Design, Synthesis, and Anticancer Activity of Novel Fused Purine Analogues

Article abstract of DOI:10.1002/jhet.2969

6-Mercaptopurine has been utilized for the synthesis of various fused purine analogues through different chemical reactions to yield [1,4]thiazino[4,3,2-gh]purines 2, 3, 10a,b, 14, (8-oxo-[1,4]thiazino[4,3,2-gh]purin-7(8H)-ylidene) acetate 4, [1,4]thiazepino[4,3,2-gh]purine 6, thiazolo[3,4,5-gh]purine 7, imidazo[1,5,4-gh]purin-5-amine 8, 5-methylimidazo[1,5,4-gh]purine 9, [1,2,4]triazino[4,3-i]purines 16, 18, 21, [1,2,4]triazino[4,5,6-gh]purine 20, 5-methyl-2-(7H-purin-6-yl)-1H-pyrazol-3(2H)-one 22, [1,2,4]triazolo[4,3-i]purine 23, [1,2,5]triazepino[5,4,3-gh]purine 24, and ethyl 6-(2-(ethoxycarbonyl)hydrazinyl)-9H-purine-9-carboxylate 26. Seventeen of the newly synthesized compounds were selected by the NCI, Maryland, USA, and were tested for their anticancer activity in an initial single high dose in the full NCI 60 cell line panel among which [1,4]thiazino[4,3,2-gh]purine-7,8-dione 2, 7-benzyl-[1,4]thiazino[4,3,2-gh]purine 10b, and 3-(2,4-dimethoxyphenyl)-7H-[1,2,4]triazolo[4,3-i]purine 23 were found to possess very potent anticancer activity against most of the cancer cell lines.

Full text of DOI:10.1002/jhet.2969

Month 2017
Design, Synthesis, and Anticancer Activity of Novel Fused Purine
Analogues
a
b
A. Y. Hassan, M. T. Sarg,
A. H. Bayoumi,c and F. G. A. Kalafb
*
aDepartment of Organic Chemistry, Faculty of Science (Girls), Al-Azhar University, Cairo, Egypt
bDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
cDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo, Egypt
*
E-mail: m.t.sarg@hotmail.com
Received April 11, 2017
DOI 10.1002/jhet.2969
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
6-Mercaptopurine has been utilized for the synthesis of various fused purine analogues through different
chemical reactions to yield [1,4]thiazino[4,3,2-gh]purines 2, 3, 10a,b, 14, (8-oxo-[1,4]thiazino[4,3,2-gh]
purin-7(8H)-ylidene) acetate 4, [1,4]thiazepino[4,3,2-gh]purine 6, thiazolo[3,4,5-gh]purine 7,
imidazo[1,5,4-gh]purin-5-amine 8, 5-methylimidazo[1,5,4-gh]purine 9, [1,2,4]triazino[4,3-i]purines 16, 18,
21, [1,2,4]triazino[4,5,6-gh]purine 20, 5-methyl-2-(7H-purin-6-yl)-1H-pyrazol-3(2H)-one 22, [1,2,4]
triazolo[4,3-i]purine 23, [1,2,5]triazepino[5,4,3-gh]purine 24, and ethyl 6-(2-(ethoxycarbonyl)hydrazinyl)-
9H-purine-9-carboxylate 26. Seventeen of the newly synthesized compounds were selected by the NCI,
Maryland, USA, and were tested for their anticancer activity in an initial single high dose in the full NCI
60 cell line panel among which [1,4]thiazino[4,3,2-gh]purine-7,8-dione 2, 7-benzyl-[1,4]thiazino[4,3,2-gh]
purine 10b, and 3-(2,4-dimethoxyphenyl)-7H-[1,2,4]triazolo[4,3-i]purine 23 were found to possess very
potent anticancer activity against most of the cancer cell lines.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
NMR spectrum of compound 2 showed only two singlets
at δ 8.17 and 8.35 ppm corresponding to purine C2 and C8
protons, respectively. Also, the target compound
thiazino[4,3,2-gh]purine derivative 3 was synthesized
Purine is the most ubiquitous nitrogen-containing
heterocycle in nature as it is the core structure of adenine
and guanine in RNA and DNA [1]. Purine is one of the
most important rings in the class of heterocycles as it
exhibits a wide range of biological activities including
antitumor [2,3], antiviral [4,5], immunosuppressant [6,7],
through refluxing 6-mercaptopurine
1 with maleic
anhydride in methanol containing drops of concentrated
sulfuric acid. The reaction is postulated to proceed via
initial addition of the thiol group on the maleic anhydride
double bond followed by intramolecular cyclization
through the attack of purine NH function on the anhydride
carbonyl group. 1H NMR spectrum of compound 3
showed a singlet signal at δ 1.90 ppm corresponding to
CH2 protons and a deuterium oxide exchangeable singlet
at δ 8.16 ppm corresponding to carboxylic proton.
antimicrobial
[8],
anti-inflammatory
[9],
and
antileishmanial [10] activities.
Therefore, it was aimed to synthesize various novel
purine derivatives comprising different five-membered and
six-membered rings fused to the purine system through
[gh] or [i] sites and the study of their anticancer activity.
The reaction of 6-mercaptopurine 1 with dimethyl
acetylenedicarboxylate yielded both thiazine derivative 4
and the open chain analogue 5. The reaction mechanism
is suggested to proceed through the initial nucleophilic
addition of the thiol function of 6-mercaptopurine 1 on
the acetylinic carbons followed by nucleophilic attack of
purine-N7 NH function on carbonyl ester group with
subsequent elimination of a methanol molecule to yield
the thiazine derivative 4. 1H NMR spectrum of
compound 5 displayed a singlet signal at δ 3.86 ppm
integrated for six protons due to two methyl ester
protons, as well as, a singlet signal at δ 5.90 ppm
corresponding to olefinic proton, in addition to a
RESULTS AND DISCUSSION
Chemistry.
Literature survey revealed that [1,4]
thiazine ring was widely reported as potent anticancer
agent [11,12]. So novel [1,4]thiazino[4,3,2-gh]purine-7,8-
dione 2 was obtained by fusion of equivalent amounts of
diethyl oxalate and 6-mercaptopurine (Scheme 1). 1H
© 2017 Wiley Periodicals, Inc.

A. Y. Hassan, M. T. Sarg, A. H. Bayoumi, and F. G. A. Kalaf
Vol 000
Scheme 1
deuterium oxide exchangeable singlet at δ 13.60 ppm
corresponding to NH proton.
Furthermore, 6-mercaptopurine 1 was reacted with
cinnamoyl chloride in benzene/pyridine mixture to
produce 7-phenyl-7H-[1,4]thiazepino[4,3,2-gh]purin-9(8H)-
one 6 (Scheme 2). The reaction is suggested to proceed
first through nucleophilic attack of the thiol function on
the acid chloride moiety of cinnamoyl chloride with
subsequent elimination of a hydrochloride molecule to
yield the α, β-unsaturated carbonyl intermediate followed
by intramolecular addition of purine-N7-NH function on
the double bond of the α, β-unsaturated carbonyl side
Scheme 2
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Design, Synthesis, and Anticancer Activity of Novel Fused Purine Analogues
chain to yield the desired product. The most informative
proof for the structure of compound 6 was 1H NMR
spectrum, as it revealed two singlets at δ 2.93 and
3.16 ppm corresponding to thiazepine CH2 and CH–C6H5
protons, respectively. In 1964, Dyer and Bender [13]
reported the synthesis of ethyl purin-6-yl thiocarbonate
compounds via their prolonged reflux in acidic medium
in case of aliphatic methyl ketones [16,17] or by heating
the S-alkylated derivative obtained from aromatic methyl
ketones either with polyphosphoric acid (PPA) at 100°C
[18,19] or in glacial acetic acid/acetic anhydride mixture
containing few drops of concentrated sulfuric acid [12].
However, the reaction of of 6-mercaptopurine 1 with
methyl propyl ketone and methyl benzyl ketone in glacial
acetic acid containing few drops of concentrated sulfuric
acid yielded both the cyclic thiazinopurines 10a,b and the
open chain S-alkylated derivatives 11a,b (Scheme 3). The
reaction was postulated to proceed via the initial
formation of S-alkylated analogues followed by
intramolecular condensation through the nucleophilic
attack of the purine N7–H on the ketonic carbonyl
function. 1 H NMR spectrum of compound 10b
displayed a singlet signal at δ 7.16 ppm corresponding
to thiazine C–H protons. While 1H NMR spectrum of
compounds 11a and 11b revealed two singlet signals at
δ 1.91and 2.06 ppm due to CH2 protons beside to two
deuterium oxide exchangeable singlet signals at δ 13.60
and 13.75 ppm attributed to purine NH proton,
respectively.
through stirring of 6-mercaptopurine
1 with ethyl
chloroformate in dry dimethylformamide containing
potassium carbonate as a catalyst. In this work, 6-
mercaptopurine 1 was refluxed with ethyl chloroformate
in presence of sodium ethoxide as a base to yield the
thiazolo[3,4,5-gh]purin derivative 7.
Our scope was extended to study the effect of various
five-membered rings fused to the purine nucleus and
encouraged by the fact that various reports stated potent
anticancer activities for imidazole rings [14,15]. So
compound
1 was reacted with different reagents:
cyanamide and thioacetamide to produce imidazo[1,5,4-
gh]purin-5-amine 8 and 5-methylimidazo[1,5,4-gh]purine
9, respectively.
The reactions were suggested to proceed through the
initial nucleophilic substitution of 6-mercaptopurine SH
function with subsequent elimination of a hydrogen
sulfide molecule followed by intramolecular addition of
purine-N7-NH on the cyano and thioxo functions to yield
6-Mercaptopurine
1
was reacted with different
halocompounds, such as chloroacetone, phenacyl
chloride, and chloroacetonitrile, to afford compounds
1
the target compounds 8 and 9; respectively. The H NMR
1
spectrum of compound 8 revealed a deuterium oxide
exchangeable singlet signal at δ 7.21 ppm corresponding
12a,b, 13a,b, and 14, respectively. H NMR spectrum of
compound 12b showed a singlet signal at δ 5.01 ppm
corresponding to thiazine–CH proton, while 1H NMR
spectrum of compound 14 revealed a deuterium oxide
exchangeable singlet signal at δ 4.50 ppm corresponding
to NH2 protons and a singlet signal at δ 5.59 ppm
corresponding to thiazine–CH proton.
1
to NH2 protons, while H NMR spectrum of compound 9
revealed a singlet signal at δ 1.97 ppm corresponding to
CH3 protons.
Moreover, the reaction of thiol-bearing compounds with
different methyl ketones was reported to yield cyclic
Scheme 3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Y. Hassan, M. T. Sarg, A. H. Bayoumi, and F. G. A. Kalaf
Vol 000
Furthermore, Montgomery and Holum [16] reported the
synthesis of 6-hydrazinyl-9H-purine 15 from the reaction
of 6-chloropurine with anhydrous hydrazine by stirring at
room temperature. However, the target 6-hydrazinyl-9H-
purine 15 was synthesized via fusion of 6-mercaptopurine
with hydrazine hydrate 99% (Scheme 4), which was
further utilized for the synthesis of various purine
derivatives among which its fusion with chloroacetone to
yield tetrahydro[1,2,4]triazino[4,3-i]purin-4-ol derivative
3.60 ppm corresponding to CH3–C=N and CH3–C=O
protons, respectively, besides to a singlet signal at δ
3.80 ppm due to CH2 protons. In addition to two
deuterium oxide exchangeable singlets at δ 7.08 and
13.70 ppm due to purine C6–NH and purine NH protons,
respectively. Furthermore, [1,2,4]triazino[4,5,6-gh]purin-
3-amine 20 was obtained via the reaction of the
6-hydrazinylpurine 15 with phenyl isothiocyanate in
pyridine. 1H NMR spectrum of compound 20 revealed
two deuterium oxide exchangeable singlet signals at δ
4.90 and 9.81 ppm attributed to phenyl–NH and triazine–
NH protons, respectively.
Moreover, diethyl oxalate was reported to react with
hydrazinyl analogues through nucleophilic attack on its
carbonyl ester functions with the subsequent elimination
of two ethanol molecules yielding their corresponding
dioxo heterocyclic rings [20,21]. So 6-hydrazinylpurine
15 was fused with diethyl oxalate to yield [1,2,4]
triazino[4,3-i]purine-3,4(2H,10H)-dione 21. The IR
spectrum of compound 21 revealed a strong absorption
band at 1676 cmÀ1 corresponding to carbonyl functions.
Furthermore, the work was extended to fuse the hydazinyl
purine derivative 15 with ethyl acetoacetate to yield
7H-purin-6-yl-1H-pyrazolone analogue 22 (Scheme 5). 1H
NMR spectrum of compound 22 revealed two singlet
signals at δ 2.27 and 7.56 ppm attributed to methyl protons
and pyrazolone–C4–H, respectively. Also, reaction of
1
16 and hydrazinylpropan-2-one purine analogue 17. H
NMR spectrum of compound 16 revealed a singlet at δ
5.01 corresponding to triazine–CH2 protons in addition to
three deuterium oxide exchangeable singlet signals at δ
5.98, 9.22, and 9.70 ppm attributed to OH, triazine NH,
and purine NH protons, respectively (Fig. 1).
Compound 15 was also fusion with chloroacetonitrile to
1
yield [1,2,4]triazino[4,3-i]purin-4-amine analogue 18. H
NMR spectrum of compound 18 showed three deuterium
oxide exchangeable singlet signals at δ 5.05, 10.00,
and 13.72 ppm corresponding to NH2, NH–triazine, and
purine NH proton, respectively. Attempts to prepare
dimethyl pyrazolopurine analogue through the reaction of
compound 15 and acetyl acetone were unsuccessful.
However, with equimolar amounts of 6-hydrazinylpurine
15 and acetylacetone, the open chain hydrazinopentane-2-
one analogue 19 was obtained. 1H NMR spectrum of
compound 19 showed two singlet signals at δ 3.18 and
Scheme 4
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Month 2017
Design, Synthesis, and Anticancer Activity of Novel Fused Purine Analogues
Figure 1. Proposed mechanism for synthesis of compound 16.
compound 15 with 2,4-dimethoxybenzoyl chloride in dry
pyridine afforded [1,2,4]triazolo[4,3-i]purine derivatives
23. IR spectrum of compound 23 was devoid of any
absorption bands due to carbonyl function. While its H
NMR spectrum revealed a singlet signal at δ 4.12 ppm
integrated for six protons of the two methoxy groups.
Moreover 6-hydrazinyl-9H-purine 15 was heated under
reflux with phenacyl chloride to yield the N-alkylated
analogue 25 as well as the dihydro[1,2,5]triazepino[5,4,3-
gh]purine 24, which was suggested to be obtained via
intramolecular cyclocondensation of compound 25. 1H NMR
spectrum of compound 24 revealed a deuterium oxide
exchangeable singlet signal at δ 7.20 ppm attributed to
triazepine–NH proton, besides to a singlet signal at δ
7.83 ppm due to triazepine–C3–H. However, 1H NMR
spectrum of compound 25 displayed a singlet signal at δ
2.09 ppm due to CH2 protons. In addition to three
deuterium oxide exchangeable singlet signals at δ 3.52,
8.11, and 12.23 ppm attributed to NH, purine C4–NH, and
purine NH protons, respectively.
1
Finally, compound 15 was fused with excess ethyl
chloroformate to give ethyl 6-(2-(ethoxycarbonyl)
hydrazinyl)-9H-purine-9-carboxylate
26
that
was
rationalized via dehydrohalogenation reactions between
the C6–hydrazinyl group and N9–H of the purine ring and
ethyl chloroformate. IR spectrum of compound 26
Scheme 5
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Y. Hassan, M. T. Sarg, A. H. Bayoumi, and F. G. A. Kalaf
showed two absorption bands at 1746 and 1700 cmÀ1 due
Vol 000
Anticancer screening.
Seventeen of the newly
to two ester carbonyl functions. While, its electron impact
mass spectrum revealed a peak corresponding to M+. + 1
at m/z 295 (0.31).
synthesized compounds were selected by development
therapeutic program (DTP), division of cancer treatment
and diagnosis, National Cancer Institute (NCI), Bethesda,
Table 1
Themeangrowthinhibitionpercent,deltavalues,andthepercentgrowthinhibitionagainstsomesubpanelcelllinesofselectedcompoundsofSchemes1and2.
Comp. no.
(NCI-No.)
Mean growth %
(delta)
Panel: subpanel cell line (growth inhibition percent)
2 (778899)
57.69 (33.57)
Leukemia: CCRF-CEM (60.41), HL-60 (TB) (62), K-562 (72.62), MOLT-4 (75.88), RPMI-8226 (64.49),
SR (51.85).
Non-small cell lung cancer: HOP-62 (57.59), HOP92 (13.56), NCI-H226 (35.81), NCI-H23 (27.53),
NCIH322 (32.86), NCI-H460 (25.94), NCI-H522 (28.87).
Colon cancer: COLO 205(22.57), HCC-2998(62.8), HCT-116 (60.86), HCT-15 (39.08), HT29 (44.11),
KM12 (42.66), SW620 (43.1).
CNS cancer: SF268 (38.23), SF295 (61.36), SF539 (56.44), SNB-75 (37.12), U251 (20.03).
Melanoma: LOXIMVI (75.4), MALME-3M (16.16), M14 (61.26), MDA-MB-435(69.93), SK-MEL-
2(46.79), SK-MEL5 (15.59), UACC257 (34.68), UACC-62 (65.68).
Ovarian cancer: IGROVI (20.07), OVCAR-3(44.84), OVCAR-4 (18.63), OVCAR-5 (35.48), OVCAR-8
(44.94), NCI/ADR-RES (50.46), SK-OV-3 (70.77).
Renal cancer: 786-0(65.14), A498 (20.44), ACHN (29.15), CAKI-1 (60.07), TK-10 (52.37), UO-
31(56.55).
Prostate cancer: PC-3 (17.54), DU-145 (45.12).
Breast cancer: MCF7 (70.29), MDA-MB-231/ATCC (48.68), HS578T (15.53), BT-549 (21.37), T-47D
(49.05), MDA –MB-468 (58.14).
6 (778890)
7 (778896)
98.97 (20.39)
58.66 (49.83)
Non-small cell lung cancer: HOP92 (10.66).
CNS cancer: SNB-75 (21.42).
Melanoma: MALME-3M (10.52).
Renal cancer: UO-31 (20.79).
Breast cancer: MCF7 (10.51), T-47D (10.85), MDA –MB-468 (12.31).
Leukemia: CCRF-CEM (45.22), HL-60 (TB) (24.90), K-562 (74.80), MOLT-4 (64.11), RPMI-8226
(74.44), SR (66.78).
Non-small cell lung cancer: HOP-62 (58.21), HOP92 (50), NCI-H226 (38.57), NCI-H23 (33.93), NCIH
322 (22.10), NCI-H460 (14.33).
Colon cancer: COLO 205 (16.48), HCC-2998 (20.46), HCT-116 (49.06), HCT-15 (22.02), HT29 (43.76),
KM12 (29.96), SW 620 (26.89).
CNS cancer: SF268 (17.98), SF295 (61.65), SF539 (54.21), SNB-75 (53.85), U251 (12.59).
Melanoma: LOXIMVI (87.32), MALME-3 M (26.4), M14 (85.46), MDA-MB-435 (70.5), SK-MEL-2
(18.06), SK-MEL5 (10.68), UACC257 (11.88), UACC-62 (43.2).
Ovarian cancer: OVCAR-3(82.39), OVCAR-4(51.34), OVCAR-5(34.71), OVCAR-8 (21.71), NCI/
ADR-RES (29.89), SK-OV-3 (77.25).
Renal cancer: 786-0(65.40), ACHN (19.72), CAKI-1 (56.91), SN12C (15.05), TK-10 (73.05), UO-
31(47.43).
Prostate cancer: PC-3 (24.17), DU-145 (66.35).
Breast cancer: MCF7 (77.83), MDA-MB-231/ATCC (56.31), HS578T (27.33), T-47D (65.46), MDA –
MB-468 (91.17).
8 (778897)
87.74 (59.55)
Leukemia: CCRF-CEM (22.11), K-562 (36.33), MOLT-4 (12.36), RPMI-8226 (31.78), SR (33.2).
Non-small cell lung cancer: HOP-62(13.94), HOP92 (50.01), NCI-H226 (16.2), NCI-H23(14.67).
Colon cancer: HCT-116 (11.5), SW 620 (9.2).
CNS cancer: SF295 (11.57), SNB-75 (29.44).
Melanoma: LOXIMVI (40.87), M14 (36.43), MDA-MB-435 (10.48).
Ovarian cancer: OVCAR-3 (15.54), OVCAR-4 (34.54), OVCAR-5 (10.13), NCI/ADR-RES (9.91), SK-
OV-3 (9.58).
Renal cancer: ACHN (9.22), CAKI-1 (12.93), UO-31(17.49).
Leukemia: CCRF-CEM (22.28), K-562 (64.24), MOLT-4 (38.74), RPMI-8226 (37.57), SR (35.84).
Non-small cell lung cancer: HOP-62(18.81), HOP92 (30.75), NCI-H226 (9.47).
Colon cancer: HCT-2998(12.4).
9 (778904)
85.84 (53.31)
CNS cancer: SNB-75 (32.98).
Melanoma: LOXIMVI (60.25), M14 (53.82), MDA-MB-435 (11.07), UACC62 (12.57).
Ovarian cancer: OVCAR-3 (21.72), OVCAR-4 (29.91), SK-OV-3 (38.44).
Renal cancer: 786-0 (14.31), A498 (10.04), CAKI-1 (17.86), UO-31(10.64).
Prostate cancer: DU145 (25.12).
Breast cancer: MCF7 (27.92), MDA-MB-231/ATCC (12.35), T-47D (32.O2), BT-549 (12.98), MDA –
MB-468 (67.47).
Journal of Heterocyclic Chemistry
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Month 2017
Design, Synthesis, and Anticancer Activity of Novel Fused Purine Analogues
Maryland, USA, to be evaluated in vitro in a single high
dose (10 μmol) against 60 human tumor cell lines for
their primary anticancer activities [22]. The mean growth
inhibition percent, delta values, and percent growth
inhibition against some subpanel cell lines, of the
selected compounds, are presented in Tables 1–3.
As revealed from the results presented in Tables 1–3,
and as a result of studying the effect of fusion of different
heterocyclic rings of different sizes to the purine system,
it could be concluded that fusion of six-membered rings
as in compound 2 that possesses 2,3-dioxo[1,4]thiazine
ring system fused via the [gh] junction exhibited
remarkable growth inhibition anticancer activity against
most cell lines. Furthermore, upon studying the effect of
various substituents attached to [1,4]thiazine ring fused to
the purine ring as in compounds 10b, 12a,b, and 14. It
was found that the presence of a benzyl group on seven
position of the [1,4]thiazino[4,3,2-gh]purine as in
compound 10b exhibited strong anticancer activity
against some cancer cell lines. However, replacement of
benzyl group of compound 10b with methyl, phenyl, or
amino functions as in compounds 12a, 12b, and 14,
respectively, diminished the anticancer activity against all
cell lines.
Moreover, investigating the effect of fusion of different
substituted [1,2,4]triazine ring through the [4,3-i] position
of the purine nucleus as in compounds 16, 18, and 21
resulted in inactive anticancer agents. It is to be noted
Table 2
The mean growth percent, delta values, and the percent growth inhibition against some subpanel cell lines of selected compounds of Schemes 3 and 4.
Comp. no.
(NCI-No.)
Mean growth %
(delta)
Panel: subpanel cell line (growth inhibition percent)
10b (778894)
55.09 (66.93)
Leukemia: CCRF-CEM (41.12), HL-60 (TB) (24.89), K-562 (85.61), MOLT-4 (68.86), RPMI-8226
(72.03), SR (63.19).
Non-small cell lung cancer: A549/ATCC (12.7), HOP-62(60.09), HOP92 (43.37), NCI-H226 (46.33),
NCI-H23 (38.70), NCIH322 (27.03), NCI-H460 (16.20).
Colon cancer: COLO 205(17.99), HCC-2998 (29.84), HCT-116 (42.92), HCT-15 (28.22), HT29 (44.33),
KM12 (30.34), SW 620 (31.01).
CNS cancer: SF268 (28.14), SF295 (70.03), SF539 (56.78), SNB-75 (54.21), U251 (21.23).
Melanoma: LOXIMVI (90.69), MALME-3M (31.95), M14 (86.8), MDA-MB-435 (73.48), SK-MEL-2
(24.98), SK-MEL- 5 (13.87), UACC257 (25.07), UACC-62 (45.52).
Ovarian cancer: IGROV1 (13.24), OVCAR-3 (84.19), OVCAR-4(59.86), OVCAR-5(29.84), OVCAR-8
(26.5), NCI/ADR-RES (43.29), SK-OV-3 (51.34).
Renal cancer: 786-0(70.74), ACHN (12.76), CAKI-1 (60.43), SN-12C (13.56), TK-10 (71.43), UO-31
(51.74).
Prostate cancer: PC-3 (21.83), DU-145 (72.73).
Breast cancer: MCF7 (79.72), MDA-MB-231/ATCC (64.94), HS578T (24.71), T-47D (70.42).
Lethality against MDA –MB-468 (11.84).
12a (778889)
12b (778892)
102.32 (22.50)
97.71 (23.35)
Non-small cell lung cancer: HOP92 (20.18).
CNS cancer: SNB-75 (11.74).
Breast cancer: T-47D (14.51).
Non-small cell lung cancer: HOP92 (25.64), NCI-H226 (10.36), NCI-H23 (10.20).
Colon cancer: SW 620 (9.73).
CNS cancer: SNB-75 (23.1).
Renal cancer: UO-31(21.11).
Prostate cancer: PC-3 (10.43).
Breast cancer: MCF7 (11.43), MDA –MB-468 (17.97).
Non-small cell lung cancer: HOP92 (25.12), NCI-H322 (9.97), NCI-H23 (10.63).
CNS cancer: SNB-75 (18.23).
14 (778891)
99.26 (24.38)
Renal cancer: UO-31(14.4).
Prostate cancer: PC-3(9.41).
Breast cancer: MDA –MB-468 (17.45).
16 (778901)
18 (778902)
19 (778903)
20 (778893)
102.37 (11.79)
105.51 (20.29)
103.48 (14.07)
93.16 (57.04)
Renal cancer: UO-31(9.42).
Renal cancer: A498 (14.78).
Renal cancer: A498 (10.59).
Leukemia: MOLT-4 (63.88), SR (32.34).
Non-small cell lung cancer: HOP-92 (35.04), NCI-H23 (21.84).
Colon cancer: HCT-15(11.97), KM12 (30.36).
CNS cancer: SNB-75 (30.36).
Melanoma: LOXIMVI (9.24), MALME-3M (9.37).
Ovarian cancer: OVCAR-3(10.07), OVCAR-4(11.63), NCI/ADR-RES (9.40).
Renal cancer: UO-31(26.61).
21 (778898)
104.56 (22.49)
Prostate cancer: PC-3 (24.02).
Breast cancer: MCF7 (9.01), MDA-MB-231/ATCC (13.33), MDA –MB-468 (16.88), HS 578T (17.93).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Y. Hassan, M. T. Sarg, A. H. Bayoumi, and F. G. A. Kalaf
Vol 000
Table 3
The mean growth percent, delta values, and the percent growth inhibition of some subpanel cell lines of selected compounds of Scheme 5.
Comp. no.
(NCI-No.)
Mean growth
percent (delta)
Panel: subpanel cell line (growth inhibition percent)
23 (778895)
29.22 (45.46)
Leukemia: CCRF-CEM (87.39), HL-60 (TB) (87.2), K-562 (88.47), MOLT-4 (91.27), RPMI-8226
(90.68), SR (84.52).
Non-small cell lung cancer: A549/ATCC (28.40), HOP-62(74.18), HOP92 (47.10), NCI-H226 (56.50),
NCI-H23 (77.65), NCIH322 (67.11), NCI-H460 (60.64).
Colon cancer: COLO 205(67.49), HCC-2998 (73.73), HCT-116 (85.22), HCT-15 (70.77), HT29
(80.84), KM12 (77.48), SW 620(76.78).
CNS cancer: SF268 (67.02), SF295 (79.88), SF539 (85.44), SNB-75 (60.85), U251 (58.95).
Melanoma: LOXIMVI (94.37), MALME-3M (62.62), M14 (89.52), MDA-MB-435 (86.83), SK-MEL-
2 (70.13), SK-MEL- 28 (15.27), SK-MEL-5 (51.05), UACC257 (59.37), UACC-62 (76.31).
Ovarian cancer: IGROV1 (54.19), OVCAR-3(86.61), OVCAR-4(63.15), OVCAR-5(83), OVCAR-8
(81.56),NCI/ADR-RES(82.39) , SK-OV-3(88.61).
Renal cancer: 786-0(78.69), A498 (19.85), ACHN (71.81), CAKI-1 (79.07), SN-12C (44.74), TK-10
(89.20), UO-31(76.68).
Prostate cancer: PC-3 (59.29), DU-145 (73.47).
Breast cancer: MCF7 (92.81), MDA-MB-231/ATCC (70.99), HS578T (45.65), T-47D (71.45).
Lethality against MDA –MB-468 (16.24).
24 (778905)
25 (778906)
100.29 (23.99)
74.93 (52.94)
Non-small Cell Lung cancer: HOP-92 (15.4).
Colon cancer: HCC-2998 (23.7).
Renal cancer: UO-31(16.55).
Breast cancer: MCF7 (11.51), MDA-MB-231/ATCC (14.19), T-47D (10.02).
Leukemia: CCRF-CEM (25.41), HL-60 (TB) (21.26), K-562 (75.72), MOLT-4 (48.35), RPMI-8226
(43.74), S R (52.32).
Non-small cell lung cancer: HOP-62(44.71), NCI-H226 (14.94), NCI-H23 (9.97), NCIH522 (44.42).
Colon cancer: HCT-116 (24.10), HT29 (20.92), KM12 (14.16).
CNS cancer: SF295 (32.87), SF539 (25.19), SNB-75 (40.80).
Melanoma: LOXIMVI (78.01), MALME-3M (14.06), M14 (67.59), MDA-MB-435 (46.59), SK-MEL-
2(10.99), UACC-62 (21.35).
Ovarian cancer: OVCAR-3(44.26), OVCAR-4(30.30), OVCAR-5(17.8), OVCAR-8 (16.39), NCI/
ADR-RES (19.91), SK-OV-3(64.73).
Renal cancer: 786-0(42.62), CAKI-1 (31.79), TK-10 (55.49),UO-nnnn(29.99).
Prostate cancer: DU-145 (45.65).
Breast cancer: MCF7 (52.88), MDA-MB-231/ATCC (29.08), BT-549 (22.04), T-47D (52.05), MDA –
MB-468 (67.51).
that changing the site of fusion of triazine ring to [4,5,6-gh]
as in compound 20 led to strong growth inhibitory activity
against leukemia MOLT-4 cell line.
Finally, the attachment of different side chains to the
purine C6 position as in compounds 19 and 25 bears
hydrazonopentanone and hydrazinylphenylethanone side
chains, respectively. Compound 19 was abolished of
any anticancer activity, while compound 25 showed
strong growth inhibitory activity against few cancer cell
lines.
However, fusion of seven-membered rings as [1,4]
thiazepine as in compound 6 or [1,2,5]triazepine ring as
in compound 24 resulted in inactive anticancer agents.
Furthermore, fusion of five-membered rings to the
purine nucleus led to great variation in the anticancer
activity, in which fusion of 2-thiazolone ring as in
compound 7 showed strong anticancer activity against
some cell lines. While, fusion of 2-aminoimidazole ring
to the purine nucleus as in compound 8 resulted in weak
anticancer activity. However, replacing the 2-amino
function by 2-methyl group as in compound 9 exerted
strong growth inhibitory activity against few cell lines.
Besides, fusion of [1,2,4]triazole ring to the purine
backbone as in compound 23 resulted in strong growth
inhibitory anticancer activity against most of the cell
lines, in addition to lethal activity against breast cancer
MDA-MB-468 cell line.
CONCLUSIONS
It can be concluded that, by studying the effect of
fusion of different five-membered and six-membered
rings to the purine system, fusion of 1,4-thiazine ring to
the purine nucleus via the [gh] junction bearing either
two oxo functions or a benzyl group as in compounds
2 and 10b, respectively, led to highly potent anticancer
activity against almost all cell lines. Besides, fusion of
1,2,4-triazole ring through [4,3-i] position to purine
Journal of Heterocyclic Chemistry
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Design, Synthesis, and Anticancer Activity of Novel Fused Purine Analogues
backbone as in compound 23 exerted strong activity
purine C2–H). Anal. Calcd for: C9H6N4O3S: C, 43.20; H,
against many cell lines.
2.42; N, 22.39; S, 12.81. Found: C, 43.38; H, 2.40; N,
22.51; S, 12.94.
Methyl 2-(7-oxo[1,4]thiazino[4,3,2-gh]purin-8(7H)-ylidene)
acetate (4) and dimethyl 2-(9H-purin-6-ylthio)maleate (5).
EXPERIMENTAL
A mixture of 6-mercaptopurine 1 (0.38 g, 2.5 mmol) and
dimethyl acetylene dicarboxylate (0.28 g, 0.24 mL,
2 mmol) in (30 mL) toluene was heated under reflux for
90 h, and the obtained precipitate was filtered off to yield
compound 4, while the filtrate was concentrated to afford
compound 5.
Methyl 2-(7-oxo[1,4]thiazino[4,3,2-gh]purin-8(7H)-
ylidene)acetate (4). Dark brown powder, washed by
several solvents, 0.1 g (15%), mp > 360°C. IR (KBr,
cmÀ1): 2942 (CH aliphatic), 1724 (C=O), 1602 (C=N),
1290, 1024 (C–O–C). MS: m/z (%): 261 (M+. À1, 9.09),
55 (100). Anal. Calcd for: C10H6N4O3S: C, 45.80; H,
Chemistry. All melting points were determined in
open glass capillaries on Electro thermal LA 9000 SERIS
and are uncorrected. IR spectra were recorded, for
potassium bromide discs, on Nikolet IR 200 FTIR
spectrophotometer at pharmaceutical analytical unit,
Faculty of Pharmacy, Al-Azhar University, and the values
are represented in cmÀ1
.
1H NMR spectra were
determined on Varian Gemini EM-300 MHz, NMR
spectrometer at Research service unit, Faculty of Science,
Cairo University. DMSO-d6 was used as solvent;
chemical shifts were measured in δ ppm, relative to TMS
as an internal standard. Mass spectra were carried out
using a Schimadzu GC/MS-QP-5050A mass spectrometer
at 70 eV at Regional Center for Mycology and
Biotechnology, Al-Azhar University. Elemental analyses
were performed on Elementar Vario EI III CHN analyzer
at Micro-analytical, Regional Center for Mycology and
Biotechnology, Al-Azhar University. Progress of the
reactions was monitored by thin-layer chromatography
(TLC) on silica gel sheets (60 GF 254, Merk), and the
spots were visualized by exposure to UV lamp at λ 254
and 365 nm for few seconds.
2.31; N, 21.36; S, 12.23. Found: C, 45.93; H, 2.28; N,
21.50; S, 12.34.
Dimethyl 2-(9H-purin-6-ylthio)maleate (5). Gray
powder, recrystallized from ethanol, 0.28 (37.8%), mp
173–175°C. IR (KBr, cm1): 3120 (NH), 3041 (CH
aromatic), 2941 (CH aliphatic), 1730 (C=O), 1604
(C=N), 1250, 1012 (C–O–C). 1H NMR (DMSO-d6, δ
ppm): 3.86 (s, 6H, two O–CH3), 5.90 (s, 1H, CH=C),
8.16 (s, 1H, purine C2–H), 8.38 (s, 1H, purine C8–H),
13.60 (s, 1H, purine NH, D2O exchangeable).
7-Phenyl-7H-[1,4]thiazepino[4,3,2-gh]purin-9(8H)-one
(6). A solution of cinnamoyl chloride (0.33 g, 2 mmol) in
benzene (10 mL) was added to a solution of 6-
mercaptopurine 1 (0.30 g, 2 mmol) in pyridine (10 mL)
at 20°C. The reaction mixture was then heated under
reflux for 20 h, concentrated, and then left to cool, and
the precipitated product was filtered off, washed with
ethanol, and crystallized from ethanol. Colorless crystals,
0.56 g (63%), mp 83–85°C. IR (KBr, cmÀ1): 3041 (CH
aromatic), 2927 (CH aliphatic), 1649 (C=O), 1597
(C=N), 1500 (C=C), 1256, 1090 (C–S–C). 1H NMR
(DMSO-d6, δ ppm): 2.93 (s, 2H, CH2–C=O), 3.16 (s, 1H,
ÀCH–C6H5), 7.16 (s, 1H, purine C8–H), 7.21 (s, 1H,
purine C2–H), 7.37–7.43 (m, 3H, C6H5–C3,4,5–H), 7.69
(d, 2H, J = 7.4 Hz, C6H5–C2,6–H). MS: m/z (%): 281(M+.
À1, 2.21), 132 (100). Anal. Calcd for C14H10N4OS: C,
59.56; H, 3.57; N, 19.85; S, 11.36. Found (%): 59.71; H,
3.53; N, 20.02; S, 11.43.
[1,4]Thiazino[4,3,2-gh]purine-7,8-dione
(2).
An
equimolar mixture of 6-mercaptopurine 1 (0.30 g,
2 mmol) and diethyl oxalate (0.29 g, 0.27 mL, 2 mmol)
was fused for 20 h. The reaction mixture was cooled,
triturated with ethanol, and the obtained product was
filtered off, dried, and crystallized from ethanol as brown
powder, 0.41 g (51%), mp > 360°C. IR (KBr, cmÀ1):
1
3000 (CH aromatic), 1735 (C=O), 1604 (C=N). H NMR
(DMSO-d6, δ ppm): 8.17 (s, 1H, purine C2–H), 8.35 (s,
1H, purine C8–H). MS: m/z (%): 206 (M+., 18.02), 139
(100). Anal. Calcd for C7H2N4O2S: C, 40.78; H, 0.98; N,
27.17; S, 15.55. Found: C, 40.86; H, 1.01; N, 27.26; S,
15.62.
2-(7-Oxo-7,8-dihydro[1,4]thiazino[4,3,2-gh]purin-8-yl)acetic
acid (3).
A mixture of 6-mercaptopurine 1 (0.30 g,
2 mmol), maleic anhydride (0.2 g, 2 mmol), and 3–5
drops conc. H2SO4 in methanol (30 mL) was heated
under reflux for 20 h. The reaction mixture was triturated
with 5% sodium bicarbonate solution, concentrated, and
cooled, and the precipitated solid was filtered off, dried,
and recrystallized from ethanol. Colorless crystals, 0.25 g
(50%), mp >360°C. IR (KBr, cmÀ1): 3447 (br. OH),
3000 (CH aromatic), 2930 (CH aliphatic), 1732 (C=O),
7H-thiazolo[3,4,5-gh]purin-7-one (7).
An equimolar
mixture of 6-mercaptopurine 1 (0.30 g, 2 mmol) and
ethyl chloroformate (0.22 g, 0.19 mL, 2 mmol) was
refluxed in sodium ethoxide (30 mL) [prepared by
dissolving sodium metal (0.05 g, 2 mmol) in 30 mL
absolute ethanol] for 20 h. The reaction mixture was
cooled, poured onto crushed ice, and the obtained
precipitate was filtered off, dried, and washed with
1
1596 (C=N). H NMR (DMSO-d6, δ ppm): 1.90 (s, 2H,
CH2), 5.92 (s, 1H, thiazine CH), 8.13 (s, 1H, purine C8–
H), 8.16 (s, 1H, OH, D2O exchangeable), 8.25 (s, 1H,
several solvents. White powder, 0.3
g
(84%),
mp > 360°C. IR (KBr, cmÀ1): 2992 (CH aromatic), 1720
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A. Y. Hassan, M. T. Sarg, A. H. Bayoumi, and F. G. A. Kalaf
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Gray
1
(C=O), 1633 (C=N), 1454 (C=C). H NMR (DMSO-d6, δ
7-Benzyl[1,4]thiazino[4,3,2-gh]purine (10b).
powder, 0.15 g (28%), mp 328–330°C. IR (KBr, cmÀ1):
ppm): 7.87 (s, 1H, purine C2–H), 8.00 (s, 1H, purine C8–
H). MS: m/z (%):179 (M + 1, 4.92), 66 (100). Anal.
Calcd for C6H2N4OS: C, 40.45; H, 1.13; N, 31.45; S,
18.00. Found (%): C, 40.62; H, 1.11; N, 31.59; S, 18.13.
2920 (CH aromatic), 2855 (CH aliphatic), 1606 (C=N),
1461 (C=C), 1290, 1030 (C–S–C). H NMR (DMSO-d6,
1
δ ppm): 1.81 (s, 2H, CH2–C6H5), 7.16 (s, 1H, thiazine
CH), 7.30–7.55 (m, 1H, C6H5–C4–H), 7.59–7.65 (m, 2H,
C6H5–C3,5–H), 7.84 (d, 2H, J = 6.3 Hz, C6H5–C2,6–H),
8.16 (s, 1H, purine C8–H), 8.35 (s, 1H, purine C2–H).
MS: m/z (%): 265 (M+.À1, 6.44), 65 (100). Anal. Calcd
for C14H10N4S: C, 63.14; H, 3.78; N, 21.04; S, 12.04.
Found: C, 63.28; H, 3.80; N, 21.22; S, 12.21.
Imidazo[1,5,4-gh]purin-5-amine (8).
An equimolar
mixture of 6-mercaptopurine 1 (0.30 g, 2 mmol) and
cyanamide (0.08 g, 2 mmol) was refluxed in absolute
ethanol (30 mL) containing piperidine (0.17 g, 0.20 mL,
2 mmol) for 25 h till evolution of H2S ceased. The
reaction mixture was then concentrated, allowed to cool,
and the precipitated solid was filtered off, dried, and
crystallized from ethanol. Brown powder, 0.32 g (66%),
mp > 360°C. IR (KBr, cmÀ1): 3330, 3127 (NH2), 2933
(CH aromatic), 1599 (C=N). 1H NMR (DMSO-d6, δ
ppm): 7.21 (s, 2H, NH2, D2O exchangeable), 8.49 (s, 1H,
purine C8–H), 8.81 (s, 1H, purine C2–H). MS: m/z (%):
160 (M+., 4.41), 54 (100). Anal. Calcd for: C6H4N6: C,
45.00; H, 2.52; N, 52.48. Found: C, 45.13; H, 2.55; N,
52.56.
1-(7H-purin-6-ylthio)pentan-2-one
(11a).
Brown
powder, crystallized from ethanol, 0.27 g (57%), mp
278–280°C. IR (KBr, cmÀ1): 3180 (NH), 3089 (CH
aromatic), 2925, 2853 (CH aliphatic), 1720 (C=O), 1610
(C=N), 1212, 1095 (C–S–C). 1H NMR (DMSO-d6, δ
ppm): 0.70–0.90 (m, 2H, ÀCH2–CH2–CH3), 1.04 (t, 3H,
J = 6.9 Hz, CH2–CH2–CH3), 1.10–1.30 (m, 2H, ÀCH2–
CH2–CH3), 1.91 (s, 1H, S–CH2–CO), 8.16 (s,1H, purine
C8–H), 8.35 (s, 1H, purine C2–H), 13.60 (s, 1H, purine
NH, D2O exchangeable). MS: m/z (%): 237 (M+. À1,
24.53), 56 (100). Anal. Calcd for C10H12N4OS: C, 50.83;
H, 5.12; N, 23.71; S, 13.57. Found: C, 50.37; H, 5.98; N,
23.59; S, 13.55.
5-Methylimidazo[1,5,4-gh]purine (9).
An equimolar
mixture of 6-mercaptopurine 1 (0.30 g, 2 mmol) and
thioacetamide (0.15 g, 2 mmol) was refluxed in absolute
ethanol (50 mL) for 25 h until it stopped evolution of H2S.
The reaction mixture was concentrated, allowed to attain
room temperature, and the obtained solid was filtered off,
washed with ethanol, dried, and crystallized from ethanol.
Brown powder, 0.15 g (47%), mp > 360°C. IR (KBr,
cmÀ1): 3038 (CH aromatic), 2936 (CH aliphatic), 1596
1-(7H-purin-6-ylthio)-3-phenylpropan-2-one (11b).
Brown powder, 0.24 g (42%), mp 238–240°C. IR (KBr,
cmÀ1): 3132 (NH), 3050 (CH aromatic), 2811 (CH
1
aliphatic), 1707 (C=O), 1595 (C=N). H NMR (DMSO-
d6, δ ppm): 1.84 (s, 2H, CH2–C6H5), 2.06 (s, 2H,
S–CH2), 7.92–8.15 (m, 5H, C6H5), 8.16 (s, 1H, purine
C2–H), 8.33 (s, 1H, purine C8–H), 13.75 (s, 1H, purine
NH, D2O exchangable). MS: m/z (%): 286 (M+. +2,
6.95), 102 (100). Anal. Calcd for C14H12N4OS: C, 59.14;
H, 4.25; N, 19.70; S, 11.28. Found: C, 59.36; H, 4.31; N,
1
(C=N), 1466 (C=C). H NMR (DMSO-d6, δ ppm): 1.97
(s, 3H, CH3), 8.22 (s, 1H, purine C8–H), 8.38 (s, 1H,
purine C2–H). MS: m/z (%): 159 (M+., 1.22), 56 (100).
Anal. Calcd for C7H5N5: C, 52.83; H, 3.17; N, 44.01.
Found: C, 52.89; H, 3.21; N, 44.13.
7-Substituted[1,4]thiazino[4,3,2-gh]purines (10a,b) and
19.87; S, 11.36.
6-substitutedmercapto-7H-purines (11a,b).
An equimolar
7-Substituted[1,4]thiazino[4,3,2-gh]purines (12a,b) and
6-substitutedmercapto-7H-purines (13a,b).
An equimolar
mixture of 6-mercaptopurine 1 (0.30 g, 2 mmol) and the
appropriate methyl ketone, namely, methyl propyl ketone
and benzyl methyl ketone (2 mmol) was heated under
reflux in glacial acetic acid (30 mL) containing (3–5)
drops of c. H2SO4 for 30 h. The reaction mixture was
cooled, neutralized with NH4OH, and the obtained
precipitate was filtered off, washed with water, dried, and
crystallized from ethanol to yield two products. The
insoluble part in ethanol was filtered off to give
compounds 10a,b, while the filtrate was concentrated to
afford compounds 11a,b.
mixture of 6-mercaptopurine 1 (0.30 g, 2 mmol) and the
appropriate chlorocompound, namely, chloroacetone and
phenacyl chloride (2 mmol) in sodium ethoxide (30 mL)
[prepared by dissolving sodium metal (0.05 g, 2 mmol)
in 30 mL absolute ethanol] was heated under reflux for
20 h. The reaction mixture was cooled, poured onto
crushed ice, and the obtained precipitate was filtered off
and crystallized from ethanol to yield two products. The
insoluble part in boiling ethanol was filtered off to give
the cyclic compounds 12a,b, while the filtrate was
concentrated and allowed to cool to yield the open chain
compounds 13a,b.
7-Propyl[1,4]thiazino[4,3,2-gh]purine (10a).
Colorless
needle crystals, 0.2 g (46%), mp 343–345°C. IR (KBr,
cmÀ1): 3035 (CH aromatic), 2933, 2854 (CH aliphatic),
1556 (C=N), 1409 (C=C), 1300, 1091(C–S–C). MS: m/z
(%): 218 (M+. + 1, 6.37), 84 (100). Anal. Calcd for:
C10H10N4S: C, 55.02; H, 4.62; N, 25.67; S, 14.69.
Found: C, 55.14; H, 4.70; N, 25.74; S, 14.80.
7-Methyl[1,4]thiazino[4,3,2-gh]purine (12a).
White
powder, 0.08 g (21%), mp > 360°C [12]. IR (KBr,
cmÀ1): 3085, 2993 (CH aromatic), 2839 (CH aliphatic),
1627 (C=N), 1330, 1018 (C–S–C). MS: m/z (%): 191
(M+. +1, 40.99), 72 (100). Anal. Calcd for C8H6N4S: C,
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Design, Synthesis, and Anticancer Activity of Novel Fused Purine Analogues
50.51; H, 3.18; N, 29.45; S, 16.86. Found: C, 50.68; H,
3.22; N, 29.58; S, 17.01.
with ethanol, dried, and recrystallized from ethanol.
White crystals, 1.23 g (82%), mp 244–246°C [16]. IR
(KBr, cmÀ1): 3386, 3244, 3151 (NH2, NH), 3023 (CH
aromatic), 1593 (C=N), 1527 (C=C). MS (%): m/z (%):
150 (M+., 17.17), 58 (100). Anal. Calcd for C5H6N6: C,
40.00; H, 4.03; N, 55.97. Found: C, 40.08; H, 4.11; N,
7-Phenyl[1,4]thiazino[4,3,2-gh]purine (12b).
White
powder; 0.20 (40%), mp > 360°C. IR (KBr, cmÀ1): 3051
(CH aromatic), 1592 (C=N), 1444 (C=C). 1H NMR
(DMSO-d6, δ ppm): 5.01 (s, 1H, thiazine CH), 7.00–7.60
(m, 5H, C6H5), 7.97 (s, 1H, purine C2–H), 8.18 (s, 1H,
purine C8–H). MS: m/z (%): 254 (M+. +2, 9.26), 56
(100). Anal. Calcd for C13H8N4S: C, 61.89; H, 3.20; N,
22.21; S, 12.71. Found: C, 62.13; H, 3.25; N, 22.37; S,
12.82.
56.13.
4-Methyl-2,3,4,10-tetrahydro[1,2,4]triazino[4,3-i]purin-4-ol
(16) and 1-[2-(7H-purin-6-yl)hydrazinyl]propan-2-one (17).
Equivalent amounts of 6-hydrazinyl-9H-purine 15 (0.3 g,
2 mmol) and chloroacetone (0.18 g, 0.16 mL, 2 mmol)
were fused for 18 h. The reaction mixture was then
triturated with ethanol, and the obtained precipitate was
filtered off, washed with ethanol, and then crystallized
from ethanol to yield two products. The insoluble part
in hot ethanol was filtered off to give dark brown
powder that was found to be compound 16, while the
filtrate was concentrated and allowed to cool to yield
compound 17.
1-(7H-purin-6-ylthio)propan-2-one
(13a).
Brown
powder, 0.17 g (41%), mp 213–215°C [17]. IR (KBr,
cmÀ1): 3428 (NH), 3012 (CH aromatic), 2813 (CH
aliphatic), 1673 (C=O), 1606 (C=N), 1216, 1010 (C–S–
1
C). H NMR (DMSO-d6, δ ppm): 2.29 (s, 3H, CH3), 4.32
(s, 2H, S–CH2), 8.17 (s, 1H, purine C8–H), 8.36 (s, 1H,
purine C2–H), 13.73 (s, 1H, purine NH, D2O
exchangeable). MS: m/z (%): 211 (M+. +3, 2.92), 126
(100). Anal. Calcd for C8H8N4OS: C, 46.14; H, 3.87; N,
26.90; S, 15.40. Found: C, 46.31; H, 3.94; N, 26.87; S,
15.47.
4-Methyl-2,3,4,10-tetrahydro[1,2,4]triazino[4,3-i]purin-
4-ol (16).
Dark brown powder, 0.19 g (46.3%),
mp > 360°C. IR (KBr, cmÀ1): 3402 (br. OH), 3210 (NH),
3080 (CH aromatic), 2930 (CH aliphatic), 1626 (C=N).
1H NMR (DMSO-d6, δ ppm): 1.44 (s, 3H, CH3), 5.01 (s,
2H, triazine CH2), 5.98 (s, 1H, OH, D2O exchangeable),
8.07 (s, 1H, purine C2–H), 8.45 (s, 1H, purine C8–H),
9.22 (s, 1H, triazine NH, D2O exchangeable), 9.70 (s, 1H,
purine NH, D2O exchangeable). MS (%): m/z (%): 206
(M+., 3.17), 203 (M+. À3,100). Anal. Calcd for
C8H10N6O: C, 46.60; H, 4.89; N, 40.76. Found: C, 46.78;
H, 4.94; N, 40.99.
2-(7H-purin-6-ylthio)-1-phenylethanone (13b).
Brown
crystals, yield 0.33 g (61%), m.p.138–140°C 23. IR (KBr,
cmÀ1): 3431 (NH), 3002 (CH aromatic), 2828 (CH
aliphatic), 1679 (C=O), 1609 (C=N), 1344, 1014 (C–S–
1
C). H NMR (DMSO-d6, δ ppm): 4.80 (s, 2H, S–CH2),
7.17–7.44 (m, 5H, C6H5), 8.12 (s, 1H, purine C2–H),
8.36 (s, 1H, purine C8–H), 13.75(s, 1H, purine NH, D2O
exchangeable).
[1,4]Thiazino[4,3,2-gh]purin-7-amine
(14).
An
1-(2-(7H-purin-6-yl)hydrazinyl)propan-2-one (17).
Gray powder, 0.22 g (54%), mp 140–142°C. IR (KBr,
cmÀ1): 3360, 3182 (NH), 3011 (CH aromatic), 2932,
2880 (CH aliphatic), 1695 (C=O), 1624 (C=N). MS (%):
m/z (%): 205 (M+. À1, 8.09), 69 (100). Anal. Calcd for
C8H10N6O: C, 46.60; H, 4.89; N, 40.76. Found: C,
equimolar mixture of 6-mercaptopurine 1 (0.30 g,
2 mmol) and chloroacetonitrile (0.15 g, 0.13 mL,
2 mmol) was heated under reflux in sodium ethoxide
(30 mL) [prepared by dissolving sodium metal (0.05 g,
2 mmol) in 30 mL absolute ethanol] for 30 h. The
reaction mixture was allowed to cool and poured onto
crushed ice. The obtained precipitate was filtered off,
washed with water, dried, and crystallized from ethanol.
Brown powder, 0.29 g (76%), mp 163–165°C. IR (KBr,
cmÀ1): 3444, 3123 (NH2), 3010 (CH aromatic), 1568
(C=N), 1335, 1054 (C–S–C). 1H NMR (DMSO-d6, δ
ppm): 4.50 (s, 2H, NH2, D2O exchangeable), 5.59 (s, 1H,
thiazine CH), 8.62 (s, 1H, purine C8–H), 8.94 (s, 1H,
purine C2–H). MS (%): m/z (%): 192 (M+. +1, 5.58), 59
(100). Anal. Calcd for C7H5N5S: C, 43.97; H, 2.64; N,
36.63; S, 16.77. Found: C, 44.08; H, 2.59; N, 36.74; S,
16.83.
46.76; H, 4.92; N, 40.97.
2,10-Dihydro[1,2,4]triazino[4,3-i]purin-4-amine (18). An
equimolar mixture of 6-hydrazinyl-9H-purine 15 (0.3 g,
2 mmol) and chloroacetonitrile (0.15 g, 0.13 mL,
2 mmol) was fused for 20 h. The reaction was then
triturated with ethanol, and the obtained solid was filtered
off, washed with ethanol, and crystallized from ethanol.
Brown powder, 0.2 g (52%), mp > 360°C. IR (KBr,
cmÀ1): 3360, 3135 (NH2, NH), 3008 (CH aromatic),
1
1610 (C=N), 1532 (C=C). H NMR (DMSO-d6, δ ppm):
4.59 (s, 1H, triazine CH), 5.05 (s, 2H, NH2, D2O
exchangeable), 8.17 (s, 1H, purine C2–H), 8.37 (s, 1H,
purine C8–H), 10.00 (s, 1H, triazine N1–H, D2O
exchangeable), 13.72 (s, 1H, purine N7–H, D2O
exchangeable). MS (%): m/z (%): 189 (M+., 0.63),
76(100). Anal. Calcd for C7H7N7: C, 44.44; H, 3.73; N,
51.83. Found: C, 44.71; H, 3.71; N, 52.01.
6-Hydrazinyl-9H-purine (15).
6-Mercaptopurine
1
(1.52 g, 10 mmol) was subjected to hydrazinolysis by
fusion with an equimolar amount of hydrazine hydrate
99% (0.32 g, 0.31 mL, 10 mmol) for 30 h. Then the
reaction mixture was allowed to cool, triturated with
ethanol, and the obtained solid was filtered off, washed
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Y. Hassan, M. T. Sarg, A. H. Bayoumi, and F. G. A. Kalaf
Vol 000
1
4-(2-(7H-purin-6-yl)hydrazono)pentan-2-one
(19).
(C=C). H NMR (DMSO-d6, δ ppm): 2.27 (s, 3H, CH3),
7.43 (s, 1H, pyrazolone N1–H, D2O exchangeable), 7.56
(s, 1H, pyrazolone C4–H), 8.17 (s, 1H, purine C2–H),
8.32 (s, 1H, purine C8–H), 13.30 (s, 1H, purine N7–H,
D2O exchangeable). MS (%): m/z (%): 216 (M+., 11.32),
185 (100). Anal. Calcd for C9H8N6O: C, 50.00; H, 3.73;
Equivalent amounts of 6-hydrazinyl-9H-purine 15 (0.3 g,
2 mmol) and acetyl acetone (0.20 g, 0.21 mL, 2 mmol)
were refluxed in absolute ethanol (30 mL) for 20 h. The
reaction mixture was concentrated, then allowed to cool,
and the obtained precipitate was filtered, washed with
ethanol, dried, and recrystallized from ethanol. Buff
crystals, 0.28 g (60%), mp 268–270°C. IR (KBr, cmÀ1):
3118 (NH), 3030 (CH aromatic), 2889, 2779 (CH
aliphatic), 1732 (C=O), 1598 (C=N), 1519 (C=C). 1H
NMR (DMSO-d6, δ ppm): 3.18 (s, 3H, C–CH3), 3.60 (s,
3H, COCH3), 3.80 (s, 2H, CH2), 7.08 (s, 1H, NH, D2O
exchangeable), 8.17 (s, 1H, purine C2–H), 8.37 (s, 1H,
purine C8–H), 13.70 (s, 1H, purine N7–H, D2O
exchangeable). MS (%): m/z (%): 231 (M+. À1, 1.56), 84
(100). Anal. Calcd for C10H12N6O: C, 51.72; H, 5.21; N,
N, 38.8. Found: C, 50.18; H, 3.76; N, 38.76.
3-(2,4-Dimethoxyphenyl)-7H[1,2,4]triazolo[4,3-i]purine (23).
Equimolar amounts of 6-hydrazinyl-9H-purine 15 (0.3 g,
2 mmol) and 2,4-dimethoxybenzoyl chloride (0.4 g,
2 mmol) were refluxed in dry pyridine (30 mL) for 8 h.
The reaction mixture was then allowed to attain room
temperature, poured onto crushed ice, and neutralized
with dilute HCl, to yield an oily layer, which was
isolated, left in refrigerator to give low melting point
crystals. Colorless crystals, 0.40 g (68%). 1H NMR
(DMSO-d6, δ ppm): 4.12 (s, 6H, two OCH3), 7. 20 (s,
1H, 2,4-di(OCH3)2–C6H3–C3–H), 7.64–7.82 (m, 1H, 2,4-
di(OCH3)2–C6H3–C5–H), 7.90–8.18 (m, 1H, 2,4-
di(OCH3)2–C6H3–C6–H), 8.48 (s, 1H, purine C8–H), 8.70
(s, 1H, purine C2–H), 12.30 (s, 1H, purine N7–H, D2O
exchangeable). MS (%): m/z (%): 296 (M+., 0.20), 52
(100). Anal. Calcd for C14H12N6O2: C, 56.75; H, 4.08;
36.19. Found: C, 52.29; H, 4.41; N, 36.62.
N-phenyl-1H-[1,2,4]triazino[4,5,6-gh]purin-3-amine (20).
A mixture of 6-hydrazinyl-9H-purine 15 (0.3 g, 2 mmol)
and phenyl isothiocyanate (0.27 g, 0.24 mL, 2 mmol) in
pyridine (20 mL) was heated under reflux for 18 h. The
reaction mixture was then concentrated, and the obtained
precipitate was filtered off, washed with ethanol, and
washed with several solvents. Gray powder, 0.08 g
(16%), mp 258–260°C. IR (KBr, cmÀ1): 3215, 3120
N, 28.36. Found: C, 56.89; H, 4.13; N, 28.35.
4-Phenyl-1,4-dihydro[1,2,5]triazepino[5,4,3-gh]purine (24)
and 2-(2-(7H-purin-6-yl)hydrazinyl)-1-phenylethanone (25).
1
(NH), 2976 (CH aromatic), 1600 (C=N), 1483 (C=C). H
Equivalent amounts of 6-hydrazinyl-9H-purine 15 (0.3 g,
2 mmol) and phenacyl chloride (0.31 g, 2 mmol) were
heated under reflux in absolute ethanol (30 mL) for 18 h.
The reaction mixture was then concentrated, allowed to
cool, and the obtained precipitate was filtered off, washed
with ethanol, and crystallized from ethanol to yield two
products. The insoluble part in boiling ethanol was
filtered off to give the triazepine derivative 24, while the
filtrate was concentrated and allowed to cool to yield the
open chain analogue 25.
NMR (DMSO-d6, δ ppm): 4.90 (s, 1H, C6H5–NH, D2O
exchangeable), 6.94 (t, 1H, J = 7.5 Hz, C6H5–C4–H),
7.18–7.38 (m, 2H, C6H5–C3,5–H), 7.55 (d, 2H,
J = 7.5 Hz, C6H5–C2,6–H), 8.15 (s,1H, purine C2–H),
8.65 (s, 1H, purine C8–H), 9.81 (s, 1H, triazine NH, D2O
exchangeable). MS (%): m/z (%): 251 (M+., 100). Anal.
Calcd for C12H9N7: C, 57.37; H, 3.61; N, 39.02. Found:
C, 57.44; H, 3.63; N, 39.17.
[1,2,4]Triazino[4,3-i]purine-3,4(2H,10H)-dione (21).
An
equimolar mixture of 6-hydrazinyl-9H-purine 15 (0.3 g,
2 mmol) and diethyl oxalate (0.29 g, 0.27 mL, 2 mmol)
was fused for 20 h. The reaction mixture was triturated
with ethanol. The obtained solid was filtered and washed
with several solvents to give compound 23. White
powder, 0.22 (54%), mp > 360°C. IR (KBr, cmÀ1):
3280, 3100 (NH); 3048 (CH aromatic), 1676(C=O), 1610
(C=N), 1526 (C=C). MS (%): m/z (%): 205 (M+. +1,
2.74), 57 (100). Anal. Calcd for C7H4N6O2: C, 41.18; H,
4-Phenyl-1,4-dihydro[1,2,5]triazepino[5,4,3-gh]purine
(24).
White powder, 0.17 g (34%), mp 198–200°C. IR
(KBr, cmÀ1): 3390, 3120 (NH), 2916 (CH aromatic),
1
1640 (C=N), 1551 (C=C). H NMR (DMSO-d6, δ ppm):
7.20 (s, 1H, NH, D2O exchangeable), 7.42 (d, 1H,
J = 7.4 Hz, triazepine C4–H), 7.45–7.59 (m, 5H, C6H5),
7.83 (d, 1H, J = 7.4 Hz, triazepine C3–H), 8.22 (s, 1H,
purine C8–H), 8.33 (s, 1H, purine C2–H). MS (%): m/z
(%): 253 (M + 3, 6.32), 180 (100). Anal. Calcd for
C13H10N6: C, 62.39; H, 4.03; N, 33.58. Found: C, 62.53;
H, 4.11; N, 33.67.
1.98; N, 41.17. Found: C, 41.34; H, 2.02; N, 41.38.
5-Methyl-2-(7H-purin-6-yl)-1H-pyrazol-3(2H)-one (22).
6-Hydrazinyl-9H-purine 15 (0.3 g, 2 mmol) was fused
with equivalent amount of ethyl acetoacetate (0.26 g,
0.25 mL, 2 mmol) for 20 h. The reaction mixture was
then triturated with ethanol, and the obtained solid was
filtered, washed with ethanol, dried, and recrystallized
from ethanol. Buff crystals, 0.29 g (67%), mp > 360°C.
IR (KBr, cmÀ1): 3352 (NH), 3083 (CH aromatic), 2924,
2860 (CH aliphatic), 1721 (C=O), 1638 (C=N), 1551
2-(2-(7H-purin-6-yl)hydrazinyl)-1-phenylethanone
(25).
Brown powder, 0.28 g (52%), mp > 360°C. IR
(KBr, cmÀ1): 3439 (br. OH tautomer), 3120 (NH), 3057
(CH aromatic), 2941 (CH aliphatic), 1677 (C=O), 1590
1
(C=N). H NMR (DMSO-d6, δ ppm): 2.09 (s, 2H, CH2),
3.52 (s, 1H, NH–CH2, D2O exchangeable), 7.92–8.00 (m,
3H, C6H5–C3,4,5–H), 8.11(s, 1H, purine C4–NH, D2O
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Design, Synthesis, and Anticancer Activity of Novel Fused Purine Analogues
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exchangeable), 8.16 (s,1H, purine C8–H), 8.30–8.40 (m,
3H, C6H5–C2,6–H), 8.47 (s, 1H, purine C2–H), 12.23 (s,
1H, purine N7–H, D2O exchangeable). MS (%): m/z (%):
268 (M+. +2, 4.61), 132 (100). Anal. Calcd for
C13H12N6O: C, 58.20; H, 4.51; N, 31.33. Found: C,
58.89; H, 3.86; N, 31.72.
Ethyl
6-[2-(ethoxycarbonyl)hydrazinyl]-7H-purine-7-
carboxylate (26).
6-Hydrazinyl-9H-purine 15 (0.3 g,
2 mmol) was fused with excess ethyl chloroformate
(0.3 mL) for 18 h. The reaction mixture was then
triturated with ethanol, and the obtained precipitate was
filtered off, washed with ethanol, and recrystallized
from ethanol. Yellow crystals, 0.35 g (59%), mp 118–
120°C. IR (KBr, cmÀ1): 3256 (NH), 3038 (CH
aromatic), 2995, 2863 (CH aliphatic), 1746, 1700
(C=O), 1536 (C=C), 1246, 1065 (C–O–C). MS (%): m/z
(%): 295 (M+. +1, 0.31), 54 (100). Anal. Calcd for
C11H14N6O4: C, 44.90; H, 4.80; N, 28.56. Found: C,
44.87; H, 4.86; N, 28.71.
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Acknowledgments. We would like to thank the National Cancer
Institute (NCI), Bethesda, Maryland, USA, for performing the
anticancer screening of the newly synthesized compounds.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet