Structural and Biological Investigations for a Series of N-5 Substituted Pyrrolo[3,2-d]pyrimidines as Potential Anti-Cancer Therapeutics

Article abstract of DOI:10.3390/molecules24142656

Pyrrolo[3,2-d]pyrimidines have been studied for many years as potential lead compounds for the development of antiproliferative agents. Much of the focus has been on modifications to the pyrimidine ring, with enzymatic recognition often modulated by C2 and C4 substituents. In contrast, this work focuses on the N5 of the pyrrole ring by means of a series of novel N5-substituted pyrrolo[3,2-d]pyrimidines. The compounds were screened against the NCI-60 Human Tumor Cell Line panel, and the results were analyzed using the COMPARE algorithm to elucidate potential mechanisms of action. COMPARE analysis returned strong correlation to known DNA alkylators and groove binders, corroborating the hypothesis that these pyrrolo[3,2-d]pyrimidines act as DNA or RNA alkylators. In addition, N5 substitution reduced the EC₅₀ against CCRF-CEM leukemia cells by up to 7-fold, indicating that this position is of interest in the development of antiproliferative lead compounds based on the pyrrolo[3,2-d]pyrimidine scaffold.

Full text of DOI:10.3390/molecules24142656

molecules
Article
Structural and Biological Investigations for a Series of
N-5 Substituted Pyrrolo[3,2-d]pyrimidines as
Potential Anti-Cancer Therapeutics
1
1
2
2
Brian M. Cawrse , Nia’mani M. Robinson , Nina C. Lee , Gerald M. Wilson and
1
,
Katherine L. Seley-Radtke *
1
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore,
MD 21250, USA
2
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore,
MD 21201, USA
*
Correspondence: kseley@umbc.edu; Tel.: +1-410-455-8684
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Christian Ducho
Received: 1 July 2019; Accepted: 22 July 2019; Published: 23 July 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Pyrrolo[3,2-d]pyrimidines have been studied for many years as potential lead compounds
for the development of antiproliferative agents. Much of the focus has been on modifications to the
pyrimidine ring, with enzymatic recognition often modulated by C2 and C4 substituents. In contrast,
this work focuses on the N5 of the pyrrole ring by means of a series of novel N5-substituted
pyrrolo[3,2-d]pyrimidines. The compounds were screened against the NCI-60 Human Tumor Cell
Line panel, and the results were analyzed using the COMPARE algorithm to elucidate potential
mechanisms of action. COMPARE analysis returned strong correlation to known DNA alkylators
and groove binders, corroborating the hypothesis that these pyrrolo[3,2-d]pyrimidines act as DNA or
RNA alkylators. In addition, N5 substitution reduced the EC50 against CCRF-CEM leukemia cells by
up to 7-fold, indicating that this position is of interest in the development of antiproliferative lead
compounds based on the pyrrolo[3,2-d]pyrimidine scaffold.
Keywords: pyrrolo[3,2-d]pyrimidine; anti-cancer; antiproliferative; DNA damage; DNA alkylator
1
. Introduction
Pyrrolo[3,2-d]pyrimidines, or 9-deazapurines, represent a class of compounds that are sterically
and electronically similar to the naturally occurring purine nucleobases, with the exception of a
hydrogen-bond donating moiety at N5 (Figure 1) [ ]. These compounds have seen widespread
biological activity and have been extensively utilized in the design of small molecule inhibitors of purine
nucleoside phosphorylase (PNP) [ ], dihydrofolate reductase (DHFR) [ ], the transient receptor
potential channel family [10], and various kinases [11 13]. In addition, pyrrolo[3,2-d]pyrimidines
have shown promise in the development of antitumor agents [ 14 16]. The research detailed herein
1–3
4–
9
3
–
1, –
examined the effect of N5 substitution on the pyrrolo[3,2-d]pyrimidine scaffold, with ten novel
compounds synthesized and subsequently tested for antiproliferative activity.
7
5
6
4
H
5
3
4
5
4
N
N
N
1
N
3
2
8
N
3
6
2
8
N
2
N
3
N
1
N
H
2
N
5
6
N
H
H
7
4
N
1
9
1
9
Purine
Pyrrolo[3,2-d]pyrimidine Dihydro-pyrrolo[2,3-b]quinoline Pyrimido[4,5-b]indole
Figure 1. Structures of biologically relevant heterocycles.
Molecules 2019, 24, 2656; doi:10.3390/molecules24142656
www.mdpi.com/journal/molecules

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N5 and C7 substitutions have been shown by multiple researchers to play important
roles in the potency of pyrrolo[3,2-d]pyrimidines designed as enzyme inhibitors and potential
therapeutics. Recently, the pyrrolo[3,2-d]pyrimidine scaffold has been shown to inhibit Multi-drug
Resistance-Associated Protein 1 (MRP1), an active efflux transporter responsible for resistance
to a wide variety of antiproliferative agents [
have been investigated as potent inhibitors of MRP1 [18
dihydropyrroloquinoline [20], indolopyrimidine [21], and pyrrolopyrimidine [
Figure 1).
Schmitt et al. synthesized a large series of 7-cyano-pyrrolo[3,2-d]pyrimidines that mapped the
effects of various substituents at C4, N5, and C6 [ 17 22]. Their findings suggested that an aliphatic
group at N5 increased activity against MRP1, but this activity was not further increased by the addition
of aromatic rings at the end of the aliphatic N5 chains (Figure 2 ) [22]. A second study by the same
1
,
17]. Similarly, multiple quinazolinone scaffolds
19], which led to the development of
17 18 21] inhibitors
,
1, , ,
(
1, ,
A
group verified the increase in MRP1 activity with N5 aliphatic substitutions, with a cyclopropyl group
imparting the greatest effect (Figure 2B) [17].
Figure 2. Structures of active pyrrolo[3,2-d]pyrimidines. (
protein 1 (MRP1), P-glycoprotein, and breast cancer resistance protein (BCRP). (
of dipeptidyl peptidase IV (DPP-IV). (
against MIA PaCa-2 pancreatic cells.
A
and
B
) Inhibitors of multidrug resistance
and ) Inhibitors
E) Broad-spectrum antiproliferative agent, with potent activity
C
D
Zeng et al. synthesized C2/N3 substituted pyrrolo[3,2-d]pyrimidine inhibitors of dipeptidyl
peptidase IV (DPP-IV) that explored the effect of various quinolone and quinazoline substituents, finding
that different N5 bicyclic substituents had widely differential activities (1.5–139 nM, Figure 2C) [23].
Related to this, Xie et al. synthesized N2/N3 substituted pyrrolo[3,2-d]pyrimidines as potent and
selective inhibitors of dipeptidyl peptidase IV (DPP-IV) that explored the effect of C7 substitution,
finding that C7-bromine imparted the greatest desired activity (Figure 2D) [24].
Finally, Temburnikar et al. previously determined that 2,4-dichloro-pyrrolo[3,2-d]pyrimidines
showed broad-spectrum antiproliferative activity, which was greatly enhanced via C7 halogenation
(
Figure 2E) [16,25,26]. In addition, N5 substitution led to a marked decrease in toxicity, lending credence
to the hypothesis that the toxicity of these halogenated pyrrolo[3,2-d]pyrimidines can be modulated in
order to take full advantage of their potent antiproliferative properties [15].
These studies all suggested that the N5 position of pyrrolo[3,2-d]pyrimidines was of interest
in potentially improving activity and modulating toxicity. As a result, a series of N5 substituted
compounds were synthesized and characterized as part of an ongoing structure-activity relationship
study, designed to investigate the effect of various substituents at N5. The target compounds are shown
in Figure 3. The sulfonamide compounds
N5 sulfonamide substituents decreased toxicity without adversely affecting activity [15]. The benzyl
compound series 11 provided an opportunity to explore whether the N5 substituent requires
hydrolytic cleavage prior to antiproliferative activity, as was indicated in earlier pharmacokinetic
testing [15]. Amide substitution products 12 14 represent a series of compounds possessing the
6–8 are a continuation of a previous study that indicated that
9–
–

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naturally occurring and medicinally prevalent amide group. Finally, the addition of a polar aliphatic
alcohol decreased log P from 1.66 for parent compound 5 to 1.38 for compound 15, allowing investigation
of the effects of increased aqueous solubility.
Figure 3. Target compounds, starting from parent compound 5. Compounds 6–15 were submitted to
the National Cancer Institute for NCI-60 cell line screening.
The most active compounds from the NCI-60 cell line screen (7, 9, 10, and 14) were subjected to
cell assays to determine their activity against the cell line CCRF-CEM, an acute lymphoblastic leukemia
cell line, derived from T lymphoblast cells [27]. Finally, chemical and pharmacokinetic properties were
calculated using Schrodinger QikProp software (Schrodinger, LLC., New York, NY, USA) [28].
2
. Results
2
.1. National Cancer Institute-60 Human Tumor Cell Line Screen and COMPARE Analysis
In 1990, the National Cancer Institute (NCI) established a 60 human cell line screen for the
evaluation of antiproliferative agents [29
lung, colon, nervous system, ovarian, renal, prostate, and breast cancers. Compounds
screened at 10 M concentrations, and the results indicated that N5 substitution has a significant effect
–31]. Represented cell lines encompass leukemia, melanoma,
6–
15 were
µ
on both activity and selectivity. The results are presented in terms of growth percent (GP), which is the
growth of a treated culture compared to the growth of an untreated culture, i.e., 60% GP represents
40% growth inhibition, compared to a negative control cell culture [30]. GP between 0–99 represents
cytostatic properties; between 100–0 represents cytotoxic properties [30]. Table 1 shows the best
−
growth percent for each compound, which represents the maximal inhibitory effect against the most
affected cell line. The complete NCI-60 data can be found in the Supplemental Information.
Overall, the compounds were minimally active as broad-spectrum antiproliferatives, but did
have specificity leukemia cell lines, with good activity for compounds
affected the cell growth in leukemia (CCRF-CEM, K-562, and RPMI-8226) cell lines, with an average
GP of 63.6% (Supplemental Information). Compound showed best growth inhibition against
NCI-H522 non-small cell lung cancer (49.5% GP) but did not affect other non-small cell lung cancer
lines. The two other sulfonyl substituted compounds ( and ) did not show significant activity. Benzyl
substituted compounds 11 showed the best activity against the CCRF-CEM leukemia cell line, with
7, 9, 10, and 14. Compound 7
7
6
8
9–
GP ranging from 22.88–65.83 (Table 1). Compound 10 also displayed potent activity against NCI-H522
non-small cell lung cancer, with a 35.1% GP (Supplemental Information). Within the amide substituted
compounds 12
potent. Notably, this compound was the only one to show lethality against breast cancer cell lines, with
0.95% GP against MDA-MB-468 (Table 1). Finally, hydroxyethyl substituted 15 showed decreased
activity compared to parent 5.
–14, only 14 showed appreciable activity, and in general, this compound was the most
−

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Table 1. Best Growth Percent and Target Cell Line for Compounds 5–15.
Compound
Best Growth Percent *
Most Affected Cell Line
5
6
7
8
9
60.77
84.49
49.57
93.16
36.31
22.88
65.83
81.36
75.18
−0.95
89.73
LOX-IMVI (Melanoma)
NCI-H522; NSC Lung
NCI-H522; NSC Lung
UO-31; Renal
CCRF-CEM; Leukemia
CCRF-CEM; Leukemia
CCRF-CEM; Leukemia
UO-31; Renal
1
0
1
2
3
4
5
1
1
1
1
1
SR, Leukemia
MDA-MB-468; Breast
NCI-H522; NSC Lung
*
Represents the growth percent in the most affected cell line (i.e., highest antiproliferative activity exhibited in the
NCI-60 cell screening).
COMPARE analysis [31–36] was then performed for compounds 5–15 against the NCI Standard
Agents list, an aggregate of 171 compounds that have shown clinical antiproliferative activity.
The analysis returns a Pearson correlation coefficient (PCC; [37 38]) that ranges from 1.0 to 1.0,
representing the statistical similarity of a compound, compared to the activity of the known standard
with 1.0 representing a perfect match, and 1.0 representing a perfect mirror image of the compared
activities [31]. Moderate correlation is represented by PCC values between 0.5–0.7, and strongly
analogous compounds generally have a PCC of 0.7 [39]. The top five correlation results all had PCC
0.6, indicating a moderate correlation (Table 2).
,
−
−
≥
≥
Table 2. Pearson Correlation Coefficient of Selected Compounds with Target Vector and Common Cell
Line Count.
Common Cell Line Count b
Compound
Correlation (PCC)
Target Vector a
9
7
9
9
9
0.717
0.661
0.640
0.622
0.622
D-Tetrandrine
Pibenzimol HCl
Fluorodopan
Melphalan
Carmustine (BCNU)
55
57
58
58
59
a
Known compound with activity most comparable to experimental compound; b Number of cell lines displaying
similar activity between experimental compound and target vector.
2
.2. In Vitro Cell Growth Inhibition
Based on the results of the NCI-60 screen, compounds 7, 9, 10, and 14 were selected for in vitro
assays against CCRF-CEM leukemia cells, using a commercially available MTT assay kit [27]. The most
active compound in this assay was 10, with an EC50 = 3.3 1.2 M; the least active compound was
, with EC50 = 23.2 2.0 M. The single digit micromolar EC values observed for 10, and 14
±
µ
9
±
µ
7,
50
represent an approximately 7-fold increase in activity compared to parent unsubstituted compound 5,
which has low activity against CCRF-CEM cells (EC50CCRF-CEM = 25
±
2 µM) [16]. The results of the
contemporary assays are shown in Figure 4.

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Figure 4. CCRF-CEM MTT assay results.
3
. Discussion
Previous studies on pyrrolo[3,2-d]pyrimidines indicate that the scaffold holds promise in
the development of potent antiproliferative agents [16]. Halogenated pyrrolo[3,2-d]pyrimidines,
synthesized previously by the Seley-Radtke laboratory, exhibited low micromolar to nanomolar activity
against a wide variety of cancer cell lines, with results indicating that N5 substitution allows modulation
of toxicity and activity [15
cell line screening showing good activity and specificity against CCRF-CEM leukemia.
The results observed for compounds 11 strongly suggest that enzymatic cleavage of the N5
,16]. The SAR study presented here corroborated those results, with NCI-60
9–
substituent is not required for biological activity, as the benzyl substituents are assumed to be stable
under physiological conditions. Interestingly, this series showed the best activity as a group, with
preferential inhibition in leukemia cultures. Leukemia cells have been associated with an abnormally
high rate of cell proliferation (cell birth rates have been measured at 0.07–1.31% per day), as well as
a low rate of cell death [40
,41]. In addition, acute myeloid leukemia has been demonstrated to have
an increased expression of non-coding RNA (miRNA), associated with leukemia pathogenesis and
leukemogenesis, providing additional nucleic acid targets for DNA/RNA alkylating molecules [42
This correlates with the trend for preferential leukemia growth inhibition observed for 9–11.
,43].
The necessity for aromatic substituents was further demonstrated in the acyl/amide substituted
compounds 12 14, with the acetyl- and isobutyryl-substituted compounds 12 and 13 showing little
growth inhibition, compared to the significant inhibitory activity of N,N-diphenylcarboxamide 14
–
.
Compound 14 showed the greatest inhibitory activity overall, suggesting that the single aromatic ring
present in 7–11 may not be sufficient to impart maximal inhibitory activity.
Hydroxyethyl substituted 15 showed the least inhibitory action of all of the compounds tested,
suggesting that increased hydrophilicity is not a factor in the activity of these compounds.
COMPARE analysis returned moderate to strong correlation to d-tetrandrine, pibenzimol
hydrochloride, fluorodopan, melphalan, and carmustine. d-tetrandrine is a naturally occurring
benzylisoquinoline alkaloid that has been shown to associate with DNA and RNA in sarcoma cell
lines [44]; pibenzimol HCl is a benzimidazole DNA minor groove binder and inhibitor of topoisomerase
I [45
for colorectal cancer [47]; melphalan and carmustine are both alkylating nitrogen mustards used to
treat multiple myeloma, and glioblastoma, respectively [48 49]. These compounds all share a similar
,46]; fluorodopan is a halogenated pyrimidine-2,4-dione once considered as a possible treatment
,

Molecules 2019, 24, 2656
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mechanism of action—direct association or alkylation of nucleic acids, suggesting that compounds
15 may follow a similar mechanism. This observation supports previous studies that indicated the
compounds operate via an alkylation mechanism. Briefly, flow cytometry revealed an accumulation
in the G2/M phase, consistent with a DNA damage checkpoint, and -H2Ax staining of cell cultures
treated with an analogue of parent revealed DNA damage [15 16]. In addition, a proteomic study
6
–
γ
5
,
did not reveal any covalent protein targets, which suggested that the inhibition of enzymes such as
topoisomerases is unlikely (unpublished results).
The in vitro cell proliferation assays showed that compound 10 was the most efficacious at
preventing cell proliferation, with an EC50 = 3.3
EC50 = 23 M. The low EC50 observed for compound
this compound, in particular the solvent accessible surface area (SASA) and electron affinity (EA). Of
the four compounds tested, has the lowest SASA and EA, which would hinder the bioavailability
and ability to accept an electron during an alkylation reaction, (Table 3) [50]. However, compared
±
1.2
µ
M. Compound
9 was the least effective, with an
±
2
µ
9
may be due to the chemical properties of
9
to the parent compound
5
(2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine), compounds 7, 10, and 14 all
CCRF-CEM
showed better activity in CCRF-CEM cells by up to a factor of ~7 (EC50
compound
antiproliferative lead compounds.
= 25
±
2
µM for
5) [16], indicating that N5 substitution may offer a viable path for development of potent
Table 3. Physical and pharmacokinetic properties of active pyrrolo[3,2-d]pyrimidines.
SASA a (Å2)
EA b (eV)
QPPCaco c (nm/sec)
QPlogS d (log[mol/dm ])
3
Compound
7
9
514.262
476.642
509.662
601.2
9.613
8.878
8.947
9.071
243.855
3989.857
4018.835
1349.875
−2.017
−4.405
−5.547
−4.892
1
1
0
4
a
solvent accessible surface area; b electron affinity; c predicted apparent Caco-2 cell permeability; d predicted
aqueous solubility.
In summary, the structure-activity relationship study of compounds 6–15 revealed that aromatic
substitutions impart increased activity, with a trend towards inhibition of leukemia. The addition of
a small polar substituent (compound 15) resulted in activity similar to that of the parent compound
5
, suggesting that hydrophilicity does not affect activity. These compounds do not appear to have a
particular protein target, and instead mirror the activity of known DNA alkylators and groove binders.
This is in line with earlier findings of DNA damage by analogues of parent compound In vitro assays
show low micromolar EC50 values for three of the four most active compounds, which represents
a 7-fold increase in activity, compared to the unsubstituted parent compound Together, these data
5.
.
support that halogenated pyrrolo[3,2-d]pyrimidines act as non-specific antiproliferative agents with
tunable activity via N5 substitution, and that further study of N5 substituents holds the potential to
improve activity and cell line specificity.
4
. Materials and Methods
4
.1. NCI-60 Human Tumor Cell Lines Screen and COMPARE Analysis
The standard operating procedures for the NCI-60 cell line screen have been well-documented and
reported in multiple sources [29,30,33,36]. Briefly, accepted antiproliferative compounds are dissolved
in DMSO, then stored frozen prior to use. All cultures are grown in RPMI 1640 medium, supplemented
with 5% bovine serum albumin and 2 mM l-glutamine. Prior to evaluation, cultures are dispensed into
◦
9
6-well microtiter plates at densities between 5000–40,000 cells/well, then incubated for 24 h at 37 C, 5%
CO , 95% air, 100% humidity. Time zero cell density is determined by fixing two wells with trichloroacetic
2
acid (TCA). Experimental compounds are diluted to 10 µM in complete medium containing 50 µg/mL
gentamicin and administered to plate wells. Plates are incubated for 48 h, followed by addition of TCA
◦
to a final concentration of 10% and held at 60 C for 4 h. Supernatant is discarded, and protein content

Molecules 2019, 24, 2656
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of wells is determined by sulforhodamine B staining. Percentage growth inhibition is determined
relative to DMSO-treated cells and time-zero control wells. A list of current cell lines included in the
NCI-60 screen can be found at https://dtp.cancer.gov/discovery_development/nci-60/cell_list.htm, and
is also detailed in the NCI-60 data in the Supplementary Information.
COMPARE analysis was performed using the publicly available online COMPARE algorithm [51].
Using compounds 6–15 as seed compounds, and by comparing to the NCI Standard Agents list, growth
inhibition patterns can be compared across the NCI-60 screening results. The results were quantitated
as a Pearson correlation coefficient (PCC) with high-ranking compounds, suggesting a mechanism
of action similar to that of the seed compounds [33]. Parameters selected for analysis were: Target
Set Name: Standard Agents; Target Set Endpoint: GI ; Minimum Correlation: 0.4; Minimum Count
50
Common Cell Lines: 40; Minimum Standard Deviation: 0.05.
4
.2. In Vitro Cell Growth Inhibition
A total of 4 mM stock solutions of compounds 7, 9, 10, and 14 were prepared in DMSO to
be used in serial dilutions to achieve final concentrations of 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and
00 M in cell-seeded 96-well plates. Human lymphocytic CCRF-CEM cells were obtained from
the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI 1640
1
µ
(
1
Corning Life Sciences, Tewksbury, MA, USA) containing 1
0% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA) at 37 C in a humidified, CO -controlled
×
penicillin/streptomycin (Corning) and
◦
2
4
incubator. The cytostatic assays were performed by incubating 5
×
10 CCRF-CEM cells in 0.1 mL
media with the described concentration of test compounds or vehicle (DMSO) for 48 h. Cell viability
was then measured using the MTT Cell Proliferation Assay kit (ATCC) according to the manufacturer’s
instructions, with absorbance measured at 595 nm. Percent cell viability was calculated relative to
cells incubated with vehicle alone, then plotted as a function of drug concentration. EC50 values were
resolved by analyzing viability curves using a 4-parameter dose response function utilizing PRISM
v3.03 software (GraphPad, San Diego, CA, USA).
4
.3. Chemistry
4
.3.1. General Experimental
All solvents and reagents purchased through Fisher Scientific (Hampton, NH, USA) or
1
13
Sigma-Aldrich (St. Louis, MO, USA). All H- and C-NMR were obtained on a JEOL ECX 400 MHz
NMR. All NMR spectra were referenced to 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 (multiplet), 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 silica glass
plates. All column chromatography was run 32–63 micron silica gel obtain from Dynamic Adsorptions
Inc. (Norcross, GA, USA). Yields refer to chromatographically and spectroscopically homogenous
materials. All mass spectra were recorded and obtained from the UMBC Mass Spectrometry Facility
using a Bruker amazon Speed Ion Trap fitted with an electrospray (ESI) or atmospheric pressure
chemical ionization (APCI) injection port. Elemental analysis was conducted by Atlantic Microlabs
(Norcross, GA, USA) via combustion analysis.
4
.3.2. Synthesis of Target Compound 5
Parent compound was synthesized starting from commercially available 6-methyluracil (1),
5
shown in Scheme 1. Nitration at C5 was accomplished by adding 6-methyluracil to a solution of
◦
concentrated sulfuric and nitric acids at 0 C, followed by quenching in ice water to give
2 as a
pale yellow solid [24]. This was followed by a modified Batcho-Leimgruber indole synthesis to
first prepare enamine 52 53]. Ring cyclization is accomplished by reduction of the nitro moiety
to an amino group, with subsequent nucleophilic attack at C8 by the amino lone pair with loss of
3
[ ,

Molecules 2019, 24, 2656
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dimethylamine. The reduction of the nitro group is accomplished by stirring
dust overnight at 80 C, followed by filtration, formation of the N5 sodium salt via NaOH dissolution,
3
in acetic acid and zinc
◦
then re-acidification and collection of solid 4 [23,54].
O
O
O
O
H
O N
2
O N
2
N
NH
a
NH
b
NH
c
NH
N
H
O
N
H
O
(Me) N
N
H
O
N
H
O
2
1
2
3
4
Cl
Cl
H
R
N
N
N
N
d
e
N
Cl
N
Cl
5
6-15
Reagents and Conditions: a) H SO , HNO , rt, 2h, 80%; b) DMF-DMA, DMF, 80 to 140
2
4
3
°
C, 1h, 70%; c) AcOH, Zn (dust), rt, 12h, 70%; d) PhPOCl , reflux, 6h, 60%; e)
2
DMF:THF 1:1, NaH, R-X, rt, 16h, 60-80%
Scheme 1. Synthesis of parent 2,4-dichloro-pyrrolo[3,2-d]pyrimidine 5 and N5 substituted analogues
6–15.
The parent compound
compound
5
was synthesized by first reforming the sodium salt of
4
, by stirring
◦
◦
4
in 1 M NaOH at 40 C for one hour, then followed by crystallization at 0 C and
repeated washes with cold water and acetone. Chlorination of the sodium salt of 4 was achieved by
refluxing for 5 h in phenylphosphonic dichloride under a nitrogen atmosphere. Substitution at N5
◦
was accomplished by N5 deprotonation using sodium hydride at 0 C, followed by addition of the
appropriate halogenated reagent to obtain target compounds 6–15.
4
.3.3. General Procedure for Synthesis of Compounds 6–15
,4-dichloro-1H-pyrrolo[3,2-d]pyridine ( ) was dissolved in a solution of 1:1 THF:DMF under
N . Sodium hydride (60% suspension in oil) was added directly, and the mixture was stirred
2
5
2
at room temperature for 1 h. Appropriate halogenated reagent (methanesulfonyl chloride (
-nitrobenzenesulfonyl chloride ( ), 2,4,6-Triisopropylbenzenesulfonyl chloride ( ), benzyl bromide
), 2,4-dichlorobenzyl bromide (10), 4-methoxybenzyl chloride (11), acetyl chloride (12), isobutyryl
6),
2
7
8
(9
chloride (13), diphenylcarbamyl chloride (14), iodoethane (15)) was added and the mixture was stirred
at r.t. for 18 h. The solvent was removed and residue dissolved in EtOAc and washed with water
and brine, then dried over MgSO . Organics were loaded onto Celite and product purified using
4
silica column chromatography eluting with 4:1 hexanes:EtOAc or 9:1 DCM:MeOH to obtain product,
generally as a white to off-white solid.
4
.3.4. Characterization
,4-dichloro-5-(methylsulfonyl)-5H-pyrrolo[3,2-d]pyrimidine (
s, 3H); 6.83 (d, J = 4.12 Hz, 1H); 8.10 (d, J = 4.12 Hz, 1H). C-NMR (100 MHz, DMSO-d6):
53.40, 152.14, 144.44, 139.10, 137.58, 124.23, 123.05, 105.97, 102.31, 44.88. ESI-MS m/z for C H Cl N O S
1
2
(
1
6
). H-NMR (400 MHz, CDCl ) (ppm)
δ
: 3.66
3
13
δ158.79,
7
5
2
3
2
+
calcd. at 264.95, found at 266.01 [M + H] . Anal. calcd. for C H Cl N O S: C, 31.60; H, 1.89; N, 15.79.
7
5
2
3
2
Found: C, 31.58; H, 1.89; N, 15.78.
,4-dichloro-5-((2-nitrophenyl)sulfonyl)-5H-pyrrolo[3,2-d]pyrimidine (
ppm)
Hz, 1H); 8.25 (d, J = 7.80 Hz, 1H); 8.60 (d, J = 2.76 Hz, 1H). C-NMR (100 MHz, DMSO-d6):
52.97, 147.51, 144.41, 140.13, 137.28, 134.88, 131.37, 130.61, 126.68, 108.04, 102.79. ESI-MS m/z for
1
2
7
). H-NMR (400 MHz, DMSO-d )
6
(
δ: 7.20 (t, J = 2.32 Hz, 1H); 7.75 (d, J = 8.24 Hz, 1H); 7.82 (t, J = 7.80 Hz, 1H); 8.04 (q, J = 7.32
13
δ158.30,
1
+
C H Cl N O S calcd. at 371.95, found at 373.00 [M + H] . Anal. calcd. for C H Cl N O S: C, 38.62;
12
6
2
4
4
12
6
2
4
4
H, 1.62; N, 15.01. Found: C, 38.61; H, 1.62; N, 15.01.

Molecules 2019, 24, 2656
9 of 13
1
2,4-dichloro-5-((2,4,6-triisopropylphenyl)sulfonyl)-5H-pyrrolo[3,2-d]pyrimidine (
8). H-NMR (400 MHz,
CDCl ) (ppm) : 1.08 (d, J = 6.84 Hz, 12H); 1.24 (d, J = 6.88 Hz, 6H); 2.92 (septet, 1H); 3.85 (septet, 2H);
δ
3
13
6
.83 (d, J = 3.64 Hz, 1H); 7.17 (s, 2H); 8.26 (d, J = 3.68 Hz, 1H). C-NMR (100 MHz, CDCl3): δ157.78,
1
55.99, 153.18, 151.22, 145.17, 136.98, 131.89, 124.12, 123.62, 105.89, 34.42, 30.26, 24.38, 23.55. ESI-MS m/z
+
for C H Cl N O S calcd. at 453.10, found at 454.10 [M + H] . Anal. calcd. for C H Cl N O S: C,
21
25
2
3
2
21 25
2
3
2
5
5.51; H, 5.55; N, 9.25. Found: C, 55.49; H, 5.54; N, 9.24.
1
2
6
,4-dichloro-5-benzyl-5H-pyrrolo[3,2-d]pyrimidine (
9
). H-NMR (400 MHz, CDCl ) (ppm)
δ
: 5.67 (s, 2H);
3
13
.71 (d, J = 3.2 Hz, 1H); 7.05 (d, J = 6.44 Hz, 2H); 7.34 (m, 3H); 7.53 (d, J = 3.2 Hz, 1H). C-NMR
(
100 MHz, CDCl ): 154.4,4, 150.61, 143.16, 138.92, 136.36, 129.25, 128.48, 126.63, 123.15, 103.00, 52.41.
3
+
ESI-MS m/z for C H Cl N calcd. at 277.02, found at 278.03 [M + H] . Anal. calcd. for C H Cl N :
13
9
2
3
13
9
2
3
C, 56.14; H, 3.26; N, 15.11. Found: C, 56.11; H, 3.26; N, 15.10.
1
2
,4-dichloro-5-(2,4-dichlorobenzyl)-5H-pyrrolo[3,2-d]pyrimidine (10). H-NMR (400 MHz, CDCl ) (ppm)
3
δ
: 5.72 (s, 2H); 6.42 (d, J = 8.68 Hz, 1H); 6.75 (d, J = 3.24 Hz, 1H); 7.15 (d, J = 8.28 Hz, 1H); 7.46 (d,
J = 2.28 Hz, 1H); 7.51 (d J = 3.20 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6):
δ
154.39, 149.55, 142.77,
1
3
4
42.23, 135.14, 133.24, 132.01, 129.52, 128.13, 122.50, 103.03, 49.67. ESI-MS m/z for C H Cl N calcd. at
13
7
4
3
+
44.94, found at 345.95 [M + H] . Anal. calcd. for C H Cl N : C, 45.00; H, 2.03; N, 12.11. Found: C,
13
7
4
3
4.96; H, 2.03; N, 12.09.
1
2
3
2
,4-dichloro-5-(4-methoxybenzyl)-5H-pyrrolo[3,2-d]pyrimidine (11). H-NMR (400 MHz, CDCl ) (ppm) δ:
3
.78 (t, J = 7.36 Hz, 3H); 5.60 (d, J = 3.68 Hz, 2H); 6.67 (s, 1H); 6.86 (q, J = 4.12 Hz, 2H); 7.03 (t, J = 3.68 Hz,
H); 7.50 (s, 1H). 13C-NMR (100 MHz, DMSO-d6):
δ
159.48, 155.10, 149.01, 142.96, 141.49, 141.00, 130.06,
1
3
28.35, 123.01, 114.97, 102.07, 55.61, 51.20. ESI-MS m/z for C H Cl N O calcd. at 307.03, found at
07.93 [M + H] . Anal. calcd. for C H C N O: C, 54.57; H, 3.60; N, 13.64. Found: C, 54.54; H, 3.60;
14 11 l2 3
14
11
2
3
+
N, 13.63.
1
1
2
-(2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethan-1-one (12). H-NMR (400 MHz, CDCl ) (ppm) δ:
3
13
.73 (s, 3H); 6.77 (d, J = 3.64 Hz, 1H); 7.92 (d, J = 4.12 Hz, 1H). C-NMR (100 MHz, DMSO-d6): δ168.26,
1
58.29, 152.17, 145.87, 140.28, 123.01, 107.93, 24.18. ESI-MS m/z for C H Cl N O calcd. at 228.98, found
8 5 2 3
+
at 229.54 [M + H] . Anal. calcd. for C H Cl N O: C, 41.77; H, 12.19; N, 18.27. Found: C, 41.75; H, 2.19;
8
5
2
3
N, 18.25.
1
1
-(2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-2-methylpropan-1-one (13). H-NMR (400 MHz, CDCl )
3
(
ppm)
δ: 1.37 (d, J = 6.84 Hz, 6H); 3.33 (septet, J = 6.88 Hz, 1H); 6.77 (d, J = 3.68 Hz, 1H); 7.92 (d,
J = 3.64 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6):
δ
174.60, 158.78, 152.44, 146.37, 138.82, 124.21, 106.45,
+
3
4.17, 19.07. ESI-MS m/z for C H Cl N=O calcd. at 257.01, found at 257.68 [M + H] . Anal. calcd. for
10
9
2
C H Cl N O: C, 46.54; H, 3.51; N, 16.28. Found: C, 46.51; H, 3.51; N, 16.27.
10
9
2
3
1
2
(
,4-dichloro-N,N-diphenyl-5H-pyrrolo[3,2-d]pyrimidine-5-carboxamide (14). H-NMR (400 MHz, DMSO-d )
6
13
ppm)
DMSO-d6):
C H Cl N O calcd. at 382.04, found at 383.04 [M + H] . Anal. calcd. for C H Cl N O: C, 59.55; H,
δ
: 6.76 (d, J = 3.20 Hz, 1H); 7.23–7.34 (m, 10H); 8.37 (d, J = 3.20 Hz, 1H). C-NMR (100 MHz,
δ
155.30, 151.12, 149.65, 144.15, 141.73, 139.55, 130.09, 123.10, 104.63. ESI-MS m/z for
+
19
12
2
4
19 12
2
4
3
.16; N, 14.62. Found: C, 59.53; H, 3.16; N, 14.61.
1
2
-(2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethan-1-ol (15). H-NMR (400 MHz, DMSO-d ) (ppm) δ:
6
3
(
.70 (d, J = 5.52 Hz, 2H); 4.50 (t, J = 5.48 Hz, 2H); 6.68 (d, J = 3.24 Hz, 1H); 8.05 (d, J = 3.20 Hz, 1H); 8.08
153.40, 142.99, 137.59, 124.25, 102.56, 101.54, 61.40, 51.47.
s, 1H). 13C-NMR (100 MHz, DMSO-d6):
δ
+
ESI-MS m/z for C H Cl N O calcd. at 231.00, found at 231.93 [M + H]
8
7
2
3

Molecules 2019, 24, 2656
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Supplementary Materials: The following are available online, Figure S1: Complete NCI-60 Mean Growth Charts,
1
13
Figure S2: H-NMR of Compounds 6–15, Figure S3: C-NMR of Compounds 6–15.
Author Contributions: K.L.S.-R. and B.M.C. designed the synthesis of the experimental compounds, analyzed
the NCI and COMPARE data, and prepared the manuscript. N.M.R. assisted in the synthesis of the experimental
compounds. N.C.L. and G.M.W. conducted the MTT assays and prepared the resulting biological data. All authors
edited and contributed to drafts of the manuscript.
Acknowledgments: The authors thank the National Institutes of Health and NIGMS for financial support through
the Chemistry-Biology Interface Program (T32 GM066706, K.L.S.-R. and B.M.C.). N.C.L. was supported by the
Nathan Schnaper Intern Program funded in part through NCI grant R25CA186872 to Bret A. Hassel.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 5–15 are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).