INTRODUCTION:

Pyrazolines have been studied extensively because of their diverse, broad-spectrum biological activities including antibacterial¹, antifungal², anti-inflammatory³, antidepressant⁴and anticancer activities⁵. The Nitrogen mustards are well known anti-neoplastic agents. When both, pyrazoline and nitrogen mustard moieties are brought together in a single molecular moiety the resultant molecular systems may play an important role in the class of DNA-intercalating agents. The critical cellular target of these drugs is DNA, which is alkylated primarily at the N - 7 position of guanine with lesser reaction at the N - 3 position of adenine. Nitrogen mustards are bi-functional and have the ability to produce cross-links on DNA. The cross link formed between the two complimentary strands of DNA is believed to be the major cytotoxic lesion. In view of above findings we aimed to synthesize a novel series of pyrazoline derivatives bearing a nitrogen mustard group as an important pharmacophore.

The pyrazolines were subjected to N - acylation reaction with chloroacetyl chloride by standard procedure and the resultant N - chloroacetyl - derivatives have been subjected to a nucleophilic substitution reaction with diethanolamine by heating under reflux in pyridine to get

1-[Bis - N,N -(hydroxyethyl) aminoacetyl] -3,5 -disubstituted-1,2 - pyrazolines, then subsequent chlorination with phosphorous oxychloride could afford the respective 1 - [ Bis - N,N - (chloroethyl) aminoacetyl ] - 3,5 - disubstituted -1,2 - pyrazolines .The purity of the compounds was checked by TLC and was characterized with the help of their IR, NMR and Mass spectral data. These compounds were also subjected to elemental analyses and the data was found to be in agreement with their molecular formula.

EXPERIMENTAL:

The melting points were determined in open capillaries using Veego VMP-1 melting point apparatus expressed in ^oC and are uncorrected. The reactions were monitored by TLC (pre-coated - Merck) using the solvent system of methanol: ethyl acetate (1:1) and detected by UV chamber method and also using iodine as visualizing agent. The IR spectra of the compounds were recorded on Perkin-Elmer infrared-283 spectrophotometer using KBr and are expressed in cm^-1. Nuclear magnetic spectra were obtained from dpx - 300 MHz spectrophotometer using d₆– DMSO as solvent with TMS as an internal standard (chemical shifts in d, ppm).

Synthesis of substituted Chalcones (I)- General procedure:

To a solution of sodium hydroxide (0.55 mol), a mixture of water (200 ml), rectified spirit (122.5 ml), acetophenone or substituted acetophenone (0.43mol) and benzaldehyde or substituted benzaldehyde (0.43 mol) were added and the reaction mixture was stirred while cooling in an ice-bath until the stirring was almost impossible owing to its increased viscosity. The flask was left overnight in a refrigerator and then acidified with dilute hydrochloric acid. The resultant product was filtered, washed with small portions of ice-cold water and dried. It was purified by recrystallization from acetone. IR (KBr) cm ^–1 : 1678 (C= O) and 1565 (CH=CH).

Synthesis of 3,5 - disubstituted 1,2 - pyrazolines (II)⁶^--General procedure:

A mixture of chalcone (0.01 mol) and hydrazine hydrate (0.02 mol) in 20 ml of ethanol was heated under reflux for 4 to 6 h and cooled, the crystalline solid that separated was pure enough for the next step. IR (KBr) cm ^–1: 3335 (NH),1587 (C=N) and 1052 ( CH₂ ) ; ¹H NMR (d₆- DMSO ) d : 3.36 ( 2H, s, CH₂), 5.71 ( IH, s, - NH - ) and 7.22 ( 4H, m, aromatic) and 7.86 ( 5H, m, aromatic ).

Synthesis of N - Chloroacetyl - 3, 5 - disubstituted - 1, 2 - pyrazolines (III)⁶–

General procedure:

To the pyrazoline (0.01 mol) obtained from step (II) in dry benzene, chloroacetyl chloride (0.012 mol) was added with stirring and stirring was continued for further 2 to 3 h to complete the reaction. Excess chloroacetyl chloride was distilled off as far as possible and the product was poured onto crushed ice. The product was filtered and washed with small portions of cold water and dried. The product was recrystallized from suitable solvents. IR (KBr) cm^-1: 1639 (C=O), 1601 (C=N), 1048 ( CH₂), and 753 (C –Cl) ; ¹H NMR(d₆-DMSO) d: 4.3-4.5(2H, s, -COCH₂Cl ), 3.28 (2H, s, -CH₂) 7.33 (4H, m, aromatic ) and 7.9 (5H,m, aromatic).

Synthesis of 1- [Bis - N, N - (hydroxyethyl) aminoacetyl] - 3, 5- disubstituted - 1, 2 - pyrazolines ( IV )⁷ –

General procedure:

A mixture of appropriate chloroacetyl derivative (III, 0.01 mol) and diethanolamine (0.012 mol) in pyridine was heated under reflux for 3 h, then pyridine was distilled off as far as possible under reduced pressure and the residue was poured onto crushed ice containing few drops of hydrochloric acid while stirring. It was kept aside for overnight and the product resulted was filtered and washed with small portions of cold water and dried. The product was recrystallized from appropriate solvent to get pure compound. . IR (KBr) cm^-1; 3446 (OH), 1654 (C=O), 1584 (C=N) and 1048 (CH₂) ; (C=O), ¹H NMR spectrum (DMSO) d: 2.50 (2H, s , -CH₂- ,N), 3.32 (2H, S, -CH₂-), 4.46 (4H, t, OH, CH₂- CH₂- N - CH₂ - CH₂ - OH), 5.06 (4H, t, OH - CH₂- CH₂- N - CH₂-CH₂-OH), 6.04 (brs, 2H , 2 X OH), 7.29 (4H, m , aromatic) and 7.88 (5H, m, aromatic ).

Synthesis of 1 - [Bis - N, N - (chloroethyl) aminoacetyl ]- 3,5 - disubstituted pyrazolines ( V )⁷-

General procedure:

Compound (IV, 0.01 mol) and Phosphorous oxychloride (40 ml) was gently heated under reflux for 2 h. The excess Phosphorous oxychloride was removed under vacuum and the residue was triturated with crushed ice. The product was filtered washed with cold water and dried. The product was recrystallized from suitable solvents. IR (KBr) cm^-1; 3012 (C - H, aromatic), 1670 (C = O), 1634 (C = N), 1548 (C = C), 1324 (C - N), 1092 (CH₂-) and 793 (C - Cl).

¹H NMR: 2.2 (2H,s, COCH₂), 3.1 (2H,s, CH₂), 4.2 (1H,s, CH), 4.4 [4H, t, (CH₂Cl)₂], 5.1 [4H, t, N(CH₂)₂ ] and 8.2 – 8.8 (10 H, m, aromatic).

Anticancer Screening:

The title compounds were evaluated for their anticancer activity in vitro and in vivo model. Student-t-test was performed for all the parameters to ascertain the significance of the activities exhibited.

Short term in vitro anticancer activity in DLA cells:⁸

All the title compounds were subjected for in vitro cytotoxicity study using DLA cells. Ten thousand cells per 0.1ml were seeded in 96 well microtitre plates and after 3h of incubation at 37^oC, the cultures were exposed to various concentrations of the compounds ranging from 7.81 to 500 mg/ ml as per the standard procedures. The cytotoxic activity was determined by tryphan blue dye exclusion technique.

Percentage cytotoxicity =

100 – (Total cells – dead cells) X 100

Total cells

In vivo anticancer screening:⁹

In vivo anticancer activity was assayed by using Dalton Lymphoma ascite tumor model. The test compounds and the standard drug were administered as suspension in 0.3% Carboxy methyl cellulose as vehicle orally to groups of animals. Each group consisted of 5 animals to access the potency of the test compounds. Mean survival time (MST), percentage increase in life span (% ILS) and hematological parameters were recorded as per the standard procedure.

% ILS =

MST of treated group – MST of control group X 100

MST of control group

RESULTS AND DISCUSSIONS:

Compound v_h 1 - [Bis - N,N -(chloroethyl) aminoacetyl ] -3- (2 -hydroxyphenyl) -5 - (3-chlorophenyl) - 1, 2 - pyrazoline was the most potent inhibitor of tumour cells with its CTC⁵⁰ : 9.37mg /ml followed by compound V_e and V_i with CTC ₅₀ :12.49 mg /ml and closely followed by compound V_g with CTC₅₀ : 14.05 mg /ml. The rest of the compounds could exhibit a moderate to weak anti-tumour activity with their CTC ₅₀ values being in the range of 23.43 to 378.12 mg /ml.

Table1: Physical data of 1-[ Bis - N, N- (chloroethyl)aminoacetyl ]-3,5-disubstitued -1,2-pyrazolines (V_a- _j)

Compound No	Substituents		Molecular Formula	Molecular Weight	Melting point (⁰c)	Yield ( %)
Compound No	Ar₁	Ar₂	Molecular Formula	Molecular Weight	Melting point (⁰c)	Yield ( %)
V_a	Phenyl	Phenyl	C₂₁H₂₃N₃OCl₂	403	74-76	83
V_b	Phenyl	3-chloro phenyl	C₂₁H₂₂N₃OCl₃	437	78-79	85
V_c	Phenyl	3-nitro phenyl	C₂₁H₂₂N₄O₃Cl₂	448	85-86	78
V_d	Phenyl	2-furyl	C₁₉H₂₁N₃O₂Cl₂	393	83-85	75
V_e	Phenyl	2-hydroxy phenyl	C₂₁H₂₃N₃O₂Cl₂	419	80-82	67
V_f	Phenyl	3-pyridyl	C₂₀H₂₂N₄OCl₂	405	71-72	72
V_g	2-hydroxy phenyl	Phenyl	C₂₁H₂₃N₃O₂Cl₂	419	76-78	80
V_h	2-hydroxy phenyl	3-chloro phenyl	C₂₁H₂₂N₃O₂Cl₃	454	81-83	88
V_i	2-hydroxy phenyl	3-nitro phenyl	C₂₁H₂₂N₄O₄Cl₂	464	75-77	86
V_j	2-hydroxy phenyl	2-furyl	C₁₉H₂₁N₃O₃Cl₂	409	72-73	71

Table 2. Short term in-vitro Anticancer screening of 1- [Bis - N, N - (Chloroethyl) aminoacetyl ] - 3, 5 - Disubstituted - 1,2 - Pyrazolines.

Compound No	% Growth Inhibition of test compounds at different concentration in mg/ml on DLA cells.							CTC₅₀ (mg/ml)
Compound No	500	250	125	62.5	31.25	15.62	7.81	CTC₅₀ (mg/ml)
V_a	97.14	88.23	80.00	55.88	41.17	20.00	2.85	50.00
V_b	94.28	85.71	68.57	62.85	57.14	42.85	17.14	23.43
V_c	70.14	66.66	62.85	57.14	41.17	40.00	11.42	48.43
V_d	60.00	37.14	29.57	28.57	25.71	2.85	0. 00	378.12
V_e	94.44	91.17	88.23	85.71	76.47	58.82	40.00	12.49
V_f	80.00	73.52	68.57	62.85	50.00	34.28	17.14	31.25
V_g	100.00	82.85	80.00	80.00	77.14	51.61	41.17	14.05
V_h	91.42	88.57	85.71	82.85	80.00	65.71	48.57	9.37
V_i	97.14	85.71	80.00	80.00	68.57	51.72	47.22	12.49
V_j	94.28	82.85	75.00	65.71	54.28	47.05	44.11	23.43

Standard: Cyclophosphamide.

Table 3. Effect of test compounds on Mean survival time and % increase in Life span of mice inoculated with DLA cells ( 1 X 10⁶)

Test compound	Mean survival time in days	% ILS
CMC ( vehicle )	18.00 ± 0.707	---
Cyclophosphamide	28.20 ± 0.734	56.66
V_b	26.16 ± 0.307	45.33
V_e	24.00 ± 0.683	33.33
V_g	26.66 ± 0.333	48.11
V_h	22.00 ± 1.238	22.22

Table:4 Effects of test compounds on Haematological parameters in DLA (1X10⁶) cells induced Swiss albino mice

Test compound⁺	*RBC 10⁶/mm³**	WBC 10³/mm³	*HGB g/dl**	*HCT %**	PLT 10³/mm³
Normal	9.77± 0.445	10.46±0.233	15.46±0.688	50.46±1.822	665.00±130.149
Negative control	10.10±0.440	34.84±2.153	11.64±0.628	39.68±2.440	293.00±27.453
Cyclosphosphamide (Standard drug)	10.86±0.405	10.32±0.281	14.32±0.582	45.66±1.220	445.80±11.719
Compound V_b	10.99±0.245	13.36±1.166	13.30±0.230	43.43±0.480	252.30±5.547
Compound V_e	9.90±0.935	14.40±0.585	11.96±0.744	38.43±1.937	215.66±22.400
Compound V_g	10.63±0.314	11.43±0.635	12.96±0.409	43.20±0.519	251.33±63.237
Compound V_h	10.12±0.512	16.80±0.621	10.41±0.532	32.71±0.761	228.70±12.812
Compound V_i	9.72±0.441	20.70±0.428	9.21±0.341	30.91±0.621	210.62±11.831

+: A group of 6 animals were used for each compound, All the test compounds were administered orally in a dose of 50mg /Kg, The standard drug was administered orally in a dose of 27.3mg/ Kg, The negative control was maintained on an oral administration of 10ml/Kg.