Synthesis and antiproliferative activity of 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides

Article abstract of DOI:10.1016/j.bmc.2009.05.078

A series of 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides were synthesized and in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780, HBL-100, HeLa, SW1573, T-47D, and WiDr. The most potent analog induced considerably growth inhibition in the range 0.35-2.0 μM. Cell cycle studies in the breast and lung cancer cells revealed arrest in the G₂/M compartment. The results showed that the title compounds bearing alkylamino or dialkylamino moieties in position 2 of the pyrimidine ring are more active than those bearing hydrogen or methylthio groups.

Full text of DOI:10.1016/j.bmc.2009.05.078

Bioorganic & Medicinal Chemistry 17 (2009) 4955–4960
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiproliferative activity of 2,4-disubstituted
6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides
b,c,
Erika Pudziuvelyte a, Carla Ríos-Luci b,c, Leticia G. León b,c, Inga Cikotiene a, , José M. Padrón
*
*
a Department of Organic Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko 24, Vilnius LT-03225, Lithuania
b Instituto Universitario de Bio-Orgánica ‘Antonio González’ (IUBO-AG), Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
c BioLab, Instituto Canario de Investigación del Cáncer (ICIC), C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides were synthesized and
in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780, HBL-100,
HeLa, SW1573, T-47D, and WiDr. The most potent analog induced considerably growth inhibition in
the range 0.35–2.0 lM. Cell cycle studies in the breast and lung cancer cells revealed arrest in the G2/
M compartment. The results showed that the title compounds bearing alkylamino or dialkylamino moi-
Received 29 April 2009
Revised 25 May 2009
Accepted 31 May 2009
Available online 6 June 2009
eties in position 2 of the pyrimidine ring are more active than those bearing hydrogen or methylthio
groups.
Keywords:
Pyrrolo[3,2-d]pyrimidine
Privileged structure
Antitumor drugs
Cell cycle arrest
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The pyrrolo[3,2-d]pyrimidine heterocyclic framework consti-
tutes the basis of an important class of compounds possessing
remarkable biological activities. These compounds are 9-deazaan-
alogs of biogenic purines and have been reported to be inhibitors
of purine nucleoside phosphorilase1 and thymidylate synthase;2
in addition to antagonists of the neuropeptide Y5,3 and the A1
and A2 adenosine receptors.4
Recently, we have developed a novel, concise, and high-yielding
formation of pyrrolo[3,2-d]pyrimidin-7-one 5-oxides via smooth
cycloisomerization of 4-amino-6-arylethynyl-5-nitropyrimidines
in the presence of pyridine.5 Moreover, it was showed that the sub-
stituent at the C-2 position of the pyrrolo[3,2-d]pyrimidine hetero-
cyclic system could be easily modified via one-pot oxidation/
substitution of a methylthio group.6
We report herein on the synthesis of a series of 2,4-disubsti-
tuted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides and the
evaluation of the cytotoxicity of the title compounds. The antipro-
liferative profile of the obtained derivatives was evaluated in vitro
against a panel of six human solid tumor cell lines: A2780 (ovar-
ian), HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell
lung), T-47D (breast), and WiDr (colon). The most active derivative
was further studied to determine disturbances on the cell cycle.
2.1. Chemistry
Our strategy to synthesize 2,4-disubstituted 6-aryl-7H-pyrrol-
o[3,2-d]pyrimidin-7-one 5-oxides was based on our previous
reported methods.5,6 Scheme 1 outlines the general synthetic
pathway. The 2,4-disubstituted 6-arylpyrrolo[3,2-d]pyrimidin-7-
one 5-oxides (2a–i) were prepared via pyridine initiated smooth
cycloisomerization of 2,4-disubstituted 5-nitro-6-arylethynylpyr-
imidines (1a–i). Reactions were performed in boiling 2-propanol
for 10–30 min and the resulting compounds 2a–i were obtained
in high yields (Table 1). The modification of the C-2 position of
the pyrrolo[3,2-d]pyrimidine heterocyclic system was achieved
by oxidation of methylthio moiety of compound 2d and subse-
quent nucleophilic displacement of the resulting methylsulfonyl
group by the appropriate alkylamine or dialkylamine. With this
straightforward strategy we obtained the corresponding deriva-
tives 4a–k in yields ranging 52–92% (Scheme 1, Table 1).
2.2. Antiproliferative activity
Our synthesized 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyr-
imidin-7-one 5-oxides 2–4 were tested for antiproliferative activ-
ity against the representative panel of human solid tumor cell
lines A2780 (ovarian cancer), HBL-100 (breast), HeLa (cervix),
SW1573 (non-small cell lung), T-47D (breast), and WiDr (colon).
The in vitro activity was evaluated with the National Cancer
* Corresponding authors. Tel.: +34 922316502x6126; fax: +34 922318571 (J.M.P).
E-mail addresses: inga.cikotiene@chf.vu.lt (I. Cikotiene), jmpadron@ull.es (J.M.
Padrón).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.078

4956
E. Pudziuvelyte et al. / Bioorg. Med. Chem. 17 (2009) 4955–4960
Table 1
2,4-Disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides (2–4)
Compd
R
R1
R2
Yield (%)
2a
2b
2c
2d
2e
2f
H
H
H
SCH3
SCH3
SCH3
SCH3
NH2
NH2
NH2
NH2
NH2
NH2
NHCH2C6H5
H
95
90
97
95
89
92
91
CH3
C2H5
H
CH3
C2H5
H
2g
2h
2i
SCH3
SCH3
H
79
H
95
3
SOCH3
NH2
NH2
NH2
NH2
NH2
H
H
H
H
H
85
79
52
78
80
4a
4b
4c
4d
NHCH2CH@CH2
NHCH2CO2CH3
NHCH2C6H5
NH(CH2)2C6H5
4e
4f
NH2
NH2
H
H
79
75
Scheme 1. Reagents and conditions: (a) pyridine (cat), 2-PrOH, reflux, 10–30 min,
79–95%; (b) mCPBA, DCM, rt, 1 h, 85%; (c) amine, rt, 3–8 h, 52–92%.
4g
4h
NH(CH2)3N(CH3)2
NH2
NH2
H
H
69
79
Institute (NCI) protocol7 after 48 h of drug exposure using the sul-
forhodamine B (SRB) assay.8 The sensitivities expressed as GI50 are
listed in Table 2. In addition to the antitumor activity, the lipophil-
icity (Clog P) of the compounds was evaluated by in silico calcula-
tion based on their chemical structure. Clog P values were
calculated to correlate lipophilicity with antitumor activity.9 The
Clog P values for the compounds reported in this study are in the
range À1.1 to 3.2. Taken as a whole, lipophilicity is not sufficient
to explain the observed differences in growth inhibition.
4i
NH2
NH2
NH2
H
H
H
83
85
92
4j
4k
The GI50 values allow classifying the compounds in two groups.
The first group is comprised of 2,4-disubstituted 6-aryl-7H-pyrrol-
o[3,2-d]pyrimidin-7-one 5-oxides 2a–i and 3. These compounds
showed overall a modest antiproliferative activity with GI50 values
active against all cell lines; the derivatives 2c, 2e, and 2g, with
GI50 values in the range 26–82 M.
l
The second group is formed by all derivatives bearing N-alkyl-
higher than 24 lM. From this series, only three compounds were
amino (4a–g) or N,N-dialkylamino (4h–k) substituents at C-2
Table 2
Lipophilicity and GI50 values of 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides (2–4) against human solid tumor cellsa
Compd
CLog P
A2780 (ovarian)
HBL-100 (breast)
HeLa (cervix)
SW1573 (lung)
T-47D (breast)
WiDr (colon)
2a
2b
2c
2d
2e
2f
2g
2h
2i
À0.2
0.3
0.8
1.0
1.5
2.0
3.2
2.0
2.5
À1.1
1.3
0.3
2.0
2.7
1.4
0.8
1.1
1.9
0.3
À0.1
2.4
30 ( 11)
>100
39 ( 9.6)
nt
93 ( 6.8)
54 ( 7.1)
59 ( 5.4)
>100
72 ( 30)
>100
54 ( 7.6)
nt
>100
>100
75 ( 29)
nt
>100
80 ( 20)
36 ( 4.7)
>100
>100
>100
7.6 ( 1.3)
19 ( 4.1)
29 ( 4.0)
>100
16 ( 1.9)
15 ( 5.9)
20 ( 4.3)
3.5 ( 0.6)
38 ( 12)
>100
85 ( 25)
>100
40 ( 6.7)
nt
31 ( 7.3)
37 ( 5.3)
26 ( 4.3)
>100
>100
>100
82 ( 17)
nt
>100
43 ( 13)
81 ( 27)
>100
>100
>100
2.8 ( 0.9)
14 ( 1.6)
4.0 ( 1.1)
41 ( 30)
13 ( 0.7)
17 ( 1.3)
16 ( 3.1)
2.7 ( 0.8)
7.5 ( 0.6)
>100
>100
>100
70 ( 23)
nt
>100
53 ( 4.6)
79 ( 30)
>100
>100
>100
4.7 ( 0.03)
18 ( 2.1)
24 ( 10)
71 ( 41)
14 ( 0.6)
16 ( 0.4)
19 ( 1.0)
3.7 ( 1.2)
20 ( 7.3)
46 ( 3.3)
1.8 ( 0.8)
28 ( 7.9)
37 ( 3.3)
27 ( 7.8)
24 ( 0.9)
>100
32 ( 6.1)
2.7 ( 0.9)
2.8 ( 0.02)
5.1 ( 1.0)
17 ( 4.3)
1.8 ( 0.3)
3.4 ( 0.8)
2.0 ( 0.6)
1.7 ( 0.3)
22 ( 4.9)
18 ( 1.1)
1.4 ( 0.6)
>100
>100
>100
3
79 ( 19)
1.9 ( 0.06)
1.9 ( 0.4)
3.7 ( 0.7)
37 ( 7.3)
2.6 ( 0.3)
2.9 ( 1.0)
2.3 ( 0.2)
2.9 ( 0.2)
37 ( 0.4)
24 ( 7.1)
1.8 ( 0.2)
4a
4b
4c
4d
4e
4f
4g
4h
4i
6.8 ( 0.7)
12 ( 6.4)
30 ( 7.9)
22 ( 4.6)
6.2 ( 2.3)
14 ( 1.5)
2.1 ( 0.5)
3.3 ( 0.7)
27 ( 2.3)
23 ( 2.1)
1.3 ( 0.1)
4j
4k
2.0 ( 0.3)
0.35 ( 0.19)
a
Values are given in lM and are means of two to six experiments, standard deviation is given in parentheses (nt = not tested).

E. Pudziuvelyte et al. / Bioorg. Med. Chem. 17 (2009) 4955–4960
4957
Figure 1. Cell cycle phase distribution in untreated cells (C) and cells treated with compound 4k for 24 h at 2, 5, and 10 lM.
position of the pyrrolo[3,2-d]pyrimidine heterocyclic framework.
This set of compounds exhibited the best results. The vast majority
of the compounds were able to induce antiproliferative effects in
all cell lines. Only derivatives 4d and 4j showed inactive against
one or two of the cell lines. The most potent derivatives were
compounds 4a, 4h, and 4k, bearing allylamino, piperidino, and
azepanyl substituents, respectively. Their antiproliferative activity
was similar against the six cell lines, showing GI50 values within
particular context, HBL-100 cells are more sensitive to compound
4k as observed in the increased sub G1 compartment at 5 and
10
was observed for the breast cancer cell lines HBL-100 and T-47D,
and for the lung cancer cells SW1573 when exposed at 2 M of
lM. Additionally, a clear cell cycle arrest in the G2/M phase
l
4k (Fig. 2). This increase was concomitant with a decrease in both
the G1 and S compartments. On the contrary, cell cycle arrest was
not apparent in HeLa and WiDr cells.
the range 1.9–7.6
l
M for 4a, 1.7–3.7
l
M for 4h, and 0.35–2.0
l
M
Although the mechanism of action of the title compounds
remains unclear, the first results suggested a significant role of
the lipophilic 2-alkylamino and 2-dialkylamino fragment in the
enhancement of the antiproliferative activity of 6-aryl-7H-pyrrol-
o[3,2-d]pyrimidin-7-one 5-oxides. The observation that the major
part of the N-alkylamino (4a–g) or N,N-dialkylamino (4h–k)
derivatives evaluated in this study present antiproliferative activ-
ity lead us to consider the 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-
one 5-oxide as a privileged structure with the substituents on
the pyrimidine ring (R and R1) modulating the biological activity.
Privileged structures, with their inherent drug-likeness, repre-
sent an ideal source of core scaffolds to obtain combinatorial
libraries targeted at various receptors. A number of these libraries
have proved to be an extremely powerful tool to aid the rapid dis-
covery and optimization of potent and selective drugs for a wide
variety of cellular targets.12 The straightforward synthesis of the
6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides reported in this
study, together with the high chemical yields, make these com-
pounds valuable scaffolds for obtaining combinatorial libraries in
which the cytotoxic activity of the molecules is anticipated.
In conclusion, a series of 2,4-disubstituted 6-aryl-7H-pyrrol-
o[3,2-d]pyrimidin-7-one 5-oxides were prepared and in vitro anti-
proliferative activities were examined in the human solid tumor
cell lines A2780, HBL-100, HeLa, SW1573, T-47D, and WiDr. The
in vitro experiments showed that the analogs containing N-alkyl-
amino or N,N-dialkylamino substituents in position 2 of the pyrrol-
o[3,2-d]pyrimidine heterosystem induced considerable growth
inhibition in the panel of six diverse human solid tumor cells. Cell
cycle studies demonstrated arrest in the G2/M phase when the
breast and lung cancer cells were exposed to compound 4k. The ti-
tle products appear as good lead molecules for the development of
novel antitumor agents.
for 4k. This is a remarkable effect, since the general observation for
conventional antitumor drugs is that WiDr, T-47D, and HeLa cancer
cells are more drug resistant than A2780 and HBL-100 cancer cells.10
From the biological activity data, some structure–activity rela-
tionships can be inferred. The presence at C-2 position of the
pyrimidine ring of hydrogen (2a–c), methylthio (2d–i) or methyl-
sulfonyl (3) moieties resulted in modest or inactive compounds.
Neither the alkyl substituent at the phenyl group (R2) nor the
amines at C-4 position of the pyrimidine ring (R1) appear to be cru-
cial for the modulation of the antiproliferative activity. In contrast,
the N-alkylamino (4a–g) or N,N-dialkylamino (4h–k) substituents
at C-2 position induced an enhancement of the biological activity.
Overall, the derivatization at C-2 of the pyrimidine ring with phen-
ylethylamino (4d), morpholino (4i) or N-methylpiperazino (4j)
moieties produced a decrease of the biological activity. The amines
that led to the most potent derivatives were allylamine (4a), piper-
idine (4h), and azepane (4k).
2.3. Cell cycle disturbances
When exposed to cytotoxic agents, damaged cells may suffer a
stop in their cell cycle. If the cell cannot recover from the damage,
cell death occurs through apoptosis. This stop in cell division is
known as cell cycle arrest and can occur at any of the cell cycle
phases, namely G0/G1, S, or G2/M phase.
We studied cell cycle phase distribution by flow cytometry to
determine if cell growth inhibition involved cell cycle changes.
For these studies we selected 4k, the most active compound from
the series. The effect on the cell cycle was investigated after 24 h
exposure. Cells were exposed to compound 4k at three different
drug concentrations: 2, 5, and 10 lM. The drug doses were chosen
based on two premises.11 On the one hand, the GI50 values against
each cell line. On the other hand, the sensitivity of the cell line to
drug treatment, since at higher drug doses large cell death pre-
vents examination of the cell cycle phase distribution. Control cells
were incubated in the absence of test drug. The results are shown
in Figure 1.
3. Experimental
3.1. General
Melting points were determined in open capillaries and are
uncorrected. IR spectra were run in Nujol mulls or in KBr discs
on a Perkin–Elmer FT spectrophotometer Spectrum BX II. 1H and
13C NMR spectra were recorded with a Varian Unity INOVA
spectrometer (300 MHz) using tetramethylsilane as internal stan-
dard. Elemental analysis (C, H, N) results were found to be in good
agreement ( 0.4%) with the calculated values. All reactions and
Overall, cell cycle distributions of samples collected from
control and treated cells show in all cell lines an increase of the
sub G1 compartment in a dose dependent manner. The results indi-
cate that compound 4k produces net cell killing. However, the data
revealed a diverse pattern of sensitivity between HBL-100 cells and
the remaining cell lines HeLa, SW1573, T-47D, and WiDr. In this

4958
E. Pudziuvelyte et al. / Bioorg. Med. Chem. 17 (2009) 4955–4960
Figure 2. Cell cycle phase distribution in control and treated HBL-100, T-47D, and SW1573 cells, after 24 h exposure to compound 4k at 2 lM.
purity of the synthesized compounds were monitored by TLC using
Silica Gel 60 F254 aluminium plates (Merck). Visualization was
accomplished by UV light.
Compounds2a–i, 3, and 4a–kwere prepared accordingthemeth-
ods we have described earlier.5,6 Data for compounds 2a–b,5a 2d,5b
2g,6 2i,6 3,6 4c,6 4f,6 4h–i6 have been described previously.
J = 6.3 Hz, CH3), 2.62 (3H, s, SCH3), 2.72 (2H, q, J = 6.3 Hz, CH2),
5.56 (1H, br s, NH), 7.00 (1H, br s, NH), 7.31 (2H, d, J = 7.8 Hz,
ArH), 8.43 (2H, d, J = 7.8 Hz, ArH); 13C NMR (DMSO, 75 MHz): d
13.9, 15.2, 28.2, 119.5, 123.2, 126.7, 127.9, 146.1, 150.8, 173.6,
186.3. Anal. Calcd for C15H14N4O2S (314.36): C, 57.31; H, 4.49; N,
17.82. Found: C, 57.19; H, 4.46; N, 18.01.
3.1.1. 4-Amino-6-(4-ethylphenyl)-7H-pyrrolo[3,2-d]pyrimidin-
3.1.4. 2-Methylthio-4-[(2-morpholin-4-ylethyl)amino]-6-
7-one 5-oxide (2c)
phenyl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxide (2h)
Mp 235–236 °C (toluene); IR (KBr)
m
cmÀ1 3341, 3291 (NH2),
Mp 208–210 °C (2-PrOH); IR (KBr) m
cmÀ1 3401, 3390, 3329
1724 (CO); 1H NMR (DMSO, 300 MHz): d ppm: 1.22 (3H, t,
J = 7.5 Hz, CH3), 2.67 (2H, q, J = 7.5 Hz, CH2), 7.42 (2H, d,
J = 8.0 Hz, ArH), 7.47 (1H, br s, NH), 8.23 (1H, br s, NH), 8.33 (2H,
d, J = 8.0 Hz, ArH), 8.72 (1H, s, CH); 13C NMR (DMSO, 75 MHz): d
15.0, 28.3, 119.1, 123.5, 126.7, 127.8, 146.2, 150.8, 160.6, 185.3.
Anal. Calcd for C14H12N4O2 (268.27): C, 62.68; H, 4.51; N, 20.88.
Found: C, 62.49; H, 4.40; N, 20.71.
(NH2, NH), 1712 (CO); 1H NMR (DMSO, 300 MHz): d ppm: 2.58
(3H, s, SCH3), 2.70–2.79 (4H, m, N(CH2)2), 2.83 (2H, t,
J = 6.8 Hz, NCH2), 3.66–3.69 (4H, m, O(CH2)2), 3.89 (2H, td,
J = 6.8, 1.5 Hz, NHCH2), 7.51–7.56 (3H, m, ArH), 8.07 (1H, br s,
NH), 8.36 (2H, d, J = 7.5 Hz, ArH); 13C NMR (DMSO, 75 MHz): d
14.9, 37.4, 53.7, 57.1, 66.8, 120.6, 126.3, 127.5, 128.2, 129.2,
130.7, 149.6, 150.2, 174.9, 186.8. Anal. Calcd for C19H21N5O3S
(399.47): C, 57.13; H, 5.30; N, 17.53. Found: C, 57.10; H, 5.40;
N, 17.33.
3.1.2. 4-Amino-6-(4-methylphenyl)-2-methylthio-7H-
pyrrolo[3,2-d]pyrimidin-7-one 5-oxide (2e)
Mp 230–232 °C (2-PrOH); IR (KBr)
m
cmÀ1 3427, 3246 (NH2),
3.1.5. 2-Allylamino-4-amino-6-phenyl-7H-pyrrolo[3,2-
d]pyrimidin-7-one 5-oxide (4a)
1721 (CO); 1H NMR (DMSO, 300 MHz): d ppm: 2.33 (3H, s, CH3),
2.53 (3H, s, SCH3), 7.29 (2H, d, J = 7.8 Hz, ArH), 7.36 (1H, br s,
NH), 8.18 (1H, br s, NH), 8.25 (2H, d, J = 7.8 Hz, ArH); 13C NMR
(DMSO, 75 MHz): d 13.9, 25.2, 119.7, 123.0, 126.9, 127.9, 146.0,
151.8, 174.6, 187.3. Anal. Calcd for C14H12N4O2S (300.34): C,
55.99; H, 4.03; N, 18.65. Found: C, 56.17; H, 4.16; N, 18.79.
Mp 228–230 °C (2-PrOH); IR (KBr)
m
cmÀ1 3400, 3393, 3322
ppm:
(NH2, NH), 1714 (CO); 1H NMR (DMSO, 300 MHz):
d
3.94–3.99 (2H, m, NHCH2), 5.05 (1H, d, J = 10.2 Hz, CH), 5.16
(1H, d, J = 17.7 Hz, CH), 5.83–5.92 (1H, m, CH), 6.88 (1H, br s,
NH), 7.35–7.40 (3H, m, ArH), 7.81 (1H, br s, NH), 7.94 (1H, br
s, NH), 8.29 (2H, d, J = 7.5 Hz, ArH); 13C NMR (DMSO, 75 MHz):
d 40.8, 112.5, 120.4, 125.9, 126.4, 126.7, 128.3, 128.9, 147.0,
151.2, 152.7, 162.0, 174.5, 185.9. Anal. Calcd for C15H13N5O2
(295.30): C, 61.01; H, 4.44; N, 23.72. Found: C, 61.10; H, 4.45;
N, 24.01.
3.1.3. 4-Amino-6-(4-ethylphenyl)-2-methylthio-7H-
pyrrolo[3,2-d]pyrimidin-7-one 5-oxide (2f)
Mp 211–213 °C (2-PrOH); IR (KBr)
1713 (CO); 1H NMR (CDCl3, 300 MHz): d ppm: 1.29 (3H, t,
m
cmÀ1 3405, 3398 (NH2),

E. Pudziuvelyte et al. / Bioorg. Med. Chem. 17 (2009) 4955–4960
4959
3.1.6. 4-Amino-2-methoxycarbonylmethylamino-6-phenyl-7H-
pyrrolo[3,2-d]pyrimidin-7-one 5-oxide (4b)
126.2, 126.3, 128.3, 128.9, 151.4, 152.5, 162.0, 186.9. Anal. Calcd
for C18H19N5O2 (337.38): C, 64.08; H, 5.68; N, 20.76. Found: C,
64.30; H, 5.55; N, 20.78.
Mp 202–203 °C (2-PrOH); IR (KBr)
m
cmÀ1 3469, 3318, 3251
(NH2, NH), 1741, 1710 (CO); 1H NMR (DMSO, 300 MHz): d ppm:
3.82 (3H, s, OCH3), 4.25 (2H, t, J = 7.8 Hz, NHCH2), 6.89 (1H, br s,
NH), 7.37–7.40 (3H, m, ArH), 7.82 (1H, br s, NH), 7.97 (1H, br s,
NH), 8.31 (2H, d, J = 7.5 Hz, ArH); 13C NMR (DMSO, 75 MHz): d
39.4, 54.8, 118.4, 125.5, 126.6, 126.3, 128.7, 128.9, 151.4, 152.5,
161.5, 164.3, 183.7. Anal. Calcd for C15H13N5O4 (327.29): C,
55.05; H, 4.00; N, 21.40. Found: C, 54.96; H, 4.18; N, 21.31.
3.2. Clog P
Software-predicted lipophilicity of the compounds (Clog P) was
calculated with the program CS ChemFinder Ultra, version 9.0,
Cambridge-Soft Corporation. Alternatively, Clog P values can be
calculated with the free program New and Improved Clog P calculator
accessible via Internet (http://intro.bio.umb.edu/111-112/OLLM/
111F98/newclogp.html).
3.1.7. 4-Amino-2-phenylethylamino-6-phenyl-7H-pyrrolo[3,2-
d]pyrimidin-7-one 5-oxide (4d)
Mp 239–241 °C (2-PrOH); IR (KBr)
m
cmÀ1 3464, 3386, 3327
3.3. Biological tests
(NH2, NH), 1709 (CO); 1H NMR (DMSO, 300 MHz): d ppm: 2.87
(2H, t, J = 6.9 Hz, PhCH2), 3.37–3.53 (2H, m, NHCH2), 6.99 (1H, br
s, NH), 7.31 (H5, br s, ArH), 7.42–7.51 (3H, m, ArH), 7.72 (1H, br
s, NH), 8.11 (1H, br s, NH), 8.30 (2H, d, J = 7.5 Hz, ArH); 13C NMR
(DMSO, 75 MHz): d 37.2, 39.0, 117.4, 125.8, 125.9, 126.2, 126.3,
127.1, 127.8, 128.3, 128.5, 135.9, 152.1, 152.5, 162.3, 185.8. Anal.
Calcd for C20H17N5O2 (359.38): C, 66.84; H, 4.77; N, 19.49. Found:
C, 67.00; H, 4.89; N, 19.70.
3.3.1. Cells, culture, and plating
The humansolid tumor cell lines A2780, HBL-100, HeLa, SW1573,
T-47D, and WiDr were used in this study. These cell lines were a kind
gift from Professor Godefridus J. Peters (VU Medical Center, Amster-
dam, The Netherlands). Cells were maintained in 25 cm2 culture
flasks in RPMI 1640 supplemented with 5% heat inactivated fetal calf
serum and 2 mM -glutamine in a 37 °C, 5% CO2, 95% humidified air
L
incubator. Exponentially growing cells were trypsinized and resus-
pended in antibiotic containing medium (100 units penicillin G
and 0.1 mg of streptomycin per mL). Single cell suspensions display-
ing >97% viability by trypan blue dye exclusion were subsequently
counted. After counting, dilutions were made to give the appropriate
cell densities for inoculation onto 96-well microtiter plates. Cells
were inoculated in a volume of 100 lL per well at densities of
15,000 (WiDr, T-47D, and HeLa) and 10,000 (A2780, SW1573, and
HBL-100) cells per well, based on their doubling times.
3.1.8. 4-Amino-6-phenyl-2-[(2-pyrrolidin-4-ylethyl)amino]-7H-
pyrrolo[3,2-d]pyrimidin-7-one 5-oxide (4e)
Mp 210–213 °C (2-PrOH); IR (KBr)
m
cmÀ1 3400, 3317, 3251
(NH2, NH), 1714 (CO); 1H NMR (DMSO, 300 MHz): d ppm: 1.69
(4H, br s, (CH2)2), 1.74 (4H, br s, (CH2)2), 2.49 (4H, br s, (CH2)2),
2.61 (4H, br s, (CH2)2), 3.43–3.45 (4H, m, N(CH2)2), 6.99 (1H, br s,
NH), 7.35–7.50 (3H, m, ArH), 7.60 (1H, br s, NH), 7.89 (1H, br s,
NH), 8.30 (2H, d, J = 7.5 Hz, ArH); 13C NMR (DMSO, 75 MHz): d
23.1, 40.6, 53.5, 54.3, 113.1, 124.7, 125.8, 126.2, 128.3, 128.9,
151.5, 152.9, 163.2, 186.7. Anal. Calcd for C18H20N6O2 (352.39): C,
61.35; H, 5.72; N, 23.85. Found: C, 61.21; H, 5.88; N, 24.00.
3.3.2. Antiproliferative tests
Chemosensitivity testswereperformedusingtheSRBassay of the
NCI with slight modifications. Briefly, purecompoundswere initially
dissolved in DMSO at 400 times the desired final maximum test con-
centration. Control cells were exposed to an equivalent concentra-
tion of DMSO (0.25% v/v, negative control). Each agent was tested
3.1.9. 4-Amino-2-[(3-dimethylaminopropyl)amino]-6-phenyl-
7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxide (4g)
Mp 201–202 °C (2-PrOH); IR (KBr)
m
cmÀ1 3405, 3319, 3259
(NH2, NH), 1710 (CO); 1H NMR (CDCl3, 300 MHz): d ppm: 1.77–
1.86 (2H, m, CH2), 2.31 (6H, s, N(CH3)2), 2.46 (2H, t, J = 9.2 Hz,
NCH2), 3.56 (2H, br s, NHCH2), 6.75 (1H, br s, NH), 7.42–7.51 (3H,
m, ArH), 7.88 (2H, br s, NH2), 8.44 (2H, dd, J = 7.5, 4.8 Hz, ArH);
13C NMR (DMSO, 75 MHz): d 26.2, 29.7, 45.3, 57.8, 114.0, 125.8,
126.9, 127.1, 128.4, 129.6, 140.6, 151.8, 163.7, 187.0. Anal. Calcd
for C17H20N6O2 (340.38): C, 59.99; H, 5.92; N, 24.69. Found: C,
60.28; H, 5.78; N, 24.55.
in triplicates at different dilutions in the range 1–100
treatment was started on day 1 after plating. Drug incubation times
were 48 h, after which time cells were precipitated with 25 L ice-
lM. The drug
l
cold 50% (w/v) trichloroacetic acid and fixed for 60 min at 4 °C. Then
the SRB assay was performed. The optical density (OD) of each well
was measured at 492 nm, using BioTek’s PowerWave XS Absorbance
Microplate Reader. Values were corrected for background OD from
wells only containing medium. The percentage growth (PG) was cal-
culated with respect to untreated control cells (C) at each of the drug
concentration levels based on the difference in OD at the start (T0)
and end of drug exposure (T), according to NCI formulas. Therefore,
if T is greater than or equal to T0 the calculation is 100 Â [(T À T0)/
(C À T0)]. If T is less than T0 denoting cell killing the calculation is
100 Â [(T À T0)/(T0)]. The effect is defined as percentage of growth,
where 50% growth inhibition (GI50) represents the concentration
at which PG is +50. With these calculations a PG value of 0 corre-
sponds to the amount of cells present at the start of drug exposure,
while negative PG values denote net cell kill.
3.1.10. 4-Amino-2-(4-methylpiperazin-1-yl)-6-phenyl-7H-
pyrrolo[3,2-d]pyrimidin-7-one 5-oxide (4j)
Mp 207–209 °C (2-PrOH); IR (KBr)
m
cmÀ1 3472, 3324 (NH2),
1706 (CO); 1H NMR (DMSO, 300 MHz): d ppm: 2.23 (3H, s,
NCH3), 2.38 (4H, br s, N(CH2)2), 3.82 (4H, br s, N(CH2)2), 7.00 (1H,
br s, NH), 7.40–7.53 (3H, m, ArH), 7.70 (1H, br s, NH), 8.30 (2H,
d, J = 7.5 Hz, ArH); 13C NMR (DMSO, 75 MHz): d 44.5, 46.4, 55.1,
14.5, 120.7, 126.8, 126.9, 127.1, 129.1, 129.9, 152.2, 153.1, 162.6,
187.5. Anal. Calcd for C17H18N6O2 (338.36): C, 60.34; H, 5.36; N,
24.84. Found: C, 60.30; H, 5.24; N, 24.79.
3.3.3. Cell-cycle analysis
3.1.11. 4-Amino-2-azepan-1-yl-6-phenyl-7H-pyrrolo[3,2-
d]pyrimidin-7-one 5-oxide (4k)
Cells were seeded in a six well plates at a density of 2.5–
5 Â 105 cells/well. After 24 h the products were added to the respec-
tive well and incubated for an additional period of 24 h. Cells were
trypsinized, harvested, transferred to test tubes (12 Â 75 mm) and
centrifuged at 1500 rpm for 10 min at 5 °C. The supernatant was
discarded and the cell pellets were resuspended in 200 lL of cold
PBS and fixed by the addition of 1 mL ice-cold 70% ethanol. Fixed
cells were incubated overnight at À20 °C after which time was
Mp 215–217 °C (2-PrOH); IR (KBr)
m
cmÀ1 3470, 3340 (NH2),
1704 (CO); 1H NMR (DMSO, 300 MHz): d ppm: 1.52 (4H, br s,
(CH2)2), 1.74 (4H, br s, (CH2)2), 3.74–3.80 (4H, m, N(CH2)2), 6.94
(1H, br s, NH), 7.42 (1H, t, J = 7.5 Hz, ArH), 7.50 (2H, d, J = 7.5 Hz,
ArH), 7.62 (1H, br s, NH), 8.29 (2H, d, J = 7.5 Hz, ArH); 13C NMR
(DMSO, 75 MHz): d 26.4, 26.9, 27.7, 46.8, 47.5, 113.4, 125.8,

4960
E. Pudziuvelyte et al. / Bioorg. Med. Chem. 17 (2009) 4955–4960
centrifuged at 1500 rpm for 10 min. The cell pellets were resus-
pended in 500 L PBS. Then, 5 L of DNAse-free RNAse (10 mg/mL)
was incubated in the dark at 37 °C for 30 min. After incubation
L of propidium iodide (0.5%) was added. Flow cytometric deter-
mination of DNA content (25,000 cells/sample) was analyzed by
LSR II Flow Cytometer (Becton Dickinson, San José, CA, USA). The
fractions of the cells in sub G1, G0/G1, S, and G2/M phase were
analyzed using cell cycle analysis software, FACSDiva 6.0 (Becton
Dickinson, San José, CA, USA).
References and notes
l
l
1. (a) Farutin, V.; Masterson, L.; Andricopulo, A. D.; Cheng, J.; Riley, B.; Hakimi, R.;
Frazer, J. W.; Cordes, E. H. J. Med. Chem. 1999, 42, 2422; (b) Evans, G. B.;
Furneaux, R. H.; Gainsford, G. J.; Hanson, J. C.; Kicska, G. A.; Sauve, A. A.;
Schramm, V. L.; Tyler, P. C. J. Med. Chem. 2003, 46, 155.
2. (a) Gangjee, A.; Li, W.; Yang, J.; Kisliuk, R. L. J. Med. Chem. 2008, 51, 68; (b)
Bavetsias, V.; Jackman, A. L. Curr. Med. Chem. 1998, 5, 265.
3. Norman, M. H.; Chen, N.; Chen, Z.; Fotsch, C.; Hale, C.; Han, N.; Hurt, R.; Jenkins,
T.; Kincaid, J.; Liu, L.; Lu, Y.; Moreno, O.; Santora, V. J.; Sonnenberg, J. D.; Karbon,
W. J. Med. Chem. 2000, 43, 4288.
4. (a) Grahner, B.; Winiwarter, S.; Lanzner, W.; Muller, C. E. J. Med. Chem. 1994, 37,
1526; (b) Esteve, C.; Nueda, A.; Díaz, J. L.; Beleta, J.; Cárdenas, A.; Lozoya, E.;
Cadavid, M. I.; Loza, M. I.; Ryder, H.; Vidal, B. Bioorg. Med. Chem. Lett. 2006, 16,
3642.
5
l
Acknowledgments
5. (a) Susvilo, I.; Brukstus, A.; Tumkevicius, S. Synlett 2003, 1151; (b) Cikotiene, I.;
Pudziuvelyte, E.; Brukstus, A.; Tumkevicius, S. Tetrahedron 2007, 63, 8145.
6. Cikotiene, I.; Pudziuvelyte, E.; Brukstus, A. J. Heterocycl. Chem. 2008, 45, 1615.
7. Skehan, P.; Storeng, P.; Scudeiro, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
This research was supported by the Lithuanian State Science
and Studies Foundation (reg. nr. T-09027, grant nr. T-67/09); the
Spanish MICIIN (CTQ2008-06806-C02-01/BQU) and the Canary
Islands Agencia Canaria de Investigación, Innovación y Sociedad
de la Información (PI 2007/021) co-financed by the European FED-
ER; the Spanish MSC (RTICC RD06/0020/1046) and the Canary
Islands FUNCIS (PI 01/06 and 35/06). L.G.L. thanks the Spanish
MSC-FIS for a postdoctoral contract. J.M.P. thanks the Spanish
MEC-FSE for a Ramón y Cajal contract.
8. Miranda, P. O.; Padrón, J. M.; Padrón, J. I.; Villar, J.; Martín, V. S. ChemMedChem
2006, 1, 323.
9. (a) Hallgas, B.; Dobos, Z.; Osz, E.; Hollósy, F.; Schwab, R. E.; Szabó, E. Z.; Eros, D.;
Idei, M.; Kéri, G.; Lóránd, T. J. Chromatogr., B 2005, 819, 283; (b) Hollósy, F.;
Seprödi, J.; Orfi, L.; Erös, D.; Kéri, G.; Idei, M. J. Chromatogr., B 2002, 780, 355; (c)
Hollósy, F.; Lóránd, T.; Orfi, L.; Erös, D.; Kéri, G.; Idei, M. J. Chromatogr., B 2002,
768, 361.
10. Pizao, P. E.; Peters, G. J.; van Ark-Otte, J.; Smets, L. A.; Smitskamp-Wilms, E.;
Winograd, B.; Pinedo, H. M.; Giaccone, G. Eur. J. Cancer 1993, 29A, 1566.
11. Padrón, J. M.; Peters, G. J. Invest. New Drugs 2006, 24, 195.
12. (a) Guo, T.; Hobbs, D. W. Assay Drug Dev. Technol. 2003, 1, 579; (b) Horton, D.
A.; Bourne, G. T.; Smythe, M. L. Mol. Diversity 1999, 5, 289.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.05.078.