Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach

Article abstract of DOI:10.1021/acs.jmedchem.7b00883

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC₅₀ values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.

Full text of DOI:10.1021/acs.jmedchem.7b00883

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Article
Discovery of a Novel Series of Tankyrase
Inhibitors by a Hybridization Approach
Upendra Rao Anumala, Jo Waaler, Yves Nkizinkiko, Alexander Ignatev, Katina Lazarow, Peter
Lindemann, Petter Angell Olsen, Sudarshan Murthy, Ezeogo Obaji, Alexander G. Majouga,
Sergey V. Leonov, Jens Peter von Kries, Lari Lehtiö, Stefan Krauss, and Marc Nazaré
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00883 • Publication Date (Web): 20 Nov 2017
Downloaded from http://pubs.acs.org on November 20, 2017
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Discovery of a Novel Series of Tankyrase
Inhibitors by a Hybridization Approach
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Upendra Rao Anumala,†,∫ Jo Waaler,§ Yves Nkizinkiko,¥ Alexander Ignatev,¥ Katina
Lazarow,† Peter Lindemann†, Petter Angell Olsen,§ Sudarshan Murthy,¥ Ezeogo Obaji,¥
,
◊
Alexander G. Majouga,∇ Sergey Leonov,⊥ Jens Peter von Kries,†,‡ Lari Lehtiö,¥ Stefan
Krauss,*§ and Marc Nazaré*†,‡
†LeibnizꢀForschungsinstitut für Molekulare Pharmakologie (FMP), Campus BerlinꢀBuch,
13125 Berlin, Germany.
‡Berlin Institute of Health (BIH), AnnaꢀLouisaꢀKarschꢀStr. 2, 10178 Berlin, Germany.
§Unit for Cell Signaling, Institute of Medical Microbiology, Oslo University Hospital,
Gaustadalleen 34, 0372 Oslo, Norway and Hybrid Technology Hub ꢀ Centre of Excellence,
Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.
¥Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, PO
Box 5400, 90014 Oulu, Finland.
∇Moscow State University, Chemistry Department, Leninskie Gory 1/3, Moscow 119991,
Russia.
⊥National University of Science and Technology MISiS, Leninsky Avenue 4, Moscow
119049, Russia.
◊Moscow Institute of Physics and Technology (State University), Institutskiy Lane 9, 141700
Dolgoprudny, Russia.
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ABSTRACT
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A structureꢀguided hybridization approach using two privileged substructures gave instant
access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target
affinity on tankyrase 1 and 2 with a biochemical and cellular IC50 values of 29 nM, 6.3 nM
and 19 nM, respectively, and high selectivity towards other Poly(ADPꢀribose) polymerase
enzymes. The identified inhibitor shows a favorable inꢀvitro ADME profile as well as good
oral bioavailability in mice, rats and dogs. Critical for the approach was the utilization of an
appropriate linker between 1,2,4ꢀtriazole and benzimidazolone moieties, whereby a
cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.
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Introduction
Catalytic modification of proteins using the redox metabolite nicotinamide adenine
dinucleotide (NAD+) as a substrate to successively add ADPꢀribose moieties onto the target
protein is known as PARsylation.1 In 1960s, this posttranslational protein modification was
reported with the identification of PARP1 and its role in DNA repair.2ꢀ4 There are two classes
of PARP enzymes: mono (ADPꢀribosyl)ating and (oligoꢀ)poly(ADPꢀribosyl)ating proteins.5ꢀ6
The polyꢀADPꢀribose polymerases tankyrase 1 and tankyrase 2 (TNKS1, PARP5a and
TNKS2, PARP5b) share almost 82% sequence identity and show predominantly cytoplasmic
differential expression in a variety of tissues.7 Both tankyrase paralogs have three functional
segments: the carboxy terminal catalytic ARTD domain (ADPꢀribosyltransferase with
Diphtheria toxin homology), five ankyrin domains involved in protein/protein interactions
with target proteins such as AXIN, NuMA, TRF1, GRB and IRAP8 and a sterile alpha motif
(SAM) domain that is involved in tankyrase polymerization and supports the catalytic activity
of the PARP domain.9 The catalytic ARTD domains of both tankyrases show high similarity
with 89% sequence identity.
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Tankyrases can control in a context dependent manner several cellular pathways including,
the WNT/βꢀcatenin signaling pathway that executes key functions in embryonic
development, stem cell biology, cell fate specification, energy metabolism and cell migration.
Other roles of tankyrases comprise the involvement in e.g. mitosis, and cherubism.6, 10ꢀ14
Tankyrase inhibition may have therapeutic potential in several diseases like selected cancers
such as colorectal carcinoma and nonꢀsmall cell lung cancer as well as in fibrotic diseases,
and herpes simplex virus (HSV) infections.15ꢀ17
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Several tankyrase inhibitors emerging from high throughput screening or chemical
optimization efforts have recently been reported and inhibitor binding modes in the active site
has been effectively elucidated by Xꢀray crystallography and complemented by inꢀsilico
docking studies.13, 18ꢀ28 Two distinct binding sites, the nicotinamide and the adenosine subꢀ
pocket have been described to accommodate small molecule ligands. Compound 1
(XAV939), binding to the nicotinamide subꢀpocket, was the first reported tankyrase inhibitor
which showed an impact on WNT/βꢀcatenin signaling by stabilizing AXIN and decreasing βꢀ
catenin levels.13 Due to the conservation of the nicotinamide subꢀpocket throughout the entire
PARP family, 1, and related inhibitors 2-4 show variable selectivity towards the other
members or the PARP family.
In contrast to the nicotinamide binding site, the architecture of the adenosine binding cavity
is more varied between PARPs. Consequently, inhibitors addressing the adenosine subꢀ
pocket of TNKS1/2 appear to be intrinsically more selective over the other members of the
PARP family. Notably only tankyrase has been reported to be effectively inhibited by a
compound which is solely binding to this subꢀpocket. Recently, dual site tankyrase inhibitors
that span both, the nicotinamide and the adenosine binding site have shown to be potent and
tankyrase selective.22, ,11, 26ꢀ28, Examples of reported tankyrase inhibitors are given in Figure 1.
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We previously reported the discovery of, a 1,2,4ꢀtriazole based specific tankyrase inhibitor
5 (JW74) which culminated in the development of 6 (G007ꢀLK) which inhibits TNKS1/2 in
vitro and in vivo with high specificity.18 However, albeit being highly potent and selective,
and showing an excellent oral bioavailability in mice, 6 had poor PK in rats, hampering its
further preclinical development. Here we report the design of a new class of tankyrase
inhibitors based on a hybridization approach of privileged fragments from two distinct
inhibitor series18 6 and compound 8 described by Bregman et al..20 We show that the lead
compound of the series shows a further improved affinity and cellular activity, good
solubility, good target specificity within the PARP family, low adverse inhibition in a kinase
panel, good oral bioavailability in mouse, rat and dog as well as efficacy in mouse xenograft
models.
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Figure 1. Examples of chemical structures of known tankyrase inhibitors; A) Inhibitors
addressing the nicotinamide pocket (113, 223, 312, 421), B) Inhibitors addressing the adenosine
pocket (518, 618, 724, 820, 920, 1019), C) Dual inhibitors addressing the adenosine and
nicotinamide pocket (1126, 1222, 1327).
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Results and Discussion
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Design of the Hybrid Inhibitors.
Our previous studies of the diaryl 1,2,4ꢀtriazole series yielded potent and selective
tankyrase inhibitors.18, 29 However, the lead compound 6 suffered from an extended, highly
conjugated, aromatic system incorporating a vinylic bond in combination with the
intrinsically high lipophilicity, a negligible low Fsp3 content and a critically high molecular
weight for some derivatives. Collectively, this suggested that a further classical optimization
with incremental structural changes may not solve these issues. We speculated that due to the
resemblance of the Nꢀdisubstituted glycine pharmacophore in 8 with a diaryl 1,2,4ꢀtriazole
the distal benzimidazolone could be grafted on this scaffold with the appropriate spacer.
Thus, we resorted to a hybridization approach by joining the diaryl substituted 1,2,4ꢀtriazole
and the benzimidazolone from the two inhibitors 6 and 8 (Figure 2). The available coꢀcrystal
structures of TNKS2ꢀ6 and TNKS1ꢀ8 analogs were used to guide the hybrid and linker design
(Figure 3 aꢀc). In these crystal structures the hydrogen bonding pattern is conserved and
importantly the atoms of the triazole and glycine moieties superpose well and the vinyl and
cyclohexane linkers occupy the same space in the pocket (Figure 3c). An appropriate linker
for the hybridization approach would thus enable us to preserve the overall binding mode and
maintain distinct interaction anchor points of the merged fragments. Three different linkers
were chosen to provide the appropriate distance and conformational adaptability within this
new class of tankyrase inhibitors (Figure 2). Design was based on the parent compound
crystal structures and docking was used to check compatibility with the binding pocket. A
phenyl linker group present in 14 has been used in many tankyrase inhibitors at this position
(Figure 1, compounds 7, 9, 11), while a saturated cyclohexyl in 15 is equidistant but provides
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more flexibility to the linker. A shorter and more rigid 1,3ꢀtrans substituted cyclobutyl (16)
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was designed as a novel linker in the hybrid compound (Figure 2).
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Figure 2. Hybridization design strategy for the new tankyrase inhibitors 14-16.
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Figure 3. Coꢀcrystal structures of TNKS1/2 and inhibitors. (a) Binding mode of 6 in TNKS2
catalytic domain (PDB: 4HYF). (b) Binding mode of 8 in TNKS1 catalytic domain (PDB:
4K4E). (c) superposition of 6 and 8 coꢀcrystal structures. Only TNKS2 protein is shown for
clarity. (d) Coꢀcrystal structure of 14 with TNKS2 (PDB: 5NSP Sigma A weighted 2Fo – Fc
electron density map around the ligand is contoured at 1.0 σ. (e) Coꢀcrystal structure of 16
with TNKS2. Sigma A weighted 2Fo – Fc electron density map around the ligand is
contoured at 1.5 σ. (PDB: 5NOB). (f) Superposition of 6 and 16 coꢀcrystal structures showing
the compounds and TNKS2 protein corresponding to TNKS2ꢀ6 coꢀcrystal. The black dash
lines represent hydrogen bonds and the red spheres represent water molecules.
Synthesis of the Designed Tankyrase Inhibitors.
The phenyl derivative 14 was synthesized by coupling of pyrimidyl imidohydrazide
derivative 20 with the benzimidazolone acid 22 in a key condensation step. The pyrimidyl
imidohydrazide derivative 20 was obtained via activation of the amide as imidoyl cloride
intermediate 19 and subsequent substitution with hydrazine hydrate. This building block 20
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was then coupled with benzimidazolone acid 22 using EDCI and cyclized by dehydration in
refluxing toluene to afford the phenyl triazole 14 (Scheme 1).
Scheme 1. Synthesis of compound 14
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Reagents and conditions: (a) HATU, DIPEA, dichloromethane, rt, 14 h, 68%; (b) PCl5,
POCl3, Toluene, reflux, 7h, not isolated; (c) N2H4.H2O, THF, 3 h, 48%; (d) SnCl2, methanol,
reflux, 45 min, 92%; (e) triphosgene, dichloromethane, rt, 24 h, then reflux, 24 h, 72%; (f)
LiOH, THF:H2O (4:1), rt, overnight, 94%; (g) 20, EDCI.HCl, HOBt, triethylamine, THF, rt,
72 h, 8%; (h) Toluene, reflux, 72 h, 31%.
The cyclohexyl and cyclobutyl derivatives 15, 15a, and 16, 16a were synthesized by
analogous convergent routes. Carbimidothioate building block 26 was prepared from Nꢀ(2ꢀ
chlorophenyl)pyrimidineꢀ4ꢀcarboxamide 24 by thionation with Lawesson’s reagent to afford
25, subsequent methylation with methyl tosylate in the presence of potassium tꢀbutoxide
resulted in 26. The benzimidazolone ester intermediates (30a-d) were constructed using a
threeꢀstep sequence consisting of nucleophilic aromatic substitution, reduction and
cyclization. The nucleophilic aromatic substitution reaction of 4ꢀfluoroꢀ3ꢀnitrobenzonitrile
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(28a), as well as for 1ꢀfluoroꢀ2ꢀnitrobenzene (28b), with 27a-b respectively, was followed by
a hydrogenation under standard conditions in the presence of palladium on charcoal to afford
the corresponding diamines. Treatment with triphosgene yielded the benzimidazolone ester
derivatives 30a-d. These esters 30a-d were then converted into the hydrazide 31a-d using
hydrazine hydrate and were then condensed with the carbimidothioate 26 to afford the final
triazole derivatives 15, 15a and 16, 16a.
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Scheme 2. Synthesis of compounds 15, 15a and 16, 16a
Reagents and conditions: (a) Lawesson’s reagent, toluene, 7h, reflux, 63%; (b) tꢀBuOK,
methyl tosylate, THF, rt, 18 h, 94%; (c) DIPEA, acetonitrile, reflux, 24 h, 29a: 77%, 29b:
91%, 29c: 96%, 29d: 95%; (d) Pd/C, H2, EtOH, 2 h, a 86%, b 76%, c 72%, d 76%; (e)
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triphosgene, dichloromethane, rt 24 h, reflux 24 h, 30a: 93%, 30b: 92%, 30c: 95%, 30d:
.
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92%; (f) N2H4 H2O, methanol, 20 oC, 20 h, 31c: 64%; (g) N2H4 H2O, ethanol, 80 oC, 3 h, 31a:
o
90%, 31b: 92%, 31d: 90%; (h) 26, TFA, DMA, 120 C, 14 h, 15: 10%, 15a: 21%, 16: 15%,
16a: 16%.
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Biochemical and Cellular Activity of the Inhibitors.
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The five hybrid compounds were tested for their ability to inhibit TNKS2 in a biochemical
assay and in the human embryonic kidney HEK293 as well as the in human colon SW480
cell line as functional assays of the WNT/βꢀcatenin signaling pathway. While compound 14
with a phenyl linker displayed moderate activity in the biochemical assays (TNKS2: IC50 340
nM) only a weak activity was measured in the cellular assays. The transꢀcyclohexane linker
in 15a displayed moderate affinity versus tankyrase in the biochemical assay (TNKS2: IC50
430 nM), however, it showed moderate activity in the cellular assay (HEK293: IC50 1.06 µM;
SW480: IC50 1.8 µM). By incorporation of a nitrile moiety at the benzimidazolone in 15, a
10ꢀfold increase in cellular potency (HEK 293: IC50 0.12 µM; SW480: IC50 1.49 µM) was
observed, while the biochemical potency was only slightly improved. A trans configured
cyclobutane linker in 16a improved the cellular IC50 by an order of magnitude compared to
15a (HEK293: IC50 0.2 µM; SW480: IC50 0.41 µM). When incorporating a nitrile group in 16
a further 10ꢀfold increase of activity was observed (HEK293: IC50 19 nM; SW480: IC50 70
nM) accompanied by a favorable biochemical IC50 (TNKS2: IC50 6.3 nM), suggesting a
preserved overall binding mode with similar underlying contacts for 15 and 16 which was
subsequently confirmed by Xꢀray crystallography (Figure 3).
Table 1. Activity data in biochemical and cellular assays of 14-16
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STꢀLuc/Ren STꢀLuc/Ren
(HEK293) (SW 480)
TNKS2
Cmpd
R
L
IC50 (ꢀM)
IC50 ( M) IC50 ( M)
ꢀ
ꢀ
818 (Reference) ꢀ
ꢀ
0.025
0.34
0.05 4.2
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ꢀCN
ꢀH
>10
N.D.
1.80
14
0.43
0.33
1.06
15a
1.49a
0.41
ꢀCN
0.12
0.2
15
ꢀH
0.0098
0.0063
16a
16
ꢀCN
0.019
0.070
a partial precipitation under assay conditions.
Binding Mode of the Inhibitors 14 and 16.
We obtained the coꢀcrystal structures of 14 and 16 with the TNKS2 catalytic domain and
these confirmed our hypothesis of the binding mode of these hybrid compounds. Both
compounds were clearly visible in the electron density although the soaking approach used
for obtaining 14 coꢀcrystals was detrimental to the apo crystals and resolution of this data set
was lower (Supplementary Table S1). Electron density for the compound is clear, but the
density for the CN group is essentially missing (Figure 3d). 14 was also only found in one out
of two TNKS2 proteins present in the crystallographic asymmetric unit. In comparison to coꢀ
crystal structure of 6 (Figure 3a), the pyrimidine of 14 has rotated and forms a hydrogen bond
to a water molecule. This feature is also conserved in the binding mode of 16 (Figure 3e).
The rigid phenyl linker in 14 positions the triazole deeper in the pocket and closer to the
nicotinamide sub pocket. Despite the conserved hydrogen bond to Tyr1060 (TNKS2)
backbone amide the distorted placement on this side of the compound is likely causing the
observed lower potency (Table 1). The shorter linker found in 16 directs the triazole moiety
in a slightly different orientation than in 14 and the binding mode is more similar to the
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template compound 6 (Figure 3f). The pyrimidine is in a better position to efficiently form a
displaced πꢀπ stacking interaction with the sidechain of Tyr1060 yielding a better potency
over the other tested linker moieties.
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Profile of Tankyrase Inhibitor 16.
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To assess the biotarget specificity of 16, biochemical inhibition of a panel of human PARP
family members was tested. Potency for TNKS1 (29 nM) was slightly lower than for TNKS2
(6.3 nM) and none of the other tested PARP enzymes were inhibited by 16 (Table 2).
Table 2. Summary of activity and selectivity data as well as physicochemical and ADME
properties of the tankyrase inhibitor 16. For further details see Supporting Information.
Molecular weight
468.9
cLogP
3.39
Hydrogen bond donors
Hydrogen bond acceptors
Number of rotatable bonds
PSA
1
6
4
112.6 Å2
Aqueous solubility (pH =7.4, 25°C)
Ligand efficiency (LE)
IC50 TNKS1 (pIC50 ± SD)c
IC50 TNKS2 (pIC50 ± SD)c
IC50 (STꢀLuc/Ren in HEK293)
IC50 (STꢀLuc/Ren in SW480)
IC50 ARTD1/PARP1
31.2 µM
0.34
0.029 µM (7.54 ± 0.07)
0.0063 µM (8.20 ± 0.03)
0.019 µM
0.070 µM
> 100 µM
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IC50 ARTD2/PARP2
IC50 ARTD3/PARP3
IC50 ARTD4/PARP4
IC50 ARTD7/PARP15
IC50 ARTD8/PARP14
IC50 ARTD10/PARP10
IC50 ARTD12/PARP12
IC50 ARTD15/PARP16
> 100 µM
> 100 µM
> 100 µM
> 10 µMa
> 10 µMa
> 10 µMa
> 10 µMa
> 10 µMa
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Kinase selectivity: kinases with >50% 4/320
inhibition at 10
μM
Clearance human liver microsomes 14.8 (µL/min/mg protein)
(Clint)
Clearance human hepatocyte (Clint)
MDCKꢀMDR1 permeability
Papp at 10 µM
6.44 (µL/min/106 cells)
5.3 ×10ꢀ7 cm/s (AꢀB)
28.2×10ꢀ7 cm/s (BꢀA)
CYP (3A4/2C9/2C19/2D6/1A)
Mouse PK (p.o., 5 mg/kg)
1.3 µM/11.9 µM/>25 µM/>25 µM/>25 µM
F 47%; t1/2 1.5 h; Cmax 123.5 ng/mL; AUC(0ꢀt)
144.7 hr×ng/mL; CL 34.02 L/kg
Rat PK (p.o., 14 mg/kg)
Dog PK (p.o., 7 mg/kg)
F 35%; t1/2 N.A.; Cmax 843 ng/mL; AUC(0ꢀt)
2765 hr×ng/mL; CL N.A.
F 91%; t1/2 4.7 h; Cmax 5851 ng/mL; AUC(0ꢀt)
27134 hr×ng/mL; CL 0.14 L/kg
aNo inhibition; concentration limited by low DMSO tolerance of the enzymes. Calculated
b
properties using ChemAxon package version 16.1., 2010ꢀ2016. cSD = standard deviation.
Furthermore, a kinase selectivity profiling on 320 wildꢀtype protein kinases of 16 at 10 µM
concentration was carried out revealing only 4 kinase hits with > 50% inhibition at 10 µM
concentration (CLK2: 73%; MELK: 70%; PRKG1: 66% and TSF1: 52%), but no kinase with
a > 90% inhibition (Supplementary Table S2). Cytochrome P450 inhibition was tested with a
panel of CYP isoforms and in vitro metabolic stability was evaluated in human hepatocytes
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and human liver microsomes. Of the tested CYP isoforms, 3A4 and 2C9 were significantly
inhibited possibly due to the pyrimidine ring in 16, while CYP2C19, CYP1A and CYP2D6
were not affected. Human hepatocyte clearance (Clint) was modest at 6.44 (µL/min/106 cells)
and 14.8 (µL/min/mg protein), respectively (Table 2). MDCKꢀMDR1 permeability of 16
showed a moderate influx ratio and a high mean efflux ratio leading to a significant efflux
ratio of 53.2 indicating an active efflux and thus a low predicted bloodꢀbrainꢀbarrier
penetration (Table 2). However, an overall good oral bioavailability in mouse (F: 47%), rat
(F: 35%) and dog (F: 91%) including a surprisingly low compound excretion in urine and
feces in rat, underscored the suitability of 16 as a chemical tool for pharmacological inꢀvivo
evaluation.
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Figure 4. Antiꢀtumor activity of 16 in xenograft models. A) COLO320 colon cancer
xenograft B) isogenic p388 leukemia mice model. Reduction of tumor volume (mm3) versus
vehicle treated controls (blue) after once daily oral dosing of 16 at various depicted doses.
Statistical significance is indicated: ANOVA on Ranks/Dunn's method, P < 0.05 (*), One
Way ANOVA/HolmꢀSidak method, P < 0.001 (**) and Oneꢀtailed Pꢀvalue < 0.05 (***).
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To evaluate the antiꢀtumor effects of 16 in vivo, we established xenografts using the human
colorectal cancer cell line COLO 320DM cells in male Balb/c nude mice. The tumorꢀbearing
mice were randomized into 4 treatment groups, 2 days after inoculation: i) vehicle control
(1% starch, n = 4), ii) 15 mg/kg 16 (n = 5), iii) 30 mg/kg 16 (n = 5) and iv) 60 mg/kg 16 (n =
5). After 10 days of once daily oral drugꢀadministration, and caliperꢀbased tumor size
measurements on day 12, 17 and 21, the experiment was terminated. Compared to vehicle
control, treatment with 16 resulted in 53%, 63% and 63% statistically significant tumor size
reductions at 15 mg/kg, 30 mg/kg and 60 mg/kg once daily oral administration, respectively
(Figure 4A). In a second tumor model, we used the syngeneic leukemic p388 mouse model.
Immunocompetent BDF1 (DBA2×C57Bl6j) mice were implanted with p388 cells and
randomized into 3 treatment groups consisting of 6 mice each after 2 days: Vehicle (1%
starch, n = 6) and two treatment groups for 16: 10 mg/kg (n = 5) and 30 mg/kg (n = 5). After
10 days of once daily oral administration, the tumors sizes were measured using caliper and
the experiment was ended. Compared to vehicle control, treatment with 16 resulted in 32%
and 57% statistically significant tumor size reductions for 15 mg/kg and 30 mg/kg dosing,
respectively (Figure 4B). No animal discomforts or body weight differences were registered
throughout the experiment period. Collectively, the results show that 16 can significantly
decrease the growth of colorectal cancer and leukemia in immunodeficient and
immunocompetent models, respectively.
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CONCLUSIONS
A structure guided hybridization approach based on two known inhibitors allowed us to
design successfully a novel series of tankyrase inhibitors. The lead compound 16 shows high
selectivity towards TNKS1/2, enhanced IC50 in biochemical assays (IC50: TNKS1 29 nM,
TNKS2 6.3 nM) and in vitro cellular assays (HEK293: IC50 19 nM; SW480: IC50 70 nM). In
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addition, good pharmacokinetic properties in mice, and dogs and efficacy in a tumor
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xenograft model were achieved. The novel tankyrase inhibitor 16 expands the available small
7
8
molecules space to selectively inhibit TNKS1/2 in vitro and in vivo
.
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EXPERIMENTAL SECTION
Chemistry. All chemicals were purchased from commercial suppliers: Activate Scientific,
SigmaꢀAldrich and Alfa Aesar and used as received unless otherwise specified. NMR spectra
were recorded at either 295 K (300 MHz) or 300 K (600 MHz) at either Bruker AV 300 (300
MHz, 75 MHz) or Bruker AV 600 (600 MHz, 151 MHz) spectrometers. Chemical shifts are
reported in ppm (δ) referenced to TMS (δ = 0.00 ppm), DMSO (2.50 ppm) and CHCl3 (7.26
ppm). Melting points were recorded in open capillaries on a Büchi Bꢀ545 Melting Point
Apparatus. Temperatures are expressed in degrees Celsius (°C) and are uncorrected.
LC/MS analysis was performed on an Agilent LC/MS 1260 analytical HPLC with DAD
coupled to an Agilent 6120 single quadrupole mass spectrometer (ESIꢀSQ) equipped with a
Thermo Fisher Scientific Accucore C18 column, 2.1 x 30 mm, 2.6 μm. Method: ESI+, flux:
0.8 ml/min, 5ꢀ95% CH3CN in H2O + 0.1% FA, total runtime: 2.5 min. High resolution mass
spectra were recorded on an Agilent 6220A accurateꢀmass timeꢀofꢀflight mass spectrometer
(ESIꢀTOF) with Agilent 1200 HPLC/DAD frontꢀend. The HPLC was equipped with an
Agilent Poroshell 120, C18 column, 2.1 x 100 mm, 1.8 µm. Method: ESI+, flux: 0.6 ml/min,
5ꢀ99% CH3CN in H2O + 0.1% FA, total runtime: 4.5 min.
Purity and characterization of all final compounds was established by a combination of LCꢀ
MS, LCꢀHRMS and NMR analytical techniques. All compounds were found to be >95% pure
by LCꢀMS and LCꢀHRMS analysis.
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Preparation
of
1-(4-(4-(2-Chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3-
4
5
6
yl)phenyl)-2,3-dihydro-2-oxo-1H-benzo[d]imidazole-5-carbonitrile (14).
7
8
9
(i) N-(2-Chlorophenyl)pyrimidine-4-carboxamide (24). To a flask charged with
pyrimidineꢀ4ꢀcarboxylic acid (17) (1.4 g, 11.28 mmol) were added dichloromethane (50 ml),
N,Nꢀdiisopropylethylamine (5.89 ml, 33.84 mmol), HATU ( 4.75 g, 12.4 mmol) and 2ꢀchloro
benzenamine (18) (1.18 ml, 11.28 mmol) respectively. The resulting mixture was stirred
overnight at room temperature. Water was added to reaction mixture and the organic layer
was separated using a separating funnel. The organic layer was dried over magnesium sulfate,
filtered and concentrated in vacuo, and subjected to column chromatography on silica gel
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using
a
cyclohexane and ethyl acetate gradient as eluent to afford Nꢀ(2ꢀ
chlorophenyl)pyrimidineꢀ4ꢀcarboxamide (24) as an amorphous solid. Yield 1.79 g (68%). MS
1
(ESI) for C11H8ClN3O +H+, [M+H]+ m/z, calcd 234.0, found 234.1. H NMR (300 MHz,
CDCl3)
8.22 (d,
δ
10.57 (s, 1H), 9.37 (s, 1H), 9.05 (d, J = 5.0 Hz, 1H), 8.61 (dd, J = 8.2, 1.6 Hz, 1H),
= 5.0 Hz, 1H), 7.44 (dd, J = 8.1, 1.5 Hz, 1H), 7.35 (td, J = 7.9, 1.6 Hz, 1H), 7.19 –
J
7.06 (m, 1H). 13C NMR (75 MHz, CDCl3)
125.3, 123.6, 121.1, 118.5.
δ 160.3, 159.4, 157.8, 156.1, 133.9, 129.2, 127.7,
(ii) (Z)-N''-(2-Chlorophenyl)pyrimidine-4-carboximidhydrazide (20). To a solution of
Nꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀcarboxamide (24) (450 mg, 1.92 mmol) in toluene (20 ml)
was added phosphorous pentachloride (1.2 g, 5.77 mmol) and phosphoryl chloride (540 µl,
5.77 mmol) and the mixture stirred at reflux for 7 hours. The solvents were then evaporated
to achieve (Z)ꢀNꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀcarbimidoyl chloride (19) as intermediate
and 3 ml of hydrazine hydrate was added and stirred for 4 hours at room temperature.
Solvents were evaporated and subjected to column chromatography on silica gel using a
cyclohexane and ethyl acetate gradient as eluent to afford (Z)ꢀN''ꢀ(2ꢀ
chlorophenyl)pyrimidineꢀ4ꢀcarboximidhydrazide (20) as a yellow foam. Yield: 230 mg
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(48%) 1H NMR (300 MHz, DMSOꢀd6
) δ 9.05 – 8.98 (m, 1H), 8.66 (d, J = 5.5 Hz, 1H), 7.86
5
6
7
8
(dd, J = 5.5, 1.0 Hz, 1H), 7.44 – 7.35 (m, 1H), 7.30 (s, 2H), 7.18 – 7.04 (m, 2H), 6.87 – 6.72
(m, 1H), 6.22 (d, J = 7.9 Hz, 1H). 13C NMR (75 MHz, DMSOꢀd6
) δ 160.2, 158.5, 156.6,
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138.8, 133.2, 129.5, 127.9, 120.6, 120.5, 116.6, 116.1.
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(iii) 4-(6-Cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)benzoic acid (22). To a
solution of ethyl 4ꢀ(4ꢀcyanoꢀ2ꢀnitrophenylamino)benzoate (21) (891 mg, 2.86 mmol) in
EtOH (20 mL) was added SnCl2 (5.55 g, 29.2 mmol). The orange mixture was refluxed for 45
min. It was then poured into EtOAc. The organic layer was washed with 1N HCl (50 mL),
brine, and satd. NaHCO3, and dried over Na2SO4. After evaporation of the solvents under
reduced pressure the residue was purified by column chromatography on silica gel with 25%
EtOAc in hexanes to afford ethyl 4ꢀ(2ꢀaminoꢀ4ꢀcyanophenylamino)benzoate as a pale yellow
foam. Yield: 743 mg (92%). 1HꢀNMR (300 MHz, CDCl3)
δ ppm 7.98ꢀ7.95 (m, 2H), 8.25 (d,
J = 6.0 Hz, 1 H), 7.11ꢀ7.06 (m, 2H), 6.89ꢀ6.85 (m, 2H), 5.71 (s, 1H), 4.38ꢀ4.32 (m, 2H),
4.00ꢀ3.70 (m, 2H), 1.40ꢀ1.36 (m, 3H). To a mixture of ethyl 4ꢀ(2ꢀaminoꢀ4ꢀ
cyanophenylamino)benzoate (320 mg, 1.21 mmol) in dichloromethane (50 mL) under iceꢀ
water bath cooling triphosgene (573 mg, 1.93 mmol) was added. The mixture was stirred at 0
°C for 1 h. Then it was allowed to warm to room temperature and stirred overnight. The
resulting mixture was then refluxed overnight. It was diluted with dichloromethane, washed
with satd. NaHCO3, and brine, and evaporated to give a white solid ethyl 4ꢀ(6ꢀcyanoꢀ1,2ꢀ
1
dihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀyl)benzoate. Yield: 254 mg (72%). HꢀNMR (300 MHz,
CDCl3)
δ ppm 9.55 (s, 1H), 8.29ꢀ8.26 (m, 2H), 7.66ꢀ7.30 (m, 2H), 7.45ꢀ7.42 (m, 2H), 7.18ꢀ
7.15 (m, 1H), 4.45 (q, J = 5.4 Hz, 2H), 1.44 (t, J = 5.4 Hz, 3H). To a mixture of ethyl 4ꢀ(6ꢀ
cyanoꢀ1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀyl)benzoate (400 mg, 1.30 mmol) in THF/H2O
(4:1 v/v, 15 mL) was added LiOH monohydrate (133 mg, 3.16 mmol). The mixture was
stirred overnight at room temperature and was then acidified with 1N HCl. The resulting
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precipitate was collected by filtration to give 4ꢀ(6ꢀcyanoꢀ1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ
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5
6
3ꢀyl)benzoic acid (22) as a greasy solid. Yield: 340 mg (94%). 1HꢀNMR (300 MHz, CDCl3)
δ
7
8
9
ppm 8.09 (d, J = 6.6 Hz, 2H), 7.67 (d, J = 6.6 Hz, 2H), 7.50ꢀ7.44 (m, 2H), 7.19 (d, J = 6.0
Hz, 1H), 7.08ꢀ7.02 and 6.82ꢀ6.76 (m, 1H).
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(iv) N'-((Z)-(2-Chlorophenylimino)(pyrimidin-4-yl)methyl)-4-(6-cyano-1,2-dihydro-
2-oxobenzo[d]imidazol-3-yl)benzohydrazide (23). To a mixture of 4ꢀ(6ꢀcyanoꢀ1,2ꢀdihydroꢀ
2ꢀoxobenzo[d]imidazolꢀ3ꢀyl)benzoic acid (22) (271 mg, 0.97 mmol) in THF (35 mL) was
sequentially added (Z)ꢀN''ꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀcarboximidhydrazide (20) (331 mg,
1.34 mmol), EDCI•HCl (323 mg, 1.68 mmol), HOBT (175 mg, 1.30 mmol) and Et3N (1.5
mL, 10.8 mmol). The mixture was stirred for 3 d. It was diluted with dichloromethane,
washed with water, satd. NaHCO3, dried over Na2SO4. After filtration and evaporation of the
solvents under reduced pressure the residue was purified by column chromatography on silica
gel with 5% MeOH in dichloromethane as an eluent to afford N'ꢀ((Z)ꢀ(2ꢀ
chlorophenylimino)(pyrimidinꢀ4ꢀyl)methyl)ꢀ4ꢀ(6ꢀcyanoꢀ1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ
1
3ꢀyl)benzohydrazide (23) as a pale yellow foam. Yield: 40 mg (8%). HꢀNMR (300 MHz,
DMSOꢀd6) δ ppm 11.65 (s, 1H), 9.04 (s, 1H), 8.89ꢀ8.87 (m, 1H), 8.01ꢀ7.92 (m, 1H), 7.91ꢀ
7.82 (m, 2 H), 7.64ꢀ7.58 (m, 3H), 7.50ꢀ7.35 (m, 4H), 7.18ꢀ7.02 (m, 2H), 7.00ꢀ6.92 (m, 1H).
6.75ꢀ6.68 (m, 1H).
(v) 1-(4-(4-(2-Chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3-yl)phenyl)-2,3-
dihydro-2-oxo-1H-benzo[d]imidazole-5-carbonitrile (14). A mixture of N'ꢀ((Z)ꢀ(2ꢀ
chlorophenylimino)(pyrimidinꢀ4ꢀyl)methyl)ꢀ4ꢀ(6ꢀcyanoꢀ1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ
3ꢀyl)benzohydrazide (23) (20 mg, 0.039 mmol) in toluene (10 mL) was refluxed for 3 d. The
mixture was evaporated and subjected to column chromatography on silica gel with 2%
MeOH in dichloromethane to give the crude product, which was washed with Et2O to afford
the
title
compound
1ꢀ(4ꢀ(4ꢀ(2ꢀchlorophenyl)ꢀ5ꢀ(pyrimidinꢀ4ꢀyl)ꢀ4Hꢀ1,2,4ꢀtriazolꢀ3ꢀ
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yl)phenyl)ꢀ2,3ꢀdihydroꢀ2ꢀoxoꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarbonitrile (14) as a pale yellow
4
5
6
7
solid. Yield: 6 mg (31%); mp 182 °C. MS for C26H15ClN8O ꢀH+, [MꢀH]ꢀ m/z, calcd 489.1,
1
found 489.3. H NMR (300 Hz, CDCl3+CD3OD)
δ 8.82ꢀ8.00 (m, 2H), 8.28ꢀ8.26 (m, 1H),
8
9
7.66ꢀ7.60 (m, 2H), 7.54ꢀ7.44 (m, 4H), 7.40ꢀ7.32 (m, 4H), 7.08ꢀ7.06 (m, 1H).
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Preparation of 1-((trans)-4-(4-(2-Chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3-
yl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-one (15a).
(i) N-(2-Chlorophenyl)pyrimidine-4-carbothioamide (25). To a solution of Nꢀ(2ꢀ
chlorophenyl)pyrimidineꢀ4ꢀcarboxamide (24) (2.0 g, 8.55 mmol) in toluene (20 ml)
Lawesson’s reagent (2.42 g, 5.99 mmol) was added and the mixture was refluxed for 7 hours.
After the reaction mixture was cooled to room temperature, the solvents were evaporated.
The crude product was purified by chromatography on silica gel eluting with a gradient of
cyclohexane /ethyl acetate. The fractions containing the product were combined and the
solvent evaporated under reduced pressure to yield Nꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀ
carbothioamide (25) as an amorphous solid. Yield: 1.34 g (63%). HRMS for C11H8ClN3S
1
+H+, [M+H]+ m/z, calcd 250.0200, found 250.0206. H NMR (300 MHz, CDCl3)
δ 12.39 (s,
1H), 9.32 (s, 1H), 9.09 (d, J = 8.2 Hz, 1H), 9.02 (d, J = 5.2 Hz, 1H), 8.65 (d, J = 5.2 Hz, 1H),
13
7.54 (dd, J = 8.0, 1.5 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.27 (td, J = 7.7, 1.5 Hz, 1H). C
NMR (75 MHz, CDCl3)
123.4, 120.3.
δ 185.5, 158.6, 157.0, 156.5, 134.9, 129.7, 127.5, 127.1, 126.7,
(ii) Methyl(Z)-N-(2-chlorophenyl)pyrimidine-4-carbimidothioate (26). To a flask
charged with Nꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀcarbothioamide (25) (249.7 mg, 1.0 mmol) in
tetrahydrofuran (6 ml) was added potassium tertꢀbutoxide (112.2 mg, 1.0 mmol) and stirred
for 15 min. Methyl tosylate (151 µl, 1.0 mmol) was then added dropwise and the reaction
mixture was stirred at room temperature for further 18 hours. The reaction mixture was then
partitioned between water and ethyl acetate, the layers were separated and the organic layer
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was washed twice with water and brine, dried over magnesium sulfate, filtered and
evaporated under reduced pressure to provide crude product. The crude greasy product
methyl(Z)ꢀNꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀcarbimidothioate (26) was directly used in the
next reaction step without any purification. Yield: 248 mg (94%). HRMS for C12H10ClN3S
+H+, [M+H]+ m/z, calcd 264.0357, found 264.0369.
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(iii) (trans)-Methyl-4-(2-nitrophenylamino)cyclohexane carboxylate (29b). To a
solution of (trans)ꢀmethylꢀ4ꢀaminocyclohexane (27a) carboxylate hydrochloride (1 g, 5.16
mmol) and 1ꢀfluoroꢀ2ꢀnitrobenzene (28b) (544 µl, 5.16 mmol) in acetonitrile (50 ml)
potassium carbonate (1.06 g, 77.4 mmol) was added at ambient temperature. The resulting
reaction mixture was heated for 8 hours at reflux. After cooling to room temperature, the
acetonitrile was removed under reduced pressure and the crude solid was dissolved in
dichloromethane. The dichloromethane layer was washed with water, dried over magnesium
sulfate, filtered and then concentrated under reduced pressure. The crude solid product was
washed with cold methanol to yield (trans)ꢀmethylꢀ4ꢀ(2ꢀnitrophenylamino)ꢀcyclohexane
carboxylate (29b) as a yellow solid which was used in the next reaction step without any
further purification. Yield: 1.3 g (91%); mp 81 ꢀ 83 °C. HRMS for C14H18N2O4 +H+, [M+H]+
m/z, calcd 279.1339, found 279.1350. 1H NMR (600 MHz, DMSOꢀd6
7.89 (d, = 8.0 Hz, 1H), 7.51 (q, = 9.4, 8.8 Hz, 1H), 7.14 (t, = 9.2 Hz, 1H), 6.67 (q,
) δ 8.08 – 8.00 (m, 1H),
J
J
J
J
J
=
=
10.4, 9.1 Hz, 1H), 3.67 – 3.53 (m, 4H), 2.40 – 2.31 (m, 1H), 2.09 – 2.03 (m, 2H), 1.95 (d,
13
14.4 Hz, 2H), 1.61 – 1.51 (m, 2H), 1.38 (q,
DMSOꢀd6
(iv) (trans)-Methyl-4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)cyclohexane
carboxylate (30b). reaction flask with mixture of (trans)ꢀmethylꢀ4ꢀ(2ꢀ
J
= 15.4, 13.0 Hz, 2H). C NMR (151 MHz,
)
δ 175.1, 144.3, 136.6, 130.8, 126.3, 115.3, 114.9, 51.3, 49.8, 41.3, 31.0, 27.2.
A
a
nitrophenylamino)ꢀcyclohexane carboxylate (29b) (1.85 g, 9.55 mmol) and a catalytic
amount of 10% palladium on charcoal in ethanol (400 ml) was equipped with a hydrogen
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balloon and hydrogen gas was bubbled into reaction mixture at atmospheric pressure. After 2
hours, the balloon was removed and the reaction mixture purged with nitrogen, filtered
through a pad of celite and washed twice with ethanol. The filtrate was concentrated under
reduced pressure to afford (trans)ꢀmethylꢀ4ꢀ(2ꢀaminophenylamino)cyclohexane carboxylate
as a yellow greasy solid. Yield: 1.25 g (76%). HRMS for C14H20N2O2 +H+, [M+H]+ m/z,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
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45
46
47
48
49
50
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60
calcd 249.1598, found 249.1599. 1H NMR (600 MHz, DMSOꢀd6
)
δ
6.58 (d,
J
J
= 7.7 Hz, 1H),
= 4.4 Hz, 3H),
6.51 (t,
3.17 (q,
J
= 4.1 Hz, 2H), 6.42 (td,
= 7.8, 4.7 Hz, 1H), 2.31 (tt,
J = 7.9, 3.8 Hz, 1H), 4.88 (s, 3H), 3.60 (t,
J
J
= 15.4, 4.8 Hz, 1H), 2.07 – 2.00 (m, 2H), 1.97 – 1.91
(m, 2H), 1.47 (qd,
MHz, DMSOꢀd6 δ 175.3, 134.7, 134.3, 118.1, 117.2, 115.1, 111.5, 51.3, 50.8, 42.0, 31.5,
J = 13.3, 3.9 Hz, 2H), 1.21 (dq, J
= 17.8, 7.2, 4.6 Hz, 2H). 13C NMR (151
)
27.6. To a mixture of (trans)ꢀmethylꢀ4ꢀ(2ꢀaminophenylamino)cyclohexane carboxylate (1.00
g, 4.02 mmol) in dichloromethane (70 ml) at 0°C (ice bath) was added triphosgene (1.79 g,
6.03 mmol). The mixture was stirred at 0oC for 1 h. Then it was allowed to warm to room
temperature and stirred for 24 hours. Next the resulting mixture was refluxed for 24 hours.
The reaction mixture was diluted with dichloromethane and washed with saturated sodium
bicarbonate, and brine, dried over magnesium sulfate filtered and evaporated to give (trans)ꢀ
methylꢀ4ꢀ(1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀyl)cyclohexane carboxylate (30b) as an
amorphous solid. Yield: 950 mg (86%). HRMS for C15H18N2O3 +H+, [M+H]+ m/z, calcd
275.1390, found 275.1403. 1H NMR (600 MHz, DMSOꢀd6
)
δ
10.80 (d,
= 5.6 Hz, 3H), 4.19 – 4.11 (m, 1H), 3.64 – 3.58 (m, 3H), 2.21
= 13.2 Hz, 2H), 1.72 (d, = 12.5 Hz, 2H), 1.53 (q, = 12.9
175.1, 153.7, 129.2, 128.3, 120.4, 120.2, 108.9,
J = 5.1 Hz, 1H), 7.30
(t,
J
= 6.6 Hz, 1H), 6.95 (d,
J
(q,
J
= 12.8 Hz, 2H), 2.04 (d,
J
J
J
Hz, 2H). 13C NMR (151 MHz, DMSOꢀd6
) δ
108.7, 51.4, 50.7, 41.0, 28.1.
(v) (trans)-4-(1,2-Dihydro-2-oxobenzo[d]imidazol-3-yl)cyclohexanecarbohydrazide
(31b). To
a
mixture of (trans)ꢀmethylꢀ4ꢀ(1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀ
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yl)cyclohexane carboxylate (30b) (500 mg, 1.82 mmol) in ethanol was added hydrazine
hydrate and heated at 120oC for 3 hours under microwave irradiation in a sealed vial (Biotage
initiator+). Upon completion of reaction, the solvent was removed under reduced pressure and
the crude solid was washed with a mixture of dichloromethane and methanol to yield (trans)ꢀ
4ꢀ(1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀyl)cyclohexanecarbohydrazide (31b) as a solid
powder which was used in the next reaction step without further purification. Yield: 460 mg
(92%), mp 273 °C (decomposition). HRMS for C14H18N4O2 +H+, [M+H]+ m/z, calcd
275.1503, found 275.1511.
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11
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15
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(vi) 1-((trans)-4-(4-(2-Chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3-
yl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-one (15a). The (trans)ꢀ4ꢀ(1,2ꢀdihydroꢀ2ꢀ
oxobenzo[d]imidazolꢀ3ꢀyl)cyclohexanecarbohydrazide (31b) (200 mg, 0.729 mmol) was
added to a solution of methyl(Z)ꢀNꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀcarbimidothioate (26) (211
mg, 0.801 mmol) in N,Nꢀdimethylacetamide (1 ml). Trifluoroacetic acid (27.8 µl) was added
and the reaction mixture was heated at 120oC for 14 hours. After cooling down to room
temperature, the reaction mixture was filtered and the residue washed with dichloromethane
and methanol. The filtrate was concentrated under reduced pressure to remove the methanol
and dichloromethane. A mixture of water and dichloromethane was added and the reaction
mixture portioned using a separating funnel. The organic layer was washed with water, brine,
dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude
product was purified by chromatography on silica gel eluting with a gradient of
dichloromethane / methanol. The fractions containing the product were combined and the
solvent evaporated under reduced pressure to yield the title compound 15a as a white solid.
Yield: 72 mg (21%); mp 274 ꢀ 276 °C. HRMS for C25H22ClN7O +H+, [M+H]+ m/z, calcd
1
472.1647, found 472.1654. H NMR (600 MHz, DMSOꢀd6
)
δ
10.87 (s, 1H), 9.00 (d,
J = 5.3
Hz, 1H), 8.93 (d,
J
= 1.4 Hz, 1H), 8.27 (dd,
J
= 5.3, 1.4 Hz, 1H), 7.86 (dd, = 7.8, 1.7 Hz,
J
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1H), 7.81 (dd,
1H), 7.35 (td,
J
= 8.0, 1.4 Hz, 1H), 7.72 (td,
J
= 7.8, 1.7 Hz, 1H), 7.67 (td, = 7.6, 1.5 Hz,
J
J
= 4.7, 4.1, 2.2 Hz, 1H), 7.01 (d,
J
= 2.7 Hz, 3H), 4.29 (tt, J = 12.2, 3.9 Hz,
6
7
8
1H), 2.62 (tdd,
J
= 11.9, 7.3, 3.7 Hz, 1H), 2.19 (dtd,
J = 30.7, 14.8, 13.9, 3.5 Hz, 3H), 2.02 –
9
1.90 (m, 3H), 1.81 (ddd,
J
= 15.6, 8.8, 2.9 Hz, 2H). 13C NMR (151 MHz, DMSOꢀd6
) δ 160.1,
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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28
29
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59
60
158.3, 158.0, 153.7, 153.2, 150.0, 132.5, 131.6, 131.0, 130.1, 129.8, 129.4, 128.5, 128.2,
120.4, 120.2, 118.9, 108.7, 108.6, 50.8, 32.7, 30.6, 30.1, 28.5.
Preparation of 1-((trans)-4-(-4-(2-Chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-
3-yl)cyclohexyl)-2,3-dihydro-2-oxo-1H-benzo[d]imidazole-5-carbonitrile (15)
(i) (trans)-Methyl-4-(4-cyano-2-nitrophenylamino)cyclohexane carboxylate (29a). To
a solution of methylꢀ4ꢀaminocyclohexane carboxylate hydrochloride (27a) (1.5 g, 7.74
mmol) and 4ꢀfluoroꢀ3ꢀnitrobenzonitrile (28a) (1.28 g, 7.74 mmol) in acetonitrile (50 ml) was
added N,Nꢀdiisopropylethylamine (2.69 ml, 15.49 mmol) at room temperature. The resulting
reaction mixture was stirred for 18 hours at room temperature. Acetonitrile was removed
under reduced pressure and the crude solid was dissolved in dichloromethane and the
dichloromethane layer was washed with water, dried over magnesium sulfate, filtered and
then concentrated under reduced pressure. The crude solid (trans)ꢀmethylꢀ4ꢀ(4ꢀcyanoꢀ2ꢀ
nitrophenylamino)cyclohexane carboxylate (29a) was washed with cold methanol and used in
the next step without any further purification. Yield 1.8 g (77%); mp 138 ꢀ 140 °C. HRMS
1
for C15H17N3O4 +H+, [M+H]+ m/z, calcd = 304.1292, found 304.1307. H NMR (600 MHz,
DMSOꢀd6
7.30 (d,
)
δ
8.49 (d,
J
= 2.1 Hz, 1H), 8.20 (d,
J
= 7.9 Hz, 1H), 7.81 (dd,
J
= 9.1, 2.1 Hz, 1H),
= 11.9,
J = 13.0, 3.1 Hz, 2H), 1.49 –
J
= 9.2 Hz, 1H), 3.72 (tdt,
J
= 11.3, 7.8, 3.9 Hz, 1H), 3.61 (s, 3H), 2.35 (tt, J
3.6 Hz, 1H), 2.05 – 1.99 (m, 2H), 1.98 – 1.92 (m, 2H), 1.56 (qd,
13
1.41 (m, 2H). C NMR (151 MHz, DMSOꢀ d6
)
δ
175.0, 146.0, 137.7, 132.0, 130.6, 118.2,
116.1, 96.5, 51.4, 50.3, 41.2, 30.6, 27.1.
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(ii) (trans)-Methyl-4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)cyclohexane
carboxylate (30a). A reaction flask with a mixture of (trans)ꢀmethylꢀ4ꢀ(4ꢀcyanoꢀ2ꢀ
nitrophenylamino) cyclohexane carboxylate (29a) (2.38 g, 7.85 mmol), catalytic amount of
10% palladium on charcoal in ethanol (450 ml) was equipped with a hydrogen balloon and
hydrogen gas bubbled through the reaction mixture at atmospheric pressure. After 2 hours,
the balloon was removed and the reaction mixture purged with nitrogen, filtered through a
pad of celite and washed twice with ethanol. Then the filtrate was concentrated under reduced
pressure to afford (trans)ꢀmethylꢀ4ꢀ(2ꢀaminoꢀ4ꢀcyanophenylamino)cyclohexane carboxylate
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13
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58
59
60
as an amorphous solid. Yield: 1.84 g (86%). HRMS for C15H19N3O2 +H+, [M+H]+ m/z, calcd
1
274.1550, found 274.1553. H NMR (600 MHz, DMSOꢀd6
)
δ
6.89 (d,
J = 8.8 Hz, 1H), 6.75
(d,
= 12.0 Hz, 3H), 2.32 (q,
2H), 1.49 (dt,
DMSOꢀd6
27.5. To
J
= 9.7 Hz, 1H), 6.51 (t,
J
= 9.8 Hz, 1H), 5.09 (d,
J
= 8.5 Hz, 1H), 4.97 (s, 2H), 3.59 (q, J
J
= 11.2, 10.0 Hz, 1H), 2.04 – 1.97 (m, 2H), 1.93 (t, J = 11.3 Hz,
13
J
= 22.1, 12.1 Hz, 2H), 1.23 (dt,
J
= 22.3, 12.1 Hz, 2H). C NMR (151 MHz,
)
δ
175.2, 138.8, 135.0, 122.7, 121.1, 115.1, 108.9, 96.1, 51.3, 50.0, 41.8, 31.3,
a
mixture of (trans)ꢀmethylꢀ4ꢀ(2ꢀaminoꢀ4ꢀcyanophenylamino)cyclohexane
carboxylate (1.87 g, 6.84 mmol) in dichloromethane (65 ml) was added triphosgene (3.06 g,
10.31 mmol) at 0°C (ice water bath). The mixture was stirred at 0 °C for 1 h. Then it was
allowed to warm to room temperature and stirred for 24 hours. Next the resulting mixture was
heated to reflux for 24 hours. The reaction mixture was diluted with dichloromethane and
washed with saturated sodium bicarbonate, and brine, dried over magnesium sulfate and
evaporated to give (trans)ꢀmethylꢀ4ꢀ(6ꢀcyanoꢀ1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀ
yl)cyclohexane carboxylate (30a) as a glassy solid. Yield: 1.90 g (93%). HRMS for
1
C16H17N3O3 +H+, [M+H]+ m/z, calcd 300.1343, found 300.1356. H NMR (600 MHz,
DMSOꢀd6
) δ 11.30 (s, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.44 (dd, J = 8.3, 1.6 Hz, 1H), 7.35 (d, J
= 1.6 Hz, 1H), 4.20 (tt,
J
= 12.4, 4.0 Hz, 1H), 3.62 (s, 3H), 2.20 (qd, J = 13.0, 3.7 Hz, 2H),
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2.03 (ddd,
J
= 11.9, 5.0, 2.8 Hz, 2H), 1.74 (dt,
J
= 13.4, 3.7 Hz, 2H), 1.53 (qd,
J = 13.2, 3.5
4
5
6
13
Hz, 2H). C NMR (151 MHz, DMSOꢀd6) δ 175.1, 153.6, 133.0, 128.6, 125.5, 119.7, 111.5,
7
8
109.5, 102.3, 51.4, 51.2, 40.9, 28.0, 27.9.
9
(iii) (trans)-4-(6-Cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-
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13
14
15
16
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yl)cyclohexanecarbohydrazide (31a). To a mixture of (trans)ꢀmethylꢀ4ꢀ(6ꢀcyanoꢀ1,2ꢀ
dihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀyl)cyclohexane carboxylate (30a) (299.3 mg, 1.00 mmol)
in ethanol (7 ml) was added hydrazine hydrate (7 ml) and heated to 80 °C for 3 hours under
microwave irradiation in a sealed vial (Biotage initiator+). Upon completion of reaction, the
reaction mixture was filtered and the crude solid was washed with methanol, filtered and
dried
to
afford
(trans)ꢀ3ꢀ(1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀ
yl)cyclobutanecarbohydrazide (31a) as a white solid. Yield: 269 mg (90%); mp 348 °C
(decomposition). HRMS for C15H17N5O2 +H+, [M+H]+ m/z, calcd 300.1455, found 300.1466.
(iv) 1-((trans)-4-(-4-(2-Chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3-
yl)cyclohexyl)-2,3-dihydro-2-oxo-1H-benzo[d]imidazole-5-carbonitrile (15). (trans)ꢀ4ꢀ(6ꢀ
Cyanoꢀ1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀyl)cyclohexanecarbohydrazide (31a) (142 mg,
0.47 mmol) was added to a solution of methyl(Z)ꢀNꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀ
carbimidothioate (26) (138 mg, 0.52 mmol) in N,Nꢀdimethylacetamide (2 ml). Trifluoroacetic
acid (18.2 µl, 0.23 mmol) was added and the reaction mixture was heated at 120 °C for 14
hours. The reaction mixture was then cooled down to room temperature and filtered. Water
was added to the filtrate and the reaction mixture was extracted with dichloromethane. The
combined organic layers were washed with water, brine, dried over magnesium sulfate,
filtered and concentrated under reduced pressure. The crude solid was then washed with an
acetonitrile/water mixture (1:1, 3 ml) followed by cold methanol to afford compound 1ꢀ
(
(trans)ꢀ4ꢀ(4ꢀ(2ꢀChlorophenyl)ꢀ5ꢀ(pyrimidinꢀ4ꢀyl)ꢀ4Hꢀ1,2,4ꢀtriazolꢀ3ꢀyl)cyclohexyl)ꢀ2,3ꢀ
dihydroꢀ2ꢀoxoꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarbonitrile (15) as a light yellow solid. Yield: 24
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mg (10%); mp 267 °C. HRMS for C26H21ClN8O, +H+, [M+H]+ m/z, calcd = 497.1600, found
4
5
6
7
8
9
497.1609. 1H NMR (600 MHz, DMSOꢀd6
= 1.4 Hz, 1H), 8.34 (dd, = 5.3, 1.4 Hz, 1H), 7.93 (dd,
8.1, 1.4 Hz, 1H), 7.80 (td, = 7.8, 1.7 Hz, 1H), 7.75 (td,
Hz, 1H), 7.58 (dd, = 8.2, 1.6 Hz, 1H), 7.48 (d, = 1.5 Hz, 1H), 4.43 (tt,
)
δ
11.44 (s, 1H), 9.08 (d,
= 7.8, 1.7 Hz, 1H), 7.89 (dd,
= 7.7, 1.5 Hz, 1H), 7.67 (d, = 8.3
J = 5.3 Hz, 1H), 9.01 (d,
J
J
J
J =
J
J
J
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13
14
15
16
17
18
19
20
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22
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26
27
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J
J
J
= 12.3, 3.9 Hz,
13
1H), 2.73 – 2.66 (m, 1H), 2.31 – 2.19 (m, 3H), 2.10 – 1.98 (m, 3H), 1.95 – 1.85 (m, 2H). C
NMR (151 MHz, DMSOꢀd6) δ 160.0, 158.3, 158.0, 153.6, 153.2, 150.0, 133.2, 132.5, 131.6,
131.0, 130.1, 129. 8, 128.5, 128.4, 125.5, 119.7, 118.9, 111.4, 109.4, 102.3, 51.3, 32.6, 30.5,
30.0, 28.2.
Preparation of 1-((trans)-3-(-4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3-
yl)cyclobutyl)-1H-benzo[d]imidazol-2(3H)-one (16a).
(i) (trans)-Methyl-3-(2-nitrophenylamino)cyclobutanecarboxylate (29d). To
a
solution of (trans)ꢀmethylꢀ3ꢀaminocyclobutanecarboxylate hydrochloride (27b) (1.0 g, 6.05
mmol) and 1ꢀfluoroꢀ2ꢀnitrobenzene (28b) (1.09 g, 7.74 mmol) in acetonitrile (50 ml) was
added potassium carbonate (1.60 g, 11.6 mmol) at ambient temperature. The resulting
reaction mixture was heated for 16 hours at reflux. Acetonitrile was removed under reduced
pressure and the crude solid was dissolved in dichloromethane and the dichloromethane layer
was washed with water, dried over magnesium sulfate and then concentrated under reduced
pressure. Unreacted 1ꢀfluoroꢀ2ꢀnitrobenzene was removed by coꢀdistillation with toluene.
The crude product was purified by chromatography on silica gel eluting with a gradient of
cyclohexane /ethyl acetate. The fractions containing the product were combined and the
solvent evaporated under reduced pressure to yield 1.43g of (trans)ꢀmethylꢀ(2ꢀ
nitrophenylamino)cyclobutanecarboxylate (29d) as a greasy solid. Yield: 1.43 g (95%).
HRMS for C12H14N2O4 +H+, [M+H]+ m/z, calcd 251.1026, found 251.1044. H NMR (300
1
MHz, DMSOꢀd6) δ 8.10 – 8.04 (m, 1H), 8.01 (d, J = 5.8 Hz, 1H), 7.53 (ddd, J = 8.7, 6.9, 1.9
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Hz, 1H), 6.81 (d,
J
= 8.7 Hz, 1H), 6.79 – 6.67 (m, 1H), 4.24 (h,
J
= 7.1 Hz, 1H), 3.66 (s, 3H),
= 12.9, 6.9, 2.5 Hz, 2H). 13C
NMR (75 MHz, DMSOꢀd6) δ 175.3, 143.7, 136.7, 131.4, 126.2, 115.9, 114.9, 51.7, 45.5,
3.18 (tt,
J
= 9.6, 4.4 Hz, 1H), 2.75 – 2.60 (m, 2H), 2.34 (dtd, J
9
32.3, 32.2.
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(ii) (trans)-Methyl-3-(1,2-dihydro-2-oxobenzo[d]imidazol-3-
yl)cyclobutanecarboxylate (30d). A reaction flask with a mixture of (trans)ꢀmethylꢀ3ꢀ(2ꢀ
nitrophenylamino)cyclobutane carboxylate (29d) (1.4 g, 5.59 mmol), a catalytic amount of
10% palladium on charcoal in ethanol was equipped with a hydrogen balloon and hydrogen
gas was bubbled into reaction mixture at atmospheric pressure. After 2 hours, the balloon was
removed and the reaction mixture purged with nitrogen, filtered through a pad of celite and
washed twice with ethanol. The filtrate was concentrated under reduced pressure to afford
(trans)ꢀmethylꢀ3ꢀ(2ꢀaminophenylamino)cyclobutanecarboxylate as a solid. The crude product
was used in next step without any further purification. Yield: 936 mg. (76%). HRMS for
C12H16N2O2 +H+, [M+H]+ m/z, calcd 221.1285, found 221.1286. H NMR (600 MHz,
1
DMSOꢀd6
)
δ
6.53 (d,
J
= 6.8 Hz, 1H), 6.44 (dt,
J
= 19.4, 7.2 Hz, 2H), 6.21 (d,
J
= 7.5 Hz,
1H), 4.71 (d,
J
= 6.5 Hz, 1H), 4.50 (s, 2H), 3.93 (q,
J
= 6.9 Hz, 1H), 3.65 (s, 3H), 3.14 (dt, J =
9.8, 5.1 Hz, 1H), 2.56 (ddd,
J
= 12.5, 7.4, 4.3 Hz, 2H), 2.15 (ddd, J = 12.6, 9.5, 6.0 Hz, 2H).
13C NMR (151 MHz, DMSOꢀd6
)
δ 175.7, 135.5, 134.3, 117.4, 114.0, 110.5, 51.5, 46.3, 32.6,
32.5. To a mixture of (trans)ꢀmethylꢀ3ꢀ(2ꢀaminophenylamino)cyclobutanecarboxylate (1.12
g, 5.08 mmol) in dichloromethane was added triphosgene (2.26 g, 7.63 mmol) at 0°C (ice
o
water bath). The mixture was stirred at 0 C for 1 h. Then it was allowed to warm to room
temperature and stirred for 24 hours. Next the resulting mixture was refluxed for 24 hours.
The reaction mixture was diluted with dichloromethane and washed with saturated sodium
bicarbonate, and brine, dried over magnesium sulfate and evaporated under reduced pressure
to give (trans)ꢀmethylꢀ3ꢀ(1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀyl)cyclobutanecarboxylate
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Page 29 of 41
Journal of Medicinal Chemistry
1
2
3
(30d) as a white foam. Yield: 1.15 g (92%). HRMS for C13H14N2O3 +H+, [M+H]+ m/z, calcd
4
5
6
7
247.1077, found 247.1076. 1H NMR (300 MHz, CDCl3)
5.36 – 5.03 (m, 1H), 3.80 (s, 3H), 3.33 (q,
δ
10.60 (s, 1H), 7.22 – 7.04 (m, 4H),
13
J
= 7.5 Hz, 3H), 2.82 – 2.65 (m, 2H). C NMR
8
9
(75 MHz, CDCl3)
30.3.
δ 176.0, 155.5, 129.3, 128.0, 121.4, 121.0, 109.9, 108.3, 52.0, 45.3, 32.3,
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(iii) (trans)-3-(1,2-Dihydro-2-oxobenzo[d]imidazol-3-yl)cyclobutanecarbohydrazide
(31d). To mixture of (trans)ꢀmethylꢀ3ꢀ(1,2ꢀdihydroꢀ2ꢀoxobenzo[d]imidazolꢀ3ꢀ
a
yl)cyclobutanecarboxylate (30d) (400 mg, 1.62 mmol) in ethanol (7 ml) was added hydrazine
o
hydrate (7 ml) and heated to 100 C for 3 hours under microwave irradiation in a sealed vial
(Biotage initiator+). Upon completion of reaction, the solvent was removed under reduced
pressure and the crude solid was washed with methanol resulted in (trans)ꢀ3ꢀ(1,2ꢀdihydroꢀ2ꢀ
oxobenzo[d]imidazolꢀ3ꢀyl)cyclobutanecarbohydrazide (31d) as a solid product which was
used in the next step without any further purification. Yield: 360 mg. (90%); mp > 250 °C
(decomposition). HRMS for C12H14N4O2 +H+, [M+H]+ m/z, calcd 247.1190, found 247.1202.
(iv) 1-((trans)-3-(-4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3-
yl)cyclobutyl)-1H-benzo[d]imidazol-2(3H)-one
(16a).
(trans)ꢀ3ꢀ(1,2ꢀDihydroꢀ2ꢀ
oxobenzo[d]imidazolꢀ3ꢀyl)cyclobutanecarbohydrazide (31d) (123 mg, 0.5 mmol) was added
to a solution of methyl(Z)ꢀNꢀ(2ꢀchlorophenyl)pyrimidineꢀ4ꢀcarbimidothioate (26) (158 mg,
0.6 mmol) in N,Nꢀdimethylacetamide (1 ml). Trifluoroacetic acid (19 µl, 0.25 mmol) was
added and the reaction mixture was heated to 120oC for 14 hours. Water was added and the
reaction mixture was extracted with dichloromethane. The organic layer was washed with
water, brine, dried over magnesium sulfate, filtered and concentrated. The crude solid was
purified by preparative HPLC (C18 reverse phase column, elution with a water/MeCN
gradient with 0.1% TFA). The fractions containing the product were evaporated and
lyophilized to yield 1ꢀ((trans)ꢀ3ꢀ(4ꢀ(2ꢀchlorophenyl)ꢀ5ꢀ(pyrimidinꢀ4ꢀyl)ꢀ4Hꢀ1,2,4ꢀtriazolꢀ3ꢀ
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