Synthesis and Biological Studies of Pyrazolo [3, 4-d] Pyrimidines

Introduction

pyrazolo [3,4-d] pyrimidines are of considerable chemical and pharmaceutical importance as purines analogs ^[1-3] of naturally occurring fused uracils that possess diverse biological activities⁴.These derivatives^[5-8] were found to be selective ligands with antagonist activity  for A₁ adenosine receptors(A₁AR). They may have therapeutically use as cognitive enhancers , antidementia drugs(e.g. for Alzheimer’s disease and cerebrovascular dementia),psycostimulants, antidepressant drugs and ameliorants of cerebral fuction^[9],Further more , a large number of pyrimidine derivatives are reported to exhibit antimycobacterial ^[10], antitumor^[11], antiviral^[12], anticancer^[13], antiinflammatory^[14], analgesic^[15], antifolate^[16] ,  antimicrobial^[17], antifungal^[18] , antiproliferative^[19] and antihistaminic^[20] activities.

Due to various biodynamic activities of  pyrazolo [3,4-d] pyrimidines ,one pot synthesis of 3-Amino-4-Aryl-6-Mercapto-3a,4-Dihydro-1H-Pyrazolo[3,4-d] Pyrimidines[IIIa-n] have been undertaken by the condensation of 4-amino-5-cyano-6-aryl-2- mercapto-5,6-dihydro pyrimidines with hydrazine hydrate in ethanol. The product(IIIa-n) were assayed for their in vitro biological assay like antibacterial activity towards Gram positive and Gram negative bacterial strain and antifungal activity towards Aspergillus nigor and Candida albicans at different concentration for their MIC values. The biological activities of the synthesized compounds were compared with standard drugs. [Table II]. The physical constant, antimicrobial and antimicobacterial activities of compounds (IIIa-n) recorded in table I, II and III respectively.

Materials and Methods

Melting points were determined routinely in open capillary tube and are uncorrected. The completion of reaction was routinely checked by TLC on silica gel-G plates of 0.5mm thickness and spots were located by iodine. Elemental analyses of the newly synthesized compounds was carried out on Carlo Reba 1108 analyzer and are found within the range of theoretical value. IR spectra were recorded on Shimadzu-8400 FT-IR spectrometer in Ker ( in cm^-1). ¹H NMR spectra were recorded in CDCl₃ on a Bruckner DRX-300 at 300 MHz. EI-MS spectra were recorded on Shimadzu GC-MS QP-2010 by Electron Impact method. In all the compounds, the molecular weights were found to be 43 m/z less than the molecular ion peak. No particular fragmentation pattern is observed from the spectra.

General Method for Synthesis of 3-Amino-4-Aryl-6-Mercapto-3a, 4-Dihydro-1H-Pyrazolo [3, 4-d] Pyrimidines (III a-n)

Amixture of 4-amino-5-cyano-6-aryl-2- mercapto-5, 6-dihydro pyrimidines (0.01mole) with hydrazine hydrate (0.01mole) in ethanol (30ml) under reflux for a specific period.The reaction mixture was kept at room temperature for 3 hrs. The product was isolated and crystallized from a suitable solvent to give the desire product (IIIa-n).

3-Amino-4-(1’-N-Phenyl-3’-Methyl-5’-Chloro-Pyrazol-4’-YL)-6-Mercapto-3a,4-Dihydro-1H-Pyrazolo[3,4-d] Pyrimidines(IIIn).

IR: 3042(C-H) str.Aromatic), 1506 (C=C ring skeletal vib. Of pyrimidine) ,1454 (C=N ring skeletal vib. pyrimidine), 2929(C-H str.asym.), 2839 (C-H sym.), 1409(C-H sym.), 1454(C-H def.asym.), 3417(N-H str.), 3375 (N-H str.), 1598 (N-H def.) ,1290 (C-N str.), 1598 (C=N str.of pyrazol), 1572 (N-N def.of pyrazol), 1109(C-N str.of Pyrazol), 813 (C-Cl str.)

¹ H –NMR (DMSO+ CDCl3, δ ppm): 2.41(3H,-CH₃), 5.73 (1H, -CH), 7.16-7.63(6H, Ar-H+CH+NH+SH), 7.96-8.02(2H,-NH₂)

MASS spectra: The mass spectrum fragmentation shows molecular ion (M⁺) peak at m/z=360 was consistent with molecular formula C₁₅H₁₄N₇SCl

3-amino-3a, 4-dihydro-4-phenyl-1H-pyrazolo [3, 4-d] pyrimidine-6-thiol(III-a)

IR: 3004(C-H) str.Aromatic), 1504 (C=C ring skeletal vib. Of pyrimidine) ,1456(C=N ring skeletal vib. pyrimidine), 2925(C-H str.asym.), 2837 (C-H sym.), 1365(C-H sym.), 3471(N-H str.), 3135 (N-H str.), 1581 (N-H def.) ,1355 (C-N str.), 1651 (C=N str.of pyrazol), 1620(N-N def.of pyrazol), 1165(C-N str.of Pyrazol)

¹ H –NMR (DMSO+ CDCl3, δ ppm): 5.62 (1H, -CH), 6.74-7.93(9H, Ar-H+CH+NH₂+SH)

MASS spectra: The mass spectrum fragmentation shows molecular ion (M⁺) peak at m/z=245.0 was consistent with molecular formula C₁₁H₁₁N₅S 

3-amino-3a,4-dihydro-4-(methylthio)-1H-pyrazolo[3,4-d]pyrimidine-6-thiol(III-j)

IR: 3030(C-H) str.Aromatic), 1512 (C=C ring skeletal vib. Of pyrimidine) ,1456(C=N ring skeletal vib. pyrimidine), 2920(C-H str.asym.), 2852 (C-H sym.), 1383(C-H sym.), 3435(N-H str.), 3375 (N-H str.), 1311 (N-H def.) ,1178 (C-N str.), 1662 (C=N str.of pyrazol), 1583(N-N def.of pyrazol), 1178(C-N str.of Pyrazol)

¹ H –NMR (DMSO+ CDCl3, δ ppm): 2.47(3H, SCH₃), 5.95 (1H, -CH), 6.71-8.70(9H, Ar-H+CH+NH₂+SH)

MASS spectra: The mass spectrum fragmentation shows molecular ion (M⁺) peak at m/z=271.0 was consistent with molecular formula C₁₃H₁₃N₅S.

Antimicrobial Activity

Antimicrobial was carried out by using cup-plate method [90].which has been described as under.

Antibacterial Activity

Gram positive bacteria were grown in nutrient broth and Gram negative bacteria in Peptone water (PW, 1% bacteriological peptone and 0.5% NaCl) for 24 hours; this gave an optimum growth of the test bacteria. Each purified compound was dissolved in DMF sterilized by filtration by using sintered glass filter and stored at 4⁰C.Each agent was then added to molten nutrient agar in the following concentration(µg/ml): 0 (control), 25,50,100,200,500,800and poured into sterile Petri dished. The pH of the media was maintained at 7.2-7.4. The inoculums consisted of an overnight growth broth culture of a bacterium diluted in such a manner that a 2mm (internal diameter) loopful of the culture contain 10⁰ colony-forming unit (CFU). These were then spot inoculated on nutrient agar plates containing increasing amount of a compound, incubated at 37⁰C up to 24 hrs. for determination of the minimum inhibitory concentration (MIC) ^91-92.The antibacterial activity of the compounds (III a-n) was compared with known standard reference drugs like Ampicillin, Ciprofloxacin, Chloramphenical, Griseofulvin, at same concentration. The moderate and comparable antibacterial activities of compound are recorded.

Antifungal Activity

Aspergillus Niger MTCC-282 and Candida albicans MTCC-227 were employed for testing fungicidal activity using cup plate method. The cultures were maintained on Sabouraud’s agar for72 hours this gave an optimum growth of the test fungal spores Each purified compound was dissolved in DMF sterilized by filtration by using sintered glass filter and stored at 4⁰C.Each agent was then added to Sabouraud’s agar in the following concentration(µg/ml): 0 (control), 25,50,100,200,500,800 and poured into sterile Petri dished.. The inoculums consisted of an overnight growth broth culture of a bacterium diluted in such a manner that a 2mm (internal diameter) loopful of the culture contain 10⁵ colony-forming units (CFU). These were then spot inoculated on Sabouraud’s agar plates containing increasing amount of a compound, incubated at 37⁰C up to 48 hrs. For determination of the minimum inhibitory concentration (MIC) ^91-92.Th MIC value of test solutions are recorded in table No 2.

Conclusion

It was interesting to note that the reaction occurred immediately. This work demonstrates a very simple and efficient method for the synthesis of a well functionalized pyrazolo [3, 4-d] pyrimidines of biological importance in excellent yields.

Table 1: Physical and analytical data:

Sr. No.	R	X	Molecular Formula	m.p. OC	Yield (%)	Rf Value	% of  Nitrogen Calcd.    Found
III a	C6H5	S	C11H11N5S	140	61	0.62	28.57/28.52
III b	2-Cl-C6H4	S	C11H10N5SCl	144	63	0.58	25.04/25.00
III c	4-Cl-C6H4	S	C11H10N5SCl	158	48	0.49	25.04/24.99
III d	3-Br-C6H4	S	C11H10N5SBr	165	51	0.51	21.67/21.61
III e	3-NO2-C6H4	S	C11H10N6O2S	175	46	0.53	27.63/27.57
III f	3-C6 H5-O-C6H4	S	C17H15N5OS	138	54	0.50	20.77/20.71
III g	4-OCH3-C6H4	S	C12H13N5OS	147	50	0.63	25.45/25.40
III h	2-OH-C6H4	S	C11H11N5OS	172	67	0.64	26.81/26.76
III i	4-OH-C6H5	S	C11H11N5SO	230	52	0.52	26.81/26.8
III j	C6H4-CH=CH	S	C13H13N5S	151	56	0.55	25.83/25.80
III k	4-CH3S-C6H4	S	C12H13N5S2	168	48	0.57	24.05/23.97
III l	α- C4H3O	S	C9H9N5OS	250	68	0.58	29.78/29.73
III m	4-N-(CH3)2-C6H4	S	C13H16N6S	180	74	0.61	29.16/29.11
III n	1-N-C6 H 5-3-CH3-5Cl-C3 N 4	S	C15H14N7SCl	128	68	0.62	27.26/27.20

TLC Solvent systems: Acetone: Benzene= 1:9

Table 2: Antimicrobial Activity of 3-Amino-4-Aryl-6-Mercapto-3a, 4-Dihydro-1H-Pyrazolo [3, 4-d] Pyrimidines (III a-n)

Compound	R				Antibacterial activity			Antifungal activity
			S.pyogensMTCC-442		S.aureusMTCC-96	E.ColiMTCC-443	B.subtillisMTCC-441	C.alibicansMTCC-227	A.nigerMTCC-282
III a	C6H5		200		200	500	800	–	200
III b	2-Cl-C6H4		25		100	200	500	500	800
III c	4-Cl-C6H4		–		50	800	–	100	50
III d	3-Br-C6H4		800		100	25	200	200	500
III e	3-NO2-C6H4		50		50	100	–	50	–
III f	3-C6 H5-O-C6H4		200		200	800	100	800	200
III g	4-OCH3-C6H4		25		25	–	25	–	100
III h	2-OH-C6H4		–		500	50	50	200	800
III i	4-OH-C6H5		800		100	200	200	200	–
III j	C6H4-CH=CH		100		–	–	800	800	500
III k	4-CH3S-C6H4		500		500	100	200	–	–
III l	α- C4H3O		100		200	200	500	500	800
III m	4-N-(CH3)2-C6H4		200		200	100	200	800	–
III n	1-N-C6 H 5-3-CH3-5Cl-C3 N 4		100		200	25	500	500	–
Comparative activity of (III a-n) with known choosen standard drugs
Standard Drug				Antibacterial activity				Antifungal activity
		S.pyogensMTCC-442		S.aureusMTCC-96		E.ColiMTCC-443	B.subtillisMTCC-441	C.alibicansMTCC-227	A.nigerMTCC-282
		IIIb(25),  IIIe(50)		IIIc(50), IIIe(50)		IIId(25)	IIIg(25)	IIIe(50)	IIIc(50)
		IIIg(25)		IIIg(25)		IIIh(50)	IIIh(50)	–	–
Ampicillin		30		20		30	30	–	–
Amoxycillin		20		20		20	20	–	–
Cifalexin		20		30		35	20	–	–
Erythromycin		30		35		20	20	–	–
Chlotrimazole		–		–		–	–	20	20
Griseofulvin		–		–		–	–	30	20

N.B. :(-): No activity

 

Scheme 1 Click here to View scheme

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