Pyrazolo[4,3-e][1,2,4]triazines: Purine analogues with electronic absorption in the visible region

Article abstract of DOI:10.3390/10101298

Synthesis of several pryrazolo[4,3-e][1,2,4]-triazines is described. The absorption spectrum of some 5-substituted derivatives was found to extend to the visible region. These compounds were found to inhibit some enzymes of purine metabolism, like xanthine oxidase or bacterial purine-nucleoside phosphorylase with K₁ values in the 10^-3 10^-5 M range.

Full text of DOI:10.3390/10101298

Molecules 2005, 10, 1298-1306
molecules
ISSN 1420-3049
http://www.mdpi.org
Pyrazolo[4,3-e][1,2,4]triazines: Purine Analogues with
Electronic Absorption in the Visible Region
Mariusz Mojzych 1, Andrzej Rykowski 1,* and Jacek Wierzchowski 2
1
Department of Chemistry, University of Podlasie, 3-Maja 54, Siedlce, Poland, PL-08-110, Tel.+48-
6431090, Fax +48-6442045
2
Department of Biophysics, University of Warmia & Mazury, 4 Oczapowskiego St., Olsztyn, Poland,
PL-10-789, Tel. +48-89-523-3406, Fax +48 89 524-04-08, e-mail: jacek.wie@uwm.edu.pl
* Author to whom correspondence should be addressed; e-mail: rykowski@ap.siedlce.pl
Received: 12 April 2005 / Accepted: 10 July 2005 / Published: 31 October 2005
Abstract: Synthesis of several pryrazolo[4,3-e][1,2,4]-triazines is described. The
absorption spectrum of some 5-substituted derivatives was found to extend to the visible
region. These compounds were found to inhibit some enzymes of purine metabolism, like
xanthine oxidase or bacterial purine-nucleoside phosphorylase with Ki values in the 10-3 –
10-5 M range.
Keywords: Pyrazolo[4,3-e][1,2,4]triazines, purine analogues, enzyme inhibitors.
Introduction
Modified purine nucleosides and their analogues have found numerous applications in biological
research and pharmacology [1-3]. In particular, the isomeric pyrazolopyrimidines and
triazolopyrimidines (8-azapurines) exhibit favourable spectroscopic and biochemical properties and
have potential for use in cancer and viral chemotherapy [3-6]. It has been found recently that another
class of naturally occurring purine analogues produced by Pseudomonas fluorescens var.
pseudoiodininum and Nostoc spongiaeforme such as pseudoiodinine and nostocine A (Figure 1)
display even more interesting spectral characteristics, i.e. their electronic absorption spectra extend
well to the visible region [7,8].

Molecules 2005, 10
1299
Figure 1
CH3
N
N
N
N
N
N
N
H N
H3C N
MeO
Pseudoiodinine
N
O
Nostocine A
Recent advances in nucleophilic substitution of hydrogen in heteroaromatics [9] and their
successful application to the preparation of functionalized 1,2,4-triazines [10,11] prompted us to
exploit this approach to make the N-unsubstituted pyrazolo[4,3-e][1,2,4]triazine 1 [12]. In this paper
some derivatives of such a system (compounds 2-9) have been synthesized from the common
intermediate 1, as depicted in Scheme 1. Furthermore we show that these compounds both exhibit
interesting spectral behaviour and also interact with enzymes of purine metabolism, and therefore may
find applications as spectroscopic probes.
Scheme 1
H
H
N
N
N
7
N
N
N
N
N
N
N
NH2
H
H3C
H3C
4
2) HCl / MeOH/ 12h / rt
1) NH3 liq
HCl / MeOH/ 12h / rt
O
O
O
O
KMnO4 / Bu4NBr / CH3COOH
benzene / water / 2h / 95%
HgO / EtOH / 40oC
NH-NH2
H2N-NH2 / THF / 90%
SO2CH3
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
SCH3
H
H3C
H3C
H3C
H3C
2
3
5
6
benzene / HCl / 40 oC/ 8H / 90%
O
H
H3C
H3C
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
SCH3
H
O
H3C
H3C
HO
1
8
9
H2N-NH2 . HCl / EtOH / 78oC
N
N
O
N
SCH3
H3C
10

Molecules 2005, 10
Results and Discussion
Chemistry
1300
The starting material 3-methyl-5-methylsulfanyl-1H-pyrazolo[4,3-e][1,2,4]triazine (1) was
synthesized from 5-acetyl-3-methylsulfanyl-1,2,4-triazine (10) [13] in a one-pot reaction by
condensation with hydrazine hydrochloride, followed by acid-promoted ring closure of the resulting
intermediate. According to expectations, compound 1, bearing an NH-fragment, appeared to be
unreactive towards nucleophilic displacements and attempts to perform direct nucleophilic
substitutions of the methylsulfanyl group with either ammonia or hydrazine failed.
To avoid these problems another possibility to synthesize N-unsubstituted pyrazolo
[4,3-e][1,2,4]triazines 4 and 7 is to use a N-protecting group. We decided to exploit ethyl vinyl ether as
a new protecting group for NH-pyrazoles [14]. Exposure of pyrazolo[4,3-e][1,2,4]triazine 1 to ethyl
vinyl ether in benzene for 8 hours at 40oC in the presence of traces of concentrated HCl gave the
intermediate 2 in 90% yield. This was isolated by chromatography and treated with potassium
manganate (VII) under phase transfer catalytic conditions at room temperature for 1h to give sulfone 3
in nearly quantitative yield.
Compound 3 was next reacted with liquid ammonia at -33oC. The product was cleanly deprotected
with concentrated HCl in methanol at room temperature to give the corresponding amino compound 4,
as a yellowish crystalline solid, in 80% yield. Similarly, the treatment of 3 with anhydrous hydrazine
in tetrahydrofuran provides 5-hydrazino derivative 5 in 90% yield. Oxidation of the latter with yellow
mercury (II) oxide results in replacement of the hydrazine group by hydrogen giving compound 6.
Deprotection of 6 in methanol with concentrated HCl at room temperature for 12 hours gave the
5-unsubstituted pyrazolo[4,3-e][1,2,4]triazine 7 in excellent yield (see Scheme 1).
Compound 8 was synthesized in four steps from 3-methyl-5-methylsulfanyl-1H-pyrazolo[4,3-
e][1,2,4]triazine (1) by methylation with iodomethane, oxidation with potassium manganate (VII),
hydrazinolysis with anhydrous hydrazine and treatment of the resulting 5-hydrazino-1,3-dimethyl-1H-
pyrazolo[4,3-e][1,2,4]triazine with yellow mercury (II) oxide. Bromination of 8 with N-bromo-
succinimide followed by nucleophilic displacement of bromide in the resulting C-bromomethyl
derivative by ethylene glycol anion gave aza analogue of acylopurine nucleoside 9.
Spectral properties
In agreement with previous reports [7, 8, 15], the electronic absorption spectra of 1-H-pyrazolo-
[4,3-e][1,2,4]-triazines differ very markedly from those of the analogous purines. Low-yield
fluorescence in the visible region (ca. 500 nm) was observed for some compounds in neutral aqueous
medium. The basic spectral characteristics of the synthesized compounds are summarized in Table 1.

Molecules 2005, 10
Table 1. Spectral parameters (electronic absorption and/or fluorescence) for
1301
selected pyrazolo[4,3-e]-triazines in aqueous solution. Conditions: 50
mM phosphate buffer (pH 7) or 10 mM KOH (pH 12).
λmax
[nm]
346
418
412
453
408
438
356
εmax
[cm-1M-1]
2270
fluorescence
Compound
7
pH
λmax [nm]
480wa
nfb
7
12
1450
4
1
7
1570
490w
nf
12
780
7
2300
nf
12
1370
nf
9
8
7 - 12
2460
490w
7 - 12
370
~2400
ndc
aweak fluorescence (φ ~ 0.01)
bno fluorescence detected (φ < 0.001)
cnd – not determined
Enzymatic assays
We have examined several purine metabolism enzymes to check if any of these effectively interact
with the new compounds. It was found that enzymatic phosphorolysis of m7Guo [16], catalyzed by E.
coli purine nucleoside phosphorylase (PNP), was inhibited by selected pyrazolotriazines at
concentrations of 30-500 µM. The strongest inhibitor was the 5-methylsulfanyl derivative 1, with an
IC50 of ~40 µM. Compounds methylated on the N-1 pyrazole ring nitrogen exhibited much weaker
inhibitory activity, resembling that reported for the analogous pyrazolopyrimidines [17].
Plots of 1/v vs. inhibitor concentration (not shown), obtained at constant substrate concentrations,
were in some instances apparently nonlinear, suggesting complex mode(s) of inhibition and/or
cooperative effects [18]. The corresponding IC50 values, given in Table 2, are virtually insensitive to
concentration of the m7Guo substrate, indicating noncompetitive type of inhibition. This behaviour is
not exceptional among enzymes of oligomeric structure, like bacterial PNP’s [18].
Calf spleen PNP, examined under identical conditions, was not inhibited by the pyrazolotriazines.
This result is not surprising since the bovine enzyme is known to exhibit much higher specificity
toward purines and purine moieties of nucleosides than the E. coli enzyme [19].
Since the analogous pyrazolopyrimidines, e.g. allopurinol, have therapeutic applications as known
strong inhibitors of the xanthine oxidase (Xox) enzyme, [20], we examined the inhibitory activities of
some of pyrazolotriazines toward commercially available Xox from buttermilk. Only weak inhibition
was detected in the case of the unsubstituted compound 7, but somewhat surprisingly, moderate
inhibitory activity was detected for the 5-thiomethyl derivative 1. The title compound was apparently
not a substrate for Xox, at least at the moderate enzyme concentrations employed in this work.

Molecules 2005, 10
1302
Table 2. Inhibition of bacterial (E. coli) purine-nucleoside phosphorylase and xanthine
oxidase from buttermilk by the investigated pyrazolotrazines.
Inhibition of the E. coli PNP Inhibition of Xox
Compound
substrate concentr.
IC50
[µM]
~700
~700
~900
>1000
~40
substrate concentr.
IC50
[µM]
[µM]
[µM]
32
m7Guo
7
4
Hx
Hx
48
~800
NIa
131
8.8
m7Guo
145
65.7
32
m7Guo
1
8
9
Hx
Hx
-
145
48
-
~300
NI
131
~30
-
-
ndb
32
NI
NI
m7Guo
nd
131
aNI – no inhibition detected with inhibitor concentrations up to 300 µM.
bnd – not determined
2-Amino substituted pyrazolotriazine 4, which may be regarded as an analogue of guanine, was
examined against a rabbit muscle guanine deaminase (GDA), but did not show any activity either as a
substrate, nor as an inhibitor, at least under the conditions applied in this experiment (concentrations
up to 150 µM, 50 mM phosphate pH 6.1, 25oC).
Conclusions
Pyrazolotriazines are moderate inhibitors of bacterial (E. coli) PNP, an enzyme employed recently
in cancer-oriented gene therapy experiments [21]. There is also a detectable inhibition of xanthine
oxidase by the parent 3-methyl-7-azapyrazolo[4,3-d]pyrimidine, as well as its S-methyl derivative.
These compounds are unique among purine analogues as having UV absorbtion spectra extending into
the visible region (ca. 450 nm), some of them being also weakly fluorescent in aqueous solution (cf.
Table 1), and thus are potentially applicable as spectroscopic probes for enzymes of purine
metabolism.
Acknowledgements
This work enjoyed the financial support of grant No. 3P04A02425 of the State Committee for
Scientific Research (K.B.N.).

Molecules 2005, 10
Experimental
General
1303
All melting points are uncorrected and were determined using a Boetius melting point apparatus.
Nuclear magnetic resonance (1H-NMR) spectra were recorded on a Varian Gemini 200 MHz
spectrometer in a suitable deutered solvent using TMS as internal standard. Mass spectra were
obtained on AMD 604 [electron impact (EI)] and API 350 [electrospray ionization (ESI)]
spectrometers. IR spectra were measured with a Magna IR-760 spectrophotometer, and UV on a Cary
319. Fluorescence spectra were recorded using a Perkin-Elmer LS-50B spectrofluorometer, equipped
with a pulsed xenon light source. Enzymes: xanthine oxidase (Xox) from buttermilk and purine
nucleoside phosphorylase (PNP) from calf spleen were purchased from Sigma, guanine deaminase
(GDA) was from ICN, and the E. coli PNP was a gift from Dr. W. Koszalka. Hypoxanthine and 7-
methylguanosine (m7Guo) were from Sigma. All other reagents and chemicals were obtained from
Aldrich Chemical Company and were used as received, unless otherwise noted. Enzymatic assays and
UV spectra were run using a Cary-319 UV spectrophotometer (Varian), equipped with a thermostatic
unit. Unless otherwise indicated, all the reactions were carried out in 50 mM phosphate, pH 7.0, at
25˚C. Inhibitor concentrations were evaluated spectrophotometrically, using data from Table 1. It was
possible to run assays with inhibitor concentrations up to ~300 µM. Activity of PNP was assayed by
following phosphorolysis of m7Guo in 50 mM phosphate, pH 7.0 [16], the reaction monitored
spectrophoto-metrically at 260 nm. Typical substrate concentration was ca. 9 - 135 µM. Xox activity
was measured using the hypoxanthine oxidation test, with substrate concentrations 48 – 150 µM [22],
monitored spectrophotometrically at 300 nm. Activity of GDA was assayed at pH 6.1, using ~50 µM
8-azaguanine as a substrate, the reaction monitored at 265 nm.
Syntheses
3-Methyl-5-methylsulfanyl-1H-pyrazolo[4,3-e][1,2,4]triazine (1) was obtained according to the
published procedure. [12] Details of the preparation of 1,3-dimethyl-1H-pyrazolo[4,3-e][1,2,4]triazine
(8) and 3-(2-hydroxyethoxymethyl)-1-methyl-1H-pyrazolo[4,3-e][1,2,4]triazine (9) will be published
elsewhere [23].
1-(1-Ethoxyethyl)-3-methyl-5-methylsulfanyl-1H-pyrazolo[4,3-e][1,2,4]triazine (2)
To a solution of 1 (1.81g, 10 mmol) in benzene (140 mL), ethyl vinyl ether (2 ml, 20 mmol) and
o
one drop of 38% of HCl were added. The reaction mixture was stirred at 40 C for 8 hours, at which
time a saturated solution of NaHCO3 was added. The benzene layer was separated, dried over
anhydrous MgSO4 and concentrated in vacuo. The product, compound 2, was purified by column
o
chromatography using silica gel and chloroform as eluent. Yield 90 % (2.27 g, 9 mmol); m.p. 51 C;
1H-NMR (CDCl3) δ: 1.14 (t, 3H, J=7.0 Hz), 1.92 (d, 3H, J=6.0 Hz), 2.65 (s, 3H), 2.74 (s, 3H), 3.19-
3.34 (m, 1H), 3.49-3.64 (m, 1H), 6.30 (q, 1H, J=6.0 Hz); HRMS (ESI) for C10H15N5OSNa [M+Na]:
Calcd 276.0890; Found 276.0910.
1-(1-Ethoxyethyl)-3-methyl-5-methylsulfanyl-1H-pyrazolo[4,3-e][1,2,4]triazine (3)

Molecules 2005, 10
1304
To a solution of 2 (253 mg, 1 mmol) in benzene (20 mL) water (30 mL), potassium
manganate(VII) (474 mg, 3 mmol), catalytic amounts of tetrabutylammonium bromide (65 mg, 0.2
mmol) and acetic acid (1.5 mL) were added. The reaction mixture was stirred at rt for 1 h. A saturated
solution of Na2S2O5 in water was then added to the mixture until the purple color disappeared. The
organic layer was separated and the aqueous phase was extracted with benzene (3x10 mL). The
combined organic extracts were dried over anhydrous MgSO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (eluent: chloroform) to afford 270 mg (95%) of 3
1
as a yellowish oil. H-NMR (CDCl3) δ: 1.17 (t, 3H, J=7.0 Hz), 1.98 (d, 3H, J=6.0 Hz), 2.79 (s, 3H),
3.20-3.35 (m, 1H), 3.52-3.68 (m, 1H), 3.58 (s, 3H), 6.43 (q, 1H, J=6.0 Hz); IR (KBr) cm-1: 2980, 1330,
1140; HRMS (ESI) for C10H15N5O3SNa [M+Na]: Calcd 308.0788; Found 308.0780.
5-Amino-3-methyl-1H-pyrazolo[4,3-e][1,2,4]triazine (4)
Compound 3 (206 mg, 1 mmol) was dissolved in dry liquid ammonia (15 mL) with exclusion of
moisture. The mixture was stirred at _33 oC for 1 h. After removal of the ammonia a mixture consisting
of methanol (10 mL) and concentrated HCl (0.5 mL) was added. The resulting solution was stirred at rt
for 12 h, then the solvent was removed and the crude product was purified by column chromatography
(silica gel, eluent: chloroform-ethanol, 20:1) to give compound 7 in 80% yield. This compound slowly
o
1
decomposed at approximately 256 C. H-NMR (DMSO) δ: 2.38 (s, 3H), 6.97 (s, 2H), 11.05 (broad,
pyrazole NH); IR (KBr) cm-1: 3340, 2850, 1650; HRMS (EI) for C5H6N6 [M+]: Calcd 150.06539;
Found 150.06582.
1-(1-Ethoxyethyl)-3-methyl-5-hydrazino-1H-pyrazolo[4,3-e][1,2,4]triazine (5)
To the solution of 3 (285 mg, 1 mmol) in dry THF (10 mL) cooled to 0-5 oC, anhydrous hydrazine
(0.1 mL, 3 mmol) was added. The reaction was stirred at 0-5 oC for 30 min and an additional 5 h at rt.
After that time the solvent was evaporated in vacuo and the crude product was recrystallized from
1
ethanol to give 213 mg (90%) of 5 as an orange solid. M.p. 120-123 oC; H-NMR (CDCl3) δ: 1.14 (t,
3H, J=7.0 Hz), 1.91 (d, 3H, J=6.0 Hz), 2.55 (s, 3H), 3.21-3.36 (m, 1H), 3.48-3.63 (m, 1H), 6.22 (q,
1H, J=6.0 Hz), 6.84 (s, 1H); HRMS (ESI) for C9H15N7ONa [M+Na]: Calcd 260.1230; Found
260.1226.
1-(1-Ethoxyethyl)-3-methyl-1H-pyrazolo[4,3-e][1,2,4]triazine (6)
To the solution of compound 4 (237 mg, 1 mmol) in dry ethanol (20 mL) yellow mercury oxide(II)
(1.08 g, 5 mmol) was added and the mixture was heated at 40 oC for 0.5 h. After that time the reaction
mixture was filtered off and the solvent was evaporated in vacuo. The crude product was purified by
column chromatography (silica gel, eluent: chloroform) to give 124 mg (60 %) of 7 as a yellow oil. 1H-
NMR (CDCl3) δ: 1.14 (t, 3H, J=7.0 Hz), 1.93 (d, 3H, J=6.0 Hz), 2.72 (s, 3H), 3.19-3.34 (m, 1H),
3.51-3.66 (m, 1H), 6.38 (q, 1H, J=6.0 Hz), 9.76 (s, 1H); IR (KBr) cm-1: 2980, 1130; HRMS (ESI) for
C9H13N5ONa [M+Na]: Calcd 230.1012; Found 230.1030.
3-Methyl-1H-pyrazolo[4,3-e][1,2,4]triazine (7)

Molecules 2005, 10
1305
A mixture of 5 (206 mg, 1 mmol) and concentrated HCl (0.5 mL) in methanol (10 mL) was stirred
at room temperature for 12 h. Then the solvent was removed, and the residue was separated by column
chromatography (silica gel, eluent: chloroform-ethanol, 20:1) to afford 124 mg (92 %) of 6 as a yellow
o
1
solid. M.p. 159-161 C; H-NMR (CDCl3) δ: 2.76 (s, 3H), 9.82 (s, 1H), 11.10 (broad, pyrazole-NH);
HRMS (EI) for C5H5N5[M+]: Calcd 135.05450; Found 135.05403.
References
1. Montgomery, J. A. Studies on the biologic activity of purine and pyrimidine analogs. Med. Res.
Rev. 1982, 2, 271-308.
2. Plunkett, W.; Saunders, PP. Metabolism and action of purine nucleoside analogs. Pharmacol.
Therapeut. 1991, 49, 239-68.
3. Parker, W. B.; Secrist, J. A., 3rd.; Waud, W. R. Purine nucleoside antimetabolites in development
for the treatment of cancer. Curr. Op. Invest. Drugs 2004, 5, 592-596.
4. Albert, A. A. Chemistry of 8-azapurines. Adv. Heterocycl. Chem. 1986, 39, 117-178.
5. Wierzchowski, J.; Wielgus-Kutrowska, B.; Shugar, D. Fluorescence emission properties of 8-
azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction
of purine nucleoside phosphorylase. Biochim. Biophys. Acta 1996, 1290, 9-17.
6. Sugiyama, T.; Schweinberger, E.; Kazimierczuk, Z.; Ramzaeva, N.; Rosemeyer, H.; Seela, F. 2-
aza-2'-deoxyadenosine: synthesis, base-pairing selectivity, and stacking properties of oligo-
nucleotides. Chem. Eur. J. 2000, 6, 369-78.
7. Smirnov, V. V.; Kiprianova, E. A.; Garagulya, A. D.; Esipov, S. E.; Dovjenko, S. A. Fluviols,
bicyclic nitrogen-rich antibiotics, produced by Pseudomonas Fluorescens. FEMS Microbiol. Lett.
1997, 53, 357-361.
8. Hirata, K.; Nakagami, H.; Takashina, J.; Mahmud, T.; Kobayashi, M.; In, Y.; Ishida, T.;
Miyamoto, K. Novel violet pigment, nostocine A, an extracellular metabolite from
cyanobacterium Nostoc spongiaeforme. Heterocycles 1996, 43, 1513-1519.
9. Mąkosza, M.; Wojciechowski, K. Nucleophilic aromatic substitution of hydrogen as a tool for the
synthesis of indole and quinoline derivatives. Heterocycles 2001, 54, 445-474.
10. Rykowski, A.; Olender, E.; Branowska, D.; van der Plas, H. C. A novel synthesis of 2-
acylpyridines via inverse electron demand diels-alder reaction of 5-acyl-1,2,4-triazines. Org.
Prep. Proced. Int. 2001, 33, 501-504.
11. Branowska, D.; Ostrowski, S.; Rykowski, A. Tandem vicarious nucleophilic substitution of
hydrogen/intramolecular Diels-Alder reaction of 1,2,4-triazines into functionalized
cycloalkenopyridines. Chem. Pharm. Bull. 2002, 50, 463-466.
12. Mojzych, M.; Rykowski, A. One-step synthesis and regioselective alkylation of substituted 1H-
pyrazolo-[4,3-e][1,2,4]triazine. Polish J. Chem. 2003, 77, 1797-1804.
13. A. Rykowski, A.; Lipińska, T. A concise router to a key intermediate In the total synthesis of
sempervirine. Synth. Commun. 1996, 26, 4409.
14. Vasilevsky, S. F.; Klyatskaya, S. V.; Tretyakov, E. V.; Elguero, J. Ethyl Vinyl Ether - an Agent
for Protection of the Pyrazole NH-Fragment. A Convenient Method for the Preparation of N-
Unsubstituted 4-Alkynylpyrazoles Heterocycles 2003, 60, 879-886.

Molecules 2005, 10
1306
15. Lindner, H. J.; Schaden, G. Pyrazolo-[4,3-e]-as-triazine, ein neues heterocyclisches System aus
Pseudomonas Fluorescens var. pseudo-iodinum. Chem. Ber. 1970, 105, 1949-1955.
16. Kulikowska, E.; Bzowska, A.; Wierzchowski, J.; Shugar D. Properties of two unusual, and
fluorescent substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methyl-
inosine. Biochim. Biophys. Acta 1986, 874, 355-366.
17. Kierdaszuk, B.; Modrak-Wójcik, A.; Wierzchowski, J.; Shugar D. Formycin A and its N-methyl
analogues, specific inhibitors of E. coli purine nucleoside phosphorylase: induced tautomeric shift
on binding to enzyme, and enzyme → ligand fluorescence resonance energy transfer. Biochim.
Biophys. Acta 2000, 1476, 109-128.
18. Neet, K. E. Cooperativity in enzyme function: equilibrium and kinetic aspects, Methods Enzymol.
1980, 64, 139-192.
19. Bzowska, A.; Kulikowska E.; Shugar D. Purine nucleoside phosphorylases: properties, functions
and clinical aspects. Pharmacol. Therapeut. 2000, 88, 349-425.
20. Borges, F.; Fernandes, E.; Roleira F. Progress towards discovery of xanthine oxidase inhibitors,
Curr. Med. Chem. 2002, 9, 195-217.
21. Curlee, K. V.; Parker, W. B.; Sorscher, E. J. Tumor sensitization to purine analogs by E. coli PNP.
Method. Mol. Med. 2004, 90, 223-45.
22. Heinz, F.; Reckel, S. Xanthine oxidase, in: Methods of Enzymatic Analysis, 3rd Edition;
Bergmeyer, H.U. Ed.; Verlag Chemie: Weinheim, 1983; Vol. III, pp. 210-216.
23. Mojzych, M.; Rykowski, A. Polish Pat. Appl. P372614, 2005.
Sample Availability: Available from the authors.
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