\Synthesis, Characterization of Some Novel Heterofused Thienopyrimidines for Anti-Inflammatory Activity

 

H.R. Roopa*, J. Saravanan, S. Mohan, Rekha Parmesh

Department of Pharmaceutical Chemistry, PES College of Pharmacy, Bangalore-50, Karnataka, India.

*Corresponding Author E-mail: roopad24@gmail.com

2-Amino-3-cyano-4, 5-disubstituted thiophene JSR-2 was synthesized using 2-amino-3-cyano-4,5-disubstituted thiophene. First step is preparation of 2-amino-3-cyano-4,5-disubstituted thiophene (JSR-c) which was carried out by  condensation of diethylamine, cyclohexanone, Malononitrile and sulphur this compound was refluxed with formamide and acetic anhydride to give 4-Amino-5,6-disubstituted thieno [2,3-d]  pyrimidines. Then the parent compound was treated with different substituted aryl aldehydes by microwave irradiation method to yield new series of Schiff bases (JSR 7a-7k). The compounds were characterized IR, ¹H NMR and mass spectral data and screened for anti-inflammatory activity.

 

 

 

INTRODUCTION:

Most of the therapeutic agents are heterocyclic compounds; hence heterocyclic chemistry has been the most fruitful area for drug discovery. Among the heterocyclic compounds nitrogen and sulphur containing moieties have attracted maximum attention as they have several pharmacological activities⁸ as anti- inflammatory^10,12,13, analgesic^10,13,15, antifungal⁹, antimicrobial^5,9, Adrenoreceptor^3,11, CNS depressant⁴, antioxidant, anticonvulasant, antitumor activity and so on. Similarly Schiff base derivatives also have been reported to possess biological activity as anti-inflammtory^10,12,13. The therapeutic importance of these rings prompted us to synthesize novel thienopyrimidine^1,2,7,14 and their Schiff base derivatives, characterize the compound by IR, NMR and Mass spectroscopic techniques and evaluate them for their anti-inflammatory activity.

 

MATERIAL AND METHODS:

Chemicals:

Diethylamine, cyclohexanone, Malononitrile, Sulphur, Ethanol, Formamide, Acetic anhydride, Dioxane, Isopropyl alcohol, 3,4,5-Tri methoxy Benzaldehyde, 3,4-Dimethoxy Benzaldehyde, 2-Nitro Benzaldehyde, 3-Nitro Benzaldehyde, 2-Chloro Benzaldehyde, 4-Hydroxy Benzaldehyde, 4-hydroxy-3-methoxy Benzaldehyde, 2-Hydroxy Benzaldehyde, 4-Methoxy Benzaldehyde, -N,N-Dimethyl amino Benzaldehyde,4-Chloro Benzaldehyde, Glacial acetic acid and Dimethyl formamided were obtained from local dealer. All other chemicals used were of laboratory grade.

 

Preparation of 2-Amino-3-cyano-4,5-disubstituted thiophene (JSR-c):

Diethylamine (2 ml) was added dropwise over a period of 30 min to a mixture of Methylinic ketone (cyclohexanone) (0.04 M), Malononitrile (0.04 M) and sulphur (0.04 M) in ethanol (30 ml) at room temperature (temperature should not exceed more than 40 ^oC). The reaction mixture was stirred at room temperature for sulphur to get dissolved and stirring was continued for 4-5 h at room temperature and chilled overnight. The solid obtained was filtered, washed with ethanol and recrystallized from ethanol.

 

 

 

 

Preparation of 4-Amino-5,6-disubstituted thieno [2,3-d]  pyrimidines JSR-7:

To the stirred solution of 2-Amino-3-cyano-4,5-disustituted thiophene (0.5 g) in formamide (2.9 ml) was added acetic anhydride (1 drop) at 100 ^oC. After being stirred reflux for 2 h, the reaction mixture was cooled at room temperature and diluted with water (20 ml).The crude product was collected by filtration and crystallized from dioxane to get the white crystals.

 

Preparation of 4-(Substituted benzylidene imino)-5,6-disubstituted thieno[2,3-d]pyrimidines (Schiff bases) JSR (7a-7k):

A mixture of the starting compounds (JSR 7 & 8) (0.005 M) and the required aromatic aldehydes (0.005 M) in isopropyl alcohol (30 ml) and catalytic amount of glacial acetic acid (2 ml) was taken into a conical flask and subjected to microwave irradiation for 30 sec at 900 watt. The mixture was cooled to room temperature, the solid separated was filtered, washed with isopropyl alcohol and recrystallised with DMF: Water mixture (5:1).

Anti-inflammatory studies:

All the synthesized compounds were screened for their anti-inflammatory activity by formalin induced paw oedema method¹⁴. Male or female Wistar rats with a body weight between 150- 200 g were used. The animals were starved overnight. To ensure uniform hydration, the rats received of 5 ml of water by stomach tube (controls) are the test drugs dissolved are suspended in the same volume. The vehicle / drug/ Diclofenac sodium was injected orally. One hour later the rats were challenged by injection of 0.1 ml of 1% formalin into the plantar region of the left hind paw. The paw was marked with ink at the level of the lateral malleolus and immersed in mercury up to this mark. The paw volume was measured plethysmographically immediately after injection, again at 1, 2, 3, 4hr after challenge. Mean percent change in paw volume was compared.

 

SCHEME-I

Synthesis of 4-Amino-5,6-disubstituted thieno[2,3-d]pyrimidine (JSR-7):

                                                                

 

4-Amino-5,6-disustituted thieno(2,3-d)pyrimidine                                                                                                   (JSR-7)

Where: R_1, R₂ = - (CH₂)₄ -,

 

 

SCHEME-II

Synthesis of 4-(Substituted benzylidene imino)-5,6-disubstituted thieno[2,3-d]pyrimidines (Schiff bases) JSR (7a-7k)

 

4-Amino-5,6-disustituted                             Various aromatic                                                                       JSR (7a-7k)

thieno(2,3-d)pyrimidine                                aldehydes

Where: R_1, R₂ = - (CH₂)₄ -

R=3,4,5-Tri methoxy, 3,4-Dimethoxy, 2-Nitro, 3-Nitro, 2-Chloro, 4-Hydroxy, 4-hydroxy-3-methoxy, 2-Hydroxy, 4-Methoxy, -N,N-Dimethyl amino and 4-Chloro.

Table-1 Physical data of  4-Amino-5,6-disubstituted thieno[2,3-d]pyrimidine (JSR-7)

Comp No.	Molecular Formula	M.W (g)	Recrystalization Solvent	M.P.(OC)	%Yield	TLC Solvent	Rf Value
JSR-7	C10H10SN3		Dioxane	176	64	Methanol: Chloroform(8:2)	0.68

 

Table-2 Physical data of 4-(Substituted benzylidene imino)-5,6-disubstituted thieno[2,3-d]pyrimidines (Schiff bases) JSR (7a-7k)

Comp Code	X	Molecular Formula	M.W (g)	Recrystalization Solvent	M.P.(OC)	%Yield	TLC Solvent	Rf Value
JSR-7a	3,4,5-Tri methoxy	C20H21N3SO3	383	DMF-Water	132	55	Methanol: Chloroform(9:1)	0.47
JSR-7b	3,4-Dimethoxy	C19H19N3SO2	353	DMF-Water	154	61	Methanol: Chloroform(9:1	0.44
JSR-7c	2-Nitro	C17H14N4SO2	338	DMF-Water	170	54	Methanol: Chloroform(9:1	0.38
JSR-7d	3-Nitro	C17H14N4SO2	338	DMF-Water	168	48	Methanol: Chloroform(9:1	0.61
JSR-7e	2-Chloro	C17H14N3SCl	327	DMF-Water	144	66	Methanol: Chloroform(9:1	0.53
JSR-7f	4-Hydroxy	C17H15N3SO	309	DMF-Water	151	50	Methanol: Chloroform(9:1	0.56
JSR-7g	4-hydroxy-3-methoxy	C18H17N3SO2	339	DMF-Water	176	53	Methanol: Chloroform(9:1	0.64
JSR-7h	2-Hydroxy	C17H15N3SO	309	DMF-Water	160	64	Methanol: Chloroform(9:1	0.59
JSR-7i	4-Methoxy	C18H17N3SO	323	DMF-Water	140	71	Methanol: Chloroform(9:1	0.42
JSR-7j	-N,N-Dimethyl amino	C19H20N4S	336	DMF-Water	182	68	Methanol: Chloroform(9:1	0.71
JSR-7k	4-Chloro	C17H14N3SCl	327	DMF-Water	175	59	Methanol: Chloroform(9:1	0.39

 

Table-3 Spectral data of  4-Amino-5,6-disubstituted thieno[2,3-d]pyrimidine (JSR-7) & 4-(Substituted benzylidene imino)-5,6-disubstituted thieno[2,3-d]pyrimidines (Schiff bases) JSR (7a-7k)

Comp Code	X	λ max	IR	NMR	MASS
JSR-7	--	164	3320 & 3246(-NH2); 3040(Ar-CH); 2938 (Ali-CH); 1562 (C=C); 841 (C-N); 750 (C-S).	-	-
JSR-7a	3,4,5-Tri methoxy	268	3044(Ar-CH); 2931(Ali-CH); 1540 (C=N); 1584 (C=C); 1320 (C-O); 828(C-N); 721 (C-S).	-	-
JSR-7b	3,4-Dimethoxy	272	3070(Ar-CH); 2932 (Ali-CH); 1683 (C=O); 1523 (C=C); 1289 (C-N); 824(C-N)	-	-
JSR-7c	2-Nitro	264	3072 (Ar-CH);2958(Ali-CH);1682(C=O); 1510(C=C); 1065 (C-O); 846 (C-N).704(C-S).	-	338
JSR-7d	3-Nitro	255	3132(Ar-CH); 2890 (Ali-CH); 1680 (C=O); 1524 (C=C);1227(C-0); 756 (S-C); 824 (C-N).	-	-
JSR-7e	2-Chloro	290	3090(Ar-CH);2844(Ali-CH); 1690(C=N);1535(C=C);1132(C-Cl); 786 (S-C); 835 (C-N).	-	327
JSR-7f	4-Hydroxy	242	3084(Ar-CH); 2910 (Ali-CH); 1700 (C=N); 1562.4(C=C); 1289 (C-O); 815(C-N); 728(C-S).	-	-
JSR-7g	4-hydroxy-3-methoxy	240	3134 (Ar-CH);2977(Ali-CH); 1660 (C=N); 1521 (C=C); 1225 (C-O); 824 (C-N); 728(C-S)       .	-	-
JSR-7h	2-Hydroxy	261	3050(Ar-CH);2924(Ali-CH);1680(C=O); 1554(C=C)1238(C-N);818 (C-N); 704(C-S)	-	-
JSR-7i	4-Methoxy	238	3072(Ar-CH); 2916 (Ali-CH);1678 (C=N); 1532 (C=C);1269 (C-O); 824(C-N)	-	-
JSR-7j	-N,N- Dimethyl amino	251	3088(Ar-CH);2981(Ali-CH); 1680 (C=N); 1509 (C=C); 746 (C-N).709.19(C-S).	10.89 (s, 1H, -N=CH-, g); 8.48 (s, 1H, Ar-H, 2 ); 7.88 (d, 2H, Ar-H, b,d); 7.59 (m, 2H, Ar-H, a,e); 3.95 (s, 9H, -CH2-, 5,6,7,8); 2.56(s, 6H, -N(CH3)2, C).	-
JSR-7k	4-Chloro	244	2947 (Ali-CH); 3071(Ar-CH); 1501 (C=N); 1477 (C=C); 721 (C-S); 1079 (C-O)	11.2 (s, 1H, -N=CH-, g);7.28 (s, 1H, Ar-H, 2 ); 6.98 (d, 2H, Ar-H, b,d); 6.76 (d, 2H, Ar-H, a,e); 3.25 (s, 6H, -CH2-, 5,6,8); 2.39(s, 2H, -CH2-7).	-

 

 

Table-4 Anti-inflammatory activity of Scheme-II compounds JSR (7a-7k):

Difference in paw oedema volume in ml (% Inhibition)
Groups	Dose	1 h	2 h	3 h	4 h
CONTROL		0.86 ±0.03	1.04 ±0.02	1.1±0.03	1.13 ±0.03
STANDARD (Diclofenac)	3mg/kg	0.41±0.01***   (52.32%)	0.38±0.01***   (63.46 %)	0.34±0.01***   (69.09 %)	0.32±0.01***    (71.68%)
JSR-7a	50mg/kg	0.46±0.02***    (51.49%)	0.42±0.02***    (59.62%)	0.45±0.02***     (59.09%)	0.40±0.02***    (64.6%)
JSR-7b	50mg/kg	0.44±0.02     (48.83%)	0.45±0.03***     (56.73%)	0.47±0.03***     (57.27%)	0.40±0.05***     (64.6%)
JSR-7c	50mg/kg	0.59±0.04      (31.39%)	0.61±0.05      (41.35%)	0.69±0.03      (37.27%)	0.64±0.03      (43.36%)
JSR-7d	50mg/kg	0.59±0.04      (31.39%)	0.67±0.04   (35.58%)	0.65±0.04       (40.91%)	0.51±0.04***     (54.87%)
JSR-7e	50mg/kg	0.78±0.02   (9.3%)	0.81±0.02    (22.12%)	0.90±0.03     (18.18%)	0.82±0.03   (27.43%)
JSR-7f	50mg/kg	0.66±0.01  (23.26%)	0.55±0.01   (47.12%)	0.50±0.03***     (54.55%)	0.41±0.02***  (54.87%)
JSR-7g	50mg/kg	0.41±0.02***    (52.33%)	0.42±0.02***    (59.62%)	0.49±0.02***     (55.45%)	0.43±0.02***    (61.95%)
JSR-7h	50mg/kg	0.61±0.02     (29.06%)	0.65±0.03     (37.5%)	0.69±0.03     (37.27%)	0.67±0.05     (40.71%)
JSR-7i	50mg/kg	0.54±0.04      (37.21%)	0.50±0.05***      (51.92%)	0.41±0.03***      (62.72%)	0.39±0.03***      (65.49%)
JSR-7j	50mg/kg	0.52±0.04      (39.52%)	0.56±0.04   (46.15%)	0.50±0.04***       (54.55%)	0.42±0.04***     (62.83%)
JSR-7k	50mg/kg	0.68±0.02   (20.93%)	0.60±0.02    (42.31%)	0.71±0.03     (35.45%)	0.74±0.03   (34.51%)

Data were analyzed by one-way ANOVA followed by Dunnett’s test.

Values are expressed as mean ± S.E.M. [number of animal (n) = 6]

* P<0.01 when compared to control, ns-non significant

 

RESULTS:

The results of the anti-inflammatory activity of the compounds are shown in the Table No-4. Difference in paw volume and percentage reduction in paw volume in rats. Among all the compounds JSR-7a, 7b, 7g, 7i and 7j showed the maximum reduction in paw volume of the rats and were found to be most significant compounds among all the tested compounds followed by JSR-7f. The compounds JSR-7c, 7e, 7h and 7k showed mild anti-inflammatory activity and cause mild reduction in paw volume of the rats.

 

DISCUSSION:

From the IR, ¹H NMR and Mass spectrum obtained, characterization of data has been done and given in Table No: 3. The IR spectrum of 4-Amino-5,6-disubstituted thieno[2,3-d]pyrimidine shows NH₂ peak between 3320 – 3246. The IR spectra of all the Schiff bases there is an absence of the primary aromatic amino peak at 3320-3246  cm^-1  and appearance of a new peak at 1450-1560 cm^-1 for -N=CH- (imine). Disappearance of amino peak in the title compounds compared to their starting thiophenes is sufficient to explain the formation of the new thienopyrimidines.

 

The NMR spectra of the compounds, (JSR-7j) and (JSR-7k) shows disappearance of a broad peak at δ = 5.3-5.95 of -NH₂ and also a sharp singlet peak at δ = 10.89-11.5 of –N=CH which clearly suggest the formation of expected compounds. The compounds JSR-7c and JSR-7e were also confirmed by Mass spectrum.

 

 

ACKNOWLEDGEMENT:

The authors are thankful to Management, PES College of Pharmacy for providing necessary facilities.

 

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Received on 22.01.2015         Modified on 25.02.2015

Accepted on 04.03.2015         © AJRC All right reserved