Forshey, Seen M. Philpott, Noel G. Espina, Devid E. Wentworth, Scott J. Goebel, Paul S. Masters, Barry Falgout, Ping Ren, David M. Ferguson, and Pei-Yong Shi; vol. 50, No. 4, p.1320- 1329, April-(2006).

41. Yale, H. L.; Losee, K.; Martins, J.; Holsing, M.; Perry, F. M.; Bernstein, J. Chemotherapy of Experimental Tuberculosis. VIII. J. Am. Chem. Soc. 1953, 75, 1933-1942. Molecules, 8 754, (2003).

42. Corbett, E.L., Watt, C.J., Walker, N., Maher, D., Williams, B.G. Raviglione, M.C., and Dye,C, Arch Intern Med 163, 1009-1021, (2003).

43. M.A. Ali, M. Shaharyar, A. A. Siddiqui, Eur. J. Med. Chem. 42, 268- 275, (2007).

44. M. Shaharyar, A.A.Siddiqui, M.A. Ali, D. Shriram, P.Yogeeshwari, Bioorg. Med. Chem. Lett.16, 3947- 3949, (2006).

45. J. N. Domínguez, C. León, J. Rodrigues, N. Gamboa de Domínguez, J. Gut, J. Philip, P. J. Rosenthal, Farmaco, 60, 307-10, (2005).

46. Zhang, X.H.; Wu, S.K.; Gao, Z.Q.; Lee, C.S.; Lee, S.T.; Kwong, H.L. Thin Solid Films. 371, 40-46, (2000).

47. Suwalsky M, Orellana P, Avello M, Villena F. Food and Chemical Toxicology. 45, 130-135, (2007).

48. Tice CM, Bryman LM, Roemmele RC. Eur Pat Appl. 1994;EP 733622 [Chem Abstr. 125, 275903s, (1996).

49. Verma B L and Singhal M, Indian J Heterocycl Chem., 14, 343-346, (2007).

50. Desai NC, Nayan Bhatt, Mukesh Kumar. Indian J. Heterocyclic Chem. 17, 277- 278, (2008).

51. M. A. El-Hashasn, F. M. A. Sulaiman, L. M. Souka, A. S. Salman, Rev. Roum. Chim. 40, 59, (1995).

52. G. Turan-Zitouni, P. Chevallet, F.S. Kilic¸, K. Erol, Eur. J. Med. Chem. 35, 635e641, (2000).

53. M. S. Karthikeyan, B. S. Holla, N. S. Kumari, Eur. J. Med. Chem. 42, 30, (2007).

54. Habib NS, Soliman R, Ismail K, Hassan AM, Sarg MT, Pyrimidines. Part II: Boll Chim Farm, 142, 396-405, (2003).

55. Greenlee, R.-T.; Hill-Marmon, M.-B.; Murray, T.; Thun, M., Cancer J. Clin. 51, 15-36, (2001).

Received on 10.04.2010 Modified on 06.05.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 735-744