Synthesis of Pyrazolopyrimidine Derivatives and Their Antibacterial Activity
S. Singh*, P. K. Sharma, R. Dudhe and N. Kumar
Department of Pharmaceutical Technology, Meerut Institute of Engineering and Technology, Meerut
NH-58, Bypass Road, Baghpat Crossing, Meerut- 250005, U.P., India.
*Corresponding Author E-mail: Sudha.singh758@gmail.com
ABSTRACT:
A novel series of substituted pyrazolopyrimidine derivatives have been synthesized from substituted hydrazine derivatives and ethoxy (methylene)malanonitrile gives an intermediate (5-amino-1- substituted phenyl pyrazole-4-carbonitrile) (1) which on further react with formic acid, acetic acid/HCl and formamide gives series of (1- substituted phenyl)1H pyrazole [3,4-d] pyrimidine-4(5H)-one (2). The synthesized compounds were characterized on the basis of IR, 1H NMR and mass spectral data. Furthermore, these newly synthesized compounds were screened for their antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Compounds PP-6A, PP-2B, PP-3B and PP-5C showed significant antibacterial activity.
KEYWORDS: Pyrazole, Pyrazolo[3,4-d]pyrimidines, Antibacterial activity, Heterocycles.
INTRODUCTION:
Pyrazolopyrimidines and related fused heterocycles are of interest as potential bioactive molecules. The heterocyclic fusion of pyrimidine ring and pyrazole ring resulted in the formation of pyrazolopyrimidines, the structural analogues of biogenic purine class, definitely has high consequence in the field of pharmaceutical and biotechnologcal sciences with wide spectrum of biological activities.[1, 4] They are known to display pharmacological activities such as CNS depressant,[5] neuroleptic[6], and tuberculostatic [7] Pyrazolo[3,4-d]pyrimidines were identified as a broad class of adenosine receptors[8,9]. In the literature [10,11] it was reported that replacement of 1H of the pyrazole ring of pyrazolo[3,4-d]pyrimidine derivatives by some other bioactive moieties extensively alters its pharmacological properties. The main aim of this report was to synthesize pyrazolo[3,4-d]pyrimidine derivatives bearing a heterocyclic moiety namely: (1- substituted phenyl) 1H pyrazole [3,4-d] pyrimidine-4(5H)-one which is known to exhibit pronounced biological activities. The pyrazolo [3, 4-d] pyrimidines frame work, a member of fused bicyclic heterocycles, has been a versatile scaffold for various pharmacological studies, separately from their efficacy for several diseases target, pyrazolo [3,4-d] pyrimidines have shown enormous synthetic value in the preparation of various drug and bio-active molecule12,13.
EXPERIMENTAL:
Chemistry:
The melting point of products were determined by open capillaries method and are uncorrected. IR Spectra (KBr) were recorded on FTIR Spectrophotometer (Shimadzu FTIR 84005, 4000-400cm-1). The electrospray mass spectra were recorded on a THERMO Finnigan LCQ Advantage max ion trap mass spectrometer. The 10 µl samples (dissolve in solvent such as methanol/ acetonitrile/ water) were introduced into the ESI source through Finnigen survey or autosampler. The mobile phase 90: 10
MeOH/ ACN : H2O flowed at the rate of 250 µl/ min by MS pump. Ion spray voltage was set at 5.3 KV and capillary voltage 34 V. 1H NMR were recorded on a Bruker DRX-300 MHz spectrometer in CDCl3 using TMS as an internal standard, with 1H resonance frequency of 300 MHz Chemical shift values are expressed in d ppm.
Table 1:
|
Compounds |
R |
|
P1 |
H |
|
P2 |
p-nitro |
|
P3 |
2,4-di nitro |
|
P4 |
2,4-di amino |
|
P5 |
p-amino |
|
P6 |
3,5-di chloro |
1. General procedures for the preparation of the compounds (P1- P6):
1.1 Synthesis of 5-amino-1-phenyl-1H-pyrazole-4-carbonitrile:
In reaction substituted hydrazine derivatives (1ml) in 20 ml anhydrous ethanol, ethoxymethylenemalanonitrile (1ml) was added and the reaction mixture were refluxed for 2-3 hrs, respectively. Anhydrous condition have to be maintained by using calcium chloride guard was filtered off, (compound 1) dried and recrystallized from ethanol.
1.2 Synthesis of 5-amino-1-phenyl-1H-pyrazole-4-carbonitrile P1 : Yield 64%, mp-194-196°C, IR ( KBr, cm-1): 3446.56 ( N-H Str), 2231.49 (CN str),1610.45(Ar C=C), 1598.16 ( Ar C-C), 856.34 (C -H deformation). 1H NMR: (CDCl3, δ, ppm): 7.6 (d, 1H, CH of pyrazole), 4.1 (m, 2H, Ar C-NH of (NH2), 7.0-7.14 ( m, 5H, Ar-H). MS (m/z) : (M+ =184), 105, 92.
1.3 Synthesis of 5-amino-1- (4-nitro)phenyl-1H-pyrazole-4-carbonitrile P2: Yield 92%, mp-198-200 °C, IR ( KBr, cm-1): 3444.63 ( N-H Str), 1348.15 (CN str),1611.45(Ar C=C), 844.76(C -H deformation), 1598.88 (Ar- NO2).1H NMR: (CDCl3, δ, ppm): 7.5 (d, 1H, CH of pyrazole), 4.0 (m, 2H, Ar C-NH), 7.3-8.07 ( m, 4H, Ar-H). MS (m/z) : (M+ =229), 122, 111.
1.4 Synthesis of 5-amino-1- (2,4-dinitro)phenyl-1H-pyrazole-4-carbonitrile P3 : Yield- 92 %, mp- 204-206 °C, IR (KBr, cm-1) : 3417.63 ( N-H Str), 2223.77 (CN str), 1641.31(N-H bending ), 781.12 ( p- nitro Ar substitution ), 744.47 ( o- nitro Ar substitution ). 1H NMR: (CDCl3, δ, ppm): 7.7 (d, 1H, CH pyrazole), 3.1 (m, 2H, Ar C-NH of (NH2)), 8.7-8.9 (m, 3H, Ar- H). MS (m/z) : (M+ = 274); 106, 159, 173, 140.
1.5 Synthesis of 5-amino-1- (2,4-diamino)phenyl-1H-pyrazole-4-carbonitrile P4 : Yield- 98 %, mp- 212-214°C, IR (KBr, cm-1) : 3400, ( N-H Str), 2221.55 (CN str), 1641.31(N-H bending). 1H NMR: (CDCl3, δ, ppm): 7.9 (d, 1H, CH of pyrazole), 4.8( m, 6H, Ar C-NH of (NH2)), 6.9 (d, 3H, Ar- H). MS (m/z) : (M+ = 214); 105, 119.
1.6 Synthesis of 5-amino-1- (p-amino)phenyl-1H-pyrazole-4-carbonitrile P5 : Yield -85%, mp-222-224 °C, IR (KBr, cm-1): 3411, 3400, ( N-H Str), 2411.55 (CN str), 1541.31(N-H bending), 3205.1 (-NH2), 1509 ( C=C Aromatic). 1H NMR: (CDCl3, δ, ppm): 7.5 (d, 1H, CH of pyrazole ), 4.0 (m, 4H, Ar C-NH of (NH2)), 6.1-6.8 (m, 4H, Ar-H). MS (m/z) : (M+ =199.11), 107, 91.
Table 2: Physicochemical data of intermediate substituted compounds.
|
S. No. |
Code no. |
R |
Molecular Formula |
M.W |
Rf value |
(%) Yield |
m.p. (°C) |
|
01. |
P1 |
H |
C10H8N4 |
184 |
0.71 |
64.72 |
194-196 |
|
02. |
P2 |
p-nitro |
C10H7N5O2 |
229 |
0.92 |
84.00 |
198-200 |
|
03. |
P3 |
2,4-dinitro |
C10H6N6O4 |
274 |
0.58 |
92.30 |
204-206 |
|
04 |
P4 |
2,4-diamino |
C10H10N6 |
214 |
0.79 |
98.00 |
212-214 |
|
05 |
P5 |
p-amino |
C10H9N5 |
199 |
0.69 |
85.00 |
222-224 |
|
06 |
P6 |
3,5-dichloro |
C10H6Cl2N4 |
252 |
0.71 |
78.00 |
219-221 |
1.7 Synthesis of 5-amino-1- (3,5 dichloro)phenyl-1H-pyrazole-4-carbonitrile P6 : Yield- 92%, mp- 219-221 °C, IR (KBr, cm-1): 3520.11, ( N-H Str), 2416.55 (CN str), 1521.01 (N-H bending), 3255.1 (-NH2), 1569 ( C=C Aromatic). 1H NMR: (CDCl3, δ, ppm): 7.6 (d, 1H, CH of pyrazole), 4.0 (m, 2H, Ar C-NH of (NH2)), 6.9-7.09 (d, 3H, Ar-H). MS (m/z) : (M+ = 252), 106, 159, 144.
2. General procedure for the synthesis of the compounds PPA-PPC
2.1 Formic acid: Compound 1 ( 0.01 mol) was refluxed in formic acid (30 mL, 85%) for 5 h. The reaction mixture was cooled and poured into water. The formed solid compound PPA was filtered off, dried, and recrystallized from dioxane.
2.2 Acetic acid/ HCl: Compound 1 ( 0.01 mol) was refluxed in a mixture of hydrochloric acid (3 mL) and acetic acid (9 mL) for 3 h. The reaction mixture was cooled, poured into water and the solid compound PPB formed was filtered off, dried and recrystallized from dioxane.
2.3 Formamide: Compound 1 ( 0.01 mol) was refluxed in formamide (20 mL) for 3 h. The reaction mixture was cooled and poured into water. The solid compound PPC formed was filtered off, dried and recrystallized from dioxane.
3.1 Synthesis of 1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP-1A: Yield: 60%, mp 178-180°C, IR ( KBr, cm-1) 3540.60 ( N-H secondary amine), 3346.21 (C-H Ar str), 2223.77 (C=N Ar) 1647.10 (C=O str), 1598 (C=C str). 1H NMR: (CDCl3, δ, ppm): 7.4( d, 1H, CH of pyrazole), 7.3-7.5 (m, 5H, CH), 8.0 ( m. 1H, NH). MS (m/z) : (M+= 212); 125, 196, 145.
3.2 Synthesis of 6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP-1B: Yield: 50%, 156-158 °C, IR ( KBr, cm-1) 3460.60 ( N-H secondary amine), 3396.41 (C-H Ar str), 2223.77 (C=N Ar) 1647.10 (C=O str), 1598 (C=C str), 1H NMR: (CDCl3, δ, ppm):7.5 (d, 1H, CH of Pyrazole), 7.2-7.6 (m, 5H, Ar-H ), 4.1(m, 2H, Ar C-NH of NH2 ) . MS (m/z) : (M+= 223); 200, 160, 186.
3.3 Synthesis of 1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine PP-1C: Yield: 57.40 %, 200- 202 °C, IR ( KBr, cm-1) 3375.20 (N-H Ar), 3062 (C-H Ar), 2341 ( C=N Ar), 1504.37 (C=C Ar str), 1305.72 ( NH2 str). 1H NMR: (CDCl3, δ, ppm): 7.7 (d, 1H, CH of pyrazole), 7.0- 7.3 ( m, 5H, Ar- H ), 8.4 (d, 2H, Ar- C-NH of pyrimidine). MS (m/z) : (M+= 211), 105, 118, 134.
3.4 Synthesis of 1-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP-2A: Yield: 90 % , 210-212 °C, IR ( KBr, cm-1) 3200 (NH str), 3232 (C-H str), 1616 ( C=O str), 1506 ( C=N str), 1458 ( Ar- NO2), 1496 (C=C Ar str). 1H NMR: (CDCl3, δ, ppm): 7.5 (m, 1H, CH of pyrazole), 7.5-8.2 (m, 4H, Ar-H), 8.0 (m, 1H, NH ). MS (m/z) : (M+ = 257), 230, 130.
3.5 Synthesis of 6-methyl-1-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP2- B: Yield: 81.08 % , 214-216 °C, IR ( KBr, cm-1): 3500 (NH str), 3082 (C-H str), 1616 ( C=O str), 1596 ( C=N str), 1548 ( Ar- NO2), 1506 (C=C Ar str). 1H NMR: (CDCl3, δ, ppm): 7.6 (d, 1H, CH of pyrazole), 6.6-7.3 (m, 3H, Ar- H ). MS (m/z) : (M+= 263), 249, 271.
3.6 Synthesis of 1-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine PP2-C: Yield: 81.01 % , 198-200 °C, IR ( KBr, cm-1): , 3570 (NH str), 3152 (C-H str), 1606 ( C=O str), 1545 ( C=N str), 1475 ( Ar- NO2), 1578.02 (C=C Ar str). 1H NMR: (CDCl3, δ, ppm): 7.72 (m, 1H, CH of pyrazole), 7.5-8.2 (m, 4H, Ar-H ), 8.9 (m, 1H, CH ). MS (m/z) : (M+= 256), 240, 229.
3.7 Synthesis of 1-(2,4-dinitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP3-A: Yield: 53 % , 222-224 °C, IR ( KBr, cm-1); 3456.87 (C-H Ar), 2459 (NH str), 1582 CN str, 751( p- nitro), 736 ( o- nitro Ar substitution). 1H NMR: (CDCl3, δ, ppm): 7.9 (m, 1H, CH of pyrazole), 7.9-8.8 (m, 3H, Ar-H), 8.0 (m, 1H, NH). MS (m/z) : (M+= 319.1).
3.8 Synthesis of 6-methyl-1-(2,4-dinitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP3-B: Yield: 81.00 % , 206-208 °C, 222-224 °C, IR ( KBr, cm-1); 3386.77 (C-H Ar), 2358 (NH str), 1591 CN str, 781( p- nitro), 744( o- nitro Ar substitution). 1H NMR: (CDCl3, δ, ppm): 7.3 ( m, 1H, CH of pyrazole), 7.1-8.7 (m, 3H, Ar-H ). MS (m/z) : (M+= 308), 140, 185, 277, 184.
3.9 Synthesis of 1-(2,4-dinitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine PP3-C: Yield: 64%, 196-198 °C, IR ( KBr, cm-1): 3095.54 ( CH str), 2360 ( NH str), 1593 ( C=C str), 835( p- nitro), 709 ( o- nitro Ar substitution). 1H NMR: (CDCl3, δ, ppm): 7.7 (m, 1H, CH), 7.8- 9.1 (m, 3H, Ar-H ), 4.0 ( m, 2H, ArC-NH ). MS (m/z) : (M+= 301), 242, 167, 134.
3.10 Synthesis of 1-(2,4-diaminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP4-A: Yield: 75.90%, 224-226 °C, IR ( KBr, cm-1): 3095.54 ( CH str), 2360 ( NH str), 1593 ( C=C str), 3165.3 ( -NH, 2° amide ), 3018 (= C-H, aromatic), 1733.6 (- C=O). 1H NMR: (CDCl3, δ, ppm): 7.7 (d, 1H, CH of pyrazole), 5.5-6.8 (m, 3H, Ar-H), 4.0 (m, 4H, ArC-NH). MS (m/z) : (M+= 242); 160, 147, 201.
3.11 Synthesis of 1-(4-aminophenyl)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP4 B: Yield: 61%, 222-224°C, IR(KBr,cm-1): 3127.3(-NH, 2° amide), 3014 (=C-H aromatic), 2929 ( -CH2-), 1684.9 (- C=O), 1H NMR: (CDCl3, δ, ppm): 7.6 (d, 1H, CH), 6.6-7.3 (m, 3H, Ar-H, ), 4.1 (m, 4H, ArC-NH), MS (m/z) : (M+=256), 241, 228, 172, 188.
3.12 Synthesis of 1-(4-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine PP4-C: Yield: 55%, 214-216 °C, IR(KBr,cm-1): 3205 ( NH2), 3075.1 (=C-H aromatic), 1509.2 ( C=C aromatic). 1H NMR: (CDCl3, δ, ppm): 7.72 (d, 1H, CH), 5.5-6.9 (d, 3H, Ar-H ), 4.0 (m, 4H, ArC-NH). MS (m/z) : (M+=241), 134, 107,148.
3.13 Synthesis of 1-(3,5-dichlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP-5A: Yield: 66%, 228-230 °C, IR(KBr,cm-1): cm-1): 3125.54 ( CH str), 2460 ( NH str), 1453 ( C=C str), 3255.3 ( -NH, 2° amide ), 3108 (= C-H, aromatic), 1653.6 (- C=O). 1H NMR: (CDCl3, δ, ppm): 7.6 (d, 1H, CH), 6.8-7.4 (m, 4H, Ar-H ), 3.25 (m, 2H, ArC-NH). MS (m/z) : (M+= 231), 124, 214, 122.
3.14 Synthesis of 1-(4-aminophenyl)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP-5B: Yield: 59%, 218-220 °C, IR(KBr,cm-1): 3235.2 (-NH, 2° amide), 3120.12(=C-H aromatic), 2892 ( -CH2-), 1574.9 (- C=O). 1H NMR: (CDCl3, δ, ppm): 7.7 (d, 1H, CH of pyrazole), 6.5-7.0 (d, 4H, Ar-H ), (d, 1H, ArC-NH). MS (m/z) : (M+=241), 149, 145, 161.
3.15 Synthesis of 1-(4-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine PP-5C: Yield : 65%, 188-190 °C, IR(KBr,cm-1): 3225 ( NH2), 3125.1 (=C-H aromatic), 1485.2 ( C=C aromatic). 1H NMR: (CDCl3, δ, ppm): 7.7 (d, 1H, CH of pyrazole), 6.5-7.0 (m, 4H, Ar-H ), 8.3 (d, 1H, CH of pyrimidine). MS (m/z) : (M+= 226), 134, 172.
3.16 Synthesis of 1-(3,5-dichlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP-6A: Yield : 58.13%, 212-214 °C, IR(KBr,cm-1): cm-1): 3102.54 ( CH str), 2365 ( NH str), 1353 ( C=C str), 3355.3 ( -NH, 2° amide ), 3208 (= C-H, aromatic), 1553.6 (- C=O). 1H NMR: (CDCl3, δ, ppm): 7.7 (d, 1H, CH of pyrazole), 7.1-7.2 (d, 3H, CH of pyrazole), 8.0 (d, 1H, CH of pyrazole). MS (m/z) : (M+= 279), 144, 208, 121, 209.
3.17 Synthesis of 1-(3,5-dichlorophenyl)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one PP- 6B: Yield: 65.66%, 224-226 °C, IR(KBr,cm-1): 3335.2 ( NH2), 3025.1 (=C-H aromatic), 1585.2 ( C=C aromatic). 1H NMR: (CDCl3, δ, ppm): 7.7 (s, 1H, CH ), 7.2-7.3 (d, 3H, Ar-H), 8.0 (m, 1H, NH). MS (m/z) : (M+=295), 149, 144, 197.
3.18 Synthesis of 1-(3,5-dichlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine PP-6C: : Yield: 75.53%, 218-220 °C, IR(KBr,cm-1): 3225 ( NH2), 3125.1 (=C-H aromatic), 1485.2 ( C=C aromatic). 1H NMR: (CDCl3, δ, ppm): 7.8 (d, 1H, CH of pyrazole), 6.5-7.1 (d, 3H, Ar-H), 3.2 (m, 2H, Ar C-NH). MS (m/z) : (M+= 270), 199, 262, 140, 134.
Table 3. Physicochemical data for the newly prepared compounds.
|
S. No. |
Compd no. |
R |
Molecular Formula |
M.W |
Rf value |
(%) Yield |
m.p. (°C) |
|
1 |
PP-1A |
H |
C10H9N3O |
212 |
0.71 |
60.00 |
178-180 |
|
2 |
PP-1B |
H |
C13H11N3O |
223 |
0.87 |
50.00 |
156-158 |
|
3 |
PP-1C |
H |
C11H9N5 |
211 |
0.81 |
57.40 |
200-202 |
|
4 |
PP-2A |
p-nitro |
C11H7 N5O3 |
257 |
0.86 |
90.09 |
210-212 |
|
5 |
PP-2B |
p-nitro |
C12H9N5O3 |
263 |
0.71 |
81.80 |
214-216 |
|
6 |
PP-2C |
p-nitro |
C11H8N6O2 |
256 |
0.69 |
81.10 |
198-200 |
|
7 |
PP-3A |
2,4-di nitro |
C11H6N6O5 |
319 |
0.76 |
54.00 |
222-224 |
|
8 |
PP-3B |
2,4-di nitro |
C12H8N6O5 |
308 |
0.78 |
81.00 |
206-208 |
|
9 |
PP-3C |
2,4-di nitro |
C11H7N7O4 |
301 |
0.71 |
64.00 |
196-198 |
|
10 |
PP-4A |
2,4-di amino |
C11H10N6 O |
242 |
0.82 |
75.90 |
224-226 |
|
11 |
PP-4B |
2,4-di amino |
C12H12N6O |
256 |
0.75 |
61.00 |
222-224 |
|
12 |
PP-4C |
2,4-di amino |
C11H11N7 |
241 |
0.69 |
55.00 |
214-216 |
|
13 |
PP-5A |
p-amino |
C11H9N5O |
231 |
0.81 |
66.00 |
228-230 |
|
14 |
PP-5B |
p-amino |
C12H11N5O |
241 |
0.76 |
59.00 |
218-220 |
|
15 |
PP-5C |
p-amino |
C11H10N6 |
226 |
0.75 |
65.00 |
188-190 |
|
16 |
PP-6A |
3,5-di chloro |
C11H6Cl2N4O |
281 |
0.75 |
58.13 |
212-214 |
|
17 |
PP-6B |
3,5-di chloro |
C14H9Cl2N2O |
292 |
0.68 |
65.66 |
224-226 |
|
18 |
PP-6C |
3,5-di chloro |
C13H8Cl2N3 |
276 |
0.67 |
75.53 |
218-220 |
Scheme : Schematic diagrams for the synthesis of Pyrazolopyrimidine derivatives
Antibacterial activity:
Seven of the newly synthesized target compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus as a gram positive bacteria and Pseudomonas aeruginosa as an gram negative bacteria. Agar-diffusion method was used for the determination of the preliminary antibacterial activities. The agar well diffusion test was performed using nutrient agar medium, as per the procedure described by Magaldi et al. 2004 and the medium was autoclaved at 15lbs pressure (121°C) for 15minutes then was immediately cooled to 50- 55°C in a water bath after removing it from the autoclave. The cooled medium was poured into sterile petri plates to a uniform depth of 4mm; this is equivalent to approximately 40mL in a 90mm plate. Once the medium had solidified then the culture was inoculated on the medium.
These were be performed in a laminar flow, Within 15minutes of adjusting the density of the inoculums, a sterile cotton swab was dipped into the standardized bacterial suspension or inoculated with 1mL of the organism suspension. The sterile swab was used on the surface of the nutrient agar medium to ensure an even distribution of the inoculums. The plates were undisturbed for 3 to 5 minutes to ensure absorption of excess moisture. Sterilized 7mm cork borer was used to make agar wells, and the concentrations of the 25, 50, 75, 100 and 200 µg/ml of the diluted test compound stock solutions were placed into each wells and 100% DMSO as a control [14].
The percentage of inhibition can be calculated using the formula:
%Inhibition= I (dia of inhibition zone in mm)/ 90( dia of Petri-plates in mm)× 100
Fig 4: Graph showing % inhibition and different concentrations of compounds against Staphylococcus aureus
Table 4: Zone of inhibition and % Inhibition of some synthesized compounds:
|
Gram-positive bacteria Staphylococcus aureus zone of inhibition and % inhibition |
|||||||||
|
S.NO |
Compounds code |
Concentration ( µg/ml) |
|||||||
|
25 |
% I |
50 |
%I |
100 |
% I |
200 |
% I |
||
|
1. |
PP-2B |
10 |
11.11 |
12 |
13.33 |
14 |
15.56 |
15 |
16.67 |
|
2. |
PP-3B |
13 |
14.44 |
12 |
13.33 |
13 |
14.44 |
13 |
14.44 |
|
3 |
PP-3C |
12 |
13.33 |
13 |
14.44 |
14 |
15.56 |
14 |
15.56 |
|
4 |
PP-4B |
11 |
12.22 |
12 |
13.33 |
14 |
15.56 |
14 |
15.56 |
|
5 |
PP-5B |
13 |
14.44 |
15 |
16.67 |
12 |
13.33 |
13 |
14.44 |
|
6 |
PP-5C |
10 |
11.11 |
13 |
14.44 |
14 |
15.56 |
15 |
16.67 |
|
7 |
PP-6A |
14 |
14.44 |
15 |
16.67 |
13 |
14.44 |
12 |
13.33 |
|
8 |
Standard Ampicillin |
12 |
13.33 |
11 |
12.22 |
14 |
15.56 |
16 |
17.78 |
Table 5: Zone of inhibition (mm) % Inhibition of some synthesized compounds:
|
Gram-negative bacteria Pseudomonas aeruginosa zone of inhibition (mm) |
|||||||||
|
S.NO |
Compounds code |
Concentration ( µg/ml) |
|||||||
|
25 |
% I |
50 |
% I |
100 |
% I |
200 |
% I |
||
|
1. |
PP-2B |
12 |
13.33 |
12 |
13.33 |
14 |
15.56 |
12 |
13.33 |
|
2. |
PP-3B |
13 |
14.44 |
13 |
14.44 |
15 |
16.67 |
15 |
16.67 |
|
3 |
PP-3C |
9 |
10.00 |
12 |
12.22 |
13 |
11.11 |
14 |
13.33 |
|
4 |
PP-4B |
9 |
10.00 |
12 |
13.33 |
13 |
14.44 |
14 |
15.56 |
|
5 |
PP-5B |
11 |
12.22 |
14 |
15.56 |
15 |
16.67 |
16 |
16.67 |
|
6 |
PP-5C |
9 |
10.00 |
9 |
10.00 |
11 |
12.22 |
11 |
12.22 |
|
7 |
PP-6A |
9 |
10.00 |
11 |
12.22 |
10 |
14.44 |
12 |
15.56 |
|
8 |
Standard Ampicillin |
9 |
11.11 |
11 |
12.22 |
13 |
14.44 |
14 |
15.55 |
Fig 5: Graph showing % inhibition and different concentrations of compounds against Pseudomonas aeruginosa
RESULTS AND DISCUSSION:
The examination of the data reveals that majority of the compounds showed antibacterial activity when compared with standard drug. The results of in vitro antibacterial activities of compounds of these synthesized compounds against various bacterial strains are summarized in. It has been observed that some of compounds exhibited interesting antibacterial activity. In comparison with reference antibacterial, compounds PP-6A at 25 µg/ml, PP-5B, PP6A at 100 µg/ml and PP-5C, PP-2B at 200 µg/ml concentrations showed maximum zone of inhibition and shows good activity against Staphylococcus aureus. Compounds PP-3B at 25 µg/ml, PP-5B at 50 µg/ml, PP-3B, PP-5B at 100 µg/ml and PP-5B at 200 µg/ml concentrations shows good activity against Pseudomonas aeruginosa.
REFERENCES:
1. Kim DK, Ryu DH, Lee N, Lee JY, Kim JS, Lee S, Choi JY, Ru JH, Kim NH, Im GJ, Choi WS, Kim T.K. Synthesis and Phosphodiesterase 5 Inhibitory Activity of New 5-Phenyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one Derivatives Containing an N-Acylamido Group on a Phenyl Ring. Bioorg. Med. Chem. 2001, 9, 1895–1899.
2. Ali, T.E.S. Synthesis of some novel pyrazolo[3,4-b]pyridine and pyrazolo[3,4- d]pyrimidine derivatives bearing 5,6-diphenyl-1,2,4-triazine moiety as potential antimicrobial agents. Eur. J. Med.Chem. 2009, 44, 4385–4392.
3. Aymn, E.R.; Mohamed, I.H.; Randa, E.A.M.; Nahed, F.; Farouk, M.E.A.M. Synthesis and anti-HSV-1evaluation of some pyrazoles and fused pyrazolopyrimidines. Eur. J. Med. Chem. 2009, 44, 3285–3292.
4. Quiroga, J.; Trilleras, J.; Insuasty, B.; Abonia, R.; Nogueras, M.; Marchal, A.; Cobo, J. Microwave-assisted synthesis of pyrazolo[3,4-d]pyrimidines from 2-amino-4,6-dichlorovpyrimidine- 5-carbaldehyde under solvent-free conditions. Tetrahedron Lett. 2008, 49, 3257–3259.
5. (a) Julino, M.; Stevens, M. F. G.. J. Chem. Soc., Perkin Trans. 1 1998, 1677–1684;
(b) Ibrahim Abdou, M.; Saleh, A. M.; Zohdi, H. F. Molecules 2004, 9, 109–116.
6. Filler, R. Chem. Technol. 1974, 4, 752.
7. Ghorab, M. M.; Ismail, Z. H.; Abdel-Gawad, S. M. Abdel Aziem, A. Heteroatom Chemistry 2004, 15, 57.
8. Davies, L. P.; Brown, D. J.; Chow, S. C.; Johnston, G. A.R. Neurosci. Lett. 1983, 41, 189.
9. Davies, L. P.; Chow, S. C.; Skerritt, J. H.; Brown, D. J.; Johnston, G. A. R. Life Sci. 1984, 34, 2117.
10. A.E. Rashad, A.H. Shamroukh, M.I. Hegab, H.M. Awad, Acta. Chim. Slov. 52 (2005) 429- 434.
11. B.S. Holla, M.S. Mahalinga, M.S. Karthekyan, P.M. Akberali, N.S. Shetty, Bioorg Med. Chem. 14 (2006) 2040–2047.
12. Jiang, M.X.W.; Warshakoon, N.C.; Miller, M.J. ChemoenzymaticAsymmetricTotal Synthesis of Phosphodiesterase Inhibitors:Preparation of a Polycyclic Pyrazolo[3,4-d]pyrimidine from an Acylnitroso Diels-Alder Cycloadduct-Derived Aminocyclopentenol. J. Org. Chem. 2005, 70, 2824-2827.
13. Markwalder, J.A.; Arnone, M.R.; Benfield, P.A.; Boisclair, M.; Burton, G.R.; Chang, C.H.; Cox, S.S.; Czerniak, P.M.; Dean, C.L.; Doleniak, D.; Grafstrom, R.; Harrison, B.A.; Kaltenbach, R.F.; Nugiel, D.A.; Rossi, K.A.; Sherk, S.R.; Sisk, L.M.; Stouten, P.; Trainor, G.L.; Worland, P.; Seitz, S.P. Synthesis and Biological Evaluation of 1-Aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one Inhibitors of Cyclin-Dependent Kinases. J. Med. Chem. 2004, 47, 5894-5911.
14. Magaldi S, Mata-Essayag C, Hartung de Capriles, Perez C, Collela MT, Olaizola C. Well diffusion for antifungal susceptibility testing. Int J Infect Dis 2004; 8: 39-45.
Received on 21.07.2011 Modified on 03.08.2011
Accepted on 14.08.2011 © AJRC All right reserved
Asian J. Research Chem. 4(10): Oct., 2011; Page 1594-1599