Design, synthesis, and antimicrobial activity of some new pyrazolo[3,4-d]pyrimidines

Article abstract of DOI:10.1002/hc.10187

2-Benzyl- and 2-aryloxymethyl-3-amino-1-phenyl-pyrazolo[3,4-d]pyrimidine-4-ones 5a-f have been synthesized by reacting the corresponding arylacetylamino derivatives 3a-f with hydrazine hydrate. Thionation of compounds 5d-f by action of P₂S

Full text of DOI:10.1002/hc.10187

Heteroatom Chemistry
Volume 14, Number 6, 2003
Design, Synthesis, and Antimicrobial Activity
of Some New Pyrazolo[3,4-d ]pyrimidines
Soad M. Abdel-Gawad,1 M. M. Ghorab,2 A. M. Sh. El-Sharief,3
F. A. El-Telbany,4 and M. Abdel-Alla5
1Department of Chemistry, Faculty of Science (Girl’s), Al-Azhar University, Cairo, Egypt
2Department of Drug Radiation Research, National Center for Radiation Research and Technology,
P.O. Box 29, Nasr City, Cairo, Egypt
3Department of Chemistry, Faculty of Science, Al-Azhar University, Cairo, Egypt
4Department of Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
5Faculty of Pharmacy, October 6 University, Cairo, Egypt
Received 21 January 2003; revised 7 April 2003
INTRODUCTION
ABSTRACT: 2-Benzyl- and 2-aryloxymethyl-3-
amino-1-phenyl-pyrazolo[3,4-d]pyrimidine-4-ones
5a–f have been synthesized by reacting the cor-
responding arylacetylamino derivatives 3a–f with
hydrazine hydrate. Thionation of compounds 5d–f by
action of P2S5 in pyridine yielded 2-aryloxy-methyl-
3-amino-1-pheny-lpyrazolo[3,4-d]pyrimidin-4-thions
6a–c.2,5-Diphenyl-2,3-dihydro-1H-pyrazolo[5ꢀ,1ꢀ:4:5]-
pyrazolo[3,4-d]pyrimidine-8-one (8) was also ob-
tained via reaction of ethyl-2-cinnamoylamino-1-
phenyl-pyrazole-4-car-boxylate (7) with hydrazine
hydrate. The prepared compounds were screened in
vitro for their antimicrobial activity. Some of the
tested compounds were found to be active at 100
ꢀg/ml compared with reference compounds (Ampi-
cillin and Trivid) as antibacterial agents and claforan
Several 3-substituted pyrazolo[3,4-d]pyrimidine
derivatives have shown pharmacological activity
like allopurinol, which is the inhibitor of xanthine
oxidase [1,2]. Also, some substituted pyrazolopy-
rimidines have been documented as adenosine
antagonists [3–5] and to possess antibacterial [6],
antifungal [7], and antitumor activity [8,9]. As an ex-
tension of our studies on the synthesis of some new
biologically active heterocyclic compounds [10–14],
we now wish to report the synthesis of some new
pyrazolo[3,4-d]pyrimidine derivatives as purine
isosteres to evaluate their antimicrobial activity.
CHEMISTRY
The starting material ethyl-5-amino-1-phenylpyra-
zole-4-carboxylate (1) was prepared as reported [15]
from ethyl-2-cyano-3-ethoxyacrylate and phenyl
hydrazine. The synthesis of the target compounds 2-
benzyl- or 2-aryloxymethyl-3-amino-1-phenyl-pyra-
zolo[3,4-d]pyrimidine-4-ones 5a–f and 2,5-dipenyl-
2,3-dihydro-1H-pyrazolo[5,1:4:5]pyrazolo[3,4-d]-
pyrimidine-8-one (8) was achieved by the route
depicted in Scheme 1.
ꢁ
C
as antifungal agent.
2003 Wiley Periodicals, Inc.
Heteroatom Chem 14:530–534, 2003; Published online
in Wiley InterScience (www.interscience.wiley.com). DOI
10.1002/hc.10187
Correspondence to: M. M. Ghorab; e-mail: ghorabmoustafa@
Compound 1 was acylated with various benzyl
or aryloxyacetic acids 2a–f in presence of PCl3
hotmail.com.
c
ꢁ
2003 Wiley Periodicals, Inc.
530

Design, Synthesis, and Antimicrobial Activity of Some New Pyrazolo[3,4-d ]pyrimidines 531
SCHEME 2 Rationalized formation of compound 8.
SPECTRA
The synthesized compounds were characterized by
elemental analyses and spectral and physical data.
IR spectra of compounds 3a–f show the sharp peak
around 3200–3147 cm−1 (NH), 1716–1700 (C O) es-
ter. The IR spectra of compounds 5a–f showed the
distinct peaks for amino group (NH2), and one car-
bonyl group (C O). Their 1H NMR spectra revealed
the presence of primary amine protons at δ = 5.8–
SCHEME 1 Synthesis of pyrazolopyrimidine derivatives
6.3 ppm that were D2O exchangeable. The methy-
lene protons in the side chain were found at δ = 4.8–
4.2 ppm. The mass spectra of compounds 5b, 5e,
and 5f showed the molecular ion peak and a promi-
nent M+−16 peak indicating the early loss of the free
amino function at position N3. Further fragmenta-
tion of the molecule was found to be satisfactory
(Scheme 3).
(5a–f, 6a–c, and 8). a: PCl3/xylene, reflux, 5 h; b: N2N4·H2O,
n-BuOH, reflux 6 h; c: P2S5/pyridine, reflux 16 h; d: cinnamic
acid/PCl3/xylene, reflux, 5 h.
to obtain ethyl-5-(phenyl or aryloxy)acetylamino-1-
phenyl-pyrazole-4-carboxylate 3a–f in good yield.
Cyclocondensation of 3a–f with hydrazine hy-
drate afforded 5a–f. The reaction proceeds via the
intermediate 4, which undergoes a nucleophilic ad-
dition to the carbonyl of the side chain followed by
the loss of 1 mol of water.
The IR spectra of compounds 6a–c revealed the
absence of C O and presence of C S band and
1
NH band. H NMR spectrum of 6a showed signal
at δ = 5.7 ppm (NH2), exchangeable with D2O. Mass
spectrum of compound 6b showed a molecular ion
peak m/z 399 (M+, 2.22%) with a base peak at 115
(Scheme 4).
The thione derivatives 6a–c were synthesized via
reaction of 5d–f with P2S5 in pyridine.
The ethyl-5-(cinnamoylamino)-1-phenyl-pyra-
zole-4-carboxylate (7) was obtained in good yield
via reaction of 1 with cinnamic acid in presence of
PCl3. Refluxing the amide 7 with hydrazine hydrate
in n-butanol for 6 h effected double cyclization to
give2,5-diphenyl-2,3-dihydro-1H-pyrazolo[5ꢀ,1ꢀ:4:5]-
pyrazolo[3,4-d]pyrimidine-8-one (8) (Scheme 2)
instead of the expected N-amino derivative 9,
which was eliminated from consideration on the
basis of elemental analyses and 1H NMR spec-
trum which showed the absence of an amino
group.
BIOLOGICAL ACTIVITY
The antimicrobial screening of the synthesized
compounds was undertaken using the agar dif-
fusion assay [16]. Table 1 lists the screening re-
sults of the tested compounds against the Gram-
negative bacteria Serratia marcescens and Proteus
merabities, and the Gram-positive bacteria Staphy-
lococcus aureus and Bacillus cereus, in addition
to the pathogenic fungi Aspergillus oschraceus
wilhelm.

532 Abdel-Gawad et al.
SCHEME 4 Mass fragmentation pattern of compound 6b.
From these results it can be concluded that the
biologically active compounds 3a, 3b, 3d, 3f, 5a, and
5f (MIC values were 100 ꢀg/ml) are nearly as active as
standard antibiotics Ampicillin and Trivid, and less
active than the fungicide claforan.
SCHEME 3 Mass fragmentation pattern of compound 5f.
It was found that the pyrazole derivatives 3a,
3b, and 3d having both acetamide and ester moi-
eties were found to be the most active compounds
against Gram-negative bacteria S. marcescens and P.
merabities with MICs (100 ꢀg/ml) compared with the
reference compound. Also, the amide derivatives 3e
and compound 5a having both 5-amino and 6-benzyl
moieties were found the most active compounds
against S. marcescens. In addition, the amide deriva-
tives 3c, 3f, and 5f (N-aminopyrazolopyrimidine)
showed higher activity against P. merabities (MICs
of 100 ꢀg/ml) compared with antibiotics Ampicillin
and Trivid. On the other hand, the amide derivatives
3b, 3c, and 3d were found to be the most active com-
pounds against Gram-positive bacteria S. aureus and
B. cereus (with MICs of 100 ꢀg/ml).
EXPERIMENTAL
Melting points were determined in open capil-
laries and are uncorrected. The IR spectra were
recorded in potassium bromide on a Perkin-Elmer
841 grating spectrophotometer (Perkin-Elmer, USA).
1H NMR spectra were recorded on a Varian EM
360 (240 MHz) instrument using TMS as an inter-
nal standard (chemical shift in δ ppm). Microana-
lytical data (C, H, N) were in agreement with the
proposed structure within +0.4% of the theoreti-
cal values determined at the Microanalytical Centre,
Cairo University, Egypt. Mass spectra were run using
HP Model MS-5988.
The 5-amino-4-ethoxycarbonyl-1-phenylpyra-
zole 1 was synthesized according to the literature
method [17]. All the other reagents were of reagent
grade.
Most of the synthesized compounds showed a re-
markable activity against A. oschraceus wilhelm and
less active than fungicide claforan.

Design, Synthesis, and Antimicrobial Activity of Some New Pyrazolo[3,4-d ]pyrimidines 533
TABLE 1 Antimicrobial Activity of the Synthesized Compounds
Inhibition Zonesa (mm)
Serratia
marcesens
(IMRU-70)
Proteus
merabities
(NTC-289)
Staphylococcus
aureus
(NCTC-7447)
Bacillus
cereus
(ATCC-14579)
Aspergillus
oschraceus
Wilhelm (AUCC-230)
Compound
3a
24 (100)
26 (100)
18
26 (100)
24 (100)
14
26 (100)
14
14
14
14
14
14
14
30
32
26 (100)
26 (100)
24 (100)
24 (100)
14
24 (100)
14
14
14
14
14
26 (100)
14
14
29
30
10
20 (100)
20 (100)
20 (100)
10
10
10
10
10
10
10
10
10
10
28
28
10
20 (100)
26 (100)
20 (100)
10
10
10
10
18
18
10
10
18
18
30
28
20
20
10
10
20
20
20
20
20
20
20
20
10
20
–
3b
3c
3d
3e
3f
5a
5b
5c
5d
5e
5f
7
8
Ampicillin
Trivid
–
Claforan
–
–
–
–
29
aSlight activity: 10–14 mm (+); moderate activity: 14–18 mm (++); high activity: 20–26 mm (+++); very high activity ≥26 mm (++++).
3f: Yield, 92%, m.p. 135–137◦C; IR (KBr, cm−1)
Synthesis of the Ethyl-5-(phenyl or
aryloxy)acetylamino-1-phenylpyrazole-
4-carboxylate (3a–f)
1
3180, 2920, 1720, 1690; H NMR δ 1.3 (t) 3H, CH3
ethyl; δ 4.4 (q) 2H, CH2 ethyl; δ 4.9 (s) 2H, CH2CO; δ
7.0–8.1 (m) 12H, Ar H; δ 8.4 (s) 1H, CH; δ 10.3 (s)
1H, NH.
To a solution of 2a–f (0.01 mol) and 1 (2.31 g,
0.01 mol) in xylene (50 ml), phosphorus trichloride
(3 ml) was added. The reaction mixture was heated
under reflux for 3–4 h. The crude product was re-
crystallized from ethanol to yield 76% of 3a, m.p.
131–133◦C. IR (KBr, cm−1) 3166, 2977, 1716, 1662;
1H NMR δ 1.1 (t) 3H, CH3 ethyl; δ 4.0 (q) 2H, CH2
ethyl; δ 4.6 (s) 2H, CH2CO; δ 7.2–8.0 (m) 10H, Ar H;
δ 10.1 (s) 1H, NH.
Synthesis of 2-Benzyl- or 2-Aryloxymethyl-3-
amino-1-phenyl-pyrazolo[3,4-d]pyrimidine-
4-one (5a–f)
A mixture of 3a–f (0.01 mol) and hydrazine hydrate
(95%) (0.05 mol) were dissolved in n-butanol (30 ml)
and refluxed for 3–5 h. The solvent was concentrated
and the residue was recrystallized from ethanol to
give 5a–f.
3b: Yield, 68% m.p. 123–125◦C, IR (KBr, cm−1)
1
3147, 2931, 1701, 1690; H NMR δ 1.3 (t) 3H, CH3
ethyl; δ 4.3 (q) 2H, CH2 ethyl; δ 4.6 (s) 2H, CH2CO; δ
7.0–7.8 (m) 10H, Ar H; δ 8.2 (s) 1H, CH pyrazole; δ
10.4 (s) 1H, NH.
5a: Yield, 62%, m.p. 165–167◦C; IR (KBr, cm−1)
1
3380, 3250, 2924, 1697, 1616; H NMR δ 4.4 (s) 2H,
3c: Yield, 62%, m.p. 129–131◦C; IR (KBr, cm−1)
3201, 2920, 1690, 1670.
CH2; δ 5.7 (s) 2H, NH2 (exchangeable D2O); δ 7.1–8.0
(m) 10H, Ar H; δ 8.5 (s) 1H, CH.
3d: Yield, 81%, m.p. 121–123◦C; IR (KBr, cm−1)
5b: Yield, 81%, m.p. 131–133◦C; IR (KBr, cm−1)
3313, 3201, 2910, 1666, 1612; MS (m/z): 317
(M+ − NH2), 300, 235, 181, 116, 91, 77.
1
3163, 2927, 1716, 1697; H NMR δ 1.2 (t) 3H, CH3
ethyl; δ 2.2 (s) 3H, CH3; δ; 4.4 (q) 2H, CH2 ethyl; δ 5.3
(s) 2H, CH2CO; δ 6.8, 7.1 (2d) 4H, Ar H, AB system;
δ 7.4–7.8 (m) 5H, Ar H; δ 8.1 (s) 1H, CH; δ 10.3 (s)
1H, NH.
5c: Yield, 84%, m.p. 190–192◦C; IR (KBr, cm−1)
3300, 3240, 2930, 1670, 1610.
5d: Yield, 86%, m.p. 141–143◦C; IR (KBr, cm−1)
3394, 3313, 2908, 1682, 1620; 1H NMR δ 2.2 (s)
3H, CH3; δ 4.5 (s) 2H, CH2O; δ 6.3 (s) 2H, NH2
(exchangeable D2O); δ 6.8, 7.2 (2d) 4H, Ar H, AB
system; δ 7.5–7.7 (m) 5H, Ar H; δ 8.0 (s) 1H,
CH.
3e: Yield, 68%, m.p. 127–129◦C; IR (KBr, cm−1)
1
3201, 2930, 1710, 1700, 1630; H NMR δ 1.2 (t) 3H,
CH3 ethyl; δ 4.2 (q) 2H, CH2 ethyl; δ 4.8 (s) 2H,
CH2CO; δ, 6.9–8.0 (m) 12H, Ar H; δ 8.3 (s) 1H, CH;
δ 10.4 (s) 1H, NH.

534 Abdel-Gawad et al.
5e: Yield, 88%, m.p. 154–156◦C; IR (KBr, cm−1)
3294, 3147, 2924, 1658; MS (m/z): 383 (M+), 382 (M −
1), 351, 269, 246, 174, 143, 87, 77.
(30 ml) and refluxed for 4 h. The obtained product
was recrystallized from ethanol to give 8. Yield: 81%,
m.p. 187–189◦C; IR (KBr, cm−1) 3325, 2924, 1690,
1612; 1H NMR δ 4.1 (s) 2H, CH2; δ 6.3 (s) 1H, CH; δ
7.2–8.1 (m) 10H, Ar H; δ 9.0 (s) 1H, CH pyrazole.
5f: Yield, 93%, m.p. 170–172◦C; IR (KBr, cm−1)
3309, 3201, 2930, 1680, 1627; MS (m/z): 383 (M+),
240, 224, 210, 185, 142, 116, 78 (Scheme 4).
ACKNOWLEDGMENT
Synthesis of 2-Substituted-3-amino-1-phenyl-
pyrazolo[3,4-d]pyri-midine-4-thione (6a–c)
We thank Dr. M. M. Afifi, Microbiology Department,
Faculty of Science, Al-Azhar University at Assuit,
Egypt, for doing the antimicrobial activity.
A mixture of 5d–f (0.01 mol) and phosphorus penta
sulfide (0.015 mol) in pyridine (50 ml) was refluxed
for 16 h. The reaction mixture was cooled and poured
onto ice water/HCl. The obtained product was recrys-
tallized from dioxan to give 6a–c.
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6a: Yield, 67%, m.p. 122–124◦C; IR (KBr, cm−1)
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Synthesis of Ethyl-5-(cinnamoylamino)-
1-phenylpyrazole-4-carboxylate (7)
A mixture of cinnamic acid (1.48 g, 0.01 mol) and 1
(2.31 g, 0.01 mol) in xylene (50 ml) containing phos-
phorus trichloride (3 ml) was refluxed for 5 h. The
obtained product was recrystallized from ethanol to
give 7. Yield, 77%, m.p. 122–124◦C; IR (KBr, cm−1)
1
3150, 2920, 1697, 1680, 1627; H NMR (δ, ppm) δ
1.3 (t) 3H, CH3; δ 4.3 (q) 2H, CH2; δ 6.4, 6.6 (2s) 2H,
CH CH; δ 7.3–8.0 (m) 10H, Ar H; δ 8.4 (s) 1H, CH,
10.2 (s) 1H, NH.
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Synthesis of 2,5-Diphenyl-2,3-dihydro-
1H-pyrazolo[5ꢀ,1ꢀ,4,5]-pyrazolo[3,4-d]-
pyrimidine-8-one (8)
A mixture of 7 (3.6 g, 0.01 mol) and hydrazine hy-
drate (95%) (0.05 mol) were dissolved in n-butanol