Synthesis of pyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine derivatives for antiviral evaluation

Article abstract of DOI:10.1002/ardp.200700005

6-Amino-3-methyl-4-(4-nitrophenyl)-2,4-dihydropyrano[2,3-c] pyrazole-5-carbonitrile (1) was used as a precursor for preparation of some novel 3,7-dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazolo[4′,3′:5,6] pyrano[2,3-d]pyrimidine derivatives 3-6, and some o

Full text of DOI:10.1002/ardp.200700005

236
Arch. Pharm. Chem. Life Sci. 2007, 340, 236–243
Full Paper
Synthesis of Pyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidine
Derivatives for Antiviral Evaluation
Ahmed H. Shamroukh, Magdi E. A. Zaki, Eman M. H. Morsy, Faiza M. Abdel-Motti, and
Farouk M. E. Abdel-Megeid
Photochemistry Department, National Research Centre, Dokki, Cairo, Egypt
6-Amino-3-methyl-4-(4-nitrophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (1) was used
as a precursor for preparation of some novel 3,7-dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazo-
lo[49,39:5,6]pyrano[2,3-d]pyrimidine derivatives 3–6, and some of their corresponding N2- and C5-
S-acyclic nucleosides 7 and 8. Furthermore, the preparation of 5-amino-1-[3,7-dimethyl-4-(4-nitro-
phenyl)-2,4-dihydropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-yl]-1H-pyrazole derivatives 10–16
were described. Some of the prepared products were selected and tested for antiviral activity
against Herpes Simplex Virus type-1 (HSV-1).
Keywords: Acyclic nucleosides / Antiviral activities / Pyranopyrazoles / Pyrazoles / Pyrazolopyranopyrimidines /
Received: January 11, 2007; accepted: February 14, 2007
DOI 10.1002/ardp.200700005
b-enaminonitrile
precursor:
6-amino-2,4-dihydro-3-
Introduction
methyl-4-(4-nitrophenyl)pyrano[2,3-c]pyrazole-5-carboni-
trile 1 [11, 15] as key compound for this study and for
further syntheses of other fused heterocyclic compounds.
Thus, when compound 1 was stirred at room tempera-
ture with acetic anhydride it gave 6-acetamido-3-methyl-
4-(4-nitrophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-car-
bonitrile 2. The structure of the latter compound was
confirmed on the basis of its elemental analysis and spec-
tral data (see Experimental and Scheme 1). The IR spec-
trum of compound 2 showed absorption bands assign-
The chemistry of pyrazole derivatives, especially 3-
methyl-1-phenyl-2-pyrazolin-5-one, has received great
interest, since this nucleus is the parent skeleton of pyr-
ine drugs and other medicinal compounds [1–2]. Pyr-
ano[2,3-c]pyrazole derivatives showed biological activ-
ities as bactericides [3] and virucides [4]. Moreover, a large
number of fused pyrimidine derivatives exhibited anti-
mycobacterial [5], antitumor [6], and antiviral activities
[7]. In continuation of our previous work on pyrazoles
[8–10], pyrano[2,3-c]pyrazoles [11], pyrimidines [12], and
fused pyrimidines [9, 13–14], we intended to synthesize a
pyrano[2,3-c]pyrazole ring system fused to a pyrimidine
moiety to obtain new compounds which are expected to
possess notable chemical and biological activities.
1
able to the NH groups, CN-, and CO group. Its H-NMR
spectrum showed signals at 1.77 (s, 3H, C3-CH3), 2.24 (s,
3H, O=C-CH3), 11.91, and 12.63 for 26NH (D2O exchange-
able) and MS gave the molecular ion peak at m/z (%): 339
[M+] (42.72). While carrying out the same reaction under
reflux, acetylation took place to give 2-acetyl-3,7-
dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazolo[49,39:5,6]-
pyrano[2,3-d]pyrimidin-5(6H)-one 3.
Results and discussion
The structure of the aforementioned compound was
confirmed by spectral data (see Experimental and
Scheme 1). The 1H-NMR spectrum showed a signal at 2.26
(s, 3H, C3-CH3). Actually, by surveying the alkylation and
acylation reactions of pyrazole derivatives, we can con-
clude that N2-acetylation which took place for compound
3, is in accordance with results which judged that the 1H-
NMR chemical shifts of C3-methyl group adjacent to N2-
Chemistry
This work deals with synthesis and study of the reactions
of fused pyrano[2,3-c]pyrazoles from their corresponding
Correspondence: Ahmed H. Shamroukh, Photochemistry Department,
National Research Centre, Dokki, Cairo 12622, Egypt.
E-mail: ahshamroukh@yahoo.com
Fax: +20 203 37-0931
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Arch. Pharm. Chem. Life Sci. 2007, 340, 236–243
Pyrazolopyranopyrimidine
237
Scheme 1. Synthesis route of compounds 1–8.
alkyl group are more deshielded and appear invariably at
It was reported by Arakawa et al. [18] and Kashima [19]
a lower field (ld 2.25–2.30 ppm), when compared with that formation of acyl pyrazoles is governed by the steric
that of the C3-methyl protons of N1-alkyl derivatives (ld interaction between the ring substitution groups and the
1
1.9–2.0 ppm) [16, 17]. Furthermore, the H-NMR data acyl moiety which isomerizes slowly from the N2-position
given by many authors for alkylation, acylation [11, 13, into the more thermodynamically stable N1-acetyl prod-
16, 18–20] and nucleosidation reactions [21], the single uct. However, when we attempted isomerization of prod-
crystal X-ray analysis proved no doubt that acetylation uct 3 into its corresponding N1-acetyl product under very
took place at the N2-nitrogen of the pyrazole ring (Fig. 1, mild conditions, only deacetylation started to take place
Table 1).
with formation of 3,7-dimethyl-4-(4-nitrophenyl)-2,4-
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Table 1. Crystal data, bond lengths, bond angles, and torsion angles of compound 3.
Crystal system
Monoclinic
Torsion angles (8)
Space group
a (ꢀ)
b (ꢀ)
c (ꢀ)
a (8)
P -1
C15–N4–N9–C22
N9–N4–C15–C24
N9–N4–C15–C29
C18–N4–N9–C22
N9–N4–C18–O23
N9–N4–C18–C28
C15–N4–C18–O23
C18–N4–C15–C24
C15–N4–C18–C28
C18–N4–C15–C29
C24–C15–N4–N9
C24–C15–N4–C18
C29–C15–N4–C18
O23–C18–N4–N9
O23–C18–N4–C15
C28–C18–N4–N9
C28–C18–N4–C15
–0.3 (7)
–0.3 (6)
–177.5 (11)
176.2 (9)
–171.9 (11)
7.3 (7)
24.6358 (14)
7.2915 (4)
21.7147 (14))
90.008
b (8)
93.600 (3)8
90.008
c (8)
3.9 (8)
V (ꢀ3)
3893.0 (4)ꢀ3
8
–176.3 (11)
–176.9 (12)
6.4 (7)
–0.3 (6)
–176.3 (11)
6.4 (7)
–171.9 (11)
3.9 (8)
7.3 (7)
–176.9 (12)
Z
Dx
R
wR
1.452 Mg m-3
0.043
0.076
Bond length (ꢀ)
N4–N9
1.386 (2)
1.385 (2)
1.417 (2)
N4–C15
N4–C18
Bond angles (8)
N9–N4–C15
N9–N4–C18
C15–N4–C18
N4–N9–C22
N4–C18–O23
N4–C18–C28
O23–C18–C28
112.42 (12)
118.6 (2)
128.9 (2)
100.69 (12)
119.6 (2)
115.7 (2)
124.7 (2)
Since the synthesis of acyclovir, as one of the potent
antiherpetic drug by Schaffer et al. [23], many attempts
have been directed by nucleoside chemists to prepare a
lot of related compounds with various side chains and
glycons [24, 25]. However in this connection, there is only
one recent report in the literature [21] on the N1-pyrazole
acyclic nucleosides of pyrazolo[49,39:5,6]pyrano[2,3-d]pyri-
midine derivatives which were tested for their anti-
inflammatory activity. The N2-acyclic nucleosides and S-
acyclic nucleosides of pyrazolo[49,39:5,6]pyrano[2,3-d]pyri-
midine derivatives are still not known (to the best of our
knowledge) in the literature. Thus, in continuation of
our previous work [9, 26] in preparing various cyclic and
acyclic nucleosides of different heterocyclic compounds,
in this report, we describe the synthesis of some N2- and S-
acyclic nucleosides related to pyrazolo[49,39:5,6]-pyr-
ano[2,3-d]pyrimidine derivatives by treating the sodium
salt of compounds 4 and 6 (generated in situ, see Experi-
mental) with 2-chloroethyl methyl ether; they afforded
the corresponding N2- and C5-S-acyclic nucleosides: 3,7-
dimethyl-2-(2-methoxyethyl)-4-(4-nitrophenyl)-2,4-dihy-
dropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-ol 7 and 5-
(2-methoxy-ethylsulfanyl)-3,7-dimethyl-4-(4-nitrophenyl)-
1,4-dihydropyrazolo[49,39:5,6]pyrano[3,2-d]pyrimidine 8,
respectively (Scheme 1). The structural assignments and
sites of nucleosidation of compounds 7 and 8 were
assigned on the basis of their elemental and spectral data
Figure 1. Single crystal X-ray structure of compound 3 and
dioxane solvent.
dihydropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5(6H)-
one 4 (see Experimental and Scheme 1). Compound 4 was
converted into its corresponding 5-chloro derivative 5
(Scheme 1) by refluxing with phosphorus oxychloride.
The mass spectra of compound 5 gave fragments showing
the isotopic pattern due to the presence of chlorine atom
(see Experimental). Compound 5 was converted into its
corresponding 5-thiono derivative 6 (Scheme 1) following
the procedure of Schiba et al. [22] and its 13C-NMR spec-
trum showed a signal at d 183 ppm accountable for the
C=S group (see Experimental).
1
(see Experimental and Scheme 1). The H-NMR spectrum
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Arch. Pharm. Chem. Life Sci. 2007, 340, 236–243
Pyrazolopyranopyrimidine
239
Scheme 2. Synthesis route of compounds 9–16.
of compound 7 revealed the absence of signals at d 12.18, sence of NH at d 11.30 ppm (exchangeable with D2O) in
12.40 ppm of the NH of the pyrazole and the pyrimidine 1H-NMR spectrum and the absence of the C=S group in its
ring, however a signal at d 6.79 ppm assignable to the OH 13C-NMR spectrum proved that nucleosidation took place
of the pyrimidine moiety due to the tautomerization. at the C5-S-pyrimidine. Deprotection of nucleosides 7 and
The assignment of the N2- nucleosidation was judged by
the appearance of the C3-CH3 protons at d 2.25 ppm, and afforded again compounds 4 and 6, respectively.
this is in accordance with reported results for N2-alkyla-
When compound 5 was heated with hydrazine
tion of related compounds [16]. In compound 8, the pre- hydrate, it gave a compound assigned to the structure of
8
(using ammonium hydroxide solution/ethanol)
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3,7-dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazolo-
[49,39:5,6]pyrano[2,3-d]pyrimidin-5-ylhydrazine 9 (Experi-
mental, Scheme 2). When the compound 9 was refluxed
with ethoxymethylenemalononitrile, methylethoxy-
methylenemalononitril, bis-(methylthio)methylenema-
lononitrile, tetracyanoethylene or ethyl(ethoxy-methyle-
ne)cyano-acetate, it afforded the corresponding substi-
tuted pyrazole derivatives 10–14, respectively
(Scheme 2). The structures of the latter compounds were
confirmed on the basis of their elemental analysis and
spectral data (Experimental). The IR spectra of com-
pounds 10–13 showed absorption bands characteristic
for NH2 and CN groups, while compound 14 revealed
absorption bands characteristic for NH2 and C=O. Also,
1H-NMR spectra showed signals at d = 6.79, 6.75, 6.85,
6.55, and 6.53 ppm due to NH2 (exchangeable with D2O)
for compounds 10–14, respectively. The MS gave the
molecular ion peaks at m/z (%) = 429 (23.49), 443 (20.85),
475 (3.25), 454 (18.30), and 476 (41.88) for compounds
10–14, respectively.
Figure 2. Effect of novel derivatives on HSV-1.
parison with acyclovir as control. At both concentration,
all tested compounds revealed no higher antiviral activ-
ity than acyclovir. In general, compound 4 showed the
highest effect on HSV-1 than the other tested compounds,
where its antiviral activity increased from 63% at concen-
tration of 20 lg/105 cells to 95% at concentration of
40 lg/105 cells.
Similarly, when a mixture of compound 9 and acetyla-
cetone in ethanol was refluxed, it gave 5-(3,5-dimethyl-
pyrazol-1-yl)-3,7-dimethyl-4-(4-nitrophenyl)-2,4-dihydro-
pyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidine 15 (Experimen-
1
tal and Scheme 2). The H-NMR spectrum of the latter
Conclusion
compound showed signals at d = 1.75 ppm and 2.25 ppm
for (26CH3). The MS, gave the molecular ion peak as a
base peak at m/z (%) = 417 (100).
On the other hand, when a mixture of compound 9
and excess of ethyl acetoacetate was refluxed, it lead to
an unexpected product assigned to the structure of 1-[3,7-
dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazolo[49,39:5,6]-
pyrano[2,3-d]pyrimidin-5-yl]-3,4-dimethyl-3a,7a-dihydro-
Some novel pyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidine
derivatives 3–6, and some of their corresponding N2- and
C5-S-acyclic nucleosides 7 and 8 were prepared. Further-
more, some pyrazole derivatives 10–16 were described.
Structure-activity correlation of the obtained results
revealed that the pyrazolopyranopyrimidine ring, in
compounds 3, 4, 6, and 8 showed promising antiviral
activity. When the acetyl group of compound 3 was
removed, it was noticed that the new pyrazolopyranopyr-
imidine derivative 4 was found to be more active as anti-
viral agent for HSV-1 (due to the two free NH groups).
While replacement of oxygen (in compound 4) with sul-
fur (in compound 6), decreased the activity. Addition of
another ring e.g., a pyrazole ring in compounds 10, 11,
12, 13, and 16 decreased the activity% of HSV-1 reduction.
1H-pyrano[2,3-c]pyrazol-6-one
16
(Experimental,
Scheme 2). The formation of compound 16 might have
taken place via the formation of the pyrazolone inter-
mediate which reacted further with another mole of
1
ethyl acetoacetate. The H-NMR spectrum of compound
16 showed signals at 2.35 ppm, 2.51 ppm for (26CH3),
and 5.69 ppm for (pyranyl-H), and its IR spectrum
revealed the presence of a C=O group. The MS gave the
molecular ion peak as a base peak at m/z (%) = 485 (100).
We are thankful to Dr. M. A. Ali, Virology Laboratory, National
Research Centre for antiviral evaluation.
Antiviral Screening
The plaque infectivity assay was carried out to test com-
pounds 3, 4, 6, 8, 10, 11, 12, 13, and 16 for antiviral activ-
ity. The test was performed to include the three possibili-
ties for antiviral activity – viricidal effect, virus adsorp-
tion, and effect on virus replication for HSV-1. The results
are summarized in Fig. 2. Compound 12 revealed a toxic
effect on viral growth. On the other hand, compounds 3,
4, 6, and 8 revealed the highest anti-HSV-1 activity in com-
Experimental
Chemistry
All melting points are measured using Electro-thermal IA 9100
apparatus, (Shimadzu, Japan). IR spectra were recorded as potas-
sium bromide pellets on a Perkin-Elmer 1650 spectrophot-
ometer (Perkin Elmer, USA), National Research Centre, Cairo,
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Arch. Pharm. Chem. Life Sci. 2007, 340, 236–243
Pyrazolopyranopyrimidine
241
1
Egypt. H-NMR and 13C-NMR spectra were determined on a Jeol-
5-Chloro-3,7-dimethyl-4-(4-nitrophenyl)-2,4-dihydro-
Ex-300 NMR spectrometer (JEOL, Tokyo, Japan) and chemical
shifts were expressed as part per million; ppm (d values) against
TMS as internal reference (Faculty of Science, Cairo University,
Cairo, Egypt). Mass spectra were recorded on EI + Q1 MSLMR
UPLR, National Research Centre, Cairo, Egypt. Microanalyses
were operated using Mario El Mentar apparatus, Organic Micro-
analysis Unit, National Research Centre, Cairo, Egypt and the
results were within the accepted range (l 0.40) of the calculated
values. Column chromatography was performed on Silica gel 60
(particle size 0.06–0.20 mm; Merck, Darmstadt, Germany).
Compound 1 was prepared according to a reported method
[11].
pyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidine 5
A mixture of compound 4 (0.339 g, 1 mmol) in phosphorus oxy-
chloride (10 mL) was heated for 2 h. The solution was cooled and
poured into ice-water, and the formed precipitate was filtered
off, dried, and recrystallized from dioxane to give compound 5.
1
Yield 85%, m.p. 340–3428C. H-NMR (DMSO-d6) d 2.00 (s, 3H, C3-
CH3), 2.49 (s, 3H, C7-CH3), 5.59 (s, 1H, pyran), 7.52 (d, J = 8.7 Hz,
2H, Ar-H), 8.13 (d, J = 8.7 Hz, 2H, Ar-H) and 12.29 (s, 1H, NH, D2O
exchangeable). MS m/z (%): 359 [M+] Cl37, (3.66), 357 [M+] Cl35,
(9.81). Anal. calcd. for C16H12ClN5O3: C 53.72, H 3.38, Cl 9.91, N
19.58. Found: C 53.85, H 3.31, Cl 10.02, N 19.41.
3,7-Dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazolo-
[4‘,39:5,6]pyrano[2,3-d]pyrimidine-5(6H)-thione 6
Synthesis of compounds 2, 3, 4, 5, and 6
6-Acetamido-3-methyl-4-(4-nitrophenyl)-2,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile 2
A solution of compound 5 (3.57 g, 1 mmol) and thiourea (1.52 g,
2 mmol) in ethanol (20 mL) was heated at reflux for 4 h. The
formed precipitate was filtered off, dried, and recrystallized
from dimethylformamide to give compound 6. Yield 77%, m.p.
A solution of compound 1 (2.97 g, 1 mmol) in acetic anhydride
(10 mL)was stirred at roomtemperature for 10 h. The formed pre-
cipitate was filtered off, dried, and recrystallized from ethanol to
give compound 2. Yield 89%, m.p. 240–2438C. IR (KBr, cm– 1) 3315
(NH), 3201(NH), 2200(CN) and1735 (CO).1H-NMR (DMSO-d6) d 1.77
(s, 3H, C3-CH3), 2.24 (s, 3H, CO-CH3), 4.98 (s, 1H, pyran), 7.58 (d, J =
12 Hz, 2H, Ar-H), 8.23 (d, J = 13.08 Hz, 2H, Ar-H), 11.91 (s, 1H, NH,
D2O exchangeable), and 12.63 (s, 1H, NH, D2O exchangeable). MS
m/z (%): 339 [M+] (42.72). Anal. calcd. for C16H13N5O4: C 56.64, H
3.86, N20.64. Found: C56.53, H 3.90, N20.71.
1
380–3828C. IR (KBr, cm– 1) 3250 (NH), and 3225 (NH). H-NMR
(DMSO-d6) d 2.01 (s, 3H, C3-CH3), 2.43 (s, 3H, C7-CH3), 5.47 (s, 1H,
pyran), 7.47 (d, J = 8.7 Hz, 2H, Ar-H), 8.07 (d, J = 8.7 Hz, 2H, Ar-H),
12.16 (s, 1H, NH, D2O exchangeable) and 13.97 (s, 1H, NH, D2O
exchangeable). 13C-NMR (DMSO-d6) d 9.8 (C3-CH3), 20 (C7-CH3), 37
(C-4), 98 (C-4a), 112 (C-3a), 123, 129, 136, 145 (Ar-C), 151 (C-3), 154
(C-9a), 158 (C-8a), 161 (C-7), 183 (C-5). MS m/z (%): 355 [M+] (78.38).
Anal. calcd. for C16H13N5O3S: C 54.08, H 3.69, N 19.71, S 9.02.
Found: C 54.00, H 3.75, N 19.63, S 9.12.
2-Acetyl-3,7-dimethyl-4-(4-nitrophenyl)-2,4-dihydrop-
Synthesis of compounds 7 and 8; general procedure
A mixture of compound 4 (0.34 g, 0.1 mmol) or 6 (0.36 g,
0.1 mmol), and 50% oil-immersed sodium hydride (0.05 g,
0.2 mmol) in dry dimethylformamide (30 mL) was stirred at
708C for 1 h. The solution was cooled, and then 2-chloroethyl-
methyl ether (0.1 mmol) was added and stirred at 908C for 8 h.
The reaction mixtures were evaporated under reduced pressure
and chromatographed on silica gel column using chloroform/
methanol mixture (9 : 1) as an eluent to give compounds 7 or 8,
respectively.
yrazolo[4‘,39:5,6]pyrano[2,3-d]pyrimidin-5(6H)-one 3
A solution of compound 1 (2.97 g, 1 mmol) in acetic anhydride
(10 mL) was refluxed for 3 h. The reaction mixture was evapo-
rated till dryness and the remaining solid was recrystallized
from dioxane to give compound 3. Yield 50%, m.p. 356–3588C.
IR (KBr, cm– 1) 3440 (NH), 1735 (CO) and 1650 (CO). 1H-NMR
(DMSO-d6) d 2.26 (s, 3H, C3-CH3), 2.31 (s, 3H, C7-CH3), 2.56 (s, 3H,
COCH3), 5.28 (s, 1H, pyran), 7.56 (d, J = 8.7 Hz, 2H, Ar-H), 8.14 (d, J
= 8.7 Hz, 2H, Ar-H) and 12.65 (s, 1H, NH, D2O exchangeable). 13C-
NMR (DMSO-d6) d 13.0 (C3-CH3), 21.0 (C7-CH3), 23.1 (CH3-acetyl),
33.3 (C-4), 99 (C-4a), 106.6 (C-3a), 123, 129, 140, 146 (Ar-C), 150 (C-
3), 155 (C-9a), 159.6 (C-8a), 161 (C-7), 162.4 (C-5), 171 (CO). MS m/z
(%): 381 [M+] (25.41). Anal. calcd. for C18H15N5O5: C 56.69, H 3.96, N
18.36. Found: C 56.73, H 4.01, N 18.27.
3,7-Dimethyl-2-(2-methoxyethyl)-4-(4-nitrophenyl)-2,4-
dihydropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-ol 7
1
Yield 49%. Oil. IR (KBr, cm– 1) 3420–3300 (broad, OH). H-NMR
(DMSO-d6) d 2.25 (s, 3H, C3-CH3), 2.29 (s, 3H, C7-CH3), 3.0 (s, 3H,
OCH3), 3.52–3.60 (m, 4H, CH2CH2), 5.49 (s, 1H, pyran-H), 6.79 (brs,
1H, OH, D2O exchangeable), 7.12 (d, J = 9.0 Hz, 2H, Ar-H), and 7.65
(d, J = 8.4 Hz, 2H, Ar-H). Anal. calcd. for C19H19N5O5: C 57.43, H
4.82, N 17.62. Found: C 57.51, H 4.77, N 17.59.
3,7-Dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazolo-
[49,39:5,6]pyrano[2,3-d]pyrimidin-5(6H)-one 4
A mixture of compound 3 (3.81 g, 1 mmol) and ammonium
hydroxide solution (1 mL, 25%) in ethanol (30 mL) was stirred at
room temperature for 3 h. The formed precipitate was filtered
off, washed with water several times, and recrystallized from
dioxane to give compound 4. Yield 89%, m.p. 390–3928C IR (KBr,
cm– 1) 3164 (NH), 3200 (NH) and 1653 (CO). 1H-NMR (DMSO-d6) d
1.90 (s, 3H, C3-CH3), 2.28 (s, 3H, C7-CH3), 5.14 (s, 1H, pyran), 7.46 (d, J
= 9.0 Hz, 2H, Ar-H), 8.10 (d, J = 8.7 Hz, 2H, Ar-H), 12.18 (s, 1H, NH,
D2O exchangeable) and 12.40 (s, 1H, NH, D2O exchangeable). 13C-
NMR (DMSO-d6) d 9.8 (C3-CH3), 20.9 (C7-CH3), 34 (C-4), 98 (C-4a),
99.5 (C-3a), 123, 129, 136, 145 (Ar-C), 152 (C-3), 155 (C-9a), 158 (C-
8a), 162 (C-7), 162,8 (C5). MS m/z (%): 339 [M+] (18.10). Anal. calcd.
for C16H13N5O4: C 56.64, H 3.86, N 20.64. Found: C 56.51, H 3.90, N
20.73.
5-(2-Methoxyethylsulfanyl)-3,7-dimethyl-4-(4-
nitrophenyl)–2,4-dihydropyrazolo[49,39:5,6]pyrano[3,2-d]-
pyrimidine 8
Yield 72%, m.p. 237–2408C. 1H-NMR (DMSO-d6) d 1.80 (s, 3H, C3-
CH3), 2.25 (s, 3H, C7-CH3), 3.00 (s, 3H, OCH3), 3.21-3.29 (m, 4H,
CH2CH2), 4.85 (s, 1H, pyran-H), 7.05 (d, J = 9.0 Hz, 2H, Ar-H), 7.80
(d, J = 8.4 Hz, 2H, Ar-H) and 11.30 (s, 1H, NH, D2O exchangeable).
13C-NMR (DMSO-d6) d 9.9 (C3-CH3), 25 (C7-CH3), 29 (C-4), 35.7 (S-
CH2), 57.8 (O-CH3), 70 (CH2-O), 97 (C-4a), 108 (C-3a), 123, 129, 136,
146 (Ar-C), 150 (C-3), 154 (C-9a), 162 (C-8a), 165 (C-7), 170 (C-5).
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242
A. H. Shamroukh et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 236–243
Anal. calcd. for C19H19N5O4S: C 55.20, H 4.63, N 16.94, S 7.76.
Found: C 55.25, H 4.68, N 17.00, S 7.60.
5-Amino-3-methylthio-1-[3,7-dimethyl-4-(4-nitrophenyl)-
2,4-dihydropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-
yl]-1H-pyrazole-4-carbonitrile 12
Attempted deprotection of compounds 7 and 8
To a solution of compounds 7 (0.19 g, 0.5 mmol) or 8 (0.20 g.
0.5 mmol) in ethanol (25 mL), ammonium hydroxide solution
(20 mL, 25%) was added with stirring at room temperature for
1 h. The isolated solids were filtered off, and dried to give com-
pounds identical in all aspects with compounds 4 and 6, respec-
tively.
Yield after 3 h: (84%, dioxane); m.p. 209–2118C. IR (KBr, cm– 1
)
3313, 3299 (NH2), 3200 (NH) and 2222 (CN) cm-1. 1H-NMR (DMSO-
d6) d 1.90 (s, 3H, C3'-CH3), 2.60 (s, 3H, C79-CH3), 3.76 (s, 3H, SCH3),
5.99 (s, 1H, pyran), 6.85 (s, 2H, NH2, D2O exchangeable), 7.16 (d, J
= 8.7 Hz, 2H, Ar-H), 7.95 (d, J = 8.7 Hz, 2H, Ar-H), and 12.35 (s, 1H,
NH, D2O exchangeable). MS m/z (%): 475 [M+] (3.25). Anal. calcd.
for C21H17N9O3S: C 53.05, H 3.60, N 26.51, S 6.74. Found: C 53.16,
H 3.64, N 26.38, S 6.72.
3,7-Dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazolo-
[49,39:5,6]pyrano[2,3-d]pyrimidin-5-ylhydrazine 9
5-Amino-1-[3,7-dimethyl-4-(4-nitrophenyl)-2,4-
To a solution of compound 5 (3.59 g, 1 mmol) in dry dioxane
(30 mL), hydrazine hydrate (3 mL, 99%) was added and the reac-
tion mixture was heated on a water bath for 1 h. After cooling
the precipitated material was filtered off, washed several times
with water, dried and recrystallized from ethanol to give com-
pound 9. Yield 99%, m.p. 273–2748C. IR (KBr, cm– 1) 3420–3238
(NH2, NH). 1H-NMR (DMSO-d6) d 1.97 (s, 3H, C3-CH3), 2.37 (s, 3H, C7-
CH3), 4.34 (s, 2H, NH2, D2O exchangeable), 5.37 (s, 1H, pyran), 7.99
(d, J = 9.0 Hz, 2H, Ar-H), 7.81(s, 1H, NH, D2O exchangeable), 8.13
(d, J = 9.0 Hz, 2H, Ar-H), and 12.06 (s, 1H, NH, D2O exchangeable).
MS m/z (%): 353 [M+] (20.61). Anal. calcd. for C16H15N7O3: C 54.39, H
4.28, N 27.75. Found: C 54.22, H 4.32, N 27.88.
dihydropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-yl]-1H-
pyrazole-3,4-dicarbonitrile 13
Yield after 4 h: (93%, ethyl acetate); m.p. 277–2798C. IR (KBr,
cm– 1) 3436, 3350 (NH2), 3161 (NH), and 2229 (CN). 1H-NMR
(DMSO-d6) d 1.89 (s, 3H, C39-CH3), 2.59 (s, 3H, C79-CH3), 6.12 (s, 1H,
pyran), 6.55 (s, 2H, NH2, D2O exchangeable), 7.15 (d, J = 9.0 Hz,
2H, Ar-H), 8.01 (d, J = 9.0 Hz, 2H, Ar-H) and 12.39 (s, 1H, NH, D2O
exchangeable). MS m/z (%): 454 [M+] (18.30). Anal. calcd. for
C21H14N10O3: C 55.51, H 3.11, N 30.82. Found: C 55.64, H 3.04, N
30.76.
Ethyl-5-amino-1-[3,7-dimethyl-4-(4-nitrophenyl)-2,4-
dihydropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-yl]-1H-
pyrazole-4-carboxylate 14
5-Amino-1-[3,7-dimethyl-4-(4-nitrophenyl)-2,4-dihydro-
pyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-yl]-3,4-
Yield after 4 h: (95%, dioxane); m.p. 272–2748C. IR (KBr, cm– 1
)
disubstituted-1H-pyrazoles 10–14; general procedure
To a solution of compound 7 (3.53 g, 1 mmol) in ethanol (30 mL),
ethoxymethylenemalononitrile, methylethoxymethylenemalo-
nonitril, bis(methylthio)methylene-malononitrile, tetracya-
noethylene, or ethyl(ethoxymethylene)cyanoacetate (1 mmol)
was added, respectively. The reaction mixture was heated for 2–
4 h. Then the formed precipitate was filtered off and recrystal-
lized from appropriate solvent to give compounds 10–14.
3436, 3350 (NH2), 3150 (NH) and 1683 (CO). 1H-NMR (DMSO-d6) d
1.23 (t, J = 7.2 Hz, 3H, CH2CH3), 1.90 (s, 3H, C3'-CH3), 2.60 (s, 3H, C79-
CH3), 4.15 (q, J = 6.9 Hz, 2H, CH2CH3), 6.12 (s, 1H, pyran), 6.53 (s,
2H, NH2, D2O exchangeable), 7.17 (d, J = 8.7 Hz, 2H, Ar-H), 7.75 (s,
1H, pyrazole-H), 7.96 (d, J = 8.7 Hz, 2H, Ar-H), and 12.32 (s, 1H,
NH, D2O exchangeable). MS m/z (%): 476 [M+] (41.88). Anal. calcd.
for C22H20N8O5: C 55.46, H 4.23, N 23.52. Found: C 55.52, H 4.20, N
23.54.
5-Amino-1-[3,7-dimethyl-4-(4-nitrophenyl)-2,4-
dihydropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-yl]-1H-
pyrazole-4-carbonitrile 10
5-(3,5-Dimethylpyrazol-1-yl)-3,7-dimethyl-4-(4-nitro-
phenyl)-2,4-dihydro-pyrazolo[49,39:5,6]pyrano[2,3-
d]pyrimidine 15
Yield after 2 h: (96%, dioxane); m.p. 322–3258C. IR (KBr, cm– 1
)
3430, 3300 (NH2), 3220 (NH), 2222 (CN). 1H-NMR (DMSO-d6) d 1.89
(s, 3H, C39-CH3), 2.59 (s, 3H, C79-CH3), 5.94 (s, 1H, pyran), 6.79 (s, 2H,
NH2, D2O exchangeable), 7.15 (d, J = 8.7 Hz, 2H, Ar-H), 7.82 (s, 1H,
pyrazole-H), 8.01 (d, J = 8.7 Hz, 2H, Ar-H), and 12.30 (s, 1H, NH,
D2O exchangeable). MS m/z (%): 429 [M+] (23.49). Anal. calcd. for
C20H15N9O3: C 55.94, H 3.52, N 29.36. Found: C 56.06, H 3.44, N,
29.32.
A mixture of compound 9 (3.53 g, 1 mmol), acetylacetone
(1 mmol) in anhydrous ethanol (30 mL) was refluxed for 5 h. The
reaction mixture was allowed to cool and the formed precipitate
was filtered off, dried, and recrystallized from ethanol to give
1
compound 15. Yield 65%, m.p. 337–3408C. H-NMR (DMSO-d6) d
1.75 (s, 3H, C39-CH3), 1.86 (s, 3H, C3-CH3), 2.25 (s, 3H, C59-CH3), 2.58
(s, 3H, C7-CH3), 5.80 (s, 1H, pyran-H), 5.97 (s, 1H, pyrazole-H) 7.06
(d, J = 9.0 Hz, 2H, Ar-H), 7.95 (d, J = 9.0 Hz, 2H, Ar-H), and 12.27 (s,
1H, NH, D2O exchangeable). MS m/z (%): 417 [M+] (100). Anal.
calcd. for C21H19N7O3: C 60.43, H 4.59, N 23.49. Found: C 60.33, H
4.62, N 23.56.
5-Amino-3-methyl-1-[3,7-dimethyl-4-(4-nitrophenyl)-2,4-
dihydropyrazolo[49,39:5,6]pyrano[2,3-d]pyrimidin-5-yl]-1H-
pyrazole-4-carbonitrile 11
Yield after 4 h: (93%, dioxane); m.p. 342–3448C. IR (KBr, cm– 1
)
1-[3,7-Dimethyl-4-(4-nitrophenyl)-2,4-dihydropyrazolo-
[49,39:5,6]pyrano[2,3-d]pyrimidin-5-yl]-3,4-dimethyl-3a,7a-
dihydro-1H-pyrano[2,3-c]pyrazol-6-one 16
A solution of compound 9 (3.53 g, 1 mmol) in ethyl acetoacetate
(30 mL) was refluxed for 5 h. The mixture was evaporated under
reduced pressure and the obtained oil washed several time with
diethyl ether and recrystallized from ethanol to give compound
3450, 3350 (NH2), 3235 (NH), and 2220 (CN). 1H-NMR (DMSO-d6) d
1.90 (s, 3H, C39-CH3), 1.99 (s, 3H, C3-CH3), 2.58 (s, 3H, C79-CH3), 6.04 (s,
1H, pyran), 6.75 (s, 2H, NH2, D2O exchangeable), 7.15 (d, J = 9.0 Hz,
2H, Ar-H), 7.88 (d, J = 8.7 Hz, 2H, Ar-H) and 12.42 (s, 1H, NH, D2O
exchangeable). MS m/z (%): 443 [M+] (20.85). Anal. calcd. for
C21H17N9O3: C 56.88, H 3.86, N 28.43. Found: C 56.80, H 3.92, N
28.45.
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Arch. Pharm. Chem. Life Sci. 2007, 340, 236–243
Pyrazolopyranopyrimidine
243
16. Yield 66%, m.p. 307–3108C. 1H-NMR (DMSO-d6) d 1.82 (s, 3H,
C39-CH3), 2.35 (s, 3H, C3-CH3), 2.51 (s, 3H, C4-CH3), 2.63 (s, 3H, C79-
CH3), 5.69 (s, 1H, pyran-H), 5.71 (s, 1H, pyran-H) 7.15 (d, J = 9.0 Hz,
2H, Ar-H), 7.89 (d, J = 9.0 Hz, 2H, Ar-H) and 12.35 (s, 1H, NH, D2O
exchangeable). MS m/z (%): 485 [M+] (100). Anal. calcd. for
C24H19N7O5: C 59.38, H 3.94, N 20.20. Found: C 59.51, H 4.01, N
20.00.
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addition of 10 mL antibiotic-antimycotic mixture (10000 U peni-
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African green monkey kidney-derived cells (Vero) were used.
Cells were propagated in Dulbeccos’ Minimal Essential Medium
(DMEM) supplemented with 10% fetal bovine serum, 1% antibio-
tic-antimycotic mixture. The pH was adjusted at 7.2–7.4 by 7.5%
sodium bicarbonate solution. The mixture was sterilized by fil-
tration through 0.2 lm pore size nitrocellulose membrane.
Viruses: Herpes Simplex Virus type-1 was obtained from Environ-
mental Virology Lab., Department of Water Pollution Research,
National Research Centre, Cairo, Egypt
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Cytotoxicity was assayed for both dimethylsulfoxide (DMSO) and
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maximum tolerated concentration (MTC) for each compound
was determined by both cell morphology and cell viability by
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was removed from the multiwell plate and virus-compound mix-
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i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com