Synthesis and Biological Evaluation of Chloroacetyl Derivatives of Some Schiff’s Bases

P Muthumani^*,  R Meera, Pratesh, N Chidambaranathan, P Devi and B Kameswari

K.M. College of Pharmacy, Uthangudi, Madurai – 625 107. TamilNadu, India.

*Corresponding Author E-mail: sabareesanmuthu@gmail.com

ABSTRACT

Synthesis of substituted 2-Azetidinone derivatives with chloro acetyl chloride along with their derivatives has been done. The entire synthesized compounds were characterized by UV, IR and¹HNMR spectroscopy. The antimicrobial activity of the synthesized compounds was evaluated, on Staphylococcus aureus, Pseudomonas aeruginosa. The anticonvulsant activity and anti inflammatory activities were also evaluated. The present investigation deals with the synthesized compounds possessing good anticonvulsant and anti inflammatory activity, moderate antibacterial activity.

KEYWORDS: Chloro acetyl derivative, 2-Azetidionone, Antimicrobial activity, Anti convulsant activity, Anti inflammatory activity.

INTRODUCTION:

In the design and synthesis of novel Heterocyclic compounds, recently reported the synthesis of poly heterocyclic compounds. Literature survey reveals that substituted 2-Azetidinone derivative possess broad spectrum biological activities, which include Antitubercular^1-6, Anticancer^7,8, Antimicrobia^9-l2, Antiinflammatory^13,14, Antifungal^15-18, Sedative¹⁹, Insecticidal²⁰, Antimalarial²¹, Diuretic²², Antiparkinsonism²³, CNSactive²³, Anticonvulsant^24-26, Herbicidal activity²⁷. On the basis of our observation the present research work was carried out to synthesize chloroacetyl substituted 2-Azetidinone derivative compounds and to further evaluate antimicrobial, anticonvulsant and into inflammatory activity.

MATERIALS AND METHODS:

All the chemicals are analytical grade and were purified by the established methods. Melting points and were determined by open capillary tubes method purity and homogeneity of the compounds was routinely determined by thin layer chromatography on glass plates using silica gel G as absorbent and solvent system. Benzene: Ethylacetate: Methanol (8.5:1.4:0.1). Spots were visualized by iodine vapor by irradiation with UV light.¹HNMRspectra was recorded on Bruker Ultra shield (300MHZ) spectrometer using DMSO (TMS as internal standard). The anti microbial activities of the synthesized compounds were evaluated on S.aureus and P.aeroginosa. The anti convulsant activity was determined and phenytoin used as standard. The anti inflammatory activity was evaluated by Carrageenan induced paw edema method in rats.

EXPERIMENTAL:

Synthesis of various substituted 2-azetidinone:

STEP I:

Synthesis of substituted benzoyl chloride

Weight about (0.12 mol) of substituted benzoic acids and transfer it into round bottom flask and added equivalent moles of thionyl chloride to it 3-7 ml of benzene was added to the above mixture (as a solvent).  Refluxed for 3 hrs continuously in a water bath.  Cool the mixture the solid residue obtained was substituted benzoyl chloride

STEP II:

Synthesis of substituted benzohydrazides:

Take the substituted benzoyl chloride in round bottom flask and added aquivalent moles of hydrazide (0.12 mol) and 10 ml of methanol as a solvent.  Reflux the mixture for 6 hrs, the product obtained was substituted Benzohydrazide.

Table No.1 PHYSICAL DATA OF SYNTHESIZED COMPOUNDS

S. No	Compound	Molecular Formula	Molecular  Weight	Melting Point	% Yield	Rf Value
1.	I	C16H11N2O2Cl3	369.35	180^0C	68.05%	0.73
2.	II	C17H14N2O3Cl2	364.90	210^0C	71.30%	0.71
3.	III	C19H18N2O5Cl2	424.90	170^0C	69.81%	0.75
4.	IV	C17H15N2O4Cl	346.45	160^0C	59.60%	0.65
5.	V	C18H17N2O4Cl	360.45	140^0C	57.62%	0.76
6.	VI	C19H20N3O3Cl	373.45	120^0C	63.70%	0.68
7.	VII	C19H20N3O2Cl	357.45	170^0C	79.91%	0.72
8.	VIII	C18H17N2O3Cl	344.45	165^0C	73.58%	0.78
9.	IX	C20H21N2O5Cl	404.45	150^0C	85.90%	0.81
10.	X	C16H12N2O2FCl	318.45	185^0C	76.25%	0.83
11.	XI	C17H15N2O3Cl	330.45	160^0C	71.30%	0.72
12.	XII	C19H19N2O5Cl	390.45	180^0C	53.14%	0.76

Table no -2 Anticonvulsant activity

Group	Treatment	Dose	Duration of Extension phase in seconds	% Inhibitor of Extension phase
1	Control	0.5ml DMSO	11.500.30	-
II	Std	Phenytoin 25mg/kg	1.800.42	84.3
III	I	50mg/kg	2.420.62	78.9*
IV	II	50mg/kg	2.960.35	74.3*
V	III	50mg/kg	8.921.08	22.4
VI	IV	50mg/kg	9.022.02	21.5
VII	V	50mg/kg	3.100.46	73.8*
VIII	VI	50mg/kg	9.201.18	20.0
IX	VII	50mg/kg	10.52.05	8.6
X	VIII	50mg/kg	2.680.72	76.7*
XI	IX	50mg/kg	9.41.82	18.2
XII	X	50mg/kg	9.61.48	16.5
XIII	XI	50mg/kg	2.821.02	75.5*
XIV	XII	50mg/kg	8.942.08	22.2

Values are expressed as mean SEM. P values are significantly different from control.

TABLE NO -3 ANTI- INFLAMMATORY ACTIVITY

Group	Compound	Dose mg/kg	(Mean SEM)
			0 min	60min	120min	240min
I	Control 0.5ml DMSO	0.5ml	0.7200.110	0.7180.118	072310.0213	0.7130.220
II	Std Diclofenac sodium	100mg/kg	0.7320.139	0.6290.029	0.6580.029	0.5260.104
III	I*	50mg	0.7180.218	0.6180.018	0.5860.040	0.5340.124^bb
IV	II*	50 mg	0.6960.318	0.6220.030	0.5800.221	0.5380.218
V	III	50mg	0.7120.296	0.6900.186	0.6580.244	0.642^bb0.218
VI	IV	50mg	0.7100.218	0.6840.124	0.6420.308	0.6400.216
VII	V*	50mg	0.6980.410	0.6340.401	0.5760.118	0.532^bb0.210
VIII	VI	50mg	0.6900.312	0.6780.216	0.6520.286	0.6440.286^bb
IX	VII	50mg	0.6980.216	0.6720.194	0.6580.186	0.6460.146
X	VIII*	50mg	0.7100.322	0.6460.128	0.5800.264	0.530^bb0.212
XI	IX	50mg	0.6980.218	0.6520.196	0.5920.118	0.556^b0.108
XII	X	50mg	0.7100.212	0.6820.114	0.6620.018	0.6500.216
XIII	XI*	50mg	0.7120.468	0.6520.312	0.5820.111	0.542^bb 0.264
XIV	XII	50mg	0.7020.308	0.6480.210	0.5800.110	0.558^b0.108

The values are expressed as mean SEM (n=6) bb- values are significantly different from control (p<0.01) b- values are significantly different from control (p<0.05).

STEP III:

Synthesis of Various substituted Schiff’s bases:

Take 0.01 mol of substituted benzohydrazides and to this added (o.12mol) of various substituted aromatic aldehydes in 10ml of ethanol.  The reaction mixture was refuxed for 5 hrs, then the resulting solid was collected.  Recrystallized from ethanol to give various substituted schiff’s bases.

Substituted Benzo Hydrazides Aromatic aldehydes schiff’s bases

STEP IV:

Synthesis of Various Substituted 2- Azetidinones:

Take substituted Schiff’s bases in a round bottom flask and add (0.02mol) of triethyl amine in 40ml of DMF and add chloroacetyl  chloride (0.02) mole, drop wise a period of 30 minutes.  The reaction mixture was refluxed for six hours; the product obtained was 2 Azetidinones.

R = Cl, OCH₃, CH₃ . R¹= Cl, OCH₃, (OCH₃)_3, N(CH₃)₂, OH, F

SPECTRAL DATA OF SYNTHESIZED COMPOUNDS ^28,29:

a) Compound I:

Chemical name:

4-chloro- N- (3-chloro-2 (4-chloro phenyl) -4-oxoazetidin -1-yl) benzaminde.

I.(KBr)cm^-1:3449(NH stretching), 2374 (C-H Stretching), 1685(C=O) azetidinone ring, 1592C=C stretching, 1424 primary amide stretching, 1321 C-N stretching,1091 C-Cl stretching):¹HNMR (CDCl₃): 7.2(N-CH proton), 8 (aromatic CH (Cl₂) proton), 1.3(tertiary(-CH) proton), 3(-CONH protons)

b) Compound II:

Chemical name:

4- chloro-N-3-chloro-2-(4-methoxy phenyl) -4-oxoazetidin-1-y1) benzamine.

II.(KBr)cm^-1:3429(NH stretching), 1684(C=O) azetidinone, 1592C=C stretching, 1425 CH₃ stretching, 1323 C-N stretching, 1092 C-O stretching, 851 C-Cl stretching

c) Compound III:

Chemical name:

4- Chloro- N- (3-chloro-2-oxo-4- (3,4,5- timethoxyphenyl) azetidin-1 yl) benzamide.

III.(KBr)cm^-1:3090(NH stretching), 2954 (C-H Stretching), 1462 (C=O) azetidinone ring,1592 C=C stretching, 1390 C-N stretching,1137 C-O stretching,1184  C-Cl aromatic stretching

d) Compound IV:

Chemical name:

N- (3-chloro-2-(4-hydroxy phenyl) -4-oxoazetidin -1-yl) 4-methoxy benzamide.

IV.(KBr)cm^-1: 2982 (NH stretching), 2839 (C-H Stretching), 3022 OH stretching, 1512 NH bending , 1463 (C=O) azetidinone ring, 1592C=C stretching, 1423 CH₃ stretching, 1321 C-N stretching, 1091 C-O stretching,1114 C-Cl stretching

e) Compound –V:

Chemical name:

N- (3-chloro-2- (4-methoxyphenyl) -4-oxoazetidin -1-yl) -4-methoxy benzamide.

V.(KBr)cm^-1:3426(NH stretching), 2929 (C-H Stretching), 1604 (C=O) azetidinone ring, 1686C=C stretching, 1461 CH₃ stretching, 1302 C-N stretching, 1258 C-O stretching, 773 C-Cl stretching): ¹HNMR (CDCl₃): 3.8(OCH₃proton), 7.0 (aromatic CH proton), 1.2(tertiary(-CH)proton),  2.9(-CONH protons)

f) Compound –VI

Chemical name:

N –(3-chloro-2 –(4-dimethylaminophenyl) -4-oxoazetidin-1-yl) -4- methoxy benzamide.

VI.(KBr)cm^-1:3433(NH stretching), 2374 (C-H Stretching), 1686 (C=O) azetidinone ring, 1592C=C stretching, 1427 CH₃ stretching, 1301 C-N stretching, 1262 C-O stretching,773 C-Cl stretching

g) Compound VII:

Chemical name:

N – (3-chloro-2-(4-(dimethylamino) phenyl) -4-oxoazetidin -1-yl) -4- methylbenzamide.

VII.(KBr)cm^-1: 3407 (NH stretching), 2918 (C-H stretching), 1605 (C=O) azetidinone ring, 1592C=C stretching, 1439 CH₃ stretching,1367 C-N stretching, 1228 C-O stretching,815 C-Cl stretching

h) Compound VIII

Chemical name:

N – (3-chloro-2- (4-methoxyphenyl) -4-oxoazetidin-1-yl) -4-methyl benzamide.

VIII.(KBr)cm^-1:3448(NH stretching), 2276 (C-H Stretching), 1617 (C=O) azetidinone ring, 1510C=C stretching, 1460 CH₃ stretching, 1253 C-O stretching, 1176 C-O stretching: ¹HNMR (CDCl₃): 7.3(aromatic CH proton), 3.8 OCH3 proton, 3.0 (-CONH protons) 0.8 CH3 proton, 1.2(tertiary (-CH)proton)

i) Compound – IX

Chemical name:

N –(3-chloro-2-oxo-4-(3,4,5 trimethoxyphenyl) azetidin -1-yl) 4-methyl benzamide.

IX.(KBr)cm^-1:3448(NH stretching),2937(C-H Stretching), 1583 (C=O) azetidinone ring, 1504C=C stretching, 1462 CH₃ stretching, 1328 C-N stretching, 1237 C-O stretching, 998C-Cl stretching.

j) Compound – X

Chemical name:

2-(4-fluorophenyl)-4 oxoazetidin-1-yl) benzamide

X.(KBr)cm^-1:3431(NH stretching), 2374(C-H Stretching), 1632 (C=O) azetidinone ring,1602 C=C stretching, 1295 C-N stretching,1227 C-O stretching,1115 C-F stretching):

k) Compound –XI:

Chemical name:

N – (3-chloro-2-(4-methoxyphenyl) -4-oxoazetidine-1-yl) benzamide.

XI.(KBr)cm^-1:3448(NH stretching), 2928(C-H Stretching), 1672 (C=O) azetidinone ring, 1623 C=C stretching, 1461 CH₃ stretching, 1302 C-N stretching,1250 C-O stretching, 618 C-Cl stretching): ¹HNMR (CDCl₃):7.2(N-CH proton), 1.3(tertiary(-CH) proton), 3(-CONH protons)

l) Compound XII

Chemical name:

N –(3-chloro-2-oxo-4-(3,4,5-trimethoxyphenyl) azetidin-1-yl) benzamide.

XII.(KBr)cm^-1:3448(NH stretching), 2374(C-H Stretching), 1623 (C=O) azetidinone ring, 1592C=C stretching, 1459 CH₃ stretching, 1343 C-N stretching,  1234 C-O stretching, 762 C-Cl stretching): ¹HNMR (CDCl₃):7.2(N-CH proton), 1.3(tertiary(-CH)proton),3(-CONH protons).

TABLE NO -4 ANTIBACTERIAL ACTIVITY

Name of the compound	Organism used zone of inhibition in mm
Standard Amikacin	Staphylococcus aureus	Psedomonas aeruginosa	Inference
	18	18
Test compound	10 mg/ml	10g/mg
I	8mm	0mm	NA	NA
II	0mm	0mm	NA	NA
III	0mm	0mm	NA	NA
IV	0mm	0mm	NA	NA
V	0mm	0mm	NA	NA
VI	0mm	0mm	NA	NA
VII	0mm	0mm	NA	NA
VIII	8mm	0mm	NA	NA
IX	0mm	0mm	NA	NA
X	0mm	0mm	NA	NA
XI	0mm	0mm	NA	NA
XII	0mm	0mm	NA	NA

Standard – Amikacin (10g g/disc) Control –DMSO NA – Not Active.

ANTICONVULSANT ACTIVITY^30-32:

Supra Maximal Electric Shock Method:

Healthy albino wistar rats weighing from 200-250g were selected.  They were kept in separate cages, fed with balanced diet water ad libitum. Then the animals were divided into 14 groups each group containing six animals. The first group of animals were served as control, which received 0.5 ml DMSO. Second groups served as std which received phenytoin (25 mg/kg).Third group treated with compound I (50 mg/kg). Fourth group treated with compound II (50 mg/kg). Fifth group treated with compound III (50 mg/kg).Sixth group treated with compound IV (50 mg/kg)Seventh group treated with compound V (50 mg/kg).Eight group treated with compound VI (50 mg/kg). Ninenth group treated with compound VII (50 mg/kg). Tenth group treated with compound VIII (50 mg/kg). Eleventh group treated with compound IX (50 mg/kg). Twelth group treated with compound X (50 mg/kg). Thirteenth group treated with compound XI (50 mg/kg). Fourteenth group treated with compound XII (50 mg/kg).All the test compound were dissolved in DMSO and administrative through intra peritoneal route. The evaluation was started 30 mins after administration of test compounds Pinna electrodes with the intensity of 150 mA current were used to deliver the stimuli. Inhibition of seizure relative to the control was calculated and the data shown on the Table no 2.

ANTI-INFLAMMATORY ACTIVITY³³:

Male albino rats weighing approximately, 150-200 gm were divided in to 14 groups and each of 6 animals. A mark was made on the hind paw just behind tibio-tarsal junction so that every the Paw was dipped in the mercury column up to the fixed mark to ensure constant Paw volume. The paw volume of each animal was measured before the administration of the drug. The dosage of the drug administered to different groups were as follows .Group –I control group received orally 0.5 ml of DMSO. Group II standard group received orally 10mg /kg of body weight of diclofenac sodium. Group –III to XIV received compound code I st to XII th drugs respectively. All the above test compounds were dissolved in 0.5ml solution of DMSO and given 30 minutes before the commencement of the study. After that 0.1ml of 1% w/v carrageenan solution in normal saline was injection into the sub plantar tissue of the left final paw of the rat. The volume of the mercury displaced in the plethmograph was measured at 0 min, 60min, 120min and 240min.

ANTIBACTERIAL ACTIVITY ³⁴

Assay was carried out by diffusion plate method. The method followed was spread plate technique. The plates free from contamination were spread with 50µl of 48h old culture of bacterial test organism using sterile buds.  The standard disc of Amikacin (sterile) of 5 mm diameter was in the Petri plates. Then the filter paper discs (sterile) of 5mm were soaked in 1ml (1µg/ml) of the test solution and in solvent control DMSO.  After evaporating the solvent in a sterile atmosphere the drug impregnated discs were placed in Petri plates. The plates were refrignated for 1h to arrest the growth and for easier diffusion of test compounds. Then the plates were remove from refrigerator and incubated at 37⁰C over night is an inverted position.  The clear zones of inhibition were measured using Hi media zone reader scale. The values are tabulated.  The zones of test solutions were compared with std Amikacin.

RESULTS AND DISCUSSION:

Twelve novel 2- Azetidinone derivatives were synthesized and characterized by spectral analysis.  The melting point of the synthesized compounds were found out by open capillary tube method. The structure of the synthesized compounds were characterized by its IR,H_NMR  spectral analysis in which it complies with the normal values.

BIOLOGICAL EVALUATION:

Anticonvulsant activity:

The table values shows that compound I, II, V, VIII, XI, possess significant anticonvulsant activity. But rest of the compounds such as III, IV, VI, VII, IX, X and XII does not possess anticonvulsant activity.

SCHEME OF PRESENT WORK:

Anti –Inflammatory Activity:

The table value shows that very high to significant inflammatory reduction action in compounds I, II, V, VIII, XI and compounds IX and XII possesses moderate reduction of inflammation.  Rest of the compounds III, IV, VI, VII and X does not possess anti-inflammatory activity significantly.

Antibacterial activity:

The antibacterial activity of the compounds was evaluated against gram positive organism Staphylococcus aureus and negative organism Pseudomonas aeruginosa. The zone of inhibition was measured as parameter of active Amikacin 10 µg/disc was used as standard compound.  Gram positive organism Staphylococcus aureus showed a zone of inhibition of 18mm were I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII compounds does not show any activity near Amikacin 18mm and the gram negative organism pseudomonas aeruginosa showed a zone inhibition of 18mm where as I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII compounds does not show any activity near to standard.

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Received on 10.07.2009       Modified on 13.09.2009

Accepted on 15.10.2009      © RJPT All right reserved

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 824-829