Anticancer Properties of Halogenated Pyrrolo[3,2-d]pyrimidines with Decreased Toxicity via N5 Substitution

Article abstract of DOI:10.1002/cmdc.201700641

Halogenated pyrrolo[3,2-d]pyrimidine analogues have shown antiproliferative activity in recent studies, with cell accumulation occurring in the G₂/M stage without apoptosis. However, the mechanism of action and pharmacokinetic (PK) profile of these compounds has yet to be determined. To investigate the PK profile of these compounds, a series of halogenated pyrrolo[3,2-d]pyrimidine compounds was synthesized and first tested for activity in various cancer cell lines followed by a mouse model. EC₅₀ values ranged from 0.014 to 14.5 μm, and maximum tolerated doses (MTD) in mice were between 5 and 10 mg kg^?1. This indicates a wide variance in activity and toxicity that necessitates further study. To decrease toxicity, a second series of compounds was synthesized with N5-alkyl substitutions in an effort to slow the rate of metabolism, which was thought to be leading to the toxicity. The N-substituted compounds demonstrated comparable cell line activity (EC₅₀ values between 0.83–7.3 μm) with significantly decreased toxicity (MTD=40 mg kg^?1). Finally, the PK profile of the active N5-substituted compound shows a plasma half-life of 32.7 minutes, and rapid conversion into the parent unsubstituted analogue. Together, these data indicate that halogenated pyrrolo[3,2-d]pyrimidines present a promising lead into potent antiproliferative agents with tunable activity and toxicity, and rapid metabolism.

Full text of DOI:10.1002/cmdc.201700641

Accepted Article
Title: Anticancer Properties of Halogenated Pyrrolo[3,2-d]pyrimidines
with Decreased Toxicity Via N5 Substitution
Authors: Brian M Cawrse, Rena S Lapidus, Brandon Cooper, Eun
Yong Choi, and Katherine L Seley-Radtke
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10.1002/cmdc.201700641
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Anticancer Properties of Halogenated Pyrrolo[3,2-d]pyrimidines
with Decreased Toxicity Via N5 Substitution
Brian M. Cawrsea; Rena S. Lapidusb; Brandon Cooper b; Eun Yong Choib; Katherine L. Seley-Radtke*a
[a]
[b]
Dr. K. Seley-Radtke, B. M. Cawrse
Department of Chemistry and Biochemistry
University of Maryland, Baltimore County
1000 Hilltop Circle, Baltimore MD, 21250
Email: kseley@umbc.edu
Dr. R. Lapidus, B. Cooper, E. Choi
Translational Laboratory Shared Service
University of Maryland School of Medicine
655 W. Baltimore St., Baltimore MD 21201
Supporting information for this article is given via a link at the end of the document
additions leading to increased metabolic stability.[7]
A comparison of pyrrolo[3,2-d]pyrimidines and
Abstract: Halogenated pyrrolo[3,2-d]pyrimidine analogues have
shown antiproliferative activity in recent studies, with cell
accumulation occurring in the G2/M stage without apoptosis. However,
the mechanism of action and pharmacokinetic (PK) profile of these
compounds has yet to be determined. In order to investigate the PK
profile of these compounds, a series of halogenated pyrrolo[3,2-
d]pyrimidine compounds was synthesized and first tested for activity
in various cancer cell lines followed by a mouse model. EC50 values
ranged from 0.014 – 14.5 µM, and maximum tolerated doses (MTD)
in mice were between 5-10 mg/kg. This indicates a wide variance in
activity and toxicity that necessitates further study. To decrease
toxicity, a second series of compounds was synthesized with N5 alkyl
substitutions in an effort to slow the rate of metabolism, which was
thought to be leading to the toxicity. The N-substituted compounds
demonstrated comparable cell line activity (EC50 values between 0.83
– 7.3 µM) with significantly decreased toxicity (MTD = 40 mg/kg).
Finally, the PK profile of the active N5-substituted compound shows a
plasma half-life of 32.7 minutes, and rapid conversion into the parent
unsubstituted analogue. Together, these data indicate that
halogenated pyrrolo[3,2-d]pyrimidines present a promising lead into
potent antiproliferative agents with tunable activity and toxicity, and
rapid metabolism.
their
analogous
thieno[3,2-d]pyrimidine
equivalents allowed assessment of thieno- and
pyrrolopyrimidine compounds that were identical
with the exception of the five-membered
heterocycle.[8] Results of these previous studies
indicated
that
2,4-dichloro-5H-pyrrolo[3,2-
d]pyrimidine has a cytotoxicity of 6.0 µM against
L1210 leukemia cells, whereas 2,4-dichloro-
thienopyrimidine has a cytotoxicity (CC50) of 0.32
µM in the same cell line.[9] This supported that the
pyrrolopyrimidine structure offers decreased
cellular toxicity, making it a more promising lead
compound.[8-10]
Introduction
Figure 1. Target compounds with the pyrrolo[3,2-d]pyrimidine structure outlined
in red.
Pyrrolo[3,2-d]pyrimidines, also known as 9-
deazapurines, closely resemble natural purine
nucleobases and are a medicinally important group
of heterocyclic compounds. These interesting
heterocycles have shown activity as bactericides[1],
protozoicides[2], and as anti-tumor agents through
inhibition of a variety of enzymes including
tubulin[3], NEDD8-activating enzyme (NAE)[4],
and several kinases.[5, 6] However, the properties of
these molecules that affect their pharmacokinetic
profile is under-investigated. Previous reports have
found that the pyrrole moiety in pyrrolo[3,2-
d]pyrimidines is partially responsible for their
pharmacokinetic profile, with N5 hydroxyalkyl
In an effort to further pursue efficacious
pyrrolopyrimidines
with
antiproliferative
properties, we undertook the synthesis of a series
of compounds with modifications to the pyrrole
ring in the pyrrolo[3,2-d]pyrimidine scaffold. The
effect of various pyrrole substituents on compound
pharmacokinetics were examined, as well as the
compounds’ efficacy against a wide range of
cancer cell lines. The target compounds are shown
in Figure 1.
1
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Earlier examples of pyrrolopyrimidines such as for pharmacokinetic studies and further chemical
Immunicillin H and TAK-285 have shown modification.
inhibition against various key enzymes, with
Immunicillin-H acting as a potent inhibitor of The initial pharmacokinetic results for compound 7
purine nucleoside phosphorylase (PNP), and TAK- indicated rapid metabolism, thus derivatives were
285 acting as an epidermal growth factor receptor designed that introduced N5 substituents with the
(EGFR) inhibitor (Figure 2).[11] However, these aim to slow metabolic rate compared to the
compounds have extensive modifications to the unsubstituted parent compounds. This was based
pyrimidine moiety of the molecule or the addition on previous literature that indicates the nature of
of an azasugar. In contrast, little work has been the N5 substituent can affect varied
done to explore the effects of substituents on the pharmacokinetic properties: decreased polarity can
pyrrole ring.
increase lipophilicity[6], increased polarity can
increase affinity for the membrane efflux pump P-
In that regard, pyrrolopyrimidines have been glycoprotein[13], and various substituents can alter
previously reported by our laboratory as potential or increase substrate-ligand hydrogen bonding.[14]
therapeutics against triple negative breast cancer The log P values for compounds 5, 6, and 7 are
(TNBC), leukemia, non-small cell lung cancer, and estimated at 1.66, 2.49, and 3.02, respectively. This
pancreatic
cancer.[9]
A
structure-activity increases to 5.11 and 4.95 for 8 and 9, indicating
relationship (SAR) study found that compound 5 is that the N5 substituents are significantly more
a potent inhibitor of the TNBC MDA-MB-231 cell lipophilic than the parent compounds. Further, it
line, and further, caused an accumulation of cells in was previously determined that the C7 halogen
the G2/M stage without causing significant increases cellular activity and that the C4 chlorine
apoptosis.[9] These findings are consistent with is required for activity[9], thus it was hypothesized
earlier reports of other 9-deazapurines finding use that introducing bulky N5 groups with the ability to
as antiproliferative agents.[12]
sterically hinder these positions would decrease
the metabolic rate while still the retaining
antiproliferative activity.
Results and Discussion
Chemistry
Target compounds 5-9 were synthesized starting
with commercially available 6-methyluracil,
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. This was
Figure 2. The pyrrolo[3,2-d]pyrimidine scaffold (outlined in red) in the PNP
inhibitor Immunicillin-H (left) and the EGFR inhibitor TAK-285 (right).
The introduction of a halogen at C7 resulted in
increased activity, with the IC50 against HeLa cells
decreasing from 19±3 µM for 5 to 0.92±0.04 µM
for 6. The presence of the halogen also altered the
apparent mechanism of action, with the majority of
cells now undergoing apoptosis, although the
mechanism is still unclear.[9] These findings led us
to further investigate the effect of substituents on
the pyrrole moiety of these compounds with the
aim to retain or increase activity while decreasing
toxicity. Compounds 5-9 were synthesized and
subsequently evaluated for activity against
multiple cancer cell lines. Following initial cell line
screening, compound 7 was chosen as a candidate
2
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Scheme 1. Synthesis of target compounds 5-9. Reagents and Conditions: a)
H2SO4, HNO3, rt, 80%; b) DMF·DMA, DMF, 80 °C, 60% ; c) AcOH, Zn, rt, 70%;
d) PhPOCl2, reflux, 60% ; e) NBS or NIS, THF, 0°C to rt, 85-90%; f) NaH, Tos-
Cl or BOM-Cl, DMF, 0°C to rt, 85-90%.
pancreatic carcinoma epithelial cells, and MOLM-
14 acute monocytic leukemia cells, representing a
diverse set of cancerous cell types. All of the lead
compounds demonstrated activity against the four
cell lines, suggesting that they have potential as
broad spectrum antiproliferative agents.
followed by a modified Batcho-Leimgruber indole
synthesis to prepare enamine 3.[9] The Batcho-
Leimgruber process relies on the slight acidity of
the 6-methyl group, which can be deprotonated to
form a carbocation that is resonance stabilized by
the nitro group. Nucleophilic attack on the
dimethylacetal reagent results in formation of the
amino side chain, which is deprotonated to result in
the elimination product 3.
The highest activity was against MIA Pa-Ca-2
pancreatic cancer cells by compound 6. However,
compound 7 also displayed submicromolar activity
and was subsequently determined to have
decreased toxicity compared at 6 (discussed in
following section). These combined traits led us to
utilize 7 as a lead compound, with prodrug
formulation through substitution at N5. The
activity of prodrug 8 decreased ten-fold compared
to the parent, but it was observed that 9 was
comparable (Table 1). Previous work on the
pyrrolopyrimidine analogues had indicated that the
presence of a halogen at C4 on the pyrimidine was
essential for activity and that the presence of a
halogen at C7 enhanced the activity[9] – a trend also
observed in the current study.
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 dimethylamine. The
reduction of the nitro group is accomplished by
stirring 3 in acetic acid and zinc dust overnight at
80 °C, followed by filtration, washing the solids
with water, then stirring the collected solids in 1M
NaOH for 30 minutes at 40 °C. Filtration of the
undissolved solids, then acidification of the filtrate
with acetic acid results in the precipitation of solid
4.
Table 1. Cancer cell line activity screening for target compounds 5-7.a
Compound
A549
MDA-MB-
231
MIA Pa-Ca 2
MOLM14
The parent compound 5 was synthesized by first
preparing the sodium salt of 4 by stirring
compound 4 in 1M NaOH at 40 °C for one hour,
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 hours in phenylphosphonic dichloride under a
nitrogen atmosphere. Halogenation at C7 is
achieved with N-bromosuccimimide (6) or N-
iodosuccinimide (7) in anhydrous THF at 0°C.
Substitution at N5 was accomplished by N5
deprotonation using sodium hydride at 0°C,
followed by addition of toluenesulfonyl chloride
(9) or benzylchloromethyl ether (8).
5
6
7
8
9
14.5±3.5
0.05±0.02
1.0±0.1
NDb
9.7±9.7
0.4±0.3
1.2±0.4
6.1±0.4
0.79±0.04
5.3±1.0
9.5±3.1
0.023±0.004
0.55±0.07
ND
0.014±0.001
0.37±0.05
7.3±0.8
ND
0.5±0.02
ND
[a] vales shown in µM. [b] Not Determined. Assays run in triplicate.
Maximum Tolerated Dose
The maximum tolerated dose (MTD) for
compounds 6 and 7 was 5 and 10 mg/kg,
respectively (Table 2). The higher toxicity of
compound 6 excluded it as a lead compound. In
addition, although the toxicity of 6 is only 2-fold
higher than 7, significant mortality rate was
observed in the mouse model immediately
following administration, raising concerns that
mortality would interfere with effective dose
Cancer Cell Line Screening
Initial screening of 5-7 against four cancer cell
lines showed IC50 activity as low as 0.014±0.001
µM (Table 1). The cell lines chosen were A549
adenocarcinomic human alveolar basal epithelial
cells, MDA-MB-231 triple negative human
epithelial breast cells, MIA Pa-Ca-2 human
3
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studies. Comparative values for MTD of Aqueous benchtop stability tests were carried out
pyrrolo[3,2-d]pyrimidine compounds are difficult for compounds 6 and 7, by preparing 25 µg/mL
to find in current literature, but similar compounds solutions in phosphate buffer at pH 5.0, 7.0, and 7.5.
have shown MTD of 5 mg/kg (LY231514, a Aliquots taken at 0.0, 0.25, 0.5, 1, 4, 8, and 24 hours
pyrrolo[2,3-d]pyrimidine currently in Phase II)[15] were
initially
analyzed
by
thin-layer
and 150 mg/kg (PKI-116,
a
pyrrolo[2,3- chromatography against a verified standard, then
d]pyrimidine currently in Phase I).[16] The MTD of extracted with two 250 µl portions of chloroform.
1
compound 7 is within this comparative range and Solvent removal and H NMR analysis of the
still exhibited low micromolar activity, so it was residue was performed to verify the TLC results.
chosen for further modifications in an attempt to Similar procedures were carried out with human
decrease toxicity. The addition of a toluenesulfonyl serum. These experiments indicated that the
group in 9 raised the MTD from 10 to 40 mg/kg, compounds were stable at each pH tested, and in
while maintaining submicromolar levels of activity human serum, with no evidence of hydrolysis or
against all tested cell lines.
decomposition.
Aqueous benchtop stability of 9 and 7 was also
carried out at the CCPC, by spiking 5, 12.5, 25, 50,
125, 250 and 500 ng/mL solutions in 1:1
acetonitrile/Millipore H2O and subsequent analysis
by LC/MS for both 9 and 7, quantitating using a
previously constructed calibration curve of the
same concentrations. Samples spiked with 7
showed expected concentration values within
±5.7% from the aqueous solution. Samples spiked
with 9 gave a similar result, with a recovery
deviation of ±15.8%. More importantly, there was
no observed hydrolysis of 9 to 7 in aqueous
solution.
Table 2. Maximum tolerated dose and half-lives of compounds 6, 7, and 9.
Compound
MTDa
5
t1/2
6
7
9
NDb
10
30.7 min
32.7 min
40
[a] vales shown in mg/kg. [b] Not Determined.
N5 Substitution Cell Line Activity and Toxicity
Addition of a large moiety at N5 was designed to
sterically hinder both the C4 chlorine and C7 iodine,
as these groups impart the most activity and are
likely the sites of protein-molecule interaction.
Alkyl and oxyalkyl groups are common nitrogen
substituents in pyrrolopyrimidines[17], and sulfonyl
groups are not uncommon in FDA approved
anticancer drugs, often as salts formations, lending
validity to the biological compatibility of these
groups.[18] Introduction of a benzyloxymethyl
moiety in 8 decreased the efficacy of the compound
against all cell lines, and was not pursued further.
N5 toluenesulfonyl substitution in compound 9 led
to a four-fold increase in maximum tolerated dose
compared to parent compound 7, with comparable
cell activity. This suggests that substitutions at this
position can aide in decreasing cellular toxicity, or
that the substitution allows the compound to bypass
toxic pathways during delivery to their target site.
These and other possibilities will be discussed in
later sections.
Mouse Model Pharmacokinetic Studies
Mouse studies on 7 were carried out at The Cancer
Center Pharmacology Core (CCPC, Colorado State
University) by injection of 10 mg/kg of the
compound, followed by euthanization at 15, 30, 60,
90, 120, 240, and 480 minutes. The results of the
analysis are shown in Figure 3A. The compound is
rapidly metabolized in vivo, with a half-life of 30.7
minutes (Table 2 and Figure 3A). Mouse studies on
9 were performed in the same manner and the
results are shown in Figure 3B. The half-life of 9 is
similar to the parent compound, at 32.7 minutes
(Table 2 and Figure 3B).
Aqueous Stability
4
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30
25
20
15
10
5
A
1.2E+05
8.0E+04
4.0E+04
0.0E+00
Compound 7
Compound 9
0
0
50
100
150
Time / min
0
20
40
60
80
Time / min.
30
25
20
15
10
5
B
Figure 4. In vitro conversion of compound 9 to compound 7. Data is reported in
analyte counts to control for variation between standard curves for the two
compounds.
DNA Damage Assay
It was hypothesized that the compounds are acting
as DNA alkylators since they are highly
electrophilic. This theory was tested by staining
MOLM-14 cells for γ-H2Ax (Figure 5). The
phosphorylated histone protein γ-H2Ax is formed
immediately following double-stranded DNA
cleavage, and is known to be an initial step in
recruitment of local DNA repair proteins.[19]
Fluorescently-tagged antibody staining for γ-H2Ax
allows visualization of the extent of DNA damage
in cell cultures. The cells were grown in 0.5 and 4
µM 7 for 24 hours and show significant DNA
damage (Figure 5B and C). Results indicated that
compound 7 appears to be acting as a potent DNA
damaging agent.
0
0
50
100
150
Time / min
Figure 3. Pharmacokinetics of A) compound 7, and B) compound 9. Half-life of
the prodrug 9 is similar to the parent 7.
Plasma Stability
Plasma stability was carried out at the CCPC by
spiking mouse plasma with 500 ng/mL of 9 and
analyzing for both 9 and 7 at 0, 5, 15, 30, 45, 60,
and 90 minutes. Significant conversion of 9 to 7
over the time frame of the experiment was
observed as shown in Figure 4. This is a strong
indication that plasma enzymes are capable of
metabolizing the prodrug to its active form.
Supporting this hypothesis is the analysis of 7 in
plasma, which shows excellent stability with a
deviation of ±5.7% over the time frame
investigated.
Figure 5. DNA damage assay of compound 7 by staining MOLM-14 leukemia
cells for phosphorylated γ-H2Ax. A) DMSO control; B) 500 nM 7 (1x IC50 at 24
Hours); C) 4 µM 7 (6x IC50 at 24 Hours); D) Positive control (6 gy radiation).
5
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confirm that the breakdown product of 9 is the
active compound 7.
Halogenated pyrrolo[2,3-d]pyrimidine analogues
have been shown to be potent antiproliferative
agents against a wide spectrum of cancer cell lines.
The effective dose of these compounds is in the low
micromolar to nanomolar range, representing a
promising avenue of lead compounds. Two
disadvantages for these compounds are their
toxicity and rapid metabolism. However, by
manipulation of the prodrug moieties at the N5
position, both of these have been remediated
significantly. Furthermore, this position has not
been fully explored in the literature, opening the
possibility for further modifications that can
potentially decrease the toxicity and improve the
pharmacokinetic profile. This would allow the
drugs to be more effectively delivered to the target
cells in vivo before undergoing metabolism to their
active form.
Conclusions
Initial screening of 5-7 against six cancer cell lines
showed IC50 values as low as 0.014±0.0 µM (Table
1). All of the compounds demonstrated activity
against all four cell lines, indicating that they have
potential as broad spectrum antiproliferative agents.
However, the best activity overall was displayed
against MIA Pa-Ca-2 pancreatic cancer cells.
Maximum tolerated dose of 6, 7, and 9 was
between 5-40 mg/kg, indicative of high toxicity but
not outside the range of current epigenetic
therapeutics such as paclitaxel (25 mg/kg),
docetaxel (20 mg/kg) and vinorelbine (2.5
mg/kg)[20], as well as experimental antiproliferative
compound LY231514 (5 mg/kg).[15] However, as
these other clinical compounds are all semi-
synthetic, complex molecules with different
mechanisms of action than DNA damage, it is
difficult to compare clinical results between them
and the fully synthetic pyrrolo[3,2-d]pyrimidines.
Experimental Section
Cell Lines and Culturing
The human AML cell-line, MOLM-14, was a gift
from Dr. Mark Levis from Johns Hopkins
University. The human pancreatic MIA Pa-Ca-2,
human breast MDA-MB-231 and Non-small cell
lung A549 cell lines were purchased from ATCC
(ATCC, Manassas, VA). Cell lines were grown in
37 °C with 5% CO2 atmosphere with RPMI 1640
(Life technologies, Carlsbad, CA) or DMEM
(Cellgro Mediatech; Manassas, VA) supplemented
with heat-inactivated 10% (V/V) fetal bovine
serum (Hyclone; Fisher Scientific, Pittsburgh, PA)
and 1% glutamine (Cellgro Mediatech). Cell lines
were grown and maintained according to ATCC
recommendations.
The parent compound
7
displays rapid
pharmacokinetics (Figure 3A), with little to no
detection of the compound in mouse plasma within
70 minutes. Prodrugs 8 and 9 were designed to
decrease the metabolic rate by both sterically
hindering access to the reactive C4 and C7
positions as well as by acting as a labile leaving
group following metabolism to the active
compound 7.
Compound 9 proved stable in aqueous buffer for up
to 24 hours, but was undetectable in biological
media within 1 hour (Figure 4). A pharmacokinetic
profile supported the serum stability studies,
indicating a half-life 30.7 minutes for compound 7
and a half-life of 32.7 minutes for 9. This indicates
that the N5 substituent does not act as
metabolically desired, and suggests that delivery of
the compounds to the target cells remains a limiting
factor in the use of these compounds as potential
antiproliferative agents. The plasma studies
IC50 Proliferation Assay
Cell lines were seeded into 96-well plates the
afternoon prior to treatment. Approximately 18
hours later, compounds were semi-serially diluted
in dimethyl sulfoxide (DMSO) and then growth
medium, and added to cells. Plates were incubated
for 72 hours for cell lines and 48 hours for primary
6
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cells prior to addition of water-soluble tetrazolium Compounds 7 and 9 were measured using liquid
(WST-1) (Clontech, Mountain View, CA). Plates chromatography coupled to tandem mass
were read after 4 additional hours of incubation at spectrometry (LC/MS/MS). The analysis was
37 °C using a BioTek Synergy HT plate reader carried out in the Pharmacology Shared Resource
(BioTek, Winooski, VT). Data was analyzed and (University of Colorado Cancer Center) located in
graphed using GraphPad Prism Software the Flint Animal Cancer Center at the Veterinary
(GraphPad, La Jolla, CA).
Teaching Hospital at Colorado State University.
An LC/MS/MS based assay was developed and
validated for the analysis of compounds in mouse
plasma.
Gamma-H2Ax Assay in MOLM-14 Cells
The evening prior to treatment, MOLM-14 cells
were seeded at a density of 1X105 cells/mL in Sample Extraction. Blank mouse plasma was used
RPMI 1640/10%FBS. The following morning, for the construction of calibration and quality
cells were treated with either DMSO, 7 at 1X or 6X control standards and defined amounts of 7 or 9
IC50, or 6.0 Gy gamma irradiation (followed by a added in concentrations ranging from 10-50,000
one-hour incubation period). After 24 hours (or 1 ng/mL. Samples were prepared for analysis by
hour for gamma-irradiated cells), cells were acetonitrile (ACN) precipitation of plasma
collected, washed with PBS containing 2% serum, proteins. Briefly, 5 (in 50:50 ACN:water) was
and spun onto cytospin slides at a density of added as an internal standard to prepared standard
1.5X105 cells/slide. Slides were fixed for 15 or unknown plasma samples at
a
final
minutes in 4% PFA followed by 3-15 minute concentration of 1000 ng/ml followed by the
washes in PBS. Cells were subsequently addition of an equal volume of ACN. Samples were
permeabilized with permeabilization buffer (50 then mixed on a vortex mixer for 10 minutes
mM NaCl, 3 mM MgCl2, 10 mM HEPES, 200 mM followed by centrifugation at 14,000 rpm in a
sucrose and 0.5% Triton X-100 in PBS), and microcentrifuge. The supernatants were collected
blocked overnight with 10% FBS in PBS and transferred to HPLC vials for analysis.
containing 0.1% Triton X-100. Slides were
subsequently washed, incubated with Gamma- LC/MS/MS Analysis. Aliquots (60 μL) were
H2Ax primary antibody (Millipore) and goat anti- injected for LC/MS/MS analysis. Guidelines for
mouse Dylight secondary antibody (Invitrogen) batch acceptance and assay validation were based
and mounted with Vectashield containing DAPI on
accepted
practices
for
quantitative
(VWR). After drying overnight, slides were sealed chromatographic assays (Viswanathan et al.,
with nail polish and observed under a fluorescent 2007), and the assay accuracy and precision were
scope.
calculated to be 92.6 % ± 5.0 (CV%) for 7 and
90.3% ± 10.2 for 9 (CV%). The reference standard
range used was from 1-1,000 ng/mL and quality
control samples monitored at 5, 25, and 500 ng/mL
Maximum Tolerated Dose Assay
The tolerability of 6,7, and 9 was tested in female for 7 and 10-10,000 ng/mL and quality control
nude mice ages 6 weeks old. Note this strain and samples monitored at 50, 250 and 5,000 ng/mL for
sex of mice was used since female nude mice 9. Positive ion electrospray ionization mass
would be used in future efficacy experiments. Mice spectra were obtained with a 3200 Q-TRAP triple
were dosed via intraperitoneal (IP) administration quadrupole mass spectrometer with a turbo
(10% DMSO/10% Tween 20/80% saline vehicle) 5 ionspray source interfaced to an Agilent 1200
days per week for two consecutive weeks and then Series Binary Pump SL liquid chromatography
monitored for two additional weeks. If a single system
and
HTC-PAL
autosampler.
mouse in a group lost greater than 20% body Chromatography was performed on a Waters
weight, the dose was considered toxic.
Pharmacokinetic Studies
Sunfire C18, 5 µm, 4.6 x 50 mm column using a
gradient of acetonitrile with 0.1% formic acid
(solvent A) and 0.1% formic acid (solvent B). The
initial flow rate was 40% A:60% B with a linear
7
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gradient starting at 0.1 minutes to 98% A:2% B of ice-water slurry. The resultant yellow
over 1 minute (9) or 2.5 minutes (7) followed by suspension was stirred vigorously for 30 min, then
holding at 98% A for 0.75 minutes for 7 and solids collected by vacuum filtration. Solids were
returning to the original 40% A:60% B over 2 washed with ice-cold water until the filtrate was no
minutes. The flow rate was 1 mL/minute, and the longer acidic, then dried under vacuum to yield a
1
total analysis time was 5-6 minutes. The mass pale yellow powder (10.8g, 80%). H NMR (400
spectrometer settings were (7/9): turbo ionspray MHz, DMSO-d6): δ 11.76-11.74 (d, 2H), δ 2.28 (s,
temperature, 625°C/550°C; ion-spray voltage, 3H). 13C NMR (100 MHZ, DMSO-d6): δ156.8,
4750/5500 V; with declustering potential, entrance 154.6, 149.5, 127.7, 17.2. APCI-MS m/z for
potential, collision energy, collision cell exit C5H5N3O4 calcd. at 171.03, found 171.91 (M+H)
potential, and collision cell entrance potential
optimized for each ion pair. Analytes were
quantified by area of the signal obtained by nitropyrimidine-2,4(1H,3H)-dione
(E)-6-(2-(Dimethylamino)vinyl)-5-
(3):
6-
monitoring the transitions for 7 at m/z 314.0 methyl-5-nitropyrimidine-2,4(1H,3H)-dione (5g,
→ 187.2, m/z 314.0 → 287.0, m/z 314.0 → 243.2 29.2 mmol) suspended in 25 mL of anhydrous
and m/z 316.0 → 189.3, for 9 at m/z 467.8 → 313.1 DMF, then warmed to 80 °C under N2. N,N-
and m/z 467.8 →119.2 and the internal standard dimethylformamide-dimethylacetal (8.75 mL, 65.7
(KSR01) at m/z 205.0 → 169.1 and comparison to mmol) added and the resultant orange solution
a standard curve constructed using known stirred for 15 min, then heat raised to 140 °C for 30
standards.
min. Green/yellow solids formed during this time.
Reaction cooled to room temperature, then to 0 °C,
followed by vacuum filtration to obtain green-
Aqueous Stability Studies
yellow solid that was washed with cold EtOAc and
1
Compounds 6-9 were dissolved in anhydrous dried under vacuum (3.92 g, 60%). H NMR (400
DMSO (Sigma-Aldrich, St. Louis, MO) to a final MHz, DMSO-d6): δ10.97 (s, 1H), δ10.67 (s, 1H),
concentration of 1 mg/mL. Phosphate buffer δ8.06-8.03 (d, 1H, J=13.28 Hz), δ5.35-5.32 (d, 1H,
solutions at pH 5.0, 7.0, and 7.5 were prepared and J=13.13 Hz), δ3.11 (s, 3H), δ2.85 (s, 3H). 13C NMR
spiked with at 25 µg/mL and stirred at room (100 MHZ, DMSO-d6): δ157.3, 153.7, 151.1,
temperature. 500 µl aliquots taken at 0, 0.25, 0.5, 1, 149.9, 119.1, 81.5, 45.3, 37.3. APCI-MS m/z for
4, 8, and 24 hours were initially analyzed by thin- C8H10N4O4 calcd. at 226.07, found 226.93 (M+H)
layer chromatography against a verified standard,
then extracted with two 250 µl portions of
chloroform. Solvent removal and 1H NMR analysis (4):
of the residue was performed to verify the TLC nitropyrimidine-2,4(1H,3H)-dione
1H-Pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione
(E)-6-(2-(dimethylamino)vinyl)-5-
(5g, 22.1
results. Similar procedures were carried out with mmol) was suspended in glacial acetic acid, heated
pooled human serum (K3 EDTA, Innovative to 80 °C, then -100 mesh zinc dust (7g, 110 mmol)
Research, Novi, MI).
added in five portions over 1h. The reaction was
stirred overnight to give a pale yellow to white
suspension. Solids were collected by vacuum
filtration, dried on filter for 30 minutes, then placed
in a beaker and rinsed with cold water. Undissolved
Synthesis
6-Methyl-5-nitropyrimidine-2,4(1H,3H)-dione
(2): 6-methyluracil (Sigma-Aldrich, St. Louis, solids were collected by vacuum filtration, dried on
MO) (10g, 79.3 mmol) was dissolved in filter for 45 min, then transferred to a flask with 25
concentrated sulfuric acid at 0 °C, at a rate that mL of 1M NaOH. The suspension was heated to
maintained the temperature below 20 °C. After 80 °C and stirred for 30 minutes, then insoluble
complete dissolution, concentrated nitric acid (6 material removed by filtration. Glacial acetic acid
mL) was added dropwise, keeping the temperature was added to filtrate until pH=5, then precipitated
below 15 °C. The reaction was brought to room solids collected by vacuum filtration and washed
temperature then stirred for 1 h, followed by with ice-cold water and ethanol and dried under
carefully pouring the reaction mixture into 200 mL vacuum to obtain an off-white powder (2.35g,
8
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10.1002/cmdc.201700641
ChemMedChem
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70%). %). 1H NMR (400 MHz, DMSO-d6): δ11.81 orange powder (225 mg, 90%). Rf = 0.65 (3:1
1
(s, 1H), δ10. 72 (s, 1H) δ10.54 (s, 1H), δ7.10-7.09 hexanes:EtOAc) H NMR (400 MHz, DMSO-d6):
13
(d, 1H, J=2.7 Hz)), δ5.79 (s, 1H). C NMR (100 δ13.19 (s, 1H), 8.32 (s, 1H). 13C NMR (100 MHZ,
MHZ, DMSO-d6): δ 156.3, 152.0, 135.1, 127.5, DMSO-d6): δ151.9, 150.1, 144.2, 133.0, 123.6,
110.9, 96.5. APCI-MS m/z for C6H5N3O2 calcd. at 92.2. APCI-MS m/z for C6H2BrCl2N3 calcd. at
151.04, found 151.95 (M+H)
264.88, found 265.77 (M+H). Anal. calcd. for
C6H2BrCl2N3: C, 27.00; H, 0.76; N, 15.74. Found:
C, 27.02; H, 0.78; N, 15.71.
2,4-Dichloro-5H-pyrrolo[3,2-d]pyrimidine (5):
1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione
(2g, 13.2 mmol) was dissolved in 15 mL of 1M
2,4-Dichloro-7-iodo-5H-pyrrolo[3,2-
NaOH at 40 °C and stirred for 30 min, then cooled d]pyrimidine (7): 2,4-dichloro-5H-pyrrolo[3,2-
to room temperature then 0 °C. White crystalline d]pyrimidine (250 mg, 0.937 mmol) was dissolved
solid collected by vacuum filtration and washed in anhydrous THF at room temperature under N2.
with cold methanol and acetone, then dried under N-iodosuccinimide (263 mg, 1.17 mmol) added
vacuum. Dry solids were suspended in neat directly and reaction stirred under N2 for 2 h, upon
phenylphosphonic dichloride (10 mL) and heated which TLC indicated reaction completion. The
to 175 °C to obtain a brown solution. The reaction solvent was removed and residue dissolved in
was stirred for 5 h, then carefully poured into 50 EtOAc. This organic solution was washed with aq.
mL of ice-water slurry and stirred for 30 min. The Na2S2O3, water, and brine, then dried over MgSO4.
resultant aqueous suspension was extracted with Organics were loaded onto Celite and product was
three 25 mL portions of EtOAc, then organics purified using silica column chromatography
combined and washed with multiple portions of sat. eluting with 4:1 hexanes:EtOAc to obtain a pale
sodium bicarbonate until the pH=7. Organic layer yellow powder (250 mg, 85%). Rf = 0.68 (3:1
1
then dried with brine and MgSO4, and loaded onto hexanes:EtOAc) H NMR (400 MHz, DMS0-d6):
Celite. The product was purified using silica δ13.17 (s, 1H), 8.26 (s, 1H). 13C NMR (100 MHZ,
column chromatography eluting with 4:1 DMSO-d6): δ152.9, 150.1, 143.3, 139.0, 123.9,
hexanes:EtOAc to obtain a pale yellow powder (1.5 56.3. APCI-MS m/z for C6H2Cl2IN3 calcd. at
1
g, 60%). Rf = 0.5 (3:1 hexanes:EtOAc) H NMR 312.87, found 313.77 (M+H). ). Anal. calcd. for
(400 MHz, CDCl3): δ12.75 (s, 1H), δ8.07-8.06 (d, C6H2ICl2N3: C, 22.96; H, 0.65; N, 13.39. Found: C,
1H, J=2.76 Hz), δ6.70-6.69 (d, 1H, J=3.2). 13C 22.97; H, 0.65; N, 13.37.
NMR (100 MHZ, CDCl3): δ153.2, 150.2, 143.3,
134.9, 123.9, 103.1. APCI-MS m/z for C6H3Cl2N3
5-((Benzyloxy)methyl)-2,4-dichloro-7-iodo-
calcd. at 186.97, found 187.84 (M+H). Anal. calcd. pyrrolo[3,2-d]pyrimidine (8): 2,4-dichloro-7-
for C6H3Cl2N3: C, 38.33; H, 1.61; N, 22.35; Cl, iodo-5H-pyrrolo[3,2-d]pyrimidine (100 mg, 0.318
37.71. Found: C, 38.33; H, 1.65; N, 22.27; Cl, mmol) was dissolved in 1:1 THF:DMF
37.73.
(anhydrous) at room temperature under N2. NaH
(60% in mineral oil, 16 mg, 0.398 mmol) was
added directly and reaction stirred for 1.5 h. Benzyl
7-Bromo-2,4-dichloro-5H-pyrrolo[3,2-
d]pyrimidine (6): 2,4-dichloro-5H-pyrrolo[3,2- chloromethyl ether (73 µL, 0.398 mmol) was added
d]pyrimidine (250 mg, 0.937 mmol) was dissolved and reaction stirred for 3 h, upon which TLC
in anhydrous THF at room temperature under N2. indicated reaction completion. The solvent was
N-bromosuccinimide (208 mg, 1.17 mmol) was removed and residue dissolved in EtOAc and
added directly and reaction stirred under N2 for 3 h, washed with water and brine, then dried over
upon which TLC indicated reaction completion. MgSO4. Organics were loaded onto Celite and
The solvent was removed and residue dissolved in product
purified
using
silica
column
EtOAc. This organic solution was washed with aq. chromatography eluting with 5:1 hexanes:EtOAc
Na2S2O3, water, and brine, then dried over MgSO4. to obtain a white powder (124 mg, 90%). 1H NMR
Organics were loaded onto Celite and product was (400 MHz, CDCl3): δ7.63 (s, 1H), δ7.29-7.28 (m,
purified using silica column chromatography 3H), δ7.25-7.22 (m, 2H), δ5.79 (s, 2H), δ4.54 (s,
eluting with 4:1 hexanes:EtOAc to obtain a pale 2H). 13C NMR (100 MHZ, CDCl3): δ155.4, 150.4,
9
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10.1002/cmdc.201700641
ChemMedChem
FULL PAPER
146.1, 144.8, 143.6, 128.5, 128.1, 127.9, 123.6,
86.1, 77.9, 70.4, 58.8. APCI-MS m/z for
C14H10Cl2IN3O calcd. at 432.92, found 434.06
(M+H). ). Anal. calcd. for C14H10Cl2IN3O: C,
38.74; H, 2.32; N, 9.68. Found: C, 38.74; H, 2.34;
N, 9.67.
2,4-Dichloro-7-iodo-5-tosyl-pyrrolo[3,2-
d]pyrimidine
(9):
2,4-dichloro-7-iodo-5H-
pyrrolo[3,2-d]pyrimidine (100 mg, 0.318 mmol)
was dissolved in 1:1 THF:DMF (anhydrous) at
room temperature under N2. NaH (60% in mineral
oil, 16 mg, 0.398 mmol) was added directly and
reaction stirred for 1.5 h. p-Toluenesulfonyl
chloride (76 mg, 0.398 mmol) was added and the
reaction stirred overnight. The solvent was
removed, and the residue dissolved in EtOAc and
washed with water and brine, then dried over
MgSO4. Organics were loaded onto Celite and
product
purified
using
silica
column
chromatography eluting with 5:1 hexanes:EtOAc
to obtain a white powder (126 mg, 85%). 1H NMR
(400 MHz, CDCl3): δ8.82 (s, 1H), δ7.94-7.92 (d,
2H, J=8 Hz), δ7.46-7.44 (d, 2H, J=8 Hz), δ2.43 (s,
3H). 13C NMR (100 MHZ, CDCl3): δ158.1, 154.2,
146.9, 140.9, 137.1, 134.5, 130.5, 127.9, 64.1, 21.9.
APCI-MS m/z for C13H8Cl2IN3O2S calcd. at 466.9,
found 467.97 (M+H). Anal. calcd. for
C14H8Cl2IN3O2S: C, 33.36; H, 1.72; N, 8.98.
Found: C, 33.36; H, 1.74; N, 8.96.
Acknowledgements
The authors thank the National Institutes of Health for funding -
NIH T32 GM066706 (KSR and BMC); NIH R01 GM076345 (KSR)
and the Maryland Innovation Initiative (KSR).
Keywords: Pyrrolopyrimidine • triple negative breast cancer •
anticancer • prodrug • antiproliferative
10
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Table of Contents Entry and Graphic
N-substituted pyrrolo[3,2-d]pyrimidines are demonstrated to possess low toxicity against a wide range
of cancer cell lines while retaining activity, with IC50 values ranging from 0.014 – 14.5 µM. Further, cell
studies indicate a DNA-damaging mechanism of action, and pharmacokinetic studies show rapid
metabolism (t1/2 = 30.2 min). These data suggest that N-substitution offers a path towards development
of anti-proliferative pyrrolo[3,2-d] pyrimidines with ‘tunable’ biological activity.
12
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