Synthesis of 6-azapurines by transformation of toxoflavins and reumycins (7-azapteridines) and their cytotoxicities

Article abstract of DOI:10.1071/CH14425

This paper describes a reliable and facile synthesis of 6-azapurines, 1,5-dimethyl-1H-imidazo[4,5-e][1,2,4]triazin-6(5H)-ones and 5-methyl-5H-imidazo[4,5-e][1,2,4]triazin-6(7H)-ones, by treatment of toxoflavins and reumycins with 10% aqueous or ethanolic sodium hydroxide at 5-70°C or reflux, followed by decarboxylation and oxidation by air along with a benzilic acid type rearrangement. Furthermore, heating the produced 6-azapurines in 10% ethanolic sodium hydroxide afforded the corresponding 1-methyl-5,6-dioxo-1,4,5,6-tetrahydro-1,2,4-triazines with 1-methylurea. The antitumour activities of the 6-azapurines against CCRF-HSB-2 (human T-cell acute lymphoblastoid leukemia) and KB (human oral epidermoid carcinoma) cell lines were also investigated in vitro and some of the compounds showed prospective antitumour activities.

Full text of DOI:10.1071/CH14425

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2015, 68, 203–210
Full Paper
http://dx.doi.org/10.1071/CH14425
Synthesis of 6-Azapurines by Transformation of Toxoflavins
and Reumycins (7-Azapteridines) and their Cytotoxicities
A
B
A C D
, ,
Jun Ma, Fumio Yoneda, and Tomohisa Nagamatsu
A
Department of Drug Discovery and Development, Division of Pharmaceutical
Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Okayama University, Tsushima-naka, Okayama 700-8530, Japan.
B
Fujimoto Pharmaceutical Co., Ltd, Matsubara, Osaka 580-0011, Japan.
C
Kumamoto Health Science University, Department of Medical Technology,
Faculty of Health Science, 325 Izumimachi, Kita-ku, Kumamoto City,
Kumamoto 861-5598, Japan.
D
Corresponding author. Email: nagamatu@kumamoto-hsu.ac.jp
This paper describes a reliable and facile synthesis of 6-azapurines, 1,5-dimethyl-1H-imidazo[4,5-e][1,2,4]triazin-6(5H)-
ones and 5-methyl-5H-imidazo[4,5-e][1,2,4]triazin-6(7H)-ones, by treatment of toxoflavins and reumycins with 10 %
aqueous or ethanolic sodium hydroxide at 5–708C or reflux, followed by decarboxylation and oxidation by air along with a
benzilic acid type rearrangement. Furthermore, heating the produced 6-azapurines in 10 % ethanolic sodium hydroxide
afforded the corresponding 1-methyl-5,6-dioxo-1,4,5,6-tetrahydro-1,2,4-triazines with 1-methylurea. The antitumour
activities of the 6-azapurines against CCRF-HSB-2 (human T-cell acute lymphoblastoid leukemia) and KB (human oral
epidermoid carcinoma) cell lines were also investigated in vitro and some of the compounds showed prospective
antitumour activities.
Manuscript received: 29 June 2014.
Manuscript accepted: 22 July 2014.
Published online: 5 November 2014.
Introduction
(5,7-dimethyl-5H-imidazo[4,5-e][1,2,4]triazin-6(7H)-ones)
4
Since the isolation and characterisation of the naturally
occurring antibiotics of 7-azapteridine {pyrimido[5,4-e]-1,2,4-
triazine}, there has been considerable interest in the synthe-
sis[1] and biological evaluation[2] of the 7-azapteridines, which
are known as toxoflavin (1, R ¼ H), fervenulin (2, R ¼ H) and
reumycin, (3, R ¼ H) (Scheme 1).[3] We have developed sev-
eral convenient synthetic procedures for the preparation of
toxoflavin 1 and its 3- and/or 6-substituted derivatives,[4] and
evaluated their potential antiviral[5] and antitumour activities[6]
and their application as herbicides.[7] However, we encoun-
tered difficulties when attempting to prepare the derivatives
possessing a substituent at the 1-position of the toxoflavin
skeleton 1. Because we have previously reported that toxo-
flavin and its 3-substituted derivatives 1 readily undergo
demethylation at the 1-position upon heating with some
nucleophiles, e.g. DMF and dimethylacetamide, to give the
corresponding 1-demethyltoxoflavin (reumycins 3) deriva-
tives, while the nucleophiles themselves were methylated by
the methyl group that was eliminated, novel radical species
were observed during the reactions (Scheme 1).[8] On the other
hand, the methylation of reumycin and its 3-substituted deri-
vatives 3 under alkaline conditions with dimethyl sulfate or
methyl iodide in DMF provided not toxoflavins 1 but fervenu-
lins 2, whose methyl group at the 8-position was stable.[9] We
have reported recently that heating fervenulins 2 with ethanolic
sodium hydroxide afforded the corresponding 6-azapurines
along with a benzilic acid type rearrangement.[10] We thought
previously that it was impossible to produce such 6-azapurines 5
(Scheme 2) without 1-demethylation by the rearrangement from
toxoflavins 1, which had the tendency to eliminate the methyl or
alkyl group by acid, nucleophilic solvent or heating (see ii of
Scheme 1). However, we found recently that the methyl group at
the 1-position of toxoflavins 1 is appreciably stable in some
alkaline solutions as reported in a previous paper.[9d] Conse-
quently, the toxoflavins 1 under harsher conditions such as
longer treatment or heating in alkaline solution were trans-
formed gradually to the 6-azapurines (1,5-dimethyl-1H-imidazo
[4,5-e][1,2,4]triazin-6(5H)-ones) 5 without any demethylation
(Scheme 2), and were reported as preliminary data in a com-
munication paper.[11] Dovzhenko et al. have also reported the
transformation of toxoflavin (1a, R ¼ H) into the 6-azapurine 1a
by heating with aqueous diethylamine.[12] Recent years have
seen dramatic developments in the synthesis of modified purine
derivatives and 8-azapurines as therapeutic agents[13] and anti-
viral agents,[14] but none with 6-azapurines.
On the other hand, toxoflavin, a phytotoxin produced by
Burkholderia glumae BGR1, was known to be the key factor
in rice grain rot and wilt in many field crops.[15] Toxoflavin-
degrading enzyme (TxDE) has been identified recently from
Paenibacillus polymyxa JH2, thereby providing a possible anti-
virulence strategy for toxoflavin-mediated plant diseases.[16]
We reported the crystal structure of TxDE in the substrate-free
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

204
J. Ma, F. Yoneda, and T. Nagamatsu
O
according to our previous reports.[4a–e,9d] Treatment of the
toxoflavins 1a–t with 10% aqueous sodium hydroxide at 5–708C
under the conditions described in the Experimental section, fol-
lowed by neutralisation with 10% aqueous hydrochloric acid
under cooling on ice–water afforded the 6-azapurines 5a–t in the
solution, which were concentrated to dryness under vacuum.
The solids thus obtained were crystallised from a mixture of
ethanol and water to afford the corresponding 1,5-dimethyl-1H-
imidazo[4,5-e][1,2,4]triazin-6(5H)-one (6-azapurine, 5a) and its
3-substituted derivatives 5b–e as colourless needles in 40–90%
yields. In the case of the preparation of products 5f–t, the solids
were obtained after neutralisation of the reaction solution under
cooling on ice–water. The resulting solids were collected by fil-
tration and washed with water and recrystallised from EtOH to
give the pure 6-azapurine derivatives 5f–t in 40–80 % yields as
colourless or yellow needles (Scheme 2 and Tables 1 and 2).
Furthermore, heating of the 6-azapurines 5a–k with 10 %
ethanolic sodium hydroxide under reflux for 4–6 h, followed by
neutralisation with 10 % aqueous hydrochloric acid gave the
products as solids, which were crystallised from DMF to afford
the corresponding 1-methyl-5,6-dioxo-1,4,5,6-tetrahydro-
1,2,4-triazine (6a) and its 3-substituted derivatives 6b–k as
colourless powdery crystals in 40–90 % yields, along with
methyl urea (Scheme 3 and Tables 3 and 4). The methyl urea
was identified in some cases by isolation from column chroma-
tography in 17–25 % yields. The structures of compounds 5 and
6 were confirmed on the basis of elemental analysis, IR, UV,
and 1H and 13C NMR spectra as can be seen in the Experimental
section and Tables 1–4. In particular, the structures of some
products were confirmed by the measurement of proton–carbon
interaction in 2D NMR HMQC and HMBC spectra as shown in
the Supplementary Material.
O
4
N
6
MeN
5
N
N
R
3
R
i
MeN
7
N
N
2
O
N
Me
O
N
8
N
Me
1
i
ii
toxoflavins 1
fervenulins 2
iii
O
5
Me
N
4
N
N
R
3
N
R
MeN
6
O
N
N
N
Me
7
2
O
N
H
N
1
reumycins 3
6-azapurines 4
Scheme 1. Reagents and conditions: i) MeI, K2CO3, DMF, reflux. ii) DMF
or AcOH, reflux. iii) 10 % NaOH in EtOH, 608C or reflux. Toxoflavins 1 can
be named as 3-substituted 1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7
(1H,6H)-diones (pyrimido-triazine numbering) or 6-substituted 3,8-dimethyl-
7-azapteridin-2,4(3H,8H)-diones (pteridine numbering). 6-Azapurines 4
can be named as 3-substituted 5,7-dimethyl-5H-imidazo[4,5-e][1,2,4]-
triazin-6(7H)-ones (imidazo-triazine numbering) or 2-substituted 7,9-
dimethyl-7H-6-azapurin-8(9H)-ones (purine numbering).
O
Me
N
R
N
R
N
MeN
O
N
N
N
O
N
N
N
Me
Me
Similarly, heating reumycin 3a and its 3-substituted deriva-
tives 3b–m in 10 % ethanolic sodium hydroxide solution under
reflux for 4–6 h gave the corresponding 5-methyl-5H-imidazo
[4,5-e][1,2,4]triazin-6(7H)-one (6-azapurine, 7a) and its
3-substituted derivatives 7b–m in 50–80 % yields (Scheme 4,
Tables 5 and 6). As the reumycins 3a–m were reasonably stable
in hot alkaline solution, such 1,2,4-triazines as 6a–k were not
observed in these reactions. The structures of compounds 7a–m
were also identified or confirmed on the basis of elemental
analysis, IR, UV, and 1H and 13C NMR spectra as can be seen in
the Experimental section and in Tables 5 and 6 (see also the
Supplementary Material).
toxoflavins 1a–t
6-azapurines 5a–t
a: R ꢀ H
k: R ꢀ 4-HO-C6H4
l: R ꢀ 4-Me-C6H4
m: R ꢀ 4-Pri-C6H4
b: R ꢀ Me
c: R ꢀ Et
d: R ꢀ Prn
e: R ꢀ Pri
f: R ꢀ Ph
g: R ꢀ 4-F-C6H4
h: R ꢀ 3-Cl-C6H4
i: R ꢀ 4-Cl-C6H4
j: R ꢀ 4-Br-C6H4
n: R ꢀ 4-MeO-C6H4
o: R ꢀ 3,4-(MeO)2-C6H3
p: R ꢀ 3,4,5-(MeO)3-C6H2
q: R ꢀ 4-AcO-C6H4
r: R ꢀ 4-Me2N-C6H4
s: R ꢀ 2-Furyl
t: R ꢀ 2-Thienyl
We suggest that these 6-azapurines 5 are formed from
toxoflavins (7-azapteridines) 1 via a benzilic acid type rear-
rangement in alkaline solution, followed by decarboxylation
and oxidation by air, as depicted in Scheme 5. Moreover, heating
the 6-azapurines 5 in alkaline solution gave the 5,6-dioxo-
1,4,5,6-tetrahydro-1,2,4-triazines 6 and 1-methylurea by ring
fission of the imidazole ring of the 6-azapurines 5. The ring
transformation of reumycins 3 into 6-azapurines 7 can also be
explained by a similar reaction mechanism. Because of the
reasonable stability of the 6-azapurines 7, which were formed by
ring contraction of reumycins 3 on heating in alkaline solution,
the further degradation of the 6-azapurines 7a–m was not
observed. Previously, we had reported analogous ring contrac-
tions of fervenulins 2.[10b]
Scheme 2. Reagents and conditions: 10 % aq. NaOH, 5–258C, 1–3 days
for R ¼ H and alkyl; 60–708C, 10–60 min for R ¼ aryl, furyl, and thienyl, and
then 10 % aq. HCl.
form and in complex with toxoflavin, along with the results of a
functional analysis.[17]
Herein, we wish now to report the full synthetic preparations
for 6-azapurines 5 and 7 by the transformation of toxoflavins 1
and reumycins 3, respectively, via a benzilic acid type rear-
rangement, and for 5,6-dioxo-1,4,5,6-tetrahydro-1,2,4-triazines
6 by further hydrolysis of the 6-azapurines 5, as well as the
evaluation of their antitumour activities in vitro.
Results and Discussion
Biological Activity
The desired toxoflavin 1a and its 3-substituted derivatives 1b–t
were prepared by nitrosative cyclisation of the appropriate 6-(2-
alkylidene- or 2-benzylidene-1-methylhydrazino)-3-methyluracils
A modified[18] 3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay for cellular growth and survival

Synthesis of 6-Azapurines
205

206
J. Ma, F. Yoneda, and T. Nagamatsu
application method developed by Mosmann[19] was used to
determine the growth inhibitory effects (antitumour activity) of
6-azapurines 5a–t against two human cultured tumour cell lines,
CCRF-HSB-2 (human T-cell acute lymphoblastoid leukemia)
and KB (human oral epidermoid carcinoma) cells in vitro. The
antitumour agent, arabinoside cytosine (Ara-C), was used as a
positive control. The results, i.e. 50 % inhibitory concentration
(IC50, mg mLꢀ1) of each compound against both cells are sum-
marised in Table 7. As can be seen from the table of evaluated
antitumour activities, some 3-aryl derivatives of 6-azapurines
5 exhibited moderate antitumour activities with IC50 values
of ,15.0–45.0 mg mLꢀ1 against CCF-HSB-2 cells and of
,15.0–50.0 mg mLꢀ1 against KB cells. Among them, the 3-(4-
isopropylphenyl) derivative 5m exhibited the highest anti-
tumour activity with an IC50 value of 15.6 mg mLꢀ1 against
CCF-HSB-2 cells and 31.0 mg mLꢀ1 against KB cells. The 3-(4-
chlorophenyl) derivative 5i also has high antitumour activity
with an IC50 value of 26.4 mg mLꢀ1 against CCF-HSB-2 cells
and 16.8 mg mLꢀ1 against KB cells.
5,6-dioxo-1,4,5,6-tetrahydro-1,2,4-triazines 6 by further hydro-
lysis of the 6-azapurines 5. The synthesised 6-azapurines 5 were
screened in vitro against two human cultured tumour cell lines,
CCRF-HSB-2 and KB, and some of the derivatives displayed
prospective antitumour activities, and can be new lead com-
pounds as antitumour agents. Further optimisation of these new
compounds may possibly lead to more active molecules against
tumour cells.
The reliable and facile synthetic method for preparing
6-azapurines, and the study of the chemical degradation (trans-
formation) of naturally occurring antibiotics (toxins) such as
toxoflavin, fervenulin, and reumycin are noteworthy owing to
the possible discovery of further biological activity, and may
provide explications for the detoxification for these toxins.
Experimental
Melting points were determined using a Yanaco micro-melting
point apparatus (MP-500D) and were uncorrected. IR spectra
were recorded using a JASCO FT/IR-200 spectrophotometer
1
as Nujol mulls. H and 13C NMR spectra were obtained in
Conclusion
DMSO-d6 using a Varian VXR 300 MHz spectrophotometer at
300 and 75 MHz, respectively, and chemical shift values are
expressed as d values (ppm) relative to tetramethylsilane
(TMS) as an internal standard. Coupling constants are given in
Hz and signals are quoted as follows: s, singlet; d, doublet;
t, triplet; q, quartet; br, broad. UV spectra were recorded in
EtOH with a Jasco V-600 spectrophotometer. All reagents
were of commercial quality from freshly opened containers
and were used without further purification. Reaction progress
was monitored by analytical thin-layer chromatography (TLC)
on pre-coated glass plates (silica gel 60 F254-plate-Merck) and
products were visualised by UV light. The reaction tempera-
tures are indicated as the temperature of the oil bath used
for heating. Column chromatography was accomplished on
Daisogel IR-60 (63/210 mm, Daiso Co.) and the developing
solvent used was a mixture of CH2Cl2 and EtOH. All starting
materials, toxoflavins 1 and reumycins 3, were prepared
according to previous reports.[4a–e,9d]
In this study, we demonstrated a facile and general synthesis
of 6-azapurines 5 and 7 in good yields by transformation of
toxoflavins 5 and reumycins 3 by alkaline hydrolysis involv-
ing a benzilic acid type rearrangement, and the formation of
Me
N
H
N
Me
N
R
O
O
R
NH
O
ꢁ
N
O
N
N
N
Me
N
Me
NH2
6-azapurines 5a–k
1,2,4-triazines 6a–k
a: R ꢀ H
b: R ꢀ Me
c: R ꢀ Ph
g: R ꢀ 4-HO-C6H4
h: R ꢀ 4-Me-C6H4
i: R ꢀ 4-MeO-C6H4
d: R ꢀ 4-F-C6H4
e: R ꢀ 4-Cl-C6H4
f: R ꢀ 4-Br-C6H4
j: R ꢀ 3,4-(MeO)2-C6H3
k: R ꢀ 3,4,5-(MeO)3-C6H2
Synthesis of 6-Azapurines 5a–t by Ring Contraction
of Toxoflavins 1a–t: General Procedure
Scheme 3. Reagents and conditions: 10 % NaOH in EtOH, reflux, 4–6 h,
A solution of a 1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-
5,7(1H,6H)dione 1a–t (1.4–2.6 mmol) in 10 % aqueous NaOH
and then 10 % aq. HCl.
Table 3. Physical data and UV (EtOH) spectra for compounds 6a–k
Compd
R
Yield [%] MpA [8C]
l
max [nm]
(log e [dm3 molꢀ1 cmꢀ1])
Formula
Analysis [%] Calc. (found)
H
C
N
6a
6b
6c
6d
6e
6f
H
36
41
84
72
83
86
76
87
83
78
89
205–206
216–217
244
219 (4.06), 263 (3.87)
221 (4.12), 268 (3.93)
236 (4.17), 283 (3.99)
240 (4.25), 282 (4.08)
243 (4.28), 285 (4.06)
245 (4.23), 286 (4.02)
248 (4.38), 282 (4.21)
242 (4.23), 277 (4.09)
251 (4.35), 285 (4.23)
257 (4.48), 286 (4.41)
262 (4.55), 287 (4.52)
C4H5N3O2
C5H7N3O2
37.80 (37.85) 3.97 (3.71) 33.06 (32.80)
42.55 (42.38) 5.00 (5.13) 29.77 (29.67)
57.41 (57.36) 4.66 (4.72) 20.09 (20.23)
Me
Ph
C
10H9N3O2ꢁ1/3H2O
4-F-C6H4
4-Cl-C6H4
4-Br-C6H4
4-HO-C6H4
4-Me-C6H4
4-MeO-C6H4
3,4-(MeO)2-C6H3
3,4,5-(MeO)3-C6H2
257
C10H8FN3O2ꢁ1/4H2O 53.22 (53.17) 3.80 (3.85) 18.62 (18.71)
263–264
272–274
.300
C10H8ClN3O2
50.54 (50.52) 3.39 (4.43) 17.68 (17.71)
42.58 (42.54) 2.86 (2.93) 14.90 (14.94)
53.69 (53.62) 4.28 (4.31) 18.78 (18.85)
58.87 (58.73) 5.30 (5.35) 18.72 (19.04)
55.58 (55.50) 4.88 (4.73) 17.68 (17.56)
C10H8BrN3O2
6g
6h
6i
C10H9N3O3ꢁ1/4H2O
C11H11N3O2ꢁ2/5H2O
C11H11N3O3ꢁ1/4H2O
247
256–257
266–267
280
6j
C12H13N3O4ꢁ1/10H2O 54.38 (54.24) 5.02 (5.23) 15.85 (16.12)
6k
C13H15N3O5ꢁ1/2H2O
51.65 (51.32) 5.34 (5.14) 13.90 (13.61)
AAll compounds were obtained as a colourless powder.

Synthesis of 6-Azapurines
207
solution (10–20 mL) was stirred at 5–258C for 1–3 days (for
1a–e) or 60–708C for 10–60 min (for 1f–t). The solution was
then adjusted to pH 7 with 10 % HCl under cooling on ice–water
and concentrated to dryness under vacuum. The residue
was crystallised from a mixture of EtOH/H2O to give the pure
6-azapurines 5a–t as colourless or yellow needles (see Tables 1
and 2, and the Supplementary Material).
dC ((CD3)2SO/TMS) for 5a: 26.29 (5-NCH3), 40.66
(1-NCH3), 145.34 (C3), 151.30 (C4a), 154.36 (C7a), 162.94
(C6); dC ((CD3)2SO/TMS) for 5b: 22.72 (3-CH3), 26.20
(5-NCH3), 40.46 (1-NCH3), 149.69 (C3), 153.97 (C4a), 154.40
(C7a), 163.27 (C6); dC ((CD3)2SO/TMS) for 5f: 26.42 (5-NCH3),
0
41.04 (1-NCH3), 126.89 (C4 ), 128.94 (C3 ,5 ), 130.82 (C2 ,6 ),
0
0
0
0
0
134.33 (C1 ), 150.30 (C3), 151.36 (C4a), 154.73 (C7a),
163.43 (C6).
Ring Degradation of 6-Azapurines 5a–f into
1-Methyl-5,6-dioxo-1,4,5,6-tetrahydro-1,2,4-triazines
6a–k: General Procedure
A solution of an appropriate 1,5-dimethyl-1H-imidazo[4,5-e]
[1,2,4]triazin-6(5H)-one (6-azapurine 5a–k) (300 mg, 0.8–
1.8 mmol) in 10 % ethanolic NaOH solution (10–20 mL) was
heated under reflux for 4–6 h. The solution was then adjusted to
pH 7 with 10 % HCl under cooling on ice–water to afford the
solid. The solid was collected by filtration and washed with H2O
and recrystallised from EtOH or DMF to give the corresponding
1-methyl-5,6-dioxo-1,4,5,6-tetrahydro-1,2,4-triazine 6a and its
3-substituted derivatives 6b–k as shown in Tables 3 and 4. In
order to find a by-product in the case of the degradation reaction
of 5c and 5e, the reaction solution, which removed the solid 6c
and 6e as a major product by filtration, was concentrated under
vacuum to dryness. The residue of 5c and 5e was then subjected
to column chromatography on Daisogel IR-60 using a mixture of
CH2Cl2 and EtOH as eluent to afford methyl urea in 17 (16 mg)
and 25 % (20 mg) yield, respectively.
dC ((CD3)2SO/TMS) for 6a: 38.96 (1-NCH3), 138.21 (C3),
154.88(C5), 155.91 (C6); dC ((CD3)2SO/TMS) for 6c: 39.06
0
(1-NCH3), 127.35 (C3 ,5 ), 129.50 (C2 ,6 ), 131.02 (C4 ), 131.49
0
0
0
0
0
(C1 ), 142.07 (C3), 155.28 (C5), 156.64 (C6); dC ((CD3)2SO/
TMS) for 6h: 21.09 (40-CH3), 38.37 (1-NCH3), 126.58 (C3 ,5 ),
0
0
0
0
0
0
127.42 (C4 ), 129.37 (C2 ,6 ), 140.54 (C1 ), 140.65 (C3), 154.36
(C5), 155.51 (C6).
O
Me
N
N
R
N
N
R
N
MeN
O
N
N
N
H
N
H
O
reumycins 3a–m
6-azapurines 7a–m
a: R ꢀ H
b: R ꢀ Me
c: R ꢀ Ph
d: R ꢀ 4-F-C6H4
e: R ꢀ 3-Cl-C6H4
f: R ꢀ 4-Cl-C6H4
g: R ꢀ 4-Br-C6H4
h: R ꢀ 4-Me-C6H4
i: R ꢀ 4-MeO-C6H4
j: R ꢀ 4-O2N-C6H4
k: R ꢀ 4-HO-C6H4
l: R ꢀ 3,4-(MeO)2-C6H3
m: R ꢀ 3,4,5-(MeO)3-C6H2
Scheme 4. Reagents and conditions: 10 % aq. NaOH in EtOH, reflux
1–2 h, and then 10 % aq. HCl.

208
J. Ma, F. Yoneda, and T. Nagamatsu

Synthesis of 6-Azapurines
209
O
H
ꢂO
O
O
OH
C
N
H
N
N
R
N
R
R
R
R
Hꢁ
OHꢂ
MeN
MeN
MeN
N
N
N
N
N
N
ꢂCO2
O
N
N
Me
O
N
N
Me
O
N
Me
toxoflavins 1
H
Me
Me
N
Me
N
N
N
R
N
R
N
Hꢁ
OHꢂ
ꢂO
O
O
N
N
N
N
N
ꢂH2
N
Me
N
Me
H
Me
HO
6-azapurines 5
H
O
Me
N
Nꢂ
Me
Me
N
R
N
R
OHꢂ
Hꢁ
N
O
N
O
O
H2N
O
H2N
N
N
N
H
N
Me
N
Me
N
O
Me
O
H
Me
H
N
O
R
NH
ꢁ
O
N
NH2
O
N
Me
1,2,4-triazines 6
Scheme 5. Plausible mechanism for the formation of 1,2,4-triazines via 6-azapurines produced by transformation of toxoflavins 1
(7-azapteridines).
0.9–1.7 mmol) in 10 % ethanolic NaOH solution (10–20 mL)
was heated under reflux for 1–2 h. The solution was then
adjusted to pH 7 with 10 % HCl under cooling on ice–water and
concentrated to dryness under vacuum. The residue was crys-
tallised from EtOH to give the corresponding pure 5-methyl-
5H-imidazo[4,5-e][1,2,4]triazin-6(7H)-one (6-azapurines 7a
and b) as colourless needles. On the other hand, in the case
where a solid separated after adjusting to pH 7 with 10 % HCl,
the solid was collected by filtration, washed with H2O and
recrystallised from EtOH or DMF to give the corresponding
pure 6-azapurine derivatives 7c–m as colourless or yellow
needles and as shown in Tables 5 and 6.
Table 7. Evaluation of antitumour activities in vitro of the
6-azapurines 5a–t, 50 % inhibitory concentration (IC50) against tumour
cell lines CCRF-HSB-2 (human T-cell acute lymphoblastoid leukemia)
and KB (human oral epidermoid carcinoma) in vitro
Compd
R
IC50 [mg mL]
CCRF-HSB-2
KB
5a
5b
5c
5d
5e
5f
H
.100
.100
.100
.100
.100
.100
.100
Me
Et
Prn
Pri
57.4
.100
.100
dC ((CD3)2SO/TMS) 7a: 25.38 (5-NCH3), 144.57 (C3),
146.03 (C4a), 151.24 (C7a), 153.21 (C6).
Ph
65.5
76.4
5g
5h
5i
4-F-C6H4
3-Cl-C6H4
4-Cl-C6H4
4-Br-C6H4
4-HO-C6H4
4-Me-C6H4
4-Pri-C6H4
4-MeO-C6H4
3,4-(MeO)2-C6H3
3,4,5-(MeO)3-C6H2
4-AcO-C6H4
4-Me2N-C6H4
2-Furyl
42.9
83.6
26.4
27.3
27.5
57.3
16.8
Supplementary Material
5j
.100
The selected mass, UV, 1H and 13C NMR spectra including 2D
HMQC and HMBC NMR spectra, and the detailed preparations
of 6-azapurines 5a–t are available from the Journal’s website.
5k
5l
.100
40.1
49.4
31.0
51.3
98.3
79.4
15.6
58.5
57.5
63.0
72.1
43.6
65.7
38.3
5m
5n
5o
5p
5q
5r
5s
Acknowledgements
.100
The authors are grateful to the SC-NMR Laboratory of Okayama University,
Japan, for the NMR experiments and are also indebted to Dr N. Ashida,
Biology Laboratory, Research and Development Division, Yamasa Co.,
Choshi, Chiba, Japan, for the measurement of biological activity.
57.9
24.6
70.7
58.0
5t
2-Thienyl
AraC
0.017
0.73
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