Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1

Article abstract of DOI:10.1021/acs.jmedchem.5b01741

The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.

Full text of DOI:10.1021/acs.jmedchem.5b01741

Article
pubs.acs.org/jmc
Discovery of the First Potent and Selective Inhibitors of Human dCTP
Pyrophosphatase 1
Sabin Llona-Minguez,*,†,‡ Andreas Hoglund,†,⊥,‡ Sylvain A. Jacques,†,△ Lars Johansson,†,#
̈
Jose Manuel Calderon-Montano,† Magnus Claesson,∞ Olga Loseva,† Nicholas C. K. Valerie,†
́
́
̃
Thomas Lundback,†,# Javier Piedrafita,□ Giovanni Maga,§ Emmanuele Crespan,§ Laurent Meijer,∥
̈
Estefanía Burgos Moron,† Pawel Baranczewski,†,▽ Ann-Louise Hagbjork,▽ Richard Svensson,▽
́
̈
Elisee Wiita,† Ingrid Almlof,† Torkild Visnes,† Fredrik Jeppsson,† Kristmundur Sigmundsson,†,#,○
̈
Annika Jenmalm Jensen,†,# Per Artursson,▽ Ann-Sofie Jemth,† Pal Stenmark,∞
̊
Ulrika Warpman Berglund,† Martin Scobie,† and Thomas Helleday*,†
†Division of Translational Medicine and Chemical Biology, Science for Life Laboratory, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm, Sweden
#Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology,
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
▽Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Department of Pharmacy, Science for
Life Laboratory, Uppsala University, Uppsala, Sweden
∞Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius vag 16C, SE-106 91 Stockholm, Sweden
̈
§Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
∥ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, Bretagne, France
□Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic
degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple
carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective
dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in
vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition,
deeming it a suitable compound for in vivo studies.
(1, Figure 1) ≫ CTP), leading to decreased intracellular levels
of noncanonical dNTPs.1,2 The dCTPase protein was originally
identified in bacteria3 and has recently been found to be
overexpressed in multiple human carcinomas4 and associated
with cancer stemness.2,5 Modulation of dNTP catabolism
INTRODUCTION
■
dCTPase, also known as XTP3TPA, is an all-α nucleoside
triphosphate (NTP) pyrophosphatase responsible for dNTP
pool “house-cleaning”.1 More specifically, dCTPase catalyzes
Mg2+-mediated hydrolysis of dNTPs to the corresponding
monophosphates, with higher affinity toward 5-modified
noncanonical dNTPs over canonical dNTPs and rNTPs (5-I-
dCTP > 5-formyl-dCTP > 5-Br-dCTP > 5-Me-dCTP > dCTP
Received: November 9, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b01741
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a
Scheme 1. General Syntheses of Benzimidazole Derivatives
a
Reagents and conditions: (A) AcOH, 145 °C, 20 min; (B) 65%
HNO3, 95% H2SO4, MTBE/MeCN, 0 °C to rt, 16 h; (C1) alkyl
halide, K2CO3, DMSO, rt, 16 h; (C2) boronic acid, copper acetate,
pyridine, DCM, rt, 4 days; (C3) sulfonyl chloride, Et3N, DCM, rt, 16
h; (C4) acid chloride, Et3N, DCM, rt, 16 h; (D) 22, LiOH, water/1,4-
dioxane, rt, 16 h; (E) 18, N-methyliminodiacetic acid, MgSO4,
toluene/DMSO, reflux, 1 h.
Figure 1. Structures of dCTP, 5-aza-dCTP, and selected dCTPase
1
a
inhibitors. Using dCTP as the substrate. bIC50 not available.
RESULTS AND DISCUSSION
■
To exploit the therapeutic potential of dCTPase inhibition, we
purified the full-length human dCTPase protein and screened a
proprietary compound collection (5.5K compounds, Chemical
Biology Consortium Sweden at Karolinska Institutet). This was
achieved using an HTS-adapted Malachite Green assay, a test
for the colorimetric detection of inorganic phosphate (see
Supporting Information).16 The most potent hit from the
screen was compound 6 (Table 1) which inhibited dCTPase
provides an exciting opportunity to regulate nucleotide
homeostasis under pathologic conditions such as cancer and
inflammation.6−8 We have recently shown that inhibition of the
dNTP pool-sanitizing enzyme MTH1 is an effective anticancer
strategy: inhibition of MTH1 leads to increased incorporation
of oxidized dNTPs in cancer cells, causing subsequent DNA
damage and cell death in patient-derived xenografts.9 Here we
present a new case study of anticancer therapy exploiting the
dNTP catabolic machinery. Cytidine analogues, such as
decitabine (2, Figure 1), are currently used as first-line
anticancer agents in myelodysplastic syndrome (MDS) and
acute myeloid leukemia (AML). This class of drugs requires
kinase-mediated phosphorylations to generate the correspond-
ing triphosphates which are incorporated into DNA and/or
RNA, where they exert their therapeutic effect. We hypothe-
sized that some cytidine analogue triphosphates, such as 5-aza-
dCTP (3, Figure 1), could act as noncanonical substrates of
dCTPase given their structural resemblance to the enzyme’s
known substrates.1 Inhibition of the dCTPase enzyme should
therefore suppress degradation of the drug’s active triphosphate
form and improve its anticancer effect.10
Table 1. SAR Exploration: 2-, 4-, 5-, and 6-Positions
compd
R2
R4
R5/R6
IC50 (μM)
a
6
Me
H
NO2
H
Me
H
0.057 0.022
7
>100
8
H
H
Me
H
>100
>100
22
9
Me
Me
Me
Me
H
10
11
12
H
Me
Cl
H
Despite the biological relevance of dCTPase, only a handful
of weak non-drug-like dCTPase ligands have been identified
using photo-cross-linking experiments (4 and 5, Figure 1).11,12
In this article, we report on the discovery and development of a
series of benzimidazole derivatives as the first potent and
selective dCTPase inhibitors.
H
12
NO2
1.45
a
IC50 value determined in three independent runs.
activity by 98% at 10 μM and exhibited an IC50 value of 0.057
0.02 μM, determined from three independent runs. Initially,
we probed the importance of the substituents at the 2-, 4-, 5-
and 6-positions of the benzimidazole core (Table 1). The
methyl group at the benzimidazole 2-position appeared to be
important for activity, as analogues lacking this substituent
exhibited no inhibiton of dCTPase (compounds 7 and 8). The
importance of the 2-methyl group is further manifested when
compound 7 (inactive) is compared to its 2-methyl analogue 10
(IC50 = 22 μM). However, the 2-methyl-substituted compound
9 showed no inhibition of dCTPase, demonstrating that the
substituents in the 4-, 5-, and 6-positions of the benzimidazole
ring are also important for the inhibitory properties.
Interestingly, introduction of a nitro group at the 4-position
led to a highly potent dCTPase inhibitor 6 and its replacement
by hydrogen in 10 led to a dramatic 400 fold decrease in
CHEMISTRY
■
The general synthesis of this series of benzimidazole derivatives
is shown in Scheme 1. Ring formation of the heterocyclic core
was accomplished by thermal heating of commercially available
benzene-1,2-diamines in neat carboxylic acid.13 Subsequent
regioselective mononitration gave the corresponding 4-nitro-
benzimidazoles in good yields,14 followed by alkylation,
arylation, sulfonylation, or acylation reactions to obtain the
desired final products. Reactions were regioselective, and the
corresponding 1,4-regioisomer products were formed almost
exclusively, with only small amounts of the corresponding 1,7-
regioisomers observed as alkylation side-products. N-Methyl-
iminodiacetic acid (MIDA) boronate esters can be prepared
from the parent boronic acids using Knapp’s procedure.15
B
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Table 2. SAR Exploration: N1-Position
potency. On the other hand, when both the 5- and 6-methyl
groups in 6 were replaced with hydrogens (compound 12), the
decrease in potency was approximately 25-fold. We were
intrigued to find that the 5,6-dichloro analogue 11 was about
twice as potent as the corresponding 5,6-dimethyl analogue 10.
For this reason, we next prepared a series of compounds based
on the 4-nitro-5,6-dichlorobenzimidazole scaffold to investigate
the SAR at the N1-substituent (Table 2). The unsubstituted
benzyl analogue 13 and several para-substituted benzyl
analogues (methoxy 14, methyl 16, boronic acid 18, reversed
methyl ester 21, and the derived phenol 22) were equipotent to
compound 6. Nitrile 15 and carbonyl-containing derivatives
(carboxylic acid 17, methyl ester 19, and aldehyde 20)
exhibited slightly decreased inhibitory activity, while hetero-
cycle-containing analogues displayed even lower inhibition
(compounds 23, 24, 25, and 26), with the exception of
pyrazole 27. Following up on the unusual boronic acid 18, we
determined that para was slightly preferred to ortho (28)
substitution in the benzyl moiety and equipotent to meta (29)
substitution, and boronic esters were tolerated as well (30)
(Table 2). In a subsequent exploration we retained the active
motifs present in compounds 6, 13, and 18 and explored close
analogues to these by variations of the methylene chain
connecting N1 and the aromatic ring (Table 3). Benzylic α-
methylation (33) and linker extension (32) resulted in a
significant drop in activity, whereas linker deletion (31) and
sulfonamide (34) or carboxamide (35) replacements were less
detrimental. Additional exploration of the 2-, 4-, 5-, and 6-
positions of the benzimidazole ring (Table 4) indicated that
other substituents other than 2-methyl were tolerated, for
example 2-cyclopropyl (36) and 2-trifluoromethyl (37). When
the benzyl boronic acid was present, compound 38 lacking the
4-nitro group retained submicromolar activity and so did the
5,6-difluoro analogue 39.
Compounds 14 and 18 both showed a concentration-
dependent stabilization of the purified dCTPase enzyme in a
differential scanning fluorimetry assay (Supporting Information,
Figure 1).17 However, compound 14 did not stabilize dCTPase
in a cellular thermal shift assay (Figure 2A),18 possibly due to
efflux issues, and suffered from high liver microsomal clearance
(MLM/HLM t1/2) (Table 5). Short MLM and/or HLM half-
life was an issue often observed in this chemical series (see also
analogues 25 and 27). To our delight, the boronic acid
analogue 18 enabled a strong intracellular target engagement
C
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Table 3. SAR Exploration: N1-Position and Linkers
potential of boronic acid derivatives. Given the potentially
toxicity of also present nitro moiety,26 we were pleased to
observe that compound 18 did not increase DNA replication
stress, as indicated by markers of single-strand (p-Chk1) and
double-strand DNA breaks (γ-H2AX and 53BP1), nor cleaved
caspase 3, indicative of apoptosis (Supporting Information,
Figure 3), suggesting a low-mutagenic risk for 18 at the
concentrations and time points tested. Because boronic acid-
derived inhibitors can form covalent adducts with their
biological targets,27 we decided to further characterize the
dCTPase inhibition by 18 and 36 using a jump dilution assay.28
Both inhibitors displayed a reversible off-rate at 10% of the IC50
concentration (90% activity of the control sample, see
Supporting Information).
Table 4. Additional SAR Exploration: 2-, 4-, 5-, and 6-
Positions
compd
R2
R4
R5/R6
IC50 (μM)
36
37
38
39
cPr
CF3
Me
Me
NO2
NO2
H
Cl
H
Cl
F
0.027
0.206
0.248
0.529
NO2
Given the encouraging profile of 18, we decided to test the
effect of the dCTPase inhibitor in HL60 leukemia cells, in
combination with cytidine analogues of clinical relevance, such
as 5-azacytidine (5aza), decitabine (5azadC), and gemcitabine
(Supporting Information, Figure 6). Using the Chou−Talalay
method for synergy quantification,29 compound 18 showed a
clear synergistic effect when combined with cytidine analogues
increasing the cytotoxic action of the drugs against HL60 cells
(Figure 2B). As expected, the combination treatment induced a
concentration-dependent increase in cell death against the
leukemia cells (Figure 2C).
Compounds 18 and 14 displayed excellent selectivity across a
panel of related NTPases/NUDIX hydrolases (Supporting
Information, Table 1) and other enzymes of pharmacological
relevance, such as base excision repair enzymes (Supporting
Information, Table 2), kinases (Supporting Information, Figure
4), and did not intercalate DNA (Supporting Information
Figure 5). Unfortunately, 18 displayed only moderate aqueous
solubility (52 μM), while suffering from low plasma stability
(11% left after 4 h) and high plasma protein binding (Fu =
0.1%) (Table 5). This was most probably due to the presence
of the boronic acid functionality (compare with phenol
analogue 22, Table 5). These suboptimal physicochemical
and ADME properties limit the use of 18 to in vitro
(Figure 2A) and displayed enhanced microsomal stability and
aqueous solubility (Table 5). Interestingly, replacement of the
2-methyl substituent in 18 with a cyclopropyl group in 36
improved stability in HLM but decreased in MLM (Table 5).
Attracted by the chemical novelty of 18, we decided to profile
the compound in more depth. First, we investigated the nitro
and boronic acid features of 18. Although nitro aromatic
compounds are often reported to be photoreactive,19 a solution
of 18 stored at room temperature under normal light
conditions showed no signs of degradation over a period of
five months (see Supporting Information). Then we inves-
tigated the in vitro reactivity of the electrophilic α-carbon to the
nitro group. Compounds 18 and 30 did not conjugate with the
endogenous nucleophile glutathione (GSH) in the presence of
metabolic activation system (in HLM) (see Supporting
Information). Next, we turned our attention to the boronic
acid motif. Boron-containing drugs are uncommon and only a
few compounds have reached the clinical stage in the fields of
anti-infectives (tavaborole20 and RPX700921) and oncology
(bortezomib22 and delanzomib23) (see Supporting Information,
Figure 2). Although boronic acid reagents are generally
perceived as “low-toxicity” compounds, researchers at Lilly24
and Astra Zeneca25 have recently reported on the mutagenic
D
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Figure 2. (A) Western blot images from the cellular thermal shift assay for 14 and 18. HL60 cells were incubated for 2 h with compounds 14 and 18
(increasing concentrations) and subsequently heated at 67 °C and analyzed for intracellular dCTPase stabilization. (B) Combination index (CI)
plots of 18 in combination with cytidine analogues. HL60 cells were incubated for 72 h with different concentrations of compound 18 and cytidine
analogues. Cytotoxic effect was determined by Resazurin cell viability assay. (C) Effect of compound 18 in combination with cytidine analogues on
cell death. HL60 cells were incubated for 72 h with compound 18 (2.5 μM) and increasing concentrations of cytidine analogues. Cell death was
determined by flow cytometry analysis.
Table 5. In Vitro ADME Data for Selected Compounds
14
8/30
22
25
7/6
27
36
18
30
t1/2 MLM/HLM (min)
E MLM/HLM
−
−
11
96
0.7
−
22/38
13/268
197/72
109/11
0.36/0.80
>100
86
0.88/0.59
0.89/0.83
0.73/0.61
0.83/0.14
0.24/0.38
aq solubility (μM)
plasma stability t4h (%)
protein binding (Fu %)
Papp AB/BA (cm/s)
efflux ratio
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
52
11
0.1
−
0.8
8.66 × 10−6/1.30 × 10−3
−
−
1.51
Table 6. CYP Inhibition Data for Compound 30
CYP1A2 (phenacetin) CYP2C9 (diclofenac) CYP2C19 (mephenytion) CYP2D6 (bufuralol) CYP3A4 (testosterone) CYP3A4 (midazolam)
377.2 1.72 2.93 16.75 21.94 28.12
IC50 (μM)
experiments. To develop a compound suitable for in vivo
studies, we turned our attention to the N-methyliminodiacetic
acid (MIDA) boronic acid derivative 30. This chemical motif
has gained popularity in recent years as a versatile building
block for the synthesis of heteroaromatic compounds.15,30 The
MIDA boronate 30 inhibited dCTPase comparably to 18
(Table 2) and conferred enhanced aqueous solubility and
improved plasma stability (Table 5). Compound 30 presented
suitable in vitro t1/2 in the presence of MLMs, but not HLMs,
and an overall encouraging CYP inhibition profile, with only
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then dried in vacuo to afford the desired compound of general formula
I.1.X.
1H-Benzo[d]imidazole (I-1.1), 5,6-dimethyl-1H-benzo[d]-
imidazole (I-1.2) and 2-methyl-1H-benzo[d]imidazole (I-1.3).
Commercially available.
modest inhibition by CYP2C enzymes (Table 6), deeming it a
suitable candidate for future studies in relevant MDS and AML
mouse models.
CONCLUSIONS
■
2,5,6-Trimethyl-1H-benzo[d]imidazole (I-1.4). Yield 87%. Analyt-
ical data matching the literature report.13
Here we have described the discovery and subsequent
medicinal chemistry optimization of the first potent and
selective dCTPase inhibitors. This series of N1-substituted
benzimidazole analogues relies on additional functionalities on
the 2-, 4-, 5- and 6-positions of the heterocyclic core to achieve
optimal inhibitory activity, although at least substitution at
three out of those four positions is required for sub-micromolar
dCTPase inhibition. Diverse aryl-containing substituents at the
N1-position deliver compounds with nanomolar potency.
Increased microsomal stability and intracellular target engage-
ment was achieved thanks to the addition of a boronic acid
functionality, as seen in compound 18, which effectively
synergized with cytidine analogues in killing HL60 leukemia
cells. Despite its excellent selectivity profile and cellular efficacy,
compound 18 lacked sufficient plasma stability required for in
vivo studies. This issue was circumvented by masking the
boronic acid functionality with a MIDA ester to deliver
compound 30 with a suitable profile for future experiments in
murine leukemia models. Efforts to improve human micro-
somal stability on 30 are currently ongoing and will be reported
in due course.
5,6-Dichloro-2-methyl-1H-benzo[d]imidazole (I-1.5). Reaction
performed in 21 mmol scale. Yield 93%. Analytical data matching
the literature report.13
2-Methyl-4-nitro-1H-benzo[d]imidazole (I-1.6). Yield 84%. LCMS
1
[M + H]+ m/z 178. H NMR (DMSO-d6) δ: 13.00 (br s, 1 H), 8.05
(dd, J = 8.1, 0.9 Hz, 1 H), 7.98 (dd, J = 7.9 Hz, 0.9 Hz, 1 H), 7.33 (app
t, J = 8.1 Hz, 1 H), 2.58 (s, 3 H)
5,6-Dichloro-2-cyclopropyl-1H-benzo[d]imidazole (I-1.7). Yield
1
51%. LCMS [M + H]+ m/z 227. H NMR (DMSO-d6) δ:12.62 (br
s, 1 H), 7.66 (s, 2 H), 2.16−2.06 (m, 1 H), 1.12−1.05 (m, 2 H), 1.05−
0.97 (m, 2 H)
4-Nitro-2-(trifluoromethyl)-1H-benzo[d]imidazole (I-1.8). Yield
1
51%. LCMS [M + H]+ m/z 232. H NMR (DMSO-d6) δ: 14.65 (br
s, 1 H), 8.33 (d, J = 8.1 Hz, 1 H), 8.29 (d, J = 7.3 Hz, 1 H), 7.59 (t, J =
8.1 Hz, 1 H)
4-Nitro-2-(trifluoromethyl)-1H-benzo[d]imidazole (I-1.9). Yield
68%. Analytical data matching the literature report.13
Method B. A 65% HNO3 (1.1 equiv) solution was added dropwise
to an appropriately substituted heteroaryl compound of general
formula I-1.X (1.0 mmol, 1.1 equiv) in a mixture of MTBE/MeCN
(2:1, 0.4 M) at 0 °C. The mixture was stirred at 0 °C for 1 h after
which the reaction was concentrated in vacuo. The residue was
suspended in DCM (0.4 M), and the mixture was added dropwise to
ice-cold 95% H2SO4 (10 equiv). The mixture was allowed to warm to
rt and stirred for 16 h. The mixture was poured onto ice−water and
neutralized with concd NH4OH while keeping the temperature below
5 °C. The mixture was filtered and dried to afford the desired
compound of general formula I-2.X.
2,5,6-Trimethyl-4-nitro-1H-benzo[d]imidazole (I-2.1). Yield 85%.
LCMS [M + H]+ m/z 206. 1H NMR (DMSO-d6) δ: 12.56 (br s, 1 H),
7.73 (s, 0.4H, minor tautomer), 7.47 (s, 0.6H, major tautomer), 2.47−
2.49 (m, 4.2 H), 2.40 (s, 1.2H, minor tautomer), 2.38 (s, 1.8H, major
tautomer), 2.21 (s, 1.8H, major tautomer)
These results indicate that potent and selective dCTPase
inhibitors can enhance the therapeutic effect of noncanonical
cytidine analogues. Investigations to understand the biological
relevance of dCTPase inhibition will provide invaluable insight
into dNTP pool regulation and new potential applications for
this type of therapeutic agents.
EXPERIMENTAL SECTION
■
Chemistry. All commercial reagents and solvents were used
without further purification. Analytical thin-layer chromatography was
performed on silica gel 60 F-254 plates (E. Merck) and visualized
under a UV lamp. Flash column chromatography was performed in a
Biotage SP4MPLC system using Merck silica gel 60 Å (40−63 mm
5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazole (I-2.2). Re-
action performed in 24 mmol scale. Yield 86%. LCMS [M + H]+ m/z
246. 1H NMR (DMSO-d6) δ: 13.14 (br s, 1 H), 8.10 (s, 1 H), 2.56 (s,
3 H)
mesh). 1H NMR spectra were recorded on a Bruker DRX-400 and 13
C
NMR spectra on a Bruker 600 MHz Avance III spectrometer.
Chemical shifts are expressed in parts per million (ppm) and
referenced to the residual solvent peak. Analytical HPLC-MS was
performed on an Agilent MSD mass spectrometer connected to an
Agilent 1100 system with method B1090A: column ACE 3 C8 (50 ×
3.0 mm); H2O (+ 0.1% TFA) and MeCN were used as mobile phases
at a flow rate of 1 mL/min, with a gradient time of 3.0 min; or method
0597X3: column X-Terra MSC18 (50 × 3.0 mm); H2O (containing
10 mM NH4HCO3; pH = 10) and MeCN were used as mobile phases
at a flow rate of 1 mL/min, with a gradient time of 3.0 min. Preparative
HPLC was performed on a Gilson HPLC system; basic pH: column
Xbridge Prep C18, 5 μM CBD (30 × 75 mm); H2O (containing 50
mM NH4HCO3; pH = 10) and MeCN were used as mobile phases at
a flow rate of 45 mL/min, with a gradient time of 9 min. Acidic pH:
column ACE 5 C8 (150 × 30 mm); H2O (containing 0.1% TFA) and
MeCN were used as mobile phases at a flow rate of 45 mL/min, with a
gradient time of 9 min. For HPLC-MS, detection was made by UV
using the 180−305 nM range and MS (ESI+). For preparative HPLC,
detection was made by UV at 254 or 214 nM. All intermediates and
final compounds were assessed to be >95% pure by HPLC-MS
analysis, unless stated otherwise.
5,6-Dichloro-2-cyclopropyl-4-nitro-1H-benzo[d]imidazole (I-2.3).
Yield 49%. LCMS [M + H]+ m/z 272. 1H NMR (CDCl3) δ: 10.13 (br
s, 1 H), 7.99 (s, 1 H), 2.10 (m, 1 H), 1.28 (m, 4H)
Method C1. When R1 = Arylalkyl. A mixture of the appropriate
alkyl halide (1.5 equiv), the appropriate heteroaromatic intermediate
of general formula 1.2.X or 2.X (0.1 mmol, 1.0 equiv), K2CO3 (2
equiv), and DMSO (0.2 M) was stirred at rt for 16 h (for reactions
using an alkyl chloride as the alkylating agent, one crystal of potassium
iodide was added to the reaction mixture). The mixture was purified
by preparative HPLC basic pH to afford the desired compound.
1-(4-Methoxybenzyl)-2,5,6-trimethyl-4-nitro-1H-benzo[d]-
imidazole (6). Yield 87%. LCMS [M + H]+ m/z 326. 1H NMR
(DMSO-d6) δ: 7.63 (s, 1 H), 7.09 (m, 2 H), 6.89 (m, 2 H), 6.58 (m, 2
H), 5.41 (s, 2 H), 3.70 (s, 3 H), 2.51 (s, 3 H), 2.37 (s, 3 H), 2.22 (s, 3
H)
1-(4-Methoxybenzyl)-1H-benzo[d]imidazole (7). Yield 53%.
1
LCMS [M + H]+ m/z 239. H NMR (DMSO-d6) δ: 8.38 (s, 1 H),
7.63 (m, 1 H), 7.52 (m, 1 H), 7.28 (m, 2 H), 7.19 (m, 2 H), 6.88 (m, 2
H), 5.40 (s, 2 H), 3.70 (s, 3 H)
1-(4-Methoxybenzyl)-5,6-dimethyl-1H-benzo[d]imidazole (8).
Yield 53%. LCMS [M + H]+ m/z 267. 1H NMR (DMSO-d6) δ:
8.21 (s, 1 H), 7.40 (s, 1 H), 7.29 (s, 1 H), 7.23 (m, 2 H), 6.88 (m, 2
H), 5.34 (s, 2 H), 3.70 (s, 3 H), 2.27 (s, 6 H)
Method A. The appropriate aromatic diamine (2.0 mmol, 1.0
equiv) and carboxylic acid (10 equiv) were heated in a sealed tube at
145 °C for 20 min. The resulting solution was cooled to rt and then
poured into a beaker containing ca. 10 mL of saturated Na2CO3. The
precipitate was filtered off and triturated with water. The material was
1-(4-Methoxybenzyl)-2-methyl-1H-benzo[d]imidazole (9). Yield
1
51%. LCMS [M + H]+ m/z 253. H NMR (DMSO-d6) δ: 7.52 (m,
F
DOI: 10.1021/acs.jmedchem.5b01741
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1 H), 7.47 (m, 1 H), 7.13 (m, 2 H), 7.09 (m 2 H), 6.88 (m, 2 H), 5.38
(s, 2 H), 3.70 (s, 3 H), 2.52 (s, 3 H)
7.33 (d, J = 8.0 Hz, 1 H), 7.21 (dd, J = 2.3, 8.0 Hz, 1 H), 5.34 (s, 2 H),
2.64 (s, 3 H)
5,6-Dichloro-1-(2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl)-2-methyl-
4-nitro-1H-benzo[d]imidazole (25). Yield 13%. LCMS [M + H]+ m/z
368. 1H NMR (DMSO-d6) δ: 11.92 (br s, 1 H), 8.01 (s, 1 H), 4.34 (t,
J = 6.2 Hz, 1 H), 2.73 (t, J = 6.2 Hz, 2 H), 2.23 (s, 3 H), 1.55−1.85 (br
s, 6H)
1-(4-Methoxybenzyl)-2,5,6-trimethyl-1H-benzo[d]imidazole (10).
1
Yield 29%. LCMS [M + H]+ m/z 281. H NMR (DMSO-d6) δ: 7.30
(s 1 H), 7.23 (s, 1 H), 7.04 (m, 2 H), 6.87 (m, 2 H), 5.30 (s 2 H), 3.70
(s, 3 H), 2.45 (s, 3 H), 2.27 (s, 6 H)
5,6-Dichloro-1-(4-methoxybenzyl)-2-methyl-1H-benzo[d]-
1
imidazole (11). Yield 56%. LCMS [M + H]+ m/z 321. H NMR
1-(4-(1H-Pyrrol-1-yl)benzyl)-5,6-dichloro-2-methyl-4-nitro-1H-
1
benzo[d]imidazole (26). Yield 25%. LCMS [M + H]+ m/z 401. H
(DMSO-d6) δ: 7.90 (s, 1 H), 7.80 (s, 1 H), 7.10 (m, 2 H), 6.89 (m, 2
H), 5.41 (s, 2 H), 3.70 (s, 3 H), 2.52 (s, 3 H)
NMR (DMSO-d6) δ: 8.33 (s, 1 H), 7.50−7.56 (m, 2 H), 7.40 (app t, J
= 7.8 Hz, 1 H), 7.35 (app t, J = 2.2 Hz, 2 H), 6.89 (app d, J = 7.5 Hz, 1
H), 6.27 (app t, J = 2.2 Hz, 2 H), 5.62 (s, 2 H), 4.07−4.49 (m, 1 H),
2.59 (s, 3 H)
1-(4-Methoxybenzyl)-2-methyl-4-nitro-1H-benzo[d]imidazole
1
(12). Yield 55%. LCMS [M + H]+ m/z 298. H NMR (DMSO-d6) δ:
7.99 (dd, J = 8.0, 0.9 Hz, 1 H), 7.98 (dd, J = 8.1, 0.9 Hz, 1 H), 7.36
(app t, J = 8.1 Hz, 1 H), 7.12 (m, 2 H), 6.89 (m, 2 H), 5.51 (s, 2 H),
3.70 (s, 3 H), 2.64 (s, 3 H)
1-(4-(1H-Pyrazol-1-yl)benzyl)-5,6-dichloro-2-methyl-4-nitro-1H-
1
benzo[d]imidazole (27). Yield 10%. LCMS [M + H]+ m/z 402. H
NMR (CDCl3) δ: 7.90 (app d, J = 2.5 Hz, 1 H), 7.68−7.73 (m, 3 H),
7.46 (app s, 1 H), 7.12 (d, J = 8.8 Hz, 2 H), 6.45−6.50 (m, 1 H), 5.35
(s, 2 H), 2.63 (s, 3 H)
1-Benzyl-5,6-dichloro-2-methyl-4-nitro-1H-benzo[d]imidazole
1
(13). Yield 73%. LCMS [M + H]+ m/z 336. H NMR (DMSO-d6) δ:
8.31 (s, 1 H), 7.35 (m, 3 H), 7.16 (m, 2 H), 5.59 (s, 2 H), 2.55 (s, 3
(3-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)phenyl)boronic Acid (28). Purified by preparative HPLC
(acidic pH). Yield 43%. LCMS [M + H]+ m/z 380. 1H NMR
(CD3OD) δ: 7.93 (s, 1 H), 7.57−7.54 (m, 1 H), 7.43−7.31 (m, 2 H),
7.16−7.14 (m, 1 H), 5.53 (s, 2 H), 2.60 (s, 3 H)
(2-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)phenyl)boronic Acid (29). Purified by preparative HPLC
(acidic pH). Yield 49%. LCMS [M + H]+ m/z 380. 1H NMR
(CD3OD) δ: 7.74 (s, 1 H), 7.45−7.39 (m, 1 H), 7.39−7.34 (m, 2 H),
7.10−7.05 (m, 1 H), 5.52 (s, 2 H), 2.55 (s, 3 H)
H)
5,6-Dichloro-1-(4-methoxybenzyl)-2-methyl-4-nitro-1H-benzo-
[d]imidazole (14). Yield 74%. LCMS [M + H]+ m/z 366. H NMR
1
(DMSO-d6) δ: 8.32 (s, 1 H), 7.15 (app d, J = 8.3 Hz, 2 H), 6.90 (app
d, J = 8.4 Hz, 2 H), 5.50 (s, 2 H), 3.71 (s, 3 H), 2.57 (s, 3 H). 13C
NMR (DMSO-d6) δ: 158.9 (C), 157.9 (C), 138.7 (C), 136.2 (C),
134.0 (C), 128.5 (CH), 127.6 (C), 124.8 (C), 115.3 (C), 114.6 (CH),
114.3 (CH), 55.1 (CH3), 46.5 (CH2), 14.0 (CH3)
4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)benzonitrile (15). Yield 61%. LCMS [M + H]+ m/z 361. H
1
(4-((5,6-Dichloro-2-cyclopropyl-4-nitro-1H-benzo[d]imidazol-1-
yl)methyl)phenyl)boronic Acid (36). Purified by preparative HPLC
(acidic pH). Yield 48%. LCMS [M + H]+ m/z 406. 1H NMR
(acetone-d6) δ: 8.01 (s, 1 H), 7.85−7.89 (m, 2 H), 7.24−7.27 (m, 2
H), 5.77 (s, 2 H), 2.27−2.31 (m, 1 H), 1.14−1.19 (m, 4H)
(4-((4-Nitro-2-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)-
methyl)phenyl)boronic Acid (37). Yield 58%. LCMS [M + H]+ m/z
366. 1H NMR (CD3OD) δ: 8.26−8.20 (m, 1 H), 7.94−7.92 (m, 1 H),
7.70 (br s, 1 H), 7.60 (br s, 1 H), 7.60−7.55 (m, 1 H), 7.10−7.05 (m,
2 H), 5.78 (s, 2 H)
NMR (DMSO-d6) δ: 8.31 (s, 1 H), 7.78−7.85 (app d, J = 8.3 Hz, 2
H), 7.28−7.34 (app d, J = 8.3 Hz, 2 H), 5.71 (s, 2 H), 2.53 (s, 3 H)
5,6-Dichloro-2-methyl-1-(4-methylbenzyl)-4-nitro-1H-benzo[d]-
1
imidazole (16). Yield 57%. LCMS [M + H]+ m/z 350. H NMR
(DMSO-d6) δ: 8.30 (s, 1 H), 7.16 (m, 2 H), 7.06 (m, 2 H), 5.53 (s, 2
H), 2.55 (s, 3 H), 2.26 (s, 3 H)
4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)benzoic Acid (17). Purified by preparative HPLC (acidic pH).
Yield 50%. LCMS [M + H]+ m/z 380. 1H NMR (DMSO-d6) δ: 12.98
(br s, 1 H), 8.31 (s, 1 H), 7.94−7.89 (app d, J = 8.3 Hz, 2 H), 7.28−
7.21 (app d, J = 8.3 Hz, 2 H), 5.68 (s, 2 H), 2.53 (s, 3 H)
(4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)phenyl)boronic Acid (18). Yield 67%. LCMS [M + H]+ m/z
380. 1H NMR (DMSO-d6) δ: 8.29 (s, 1 H), 8.06 (s, 2 H), 7.75 (app d,
J = 8.1 Hz, 2 H), 7.10 (app d, J = 8.1 Hz, 2 H), 5.59 (s, 2 H), 2.54 (s, 3
H). 13C NMR (DMSO-d6) δ: 158.0 (C), 138.7 (C), 137.4 (C), 136.4
(C), 134.7 (2 × CH), 134.0 (C), 125.8 (2 × CH), 124.9 (C), 115.4
(C), 114.6 (CH), 47.0 (CH2), 13.9 (CH3)
Methyl 4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-
1-yl)methyl)benzoate (19). Yield 61%. LCMS [M + H]+ m/z 394.
1H NMR (CDCl3) δ: 8.03 (app d, J = 8.3 Hz, 2 H), 7.43 (s, 1 H), 7.08
(app d, J = 8.3 Hz, 2 H), 5.38 (s, 2 H), 3.92 (s, 3 H), 2.61 (s, 3 H)
4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)benzaldehyde (20). Reaction performed in acetone. Purified
by FCC (5−75% EtOAc in hexanes). Yield 57%. LCMS [M + H]+ m/
z 364. 1H NMR (CDCl3) δ: 10.01 (s, 1 H), 7.89 (app d, J = 8.03 Hz, 2
H), 7.43 (s, 1 H), 7.18 (app d, J = 8.03 Hz, 2 H), 5.41 (s, 2 H), 2.62 (s,
3 H)
(4-((5,6-Dichloro-2-methyl-1H-benzo[d]imidazol-1-yl)methyl)-
1
phenyl)boronic Acid (38). Yield 42%. LCMS [M + H]+ m/z 335. H
NMR (CD3OD) δ: 7.71 (s, 1 H), 7.65−7.55 (m, 3 H), 7.08 (app d, J =
6.6 Hz, 2 H), 5.45 (s, 2 H), 2.55 (s, 3 H)
(4-((5,6-Difluoro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)phenyl)boronic Acid (39). Yield 76%. LCMS [M + H]+ m/z
348. 1H NMR (CD3OD) δ: 7.91−8.01 (m, 1 H), 7.74−7.62 (m, 2 H),
7.19−7.08 (m, 2 H), 5.60 (s., 2 H), 2.72 (s, 3 H)
Method C2. 5,6-Dichloro-2-methyl-4-nitro-1-phenyl-1H-benzo-
[d]imidazole (31). A mixture of I-2.2 (1.0 equiv), phenyl boronic acid
(0.1 mmol, 1.0 equiv), copper acetate (1.0 equiv), pyridine (2.0 equiv),
and DCM (0.1 M) was stirred at rt for 4 days. The mixture was cooled
and purified by FCC (5−30% EtOAc in hexanes) to afford 31. Yield
15%. LCMS [M + H]+ m/z 322. 1H NMR (CDCl3) δ: 7.60−7.69 (m,
3 H), 7.36 (s, 1 H), 7.32−7.36 (m, 2 H), 2.54 (s, 3 H)
Method C3. 5,6-Dichloro-2-methyl-4-nitro-1-(phenylsulfonyl)-
1H-benzo[d]imidazole (32). A mixture of I-2.2 (0.1 mmol, 1.0
equiv), benzenesulfonyl chloride (1.5 equiv), triethylamine (1.0
equiv), and DCM (0.1 M) was stirred at rt for 16 h. The mixture
was cooled and purified by FCC (20% EtOAc in hexanes) to afford 32.
Yield 64%. LCMS [M + H]+ m/z 322. 1H NMR (CDCl3) δ: 8.36 (s, 1
H), 7.95−7.90 (m, 2 H), 7.77−7.71 (m, 1 H), 7.64−7.57 (m, 2 H),
2.81 (s, 3 H)
Method C4. (5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]-
imidazol-1-yl) (phenyl)methanone (33). A mixture of I-2.2 (1.0
equiv), benzoyl chloride (1.5 equiv), triethylamine (1.0 equiv), and
DCM (0.1 M) was stirred at rt for 16 h. The mixture was cooled and
purified by FCC (20% EtOAc in hexanes) to afford 33. Yield 73%.
LCMS [M + H]+ m/z 350. 1H NMR (CDCl3) δ: 7.82−7.72 (m, 3 H),
7.64−7.58 (m, 2 H), 7.28 (s, 1 H), 2.65 (s, 3 H)
Method D. 4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]-
imidazol-1-yl)methyl)phenol (22). A solution of LiOH (1.5 equiv)
in water (1 M) was added to carboxylic ester 21 (0.1 mmol, 1.0 equiv)
4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)phenyl Acetate (21). Reaction performed in acetone. Yield
1
46%. LCMS [M + H]+ m/z 394. H NMR (CDCl3) δ: 7.46 (s, 1 H),
7.12−7.07 (m, 2 H), 7.05−7.00 (m, 2 H), 5.32 (s, 2 H), 2.62 (s, 3 H),
2.30 (s, 3 H)
4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]imidazol-1-yl)-
methyl)-2-methylthiazole (23). Reaction performed in acetone.
Purified by FCC (1−15% EtOAc in hexanes). Yield 29%. LCMS [M
+ H]+ m/z 394. 1H NMR (CDCl3) δ: 7.64 (s, 1 H), 6.83 (s, 1 H), 5.34
(s, 2 H), 2.74 (s, 3 H), 2.67 (s, 3 H)
5,6-Dichloro-1-((6-chloropyridin-3-yl)methyl)-2-methyl-4-nitro-
1H-benzo[d]imidazole (24). Reaction performed in acetone. Purified
by FCC (5−75% EtOAc in hexanes). Yield 15%. LCMS [M + H]+ m/
1
z 371. H NMR (CDCl3) δ: 8.30 (d, J = 2.3 Hz, 1 H), 7.45 (s, 1 H),
G
DOI: 10.1021/acs.jmedchem.5b01741
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in 1,4-dioxane (0.06 M) at rt. The mixture was stirred at rt for 16 h
Wallace at University of Vermont for donation of plasmid
materials and to the Protein Science Facility at Karolinska
Institutet for purification of proteins.
and purified by preparative HPLC basic pH to afford phenol 22. Yield
1
86%. LCMS [M + H]+ m/z 352. H NMR (DMSO-d6) δ: 9.48 (s, 1
H), 8.29 (s, 1 H), 7.08−6.99 (m, 2 H), 6.76−6.66 (m, 2 H), 5.44 (s, 2
H), 2.56 (s, 3 H)
ABBREVIATIONS USED
Method E. 2-(4-((5,6-Dichloro-2-methyl-4-nitro-1H-benzo[d]-
imidazol-1-yl)methyl)phenyl)-6-methyl-1,3,6,2-dioxazaborocane-
4,8-dione (30). A mixture of 18 (0.1 mmol, 1.0 equiv), N-
methyliminodiacetic acid (1.5 equiv), MgSO4 (10 equiv), and
toluene/DMSO (9:1, 0.05 M) was refluxed for 1 h. The mixture
was cooled and purified by FCC (0−60% diethyl ether in MeCN) to
■
XTP3TPA, XTP3-transactivated protein A; NTP, nucleoside
triphosphate; dNTP, deoxynucleoside triphosphate; rNTP,
ribonucleoside triphosphate; HTS, high throughput screen;
DSF, differential scanning fluorimetry; MLM, mouse liver
microsomes; HLM, human liver microsomes; CYP, cyto-
chrome; FCC, flash column chromatography; E, extraction
ratio; Papp, apparent permeability coefficient
1
afford 30. Yield 89%. LCMS [M + H]+ m/z 491. H NMR (DMSO-
d6) δ: 8.30 (s, 1 H), 7.46−7.37 (m, J = 8.1 Hz, 2 H), 7.20−7.12 (m, J =
8.1 Hz, 2 H), 5.60 (s, 2 H), 4.31 (d, J = 17.4 Hz, 2 H), 4.08 (d, J = 17.1
Hz, 2 H), 2.54 (s, 3 H), 2.46 (s, 3 H). 13C NMR (DMSO-d6) δ:169.3,
158.0, 138.7, 136.4, 136.3, 134.0, 133.0, 126.2, 124.9, 115.3, 114.6,
61.7, 47.5, 47.0, 14.0.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01741.
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Supplementary figures, additional data information
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: thomas.helleday@scilifelab.se.
*E-mail: sabin.llona.minguez@scilifelab.se.
■
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Present Addresses
⊥Sprint BioScience AB, Huddinge, Sweden.
△LFCS, CAMB UMR7199 CNRS/LIT UMR7200 CNRS/
́
Universite de Strasbourg, MEDALIS Drug Discovery Center,
́
Faculte de Pharmacie, 67401 Illkirch, France.
○Duke-NUS Graduate Medical School, Singapore.
Author Contributions
‡The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. S.L-M and A.H. contributed equally.
Notes
The authors declare the following competing financial
interest(s): A patent has been filed with data generated in
this manuscript where S.L-M., T.H., A.H., S.A.J., M.S., and L.J.
are listed as inventors.
ACKNOWLEDGMENTS
■
This project is primarily supported by The Knut and Alice
Wallenberg Foundation. Further support was received from the
Felix Mindus foundation for leukemia research (A.H.), the
Swiss National Science Foundation (F.G.), the Swedish
Research Council (T.H., A.JJ., P.S.), the European Research
Council (T.H.), Goran Gustafsson Foundation (T.H.), Swedish
̈
Cancer Society (T.H., P.S.), the Swedish Children’s Cancer
Foundation (T.H.), the Swedish Pain Relief Foundation
(T.H.), Carl Tryggers Foundation (P.S.), Wenner-Gren
Foundation (P.S), and the Torsten and Ragnar Soderberg
̈
Foundation (T.H.). Chemical Biology Consortium Sweden was
supported by the Swedish Research Council. We also
acknowledge ChemAxon (http://www.chemaxon.com) for
technical support. We are grateful to Hans Krokan at
Norwegian University of Science and Technology and Susan
H
DOI: 10.1021/acs.jmedchem.5b01741
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Journal of Medicinal Chemistry
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DOI: 10.1021/acs.jmedchem.5b01741
J. Med. Chem. XXXX, XXX, XXX−XXX