Synthesis of some pyrazolo[3,4-d]pyrimidine derivatives for biological evaluation

Article abstract of DOI:10.1080/104265090921074

A novel β-enaminonitrile of 1-(6-p-tolyl-pyridazin-3-yl)-pyrazole derivative 2 was formed using (6-p-tolyl-pyridazin-3-yl)-hydrazine (1) and 2-ethoxymethylenemalononitrile. The β-enaminonitrile derivative 2 was in turn used as a precursor for the preparat

Full text of DOI:10.1080/104265090921074

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Phosphorus, Sulfur, and Silicon
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Synthesis of Some Pyrazolo[3,
4]pyrimidine Derivatives for
Biological Evaluation
A. H. Shamroukh a , A. E. Rashad a & H. H. Sayed a
a Photochemistry Department , National Research
Centre, Dokki , Cairo, Egypt
Published online: 22 Aug 2006.
To cite this article: A. H. Shamroukh , A. E. Rashad & H. H. Sayed (2005)
Synthesis of Some Pyrazolo[3, 4]pyrimidine Derivatives for Biological Evaluation,
Phosphorus, Sulfur, and Silicon and the Related Elements, 180:10, 2347-2360, DOI:
10.1080/104265090921074
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Phosphorus, Sulfur, and Silicon, 180:2347–2360, 2005
Copyright © Taylor & Francis Inc.
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/104265090921074
Synthesis of Some Pyrazolo[3,4-d]pyrimidine Derivatives
for Biological Evaluation
A. H. Shamroukh
A. E. Rashad
H. H. Sayed
Photochemistry Department, National Research Centre, Dokki,
Cairo, Egypt
A
2
novel β-enaminonitrile of 1-(6-p-tolyl-pyridazin-3-yl)-pyrazole derivative
was formed using (6-p-tolyl-pyridazin-3-yl)-hydrazine (1) and 2-ethoxy-
methylenemalononitrile. The β-enaminonitrile derivative 2 was in turn used as a
precursor for the preparation of pyrazoles (4, 6), pyrazolo[3,4-d]-pyrimidines (3, 7–
12) and pyrazolo[4,3-e][1,2,4]triazolo[4,3-c]pyrimidine (13). Also, N- and S-acyclic
nucleosides 14 and 15 were prepared. Some of the prepared products showed potent
antimicrobial activity.
Keywords β-enaminonitrile; acyclic nucleosides; pyrazole; pyrazolo[3,4-d]pyrimidine;
pyrazolo-[4,3-e]-[1,2,4]triazolo[4,3-c]pyrimidine
INTRODUCTION
Pyrazolo[3,4-d]pyrimidines have received a great attentionꢀRdue to their
pharmacological activity,1−4 such as allopurinol5 (zyloric ), which is
still the drug for treatment of hyperurecemia and gouty arthritic.6−8
Various compounds with related structures also possess anti-tumor
and anti-leukemia activities.9,10 On the other hand, substituted pyri-
dazines have received great interest, especially hydrazinopyridazine
derivatives that were tested for their bactericidal, fungicidal,11 and
anti-hypertensive activity.12 In continuation of our previous work on
pyrazoles13 and pyridazines,13,14 we aimed to incorporate the pyri-
dazine moiety into the 1-position of the pyrazolo[3,4-d]pyrimidine ring
system to obtain a new compound which is expected to possess notable
chemical and biological activities.
Received July 26, 2004; accepted October 22, 2004.
We are thankful for Dr. H. M. Awad, Department of Natural and Microbial Products,
National Research Centre, for his biological evaluation.
Address correspondence to A. H. Shamroukh, National Research Centre, Photochem-
istry Department, Dokki, Cairo, Egypt. E-mail: ahshamroukh@yahoo.com
2347

2348
A. H. Shamroukh et al.
RESULTS AND DISCUSSION
This work is aimed at the synthesis of new compounds related
to β-enaminonitriles of pyridazinyl pyrazole derivatives. In particu-
lar, 5-amino-1-(6-p-tolyl-pyridazin-3-yl)-1H-pyrazole-4-carbonitrile (2)
was used as a key compound for this study and for further syn-
theses of other fused heterocyclic compounds. Thus, on reflux-
ing a mixture of (6-p-tolylpyridazin-3-yl)-hydrazine (1)15 and 2-
ethoxymethylenemalononitrile in ethanol, compound 2 was obtained.
The structure of compound 2 was confirmed with spectral data. Its IR
spectrum showed bands at (ν, cm−1): 3382, 3280 (NH2) and 2220 (CN);
1H NMR spectrum showed signals at (δ, ppm): 2.39 (s, 3H, p-CH3-tolyl),
7.39 (d, 2H, J = 7.8 Hz, H3,H5-tolyl), 8.01 (s, 1H, H3-pyrazole), 8.06 (d,
2H, J = 8.1 Hz H2,H6-tolyl), 8.17 (d, 1H, J = 9.3 Hz, H5-pyridazine),
8.22 (brs, 2H, NH2, exchangeable with D2O), 8.43 (d, 1H, J = 9.3 Hz,
H4-pyridazine); and MS gave the molecular ion peak as a base peak
at m/z = 276. The synthesis of compound 3 was achieved by refluxing
compound 2 with formamide. Its IR spectrum showed the absence of
1
the cyano group and its H NMR spectrum gave signals at (δ, ppm):
7.94 (brs, 2H, NH2, exchangeable with D2O) and 8.48–8.51 (m, 2H, H4-
pyridazine and H6-pyrimidine).
On the other hand, compound 4 was obtained by stirring compound 2
with concentrated sulfuric acid at 0◦C (Scheme 1). The spectral data of
compound 4 assigned its structure (cf. Experimental). Many reports16,17
stated that nitrozation of compounds analogous to 4 gave the corre-
sponding triazinone derivatives; however, our attempts to cyclize com-
pound 4 to 5 by stirring with nitrous acid gave the unexpected 5-amino-
1-(6-p-tolyl-pyridazin-3-yl)-1H-pyrazole-4-carboxylic acid alkyl esters
6a,b via formation of the diazonium chloride derivative, which on boil-
ing in ethanol or methanol gave the corresponding esters 6a (R = C2H5)
or 6b (R = CH3), respectively (Scheme 1). The formation of compounds
6a,b could be presumably explained by the formation of unstable dia-
zonium salt on the amino of the amide moiety and not on the 5-amino
group. Also, this could be established chemically when compound 1 was
refluxed with 2-cyano-3-ethoxy-acrylic acid alkyl esters; it gave com-
pounds 6a and 6b, respectively (Scheme 1).
When compound 4 was heated under reflux temperature with car-
bon disulphide in the presence of aqueous potassium hydroxide so-
1
lution, it gave the corresponding 6-thioxo derivative 7. Its H NMR
spectrum gave signals at (δ, ppm): 7.60 and 12.66: 2NH exchange-
able with D2O. On the other hand, when compound 4 was refluxed
with triethyl orthoformate, it afforded 1-(6-p-tolyl-pyridazin-3-yl)-1,5-
dihydro-pyrazolo[3,4-d]pyrimidin-4-one (8), which could be obtained

Synthesis of Pyrazolo[3,4-d]pyrimidine Derivatives
2349
SCHEME 1

2350
A. H. Shamroukh et al.
directly by refluxing compound 2 with formic acid. The formation of
compound 8 from compound 2 presumably took place via hydrolysis
of the cyano group to give compound 4, which on prolonged heat-
ing, converted into compound 8. The latter compound was converted
into its corresponding 4-chloro derivative 9 by refluxing in phospho-
rus oxychloride, its MS, m/z: 324 (M+, Cl37, 38.41%), 322 (M+, Cl35,
100%).
Compound 10 was obtained from refluxing compound 9 with thiourea
in dioxane according to a reported method.18 Methylation of compound
10 with methyl iodide in the presence of potassium hydroxide solution
afforded the corresponding 4-methylsulfanyl derivative 11. Compound
12 was obtained either from compounds 9 or 11 by heating with hy-
1
drazine hydrate (Scheme 2). The H NMR spectrum of compound 12
gave signals at (δ, ppm): 4.00 (brs, 1H, NH, exchangeable with D2O) and
at 7.47 (s, 2H, NH2, exchangeable with D2O); its MS gave the molecular
ion peak as a base peak at m/z = 318.
When compound 12 was heated, in triethyl orthoformate it gave a
product assigned to the structure of 7-(6-p-tolyl-pyridazin-3-yl)-7H-
pyrazolo[4,3-e][1,2,4] triazolo[4,3-c]pyrimidine (13) (cf. Experimental
Scheme 2).
Cyclic or acyclic pyrazolo[3,4-d]pyrimidine nucleosides that
attached to the pyrimidine moiety are not known (to the best of
our knowledge) in the literature. Actually, some acyclonucleosides
were reported to be potent antiherpatic drugs with selective activity
against Herpes Simplex Virus (HSV-1 and HSV-2) and Varicilla Zoster
Virus (VZV), Human Cytomegalo Virus (HCMV), and Epestein-Barr
Virus and also have shown high selectivity against Human Immun-
odeficiency Virus (HIV-1).19−24 However, there is still a great need
and interest to prepare more active acyclic pyrimidine nucleosides
with less side effects than those observed for the already-known
derivatives.
Therefore, when the sodium salts of compounds 8 and10 (generated
in situ) were treated with 2-chloroethyl methyl ether, they afforded the
corresponding N-acyclic nucleoside: 5-(2-methoxy-ethyl)-1-(6-p-tolyl-
pyridazin-3-yl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one (14) and S-
acyclic nucleoside: 4-(2-Methoxy-ethylsulfanyl)-1-(6-p-tolyl-pyridazin-
3-yl)-1H-pyrazolo[3,4-d]pyrimidine (15), respectively (Scheme 2). The
structure and sites of nucleosidation were elucidated with spectral data
(cf. Experimental). The 13C NMR spectra, as stated in similar com-
pounds previously reported,25 showed the presence of the C O group
in compound 14, while that of compound 15 showed the absence of the
C S group.

Synthesis of Pyrazolo[3,4-d]pyrimidine Derivatives
2351
SCHEME 2

2352
A. H. Shamroukh et al.
BIOLOGICAL EVALUATION
The antimicrobial activity of some newly synthesized compounds 2, 3,
4, 7, 8, 10, 11, and 15 were tested at concentration of 0.1 g/mL using
dimethylformamide as a solvent.
Microorganisms’ Species:
1. Bacteria
a) Gram-negative bacteria, Escherichia coli
b) Gram-positive bacteria, Bacillus subtilis
Yeast: Candida albicans
2. Fungi: Aspergillus Niger
Medium
The cap-assay method containing (g/L): peptone 6.0, yeast extract 3.0,
meat extract 1.5, glucose 1.0, and agar 20.0 were used. The medium
was sterilized and divided while hot (50–60◦C) in 15 mL portions among
sterile petri dishes of 9 cm diameter.
One mL of the spor suspension of each microorganism was spread
over the surface of the cold solid medium placed in the petri dish.
Method26
Each the tested compounds 0.5 g was dissolved in 5 mL of dimethylfor-
mamide. An amount of 0.1 mL of test solution was placed on watman
paper disc of 9 mm diameter and the solvent was left to evaporate.
These saturated discs were placed carefully on the surface of the inocu-
lated solid medium; each petri dish contains at least 3 discs. The petri
dishes were incubated at 5◦C for 1 h to permit good diffusion and then
transferred to an incubator of 85◦C overnight and then examined. The
results were recorded by measuring the inhibition zone diameters.
RESULTS
The antimicrobial activity of some newly synthesized compounds 2, 3,
4, 7, 8, 11 and 13 were tested and the results were shown in Table 1.
Among the compounds tested, it was noticed that compounds 4 and
7 showed more significant antibacterial and antifungal activity than
some known drugs (standers). Compounds 2 and 3 demonstrated in-
hibitory activity against Gram positive bacteria and fungi. It was obvi-
ous that compounds 8 and 10 have only an inhibitory effect on fungi.
On the other hand, compounds 11 and 15 showed activity against Gram
positive bacteria and Aspergillus Niger.

Synthesis of Pyrazolo[3,4-d]pyrimidine Derivatives
2353
TABLE I Test of Some Synthesized Compounds
Inhibition zone (mm)
Microorganism
Bacteria
Tested
compounds and
standers
Gram negative Gram positive
Fungi
Aspergillus
niger
Yeast
Candida
albicans
Escherichia
coli.
Bacillus
subtilis
Streptomycin
Erythromycin
Ampicillin
Amoxicillin
Fusidic Acid
2
3
4
7
+++
−
+++
+++
−
+
−
+++
−
++
++
−
−
−
−
−
−
−
+++
++
++
++
+++
++
++
++
++
+++
++
++
++
++
++
++
−
−
++
++
++
++
−
−
++
++
−
8
10
11
15
−
−
−
++
++
−
−
+++ Highly sensitive (inhibition zone = 21–25 mm).
++ Fairly sensitive (inhibition zone = 16–20 mm).
+ Slightly sensitive (inhibition zone = 10–15 mm).
− No sensitivity.
Experimental
All melting points are uncorrected and were measured using an Elec-
trothermal IA 9100 apparatus. Analytical data were performed by Vario
El Mentar apparatus, organic microanalysis section, National Research
Centre. Their results were found to be in agreement with the calculated
values ( 0.3). The IR spectra (KBr) were recorded on a Perkin-Elmer
1
1650 spectrophotometer. H & 13C NMR spectra were determined on
a Jeol 300 MHz in DMSO-d6 and the chemical shifts were expressed
in ppm relative to TMS as internal reference. Mass spectra were run
at 70 eV on EI + Q1 MSLMR UPLR. Column Chromatography was
performed on (Merck) Silica gel 60 (particle size 0.06–0.2).
5-Amino-1-(6-p-tolyl-pyridazin-3-yl)-1H-pyrazole-4-
carbonitrile (2)
To a solution of compound 1 (2.00 g, 0.01 mol) in ethanol (30 mL),
2-ethoxymethylene-malononitrile (1.22 g, 0.01 mole) was added. The

2354
A. H. Shamroukh et al.
reaction mixture was heated at reflux temperature for 2 h. The precip-
itate that formed was filtered while hot, dried and recrystallized from
dioxane to give 2 in 98% yield; m.p. 314–5◦C; IR spectrum (KBr, ν,
cm−1): 3382, 3280 (NH2) and 2220 (CN); 1H NMR spectrum (DMSO-d6,
δ ppm): 2.39 (s, 3H, p-CH3-tolyl), 7.39 (d, 2H, J = 7.8 Hz, H3,H5-tolyl),
8.01 (s, 1H, H3-pyrazole), 8.06 (d, 2H, J = 8.1 Hz H2,H6-tolyl), 8.17
(d, 1H, J = 9.3 Hz, H5-pyridazine), 8.22 (brs, 2H, NH2, exchangeable
with D2O), 8.43 (d, 1H, J = 9.3 Hz, H4-pyridazine); MS, m/z (%): 276
(M+, 100). Analysis for C15H12N6 (276.30): required C, 65.21; H, 4.38;
N, 30.42; found C, 65.24; H, 4.36; N, 30.40.
1-(6-p-Tolyl-pyridazin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-
ylamine (3)
A mixture of compound 2 (2.76 g, 0.01 mol) and formamide (30 mL) was
heated at 150◦C for 3 h. The reaction mixture was cooled and poured
into water. The formed solid was filtered off, dried, and recrystallized
from dioxane to give 3 in 85% yield; m.p. 333–35◦C. IR spectrum (KBr,
ν, cm−1): 3380, 3137 (NH2), no absorption in the region 2260–2240 (CN,
absent); 1H NMR spectrum (DMSO-d6, δ ppm): 2.41 (s, 3H, p-CH3-tolyl),
7.40 (d, 2H, J = 8.1 Hz, H3, H5-tolyl), 7.94 (brs, 2H, NH2, exchange-
able with D2O), 8.11 (d, 2H, J = 8.1 Hz H2, H6-tolyl), 8.34 (s, 1H, H3-
pyrazole), 8.42 (d, 1H, J = 9.3 Hz, H5-pyridazine), 8.48–8.51 (m, 2H,
H4-pyridazine and H6-pyrimidine); MS, m/z (%): 303 (M+, 100). Anal-
ysis for C16H13N7 (303.33): required C, 63.36; H, 4.32; N, 32.32; found
C, 63.20; H, 4.34; N, 32.50.
5-Amino-1-(6-p-tolyl-pyridazin-3-yl)-1H-pyrazole-4-carboxylic
acid amide (4)
To cold concentrated sulphuric acid (10 mL, 98%), compound 2 (2.76 g,
0.01 mol) was added portionwise with ice bath that was cooled and
stirred. After 10 min, the ice bath was removed and the mixture was
stirred for an additional 15 min. The resulting pale yellow solution was
carefully poured onto ice chips, neutralized with ammonium hydrox-
ide solution (5 mL, 25%), and the resulting precipitate was collected,
washed with water, dried, and recrystallized from dioxane to give 4
in 81% yield; m.p. 276–8◦C. IR spectrum (KBr, ν, cm−1): 3423–3153 (2
1
NH2), and 1662 (CO); H NMR spectrum (DMSO-d6, δ ppm): 2.40 (s,
3H, p-CH3-tolyl), 7.10 (brs, 2H, NH2, exchangeable with D2O), 7.39 (d,
2H, J = 7.8 Hz, H3, H5-tolyl), 7.63 (s, 2H, NH2, exchangeable with
D2O), 8.05–8.07 (m, 3H, H2, H6-tolyl and H3-pyrazole), 8.17 (d, 1H,
J = 9.3 Hz, H5-pyridazine), 8.40 (d, 1H, J = 9.3 Hz, H4-pyridazine);

Synthesis of Pyrazolo[3,4-d]pyrimidine Derivatives
2355
MS, m/z (%): 294 (M+, 100). Analysis for C15H14N6O (294.32): required
C, 61.22; H, 4.79; N, 28.55; found C, 61.12; H, 4.58; N, 28.75.
SYNTHESIS OF 6A AND 6B
Method A
To a suspended solution of compound 4 (2.94 g, 0.01 mol) in concentrated
hydrochloric acid (30 mL, 34%) at 0–5◦C, a solution of sodium nitrite
(3.00 g) in water (5 mL) was added over 20 min. After 2 h of stirring
at room temperature; the foamy mixture was filtered off. The resulting
solid was dried and boiled in ethanol or methanol for 10 min. The pre-
cipitates that formed were filtered on hot and dried and recrystallized
from an appropriate solvent to give 6a and 6b, respectively.
Method B
To a solution of compound 1 (2.00 g, 0.01 mol) in ethanol (30 mL),
0.01 mol of each of 2-cyano-3-ethoxy-acrylic acid ethyl ester (1.69 g, 0.01
mol) or 2-cyano-3-ethoxy-acrylic acid methyl ester (1.55 g, 0.01 mol) was
added. The reaction mixtures were heated at reflux temperature for 2 h.
The precipitates that formed were filtered while hot and dried and re-
crystallized from an appropriate solvent to give 6a and 6b, respectively.
The obtained products were identified by m.p. and tlc in comparison
with authentic samples from method A.
5-Amino-1-(6-p-tolyl-pyridazin-3-yl)-1H-pyrazole-4-carboxylic
acid ethyl ester (6a)
Yield: (86%, dioxane); m.p. 218–20◦C. IR spectrum (KBr, ν, cm−1): 3448,
3330 (NH2) and 1678 (CO); 1H NMR spectrum (DMSO-d6, δ ppm): 1.29
(t, 3H, J = 7.2 Hz, OCH2CH3), 2.39 (s, 3H, p-CH3-tolyl), 4.24 (q, 2H,
J = 7.2 Hz, OCH2CH3), 7.37 (d, 2H, J = 8.1 Hz, H3,H5-tolyl), 7.71 (brs,
2H, NH2, exchangeable with D2O), 7.88 (s, 1H, H3-pyrazole), 8.06 (d,
2H, J = 7.2 Hz, H2, H6-tolyl), 8.19 (d, 1H, J = 9.3 Hz, H5-pyridazine),
8.42 (d, 1H, J = 9.3 Hz, H4-pyridazine); MS, m/z (%): 323 (M+, 100).
Analysis for C17H17N5O2 (323.36): required C, 63.15; H, 5.30; N, 21.66;
found C, 63.28; H, 5.21; N, 21.59.
5-Amino-1-(6-p-tolyl-pyridazin-3-yl)-1H-pyrazole-4-carboxylic
acid methyl ester (6b)
Yield: (97%, benzene); m.p. 180–2◦C. IR spectrum (KBr, ν, cm−1): 3416,
3305 (NH2) and 1700 (CO); 1H NMR spectrum (DMSO-d6, δ ppm): 2.40

2356
A. H. Shamroukh et al.
(s, 3H, p-CH3-tolyl), 3.75 (s, 3H, OCH3), 7.37 (d, 2H, J = 8.1 Hz, H3,
H5-tolyl), 7.72 (brs, 2H, NH2, exchangeable with D2O), 7.89 (s, 1H, H3-
pyrazole), 8.06 (d, 2H, J = 7.2 Hz, H2, H6-tolyl), 8.20 (d, 1H, J = 9.3
Hz, H5-pyridazine), 8.43 (d, 1H, J = 9.3 Hz, H4-pyridazine); MS, m/z
(%): 309 (M+, 100).
Analysis for C16H15N5O2 (309.33): required C, 62.13; H, 4.89; N,
22.64; found C, 62.13; H, 4.89; N, 22.64.
6-Thioxo-1-(6-p-tolyl-pyridazin-3-yl)-1,5,6,7-tetrahydro-
pyrazolo[3,4-d]pyrimidin-4-one (7)
To a solution of compound 4 (2.94 g, 0.01 mol) in dimethylformamide
(30 mL), 20% potassium hydroxide solution (potassium hydroxide
1.68 g, water 7 mL) and carbon disulfide (5 mL) were added. The re-
action mixture was heated under reflux temperature for 15 h, cooled,
poured into water, neutralized with hydrochloric acid (3 mL, 34%), fil-
tered off, dried, and recrystallized from dimethylformamide to give 7 in
76% yield; m.p. 308–10◦C. IR spectrum (KBr, ν, cm−1): 3344 (NH), 3320
(NH); 1H NMR spectrum (DMSO-d6, δ ppm): 2.35 (s, 3 H, p-CH3-tolyl),
7.38 (d, 2H, J = 8.1 Hz, H3, H5-tolyl), 7.60 (s, 1H, NH, exchangeable
with D2O), 8.09 (d, 2H, J = 8.1 Hz, H2, H6-tolyl), 8.21 (d, 1H, J = 9.3 Hz,
H5-pyridazine), 8.36 (s, 1H, H3-pyrazole), 8.51 (d, 1H, J = 9.6 Hz, H4-
pyridazine) and 12.66 (s, 1H, NH, exchangeable with D2O). MS, m/z
(%): 336 (M+, 73.97), 319 (10.37), 294 (100). Analysis for C16H12N6OS
(336.38): required C, 57.13; H, 3.60; N, 24.98; S, 9.53; found C, 57.21;
H, 3.52; N, 25.01; S, 9.44.
1-(6-p-Tolyl-pyridazin-3-yl)-1,5-dihydro-pyrazolo[3,4-d]-
pyrimidin-4-one (8)
Method A
Compound 4 (2.94 g, 0.01 mol) was heated under reflux temperature in
triethyl orthoformate (30 mL) for 10 h. The product, which separated on
cooling, was filtered off, washed with ethanol, dried, and recrystallized
from dimethylformamide to give 8 in 86% yield, m.p. 383–4◦C.
Method B
Compound 2 (2.76 g, 0.01 mol) was heated under reflux temperature
in formic acid (30 mL, 85%) for 5 h. The reaction mixture was cooled
and poured into water. The formed solid was filtered off, dried, and
recrystallized from dimethylformamide to give 8 in 78% yield, which

Synthesis of Pyrazolo[3,4-d]pyrimidine Derivatives
2357
was identical in all aspects with compound 8 obtained from method A,
m.p. 383–4◦C. IR spectrum (KBr, ν, cm−1): 3102 (NH), and 1700 (CO);
1H NMR spectrum (DMSO-d6, δ ppm): 2.41 (s, 3H, p-CH3-tolyl), 7.40
(d, 2H, J = 7.8 Hz, H3, H5-tolyl), 8.11 (d, 2H, J = 7.5 Hz, H2, H6-tolyl),
8.23 (s, 1H, H3-pyrazole), 8.30 (d, 1H, J = 9.3 Hz, H5-pyridazine), 8.45–
8.48 (m, 2H, H4-pyridazine and H6-pyrimidine) and 12.51 (brs, 1H,
NH, exchangeable with D2O). MS, m/z (%): 304 (M+, 100). Analysis
for C16H12N6O (304.31): required C, 63.15; H, 3.97; N, 27.62; found C,
63.20; H, 4.05; N, 27.48.
4-Chloro-1-(6-p-tolyl-pyridazin-3-yl)-1H-pyrazolo[3,4-d]-
pyrimidine (9)
Compound 8 (1.52 g, 0.005 mol) was heated in phosphorus oxychloride
(20 mL) for 10 h. The solution was cooled and poured into ice water. The
solid was filtered off, dried, and purified on silica gel using chloroform
/n-hexane (1:1) as an eluent to give compound 9 in 55% yield, m.p. 274–
6◦C. IR spectrum (KBr, ν, cm−1): no absorption in the region of (C O);
MS, m/z (%): 324 (M+, Cl37, 32.93), 322 (M+, Cl35, 100).
1-(6-p-Tolyl-pyridazin-3-yl)-1,5-dihydro-pyrazolo[3,4-d]-
pyrimidine-4-thione (10)
Compound 9 (1.62 g, 0.005 mol) and (0.38 g, 0.005 mol) of thiourea in
dioxane (40 mL) was heated at reflux for 4 h. The precipitate that formed
was collected and dissolved in sodium hydroxide (20 mL, 10%). The mix-
ture was filtered off and the filtrate was precipitated with hydrochloric
acid (5 mL, 34%). The solid product was collected and recrystallized
from dimethylformamide to give 10 in 75% yield; m.p. 352–4◦C. IR
spectrum (KBr, ν, cm−1): 3025 (NH), and 1365 (C S); 1H NMR spectrum
(DMSO-d6, δ ppm): 2.40 (s, 3H, p-CH3-tolyl), 7.40 (d, 2H, J = 8.4 Hz, H3,
H5-tolyl), 8.11 (d, 2H, J = 7.8 Hz, H2, H6-tolyl), 8.27–8.34 (m, 2H, H3-
pyrazole and H5-pyridazine), 8.48 (d, 1H, J = 9.0 Hz, H4-pyridazine),
8.57 (s, 1H, H6-pyrimidine) and 13.96 (s, 1H, NH, exchangeable with
D2O). MS, m/z (%): 320 (M+, 100). Analysis for C16H12N6S (320.38): re-
quired C, 59.98; H, 3.78; N, 26.23; S, 10.01; found C, 60.11; H, 3.85; N,
26.10; S, 9.95.
4-Methylsulfanyl-1-(6-p-tolyl-pyridazin-3-yl)-1H-pyrazolo[3,4-
d]-pyrimidine (11)
Compound 10 (1.60 g, 0.005 mol) in ethanol (20 mL), and sodium hydrox-
ide solution (5 mL, 10%) were treated with methyl iodide (0.005 mol).

2358
A. H. Shamroukh et al.
The reaction mixture was refluxed for 5 h, cooled, and poured into wa-
ter. The separated solid was filtered off, dried, and recrystallized from
1
dioxane to give 11 in 86% yield, m.p. 238–40◦C. H NMR spectrum
(DMSO-d6, δ ppm): 2.39 (s, 3H, p-CH3-tolyl), 2.75 (s, 3H, SCH3), 7.38
(d, 2H, J = 7.8 Hz, H3, H5-tolyl), 8.09 (d, 2H, J = 8.1 Hz H2, H6-tolyl),
8.43–8.45 (m, 2H, H5, H4-pyridazine), 8.74 (s, 1H, H3-pyrazole), and
8.92 (s, 1H, H6-pyrimidine); MS, m/z (%): 334 (M+, 100).
1-(6-p-Tolyl-pyridazin-3-yl)-1H-pyrazolo[3,4-d-pyrimidin-4-yl]-
hydrazine (12)
Method A
Compound 9 (1.62 g, 0.005 mol) was heated in hydrazine hydrate
(20 mL, 98%) for 2 h, cooled, poured into water, neutralized with hy-
drochloric acid (2 mL, 34%), filtered off, dried, and purified with column
chromatography (silica gel) using chloroform: methanol (9:1), as an elu-
ent to give 12 in 57% yield, m.p. 344–6◦C.
Method B
To a solution of compound 11 (1.67 g, 0.005 mol) in dioxane (30 mL),
hydrazine hydrate (2 mL, 98%) was added and the mixture was heated
at reflux for 4 h. The reaction mixture was cooled and poured into wa-
ter and the separated solid was filtered off, dried, and recrystallized
from dioxane to give 12 in 66% yield which was identical in all aspects
with compound 12 obtained from method A, m.p. 344–6◦C. IR spectrum
1
(KBr, ν, cm−1): 3425, 3387 (NH2), and 3310 (NH); H NMR spectrum
(DMSO-d6, δ ppm): 2.40 (s, 3H, p-CH3-tolyl), 4.00 (brs, 1H, NH, ex-
changeable with D2O), 7.39 (d, 2H, J = 8.4 Hz, H3, H5-tolyl), 7.47 (s,
2H, NH2, exchangeable with D2O), 8.04–8.09 (m, 3H, H2, H6-tolyl and
H3-pyrazole), 8.21–8.28 (m, 2H, H5, H4-pyridazine), and 8.43 (s, 1H,
H6-pyrimidine); MS, m/z (%): 318 (M+, 100). Analysis for C16H14N8
(318.34): required C, 60.37; H, 4.43; N, 35.20; found C, 60.20; H, 4.50;
N, 35.29.
7-(6-p-Tolyl-pyridazin-3-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo-
[4,3-c]pyrimidine (13)
Compound 12 (1.59 g, 0.005 mol) was heated under reflux tempera-
ture in 20 mL triethyl orthoformate for 15 h. The reaction mixture was
filtered while hot and the filtrate was evaporated untill dry and the re-
maining solid was recrystallized from dioxane to give 13 in 56% yield,

Synthesis of Pyrazolo[3,4-d]pyrimidine Derivatives
2359
m.p. 308–310◦C. 1H NMR spectrum (DMSO-d6, δ ppm): 2.42 (s, 3H, p-
CH3-tolyl), 7.42 (d, 2H, J = 8.1 Hz, H3, H5-tolyl), 8.15 (d, 2H, J = 8.1
Hz, H2, H6-tolyl), 8.39 (d, 1H, , J = 9.0 Hz, H5-pyridazine), 8.53 (d, 1H,
J = 9.0 Hz, H4-pyridazine), 8.73 (s, 1H, H3-pyrazole), 8.93 (s, 1H, H8-
pyrimidine), and 9.79 (s, 1H, H6-triazole); MS, m/z (%): 328 (M+, 100).
SYNTHESIS OF 14 AND 15
General Procedure
A mixture of compound 8 (3.00 g, 0.01 mol) or 10 (3.20 g, 0.01 mol) and
50% oil-immersed sodium hydride (0.48 g, 0.02 mol) in dry dimethyl-
formamide (50 mL) was stirred at 70◦C for 1 h and then cooled to room
temperature. 2-Chloroethyl methyl ether (0.95 g, 0.01 mol) was added
and stirred at 90◦C for 8 h. The mixture was evaporated until dry un-
der reduced pressure and the solids were chromatographed on silica gel
column with chloroform: methanol mixture (9:1) to give 14 or 15.
5-(2-Methoxy-ethyl)-1-(6-p-tolyl-pyridazin-3-yl)-1,5-dihydro-
pyrazolo[3,4-d]pyrimidin-4-one (14)
Yield 77%; m.p. 269–70◦C. IR spectrum (KBr, ν, cm−1): 1691 (C O);
1H NMR spectrum (DMSO-d6, δ ppm): 2.40 (s, 3H, p-CH3-tolyl), 3.27–
3.34 (m, 5 H, OCH3 and CH2O), 3.65 (t, 2 H, CH2N), 7.39 (d, 2H, J =
7.8 Hz, H3, H5-tolyl), 8.11 (d, 2H, J = 7.8 Hz, H2, H6-tolyl), 8.44–
8.47 (m, 2H, H5, H4-pyridazine), 8.75 (s, 1H, H3-pyrazole), and 8.92
(s, 1H, H6-pyrimidine); 13C NMR spectrum (DMSO-d6, δ ppm): 21 (p-
CH3-tolyl), 32 (OCH3), 58 (CH2O), 69 (CH2N), 108 (C3a), 137 (C3), 140
(C6), 159 (C7a), 159 (C O), 122, 126, 153, and 156 (C5, C4, C6, and C3
pyridazine), 113, 127, 130, and 152 (C4, C3, C2, and C1 tolyl). Analysis
for C19H18N6O2(362.39): required C, 62.97; H, 5.01; N, 23.19; found C,
63.12; H, 4.90; N, 23.10.
4-(2-Methoxy-ethylsulfanyl)-1-(6-p-tolyl-pyridazin-3-yl)-1H-
pyrazolo[3,4-d]pyrimidine (15)
Yield 82%; m.p. 217–19◦C; 1H NMR spectrum (DMSO-d6, δ ppm): 2.40
(s, 3H, p-CH3-tolyl), 3.27–3.34 (m, 5 H, OCH3 and CH2O), 3.65 (t, 2H,
CH2S), 7.39 (d, 2H, J = 7.8 Hz, H3, H5-tolyl), 8.11 (d, 2H, J = 7.8 Hz,
H2, H6-tolyl), 8.40–8.50 (m, 2H, H5, H4-pyridazine), 8.75 (s, 1H, H3-
pyrazole), and 8.92 (s, 1H, H6-pyrimidine); 13C NMR spectrum (DMSO-
d6, δ ppm): 22 (p-CH3-tolyl), 29 (OCH3), 59 (CH2O), 72 (CH2S), 133
(C3a), 136 (C3), 141 (C6), 159 (C7a), 164 (C4), 122, 126, 153, and 156

2360
A. H. Shamroukh et al.
(C5, C4, C6, and C3 pyridazine), 114, 127, 130, and 152 (C4, C3, C2,
and C1 tolyl)) ; MS, m/z (%): 378 (M+, 8.35), 333 (17), 320 (100).
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