Synthesis and biological evaluation of new imidazole, pyrimidine, and purine derivatives and analogs as inhibitors of xanthine oxidase

Article abstract of DOI:10.1021/jm950876u

Several derivatives of 4,5-disubstituted imidazole, 2,4,5-trisubstituted pyrimidine, 2-substituted purine, thiazolo[3,2-a]purine, [1,3]thiazino[3,2- a]purine, thiazolo[2,3-i]purine, [1,3]thiazino-[2,3-i]purine, and 6- substituted pyrazolo[3,4-d]pyrimidine were synthesized and tested as inhibitors of the xanthine oxidase enzyme. Of those, some 4-(acylamino)-5- carbamoylimidazoles and 2-thioalkyl-substituted purines exhibited very good inhibitory activity, being at least 500 times more effective than allopurinol. The ineffectiveness of 6-n-alkylpyrazolo[3,4-d]pyrimidines is imputable to the alkyl chain which could hinder the coordination with molybdenum according to the known mechanism for the binding of the inhibitor allopurinol; the effectiveness of imidazole derivatives, by contrast with the ineffectiveness of 4,5-diamino-2-(thioalkyl)-6-hydroxypyrimidines, indicates the relative importance of the five-membered ring in the interaction with the enzyme. Moreover, the marked effectiveness of the angularly-cyclized [1,3]thiazino[2,3-i]purinones, which constitute an interesting new class of inhibitors, together with the weak activity of linearly-cyclized derivatives, allowed us to characterize more precisely the lipophilic region of the enzyme facing the N(1)-C(2) positions of the substrate hypoxanthine.

Full text of DOI:10.1021/jm950876u

J . Med. Chem. 1996, 39, 2529-2535
2529
Syn th esis a n d Biologica l Eva lu a tion of New Im id a zole, P yr im id in e, a n d P u r in e
Der iva tives a n d An a logs a s In h ibitor s of Xa n th in e Oxid a se
Giuliana Biagi,† Alberto Costantini,‡ Luca Costantino,‡ Irene Giorgi,† Oreste Livi,† Piergiorgio Pecorari,‡
Marcella Rinaldi,‡ and Valerio Scartoni*,†
Dipartimento di Scienze Farmaceutiche, Universita` di Modena, via Campi 183-41100 Modena, Italy, and Dipartimento di
Scienze Farmaceutiche, Universita` di Pisa, via Bonanno 6-56100 Pisa, Italy
Received December 1, 1995X
Several derivatives of 4,5-disubstituted imidazole, 2,4,5-trisubstituted pyrimidine, 2-substituted
purine, thiazolo[3,2-a]purine, [1,3]thiazino[3,2-a]purine, thiazolo[2,3-i]purine, [1,3]thiazino-
[2,3-i]purine, and 6-substituted pyrazolo[3,4-d]pyrimidine were synthesized and tested as
inhibitors of the xanthine oxidase enzyme. Of those, some 4-(acylamino)-5-carbamoylimidazoles
and 2-thioalkyl-substituted purines exhibited very good inhibitory activity, being at least 500
times more effective than allopurinol. The ineffectiveness of 6-n-alkylpyrazolo[3,4-d]pyrimidines
is imputable to the alkyl chain which could hinder the coordination with molybdenum according
to the known mechanism for the binding of the inhibitor allopurinol; the effectiveness of
imidazole derivatives, by contrast with the ineffectiveness of 4,5-diamino-2-(thioalkyl)-6-
hydroxypyrimidines, indicates the relative importance of the five-membered ring in the
interaction with the enzyme. Moreover, the marked effectiveness of the angularly-cyclized
[1,3]thiazino[2,3-i]purinones, which constitute an interesting new class of inhibitors, together
with the weak activity of linearly-cyclized derivatives, allowed us to characterize more precisely
the lipophilic region of the enzyme facing the N(1)-C(2) positions of the substrate hypoxanthine.
In tr od u ction
general observations: The heterocyclic structures al-
ways consist of two fused rings with five and six nuclear
atoms; they contain from three to five nitrogen atoms;
an oxygen or sulfur atom is bound to the carbon
corresponding to C(6) of purines; one or two couples of
heteroatoms are often present in positions correspond-
ing to C(6)dO, N(7), and N(3) or N(9) of hypoxanthine;
these couples of heteroatoms could be able to coordinate
molybdenum; unsubstituted compounds may improve
their activity when a lipophilic group, e.g., a phenyl, is
introduced. There was a 100-fold reduction in the
activity of a few compounds with two lipophilic groups
with respect to the unsubstituted ones. The positions
which more frequently bore the lipophilic group may
correspond to the 2, 8, and 9 positions of hypoxanthine.
Some information about the size of the lipophilic
region in the active site of the enzyme can be deduced.
The type-II region should be larger than the type-I
region as only lin-naphthohypoxanthine can be oxidized
by the enzyme, while lin-naphthoxanthine is not me-
tabolized.8 Recently, in an attempt to define the role
of a lipophilic substituent, on the basis of these observa-
tions, we reported the positive effect of a linear alkyl
chain of suitable length on the binding with the en-
zyme.9 This effect was recorded on both 2- and 8-n-
alkylhypoxanthines. Since 2-n-pentylhypoxanthine was
a feeble inhibitor and 8-n-pentylhypoxanthine a sub-
strate, the observation of the negative interaction of 2,8-
di-n-pentylhypoxanthine prompted us to hypothesize
that there is only one narrow lipophilic pocket, capable
of accepting an alkyl chain, in the XO active site.
In our earlier studies, we observed that the n-alkyl
chain enhances its positive effect when a bridge group,
containing an oxygen atom, binds the chain to the
heterocyclic system. The effectiveness of some 4-amino-
5-carbamoyl-1H-1,2,3-triazoles, bearing a linear acyl
substituent on N(4), showed that the five-six hetero-
The enzyme xanthine oxidase (XO) catalyzes the
hydroxylation of hypoxanthine at position 2 and of
xanthine at position 8, in the presence of molecular
oxygen as electron acceptor, to yield uric acid and
superoxide anions.1,2 XO-derived superoxide anions
have been linked to postischemic tissue injury and
edema3 as well as to changes in vascular permeability.4
XO can also oxidize synthetic purine drugs, thus neu-
tralizing their biological activity. For example, anti-
leukemic 6-mercaptopurine is converted into inactive
6-thiouric acid.5 The inhibition of this enzyme is
therefore useful in the treatment of several diseases,
such as gout, and in ischemia-reperfusion processes, in
that it prevents damage caused by free radicals.
Some potent inhibitors of this enzyme, such as al-
lopurinol, have been known for a long time. Allopurinol
is transformed by the enzyme into alloxanthine, 6-hy-
droxy-1H-pyrazolo[3,4-d]pyrimidin-4-one, which forms
a very stable complex with the reduced enzyme;6
however, given its side effects and its inability to
prevent the enzyme-forming oxygen free radicals,7 the
search for new inhibitors is a matter of some urgency.
In 1985, a comprehensive study of XO inhibitors with
a purine-like heterocyclic ring system was reported.6 In
that work, a detailed mechanism of XO oxidation of
hypoxanthine and xanthine was proposed. Two types
of binding of the two substrates, called type-II binding
and type-I binding, respectively, were described; ac-
cordingly, the inhibitors were classified on the basis of
the type of binding they can perform. Examination of
the structures of the most active compounds led to some
* Author to whom correspondence should be addressed.
† Universita` di Pisa.
‡ Universita` di Modena.
X Abstract published in Advance ACS Abstracts, J une 1, 1996.
S0022-2623(95)00876-4 CCC: $12.00 © 1996 American Chemical Society

2530 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Ta ble 1. Synthesized and Tested Compounds
Biagi et al.
cyclic system is not essential to ensure binding with the
enzyme.10 Accordingly, we synthesized some imidazoles
1 and some suitable pyrimidines 2 to ascertain the
relative importance of the two rings in active purine-
like compounds. Further, in an attempt to define the
best chemical structure for the bridge between the
heterocyclic nucleus and the alkyl chain, we synthesized
2-thioxanthine derivatives 3 and some guanine analogs
7, bearing an alkyl chain on the exocyclic heteroatom.
to verify their potential inhibitory properties. The
structures of the studied compounds are reported in
Table 1.
Ch em istr y
All the synthesized compounds are listed in Table 1.
Compound 1a was obtained from 4-amino-5-imidazole-
carboxamide and hexanoic anhydride, while compounds
1b-d were synthesized from the same starting com-
pound, and the reagent was obtained in situ by reacting
the appropriate alcohol with oxalyl chloride.10,12
Compounds 2a -f and 3a -c were obtained by alky-
lation of the corresponding mercapto derivatives with
the appropriate alkyl bromide in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU);13 starting mate-
rial 9-hydro-2-mercapto-1H-purin-6-one was prepared
according to the literature.14 Compound 3d was syn-
thesized according to ref 15 and 3f by alkylation of
2-thioxo-1,7-dihydro-9H-purine-6,8-dione. This was ob-
tained by heating a mixture of 4,5-diamino-6-hydroxy-
We also synthesized several thiazolo[3,2-a]purine and
[1,3]thiazino[3,2-a]purine derivatives 4 and thiazolo[2,3-
i]purine and [1,3]thiazino[2,3-i]purine derivatives 5,
bearing an ethylenic and propylenic bridge between N(1)
and S(2) or N(1) and S(6), respectively. The activity of
these compounds 4 and 5, together with that of the
2-linearly-substituted derivatives 3,11 allowed us to
define the lipophilic wall of the active site facing these
positions. Finally, as it was known that the length of a
C(2) n-alkyl chain could enhance the inhibitory activity
of hypoxanthines and 8-azahypoxanthines,9,10 we pre-
pared some 6-n-alkylpyrazolo[3,4-d]pyrimidin-4-ones 8

Inhibitors of Xanthine Oxidase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2531
Ta ble 2. Physical and Biological Properties of Compounds 1-8
compd
formula
anal.
mp (°C)
yield (%)
MS (M+)
IC50
12.6
4.29
1.92
Ki
1a
1b
1c
1d
2a
2b
2c
2d
2e
2f
C10H16N4O2
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
142-145
198-200
213-215
218-220
175
183
195
187
189
55
15
10
10
75
75
75
43
48
54
84
85
89
90
67
59
83
73
22
224
254
268
282
199
213
227
242
256
270
224
238
252
2.56
1.06
1.04
0.26
C
C
C
10H14N4O4
11H16N4O4
12H18N4O4
C8H13N3OS
C9H15N3OS
10H17N3OS
C9H14N4O2S
10H16N4O2S
11H18N4O2S
C9H12N4OS
1.11
inactive
inactive
inactive
>100
>100
>100
3.88
C
C
C
193
3a
3b
3c
262-264
265-266
278-279
0.176
0.155
0.00 98
4.49
77.84
0.16
2.07
5.70
0.48
24.39
131.0
C
C
10H14N4OS
11H16N4OS
2.85
0.115
3d 15
3e
>300 (>30015 )
>300
C
C
C
C
C
12H10N4O4S
11H16N4O2S
11H8N4OS
12H10N4OS
17H20N4OS
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
3f
265-267
>300
>300
>300
3g
3h
244
3i
4a 16
4b16
4c16
4d 16
4e16
4f16
4g
>100
40.25
66.80
>100
C8H8N4O4S
14H12N4OS
C,H,N
C,H,N
>300
>300
25
88
inactive
4h
C
36.52
1.86
0.89
1.26
0.33
93
5a 17
5b17
5c17
5d 17
5e
C
14H12N4OS
C,H,N
>300
58
6a 17
7a
>100
C9H11N5O2
C
C
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
>300
23
30
50
15
23
28
63
36
25
43
221
235
249
207
221
235
>100
inactive
inactive
25.5
28.3
30.5
>100
>100
>100
>100
4.2
7b
10H13N5O2
11H15N5O2
>300
7c
>300
7d 24
7e
C9H13N5O
10H15N5O
275 (286-288,24a 268-27024b
)
17.7
25.7
32.1
C
278
7f24a
8a 25
8b
C11H17N5O
280 (287-28924a
)
>300 (>30025
)
C8H10N4O
C9H12N4O
C10H14N4O
C,H,N
C,H,N
C,H,N
287
266
255
8c
8d
allopurinol
7.021
2-mercaptopyrimidine, urea, and sodium acetate. Oxi-
dation with hydrogen peroxide of 3d and 4e gave 3e
and 4g, respectively. Melting 4,5-diamino-6-hydroxy-
2-mercaptopyrimidine and benzamidine afforded 8-phen-
yl-2-thioxanthine (3g), which was converted into 3h ,i
by alkylation.
Compounds 4h and 5e were prepared from 7,8-
diamino-3,4-dihydro-2H-pyrimido[2,1-b][1,3]thiazin-6-
one16 and 8,9-diamino-3,4-dihydro-2H,6H-pyrimido[6,1-
b][1,3]thiazin-6-one,17 respectively, by reaction with
benzamidine. Compounds 7a ,b were obtained by reac-
tion of guanine and the appropriate anhydride, whereas
7c was obtained by reaction with hexanoyl chloride.
Reduction by lithium aluminum hydride of these com-
pounds gave 7d -f. The remaining compounds, 4a -f,16
5a -d , and 6a ,17 were obtained according to reported
procedures. The preparation of compounds 8a -d fol-
lowed the described method using 3-(acylamino)-4-
pyrazolecarbonitriles as intermediates.18
ing 1H-1,2,3-triazoles.10 The n-alkyl chain in both series
appeared to play the same role, probably interacting
with the same lipophilic site.
It is worth noting that the derivative 1a (R′ ) CO-
n-alkyl) was less effective than oxalyl monoesters 1b-d
(R′ ) CO-COO-n-alkyl). The imidazole compounds
1b-d and the 1H-1,2,3-triazole derivatives previously
reported10 represent one of the first examples of efficient
monocyclic inhibitors of XO.19 As regards the pyrimi-
dine derivatives 2, the activity remained very weak, in
spite of the presence of the promising 2-alkylthio
substituent.11
The monosubstituted 2-(thioalkyl)purines 3a -c were
very active, in the order 3a < 3b < 3c, which follows
increasing in the length of the alkyl linear chain. The
same effect is found in compounds of general formula
1. Compound 3c displayed the lowest Ki value (Ki
0.0098 µM) of all the compounds synthesized and was
700 times more potent than allopurinol. We experi-
mented with the possible oxidation of 3c into 3f by
xanthine oxidase; the failure of these enzymatic reac-
tions allowed us to exclude type-I binding in the case of
compounds 1 and 3 and to possibly ensure type-II
binding. Moreover, the isosteric substitution of sulfur
(compounds 3a -c) with an NH group (compounds 7d -
Biologica l Resu lts a n d Discu ssion
The biological results are summarized in Table 2. As
expected, 4-(acylamino)-5-carbamoylimidazoles 1 exhib-
ited good affinity very similar to that of the correspond-

2532 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Biagi et al.
f) caused at least a 10-fold loss of activity. The
substituted purine 3d displayed an affinity (Ki 4.5 µM)
comparable to that of allopurinol (Ki 7 µM),20 whereas
the analog with a sulfone function, 3e, was much less
active (Ki 77.8 µM). A type-II binding might similarly
be hypothesized with compounds 3a -c; the reduction
of activity would be ascribable to the presence of the
sulfonic group which could render the nitrogen atom
incapable of coordinating molybdenum. This would
indicate that complexation with the enzyme was nega-
tively affected by the electron-withdrawing group or,
alternatively, that a steric hindrance by the sulfone
group was involved. Similarly, in compound 4e, which
was only weakly active, oxidation to sulfone 4g led to a
complete lack of activity.
In agreement with the hypothesis of a single lipophilic
pocket, the introduction of a phenyl ring in position 8
of compound 3c led to the less active compound 3i,
which is in agreement with the results obtained in a
previous paper.9 In fact, 2-n-pentylhypoxanthine and
8-n-pentylhypoxanthine interacted with the enzyme,
whereas 2,8-di-n-pentylhypoxanthine was inactive.
Another interesting class of compounds (4a -g, 5a -
e, and 6a ) was structurally characterized by inserting
a thioethylene or thiopropylene bridging group on
pyrimidine to obtain linearly- or angularly-cyclized
derivatives. Of these, compounds 5 were the most
active, those with three methylene units being more
active than those with two (5b,a and 5d ,c). As in the
case of compounds 3, introducing a substituent in
position 8 of 5d produced a less active compound (5e).
This confirms the presence of only one lipophilic pocket
in this region of the enzyme.
The low affinity of compounds 4, together with the
high affinity of compounds 5, might signify that the
location of the lipophilic wall portion of the enzyme
facing the methylene units is such that it cannot receive
the linearly-cyclized derivatives. Further, the reported
XO oxidation of lin-naphthohypoxanthine to nonoxidiz-
able lin-naphthoxanthine8 showed that these molecules
can interact with the enzyme only according to type-II
binding.5 These findings led us to conclude that prod-
ucts 5a -e and the less active 4, which lacks a methyl
group, could afford a similar binding. In the case of
compounds 5, steric hindrance may also contribute to
prevent type-I binding. The loss of potency in 6a , in
contrast with the activity exerted by 5b,d , is worthy of
note and may be chiefly ascribed to the presence of the
lH-1,2,3-triazole ring.
Finally, compounds 8 proved completely inactive.
This result may be in agreement with the proposed
mechanism of action of allopurinol, which underwent
oxidation in position 6 through a type-II binding to the
more active alloxanthine which, in turn, gave a type-
III binding.6 Therefore, it appeared that type-II bind-
ing, in the case of compounds 8, might be the only way
of binding with the enzyme. The poor inclination shown
by 2-n-alkylhypoxanthines to link the enzyme9 may
agree with the biological behavior of compounds 8,
which were unable to establish a type-III binding
because of the steric hindrance due to the presence of
the linear alkyl chain on C(6).
inhibitory activity. The presence of a cyclized or linear
alkyl chain of suitable length attached to C(2) improved
binding to the enzyme as in compounds 5 and 3,
respectively. The C(2) sulfur atom afforded the best
bridge between the heterocyclic system and the n-alkyl
chain as is evident from comparison between compounds
3 and 7. The C(8) oxygen atom slightly lowered the
inhibitory activity in purine derivatives.
Exp er im en ta l Section
Ch em istr y. 4,5-Diamino-6-hydroxy-2-mercaptopyrimidine,
4-amino-6-hydroxy-2-mercaptopyrimidine, 5(4)-aminoimida-
zole-4(5)-carboxamide hydrochloride, and 2-amino-6-hydroxy-
purine (guanine) were purchased from Sigma (Sigma-Aldrich
s.r.l.). 3-Amino-4-pyrazolecarbonitrile was purchased from
Aldrich (Sigma-Aldrich s.r.l.). Melting points were determined
on a Bu¨chi 510 apparatus and are uncorrected. IR spectra
were recorded in Nujol suspension on a Perkin-Elmer Model
681 spectrophotometer. The 1H-NMR spectra were recorded
in the solvent indicated on a Bru¨cker AMX 400 (400 MHz) or
a Bru¨cker AC 200 (200 MHz) spectrometer. Chemical shifts
are reported in ppm relative to tetramethylsilane as an
internal standard. Microanalyses (C, H, N) were carried out
on a Carlo Erba elemental analyser (Model 1106) and were
within (0.4% of the theoretical values. TLC was performed
on precoated silica gel F254 plates (Merck). Flash column
chromatographies were performed using Merk Kieselgel 60
(230-400 mesh).
4(5)-Car bam oyl-5(4)-(n -pen tan oylam in o)im idazole (1a).
A mixture of 4-amino-5-imidazolecarboxamide hydrochloride
(50 mg, 0.31 mmol), hexanoic anhydride (0.366 g, 1.71 mmol),
and triethylamine (0.039 g, 0.39 mmol) was heated at 80 °C
for 1 h. After cooling, aqueous methanol was added, and the
mixture was evaporated to obtain a viscous oil which was
treated with petroleum ether to precipitate triethylamine salt.
After filtration, further petroleum ether was added to the
filtrate to give 1a as a crystallized solid: 1H NMR [(CD3)2SO]
δ 7.76 (s, 1H, ArH), 6.76 and 6.50 (2br, 2H, NH), 2.97 (t, 2H,
OCH2), 1.67-1.20 (m, 6H, (CH2)3CH3), 0.90 (t, 3H, CH3).
4(5)-Ca r b a m oyl-5(4)-[[(n -a lk yloxy)oxa lyl]a m in o]im i-
d a zoles 1b-d . A solution of oxalyl chloride (0.065g, 0.52
mmol) and the appropriate alcohol (n-butanol for 1b, n-
pentanol for 1c, and n-hexanol for 1d ; 0.52 mmol) was stirred
at 0 °C for 1 h, after which a solution of 5(4)-aminoimidazole-
4(5)-carboxamide hydrochloride (100 mg, 0.62 mmol) and
triethylamine (0.25 g, 2.48 mmol) in anhydrous DMF (5 mL)
was added. The mixture was stirred for 8 h at 0 °C and then
concentrated in vacuo, diluted with water, and extracted with
CHCl3. Evaporation of the organic layer gave a solid which
1
was crystallized from absolute EtOH. 1b: H NMR [(CD3)2-
SO] δ 7.37 (m, 3H, ArH + 2NH), 4.20 (t, 2H, OCH2), 1.61-
1.20 (m, 4H, (CH2)2CH3), 0.92 (t, 3H, CH3). 1c: 1H NMR
[(CD3)2SO] δ 7.38 (s, 1H, ArH), 7.35 (br, 1H, NH), 4.20 (t, 2H,
OCH2), 1.72-1.19 (m, 6H, (CH2)3CH3), 0.87 (t, 3H, CH3). 1d :
1H NMR [(CD3)2SO] δ 7.41 (s, 1H, ArH), 7.35 (br, 1H, NH),
4.25 (t, 2H, OCH2), 1.72-1.19 (m, 8H, (CH2)4CH3), 0.88 (t, 3H,
CH3).
2-(Alk ylt h io)-4-a m in o-6-h yd r oxyp yr im id in es 2a -c.21
To a solution of 4-amino-6-hydroxy-2-mercaptopyrimidine (1.43
g, 0.01 mol) in the minimum amount of DMF were added the
appropriate alkyl bromide (n-butyl bromide for 2a , n-pentyl
bromide for 2b, and n-hexyl bromide for 2c; 0.01 mol) and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) (0.01 mol). The mixture
was stirred at room temperature for 2 h. The pure product
that precipitated was filtered and washed with petroleum
1
ether. 2a : H NMR [(CD3)2SO + CDCl3] δ 6.19 (br, 3H, D2O
exchangeable), 4.92 (s, 1H, ArH), 3.06 (t, 2H, SCH2), 1.44-
1.63 (m, 4H, (CH2)2CH3), 0.91 (t, 3H, CH3). 2b: 1H NMR
[(CD3)2SO + CDCl3] δ 6.23 (br, 3H, D2O exchangeable), 4.92
(s, 1H, ArH), 3.04 (t, 2H, SCH2), 1.24-1.54 (m, 6H, (CH2)3-
Con clu sion s
1
CH3), 0.88 (t, 3H, CH3). 2c: H NMR [(CD3)2SO + CDCl3] δ
The imidazole ring proved to be more important than
the pyrimidine ring with a view to ensuring good
6.24 (br, 3H, D2O exchangeable), 4.95 (s, 1H, ArH), 3.04 (t,
2H, SCH2), 1.29-1.73 (m, 8H, (CH2)4CH3), 0.88 (t, 3H, CH3).

Inhibitors of Xanthine Oxidase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2533
2-(Alk ylth io)-4-a m in o-5-(for m yla m in o)-6-h yd r oxyp yr i-
m id in es 2d-f. To 4-amino-5-(formylamino)-6-hydroxy-2-mer-
captopyrimidine, obtained from 4,5-diamino-6-hydroxy-2-
mercaptopyrimidine by reflux with formic acid22 (0.20 g, 1.07
mmol), and dissolved in the minimum amount of DMF, were
added the appropriate bromide (butyl bromide for 2d , propyl
bromide for 2e, and hexyl bromide for 2f; 1.07 mmol) and DBU
(1.07 mmol); the mixture was stirred for 2 days at room
temperature. After evaporation in vacuo, water was added
to precipitate the product which was then flash-chromato-
graphed on silica gel (eluent: CHCl3-MeOH, 92:8). 2d : 1H
NMR [(CD3)2SO] δ 11.82, 8.75, and 6.00 (3br, 3H, D2O
exchangeable), 8.04 (s, 1H, COH), 3.08 (t, 2H, SCH2), 1.70-
1.19 (m, 4H, (CH2)2CH3), 0.91 (t, 3H, CH3). 2e: 1H-NMR
[(CD3)2SO] δ 11.82, 8.74, and 6.13 (3br, 3H, D2O exchangeable),
8.03 (s, 1H, COH), 3.07 (t, 2H, SCH2), 1.85-1.27 (m, 6H,
(CH2)3CH3), 0.87 (t, 3H, CH3). 2f: 1H-NMR [(CD3)2SO] δ 11.82,
8.77, and 5.89 (3br, 3H, D2O exchangeable), 8.05 (s, 1H, COH),
3.08 (t, 2H, SCH2), 1.79-1.30 (m, 8H, (CH2)4CH3), 0.88 (t, 3H,
CH3).
9-Hyd r o-2-(a lk ylth io)-1H-p u r in -6-on es 3a -c. To a solu-
tion of 2-thio-9-hydro-1H-purin-6-one (1.68 g, 0.01 mol) in the
minimum amount of DMF were added 0.01 mol of the
appropriate alkyl bromide (n-butyl bromide for 3a , n-pentyl
bromide for 3b, and n-hexyl bromide for 3c) and 0.01 mol of
DBU. The mixture was stirred at room temperature for 2.5
h. The pure product was precipitated by adding a little water
to the mixture and then filtered and dried. 3a : 1H NMR
(CDCl3) δ 6.43 and 5.56 (2br, 2H, D2O exchangeable), 3.17 (t,
2H, SCH2), 1.30-1.68 (m, 4H, (CH2)2CH3), 0.92 (t, 3H, CH3).
3b: 1H NMR (CDCl3) δ 6.43 and 5.60 (2br, 2H, D2O exchange-
able), 3.17 (t, 2H, SCH2), 1.30-1.68 (m, 6H, (CH2)3CH3), 0.89
(t, 3H, CH3). 3c: 1H NMR (CDCl3) δ 8.97 (br, 1H, D2O
exchangeable), 7.78 (s, 1H, ArH), 3.21 (t, 2H, SCH2), 1.30-
1.73 (m, 8H, (CH2)4CH3), 0.88 (t, 3H, CH3).
3H, ArH, SH), 8.20 (m, 2H, ArH), 11.92 and 12.30 (2br, 2H,
D2O exchangeable).
9-Hyd r o-2-(m eth ylth io)-8-p h en yl-1H-p u r in -6-on e (3h ).
Methyl iodide (0.13 mL, 2.1 mmol) was added to a solution of
3g (0.50 g, 2.05 mmol) in DMF (10 mL), and the reaction
mixture was mantained at room temperature for 2 days and
then poured on ice. The solid that precipitated was filtered,
washed with cyclohexane, and then crystallized from acetone/
petroleum ether: 1H NMR [(CD3)2SO] δ 2.66 (s, 3H, CH3),
7.54-7.62 (m, 3H, ArH), 8.19-8.21 (m, 2H, ArH), 12.58 and
13.70 (2br, 2H, D2O exchangeable).
9-Hydr o-2-(h exylth io)-8-ph en yl-1H-pu r in -6-on e (3i). To
a solution of 3g (0.45 g, 1.84 mmol) in a mixture of CHCl3-
water (1:1, v/v, 20 mL) were added 1 N NaOH (1.84 mL),
tetrabutylammonium bromide (200 mg), and n-iodohexane
(0.27 mL, 1.84 mmol). The resulting reaction mixture was
stirred vigorously at room temperature for 3 days. The two
phases were then separated, and from the organic layer,
treated with 1 N HCl, compound 3i separated out as a
precipitate. It was washed with water and proved to be
practically pure: 1H NMR [(CD3)2SO] δ 0.97 (t, 3H, CH3),
1.39-1.41 (m, 4H, 2CH2), 1.51 (m, 2H, CH2), 1.79 (m, 2H, CH2),
3.28 (t, 2H, CH2), 7.56-7.63 (m, 3H, ArH), 8.21-8.23 (m, 2H,
ArH), 12.54 (br, 1H, NH).
5,5-Dioxo-1,3,5,6,7,8-h exah ydr o-5-th io-1,3,4,8a-tetr aaza-
cyclop en ta [b]n a p h th a len e-2,9-d ion e (4g). Hydrogen per-
oxide (35%, 2.50 mL) was added to a suspension of 2H,10H-
7,8-dihydro-[1,3]thiazin[3,2-a]purine-2,10-dione (4e)16 (0.50 g,
2.2 mmol) in acetic acid. The reaction proceeded as described
for 3e. The insoluble compound proved to be chromatographi-
cally-pure 4g: 1H NMR [(CD3)2SO] δ 2.59 (m, 2H, CH2), 3.89
(t, 2H, CH2), 4.20 (t, 2H, CH2), 11.51 and 11.91 (2br, 2H, D2O
exchangeable).
2-P h en yl-7,8-d ih yd r o-3H,6H-5-th ia -1,3,4,8a -tetr a a za cy-
clop en ta [b]n a p h th a len -9-on e (4h ). A mixture of 7,8-di-
amino-3,4-dihydro-2H-pyrimido[2,1-b][1,3]thiazin-6-one17 (0.50
g, 2.52 mmol), benzamidine hydrochloride (2.50 g, 16.0 mmol),
and anhydrous sodium acetate (0.75 g, 9.1 mmol) was heated
at 220 °C for 45 min. The product obtained was washed with
water and proved to be chromatographically-pure: 1H NMR
[(CD3)2SO] δ 2.28 (m, 2H, CH2), 3.33 (m, 2H, SCH2), 4.21 (m,
2H, NCH2), 7.59-7.63 (m, 3H, ArH), 8.14-8.27 (m, 2H, ArH),
13.48 (br, 1H, NH).
7,9-Dih yd r o-2-t h ioxo-1H-p u r in e-6,8-d ion e. The mix-
ture, consisting of 4,5-diamino-6-hydroxy-2-mercaptopyrimi-
dine (4.00 g, 25.3 mmol), urea (6.0 g, 0.10 mol), and anhydrous
sodium acetate (4.76 g, 58.02 mmol), was heated at 220 °C for
45 min. The product was washed with water and treated with
2% NaOH. The solution was saturated with CO2 to yield the
title compound: yield 3.80 g (78%); mp >300 °C. Anal.
(C5H4N4O2S) C, H, N.
7,9-Dih yd r o-2-[(p h en ylm eth yl)th io]-1H-p u r in e-6,8-d i-
on e (3d ). To a suspension of 7,9-dihydro-2-thioxo-1H-purine-
6,8-dione (1.00 g, 5.4 mmol) in DMF (10 mL) were added K2CO3
(0.10 g) and benzyl chloride (0.62 mL, 5.4 mmol, dropwise).
The mixture was stirred at room temperature for 3 days. The
insoluble was filtered, and the crude proved to be chromato-
graphically-pure 3d : 1H NMR [(CD3)2SO] δ 4.48 (s, 2H, SCH2),
7.25-7.39 (m, 3H, ArH), 7.45-7.49 (m, 2H, ArH), 10.95, 11.50,
and 12.75 (3br, 3H, D2O exchangeable).
7,9-Dih yd r o-2-[(p h en ylm eth yl)su lfon yl]-1H-p u r in e-6,8-
d ion e (3e). Hydrogen peroxide (35%, 4.00 mL) was added to
a suspension of 3d (0.50 g, 1.8 mmol) in glacial acetic acid (5.00
mL). The reaction mixture was heated under reflux for 40
min. The insoluble product was filtered off to yield 3e: 1H
NMR [(CD3)2SO] δ 4.95 (s, 2H, SCH2), 7.20-7.40 (m, 5H, ArH),
11.50 and 12.00 (2d, 2H, D2O exchangeable), 13.50 (br, 1H,
D2O exchangeable).
7,9-Dih yd r o-2-(h exylth io)-1H-p u r in e-6,8-d ion e (3f). To
a suspension of 7,9-dihydro-2-thioxo-1H-purine-6,8-dione (1.00
g, 5.4 mmol) in DMF (10 mL) were added K2CO3 (0.10 g) and
iodohexane (0.80 mL, 5.4 mmol, dropwise). The reaction
mixture was mantained at room temperature for 3 days, and
the crude compound was then filtered off: 1H NMR [(CD3)2-
SO] δ 0.96 (m, 3H, CH3), 1.33 (m, 6H, CH2), 1.60 (m, 2H, CH2),
3.22 (m, 2H, SCH2), 10.82, 11.39, and 12.60 (3br, 3H, D2O
exchangeable).
9-Hyd r o-2-m er ca p to-8-p h en yl-1H-p u r in -6-on e (3g). A
mixture of 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (1.00
g, 6.32 mmol), benzamidine hydrochloride (4.00 g, 25.54 mmol),
and finely powdered anhydrous sodium acetate (1.20 g, 14.63
mmol) was heated at 200-210 °C for 1 h. The solid thus
obtained was disaggregated with water to yield 3g in a
practically-pure form: 1H NMR [(CD3)2SO] δ 7.53-7.90 (m,
2-P h en yl-7,8-d ih yd r o-3H,6H-9-th ia tetr a a za cyclop en ta -
[a ]n a p h th a len -5-on e (5e). 8,9-Diamino-3,4-dihydro-2H,6H-
pyrimido[6,1-b][1,3]thiazin-6-one16 (0.66 g, 3.3 mmol), benza-
midine hydrochloride (2.08 g, 13.3 mmol), and anhydrous
sodium acetate (0.62 g, 7.6 mmol) were heated at 220 °C for
45 min. The solid thus obtained was washed with water and
then with acetone and purified by flash-chromatography
(CHCl3-MeOH-cyclohexane, 4.5:0.5:5): 1H NMR [(CD3)2SO]
δ 2.35 (m, 2H, CH2), 3.34 (m, 2H, SCH2), 4.11 (m, 2H, NCH2),
7.58-7.63 (m, 3H, ArH), 8.15-8.52 (2H, m, ArH), 13.07 (br,
1H, NH).
2-(Bu ta n oyla m in o)-1H-p u r in -6-on e (7a ). A suspension
of 2-amino-6-hydroxypurine (guanine) (0.5 g, 3.31 mmol) in
butyric anhydride (2.7 mL, 16.5 mmol) was heated for 5 h at
120 °C. After cooling, the mixture was diluted with petroleum
ether and filtered and the solid washed with hot toluene.
Compound 7a precipitated from the collected filtering and
washing liquids and was recrystallized from toluene:
1H NMR
[(CD3)2SO] δ 13.15, 12.09, and 11.42 (3br, 3H, D2O exchange-
able), 7.95 (s, 1H, ArH), 2.46 (t, 2H, COCH2), 1.63 (m, 2H, CH2-
CH3), 0.91 (t, 3H, CH3).
2-(P en ta n oyla m in o)-1H-p u r in -6-on e (7b). A suspension
of guanine (0.5 g, 3.31 mmol) in pentanoic anhydride (3.26 mL,
16.55 mmol) was heated for 5 h at 160 °C. The mixture was
treated like 7a : 1H NMR [(CD3)2SO] δ 13.57, 12.01, and 11.45
(3br, 3H, D2O exchangeable), 7.93 (s, 1H, ArH), 2.44 (t, 2H,
COCH2), 1.70-1.17 (m, 4H, (CH2)2CH3), 0.91 (t, 3H, CH3).
2-(Hexa n oyla m in o)-1H-p u r in -6-on e (7c).23 A suspension
of guanine (0.2 g, 1.32 mmol), anhydrous pyridine (6.6 mL),
and hexanoyl chloride (0.55 mL, 3.96 mmol) was heated to 130
°C for 2 h. The mixture was then evaporated in vacuo and
the residue diluted with 50 mL of ethanol. This mixture was
refluxed for 1 h and then filtered to yield a solid which, on

2534 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Biagi et al.
washing with hot ethanol, proved to be pure: 1H NMR [(CD3)2-
SO] δ 13.78, 12.31, and 11.45 (3br, 3H, D2O exchangeable),
7.95 (s, 1H, ArH), 2.44 (t, 2H, COCH2), 1.68-1.17 (m, 6H,
(CH2)3CH3), 0.92 (t, 3H, CH3).
Refer en ces
(1) Parks, D. A.; Granger, D. N. Xanthine Oxidase: Biochemistry,
Distribution and Physiology. Acta Physiol. Scand. Suppl. 1986,
548, 87-99.
2-(Alk yla m in o)-1H-p u r in -6-on es 7d -f.24 LiAlH4 (0.5 g)
was added to 1.5 mmol of 7a (or 7b or 7c) in 100 mL of
anhydrous THF, and the mixture was heated to 80 °C for 48
h. After cooling to 0 °C, the excess of LiAlH4 was destroyed
by MeOH, and the mixture was evaporated in vacuo. The solid
residue was powdered, and 5% NH3 (75 mL) was added. This
mixture was stirred continuously at 60 °C for 1 h and then
filtered and the filtrate concentrated to 30 mL. HCl (2 N) was
then added to bring the mixture to pH ) 8. The solid which
slowly precipitated was flash-chromatographed (eluent: CHCl3-
MeOH, 92:8) to obtain the pure product. 7d : 1H NMR [(CD3)2-
SO + CDCl3] δ 7.75, 6.40, and 6.03 (3br, 3H, D2O exchange-
able), 7.50 (s, 1H, ArH), 3.25 (m, 2H, NHCH2), 1.50-1.21 (m,
(2) McCord, J . M.; Fridovich, I. Reduction of Cytochrome C by Milk
Xanthine Oxidase. J . Biol. Chem. 1968, 243, 5753-5760.
(3) Hearse, D. J .; Manning, A. S.; Downey, J . M.; Yellon, D. M.
Xanthine Oxidase: A Critical Mediator of Myocardial Injiury
during Ischemia and reperfusion? Acta Physiol. Scand. Suppl.
1986, 548, 65-78.
(4) Chan, P. H.; Schimidley, J . W.; Fishman, R. A.; Longar, S. M.
Brain Injury, Edema and Vascular Permeability Changes In-
duced by Oxigen-derived Free Radicals. Neurology 1984, 34,
315-320.
(5) Naeff, H. S. D.; Franssen, M. C. R.; van der Plas, H. C.
Quantitative Structure-Activity Relationship (QSAR) Studies of
the Inhibition of Xanthine Oxidase by Heterocyclic Compounds.
Recl. Trav. Pays-Bas 1991, 110, 139-150.
(6) Robins, R. K.; Revankar, G. R.; O’Brien, D. E.; Springer, R. H.;
Novinson, T.; Albert, A.; Senga, K.; Miller, J . P.; Streeter, D. G.
Purine Analog Inhibitors of Xanthine Oxidase. Structure Activity
Relationships and Proposed Binding of the Molybdenum Cofac-
tor. J . Heterocycl. Chem. 1985, 22, 601-634.
1
4H, (CH2)2CH3), 0.90 (t, 3H, CH3). 7e: H NMR [(CD3)2SO] δ
11.32, 10.22, and 6.25 (3br, 3H, D2O exchangeable), 7.52 (s,
1H, ArH), 3.27 (m, 2H, NHCH2), 1.50-1.23 (m, 6H, (CH2)3-
CH3), 0.88 (t, 3H, CH3). 7f: 1H NMR [(CD3)2SO] δ 11.41, 10.30,
and 6.25 (3br, 3H, D2O exchangeable), 7.55 (s, 1H, ArH), 3.26
(m, 2H, NHCH2), 1.50-1.20 (m, 8H, (CH2)4CH3), 0.89 (t, 3H,
CH3).
(7) Haberland, A.; Luther, H.; Schinke, I. Does Allopurinol Prevent
Superoxide Radical Production by Xanthine Oxidase (XOD)?
Agents Actions 1991, 32, 96-97.
(8) Leonard, N. J .; Sprecker, M. A.; Morrice, A. G. Defined Dimen-
sional Changes in Enzyme Substrates and Cofactors. Synthesis
of lin-Benzoadenosines and Enzymatic Evaluation of Derivative
of the Benzopurines. J . Am. Chem. Soc. 1976, 98, 3987-3994.
(9) Biagi, G.; Giorgi, I.; Livi, O.; Scartoni, V.; Tonetti, I.; Lucacchini,
A. Xanthine Oxidase (X.O.): Relative Configuration of Com-
plexes Formed by the Enzyme 2- or 8-n-Alkylhypoxanthines and
2-n-Alkyl-8-azahypoxanthines. XII. Farmaco 1993, 48, 357-374.
(10) Biagi, G.; Giorgi, I.; Livi, O.; Scartoni, V.; Tonetti, I.; Costantino,
L. Xanthine Oxidase (X.O.): 4(5)-Aminosubstituted-5(4)-carboxy-
amido-1H-1,2,3-triazoles: a New Class of Monocyclic Triazole
Inhibitors. Farmaco 1995, 50, 257-264.
(11) (a) Pecorari, P.; Costantino, L.; Rinaldi, M.; Costantini, A. New
Inhibitors of Xanthine Oxidase. Abstracts of Papers, 13th
International Symposium on Medicinal Chemistry, Paris, Sep-
tember 19-23, 1994; p 45. (b) Biagi, G.; Giorgi, I.; Livi, O.;
Scartoni, V. Recenti Acquisizioni su Analoghi Purinici come
Inibitori della Xantina Ossidasi (XO). Atti, Societa` Chimica
Italiana, Sezione Toscana, Riunione Scientifica Annuale, Pisa,
Italy, December 16, 1994; p 3.
3-(Acyla m in o)-4-p yr a zoleca r b on it r iles. These com-
pounds (acyl ) propanoyl, butanoyl, pentanoyl, hexanoyl) were
prepared by the method of Cheng and Robins.25 Acyl )
propanoyl, mp 220-221 °C; butanoyl, mp 195-196 °C; pen-
tanoyl, mp 180-182 °C; hexanoyl, mp 181-183 °C.
4-Hyd r oxy-6-a lk ylp yr a zolo[3,4-d ]p yr im id in es 8a ,25b-
d .26 The appropriate 3-(acylamino)-4-pyrazolecarbonitrile was
treated according to a known procedure25 to obtain products
8. 8a : crystallization solvent EtOH; 1H NMR [(CD3)2SO] δ
7.97 (s, 1H, ArH), 2.63 (q, 2H, CH2), 1.21 (t, 3H, CH3). 8b:
crystallization solvent EtOH; 1H NMR [(CD3)2SO] δ 8.03 (s,
1H, ArH), 2.67 (t, 2H, R-CΗ2), 1.79 (m, 2H, CH2CH3), 0.98 (t,
3H, CH3). 8c: crystallization solvent MeOH; 1H NMR [(CD3)2-
SO] δ 7.90 (s, 1H, ArH), 2.49 (t, 2H, R-CΗ2), 1.61 (m, 2Η,
â-CH2), 1.25 (m, 2H, CH2CH3), 0.85 (t, 3H, CH3). 8d : crystal-
lization solvent MeOH; 1H NMR [(CD3)2SO] δ 7.89 (s, 1H,
ArH), 2.47 (t, 2H, R-CΗ2), 1.62 (m, 2Η, â-CH2), 1.23 (m, 4H,
CH2CH2CH3), 0.81 (t, 3H, CH3).
(12) Nimitz, J . S.; Mosher, H. S. A New Synthesis of R-Keto Esters
and Acids. J . Org. Chem. 1981, 46, 211-213.
(13) (a) Ono, N.; Miyake, H.; Saito, T.; Kaji, A. A Convenient
Synthesis of Sulfides, Formaldehyde Dithioacetals, and Chlo-
romethylsulfides. Synthesis 1980, 952-953. (b) Ferreira, J .;
Tercio, B.; Comasseto, J . V.; Braga Antonio, L. An Improved
Method of Synthesis of Aryl Alkyl Sulfides. Synth. Commun.
1982, 12, 595-600. (c) Ando, W.; Furuhata, T.; Tsumaki, H.;
Sekiguchi, A. Useful Application of Thiosilanes for Preparation
of Symmetrical and Unsymmetrical Dialkyl Sulfides. Synth.
Commun. 1982, 12, 627-631.
Xa n th in e Oxid a se In h ibitor y Assa y. Xanthine was
puchased from Fluka (Sigma-Aldrich s.r.l.) and XO (from
buttermilk, 1.36 U/mg) from Boehringer (Boehringer Man-
nheim Italia s.p.a.).
XO activity was assayed spectrophotometrically in air-
saturated phosphate buffer, pH ) 7.6, I ) 0.1 at 25 ( 0.2 °C,
using a Perkin-Elmer UV/vis λ16 or Beckman DU 50 spectro-
photometer with a thermostated cuvette holder. The increase
in uric acid (product of enzymatic reaction) concentration was
evaluated at 295 nm with xanthine as substrate (8 µM) and
XO sufficient to obtain an average reaction rate for the control
reaction of 0.100 ( 0.005 absorbance units/min. The inhibitors
in question were dissolved in DMSO. DMSO concentration
in all the assays was kept at 3% (v/v), which has no effect on
XO activity. IC50 values (the concentration required to produce
50% inhibition of the enzyme-catalyzed reaction) were deter-
mined from least-squares analysis of the linear portion of log
dose-inhibition curves. Each curve was generated using at
least three concentrations of inhibitor producing an inhibition
of between 20% and 80%, with three replicates at each
concentration.
(14) Robins, R. K.; Dille, K. J .; Willits, C. H.; Christensen, B. E.
Purines. II. The Synthesis of Certain Purines and the Cyclization
of Several Substituted 4,5-Diaminopyrimidines. J . Am. Chem.
Soc. 1952, 25, 263-266.
(15) Baker, B. R.; Hendrickson, J . L. Irreversible Enzyme Inhibitors.
XCII: Inhibition of Xanthine Oxidase by Some Purines and
Pyrimidines. J . Pharm. Sci. 1967, 56, 955-959.
(16) Rinaldi, M.; Pecorari, P.; Costantino, L.; Provvisionato, A.;
Cermelli, C.; Portolani, M. Synthesis and Biological Activity of
Pyrimido[2,1-b][1,3]thiazine, [1,3]Thiazino[3,2-a]purine and [1,2,3]-
Triazolo[4,5-d][1,3]thiazino[3,2-a]pyrimidine Derivatives and
Thiazole Analogues. Farmaco 1991, 46, 899-911.
(17) Rinaldi, M.; Pecorari, P.; Costantino, L.; Provvisionato, A.;
Malogoli, M.; Cermelli, C. Synthesis and Biological Activity of
New Heterocyclic Structures:[1,3]Thiazino[2,3-i]purine, Thia-
zolo[3,2-c][1,2,3]Triazolo[4,5-e]pyrimidine and [1,2,3]Triazolo-
[4′,5′:4,5] pyrimido[6,1-b][1,3]thiazine. Farmaco 1992, 47, 1315-
1322.
(18) Cheng, C. C.; Robins, R. K. Potential Purine Antagonists. VII.
Synthesis of 6-Alkylpirazolo[3,4-d]pyrimidines. J . Org. Chem.
1958, 23, 191-200.
(19) Osada, Y.; Tsuchimoto, M.; Fukushima, H.; Takahashi, K.;
Kondo, S.; Hasegawa, M.; Komoriya, K. Hypouricemic Effect of
the Novel Xanthine Oxidase Inhibitor, TEI-6720, in Rodens. Eur.
J . Pharmacol. 1993, 241, 183-188.
Kinetic studies were performed with at least three different
concentrations of inhibitor in the presence of variable concen-
trations of xanthine (2-20 µM). Ki values (the dissociation
constants of the enzyme-inhibitor complex) were determined
from the slopes in double-reciprocal plots.27
(20) Naeff, H. S. D.; van der Plas, H. C.; Tramper, J .; Muller, F.
Synthesis and Quantitative Structure-Activity Relationship
(QSAR) Analysis of Some Novel Xanthine Oxidase Inhibitors:
6-(para-Substitutedphenyl)-pteridine-4-ones. Quant. Struct.-Act.
Relat. 1985, 4, 161-166.
Ack n ow led gm en t. The authors would like to thank
the Ministero della Ricerca Scientifica e Tecnologica
(MURST) for its financial support.

Inhibitors of Xanthine Oxidase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2535
(21) Compound 2a was previously cited in following reports: (a)
Nakashima, K.; Akiyama, S. Analytical Studies on Pyrimidine
Derivatives. IV. Iron and Cobalt Complexes with S-Substituted
6-Amino-5-nitroso-2-thiouracils. Yakugaku Zasshi 1980, 100 (5),
515-523. (b) Yoshimura, M.; Hatada, T.; Deki, M.; Sugii, M.
Analytical Studies on Thiouracil. I. Synthesis and Thin Layer
Chromatography of Thiouracils. Yakugaku Zasshi 1976, 96 (9),
1094-1102. (c) Tsuji, T.; Watanabe, S.; Nakadai, Y.; Toyoshima,
S. Synthesis and Antimicrobial Activity of N1-(2-Alkyl-thio-6-
alkoxy-4-pyrimidinyl)sulfanilamide. Chem. Pharm. Bull. 1962,
10, 9-13.
(22) Traube, W. Der Aufbau der Xanthinbasen aus der Cyanessig-
sau¨re. Synthese des Hypoxanthins und Adenins. J ustus Liebı´gs
Ann. Chem. 1904, 331, 64-88.
(23) Compound 7c was previously cited; see: Runti, C.; Colautti, A.
Potential Antiviral Agents. VI. Higher N-acyl Derivatives of
Pyrimidine and Purine bases. In Int. Congr. Chemother., Proc.,
5th.; Spitzy, K. H., Ed.; Verlag Wiener Med. Akad.: Wien, 1967;
Vol. 5, pp 307-314.
(24) (a) Hildebrand, C.; Sandoli, D.; Focher, F.; Gambino, J .; Ciar-
rocchi, G.; Spadari, S.; Wright, G. Structure-Activity Relation-
ships of N2-Substituted Guanines as Inhibitors of HSV1 and
HSV2 Thymidine kinases. J . Med. Chem. 1990, 33, 203-206.
(b) Noonan, T.; Brown, N.; Dudycz, L.; Wright, G. Interaction of
GTP Derivatives with Cellular and Oncogenic ras-p21 Protein.
J . Med. Chem. 1991, 34, 1302-1307.
(25) Cheng, C. C.; Robins, R. K. Potential Purine Antagonists. VII.
Synthesis of 6-Alkylpirazolo[3,4-d]pyrimidines. J . Org. Chem.
1958, 23, 191-200.
(26) During the preparation of compounds 8, a new synthesis was
reported; see: Reddy, K. H.; Reddy, A. P.; Veeranagaiah, V.
Versatile Synthesis of 6-Alkyl(aryl)-1H-pyrazolo[3,4-d]pyrimidin-
4-[5H]-ones. Indian J . Chem. Sect. B 1992, 31B (3), 163-166.
(27) Costantino, L.; Rastrelli, G.; Albasini, A. Inhibitory activity of
flavonols towards the xanthine oxidase enzyme. Int. J . Pharm.
1992, 86, 17-23.
J M950876U