Novel xanthine oxidase inhibitor studies. Part 3. Convenient and general syntheses of 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H)-ones as a new class of potential xanthine oxidase inhibitors

Article abstract of DOI:10.1039/a907673e

Convenient and general syntheses of 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H)-ones (12), a new class of potent xanthine oxidase inhibitors, involving the oxidative cyclisation of 6-substituted 4-alkylidenehydrazino- or 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d]pyrimidines (3 and 11) with 70% nitric acid as the key step, are reported. The hydrazones 3 and 11 were obtained by a versatile synthetic route via the key intermediates, 6-chloro-4-hydrazino-1H-pyrazolo[3,4-d]pyrimidine 2 or oxypurinol 4, starting from 2,4,6-trichloropyrimidine-5-carbaldehyde 1. Their inhibitory activities against bovine milk xanthine oxidase in vitro are also described; i.e. the pyrazolotriazolopyrimidines 12 were several hundred times more potent than allopurinol.

Full text of DOI:10.1039/a907673e

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Novel xanthine oxidase inhibitor studies. Part 3.1 Convenient and
general syntheses of 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-
triazolo[4,3-c]pyrimidin-5(6H)-ones as a new class of potential
xanthine oxidase inhibitors
1
Tomohisa Nagamatsu,*a Takayuki Fujitaa and Kazuki Endob
a Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530,
Japan
b Biology Laboratory, Research and Development Division, Yamasa Syoyu Co., Choshi,
Chiba 288-0056, Japan
Received (in Cambridge, UK) 22nd September 1999, Accepted 22nd October 1999
Convenient and general syntheses of 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H)-ones
(12), a new class of potent xanthine oxidase inhibitors, involving the oxidative cyclisation of 6-substituted 4-alkyl-
idenehydrazino- or 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d]pyrimidines (3 and 11) with 70% nitric acid as the
key step, are reported. The hydrazones 3 and 11 were obtained by a versatile synthetic route via the key intermediates,
6-chloro-4-hydrazino-1H-pyrazolo[3,4-d]pyrimidine 2 or oxypurinol 4, starting from 2,4,6-trichloropyrimidine-5-
carbaldehyde 1. Their inhibitory activities against bovine milk xanthine oxidase in vitro are also described; i.e. the
pyrazolotriazolopyrimidines 12 were several hundred times more potent than allopurinol.
Introduction
Allopurinol, a well known drug clinically used for treatment of
gout and hyperuricemia resulting from uric acid,2–4 has been
reported as a potential inhibitor of xanthine oxidase (XO),
which catalyzes the conversion of hypoxanthine and xanthine
to uric acid.5 Allopurinol is relatively non-toxic and does not
appear to interfere with anabolic processes within the cell, as
judged by its lack of inhibition of the growth of bacteria or
tumors.6 However, some allopurinol toxicities7 and a life-
threatening toxicity syndrome have been reported after its use.8
Although XO inhibitory activities have recently been discovered
in some newly synthesized compounds and previously known
compounds,9–15 no clinically effective XO inhibitors for the
treatment of hyperuricemia have been developed since allo-
purinol was introduced for clinical use in 1963.2,6 We have
recently discovered that 6-alkylidenehydrazino- or 6-aryl-
methylidenehydrazino-7H-purines (I) and the angular type
purine analogues, 9H-1,2,4-triazolo[3,4-i]purines (II), have
exhibited more potent bovine milk XO inhibitory activities than
that of allopurinol.1,16,17
In our recent communication,18 we reported the facile and
general syntheses of 3- and/or 5-substituted 7H-pyrazolo-
[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidines (III) as a new class of
potential xanthine oxidase inhibitors. Herein we report full
details of the versatile and general syntheses of the 3-
substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-
5(6H)-ones (III), involving the oxidative cyclisation of
6-substituted 4-alkylidenehydrazino- or 4-arylmethylidene-
hydrazino-1H-pyrazolo[3,4-d]pyrimidines as the key step.
Furthermore, we also report here their inhibitory activities
against bovine milk xanthine oxidase in comparison with
allopurinol in vitro.
activities than allopurinol. Therefore, in this paper we tried to
prepare 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]-
pyrimidin-5(6H)-ones (III), which are analogous to the triazol-
opurines (II), as another new class of potential XO inhibitors.
Few methods for synthesis of the pyrazolotriazolopyrimidines
(III) have been reported in the journal19,20 or patent21 literature
and several derivatives have been synthesised. However, none
of the 5-substituted derivatives has been prepared up to now.
In the first place we tried to synthesise the key intermediate,
4-hydrazino-1H-pyrazolo[3,4-d]pyrimidin-6(7H)-one 6 derived
Results and discussion
In the preceding paper,1 we have clarified that 9H-1,2,4-
triazolo[3,4-i]purines (II), especially the 5-oxo or 5-thioxo
derivatives, showed more potent bovine milk XO inhibitory
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42
This journal is © The Royal Society of Chemistry 2000
33

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from barbituric acid. The requisite starting material, 2,4,6-
trichloropyrimidine-5-carbaldehyde 1, was prepared according
to a literature method.22 Treatment of 1 with anhydrous hydra-
zine (4 equiv.) in 2-methoxyethanol at 0 ЊC afforded 6-chloro-
excess 80% hydrazine hydrate at 80–90 ЊC afforded the 4,6-
dihydrazino derivative 7 in good yields. Subsequent reaction of
compound 7 with appropriate aldehydes (3.0 equiv.) in DMF at
room temperature gave the corresponding hydrazones 8b–d,f
in excellent yields as shown in Tables 1 and 2. Next, in an
attempt to convert the 6-chloro-4-hydrazino compound 2 to the
6-oxo-4-hydrazino derivative 6, compound 2 was reacted with
urea (4.0 equiv.) in 2-ethoxyethanol under reflux for 5 hours.
However, owing to the stability of the chloro group towards
hydroxy substitution by urea or alkali, the intended compound
6 was not obtained, but 4-carbamoylhydrazino-6-chloro-1H-
pyrazolo[3,4-d]pyrimidine 9, which resulted from carbamoyl-
ation of the hydrazino group at the 4-position, was formed in
60% yield. Heating under reflux the product 9 with urea (3.0
equiv.) in 2-ethoxyethanol afforded the desired tricyclic com-
pound, 3-amino-7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrim-
idin-5(6H)-one 10, in 18% yield. This compound 10 (29% yield)
was also obtained by prolonged heating of 2 with urea under
the same reaction conditions as mentioned above.
4-hydrazino-1H-pyrazolo[3,4-d]pyrimidine
2
in 79% yield
with
(Scheme 1). Subsequent reaction of compound
2
All new compounds 2, 3 and 6–10 exhibited satisfactory
elemental combustion analyses except for 2 and 6 and FAB-MS,
IR and 1H NMR spectral data consistent with the structures. In
particular, the structure of the product 10 was confirmed by the
presence of a two-proton broad singlet signal at δ 7.94 and one-
1
proton signals at δ 12.27 and 13.10 in the H NMR spectrum
attributable to the amino and imino groups and by the presence
of peaks at 3370 (νas NH), 3260 (ν NH), 1720 (ν C᎐O) and 1685
᎐
s
(δ NH) cmϪ1 in the IR spectrum attributable to amino and
carbonyl groups. It was clarified that the substitution reaction
of the chloro group by hydroxy was difficult in the pyrazolo-
pyrimidine ring 2, while in the pyrazolotriazolopyrimidine ring
10 it was easy.
The 4-hydrazino-1H-pyrazolo[3,4-d]pyrimidin-6(7H)-one 6
as noted above was a versatile intermediate for the preparation
of the 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidine ring
system. Thus the hydrazinopyrazolopyrimidine 6 could be con-
verted to the hydrazones 11b, e–r (63–95% yields) by reaction
with an appropriate aldehyde (1.5 equiv.) in DMF at room
temperature (Scheme 2 and Tables 3 and 4). The hydrazones
11b, e–p, r were subsequently cyclised to the corresponding 3-
substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-
5(6H)-ones 12b, e–p, r by heating with 70% nitric acid (1.2
equiv.) at 100 ЊC in 60–91% yields (Method A) (Tables 5 and 6).
In the case of compound 11q possessing a 4-hydroxybenzyl-
idenehydrazino group as the substituent at the 4-position, the
3-(4-hydroxy-3-nitrophenyl) derivative 12s was obtained by
oxidative cyclisation–nitration in 52% yield. Oxidative cyclis-
ation was also accomplished by heating compounds 11e, g, h, k,
m–o, q with diethyl azodicarboxylate (DEAD) (3–7 equiv.)
under reflux in 50–67% yields (Method B). Moreover, the
3-alkyl derivatives 12a–d were synthesised by treatment of
compound 6 with an appropriate trialkyl orthoester (5.0 equiv.)
in trifluoroacetic acid at room temperature (Method C) or heat-
ing compound 6 with trialkyl orthoesters (3.0 equiv.) in DMF
at 100 ЊC (Method D) in 54–83% yields. In the light of this
multiple step synthesis, a one-pot oxidative cyclisation starting
from the 6-chloro-4-hydrazones 3a–g would be attractive.
Indeed, heating the hydrazones 3a–g with 70% nitric acid (5.0
equiv.) in DMF at 100 ЊC afforded the desired 3-substituted
7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H)-ones
12e, g, h, k, m, n, r accompanied by hydrolytic dechlorination in
60–85% yields (Method E).
Scheme 1 Reagents and conditions: i, anh. NH2NH2, 2-methoxy-
ethanol, 0 ЊC, 0.5 h; ii, RCHO, DMF, r.t., 10 h; iii, conc. HCl reflux,
1 h; iv, 10% HCl, reflux, 3 h; v, P2S5, pyridine, reflux, 2 h; vi, 50%
ethanolic NH2NH2, reflux, 10 min; vii, anh. NH2NH2, 2-methoxy-
ethanol, 100 ЊC, 5 h; viii, 80% aq. NH2NH2, 80–90 ЊC, 4 h; ix, RCHO,
DMF, r.t., 10 h; x, urea, 2-ethoxyethanol, reflux, 5 h; xi, urea, 2-ethoxy-
ethanol, reflux, 10 h; xii, urea, 2-ethoxyethanol, 36 h.
appropriate aldehydes (1.2 equiv.) in dimethylfumamide (DMF)
at room temperature gave the corresponding hydrazones 3a–g
in 60–93% yields as shown in Tables 1 and 2. Further, heating
compound 2 in concentrated hydrochloric acid (50 parts)
under reflux for 1 hour gave oxypurinol 4 (58% yield), which
was confirmed by direct comparison with an authentic
sample.23 The oxypurinol 4 was also obtained in a similar yield
by heating the hydrazone 3b (R = Ph) in 10% hydrochloric acid
(100 parts) for 3 hours. Thiation of oxypurinol 4 by phosphor-
ous pentasulfide gave the 6-oxo-4-thioxo derivative 5 (76%
yield) following a literature procedure23 and the reaction of 5
with excess 50% ethanolic hydrazine under reflux yielded the
desired intermediate, 4-hydrazino-1H-pyrazolo[3,4-d]pyrim-
idin-6(7H)-one 6, in 71% yield.
All new compounds 11 and 12 exhibited satisfactory elem-
1
ental combustion analyses and FAB MS, IR and H NMR
spectral data consistent with the structures as indicated in
Tables 3–6.
Xanthine oxidase inhibitory results
On the other hand, heating compound 1 with excess
anhydrous hydrazine at 100 ЊC or heating compound 2 with
The novel pyrazolopyrimidines 2, 3, 6, 8 and 11 and pyrazolo-
34
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42

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Table 1 Preparative, physical and analytical data for the compounds 3a–g, 8b–d,f
Found (%) (Required)
Compound
(Formula)
Yield
(%)
Recrystn. solventa
Mp/ЊC
(Rf, solvent systemb)
C
H
N
m/z MHϩ
3a
60
93
89
76
78
79
70
87
93
75
77
250
AcOEt
(0.78, A)
DMF
(0.56, A)
EtOH–DMF
(0.60, A)
EtOH–DMF
(0.64, A)
EtOH–DMF
(0.61, A)
EtOH–DMF
(0.57, A)
EtOH–DMF
(0.49, A)
EtOH–DMF
(0.60, B)
EtOH–DMF
(0.61, B)
EtOH–DMF
(0.68, B)
52.9
(53.0)
52.4
(52.85)
49.9
(49.6)
46.7
(46.9)
54.0
(54.5)
51.7
(51.6)
45.6
(45.4)
60.7
(60.95)
54.1
6.5
(6.5)
3.7
(3.3)
2.8
(2.8)
2.4
(2.6)
4.1
(3.9)
3.8
(3.7)
2.7
(2.5)
4.8
(4.85)
4.1
28.5
(28.5)
30.6
(30.8)
29.2
(28.9)
27.0
(27.4)
29.1
(29.3)
27.7
(27.8)
30.6
(30.9)
29.9
(29.9)
26.4
295/297
273/275
291/293
307/309/311
287/289
303/305
318/320
357
C13H19ClN6
3b
>300
C12H9ClN6
3c
>300
>300
>300
>300
>300
C12H8ClFN6
3d
C12H8Cl2N6
3e
C13H11ClN6
3f
C13H11ClN6O
3g
C12H8ClN7O2
8b
298–300
C19H16N8ؒH2O
8c
>300
>300
276
393
C19H14F2N8ؒ3/2 H2O
(54.4)
50.2
(50.45)
57.7
(4.1)
3.7
(3.8)
5.1
(26.7)
24.6
(24.8)
25.7
8d
425/427/429
417
C19H14Cl2N8ؒ3/2 H2O
8f
EtOH–DMF
(0.62, B)
C21H20N8O2ؒH2O
(58.1)
(5.1)
(25.8)
a All compounds 3 and 8 were obtained as colourless or pale yellow powdery crystals except for 3g (yellow). b Solvent systems: (A) AcOEt–n-hexane
(4:3 v/v), (B) AcOEt–EtOH (9:1 v/v).
Table 2 1R and 1H NMR spectroscopic data for the compounds 3a–g, 8b–d, f
Compound
νmax(Nujol)/cmϪ1
δH[60 MHz; (CD3)2SO; Me4Si]
3a
3180, 3100 (NH)
0.86 (3 H, J 6.8, CHCH2[CH2]5CH3), 1.31 (10 H, br s, CHCH2[CH2]5CH3), 2.15–2.60 (2 H, m,
CHCH2[CH2]5CH3), 7.61 (1 H, t, J 6.6, CHCH2[CH2]5CH3), 8.18 (1 H, s, 3-H), 12.00 (1 H, br,
4-NH), 12.90 (1 H, br, 1-NH)
3b
3c
3190, 3080 (NH)
3200, 3100 (NH)
3180, 3080 (NH)
3190, 3080 (NH)
3210, 3100 (NH)
3190, 3090 (NH)
3240, 3180, 3140 (NH)
3250, 3170, 3130 (NH)
7.40–7.60 (3 H, m, Ph-m,pH), 7.70–7.95 (2 H, m, Ph-oH), 8.28 (1 H, s, 3-H), 8.40 (1 H, s, CH-Ar),
12.44 (1 H, s, 4-NH), 13.75 (1 H, br s, 1-NH)
7.32 (2 H, dd, JH,H 8.8, JH,F 9.1, Ar-mH), 7.90 (2 H, dd, JH,H 8.8, JH,F 5.9, Ar-oH), 8.27 (1 H, s, 3-H),
8.40 (1 H, s, CH-Ar), 12.45 (1 H, s, 4-NH), 13.70 (1 H, br, 1-NH)
7.55 (2 H, d, J 8.6, Ar-mH), 7.85 (2 H, d, J 8.6, Ar-oH), 8.26 (1 H, s, 3-H), 8.40 (1 H, s, CH-Ar),
12.54 (1 H, br, 4-NH), 13.70 (1 H, br, 1-NH)
2.37 (3 H, s, CH3), 7.30 (2 H, d, J 7.9, Ar-mH), 7.70 (2 H, d, J 7.9, Ar-oH), 8.29 (1 H, s, 3-H), 8.38
(1 H, s, CH-Ar), 12.40 (1 H, br, 4-NH), 13.50 (1 H, br, 1-NH)
3.84 (3 H, s, OCH3), 7.06 (2 H, d, J 8.8, Ar-mH), 7.78 (2 H, d, J 8.8, Ar-oH), 8.24 (1 H, s, 3-H), 8.39
(1 H, s, CH-Ar), 12.35 (1 H, br s, 4-NH), 13.60 (1 H, br, 1-NH)
8.09 (2 H, d, J 8.8, Ar-oH), 8.32 (2 H, d, J 8.8, Ar-mH), 8.35 (1 H, s, 3-H), 8.46 (1 H, s, CH-Ar),
12.78 (1 H, br s, 4-NH), 13.90 (1 H, br, 1-NH)
7.26–7.83 (10 H, m, Ph-H × 2), 8.19 (1 H, s, 6-CH-Ar), 8.24 (1 H, s, 3-H), 8.32 (1 H, s, 4-CH-Ar),
10.81 (1 H, br s, 6-NH), 11.78 (1 H, br, 4-NH), 13.07 (1 H, br, 1-NH)
7.27 (2 H, dd, JH,H 8.8, JH,F 9.1, 6-Ar-mH), 7.32 (2 H, dd, JH,H 8.8, JH,F 9.1, 4-Ar-mH), 7.74 (2 H, dd,
JH,H 8.8, JH,F 5.9, 6-Ar-oH), 7.87 (2 H, dd, JH,H 8.8, JH,F 5.8, 4-Ar-oH), 8.20 (1 H, s, 6-CH-Ar), 8.26
(1 H, s, 3-H), 8.33 (1 H, s, 4-CH-Ar), 10.93 (1 H, br s, 6-NH), 11.80 (1 H, br, 4-NH), 12.90 (1 H, br,
1-NH)
3da
3e
3f
3ga
8b
8c
8d
8f
3250, 3180, 3120 (NH)
3250, 3170, 3140 (NH)
7.45 (2 H, d, J 8.4, 6-Ar-mH), 7.51 (2 H, d, J 8.5, 4-Ar-mH), 7.74 (2 H, d, J 8.4, 6-Ar-oH), 7.80 (2 H,
d, J 8.5, 4-Ar-oH), 8.17 (1 H, s, 6-CH-Ar), 8.28 (1 H, s, 3-H), 8.32 (1 H, s, 4-CH-Ar), 10.95 (1 H, br,
6-NH), 11.80 (1 H, br, 4-NH), 13.00 (1 H, br, 1-NH)
3.75 (3 H, s, 6-OCH3), 3.81 (3 H, s, 4-OCH3), 6.95 (2 H, d, J 8.5, 6-Ar-mH), 7.06 (2 H, d, J 8.8, 4-Ar-
mH), 7.60 (2 H, d, J 8.5, 6-Ar-oH), 7.77 (2 H, d, J 8.8, 4-Ar-oH), 8.12 (1 H, s, 6-CH-Ar), 8.20 (1 H,
s, 3-H), 8.24 (1 H, s, 4-CH-Ar), 10.59 (1 H, br s, 6-NH), 11.66 (1 H, br, 4-NH), 12.83 (1 H, br, 1-NH)
a This compound was measured at 200 MHz.
triazolopyrimidines 12 prepared in this study were tested as
inhibitors of bovine milk xanthine oxidase in a similar assay
method14 as previously reported. The inhibition (%) and IC50
(µ) values of the compounds tested against bovine milk
xanthine oxidase are shown in Table 7. Thus the introduction of
both an aryl aldehyde hydrazone at the 4-position and an oxo
group at the 6-position of the 1H-pyrazolo[3,4-d]pyrimidine
ring led to markedly better activities in xanthine oxidase inhib-
ition, these compounds being two orders of magnitude more
active than allopurinol: IC50 values for 11g–k and 11m–q were
ca. 0.08–0.4 µ, whereas that for allopurinol was 24.3 µ. Most
of the pyrazolotriazolopyrimidines 12a–s showed potent
inhibitory activities, being two or three orders of magnitude
more active than allopurinol. Of these compounds, 12k (R =
4-ClC6H4) was the most active; it showed
a 760-fold
(IC50 = 0.032 µ) more potent bovine milk XO inhibitory
activity than that of allopurinol.
Conclusion
Thus, this simple and general methodology provided a facile
and convenient route to the preparation of 3-substituted 7H-
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42
35

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apparatus and were uncorrected. Microanalyses were measured
by a Yanaco CHN Corder MT-5 apparatus. Mass spectra were
recorded at 70 eV ionizing voltage with FAB ionization using a
VG-70SE spectrometer and 3-nitrobenzyl alcohol or glycerol as
a matrix. IR spectra were recorded using a JASCO FT/IR-200
spectrophotometer as Nujol mulls. 1H NMR spectra were
obtained using Hitachi FT-NMR R-1500 (60 MHz) and Varian
VXR 200 MHz spectrometers. In all cases, chemical shifts are in
ppm relative to SiMe4 as internal standard and J values are
given in Hz. All reagents were of commercial quality from
freshly opened containers and were used without further
purification. Organic solvents were dried by standard methods
and distilled before use. Reaction progress was monitored by
analytical thin layer chromatography (TLC) on pre-coated glass
plates (silica gel 70 FM Plate-Wako) using the following solvent
systems: (A) AcOEt–EtOH (4:1 v/v), (B) EtOH, (C) MeOH
and others cited in the Tables. The products were visualized
by UV light. Column chromatography was run on Daisogel
IR-60 (63/210 µ, Daiso Co.). The reaction temperatures are
indicated as the temperature of oil bath.
6-Chloro-4-hydrazino-1H-pyrazolo[3,4-d]pyrimidine 2
To a stirring solution of 2,4,6-trichloropyrimidine-5-carb-
aldehyde 122 (3.0 g, 14.2 mmol) in 2-methoxyethanol (20 cm3) at
0 ЊC was added a solution of anhydrous hydrazine (1.82 g, 56.8
mmol) diluted with 2-methoxyethanol (18 cm3) in limited
amounts for 30 min. After the reaction was complete, the pre-
cipitated crystals were collected by filtration and washed
with water and EtOH to afford the pyrazolopyrimidine 2
(2.06 g, 79%) as pale yellow powdery crystals, mp > 300 ЊC; Rf
(A) 0.64; νmax/cmϪ1 3350 and 3260 (NH2), 3160 and 3100
(NH) and δmax/cmϪ1 1660 (NH2); δH [60 MHz; (CD3)2SO] 4.20
(2 H, br, NH2), 8.50 (1 H, s, 3-H), 9.45 (1 H, br, 4-NH)
and 13.40 (1 H, br, 1-NH); m/z (FAB, 3-nitrobenzyl alcohol
matrix) 185 (MHϩ) and 187 (MHϩ ϩ 2). The product 2
was obtained as a single compound and was used for
the following reactions without further purification because
it was difficult to purify since it was insoluble in usual
solvents.
Scheme 2 Reagents and conditions: i, RCHO, DMF, r.t. or 40 ЊC, 10 h;
ii, 70% HNO3, DMF, 100 ЊC, 1–9 h; iii, DEAD, DMF, reflux, 5–9 h; iv,
RC(OEt)3, TFA, r.t., 1 h; v, RC(OEt)3 or RC(OMe)3, DMF, 100 ЊC, 1 h.
4-Alkylidenehydrazino- and 4-arylmethylidenehydrazino-6-
chloro-1H-pyrazolo[3,4-d]pyrimidines 3a–g; General procedure
pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H)-ones (12),
which were obtained by oxidative cyclisation of the corre-
sponding 4-aldehyde hydrazones of 1H-pyrazolo[3,4-d]pyrim-
idines (3 and 11) with 70% nitric acid as the key step, as a
new class of potential xanthine oxidase inhibitors. Their
inhibitory activities against bovine milk xanthine oxidase in
vitro were investigated, and some 4-arylmethylidenehydrazino-
1H-pyrazolo[3,4-d]pyrimidin-6(7H)-ones (11) exhibited from
several times to several hundred times more potent activities
than allopurinol. In addition, the tricyclic compounds, 3-aryl-
7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H)-ones
(12) showed potent inhibitory activities, being ca. three orders
of magnitude more active than allopurinol. They did not show
any appreciable inhibition against the proliferation of T-cell
acute lymphoblastic leukemia (CCRF-HSB-2) however.† Bio-
logical testing of the compounds in vivo is now ongoing and the
results will be reported later.
A mixture of the hydrazinopyrazolopyrimidine 2 (1.0 g, 5.42
mmol) and an appropriate alkyl aldehyde or aryl aldehyde (6.50
mmol) in DMF (50 cm3) was stirred at room temperature for 10
hours. After the reaction was complete, the solution was evap-
orated under reduced pressure and the residue was triturated
with EtOH or AcOEt to give crystals, which were collected by
filtration and recrystallized from an appropriate solvent to
afford the corresponding hydrazones 3a–g as shown in Tables 1
and 2.
1H-Pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione 4
(oxypurinol)
(1) A mixture of the hydrazino derivative 2 (0.20 g, 1.08 mmol)
with concentrated hydrochloric acid (10 cm3) was heated under
reflux for 1 hour. After the reaction was complete, the solution
was treated with activated charcoal and evaporated under
reduced pressure; the residue was recrystallized from water to
Experimental
General
afford oxypurinol [95 mg, 58%; mp > 300 ЊC; Rf (A) 0.48; νmax
/
cmϪ1 3180, 3150 and 3120 (NH) and 1720 (C᎐O)], which was
᎐
identical with an authentic sample.23
Mps were obtained on a Yanagimoto micro melting point
(2) The hydrazone 3b (0.50 g, 1.83 mmol) in 10% aqueous
HCl (50 cm3) was heated under reflux for 3 hours. After the
reaction was complete, the solution was treated with activated
charcoal and cooled to afford a deposit, which was collected by
filtration. The filtrate was evaporated under reduced pressure
and the residue was recrystallized from water to get the second
† We have found that some derivatives exhibited poor inhibitory activ-
ities against the proliferation of T-cell acute lymphoblastic leukemia
(CCRF-HSB-2): the IC50 for 11j, 11 µ; for 11o, 45 µ; for 12i, 25 µ;
for 12j, 14 µ; for 12k, 35 µ; for 12l, 23 µ; for 12n, 36 µ; for 12q, 34
µ; for 12r, 36 µ; for 12s, 35 µ and for arabinosylcytosine, 0.061 µ.
36
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42

View Article Online
Table 3 Preparative, physical and analytical data for the compounds 11b, e–r
Found (%) (Required)
Compound
(Formula)
Reaction
temp/ЊC
Yield
(%)
Recrystn. solventa
Mp/ЊC
>300
(Rf, solvent systemb)
C
H
N
m/z MHϩ
11b
C7H8N6O
11e
C13H20N6Oؒ1/5 H2O
11f
C16H24N6O
r.t.
r.t.
r.t.
r.t.
r.t.
40
89
80
63
85
74
76
95
95
83
93
87
85
80
85
88
EtOH–DMF
(0.47, A)
EtOH–DMF
(0.65, A)
EtOH–DMF
(0.47, B)
water–DMF
(0.60, A)
water–DMF
(0.64, A)
EtOH–DMF
(0.64, A)
EtOH–DMF
(0.65, A)
water–DMF
(0.63, A)
EtOH–DMF
(0.65, A)
DMF
(0.67, A)
water–DMF
(0.62, A)
water–DMF
(0.67, A)
water–DMF
(0.64, C)
EtOH–DMF
(0.64, A)
EtOH–DMF
(0.60, A)
43.4
(43.75)
55.8
(55.8)
60.3
4.2
(4.2)
7.2
(7.35)
7.4
43.65
(43.7)
30.3
193
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
277
(30.0)
26.3
317
(60.7)
55.8
(55.9)
52.2
(7.65)
4.1
(4.1)
3.6
(26.6)
32.8
11g
255
C12H10N6Oؒ1/5 H2O
(32.6)
30.5
11h
273
C12H9FN6Oؒ1/4 H2O
(52.1)
49.5
(49.9)
49.3
(3.5)
3.5
(3.1)
3.4
(30.4)
28.7
(29.1)
28.9
(28.75)
28.7
(28.75)
24.9
11i
289/291
289/291
289/291
333/335
269
C12H9ClN6O
11j
40
C12H9ClN6Oؒ1/5 H2O
(49.3)
49.2
(49.3)
43.3
(3.2)
3.4
(3.2)
3.1
11k
r.t.
r.t.
r.t.
r.t.
40
C12H9ClN6Oؒ1/5 H2O
11l
C12H9BrN6O
11m
(43.3)
57.4
(57.4)
54.4
(2.7)
4.5
(4.6)
4.3
(25.2)
31.1
C13H12N6Oؒ1/5 H2O
11n
C13H12N6O2ؒ1/5 H2O
11o
C13H10N6O3
11p
C13H10N6O3
11q
(30.9)
29.2
285
(54.2)
52.1
(52.35)
52.6
(4.3)
3.6
(3.4)
3.4
(29.2)
27.7
299
(28.2)
28.2
40
299
(52.35)
53.2
(53.3)
45.45
(45.4)
(3.4)
4.0
(3.7)
3.5
(28.2)
30.7
40
271
C12H10N6O2
11r
C12H9N7O3ؒH2O
(31.1)
30.8
r.t.
300
(3.5)
(30.9)
a All compounds 11 were obtained as colourless powdery crystals except for 11m, r (pale yellow). b Solvent systems: (A) AcOEt–EtOH (4:1 v/v), (B)
AcOEt–EtOH (9:1 v/v), (C) AcOEt–n-hexane–AcOH (8:4:1 v/v).
crop. The product was identical with oxypurinol (150 mg,
54%).
8.81 (1H, s, 3-H); m/z (FAB, 3-nitrobenzyl alcohol matrix)
181 (MHϩ).
(2) To a stirring solution of 80% aqueous hydrazine hydrate
(20 cm3, 320 mmol) was added 6-chloro-4-hydrazino-1H-
pyrazolo[3,4-d]pyrimidine 2 (2.0 g, 10.8 mmol) and the
mixture was heated at 80–90 ЊC for 4 hours. After the
same work-up as noted above, recrystallization of the crude
crystals from water gave the dihydrazino derivative 7 (1.40 g,
72%).
4-Hydrazino-1H-pyrazolo[3,4-d]pyrimidin-6(7H)-one 6
To a mixture of hydrazine monohydrate (5.0 g, 99.9 mmol) and
ethanol (5 cm3) was added 4,5-dihydro-4-thioxo-1H-pyrazolo-
[3,4-d]pyrimidin-6(7H)-one 523 (1.0 g, 5.95 mmol) and the mix-
ture was heated under reflux for 10 min. After the reaction was
complete, the precipitated crystals were collected by filtration
and washed with water and EtOH to afford the hydrazino
derivative 6 (0.70 g, 71%) as colourless powdery crystals,
mp > 300 ЊC; Rf (B) 0.28; νmax/cmϪ1 3360 and 3310 (NH2),
4,6-Bis(arylmethylidenehydrazino)-1H-pyrazolo[3,4-d]pyrim-
idines 8b–d, f. General procedure
3200, 3150 and 3100 (NH), 1710 (C᎐O) and δmax/cmϪ1 1670
A mixture of the dihydrazino derivative 7 (0.60 g, 3.33 mmol)
and an appropriate aldehyde (9.99 mmol) in DMF (20 cm3) was
stirred at room temperature for 10 hours. After the reaction was
complete, the solution was evaporated under reduced pressure
and the residue was triturated with EtOH to give crystals, which
were collected by filtration and recrystallized from a mixture
of EtOH and DMF to afford the corresponding bishydrazones
8b–d, f as shown in Tables 1 and 2.
᎐
(NH2); δH [60 MHz; CF3CO2D] 8.72 (1 H, s, 3-H); m/z (FAB,
3-nitrobenzyl alcohol matrix) 167 (MHϩ). The product 6 was
obtained as a single compound and was used for the following
reactions without further purification because it was difficult to
purify since it was insoluble in usual solvents.
4,6-Dihydrazino-1H-pyrazolo[3,4-d]pyrimidine 7
(1) To a stirring solution of 2,4,6-trichloropyrimidine-5-carb-
aldehyde 122 (3.0 g, 14.2 mmol) in 2-methoxyethanol (20 cm3)
at 0 ЊC was added anhydrous hydrazine (9.1 g, 283.9 mmol)
dropwise. Then, the stirred mixture was heated at 100 ЊC for
5 hours. After the reaction was complete, the precipitated
crystals were collected by filtration, washed with water and
EtOH and recrystallized from water to afford the dihydrazino
derivative 7 (1.94 g, 76%) as colourless powdery crystals,
mp > 300 ЊC (Found: C, 33.1; H, 4.55; N, 61.5. C5H8N8ؒ1/5
4-Carbamoylhydrazino-6-chloro-1H-pyrazolo[3,4-d]pyrimidine
9
A mixture of the hydrazinopyrazolopyrimidine 2 (0.5 g, 2.71
mmol) and urea (0.65 g, 10.8 mmol) in 2-ethoxyethanol (25
cm3) was heated under reflux for 5 hours. After the reaction was
complete, the solution was evaporated under reduced pressure
to afford a solid. The solid was collected by filtration, washed
with water and recrystallized from water to afford the carb-
amoylhydrazino derivative 9 (0.37 g, 60%) as colourless powdery
crystals, mp > 300 ЊC (Found: C, 31.1; H, 2.9; N, 43.0. C6H6-
ClN7Oؒ1/7 H2O requires C, 31.3; H, 2.75; N, 42.6%); Rf (B)
H2O requires C, 32.7; H, 4.6; N, 61.0%); Rf (C) 0.20; νmax
/
cmϪ1 3335, 3270 and 3230 (NH2), 3180, 3120 and 3110 (NH)
and δmax/cmϪ1 1650 and 1640 (NH2); δH [60 MHz; CF3CO2D]
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42
37

View Article Online
Table 4 IR and 1H NMR spectroscopic data for the compounds 11b, e–r
Compound
νmax(Nujol)/cmϪ1
δH [200 MHz; (CD3)2SO; Me4Si]
11b
11e
3175, 3130, 3070 (NH);
2.01 (3 H, d, J 5.4, CHCH3), 7.69 (1 H, q, J 5.4, CHCH3), 8.35 (1 H, s, 3-H), 10.40 (1 H, br, 4-NH),
10.80 (1 H, br s, 7-NH), 12.85 (1 H, br, 1-NH)
0.86 (3 H, t, J 6.4, CHCH2CH2[CH2]4CH3), 1.29 (8 H, br s, CHCH2CH2[CH2]4CH3), 1.44–1.66
(2 H, m, CHCH2CH2[CH2]4CH3), 2.24–2.42 (2 H, m, CHCH2CH2[CH2]4CH3), 8.05 (1 H, t, J 5.4,
CHCH2CH2[CH2]4CH3), 8.28 (1 H, s, 3-H), 10.22 (1 H, br, 4-NH), 10.82 (1 H, br s, 7-NH), 12.95
(1 H, br, 1-NH)
1700 (C᎐O)
᎐
3175, 3135, 3070 (NH);
1710 (C᎐O)
᎐
11f
3175, 3135, 3070 (NH);
1.28 (10 H, br s, CHCH2CH2[CH2]5CH2CH᎐CH2), 1.44–1.64 (2 H, m, CHCH2CH2[CH2]5-
CH2CH᎐CH2), 1.90–2.08 (2 H, m, CHCH2CH2[CH2]5CH2CH᎐CH2), 2.22–2.42 (2 H, m, CHCH2-
᎐ ᎐
᎐
1700 (C᎐O)
᎐
CH2[CH2]5CH2CH᎐CH2), 4.86–5.06 (2 H, m, CHCH2CH2[CH2]5CH2CH᎐CH2), 5.64–5.90 (1 H, m,
᎐
᎐
CHCH2CH2[CH2]5CH2CH᎐CH2), 7.60–7.72 (1 H, m, CHCH2CH2[CH2]5CH2CH᎐CH2), 8.28 (1H, s,
᎐
᎐
3-H), 10.28 (1 H, br, 4-NH), 10.82 (1 H, br s, 7-NH), 12.92 (1 H, br, 1-NH)
7.40–7.53 (3 H, m, Ph-m,pH), 7.80–7.90 (2 H, m, Ph-oH), 8.40 (1 H, s, 3-H), 8.46 (1 H, s, CH-Ar),
10.40 (1 H, br, 4-NH), 11.01 (1 H, br, 7-NH), 13.10 (1 H, br, 1-NH)
11g
11h
11ia
11j
3200, 3120, 3080 (NH);
1680 (C᎐O)
᎐
3180, 3140, 3070 (NH);
7.30 (2 H, dd, JH,H 8.8, JH,F 9.0, Ar-mH), 7.30 (2 H, dd, JH,H 8.8, JH,F 5.8, Ar-oH), 8.40 (1 H, s, 3-H),
8.45 (1H, s, CH-Ar), 10.40 (1 H, br, 4-NH), 11.00 (1 H, br, 7-NH), 13.09 (1 H, br, 1-NH)
7.33–7.60 (3 H, m, 3Ј-H, 4Ј-H and 5Ј-H), 8.12–8.28 (1 H, m, 6Ј-H), 8.48 (1H, s, 3-H), 8.72 (1 H, s,
CH-Ar), 10.35 (1 H, br s, 4-NH), 11.10 (1 H, br, 7-NH), 13.40 (1 H, br, 1-NH)
7.40–7.54 (2 H, m, 4Ј-H and 5Ј-H), 7.80–7.92 (2 H, m, 2Ј-H and 6Ј-H), 8.40 (1 H, s, 3-H), 8.43 (1 H,
s, CH-Ar), 10.50 (1 H, br s, 4-NH), 11.01 (1 H, br s, 7-H), 13.10 (1 H, br s, 1-NH)
7.48 (2 H, d, J 8.6, Ar-mH), 7.87 (2 H, d, J 8.6, Ar-oH), 8.37 (1 H, s, 3-H), 8.45 (1 H, s, CH-Ar),
10.50 (1 H, br, 4-NH), 11.00 (1 H, br, 7-NH), 13.14 (1 H, br, 1-NH)
1680 (C᎐O)
᎐
3200, 3120, 3080 (NH);
1700 (C᎐O)
᎐
3170, 3140, 3060 (NH);
1720 (C᎐O)
᎐
11k
11l
3170, 3140, 3070 (NH);
1680 (C᎐O)
᎐
3170, 3140, 3060 (NH);
7.66 (2 H, d, J 8.6, Ar-mH), 7.80 (2 H, d, J 8.6, Ar-oH), 8.38 (1H, s, 3-H), 9.44 (1 H, s, CH-Ar),
10.50 (1 H, br, 4-NH), 11.00 (1 H, br s, 7-NH), 13.11 (1 H, br, 1-NH)
1685 (C᎐O)
᎐
11m
11na
11o
3170, 3140, 3060 (NH);
2.36 (3 H, s, CH3), 7.28 (2 H, d, J 7.8, Ar-mH), 7.72 (2 H, d, J 7.8, Ar-oH), 8.35 (1 H, s, 3-H), 8.44
(1 H, s, CH-Ar), 10.45 (1 H, br, 4-NH), 11.00 (1 H, br, 7-NH), 13.10 (1 H, br, 1-NH)
3.80 (3 H, s, OCH3), 6.74 (2 H, d, J 8.5, Ar-mH), 7.58 (2 H, d, J 8.5, Ar-oH), 7.94 (1 H, s, 3-H), 7.99
(1 H, s, CH-Ar), 10.06 (1 H, br s, 4-NH), 10.92 (1 H, br s, 7-NH), 12.90 (1 H, br s, 1-NH)
6.09 (2 H, s, OCH2O), 7.00 (1 H, d, J 5Ј,6Ј 8.2, 5Ј-H), 7.27 (1 H, d, J 5Ј,6Ј 8.2, J2Ј,6Ј 1.6, 6Ј-H), 7.45 (1 H,
d, J2Ј,6Ј 1.6, 2Ј-H), 8.30 (1 H, s, 3-H), 8.45 (1 H, s, CH-Ar), 10.35 (1 H, br, 4-NH), 11.03 (1 H, br s,
7-NH), 13.04 (1 H, br, 1-NH)
8.00 (4 H, br, Ar-H), 8.50 (2 H, s, 3-H and CH-Ar), 11.25 (1 H, br, 4-NH), 11.95 (1 H, br, 7-NH),
12.35 (1 H, br, COOH), 13.55 (1 H, br, 1-NH)
6.84 (2 H, d, J 8.4, Ar-mH), 7.65 (2 H, d, J 8.4, Ar-oH), 8.26 (1 H, s, 3-H), 8.41 (1 H, s, CH-Ar),
9.67 (1 H, br s, OH), 10.86 (1 H, br s, 4-NH), 11.02 (1 H, br s, 7-NH), 12.90 (1 H, br, 1-NH)
8.10 (1 H, d, J 8.8, Ar-oH), 8.30 (1 H, d, J 8.8, Ar-mH), 8.50 (1 H, s, 3-H), 8.52 (1 H, s, CH-Ar),
10.65 (1 H, br, 4-NH), 11.11 (1 H, br, 7-NH), 13.21 (1 H, br, 1-NH)
1685 (C᎐O)
᎐
3180, 3140, 3080 (NH);
1680 (C᎐O)
᎐
3180, 3140, 3070 (NH);
1655 (C᎐O)
᎐
11pa
11q
11r
3165, 3120, 3050 (NH);
1650, 1630 (C᎐O)
᎐
3160, 3140, 3050 (NH);
1640 (C᎐O)
᎐
3165, 3120, 3080 (NH);
1690 (C᎐O)
᎐
a This compound was measured at 60 MHz.
0.70; νmax/cmϪ1 3410 and 3360 (NH2), 3200, 3100 and 3040
mmol) and an appropriate alkyl aldehyde or aryl aldehyde (9.03
mmol) in DMF (50 cm3) was stirred at room temperature or
40 ЊC for 10 hours. After the reaction was complete, the solu-
tion was evaporated under reduced pressure and the residue was
triturated with EtOH or AcOEt to give crystals, which were
collected by filtration and recrystallized from an appropriate
solvent to afford the corresponding hydrazones 11b, e–r as
shown in Tables 3 and 4.
(NH), 1675 (C᎐O) and δmax/cmϪ1 1675 (NH2); δH [200 MHz;
᎐
(CD3)2SO] 6.25 (2 H, br, NH2), 7.94 (1 H, s, 3-H), 8.66 and
10.00 (each 1 H, each br s, 2 × NH) and 13.67 (1 H, br s, 1-
NH); m/z (FAB, glycerol matrix) 228 (MHϩ) and 230
(MHϩ ϩ 2).
3-Amino-7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-
5(6H)-one 10
(1) The reaction mixture in the same reaction and under the
same conditions as in the above preparation for 9 was heated
under reflux for 36 hours. After the same work-up as noted
above, recrystallization of the crude crystals from water gave
the pyrazolotriazolopyrimidine 10 (0.15 g, 29%) as colourless
powdery crystals, mp > 300 ЊC (Found: C, 37.1; H, 3.1; N, 49.9.
C6H5N7Oؒ1/4 H2O requires C, 36.8; H, 2.8; N, 50.1%); Rf (A)
0.57; νmax/cmϪ1 3370 and 3260 (NH2), 3180 and 3100 (NH),
7H-Pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H)-one
12a and its 3-substituted derivatives 12b–s. General procedure
(1) Method A: A mixture of an appropriate 4-alkylidene-
hydrazino- or 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d]-
pyrimidin-6(7H)-one 11b, e–r (2.0 mmol) with 70% nitric acid
(0.22 cm3, 2.4 mmol) in DMF (30–50 cm3) was heated at
100 ЊC for 1–9 hours. After the reaction was complete, the
precipitated crystals were collected by filtration and combined
with further material obtained by concentration of the
filtrate under reduced pressure. The crystals were recrystallized
from an appropriate solvent to afford the corresponding
pyrazolotriazolopyrimidines 12b, e–p, r, s as shown in Tables 5
and 6.
(2) Method B: A mixture of an appropriate 4-alkylidene-
hydrazino- or 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d]-
pyrimidin-6(7H)-one 11e, g, h, k, m–o, q (2.0 mmol) with
DEAD (0.35 g, 2.0 mmol) in DMF (50 cm3) was heated under
reflux. After heating for several hours, further DEAD (2.0
mmol amounts; total 3–7 equiv.) was added to the heated solu-
tion at hourly intervals until the hydrazone 11 disappeared.
After the reaction was complete, the solution was evaporated
under reduced pressure to leave a solid, which was purified by
1720 (C᎐O) and δmax/cmϪ1 1685 (NH2); δH [200 MHz;
᎐
(CD3)2SO] 7.83 (1 H, s, 9-H), 7.94 (2 H, br s, NH2), 12.27 (1 H,
br s, 6-NH) and 13.10 (1 H, br s, 7-NH); m/z (FAB, glycerol
matrix) 192 (MHϩ).
(2) A mixture of the pyrazolopyrimidine 9 (0.2 g, 0.88 mmol)
and urea (0.16 g, 2.66 mmol) in 2-ethoxyethanol (10 cm3) was
heated under reflux for 10 hours. After the same work-up as
noted above, recrystallization of the crude crystals from water
gave the pyrazolotriazolopyrimidine 10 (30 mg, 18%).
4-Alkylidenehydrazino- and 4-arylmethylidenehydrazino-1H-
pyrazolo[3,4-d]pyrimidin-6(7H)-ones 11b, e–r. General
procedure
A mixture of the hydrazinopyrazolopyrimidine 6 (1.0 g, 6.02
38
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42

View Article Online
Table 5 Preparative, physical and analytical data for the compounds 12a–s
Reaction conditionsa
Found (%) (Required)
Compound
(Formula)
Yielda
(%)
Recrystn. solventb
m/z
Method
Temp/ЊC
Time/h
Mp/ЊC
>300
(Rf, solvent systemc)
C
H
N
MHϩ
12a
(C)
(D)
r.t.
100
1
1
66
65
DMF
(0.32, A)
39.9
(39.9)
2.7
(2.5)
46.5
(46.5)
177
191
C6H4N6Oؒ1/4 H2O
12b
(A)
(C)
(D)
100
r.t
100
2.5
1
1
64
83
55
>300
EtOH–DMF
(0.38, A)
43.5
(43.4)
3.5
(3.3)
43.7
(43.4)
C7H6N6Oؒ1/5 H2O
12c
(D)
100
1
54
>300
>300
>300
EtOH–DMF
(0.45, A)
46.8
(47.1)
4.2
(3.95)
41.4
(41.2)
205
233
275
C8H8N6O
12d
(D)
100
1
55
EtOH–DMF
(0.55, A)
51.1
(50.9)
5.5
(5.3)
36.0
(35.6)
C10H12N6Oؒ1/5 H2O
12e
(A)
(B)
(E)
100
reflux
100
3
7
1
77
60
75
EtOH–DMF
(0.64, A)
55.7
(56.0)
6.7
(6.7)
30.2
(30.1)
C13H18N6Oؒ1/4 H2O
12f
(A)
100
2.5
67
>300
>300
EtOH–DMF
(0.34, B)
60.3
(60.3)
7.35
(7.1)
26.3
(26.35)
315
C16H22N6Oؒ1/4 H2O
12g
(A)
(B)
(E)
100
reflux
100
1
5
1
91
60
85
water–DMF
(0.60, A)
55.9
(56.1)
3.45
(3.3)
32.75
(32.7)
253
271
C12H8N6O/1/4 H2O
12h
(A)
(B)
(E)
100
reflux
100
1
8
3
91
65
72
>300
EtOH–DMF
(0.62, A)
52.6
(52.5)
2.9
(2.75)
30.65
(30.6)
C12H7FN6Oؒ1/4 H2O
12i
(A)
100
2
70
>300
>300
>300
water–DMF
(0.63, A)
50.0
(50.3)
2.8
(2.5)
28.9
(29.3)
287/289
287/289
287/289
C12H7ClN6O
12j
(A)
100
2
76
water–DMF
(0.64, A)
49.9
(49.65)
2.8
(2.6)
29.2
(28.95)
C12H7ClN6Oؒ1/5 H2O
12k
(A)
(B)
(E)
100
reflux
100
2
8
1
90
67
71
water–DMF
(0.63, A)
49.7
(49.65)
2.8
(2.6)
29.2
(28.95)
C12H7ClN6Oؒ1/5 H2O
12l
(A)
100
5
74
>300
>300
EtOH–DMF
(0.64, A)
43.2
(43.1)
2.5
(2.2)
24.9
(25.1)
331/333
267
C12H7BrN6Oؒ1/5 H2O
12m
(A)
(B)
(E)
100
reflux
100
9
9
3
60
54
67
water–DMF
(0.67, A)
58.0
(57.9)
4.0
(3.9)
31.3
(31.1)
C13H10N6Oؒ1/5 H2O
12n
(A)
(B)
(E)
100
reflux
100
3
9
3
88
57
61
>300
water–DMF
(0.60, A)
54.5
(54.45)
3.9
(3.7)
29.3
(29.3)
283
C13H10N6O2ؒ1/4 H2O
12o
(A)
(B)
100
reflux
1
9
81
55
>300
>300
>300
>300
>300
EtOH–DMF
(0.62, A)
52.2
(51.9)
3.1
(2.85)
28.0
(27.9)
297
297
269
298
314
C13H8N6O3ؒ1/4 H2O
12p
(A)
100
1.5
69
EtOH–DMF
(0.64, C)
51.8
(51.7)
3.2
(2.9)
27.6
(27.8)
C13H8N6O3ؒ1/3 H2O
12q
(B)
reflux
9
50
EtOH–DMF
(0.51, A)
52.8
(52.85)
2.9
(3.1)
31.0
(30.8)
C12H8N6O2ؒ1/4 H2O
12r
(A)
(E)
100
100
5
5
60
60
EtOH–DMF
(0.60, A)
48.1
(47.8)
2.8
(2.5)
32.2
(32.5)
C12H7N7O3ؒ1/4 H2O
12s
(A)
100
1
52
DMF
(0.46, A)
45.4
(45.4)
2.6
(2.4)
30.6
(30.9)
C12H7N7O4ؒ1/4 H2O
a The reaction conditions and yields depend on the particular method. b All compounds 12 were obtained as colourless powdery crystals except for
12b, h, k, l, (colourless needles) and 12r, s (yellow powder). c Solvent systems: (A) AcOEt–EtOH (4:1 v/v), (B) AcOEt–EtOH (9:1 v/v), (C) AcOEt–
n-hexane–AcOH (8:4:1 v/v).
column chromatography on silica gel using AcOEt as eluent
and recrystallized from an appropriate solvent to give the
corresponding pyrazolotriazolopyrimidines 12e, g, h, k, m–o, q
as shown in Tables 5 and 6.
ine 6 (0.60 g, 3.6 mmol) with an appropriate triethyl orthoester
(18.0 mmol) in trifluoroacetic acid (9 cm3) was stirred at room
temperature for 1 hour. After the reaction was complete,
the precipitated crystals were collected by filtration and
recrystallized from an appropriate solvent to afford the corre-
(3) Method C: A mixture of the hydrazinopyrazolopyrimid-
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42
39

View Article Online
Table 6 IR and 1H NMR spectroscopic data for the compounds 12a–s
Compound
νmax(Nujol)/cmϪ1
δH [200 MHz; (CD3)2SO; Me4Si]
12a
12b
12c
12d
3120, 3030 (NH);
8.34 (1 H, s, 9-H), 8.62 (1 H, s, 3-H), 12.58 (1 H, br s, 6-NH), 13.60 (1 H, br s, 7-NH)
2.40 (3 H, s, CH3), 8.54 (1 H, s, 9-H), 12.46 (1 H, br s, 6-NH), 13.57 (1 H, br s, 7-NH)
1740 (C᎐O)
᎐
3110, 3050 (NH);
1700 (C᎐O)
᎐
3100, 3060 (NH);
1.29 (3 H, t, J 7.6, CH2CH3), 2.77 (2 H, q, J 7.6, CH2CH3), 8.57 (1 H, s, 9-H), 12.43 (1 H, br s, 6-NH),
13.55 (1 H, br s, 7-NH)
0.92 (3 H, t, J 7.6, CH2CH2CH2CH3), 1.36 (2 H, sextet, J 7.6, CH2CH2CH2CH3), 1.72 (2 H, quintet, J 7.6,
CH2CH2CH2CH3), 2.74 (2 H, t, J 7.6, CH2CH2CH2CH3), 8.57 (1 H, s, 9-H), 12.44 (1 H, br s, 6-NH),
13.55 (1 H, br s, 7-NH)
0.86 (3 H, t, J 6.5, CH2CH2[CH2]4CH3), 1.30 (8 H, br s, CH2CH2[CH2]4CH3), 1.50–1.80 (2 H, m,
CH2CH2[CH2]4CH3), 2.50–2.95 (2 H, m, CH2CH2[CH2]4CH3), 8.53 (1 H, s, 9-H), 12.41 (1 H, br s, 6-NH),
13.50 (1 H, br, 7-NH)
1700 (C᎐O)
᎐
3090, 3060 (NH)
1700 (C᎐O)
᎐
12ea
12f
3110, 3070 (NH);
1700 (C᎐O)
᎐
3150, 3070 (NH);
1.28 (10 H, br s, CH2CH2[CH2]5CH2CH᎐CH2), 1.62–1.80 (2 H, m, CH2CH2[CH2]5CH2CH᎐CH2), 1.92–
᎐
᎐
1710 (C᎐O)
2.06 (2 H, m, CH2CH2[CH2]5CH2CH᎐CH2), 2.72 (2 H, t, J 7.3, CH2CH2[CH2]5CH2CH᎐CH2), 4.87–5.04
᎐ ᎐
᎐
(2 H, m, CH2CH2[CH2]5CH2CH᎐CH2), 5.66–5.89 (1 H, m, CH2CH2[CH2]5CH2CH᎐CH2), 8.57 (1 H, s,
᎐
᎐
9-H), 12.43 (1 H, br s, 6-NH), 13.55 (1 H, br s, 7-NH)
7.40–7.70 (3 H, m, Ph-m,pH), 7.90–8.35 (2 H, m, Ph-oH), 8.68 (1 H, s, 9-H), 12.60 (1 H, br s, 6-NH),
13.60 (1 H, br, 7-NH)
7.37 (2 H, dd, JH,H 8.8, JH,F 9.1, Ar-mH), 8.22 (2 H, dd, JH,H 8.8, JH,F 5.9, Ar-oH), 8.66 (1 H, s, 9-H), 12.59
(1 H, br s, 6-NH), 13.65 (1 H, br, 7-NH)
7.44–7.78 (3 H, m, 3Ј-H, 4Ј-H and 5Ј-H), 7.95–8.09 (1 H, m, 6Ј-H), 8.69 (1 H, s, 9-H), 12.67 (1 H, br s,
6-NH), 13.58 (1 H, br, 7-NH)
7.54–7.63 (2 H, m, 4Ј-H and 5Ј-H), 7.95–8.25 (2 H, m, 2Ј-H and 6Ј-H), 8.67 (1 H, s, 9-H), 12.62 (1 H, br s,
6-NH), 13.60 (1 H, br s, 7-NH)
7.62 (2 H, d, J 8.6, Ar-mH), 8.17 (2 H, d, J 8.6, Ar-oH), 8.70 (1 H, s, 9-H), 12.62 (1 H, br s, 6-NH), 13.66
(1 H br s, 7-NH)
7.76 (2 H, d, J 8.6, Ar-mH), 8.10 (2 H, d, J 8.6, Ar-oH), 8.69 (1 H, s, 9-H), 12.62 (1 H, br s, 6-NH), 13.65
(1 H, br s, 7-NH)
12ga
12ha
12i
3150, 3050 (NH);
1720 (C᎐O)
᎐
3110, 3090 (NH);
1710 (C᎐O)
᎐
3150, 3070 (NH);
1710 (C᎐O)
᎐
12ja
12k
12l
3150, 3100 (NH);
1720 (C᎐O)
᎐
3160, 3100 (NH);
1700 (C᎐O)
᎐
3150, 3060 (NH);
1720 (C᎐O)
᎐
12m
12na
12o
3180, 3100 (NH);
2.39 (3 H, s, CH3), 7.35 (2 H, d, J 8.0, Ar-mH), 8.05 (2 H, d, J 8.0, Ar-oH), 8.66 (1 H, s, 9-H), 12.54
(1 H, br s, 6-NH), 13.61 (1 H, br s, 7-NH)
3.85 (3 H, s, OCH3), 7.10 (2 H, d, J 8.8, Ar-mH), 8.12 (2 H, d, J 8.8, Ar-oH), 8.64 (1 H, s, 9-H), 12.60 (1 H,
br s, 6-NH), 13.50 (1 H, br, 7-NH)
6.13 (2 H, s, OCH2O), 7.08 (1 H, d, J5Ј,6Ј 8.1, 5Ј-H), 7.58 (1 H, d, J2Ј,6Ј 1.6, 2Ј-H), 7.72 (1 H, d, J5Ј,6Ј 8.1, J2Ј,6Ј
1.6, 6Ј-H), 8.68 (1 H, s, 9-H), 12.55 (1 H, br s, 6-NH), 13.62 (1 H, br s, 7-NH)
8.10 (2 H, d, J 8.8, Ar-mH), 8.32 (2 H, d, J 8.8, Ar-oH), 8.69 (1 H, s, 9-H), 12.70 (1 H, br s, 6-NH), 13.50
(2 H, br, 7-NH and COOH)
6.90 (2 H, d, J 8.8, Ar-mH), 7.99 (2 H, d, J 8.8, Ar-oH), 8.66 (1 H, s, 9-H), 9.94 (1 H, s, OH), 12.51 (1 H,
br s, 6-NH), 13.60 (1 H, br, 7-NH)
1720 (C᎐O)
᎐
3160, 3100 (NH);
1730 (C᎐O)
᎐
3160, 3050 (NH);
1720 (C᎐O)
᎐
12pa
12q
3160, 3090 (NH);
1700, 1660 (C᎐O)
᎐
3160, 3050 (NH);
1718 (C᎐O)
᎐
12ra
12s
3170, 3100 (NH);
8.39 (4 H, br s, Ar-H), 8.69 (1 H, s, 9-H), 12.70 (1 H, br s, 6-NH), 13.60 (1 H, br 7-NH)
1720 (C᎐O)
᎐
3160, 3080 (NH);
7.31 (1 H, d, J5Ј,6Ј 8.8, 5Ј-H), 8.28 (1 H, dd, J5Ј,6Ј 8.8. J2Ј,6Ј 2.2, 6Ј-H), 8.60 (1 H, d, J2Ј,6Ј 2.2, 2Ј-H), 8.70 (1 H,
s, 9-H), 11.59 (1 H, s, OH), 12.62 (1 H, br s, 6-NH), 13.65 (1 H, br s, 7-NH)
1720 (C᎐O)
᎐
a This compound was measured at 60 MHz.
sponding pyrazolotriazolopyrimidines 12a, b as shown in Tables
5 and 6.
buffer (pH 7.4) for in vitro experiments. The final concentration
of DMSO in the reaction solution was 0.1%.
(4) Method D: A mixture of the hydrazinopyrazolopyrimid-
ine 6 (0.60 g, 3.6 mmol) with an appropriate triethyl or tri-
methyl orthoester (10.8 mmol) in DMF (30–40 cm3) was heated
at 100 ЊC for 1 hour. After the reaction was complete, the solu-
tion was evaporated under reduced pressure and the residue was
triturated with EtOH or AcOEt to give crystals, which were
collected by filtration and recrystallized from an appropriate
solvent to afford the corresponding pyrazolotriazolopyrimidines
12a–d as shown in Tables 5 and 6.
Bovine milk xanthine oxidase (XO) (10 mU mlϪ1) was incu-
bated with 100 µ xanthine in the presence and absence of
the test compound (0.003–10 µ) at 25 ЊC for 15 min. Uric acid
formation was determined by absorbance at 292 nm using a
Hitachi 228-A spectrophotometer, and the inhibition rate (%)
for the formation of uric acid and IC50 values of the test com-
pounds were determined. The inhibition rate (I) of the test
compound at each concentration was calculated by eqn. (1),
(5) Method E: A mixture of an appropriate 4-alkylidene-
hydrazino- or 4-arylmethylidenehydrazino-6-chloro-1H-pyra-
zolo[3,4-d]pyrimidine 3a–g (2.0 mmol) with 70% nitric acid (0.9
cm3, 10.0 mmol) in DMF (30–50 cm3) was heated at 100 ЊC for
1–5 hours. After the reaction was complete, the precipitated
crystals were collected by filtration and further crystals were
obtained by concentration of the filtrate under reduced pres-
sure. The combined crystals were recrystallized from an
appropriate solvent to afford the corresponding pyrazolotri-
azolopyrimidines 12e, g, h, k, m, n, r as shown in Tables 5 and 6.
I (%) = 100 Ϫ [(D Ϫ DB)/T] × 100
(1)
where T is the optical density of a solution of xanthine and XO,
D is the optical density of a solution of test compound,
xanthine and XO and DB is the optical density of a solution of
test compound and XO.
The inhibitory activity of allopurinol against bovine milk
xanthine oxidase was also examined as a positive control.
Each experiment was repeated at least twice at different con-
centrations (0.003–10 µ). The values of IC50, i.e. the µ
concentration of inhibitor necessary for 50% inhibition,
were determined from the dose–response curve from the
relation of the logarithmic concentration (µ) and the inhib-
ition (%).
Xanthine oxidase assay
All test compounds and allopurinol were dissolved in dimethyl
sulfoxide (DMSO) and diluted with 50 m sodium phosphate
40
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42

View Article Online
Table 7 Inhibitory activities of the compounds 2, 3, 4, 6, 8, 11 and 12 against bovine milk xanthine oxidase in comparison with allopurinol
Inhibition (%)
10
Compound
No.
3
1
0.3
0.1
0.03
IC50/µ
>10
>10
>10
>10
>10
>10
2
21.4
7.6
11.8
8.3
3b
3c
21.0
12.0
14.2
41.0
39.8
24.1
7.2
16.0
9.5
13.8
11.5
9.4
10.8
3d
3e
3f
4a
22.1
6
>10
>10
>10
>10
>10
8b
8c
16.3
12.4
17.2
58.6
71.7
53.3
75.6
68.5
68.9
67.6
66.1
45.1
68.9
74.5
68.4
68.1
62.8
57.3
69.2
71.5
57.0
57.6
69.8
66.8
67.8
69.5
68.5
68.9
72.3
70.1
72.2
71.6
70.2
69.0
67.6
69.8
69.1
38.2
8d
8f
11b
11e
11f
11g
11h
11i
11j
11k
11lb
11m
11n
11o
11p
11q
11r
12a
12b
12cc
12dd
12e
12f
12g
12h
12i
12j
12ke
12lf
12mg
12n
12o
12p
12qh
12r
12s
Alloi
45.0
69.2
36.5
73.0
69.9
69.6
66.4
65.6
44.2
66.5
73.3
65.9
65.2
66.2
52.1
67.5
68.8
55.2
55.1
69.1
65.0
65.3
68.8
68.6
69.6
70.6
66.4
70.7
71.0
69.6
67.1
66.0
68.2
69.7
19.9
34.8
60.6
22.4
66.3
63.6
68.5
63.4
62.9
42.1
63.8
70.0
61.6
60.3
60.3
46.9
65.2
65.1
52.0
53.0
67.9
63.4
64.2
67.7
67.1
68.8
70.3
67.4
70.0
70.4
68.6
65.7
65.4
68.7
69.0
9.9
15.8
44.6
13.0
49.8
47.0
64.4
54.2
53.8
49.6
52.8
59.2
47.0
46.8
48.2
39.5
56.6
52.5
42.2
46.1
62.8
59.6
59.8
64.6
63.5
66.7
68.3
63.9
67.0
66.9
66.6
60.6
62.9
64.4
67.7
4.6
6.9
25.8
9.7
6.2
13.3
4.6
4.670
0.450
7.894
0.305
0.373
0.077
0.223
0.224
29.9
29.7
53.8
38.7
39.5
40.1
37.0
41.0
29.0
29.3
40.3
28.7
41.8
37.3
28.8
34.9
54.4
48.4
49.7
56.7
54.8
61.3
62.9
60.4
60.9
58.6
61.0
50.2
57.0
57.5
64.0
3.2
36.3
20.7
22.7
23.9
18.6
>10
0.247
0.172
0.385
0.399
0.359
1.925
0.184
0.250
0.782
0.529
0.069
0.117
0.103
0.062
0.070
0.038
0.032
0.034
0.041
0.053
0.041
0.098
0.055
0.060
0.037
14.1
14.8
16.7
14.0
23.9
20.7
17.8
18.9
40.2
32.0
32.4
39.6
38.9
47.4
49.3
48.9
46.0
42.3
46.3
34.3
42.9
39.6
46.9
24.3
a 30 µ: 53.9%, 100 µ: 63.9%. b This value is inaccurate because of insolubility in DMSO. c 0.01 µ: 12.6%. d 0.01 µ: 9.6%. e 0.01 µ: 30.4%, 0.003
µ, 16.4%. f 0.01 µ: 37.3%. g 0.01 µ: 28.2%, 0.003 µ: 13.6%. h 0.01 µ: 29.0%. i Allo: allopurinol.
8 K. R. Hande, R. M. Noone and W. J. Stone, Am. J. Med., 1984, 76,
47.
Acknowledgements
We are grateful to the SC-NMR Laboratory of Okayama
University for 200 MHz proton NMR experiments. This work
was supported in part by a Grant-in-Aid for Scientific Research
(C) (No. 09680570) from the Japan Society for the Promotion
of Science.
9 D. E. Duggan, R. M. Noll, J. E. Baer, F. C. Novello and J. J.
Baldwin, J. Med. Chem., 1975, 18, 900.
10 R. L. Wortmann, A. S. Ridolfo, R. W. Lightfoot Jr. and I. H. Fox,
J. Rheumatol., 1985, 12, 540.
11 A. Bindoli, M. Valente and L. Cavallini, Pharmacol. Res. Commun.,
1985, 17, 831.
12 T. Spector, W. W. Hall, D. J. Porter, C. U. Lambe, D. J. Nelson and
T. A. Krenitsky, Biochem. Pharmacol., 1989, 38, 4315.
13 S. Sato, K. Tatsumi and T. Takahashi, Purine and Pyrimidine
Metabolism in Man VII, Part A: Chemotherapy, ATP Depletion and
Gout, eds. R. A. Harkness, G. B. Elion and N. Zöllner, Plenum
Press, New York, 1991, p. 135.
14 Y. Osada, M. Tsuchimoto, H. Fukushima, K. Takahashi, S. Kondo,
M. Hasegawa and K. Komoriya, Eur. J. Pharmacol., 1993, 241, 183.
15 G. Biagi, I. Giorgi, O. Livi, V. Scartoni, I. Tonetti and L. Costantino,
Farmaco, 1995, 50, 257.
16 T. Nagamatsu, Y. Watanabe, K. Endo and M. Imaizumi, PCT Int.
Appl. WO 96 26,208/1996 (Chem. Abstr., 1996, 125, 247848j).
17 T. Nagamatsu, T. Abiru, Y. Watanabe and K. Endo, Jpn. Kokai
Tokkyo Koho JP 07,242,694/1995 (Chem. Abstr., 1996, 124,
117896s).
References
1 Part 2, T. Nagamatsu, H. Yamasaki, T. Fujita, K. Endo and
H. Machida, J. Chem. Soc., Perkin Trans. 1, 1999, 3117.
2 R. W. Rundles, J. B. Wyngaarden, G. H. Hitchings, G. B. Elion and
H. R. Silberman, Trans. Assoc. Am. Physicians, 1963, 76, 126.
3 T. F. Yü and A. B. Gutman, Am. J. Med., 1964, 37, 885.
4 J. R. Klinenberg, S. E. Goldfinger and J. E. Seegmiller, Ann. Intern.
Med., 1965, 62, 639.
5 G. B. Elion, Ann. Rheumat. Dis., 1966, 25, 608.
6 G. B. Elion, S. Callahan, H. Nathan, S. Bieber, R. W. Rundles and
G. H. Hitchings, Biochem. Pharmacol., 1963, 12, 85.
7 J. L. Young, R. B. Boswell and A. S. Nies, Arch. Intern Med., 1974,
134, 553.
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42
41

View Article Online
18 T. Nagamatsu and T. Fujita, Chem. Commun., 1999, 1461.
19 G. A. Bhat and L. B. Townsend, J. Chem. Soc., Perkin Trans. 1, 1981,
2387.
87508b) Ger. Offen. 2 838 029, 1979 (Chem. Abstr., 1979, 91,
39492r).
22 F. Yoneda, Y. Sakuma, S. Mizumoto and R. Ito, J. Chem. Soc.,
Perkin Trans. 1, 1976, 1805.
23 R. K. Robins, J. Am. Chem. Soc., 1956, 78,784.
20 F. Gatta, M. Luciani and G. Palazzo, J. Heterocycl. Chem., 1989, 26,
613.
21 U. D. Treuner and H. Breuer, USP 4 053 474, 1977 (Chem. Abstr.,
1977, 88, 37826s), USP 4 124 764, 1978 (Chem. Abstr., 1978, 90,
Paper a907673e
42
J. Chem. Soc., Perkin Trans. 1, 2000, 33–42