Antiproliferative activities of halogenated pyrrolo[3,2-d]pyrimidines

Article abstract of DOI:10.1016/j.bmc.2015.06.025

In vitro evaluation of the halogenated pyrrolo[3,2-d]pyrimidines identified antiproliferative activities in compounds 1 and 2 against four different cancer cell lines. Upon screening of a series of pyrrolo[3,2-d]pyrimidines, the 2,4-Cl compound 1 was foun

Full text of DOI:10.1016/j.bmc.2015.06.025

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiproliferative activities of halogenated pyrrolo[3,2-d]pyrimidines
Kartik W. Temburnikar a, Christina R. Ross b, Gerald M. Wilson b, Jan Balzarini c, Brian M. Cawrse a,
Katherine L. Seley-Radtke a,
⇑
a Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
b Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene Street, Baltimore, MD 21201, USA
c Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
In vitro evaluation of the halogenated pyrrolo[3,2-d]pyrimidines identified antiproliferative activities in
compounds 1 and 2 against four different cancer cell lines. Upon screening of a series of pyrrolo[3,
2-d]pyrimidines, the 2,4-Cl compound 1 was found to exhibit antiproliferative activity at low micromolar
concentrations. Introduction of iodine at C7 resulted in significant enhancement of potency by reducing
the IC50 into sub-micromolar levels, thereby suggesting the importance of a halogen at C7. This finding
was further supported by an increased antiproliferative effect for 4 as compared to 3. Cell-cycle and
apoptosis studies conducted on the two potent compounds 1 and 2 showed differences in their cytotoxic
mechanisms in triple negative breast cancer MDA-MB-231 cells, wherein compound 1 induced cells to
accumulate at the G2/M stage with little evidence of apoptotic death. In contrast, compound 2 robustly
induced apoptosis with concomitant G2/M cell cycle arrest in this cell model.
Received 19 April 2015
Revised 4 June 2015
Accepted 10 June 2015
Available online xxxx
Keywords:
Thieno[3,2-d]pyrimidine
Heterocyclic chemistry
Cytostatic
Apoptosis
Ó 2015 Elsevier Ltd. All rights reserved.
Cell cycle arrest
1. Introduction
thieno[3,2-d]pyrimidines were found to induce cell cycle indepen-
dent apoptosis in L1210, a mouse lymphocytic leukemia cell line.28
Pyrrolopyrimidines exist as three regioisomers, specifically
pyrrolo[2,3-d]pyrimidine, pyrrolo[3,2-d]pyrimidine and pyrrolo
[3,4-d]pyrimidine. The former two isomers more commonly
referred to as 7-deazapurine and 9-deazapurine, respectively,
(Fig. 1) have been of interest for long, due to close resemblance
to the purines. Hence these pyrrolopyrimidines have often found
use in the pharmacology field as a purine isostere.1–12 The 7-deaza-
purines (Fig. 1a) are naturally occurring and widely used in drug
design primarily due to their propensity to be ribosylated. For
example, echiguanine B and tubercidin are two naturally occurring
7-deazapurines exhibiting anticancer properties.
In contrast, the pyrrolo[3,2-d]pyrimidines do not occur naturally
and must be synthetically prepared. The 9-deazapurines have also
been explored in the past as nucleoside isosteres and these efforts
have resulted in the design of purine nucleoside phosphorylase
(PNP) inibitors,7,13–22 dihydrofolate reductase (DHFR) inhibitors23,24
and more recently for kinase inhibition (Fig. 1b).25–27
However, they showed remarkable differences in their cytotoxic
mechanisms in the aggressive triple negative breast cancer (TNBC)
cell line MDA-MB-231, which do not express estrogen or proges-
terone receptors and do not show HER-2/Neu gene amplification.
While both halogenated thieno[3,2-d]pyrimidines shown in
Figure 2 were toxic to MDA-MB-231 cells, for the analogue lacking
bromine at C7 (TP), cell death was accompanied by dramatic arrest
at the G2/M cell cycle transition (Fig. 2), a mechanism not observed
when cells were treated with the bromo analogue (Fig. 2, Br-TP).29
Together, these data indicatedthat2,4-dichlorothieno[3,2-d]pyrim-
idines were potently toxic to several tumor cell types, but also that
their antiproliferative potency and cytotoxic mechanisms varied as
a function of halogenation at C7 in a cell type-specific manner.
In an effort to further expand the repertoire of halogenated
fused pyrimidines, our studies were extended to the pyrrolo[3,
2-d]pyrimidine scaffold. Once in hand, the toxicity of halogenated
pyrrolo[3,2-d]pyrimidines was tested against four cancer cell lines
(L1210, CEM, HeLa and MDA-MB-231). This examination revealed
significant antiproliferative activities for compounds 1–4 (Fig. 3)
but also indicated that this activity was enhanced by inclusion of
iodine at C7. Subsequent testing of compounds 1 and 2 across
the NCI-60 cancer cell line panel further supported the impact of
iodine at C7 on the cytostatic/cytotoxic activity of the halogenated
pyrrolo[3,2-d]pyrimidines and underscore their spectrum of
Our laboratory’s interest in exploring the biological properties of
fused heterocyclic compounds30–34 has resulted in the identification
of antiproliferative properties of halogenated thieno[3,2-d]pyrim-
idines (TP and Br-TP) as shown in Figure 2. The halogenated
⇑
Corresponding author. Tel.: +1 410 455 8684 (O); fax: +1 410 455 2608.
E-mail address: kseley@umbc.edu (K.L. Seley-Radtke).
http://dx.doi.org/10.1016/j.bmc.2015.06.025
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 1. Deazapurines and their biological properties.
Figure 2. Antiproliferative activities of halogenated thieno[3,2-d]pyrimidines.28,29
activity. Compounds 1 and 2 were then evaluated for cell cycle
distributions and apoptosis of MDA-MB-231 cells to explore their
mechanism of antiproliferative activity. The results indicated that
both compounds trigger arrest at G2/M, although to varying
degrees.
Scheme 1. Synthesis of halogenated pyrrolo[3,2-d]pyrimidines.
2. Results
2.1. Chemistry
In order to explore the antiproliferative properties of the halo-
genated pyrrolo[3,2-d]pyrimidines as well as the effect of the halo-
gen at C7, two sets of compounds were synthesized (Schemes 1
and 2). The target compounds in Scheme 1 were designed to explore
the anticancer properties of the pyrrolo[3,2-d]pyrimidines
possessing a chlorine at C4. A second set of compounds illustrated
in Scheme 2 possessing O-benzyl groups on the C2 and C4 positions
were synthesized to evaluate the effect of C7 halogen on
antiproliferative properties independent of chlorine at C2 and C4.
The 2,4-bis-O-benzylated compounds were chosen instead of
methoxy analogues primarily because the 2,4-methoxy substitution
on thieno[3,2-d]pyrimidines lead to complete loss of activity.
Subsequently, we were unable to assess and establish unequivocally
the contribution or enhancement of activity by halogen at C7. In
order to explore an alternative substituent, 2,4-bis-O-benzylated
pyrrolo[3,2-d]pyrimidines were prepared and evaluated for antipro-
liferative properties.
Scheme 2. Synthesis of the bis-O-benzyl analogues.
The synthesis of halogenated pyrrolo[3,2-d]pyrimidines
(Scheme 1) began with chlorination of the known pyrrolo[3,
2-d]pyrimidin-2,4-dione 5.35–39 Preparation of the sodium salt,
followed by heating with phenylphosphonic dichloride (PhPOCl2)
Figure 3. Halogenated pyrrolo[3,2-d]pyrimidine targets.
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K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
at 170–175 °C gave 1.35–39 Next, we decided to introduce a halogen
at C7 that would help us evaluate halogen other than chlorine and
bromine, thereby expanding the repertoire of halogens on the
fused pyrimidine scaffold. Iodine besides being a larger halogen
is also easier to displace.40 Thus, the iodo analogue was prepared
by using N-iodosuccinimide (NIS) in tetrahydrofuran (THF) at room
temperature yielding compound 2.41
Attempts to protect the 5-NH of 2 with tert-butoxycarbonyl
(Boc), in order to evaluate the effect of the NH on the biological
activity of 2, failed due to the instability of the Boc protecting
group during the subsequent silica gel purification. Alternatively,
Boc protection of 1 was attempted using Boc2O in the presence
of 4-dimethylaminopyridine (DMAP) at room temperature to give
6.42 Iodination of pyrrolo[3,2-d]pyrimidin-2,4-dione 5 by NIS in
DMF followed by crystallization from ethanol gave 7 as a yellow
solid.43
As shown in Scheme 2, synthesis of the bis-O-benzylated
pyrrolo[3,2-d]pyrimidines was initiated by formylation of the
6-methyl group on 9 by heating with DMF-dimethylacetal in
DMF at 60–65 °C, to obtain 10 (Scheme 2).37 Subsequently, stirring
10 with zinc (Zn) in acetic acid (AcOH) enabled ring cyclization to
give 2,4-OBn pyrrolo[3,2-d]pyrimidine 3 in high yields.37,44–46
Iodination of 3 using NIS in methylene chloride (CH2Cl2) afforded
4 which was followed by the protection of 5-NH using Boc in the
presence of DMAP to obtain 10.40
Along with the compounds shown in Schemes 1 and 2, two
additional compounds, namely the 9-deaza-20-deoxynucleosides
11 and 12 were tested owing to past1–7,47–57 and recent58 interest
in the pharmacological properties of 9-deazanucleosides. The syn-
theses of the compounds shown in Figure 4 have been previously
reported by our group but their antiproliferative properties had
not yet been evaluated.40,59
nature of Boc protection on 5-NH and release of 1 in the biological
environment. On the other hand, the presence of the Boc group on
the bis-O-benzyl analogue (10) led to a complete loss in activity.
These data support the importance of 7-NH in the cytostatic role
of the 2,4-O-benzylated analogues 3 and 4. The amide groups, as
found in 5 and 7 do not impart activity against any cancer cell line
tested, which is consistent with our previous studies on the
thieno[3,2-d]pyrimidines. Similarly, the C-nucleosides 11 and 12
were both inactive and exhibited no cytotoxicity.40
2.3. NCI-60 DTP Human Tumor Cell Line screen
In order to validate the promising activities of compounds 1 and
2 and also the role of C7 iodine, their cytostatic/toxic activities
were tested at 10 lM concentrations against the NCI-60 DTP
Human Tumor Cell Line screen. These cell lines are derived from
many different cancer types including leukemia, non-small cell
lung cancer (NSCLC), colon, CNS cancer, melanoma, ovarian, renal,
prostate, and breast cancer (Fig. 5). In this screen, 10 lM of each
compound is added to cells and incubated for 48 hrs before
chemical fixation and measurement of cellular protein content
using the sulforhodamine B (SRB) dye.64,65 This assay enables dif-
ferentiation between cytostatic and cytotoxic activities based on
the magnitude and direction of endpoint deviations from initial
cell density.66
Compound 1 did not show meaningful inhibition of growth of
the cancer cell lines at 10 lM concentration (Fig. 5) however
introduction of iodine at C7 (compound 2) led to an increase in
the ability of the halogenated pyrrolo[3,2-d]pyrimidine to inhibit
the growth of all cancer cell lines tested. The results of single-
dose testing suggests that compound 2 predominately exhibits
cytostatic activity (growth percent 0–50%), but in some instances
led to a cytotoxic effect (growth percent <0%). The latter was
observed in HL-60(TB), NCI-H522, COLO 205, MDA-MB-435,
OVACAR-3, UO-31 and DU-145 (Fig. 5). These results, in addition
to the earlier testing, verify that the iodine at C7 plays a key role
in increasing the potential anticancer properties of the C7-iodi-
nated compound 2. The screening of compound 1 highlights the
cytotoxic activities against specific cell lines in various cancer
subsets, which may be due to a common or similar mechanism
of action. The study of common characteristics of these cell lines
is of our interest to elucidate the biological target and mechanism
of action.
2.2. In vitro cell growth inhibition
The cytotoxic activities of the compounds described above were
first assessed in three cultured tumor cell models: L1210, a mouse
lymphocytic leukemia cell line,60 CCRF-CEM,61 an acute lym-
phoblastic leukemia cell line, and HeLa, a human cancer cell line
derived from a human cervical adenocarcinoma.62,63 Comparisons
of resolved IC50 values (Table 1) indicate that the dichloro com-
pounds 1 and 2 show measureable cytostatic activity in all three
tumor cell lines. The presence of iodine at C7 on the 2,4-dichloro
pyrrolo[3,2-d] (2) increases the antiproliferative activity by a factor
of 5 to 20, when compared to 1. An analogous increase in cytotox-
icity for bis-O-benzylated pyrrolo[3,2-d]pyrimidine 3 was observed
upon introduction of C7 iodine (4). Replacement of the 2,4-chloro
moieties with O-benzyl groups led to a loss of activity by a factor
of 3–15, however the activity of 4 was comparable to 1. This sug-
gests a compensation for the loss of activity for the O-benzyl
groups by the presence of iodine at C7.
2.4. Cell cycle and apoptosis studies
Previously, 2,4-dichloro thieno[3,2-d]pyrimidines (see Fig. 2)
were shown to induce apoptosis independent of cell cycle arrest
in L1210 cells.28 Our recent findings show that a 2,4-dichloro
thieno[3,2-d]pyrimidine can also induce cell cycle arrest at G2/M
in the MDA-MB-231 breast cancer cell line.29 Subsequently, we
chose to study the effects of 2,4-dichloro pyrrolo[3,2-d]pyrimidi-
nes 1 and 2 on cell cycle progression and apoptosis in MDA-MB-
231 cells for two principal reasons. First, the MDA-MB-231 line is
a well-characterized cell model of TBNC, an aggressive breast can-
cer type for which no biomarker-targeted therapies are currently
available.67 Second, constitutive overexpression of anti-apoptotic
factors generally prevents chemotherapeutic induction of apopto-
sis in breast cancer cells including MDA-MB-231,68–70 so alterna-
tive triggering mechanisms such as mitotic arrest may represent
appealing therapeutic modalities for these tumors. MTT cytotoxic-
Introduction of the Boc group on 2,4-dichloro pyrrolo[3,
2-d]pyrimidine 6 did not lead to any loss in activity against CEM
and HeLa cells, while activity against L1210 was weakened by a
factor of 3. We speculate that these activities arise from the labile
ity assays resolved IC50 values of 6.0 1.3 lM and 0.51 0.10 lM
for compounds 1 and 2, respectively, in MDA-MB-231 cell cultures
(Fig. 6). These data provided dosage information critical for subse-
quent cell cycle and apoptosis assays, but also further confirm that
Figure 4. 9-Deaza-20-deoxynucleosides 11 and 12.40
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K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
Anti-tumor cell activity of test compounds
*
Compound
A
B
R1
R2
R3
X
IC50 (lM)
L1210
CEM
HeLa
1
2
3
4
5
6
7
10
Cl
Cl
OBn
OBn
O
Cl
O
OBn
Cl
Cl
OBn
OBn
O
Cl
O
OBn
—
—
—
—
H
—
H
—
—
—
—
—
H
—
H
—
H
H
H
H
H
Boc
H
H
I
H
I
H
H
I
6.8 2.8
0.93 0.0
25
4.9 0.4
86 22
17
>250
26
>250
193 81
2
19
3
0.92 0.04
98 14
17
>250
18
118
21
8
2
4
0
>250
21
1
2
3
>250
247
>250
72 47
Boc
I
4
1140
O
O
H
H
H
>250
>250
>250
1240
O
NH2
—
—
—
H
—
H
—
>250
>250
>250
5FU
—
—
0.33 0.17
18
5
0.54 0.12
*
50% inhibitory concentration. 5FU—5-fluorouracil.
introduction of iodine at C7 increases the potency by at least a fac-
tor of 10.
To determine whether the anticancer activities of pyrrolo[3,2-
d]pyrimidine compounds 1 and 2 are associated with perturbation
of the cell cycle, we monitored the effects of each compound on cell
cycle distributions using propidium iodide staining and flow cytom-
While compound 1 is clearly toxic to MDA-MB-231 cells at the
tested concentration, only a modest (although statistically signifi-
cant) increase in annexin V-positive cells was detected. There are
several possibilities that could account for this, including: (i) that
cell lysis occurs rapidly following induction of apoptosis by com-
pound 1, preventing significant accumulation of annexin V-posi-
tive bodies, or (ii) that a distinct (i.e., non-apoptotic/necrotic) cell
death pathway is triggered by 1. On the other hand, the robust
accumulation of annexin V-positive cells observed following treat-
ment with compound 2 is consistent with apoptotic cell death,
similar to that previously observed for the thieno[3,2-d]pyrimidine
compounds shown in Figure 2a in mouse lymphocytic leukemia
L1210 cells.28
etry. At the IC80 concentration of 15 lM, compound 1 induced pro-
found accumulation of the tumor cells at the G2/M stage after 48
hours (Fig. 7a), consistent with mitotic arrest and similar to the
effect of the non-brominated 2,4-dichloro thieno[3,2-d]pyrimidine
(Fig. 2, TP) on MDA-MB-231 cells reported previously.29 By contrast,
treatment with an equitoxic concentration (1.75 lM) of compound
2 yielded a much smaller but still statistically significant enrich-
ment in the G2/M fraction (Fig. 7b, P = 0.0006 vs vehicle control).
The similarities in IC50 values and the accumulation of the tumor
cells in the G2/M phase observed for compound 1 and the non-
brominated thieno[3,2-d]pyrimidine described previously29 sug-
gests that they may induce MDA-MB-231 tumor cell death via sim-
ilar mechanisms. However, the dramatic enhancement in IC50
observed with compound 2 concomitant with its decreased impact
on the cell cycle suggests that introduction of iodine at C7 might
induce cytotoxicity by a distinct molecular mechanism. That such
an impact might be directed by the iodine group on C7 is also sup-
ported by the abrogation of G2/M accumulation that was observed
when 2,4-dichloro thieno[3,2-d]pyrimidine was brominated at the
same site (Fig. 2, Br-TP).29
Next, we analyzed unfixed MDA-MB-231 cell samples by stain-
ing with annexin V and 7-amino-actinomycin D (7-AAD) to deter-
mine whether pyrrolo[3,2-d]pyrimidine compounds 1 and 2 direct
cell death using classical apoptotic or necrotic mechanisms.
Vehicle (DMSO)-treated cells yielded very low proportions of apop-
totic or necrotic cells after 48 h (Fig. 8a). Curiously, treating cells
3. Discussion
Halogenated pyrrolo[3,2-d]pyrimidines have been commonly
used as key intermediates to install various types of functional
groups on a pyrrolo[3,2-d]pyrimidine scaffold in order to evaluate
the effect of substituents for a variety of medicinal proper-
ties.26,27,39,41,43 However, the potential of halogenated pyr-
rolo[3,2-d]pyrimidines themselves as a therapeutically relevant
scaffold has not previously been evaluated. Prompted by our ear-
lier findings with the thieno[3,2-d]pyrimidines,28 2,4-dichloropy-
rrolo[3,2-d]pyrimidines
1 and 2 were evaluated for their
antiproliferative properties against a number of cancer cell lines
(Table 1 and Fig. 2). Since the importance of the C4 Cl group was
already established,28 we studied the effect of pyrrole on the
antiproliferative properties of a halogenated fused-pyrimidine
scaffold. The activity of 2,4-dichloro pyrrolo[3,2-d]pyrimidine 1 is
slightly weaker than its thieno counterpart, however, the presence
of a 7-iodo enhanced the cytotoxicity to an extent that the activity
was comparable to the halogenated thieno[3,2-d]pyrimidines
shown in Figure 2. This notable increase in cytotoxic activity for
2 led to evaluation of bis-O-benzylated pyrrolo[3,2-d]pyrimidines
3 and 4 to assess the effect of C7 iodine independent of the C2
and C4 chlorines. The enhancement of cytotoxic activity for both
2 and 4 compared with the C7-unsubstituted parent compounds
with the IC80 concentration (15 lM) of compound 1 only slightly
increased the proportion of cells recovered in early/late apoptosis
(Fig. 8b and d). By contrast, over 40% of MDA-MB-231 tumor cells
treated with compound 2 at its IC80 (1.75 lM) were annexin V-pos-
itive after 48 h, with the larger proportion of these 7-AAD negative
consistent with early apoptosis (Fig. 8c and d).
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K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
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Figure 5. Growth inhibition of cancer cell lines by compounds 1 and 2 at 10
Cell Line panel. The blue bars indicate percent changes in cell densities following incubation with 1 at 10
densities following 48 h incubation with 10 M 2. The number on each bar indicates the measured % growth.
l
M. Compounds 1 and 2 tested at 10
l
M concentrations against the NCI-60 DTP Human Tumor
lM for 48 h, while the red bars indicate percent changes in cell
l
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K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 6. MTT assays for cytotoxicity by 1 and 2 in MDA-MB-231 cells. Cells were treated with select concentrations of compounds 1 (a) and 2 (b). After 48 h, cell viability
was measured using MTT assays. Compound cytotoxicity was calculated by non-linear regression to a sigmoidal dose response function. Quoted IC50 values represent the
mean SD of three independent experiments.
Figure 7. Cell cycle analyses of MDA-MB-231 cells following treatment with 1 and 2. Cells were treated with IC80 concentrations of compound 1 (a, 15
1.75 M) for 48 h. The cell cycle distributions of drug-treated cells and vehicle (DMSO) controls were analyzed by flow cytometry of fixed, propidium iodide-stained cells. A
minimum of 3000 cells were analyzed per cell population. Each bar represents the mean SD across 4 independent cell samples.
lM) or compound 2 (b,
l
highlights the ability of the C7 iodine in imparting cytotoxic prop-
erties to the pyrrolo[3,2-d]pyrimidine scaffold independently.
Our recent studies on the cytotoxicity of 2,4-dichlorothieno[3,2-
d]pyrimidines in MDA-MB-231 cells have shown that an analogue
lacking a functional group at C7 arrests the cell cycle at the G2/M
checkpoint in this cell model. By contrast, the presence of a bro-
mine at C7 induced cell death via a distinct, cell cycle-independent
mechanism.29 Consistent with these findings, cell cycle distribu-
tion experiments conducted on MDA-MB-231 cells treated with
pyrrolo[3,2-d]pyrimidines 1 and 2 indicated that while both induce
G2/M cell cycle arrest, the presence of the C7 iodine substantially
diminished the accumulation of cells at the G2/M stage (Fig. 7).
Furthermore, the accumulation of annexin V-positive bodies
among cells treated with compound 2 (Fig. 8) indicated contribu-
tions from apoptotic pathways to cytotoxicity, an observation that
was not evident in cells treated with compound 1, which lacks the
C7 iodine. This observation is particularly intriguing in the TNBC
MDA-MB-231 cell model since elevated levels of the apoptosis
inhibitor Bcl-xL and a mutation in the tumor suppressor p53 signif-
icantly weaken its responsiveness to many apoptotic stimuli.68
Similar apoptotic resistance mechanisms are common in breast
cancers;69,70 this complication is a major motivator for the devel-
opment of novel therapeutic strategies.
underscores their utility as potential lead molecules that may be
exploited to treat cancer in a novel and effective manner.
4. Conclusion
We have identified interesting antiproliferative properties for
several 2,4-dichloro pyrrolo[3,2-d]pyrimidines. Further, the obser-
vation that the presence of iodine at C7 markedly enhances cyto-
toxic properties is notable. These studies highlight their
pharmacological relevance in addition to expanding the domain
of the halogenated fused [3,2-d]pyrimidine scaffold. These
halogenated compounds exhibited potent activity against TNBC
MDA-MB-231 cells involving arrest of the cell cycle in G2/M. We
have previously established the importance of the chlorine at C4,
however herein we have demonstrated that inclusion of an addi-
tional halogen at C7 dramatically increases cytotoxicity, which, in
the case of MDA-MB-231 cells, includes characteristics of apoptotic
cell death. Studies are underway to further expand the repertoire
of these molecules with varied substituents on the scaffold to
improve the antiproliferative activities of these halogenated pyr-
rolo[3,2-d]pyrimidines. Those results will help expand the chemi-
cal space of halogenated heterocyclic compounds with an ability
to induce death in cancer cells.
In summary, the antiproliferative activities of the halogenated-
fused pyrimidines highlight the potential of reactive species as ther-
apeutically useful scaffolds. Previously chemically labile moieties as
electrophilic covalent inhibitors have been considered undesir-
able71–78 due to their off-target interactions. However in the recent
past interest in chemically labile molecules has revived owing to
their utility to act as covalent irreversible inhibitors.79–81 The biolog-
ical activity exhibited by halogenated pyrrolo[3,2-d]pyrimidines
5. Experimental section
5.1. Synthetic methods
5.1.1. General
All chemicals and reagents listed in this section were purchased
through commercially available sources unless otherwise noted.
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K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
Figure 8. Apoptosis analyses of MDA-MB-231 cells following treatment with IC80 concentrations of compounds 1 and 2. Cells were treated with (a) an equal volume of
vehicle (DMSO), (b) 15 M compound 1, or (c) 1.75 M compound 2. After 48 h, cells were stained using annexin V and 7-AAD and analyzed by flow cytometry. A minimum of
l
l
10,000 cells were analyzed per condition, and subdivided into four categories: non-apoptotic/non-necrotic (bottom left), early apoptotic (bottom right), late apoptotic/
necrotic (top right), and necrotic (top left). Percentages of total cells detected in each quadrant are indicated. (d) Total apoptotic (early + late) cell fractions detected in each
treatment group. Bars show the mean SD values compiled across four independent experiments.
Anhydrous CH2Cl2, CH3CN, and THF were obtained from a solvent
purification system (SPS, Model: mBraun Labmaster 130).
Anhydrous DMF, MeOH and pyridine were obtained from Sigma–
Aldrich or Acros Organics. All 1H and 13C NMR spectra were
obtained from a JEOL ECX 400 MHz NMR. All 1H and 13C NMR spec-
tra were referenced to internal tetramethylsilane (TMS) at 0.0 ppm.
The spin multiplicities are indicated by the symbols s (singlet), d
(doublet), dd (doublet of doublets), t (triplet), q (quartet), m (mul-
tiplet), and br (broad). All NMR solvents were obtained from
Cambridge Isotope Laboratories. All reactions were monitored by
thin layer chromatography (TLC) on 0.25 mm precoated glass
(stabilized) (2 g) was added in two lots of 1 g with an interval of
1 h. Upon overnight stirring the yellow slurry changed to a pale
yellow to off-white slurry which was filtered and the filtrate con-
centrated in vacuo to obtain brown syrup. The product was precip-
itated from the brown syrup using ethanol to obtain 4 as white
solid (0.6 g, 89.8%). The spectral data agrees with reported data.38
1H NMR (400 MHz, DMSO-d6): d 5.82–5.83 (t, 1H, J = 2.28 Hz),
7.12–7.13 (t, 1H, J = 2.72 Hz, J = 2.96 Hz), 10.57 (s, 1H), 10.74 (s,
1H), 11.82 (s, 1H). 13C NMR (400 MHz, DMSO-d6): d 96.5, 110.9,
127.4, 135.1, 152.0, 156.3.
plates. All column chromatography was run on 32–63
l
silica gel
5.1.1.2. 2,4-Dichloropyrrolo[3,2-d]pyrimidine (1). To pyr-
rolo[3,2-d]pyrimidin-2,4-dione 5 (2.00 g, 13.2 mmol), 1 N NaOH
(15 mL), and 0.60 g NaOH in 15 mL H2O was added and the mixture
stirred at 40 °C until a clear solution was obtained. The solution
was cooled to room temperature (21–25 °C) and then placed in
an ice bath to obtain thick slurry. The slurry was then filtered to
obtain a pale yellow solid. The solid was dissolved in 1 N NaOH
(15 mL), and heated to 40 °C to obtain a clear solution that upon
cooling provided white crystals. The crystals were washed with
MeOH (20 mL) and acetone (20 mL), and then dried under vacuum.
The dry solids were taken in phenylphosphonic dichloride (10 mL)
and heated to 170–175 °C for 5 h during which the reaction mix-
ture became a brown-black solution. After 5 h the hot reaction
mixture was poured onto ice, extracted with EtOAc (200 mL) and
obtained from Dynamic Adsorbents Inc. (Norcross, GA, USA).
Melting points are uncorrected. Yields refer to chromatographi-
cally and spectroscopically (1H and 13C NMR) homogeneous mate-
rials. All mass spectra (MS) were recorded and obtained from the
University of Maryland Baltimore County Mass Spectrometry
Facility and Johns Hopkins Mass Spectrometry Facility. The FAB
mass spectra were obtained using double focusing magnetic sector
mass spectrometer equipped with a Cs ion gun and Fourier trans-
form ion cyclotron resonance equipped with ESI source.
5.1.1.1. Pyrrolo[3,2-d]pyrimidin-2,4-dione (5).
slurry of (E)-6-(2-(dimethylamino)vinyl)-5-nitropyrimidin-2,
4-dione 435,38 (1 g, 4.4 mol) in fresh glacial AcOH (50 mL), Zinc dust
To a stirred
Please cite this article in press as: Temburnikar, K. W.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.025

8
K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
the organic layer washed with satd NaHCO3 solution (3ꢀ 100 mL)
till all effervescence subsided. The organic layer was then washed
with brine and dried over MgSO4. The organic layer was concen-
trated in vacuo and loaded onto silica. The product was purified
using column chromatography eluting with 9:1 then 3:1
hexanes/EtOAc to obtain 1 as an off-white solid (1.50 g, 7.9 mmol,
60%). Rf 0.5 in 3:1 hexanes/EtOAc. Mp 228.3–232.0 °C. 1H NMR
(400 MHz, DMSO-d6): d 6.71 (d, 1H, J = 3.2 Hz), 8.09 (d, 1H,
J = 2.8 Hz), 12.75 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d
103.2, 124.3, 138.0, 143.5, 149.6, 153.9. ESI-MS m/z for C6H3Cl2N3
calculated [M+H]+ 187.9776, found 187.9777.
using column chromatography eluting with 9:1 hexanes/EtOAc to
obtain product 10 as orange-yellow solid (2 g, 75%). Rf 0.5 in 3:1
hexanes/EtOAc. Spectral data agrees with reported data.37 1H
NMR (400 MHz, CDCl3): d 2.87–2.94 (br d, 6H), 5.33–5.36 (d, 1H,
J = 12.36 Hz), 5.38 (s, 2H), 5.44 (s, 2H), 7.31–7.41 (m, 10H), 7.98–
8.01 (d, 1H, J = 12.36 Hz). 13C NMR (400 MHz, CDCl3): d 68.9,
69.5, 87.9, 127.5, 128.0, 128.1, 128.3 128.5, 135.8, 136.6, 151.8,
160.7, 161.6, 163.5. FAB-MS for C22H22N4O4 calculated [M+H]+
407.1714, found 407.1717.
5.1.1.7. 2,4-Bis-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine40 (3).
To
a suspension of 2,4-bis-O-benzyl-5-nitro-6-b-dimethy-
5.1.1.3. 7-Iodo-2,4-dichloro pyrrolo[3,2-d]pyrimidine (2). To a
laminovinyl pyrimidine 10 (2 g, 4.9 mmol) in AcOH (40 ml), Zn
(4 g) was added in lot of 2 g with an interval of 4hrs. The reaction
mixture was stirred overnight at room temperature during which
a dark yellow suspension became pale yellow suspension. The reac-
tion mixture was filtered and the filtrate concentrated in vacuo to
obtain syrup which was dissolved in CH2Cl2 then washed with sat-
urated aq. NaHCO3 followed by brine. The organic phase was dried
over MgSO4 and loaded on silica. The product was purified using
column chromatography eluting with 4:1 and 1:1 hexanes/EtOAc
to obtain product as pale-yellow solid (1.45 g, 90%). Rf 0.3 in 1:3
hexanes/EtOAc. Spectral data agrees with reported data.37 1H
NMR (400 MHz, CDCl3): d 5.47 (s, 2H), 5.54 (s, 2H), 6.50–6.51 (dd,
1H, J = 1.84, 2.28), 7.29–7.38 (m, 7H), 7.43–7.45 (m, 2H), 7.52–
7.53 (m, 2H), 8.41 (br s, 1H, NH). 13C NMR (400 MHz, CDCl3): d
68.3, 69.0, 102.6, 111.9, 127.8, 128.3, 128.4, 128.5, 128.6, 128.7,
128.8, 136.1, 137.3, 151.8, 156.79, 159.7. FAB-MS for C20H17N3O2
calculated [M+H]+ 332.1394, found 332.1398.
solution of 2,4-dichloro pyrrolo[3,2-d]pyrimidine
1 (100 mg,
0.53 mmol) in anhydrous THF (5 mL), NIS (144 mg, 0.64 mmol)
was added under N2 atmosphere and stirred for 2 h after which
TLC indicated consumption of 1. The solvent was removed in vacuo
and the residue dissolved in EtOAc. The organic phase was washed
with aq. solution of Na2S2O3 followed by water, brine and then dried
over MgSO4. The organic layer was concentrated in vacuo and
loaded on silica. The product was purified using column chromatog-
raphy eluting with 9:1 hexanes/EtOAc to obtain product as off-
white solid (90 mg, 54%). Rf 0.55 in 3:1 hexanes/EtOAc. Mp decom-
posed from 160 to 230 °C. Spectral data agrees reported data.41 1
H
NMR (400 MHz, DMSO-d6): d 8.29 (s, 1H), 13.19 (s, 1H, NH). 13C
NMR (400 MHz, DMSO-d6): d 58.2, 124.4, 140.9, 143.5, 149.8,
153.5. ESI-MS m/z for calculated [M+H]+ 313.8743, found 313.8740.
5.1.1.4.
d]pyrimidine (6). To
d]pyrimidine
N-tert-Butyloxycarbonyl-2,4-dichloro
pyrrolo[3,2-
a
mixture of 2,4-dichloro pyrrolo[3,2-
1
(50 mg, 0.26 mmol), di-tert-butyl carbonate
5.1.1.8. 7-Iodo-2,4-bis-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine40
(4). To a stirred solution of 2,4-bis-O-benzyl-5H-pyrrolo[3,2-
d]pyrimidine 3 (1.43 g, 4.3 mmol) in anhydrous CH2Cl2 (15 mL)
under N2, NIS (1.069 g, 4.7 mmol) was added at which point the
reaction mixture turned from pink to orange. The mixture was stir-
red overnight until the TLC indicated the absence of starting mate-
rial. The reaction mixture was washed with aqueous Na2S2O3
(15 mL) followed by brine (15 mL). The organic layer was dried
over MgSO4, loaded onto silica and purified using column chro-
matography eluting with 4:1 then 1:1 hexanes/EtOAc to obtain 4
as a pale-yellow solid (1.77 g, 3.88 mmol, 90%).40 Rf 0.4 in 1:3
hexanes/EtOAc. Mp 157.8–158.4 °C. 1H NMR (400 MHz, CDCl3): d
5.53 (s, 4H), 7.32–7.41 (m, 9H), 7.55–7.57 (m, 2H), 8.71 (br s, 1H,
NH). 13C NMR: 57.3, 68.7, 69.3, 111.9, 127.9, 128.3, 128.6, 128.66,
128.7, 128.9, 132.4, 135.8, 137.2, 152.0, 156.9, 160.1. FAB-MS m/z
for C20H16IN3O2 calculated [M+H]+ 458.0360, found 458.0357.
(116 mg, 0.53 mmol) and DMAP (6.5 mg, 0.053 mmol), anhydrous
THF (5 mL) was added under N2 atmosphere and stirred overnight
upon which TLC indicated consumption of 1. The reaction mixture
was concentrated and loaded on silica. The product was purified
using column chromatography eluting with 49:1 hexanes/EtOAc
to obtain product as white solid (50 mg, 65%). Rf 0.5 in 19:1
hexanes/EtOAc. Mp 101.0–103.2 °C. 1H NMR (400 MHz, DMSO-
d6): d 1.66 (s, 9H), 6.71–6.72 (d, 1H, J = 3.68 Hz), 8.01–8.02 (d,
1H, J = 3.68 Hz). 13C NMR (400 MHz, DMSO-d6): d 28.0, 87.2,
106.6, 123.4, 137.3, 146.5, 147.0, 153.1, 158.1. ESI-MS m/z for
C
11H11Cl2N3O2 calculated [M+H]+ 288.0301, found 288.0301 (2ꢀ
35Cl), 289.0335 (2ꢀ 35Cl, 13C), 290.0272 (35Cl37Cl).
5.1.1.5. 7-Iodo-pyrrolo[3,2-d]pyrimidine-2,4-dione (7). Pyrro-
lo[3,2-d]pyrimidine-2,4-dione 5 (500 mg, 3.31 mmol) was sus-
pended in anhydrous DMF (10 mL) under N2 atmosphere and
cooled to ꢁ10 to ꢁ5 °C. To this slurry, N-iodosuccinimide
(894 mg, 3.97 mmol) was added and the mixture stirred for 2 h
at ꢁ10 to ꢁ5 °C, at which point the DMF was removed in vacuo
to provide a brown sticky solid. The product was crystallized from
the residue using 50% EtOH (6 mL) to give 7 (800 mg, 87.2%) as a
yellow solid. Mp >300 °C. 1H NMR (400 MHz, DMSO-d6): 7.31 (s,
1H), 10.64 (br s, 1H), 10.76 (br s, 1H), 11.07 (br s, 1H). 13C NMR:
93.5, 111.8, 131.7, 136.9, 151.8, 155.7. FAB-MS for C6H4IN3O2 cal-
culated M+ 276.9348, found 276.9345, calculated [M+H]+
277.9421, found 277.9419.
5.1.1.9. 2,4-Bis-benzyloxy-5-N-Boc-7-iodopyrrolo[3,2-d]pyrim-
idine (10).
To a solution of 7-iodo-2,4-bis-benzyloxy-5H-pyr-
rolo[3,2-d]pyrimidine 4 (100 mg, 0.218 mmol) in dry THF (5 mL),
Boc2O (96 mg, 0.437 mmol) and DMAP (145.33 mg, 0.0437 mmol)
were added and the mixture stirred for 1 h until TLC indicated the
absence of starting material. The solvent was removed in vacuo and
the residue purified by column chromatography eluting with 49:1
hexanes/EtOAc to obtain 11 as a white solid (65.1 mg, 116 mmol,
53.4%).40 Rf 0.5 in 19:1 hexanes/EtOAc. Mp 118.5–122.5 °C. 1H NMR
(400 MHz, CDCl3): d 1.51 (d, 9H), 5.59 (s, 2H), 5.50 (s, 2H), 7.38–7.30
(m, 6H), 7.47 (d, 2H), 7.56 (d, 2H), 7.91 (s, 1H). 13C NMR: d 27.8,
64.3, 68.9, 69.5, 85.3, 110.9, 127.9, 128.4, 128.5, 128.8, 136.2, 136.5,
136.9, 147.2, 156.6, 157.9, 161.1. FAB-MS m/z for C25H24IN3O4 calcu-
lated [M+H]+ 558.0884, found MH+ 558.0885.
5.1.1.6.
2,4-Bis-benzyloxy-5-nitro-6-dimethylaminovinyl
pyrimidine (9). To a solution of 2,4-bis-O-benzyl-6-methyl-5-ni-
tro pyrimidine 937 (2.3 g, 6.5 mmol) in DMF (20 mL), DMF-
dimethyl acetal (1.74 mL, 13 mmol) was added at room tempera-
ture under N2 atmosphere. The reaction was lowered in a pre-
heated oil bath at 60–65 °C and stirred overnight upon which
TLC indicated absence of starting material. The solvents were
removed and the residue loaded on silica. The product was purified
5.1.1.10. 9-Deaza-20-deoxyxanthosine (11). White solid Mp
185.3–186.8 °C. 1H NMR (400 MHz, DMSO d6): d 1.84–1.95 (m,
2H), 3.49–3.57 (m, 2H), 3.75 (br s, 1H), 4.19 (br s, 1H), 4.96 (d,
1H, J = 3.24 Hz), 5.05 (dd, 1H, J = 5.92 Hz, J = 10.08 Hz), 5.62 (br s,
Please cite this article in press as: Temburnikar, K. W.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.025

K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
1H), 7.11 (s, 1H) 10.56 (br s, 1H, NH), 10.58 (br s, 1H, NH), 11.67 (br
s, 1H, NH). 13C NMR (400 MHz, DMSO d6): d 43.0, 62.7, 73.6, 73.7,
87.7, 111.2, 111.5, 125.2, 132.7, 151.8, 156.3. FAB-MS for
of J.B. was supported by the GOA 15/19 TMB. The authors would
like to acknowledge the National Institutes of Health for funding
(NIH T32GM066706-12, K.S.R. and C.R.R., and NIH R01CA102428,
G.M.W.). The Flow Cytometry Shared Service facility of the
University of Maryland Greenebaum Cancer Center is supported
in part by P30 CA134274.
C
11H13N3O5 calculated M+ 267.0855, found 267.0853; calculated
[M+H+] 268.0928 found 268.0930.
5.1.1.11. 9-Deaza-20-deoxyguanosine (12).
White solid Mp
>300 °C. Rf 0.5 in 4:1 CH2Cl2: MeOH. 1H NMR (400 MHz, DMSO
Supplementary data
d6):
d 1.86 (dd, 1H, J = 5.26 Hz, J = 12.62 Hz), 2.13 (td, 1H,
J = 5.04 Hz, J = 11.69 Hz), 3.37 (ddd, 2H, J = 4.12 Hz, J = 11.66 Hz,
J = 11.44 Hz), 3.69 (t, 1H, J = 4.12 Hz), 4.15 (br s, 1H), 4.85 (d, 1H,
J = 3.64 Hz), 5.05 (dd, 1H, J=5.28 Hz, J = 10.76 Hz), 5.66 (br s, 2H,
NH2), 5.81 (br s, 1H), 7.09 (d, 1H, J = 3.2 Hz), 10.41 (br s, 1H, NH),
11.33 (br s, 1H, NH). 13C NMR (400 MHz, DMSO d6): d 42.6, 63.8,
73.6, 73.9, 88.3, 113.8, 115.7, 126.1, 151.3, 155.4, 164.0. ESI-MS
m/z for C11H14N4O4 calculated [M+H+] 267.1087, found 267.1089.
Supplementary data (the 1H, 13C NMR spectra, High Resolution
Mass Spectrometry (HRMS) and single dose response date from
NCI-60 DTP Human Tumor Cell Line Screen) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmc.2015.06.025.
References and notes
5.2. Cell proliferation assays
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l
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and the cellular fractions undergoing early apoptotic, late apop-
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mycin D (7-AAD) using the BD Pharmingen PE Annexin V Apoptosis
Detection Kit I (BD Pharmingen) as described.83
Acknowledgments
We are grateful to Lizette van Berckelaer for technical
assistance with the cytostatic activity evaluations. The research
Please cite this article in press as: Temburnikar, K. W.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.025

10
K. W. Temburnikar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
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Please cite this article in press as: Temburnikar, K. W.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.025