Preparation and Biological Evaluation of 3-amino-4-aryl-4, 5- dihydro-1-N-tolyl pyrazolo [3, 4-d] pyrimidines Derivative

Introduction

Extensive studies have been made in the field of synthesis of pyrazol [3, 4-d] pyrimidines ^[1-10] Consideration research has been under taken to extend activity and reduce toxicity of pyrazolo [3, 4-d] pyrimidine. The specific biological activities have been Hyperuricemia^[11], Protozoacidal^[12], Hypoglycemic^[13], Antimalerial^[14], Anticancer^[15], Analgesic^[16],Antipyretic^[16] , Aniinflammatory^[16], Antimycotic agent^[17], Xanthine oxidaseinhibitor^[18], Antitumor^[19], Anti-HIV^[20], Anxiolytic agent^[21], Cardiovascular^[22], Antileishmanial^[23],Tranquilizer^[24],Bloodsugar lower agent^[25], Anticonvulant^[26], Antiviral^[27], Antiallergic^[28].

The product(Va-m) 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 (Va-m) recorded in Table II respectively.

 

Scheme 1 Click here to View scheme

 

Materials and Methods

Melting points were determined in open capillary tubes and are uncorrected. Formation of the compounds was routinely checked by TLC on silica gel-G plates of 0.5 mm thickness and spots were located by iodine. IR spectra were recorded Shimadzu FT-IR-8400 instrument using KBr pellet method. Mass spectra were recorded on Shimadzu GC-MS-QP-2010 model using Direct Injection Probe technique. ¹H NMR was determined in DMSO-d₆ solution on a Bruker Ac 400 MHz spectrometer. Elemental analysis of the all the synthesized compounds was carried out on Elemental Vario EL III Carlo Erba 1108 model and the results are in agreements with the structures assigned.

General Method for Synthesis of 3-amino-4-aryl-4, 5- dihydro-1-N-tolyl pyrazolo [3, 4-d] pyrimidines derivative (Va-m)

A mixture of 3-amino-1-p-tolyl-1H-pyrazol-5(4H)-one (0.01 mole), urea (0.01 mole) and aldehyde (0.01mole) in ethanol (30ml) under reflux condition for three hours. The reaction mixture was kept at room temperature for 2 hrs. The product was isolated and crystallized from a suitable solvent to give the desire product. The Physical data were recorded in Table-1

Preparation of 3-amino-4, 5-dihydro-4-(4-(methylthio) phenyl)-1-p-tolyl-1H-pyrazolo [3, 4-d] pyrimidin-6-ol (V-l)

A mixture of 3-amino-1-p-tolyl-1H-pyrazol-5(4H)-one (1.88 gm, 0.01M), urea(0.60gm,0.01M) and 4-(methylthio)benzaldehyde (1.52 ml, 0.01mole) in ethanol (30ml) under reflux condition for three hours. The reaction mixture was kept at room temperature for 2 hrs. The product was collected and recrystallized from an ethanol: dioxan (2:1).

3-amino-4, 5-dihydro-4-(4-(methylthio) phenyl)-1-p-tolyl-1H-pyrazolo [3, 4-d] pyrimidin-6-ol (V-l)

IR: 3030(C-H) str. Aromatic), 1512 (C=C ring skeletal vib. Of pyrimidine) ,1456(C=N ring skeletal vib. pyrimidine), 3030(C-H str.),1178((-C-H i.p.def.), 2920(C-H asym.), 2852(C-H sym.), 1382(C-H def. sym.), 3435(N-H str.), 3373 (N-H str.), 1588 (N-H def.) ,1311 (C-N str.), 1662 (C=N str. of pyrazol), 1583 (N-N def. of pyrazol), 1178(C-N str. of Pyrazol),3325(-OH Str.)

¹ H –NMR (DMSOd₆+ CDCl₃, δ ppm):  2.56(3H,-CH₃), 2.47(3H,-SCH₃), 5.95(1H, -CH), 6.71-8.70(16H, Ar-H+NH+NH₂+OH)

MASS spectra: The mass spectrum fragmentation shows molecular ion (M⁺) peak at m/z=365.45was consistent with molecular formula C₁₉H₁₉ON₅ OS

3-amino-4, 5-dihydro-4-(4-methoxyphenyl)-1-p-tolyl-1H-pyrazolo [3, 4-d] pyrimidin-6-ol (V-h)

IR: 3114(C-H) str. Aromatic), 1514 (C=C ring skeletal vibe. Of pyrimidine), 1459(C=N ring skeletal vib. pyrimidine), 2935(C-H str. asym.), 2845(C-H sym.), 1370(C-H sym.), 1460(C-H def. asym.), 3473(N-H str.), 3133 (N-H str.), 1587N-H def.) , 1370 (C-N str.), 1648 (C=N str. of pyrazol), 1625 (N-N def. of pyrazol), 1165(C-N str.of Pyrazol).

¹ H –NMR (DMSOd₆+ CDCl₃, δ ppm): 2.45(3H,-CH₃), 3.73(3H, 3-OCH₃), 5.19 (1H, -CH), 6.80-7.02(12 H, Ar-H+NH+NH₂+OH),

MASS spectra: The mass spectrum fragmentation shows molecular ion (M⁺) peak at m/z= 349.38was consistent with molecular formula C₁₉H₁₉N₅O₂

3-amino-4, 5-dihydro-4-(4-hydroxyphenyl)-1-p-tolyl-1H-pyrazolo [3, 4-d] pyrimidin-6-ol (V-j)

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.), 1454(C-H def. asym.), 3471(N-H str.), 3135 (N-H str.), 1581 (N-H def.) ,1365 (C-N str.), 1651 (C=N str. of pyrazol), 1620 (N-N def. of pyrazol), 1166(C-N str.of Pyrazol).

¹ H –NMR (DMSOd₆+ CDCl₃, δ ppm): 2.35(3H,-CH₃), 5.0(1H, -OH) 5.62 (1H, -CH), 6.61-7.13(12H, Ar-H+NH+NH₂+OH),

MASS spectra: The mass spectrum fragmentation shows molecular ion (M⁺) peak at m/z=335.5 was consistent with molecular formula C₁₈H₁₇N₅ O₂.

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 data 3-amino-4-aryl-4, 5- dihydro-1-N-tolyl pyrazolo [3, 4-d] pyrimidines derivative (Va-m)

Sr. No.	R	Molecular Formula	M.W.	m.p. OC	Yield (%)	Rf Value	% of  Nitrogen
							Calcd.	Found
V a	C6H5	C18H17N5O	319.36	240	68	0.68	21.80	21.75
V b	2-Cl-C6H4	C18H16ClN5O	353.80	252	72	0.63	19.69	19.63
V c	4-Cl-C6H4	C18H16ClN5O	353.80	260	67	0.59	19.69	19.65
V d	3-NO2–C6H4	C18H16N6O3	364.35	271	72	0.53	22.95	22.90
V e	2-NO2-C6H4	C18H16N6O3	364.35	245	78	0.58	22.95	22.90
V f	3-C6 H5-O-C6H4	C24H21N5O2	411.45	249	64	0.70	16.94	16.90
V g	2-OCH3-C6H4	C19H19N5O2	349.38	259	58	0.55	19.94	19.91
V h	4-OCH3-C6H4	C19H19N5O2	349.38	250	65	0.53	19.94	20.89
V i	2-OH-C6H5	C18H17N5 O2	335.35	240	72	0.51	18.56	20.51
V j	4-OH-C6H5	C18H17N5 O2	335.35	255	75	0.49	18.56	20.50
V k	C6H4-CH=CH	C26H23N5O	421.49	265	62	0.62	20.17	19.11
V l	4-CH3S-C6H4	C19H19ON5 OS	365.45	219	71	0.63	19.07	22.02
V m	α- C4H3O	C24H21N5 O2	385.45	290	77	0.61	22.50	16.45

TLC Solvent systems: Acetone: Benzene= 1:9

Antimicrobial Activity

Antimicrobial was carried out by using cup-plate method .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 units (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) .The antibacterial activity of the compounds (Va-m) 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) .The MIC value of test solutions are recorded in Table No-2 and Table No-3

 

Table 2:Antimicrobial Activity of 3-amino-4-aryl-4, 5- dihydro-1-N-tolyl pyrazolo [3, 4-d] pyrimidines derivative (Va-m)

Compound	R		Antibacterial activity			Antifungal activity
		S.pyogensMTCC-442	S.aureusMTCC-96	E.ColiMTCC-443	B.subtillisMTCC-441	C.alibicansMTCC-227	A.nigerMTCC-282
V a	C6H5	100	200	500	500	200	200
V b	2-Cl-C6H4	100	50	25	100	25	50
V c	4-Cl-C6H4	200	–	–	50	–	100
V d	3-NO2–C6H4	25	–	–	50	–	–
V e	2-NO2-C6H4	500	500	–	800	500	–
V f	3-C6 H5-O-C6H4	50	–	500	500	25	800
V g	2-OCH3-C6H4	–	50	500	–	–	500
V h	4-OCH3-C6H4	50	100	200	500	500	–
V i	2-OH-C6H5	100	200	–	500	–	800
V j	4-OH-C6H5	500	500	800	500	–	–
V k	C6H4-CH=CH	–	–	500	500	–	500
V l	4-CH3S-C6H4	–	500	500	500	500	–
V m	α- C4H3O	–	800	500	200	200	200

 

Table 3: Comparable Activity of compounds (V a-m) with known chosen standard drugs

Comparative activity of (III a-n) with known chosen standard drugs
Standard Drug		Antibacterial activity			Antifungal activity
	S.pyogens MTCC-442	S.aureus MTCC-96	E.Coli MTCC-443	B.subtillis MTCC-441	C.alibicans MTCC-227	A.niger MTCC-282
	Vd (25)	Vb(50)	Vb(50)	Vc (50)	Vb (25)	Vb (50)
	Vf (50)	Vg(50)			–	–
Ampicillin	30	20	30	30	–	–
Amoxycillin	20	20	20	20	–	–
Cifalexin	20	30	30	20	–	–
Erythromycin	30	30	20	20	–	–
Chlotrimazole	–	–	–	–	20	20
Griseofulvin	–	–	–	–	30	20

N.B. :(-): No activity

 

Acknowledgements

The author’s wish to thanks to Principal, Shree R.R. Lalan College, Bhuj for providing research facilities.

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