Hypoxia-selective O6-alkylguanine-DNA alkyltransferase inhibitors: Design, synthesis, and evaluation of 6-(benzyloxy)-2-(aryldiazenyl)- 9H -purines as prodrugs of O6-benzylguanine

Article abstract of DOI:10.1021/jm301804p

O⁶-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein which removes alkyl groups from the O-6 position of guanine, thereby providing strong resistance to anticancer agents which alkylate this position. The clinical usefulness of th

Full text of DOI:10.1021/jm301804p

Brief Article
pubs.acs.org/jmc
Hypoxia-Selective O6‑Alkylguanine-DNA Alkyltransferase Inhibitors:
Design, Synthesis, and Evaluation of 6‑(Benzyloxy)-2-
(aryldiazenyl)‑9H‑purines as Prodrugs of O6‑Benzylguanine
Rui Zhu, Raymond P. Baumann,* Philip G. Penketh, Krishnamurthy Shyam, and Alan C. Sartorelli
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, United States
ABSTRACT: O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein which removes alkyl groups from the O-6
position of guanine, thereby providing strong resistance to anticancer agents which alkylate this position. The clinical usefulness
of these anticancer agents would be substantially augmented if AGT could be selectively inhibited in tumor tissue, without a
corresponding depletion in normal tissue. We report the synthesis of a new AGT inhibitor (5c) which selectively depletes AGT
in hypoxic tumor cells.
aromatics, and N-oxides (for a review see ref 9). Although
azoreduction has been used as a therapeutic strategy in
designing prodrugs of nitrogen mustard10,11 and 5-amino-
salicylic acid,12,13 very little work, if any, has been done on the
possible use of hypoxia in solid tumors to activate azo prodrugs
for therapeutic purposes. This paper describes the synthesis and
evaluation of 6-(benzyloxy)-2-(aryldiazenyl)-9H-purines as
prodrugs of O6-BG activated under hypoxic conditions. The
2-amino group in these compounds, which is essential for the
AGT inhibitory activity of O6-BG, is latentiated as an azo
linkage. Unmasking of the 2-amino group occurs in the hypoxic
fraction of a solid tumor via reduction of the azo linkage.14
Once O6-BG is formed, it is expected to sensitize not only the
hypoxic tumor cells in which it is released but also the
surrounding aerobic tumor cells due to diffusion across
membranes.
INTRODUCTION
■
O6-Alkylguanine-DNA alkyltransferase (AGT, MGMT), a
ubiquitous DNA repair protein, effects the stoichiometric
removal of alkyl groups from the O-6 position of guanine in
DNA.1 Expression of this protein in tumors is the primary
mechanism of resistance to guanine O-6 chloroethylating
agents such as the N-(2-chloroethyl)-N-nitrosoureas (e.g.,
BCNU and CCNU) and the sulfonylhydrazine prodrugs
synthesized in our laboratory2−5 (e.g., laromustine and
KS119) and methylating agents (e.g., DTIC, Temozolomide,
and procarbazine). Inhibition of AGT activity in tumors is
currently of great interest to cancer researchers because it can
substantially increase the efficacy of antitumor guanine O-6
alkylating agents. The current therapeutic strategy of using an
AGT inhibitor such as O6-benzylguanine6 (O6-BG) to sensitize
AGT-expressing tumors to the cytotoxic effects of guanine O-6
alkylating agents has one inherent flaw. A global AGT inhibitor
not only ablates AGT in tumor tissue where the repair protein
is a hindrance to treatment, but it also significantly lowers AGT
levels in normal tissues where the protein serves a protective
function. For example, although nontoxic doses of O6-BG have
been shown in patients to deplete the AGT content of tumors,
this action also sensitizes host tissue, necessitating a
considerable decrease in the dosage of BCNU, the guanine
O-6 alkylating agent used in this trial, because of
myelosuppression, leading to an ineffective blood level of
BCNU.7,8 Therefore, for an AGT inhibitor to have a
meaningful impact on cancer therapy involving guanine O-6
alkylating agents, it is necessary for it to be delivered selectively
or preferentially to the tumor target. This will ensure that the
normal tissues are spared without severely compromising
therapeutic efficacy.
RESULTS AND DISCUSSION
■
Chemistry. 5a−e were synthesized as shown in Figure 1. 2-
Nitroso-6-benzyloxy-9-Boc-purine (3) was synthesized from 6-
benzyloxypurine (1) in four steps using published proce-
Figure 1. 5a−e were synthesized as shown. 2-Nitroso-6-benzyloxy-9-
Boc-purine (3) was synthesized from 6-benzyloxypurine (1) in four
steps using published procedures.15,16 3 was then condensed with the
appropriate aniline in dichloromethane in the presence of acetic acid at
room temperature to give the corresponding 4a−e.17 Deprotection of
the 9-t-Boc group was carried out using an equivalent amount of
piperidine in dichloromethane to give the target azo prodrug 5a−e.16
Repeated studies have demonstrated that oxygen-deficient
tumor cells resulting from the inherently abnormal tumor
vasculature create an environment conducive to reductive
processes. Several anticancer agents, particularly alkylating
agents, latentiated as bioreductive prodrugs, have been tested
and found to be preferentially toxic to oxygen deficient cells
compared to their aerobic counterparts. The substrates
examined to date have been predominantly quinones, nitro-
Received: December 7, 2012
Published: January 11, 2013
© 2013 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
dures.15,16 3 was then condensed with the appropriate aniline in
dichloromethane in the presence of acetic acid at room
temperature to give the corresponding 4a−e.17 Deprotection of
the 9-t-BOC group was carried out using an equivalent amount
of piperidine in dichloromethane to give the target azo prodrug
5a−e.16
Determination of Half-Wave Reduction Potentials.
Initially, five compounds were synthesized (5a−e, Figure 1)
and their half-wave reduction potentials determined by
differential pulse polarography (Table 1). The E1/2 values
a
Table 1. Determination of Half-Wave Reduction Potentials
concentration
half-wave reduction potential mV SE
(mV)
agent
(μM)
5d
5a
10
10
50
10
50
10
10
50
50
50
−92
−156
−213
−245
−279
−286
−307
−453
−475
−611
7
1
2
1
4
2
1
3
2
7
Figure 2. Survival (clonogenic) assays in DU145 human prostate
cancer cells pretreated for 4 h with graded concentrations of 5e then
dosed with 100 μM laromustine for a total of 24 h. Cells were treated
under either oxic ( ) or hypoxic ( ) conditions before staining and
quantification. The horizontal axis indicates the concentration of 5e in
μM. The vertical axis indicates the percent survival. All points
represent three independent determinations SEM.
2-NBP
5e
nitrofurazone
5c
■
□
mitomycin C
misonidazole
BG-M2
metronidazole
a
Polarographic half-wave reduction potentials of agents in relation to
four well-known reference compounds; nitrofurazone, a topical
bactericide; mitomycin C, a cancer chemotherapeutic agent preferen-
tially targeting hypoxic regions; misonidazole, a radiosensitizer, 18F
derivatives of which are used for oxygen deficient region imaging; and
metronidazole, an antibiotic targeting anaerobic bacteria and protozoa.
2-NBP and BG-M2 are prototype O6-BG prodrugs designed to target
hypoxic tumors. Half-wave reduction potentials were measured versus
an Ag/AgCl reference electrode. All values are the result of at least
three determinations SE.
versus an Ag/AgCl reference electrode under the tested
conditions for 5a−e were −156, −221, −286, −92, and −245
mV, respectively. Two candidates (5c and 5e, Figure 1) with
the lowest half-wave potentials (−286 and −245 mV,
respectively) were chosen for further study. This was done
not only to ensure that the candidates were relatively refractory
to reductive activation under aerobic conditions but also to
ensure facile activation under hypoxic conditions.
Figure 3. Survival (clonogenic) assays using DU145 cells pretreated
for 4 h with graded concentrations of 5c then exposed to 100 μM
laromustine for a total of 24 h. Cells were treated under either oxic
( ) or hypoxic ( ) conditions before staining and quantification. The
x axis indicates the concentration of 5c in μM. The y axis indicates the
percent survival. All points represent three independent determi-
nations SEM.
■
□
5c Sensitizes Cancer Cells to Laromustine. 5c and 5e
were tested in clonogenic assays against DU145 human
prostate carcinoma cells, which express relatively high levels
of the resistance protein AGT18 (42000 molecules per cell), to
determine their ability to sensitize these cells to laromustine by
selective release of O6-BG. 5e, which had a reduction potential
slightly higher than 5c, only weakly sensitized DU145 cells to
the guanine O-6 alkylator laromustine (Figure 2) under hypoxic
conditions and did not sensitize these cells under oxic
conditions (Figure 2). 5c was also tested in DU145 cells and
demonstrated significant sensitization of these cells to
laromustine under conditions of hypoxia with little or no effect
seen under oxygenated conditions (Figure 3). No further
increase in sensitization was observed at concentrations >10
μM (data not shown); this is likely the result of the limited
aqueous solubility of this agent.
incubated under aerobic conditions and under hypoxic
conditions in the presence of EMT6 mouse mammary tumor
cells (1 × 107/ml) at 37 °C, and samples were taken at hourly
intervals and analyzed by HPLC as described in Figure 4. Over
the course of 3 h, approximately 50% of the initial starting
amount of 50 μM of the prodrug 5c was converted to O6-BG
under hypoxic conditions with essentially no production of O6-
BG detected under oxic conditions. Under hypoxic conditions
in EMT6 cells, the loss of prodrug exceeded the production of
O6-BG, likely due to loss from nonreductive metabolism or
precipitation. Studies of other O6-BG prodrugs designed by this
laboratory have yielded similar incomplete conversions.19 5c
was also tested for the ability to generate O6-BG when
incubated with DU145 cells. As can be seen in Figure 5, DU145
cells converted over 84% of the initial input of 5c to O6-BG
under hypoxic conditions with only minimal production of O6-
5c Selectively Produces O6-Benzylguanine under
Hypoxic Conditions. 5c, which sensitizes DU145 cells to
laromustine, was further studied to determine the levels of O6-
BG released under oxic and hypoxic conditions. 5c was
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Journal of Medicinal Chemistry
Brief Article
similar conditions, than those obtained with our previously
designed O6-BG prodrugs in many cases. The yields of O6-BG
from 5c using EMT6 cells were approximately 10-fold greater
than that from 2-(4-nitrophenyl)propan-2-yl (6-(benzyloxy)-
9H-purin-2-yl)carbamate (BG-M2) and comparable to those
from the more readily reduced 2-nitro-6-benzyloxypurine (2-
NBP). In the case of DU145 cells, the yields of O6-BG from 5c
exceeded those from either BG-M2 or 2-NBP by >40-fold. The
high hypoxic azo-reduction dependent activation of 5c by
DU145 cells is particularly noteworthy as this cell line tends to
feebly activate nitro-reduction dependent prodrugs. The poor
water solubility of 5c is a potential drawback, and we are in the
process of synthesizing analogues of this agent which are
considerably more water soluble yet retain the desirable
characteristics of 5c, with the intent of conducting in vivo
studies on suitable compounds.
Figure 4. The generation of O6-BG, and parental prodrug loss (5c)
over a 3 h time interval. Prodrug was incubated with 107/mL of EMT6
mouse mammary tumor cells, and samples were analyzed at 1 h
intervals by HPLC. The x axis indicates time in h. The y axis indicates
the concentration in μM. All points are the results of at least three
determinations SEM.
EXPERIMENTAL SECTION
■
Chemical Syntheses. Melting points were determined on a
Thomas−Hoover Unimelt melting point apparatus and are
uncorrected. 1H NMR spectra were recorded on a Bruker Avance
DPX-400 spectrometer (400 MHz) with tetramethylsilane as an
internal standard. High resolution mass spectra were recorded on a
Bruker Daltonics 9.4 T APEXQe FT-ICR and Ultimate mass
spectrometer. Column chromatography was conducted with Merck
Silica Gel 60 (230−400 mesh). Thin layer chromatography was
performed on EM precoated silica gel sheets containing a fluorescent
indicator. All the reported test compounds possess a purity of at least
95% as determined by HPLC.
tert-Butyl-6-(benzyloxy)-2-(phenyldiazenyl)-9H-purine-9-carbox-
ylate (4a). To a solution of aniline (0.28 g, 3 mmol) in CH2Cl2 (10
mL) containing acetic acid (0.5 mL) was added 2-nitroso-6-benzyloxy-
9-Boc-purine (3) (0.71 g, 2 mmol) at room temperature. The reaction
mixture was stirred for 24 h. The solvent was removed by vacuum, and
the residue was purified by flash chromatography using CH2Cl2:EtOAc
(20:1) as eluent, which gave 4a as a yellow solid (0.43g, 61%); mp
230−232 °C. 1H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H), 8.20−8.01
(m, 2H), 7.68−7.50 (m, 5H), 7.45−7.30 (m, 3H), 5.80 (s, 2H), 1.71
(s, 9H).
Figure 5. The generation of O6-BG and parental prodrug (5c) loss
over a 3 h time interval. Prodrug was incubated with 107/mL of
DU145 human prostate carcinoma cells, and samples were analyzed at
1 h intervals by HPLC. The x axis indicates time in h. The y axis
indicates the concentration in μM. All points are the results of at least
three determinations SEM.
4b−4e were synthesized using procedures analogous to the one
BG under oxic conditions. These studies using high cell
densities indicate that significant quantities of the AGT
inhibitor O6-BG are released selectively under hypoxic
conditions by the novel azo prodrug 5c. The yields of O6-BG
obtained under hypoxic conditions following reductive
fragmentation of 5c are significantly higher than those obtained
from 2-(4-nitrophenyl)propan-2-yl (6-(benzyloxy)-9H-purin-2-
yl)carbamate (BG-M2) under similar conditions.19 A significant
decrease in the concentration of 5c was seen with both cell
lines under aerobic conditions, but this was not associated with
the production of O6-BG and was likely the result of parental
material lost to precipitation or nonreductive routes of
metabolism.
described above.
tert-Butyl-6-(benzyloxy)-2-((4-methoxyphenyl)diazenyl)-9H-pu-
rine-9-carboxylate (4b). 4b was obtained in a 60% yield; mp 143−144
°C. 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 8.20−8.09 (m, 2H),
7.67−7.56 (m, 2H), 7.45−7.32 (m, 3H), 7.11−7.02 (m, 2H), 5.80 (s,
2H), 3.93 (s, 3H), 1.71 (s, 9H).
tert-Butyl-6-(benzyloxy)-2-((4-(dimethylamino)phenyl)diazenyl)-
9H-purine-9-carboxylate (4c). 4c was obtained in a 55% yield; mp
226−227 °C. 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 8.10 (t, J =
9.2 Hz, 2H), 7.60 (d, J = 6.9 Hz, 2H), 7.44−7.30 (m, 3H), 6.77 (d, J =
9.2 Hz, 2H), 5.81 (s, 2H), 3.15 (s, 6H), 1.71 (s, 9H).
tert-Butyl-6-(benzyloxy)-2-((4-(ethoxycarbonyl)phenyl)diazenyl)-
9H-purine-9-carboxylate (4d). 4d was obtained in a 52% yield; mp
138−140 °C. 1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.28−8.09
(m, 3H), 7.59 (d, J = 7.7 Hz, 2H), 7.43−7.29 (m, 3H), 5.79 (s, 2H),
4.43 (q, J = 7.1 Hz, 2H), 1.71 (s, 9H), 1.43 (t, J = 7.1 Hz, 3H).
tert-Butyl-6-(benzyloxy)-2-((4-(4-methylpiperazin-1-yl)phenyl)-
diazenyl)-9H-purine-9-carboxylate (4e). 4e was obtained in a 50%
CONCLUSION
■
Compound 5c, conceived as a hypoxia-activated prodrug of O6-
BG, has the following desirable properties: (a) 5c has little or
no activity under normoxic conditions in tissue culture as
measured by its ability to sensitize AGT expressing DU145 cells
to the cytotoxic effects of laromustine, a DNA guanine O-6
alkylating agent. However, significant sensitization of DU145
cells to laromustine occurs under hypoxic conditions. (b)
Reduction of 5c under hypoxic conditions by DU145 and
EMT6 cells results in excellent yields of O6-BG. In fact, the
yields of O6-BG obtained were significantly higher, under
1
yield; mp 192−194 °C. H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H),
8.09 (d, J = 9.2 Hz, 2H), 7.67−7.54 (m, 2H), 7.43−7.29 (m, 3H), 6.98
(d, J = 9.2 Hz, 2H), 5.80 (s, 2H), 3.53−3.43 (m, 4H), 2.63−2.53 (m,
4H), 2.37 (s, 3H), 1.71 (s, 9H).
6-(Benzyloxy)-2-(phenyldiazenyl)-9H-purine (5a). To a solution of
tert-butyl 6-(benzyloxy)-2-(phenyldiazenyl)-9H-purine-9-carboxylate
(4a) (0.43 g, 1 mmol) in CH2Cl2 (5 mL) was added dropwise
piperidine (0.08 g, 1 mmol) at room temperature. The mixture was
stirred overnight. The precipitate was collected and washed with
CH2Cl2 and ether to give the target molecule as a yellow solid (0.30 g,
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1
90%); mp 242−243 °C. H NMR (400 MHz, DMSO-d6) δ13.74 (s,
H2O2 is removed rapidly by a large excess of catalase.5,20,21 It is
expected that this low transient exposure to H2O2 will have no
significant effect on the reduction of 5c during these 1 h incubations.
At various time intervals, samples were withdrawn and the cellular and
medium components were precipitated by mixing with an equal
volume of acetonitrile for 20 min at room temperature followed by
centrifugation at 10000g for 15 min. The supernatant was then
analyzed by HPLC for 5c and O6-BG.
1H), 8.55 (s, 1H), 8.05−7.98 (m, 2H), 7.74−7.56 (m, 5H), 7.41 (dt, J
= 21.6, 7.1 Hz, 3H), 5.71 (s, 2H). HRMS: calcd for C18H14N6O, m/z,
331.1302 [(M + H)+]; found, 331.1303. HPLC: tr = 35.26 min
(97.1%).
5b−5e were synthesized using procedures analogous to the one
described above.
6-(Benzyloxy)-2-((4-methoxyphenyl)diazenyl)-9H-purine (5b). 5b
was obtained in an 88% yield; mp 223−224 °C. 1H NMR (400 MHz,
DMSO-d6) δ 13.61 (s, 1H), 8.51 (s, 1H), 8.07−7.93 (m, 2H), 7.69−
7.55 (m, 2H), 7.48−7.32 (m, 3H), 7.26−7.14 (m, 2H), 5.70 (s, 2H),
3.91 (s, 3H). HRMS: calcd for C19H16N6O2, m/z, 361.1408 [(M +
H)+]; found, 361.1407.
4-((6-(Benzyloxy)-9H-purin-2-yl)diazenyl)-N,N -dimethylaniline
(5c). 5c was obtained in an 80% yield; mp 245−246 °C. H NMR
(400 MHz, DMSO-d6) δ 13.52 (s, 1H), 8.44 (s, 1H), 7.88 (d, J = 9.1
Hz, 2H), 7.61 (d, J = 7.0 Hz, 2H), 7.40 (dt, J = 21.5, 7.1 Hz, 3H), 6.88
(d, J = 9.2 Hz, 2H), 5.68 (s, 2H), 3.11 (s, 6H). HRMS: calcd for
C20H19N7O, m/z, 374.1724 [(M + H)+]; found, 374.1716.
Ethyl 4-((6-(benzyloxy)-9H-purin-2-yl)diazenyl)benzoate (5d). 5d
was obtained in a 90% yield. mp 228−230 °C. H NMR (400 MHz,
DMSO-d6) δ 13.80 (s, 1H), 8.58 (s, 1H), 8.28−8.18 (m, 2H), 8.15−
8.06 (m, 2H), 7.66−7.56 (m, 2H), 7.50−7.35 (m, 3H), 5.71 (s, 2H),
4.38 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H). HRMS: calcd for
C21H18N6O3, m/z, 403.1513 [(M + H)+]; found, 403.1513.
6-(Benzyloxy)-2-((4-(4-methylpiperazin-1-yl)phenyl)diazenyl)-9H-
purine (5e). 5e was obtained in a 74% yield; mp 219−221 °C. H
NMR (400 MHz, DMSO-d6) δ 13.57 (s, 1H), 8.46 (s, 1H), 7.88 (d, J
= 9.2 Hz, 2H), 7.61 (d, J = 6.9 Hz, 2H), 7.47−7.33 (m, 3H), 7.13 (d, J
= 9.2 Hz, 2H), 5.68 (s, 2H), 3.47−3.39 (m, 4H), 2.49−2.41 (m, 4H),
2.24 (s, 3H). HRMS: calcd for C23H24N8O, m/z, 429.2146 [(M +
H)+]; found, 429.2133. HPLC: tr = 17.55 min (99.9%).
Cell Culture. DU145 human prostate carcinoma cells were
cultured in α-MEM medium supplemented with 10% fetal bovine
serum. EMT6 mouse mammary carcinoma cells were cultured in
DMEM supplemented with 10% fetal bovine serum. Both cell lines
were maintained at 37 °C in a 5% CO2 atmosphere.
Cytotoxicity Assays. Cells survival (clonogenic) assays were
performed as previously described.5,20,21 DU145 cells were plated in
25 cm2 plastic flasks at a density of 2 × 105 cells and used when near
confluent.5 Cells were pretreated for 4 h with graded concentrations of
5c or 5e under oxic or hypoxic conditions prior to the addition of 100
μM of laromustine for a total incubation time of 24 h at 37 °C.
Hypoxia was generated by the direct depletion of oxygen in sealed
flasks using the glucose oxidase (2 units/mL, Sigma G6641) and
catalase (120 units/mL, Sigma C1345) dual enzyme system as
previously described.5,20,21
Determination of Half-Wave Reduction Potentials (E1/2). The
E1/2 values were determined by differential pulse polarography (DPP).
The supporting electrolyte was 80% by volume 100 mM potassium
chloride and 50 mM potassium phosphate (pH 7.0) and 20% by
volume of CH3CN in all cases. Agents were added as 1% by volume
solutions in DMSO. The E1/2 values of six reference compounds were
also measured. Dissolved oxygen was removed by purging with
nitrogen. DPP voltammograms were generated using a Princeton
Applied Research electrochemical trace analyzer model 394, with a
model 303A static mercury drop electrode (Princeton Applied
Research, Oak Ridge, TN, USA) utilizing a platinum counter electrode
and an Ag/AgCl reference electrode. Voltammograms were taken from
0 to −900 mV at a scan rate of 2 mV/s using a pulse amplitude of 50
mV. The E1/2 value was determined from the peak current potential
(EP) using the following equation: E1/2 = EP − pulse amplitude/2.22
Cell Dependent O6-BG Generation. Cell suspensions (107 cells/
mL) were treated with 5c (50 μM) under oxic or hypoxic conditions
in DMEM (EMT6), or α-MEM (DU145) media containing 10% FBS.
Plastic flasks (25 cm2) with shallow 4 mL layers were employed for
oxic studies and were shaken to maintain aeration. The glucose
oxidase/catalase/glucose system was used to generate hypoxic
conditions before the addition of 5c; the mixtures were stirred gently
in sealed tubes. Using this system, oxygen is depleted in ∼3 min and
AUTHOR INFORMATION
Corresponding Author
*Phone: 203-785-4524. Fax: 203-737-2045. E-mail:
baumannray@yahoo.com.
■
1
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by U.S. Public Health Service
grants CA090671, CA122112, and CA129186 from the
National Cancer Institute and a grant from the National
Foundation for Cancer Research.
1
ABBREVIATIONS USED
■
AGT, O6-alkylguanine-DNA alkyltransferase; BCNU, bis(2-
chloroethyl)-1-nitrosourea; O6-BG, O6-benzylguanine; BG-M2,
2-(4-nitrophenyl)propan-2-yl (6-(benzyloxy)-9H-purin-2-yl)-
carbamate; DMEM, Dulbecco’s Modified Eagle Medium;
MEM, minimal essential media; 2-NBP, 2-nitro-6-benzylox-
ypurine
1
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