Optimization of Tetrahydroindazoles as Inhibitors of Human Dihydroorotate Dehydrogenase and Evaluation of Their Activity and in Vitro Metabolic Stability

Article abstract of DOI:10.1021/acs.jmedchem.9b01658

Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.

Full text of DOI:10.1021/acs.jmedchem.9b01658

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Article
Optimization of tetrahydroindazoles as inhibitors of
human dihydroorotate dehydrogenase and evaluation
of their activity and in vitro metabolic stability
Gergana Popova, Marcus James Graeme Watson Ladds, Lars Johansson, Aljona Saleh, Johanna
Larsson, Lars Sandberg, Sara Häggblad Sahlberg, Weixing Qian, Hjalmar Gullberg, Neeraj
Garg, Anna-Lena Gustavsson, Martin Haraldsson, David P. Lane, Ulrika Yngve, and Sonia Lain
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01658 • Publication Date (Web): 26 Mar 2020
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Optimization of tetrahydroindazoles as inhibitors of human dihydroorotate
dehydrogenase and evaluation of their activity and in vitro metabolic stability
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Gergana Popova, Marcus J. G. W. Ladds,
Lars Johansson, Aljona Saleh, Johanna
‡
‡, ∫
£
&
Larsson, Lars Sandberg,
Sara Häggblad Sahlberg, Weixing Qian, Hjalmar
£
‡
§
§
†
Gullberg, Neeraj Garg, Anna-Lena Gustavsson, Martin Haraldsson, David Lane,
‡
†,#
Ulrika Yngve, Sonia Lain
†Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet,
Solnavägen 9, SE-171 65 Solna, Stockholm, Sweden.
#SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska
Institutet, Tomtebodavägen 23, SE-171 21 Solna, Stockholm, Sweden.
§Chemical Biology Consortium Sweden, SciLifeLab, Department of Medical
Biochemistry and Biophysics, Karolinska Institutet, SE-171 21 Stockholm, Sweden.
‖
SciLifeLab, Drug Discovery and Development Platform, ADME of Therapeutics
Facility, Department of Pharmacy, Uppsala University, Box 580, SE-751 23 Uppsala,
Sweden.
‡SciLifeLab, Drug Discovery and Development Platform, Department of Medicinal
Chemistry, Uppsala University, Box 574, SE-751 23, Uppsala, Sweden
∫
SciLifeLab, Drug Discovery and Development Platform, Department of Organic
Chemistry, Stockholm University, Box 1030, SE-171 21 Solna, Sweden.
£SciLifeLab, Drug Discovery and Development Platform, Department of Biochemistry
and Biophysics, Stockholm University, SE-171 21 Solna, Stockholm, Sweden
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&Chemical Biology Consortium Sweden, Laboratories for Chemical Biology Umeå,
Umeå University, SE-901 87 Umeå, Sweden.
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*corresponding author
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Abstract
Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo
pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and
multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we
describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH
inhibitors. Several of the HZ analogues synthesized in this study are highly potent
inhibitors of DHODH in an enzymatic assay, whilst also inhibiting cancer cell growth
and viability, and activating p53-dependent transcription factor activity in a reporter
cell assay. Furthermore, we demonstrate the specificity of the compounds towards the
de novo pyrimidine synthesis pathway through supplementation with an excess of
uridine. We also show that induction of the DNA damage marker -H2AX after
DHODH inhibition is preventable by co-treatment with the pan-caspase inhibitor Z-
VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed
compound 51 as a favorable candidate for preclinical efficacy studies.
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Introduction
Cellular pyrimidine ribonucleotide pools are maintained through both de novo
synthesis and salvage pathways. DHODH, the only monofunctional enzyme in the de
novo pyrimidine synthesis pathway, is located in the inner mitochondrial membrane.
The enzyme catalyzes the only redox reaction in the pathway; the oxidation of
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dihydroorotate to orotate using ubiquinone (Q ) as an electron acceptor. The final
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product of the pathway, uridine monophosphate (UMP), can also be synthesized via the
pyrimidine nucleotide salvage pathways that rely on uridine import, deamination of
cytidine to uridine, or by recycling of RNA.3
Depending on the cell type, growth rate, and the phase of the cell cycle, the nucleotide
requirements of cells vary.4-6 Therefore, rapidly proliferating cells, such as cancer cells
or activated T and B cells, may be highly sensitive to inhibition of nucleotide synthesis.7
Consequently, the enzymes in the de novo pyrimidine ribonucleotide synthesis
pathway, and DHODH as part of the pathway, have been considered as attractive targets
for the treatment of cancer and autoimmune diseases, as well as for treatment of viral
infections.8-11
The pro-drug leflunomide, and its active metabolite teriflunomide (A771726) (Figure
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), are used in the clinic for the treatment of rheumatoid arthritis and multiple sclerosis
respectively.12, 13 These DHODH inhibitors, however, have known off-target effects.14,
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Another well-studied inhibitor of DHODH is brequinar (Figure 1). First described in
the 1980’s, this compound showed antitumor activity in multiple cell lines and in
murine tumor models.9, 16 Phase II clinical trials using brequinar against solid tumors
revealed modest to no effect.17-19 However, recent studies in hematological
malignancies demonstrated therapeutic potential in pre-clinical models and have
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renewed interest in the use of DHODH inhibitors for the treatment of cancer. Other
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compounds that inhibit human DHODH have also been developed,20-22 and numerous
studies have been carried out to improve the specificity, potency and preclinical
properties of these inhibitors.23-27 At present, several DHODH inhibitors, including
BAY 2402234 (Clinical trial identifier: NCT03404726) and ASLAN003 (Clinical trial
identifier: NCT03451084) are in clinical trials for the treatment of myeloid
malignancies.28, 29
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Recently, chiral tetrahydroindazoles (HZ) were identified as a novel class of human
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DHODH inhibitors. (R)-HZ00, which has a pyridyl Ar substituent, and its analogue
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R)-HZ05, with a tetrahydrobenzisoxazolyl Ar substituent (Figure 1), inhibited
DHODH activity and together with an inhibitor of p53 degradation, reduced melanoma
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cell growth in vivo. The originally identified hit compound HZ00 showed good
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metabolic stability in human liver microsomes. The in vivo studies with the more
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potent analogue (R)-HZ05, however, demonstrated that further medicinal chemistry
optimization would facilitate the HZ compound class to progress towards in vivo
efficacy studies in mouse models. In this paper we report a lead compound development
through a structure-activity relationship (SAR) study of the HZ series. In addition, we
provide data suggesting that the appearance of the DNA damage marker -H2AX in
response to DHODH inhibition is not due to the induction of DNA damage, but rather
to the onset of apoptosis. Based on the activity of the compounds in cells, as well as
their solubility and in vitro metabolic stability, we identified compound 51 as a potent
and selective DHODH inhibitor from which a compound with candidate drug properties
may be developed.
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Results and Discussion
Rational for the design of new HZ analogues. The design of new HZ analogues with
potentially improved solubility and metabolic stability was based on a metabolic soft
spot identification study performed with (R)-HZ05. The disappearance of the
compound and the formation of metabolites after incubation with mouse liver
microsomes in the presence of NADPH, was monitored by LC-MS/MS. A structural
elucidation of the MS-fragments, based on the detected two major oxidation products,
suggested mono-hydroxylation of either the tetrahydroisoxazole ring or the central
tetrahydroindazole ring in approximately equal amounts (Figure 2). These results
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indicated that the metabolic profile could be improved by modifying the Ar moiety of
the HZ series.
The previously published X-ray co-crystal structure of (R)-HZ05 with DHODH served
as an important tool in the design of new HZ analogues.30 A 2D schematic
representation of the binding pocket of co-crystallized DHODH with ligand (R)-HZ05
is shown in Figure 3. According to the co-crystal structure, the major interaction
between the ligand and the protein occurs between Arg136 of the enzyme and the amide
carbonyl oxygen of (R)-HZ05 through a two-water molecule bridge. Furthermore, the
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structure indicated that a limited space might be available around the Ar tetrahydro-
benzisoxazolyl ring occupying the inner part of the binding pocket. Thus, introduction
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of small substituents on the Ar pyridyl or tetrahydro-benzisoxazolyl moieties of the
HZ inhibitors was considered a possible strategy for making this part of the compounds
less prone to oxidative metabolism. Analysis of the X-ray structure also suggested that
adding an extra hydrogen bond acceptor in the HZ compounds could allow for a new
interaction with Tyr356 (Figure 3). This hypothesis is supported by the fact that a
hydrogen bond interaction between DHODH inhibitors and Tyr356 is found in several
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of the published structures, many available in the Protein Data Bank (PDB), for
example BAY2402234 and teriflunomide.29, 31 Such modification could potentially
facilitate interactions of ligands with decreased lipophilicity, increased aqueous
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solubility, and enhanced metabolic stability. Moreover, the meta-position of the Ar2
phenyl appeared feasible for extended substituents towards the surrounding solvent,
which could allow for larger modification such as photoaffinity probe constructs.
Synthesis of target compounds 28–54. Target compounds 28–54 were prepared by
amide coupling between substituted 1-phenyl-4,5,6,7-tetrahydro-1H-indazol-4-amines
(1) and aromatic carboxylic acids using various coupling reagents such as HATU,
HBTU or 1,3,5-tripropyl-1,3,5-triphosphinane-2,4,6-trione (Scheme 1). The
substituted 1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-ones 410 were prepared from
cyclohexadione and optionally substituted phenylhydrazines (3) using the method
described by Guo et al.32 (Scheme 2). The ketones 410 were either reductively
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aminated using the method described by Guo et al. to give the racemic 1-phenyl-
,5,6,7-tetrahydro-1H-indazol-4-amines 1116 or first transformed to the chiral
intermediates 1720 using Ellman´s reagent and afterwards reduced and deprotected as
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described in Ladds et al. to give the chiral intermediates 2124. The carboxylic acids
used were commercially available, except compound 27 which was prepared via a
palladium mediated carbonylation (Scheme 3).
DHODH inhibitory activity of HZ00 analogues. The inhibitory activity of the newly
synthesized HZ analogues against DHODH was tested in vitro by an optimized
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enzymatic assay that uses 2,3–dimethoxy-5-methyl-p-benzoquinone as a substitute
for the highly insoluble coenzyme Q . The colorimetric kinetic reaction, which detects
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changes of the oxidation state of 2,6-dichlorophenolindophenol (DCIP) as final electron
acceptor, was monitored for 68 min. The IC was calculated based on the estimated
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Vmax values for each of the tested concentrations within a linear range using linear
regression. The expansion of the HZ series was primarily focused on (R)-configured
tetrahydroindazoles, i.e. analogues of the most potent HZ00 and HZ05 enantiomers (R)-
HZ00 and (R)-HZ05, respectively (Supporting Information Figures S1-S2). First, we
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examined the effect of introducing various small substituents on the Ar pyridyl ring by
synthesizing compounds 2834 (Table 1). Both the 5-methyl analogue 28 (IC = 55
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nM) and the 4-methyl analogue 29 (IC = 34 nM) showed significantly increased
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potency when compared to (R)-HZ00. Compounds with alternative small substituents
in the 4-position, CF (30), Cl (31), Br (32), and F (33), exhibited similar potencies as
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analogue with an IC value of 15 nM. The 4,5-dimethyl analogue 34 was also slightly
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more potent than the 4-methyl compound 29. Introduction of larger substituents on the
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Ar pyridyl ring, exemplified by compound 35, resulted in a decrease in potency, in
agreement with the predictions from the analysis of the co-crystal structure between
(R)-HZ05 and DHODH (Figure 3).
In an attempt to reduce the lipophilicity, and potentially increase the metabolic stability,
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an additional nitrogen was introduced in the Ar heterocyclic moiety by the synthesis
of the pyrimidinyl analogue 36 (Supporting Information Figure S1). However, this
compound showed a decrease in DHODH inhibitory potency when compared to (R)-
HZ00. Likewise, the racemic phenyl analogue 37 (Supporting Information Figure S1)
was 3–4-fold less potent than the parent racemic compound HZ00.
Finally, we investigated the potency of three analogues with alternative substitution
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patterns on the Ar phenyl moiety. Compounds 38 (Ar = 2-methylphenyl; Supporting
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Information Figure S1), 39, and 40 (Ar = 2-fluoro-5-methylphenyl), all inhibited
DHODH with similar potencies as their parent 2-fluoromethyl analogues 29 and 30,
respectively. In summary, introduction of small lipophilic substituents on the Ar1
pyridyl ring of the HZ compounds increased the potency towards DHODH, while
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replacing the Ar pyridyl with phenyl or pyrimidinyl substituents had adverse effect.
DHODH inhibitory activity of HZ05 analogues. The modifications introduced in the
HZ00 series provided a base for the SAR of the more potent parent compound (R)-
HZ05 (Supporting Information Figure S2a). Initially, we isolated the enantiomeric pair
of the closely related regioisomers of (R)-HZ05 and (S)-HZ05, compounds 41 and 42,
1
respectively, with reversed N and O positions in the Ar tetrahydro-benzisoxazolyl
moiety. Both compounds were equipotent to their respective (R)- and (S)-HZ05
counterparts (Table 2), again demonstrating the superior DHODH inhibitory properties
of HZ compounds with (R) configuration.
We also synthesized HZ05 analogues with alternative small, or no substituents on the
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Ar phenyl moiety, compounds 4350 (Supporting Information Figure S2b). In
agreement with the SAR from the HZ00 series, these compounds showed similar or
increased potencies in their inhibition of DHODH when compared to the parent HZ05
compounds.
The X-ray co-crystal structure of DHODH with (R)-HZ0530 suggested that an
1
additional hydrogen bond acceptor in the Ar moiety of the HZ compounds might allow
for a complementary interaction with the hydroxyl group of the DHODH residue
Tyr356 (Figure 3). Therefore, in an attempt to facilitate an interaction with Tyr356
(
Figure 3), and at the same time decrease the lipophilicity and potentially increase the
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metabolic stability of the compounds, the imidazopyridinyl analogues 51 (Supporting
Information Figure S2b) and 52 were synthesized. In agreement with our theory, both
compounds 51 and 52 were highly potent DHODH inhibitors, with IC values below
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that of (R)-HZ05. We have, however, no conclusive evidence that the improved potency
of these analogues is due to an actual additional interaction between the inhibitors and
Tyr356 residue of the enzyme. Nevertheless, the ligand-lipophilicity efficiency
(Supporting Information Table S1), defined as the difference between the ligand pIC50
and logP, increases considerably from 4.6 for (R)-HZ05 to 5.6 for compound 52 and
5
.7 for compound 51 due to a concurrent increase in pIC and decrease in logP.
50
2
The co-crystal structure also suggested that the meta position of the Ar phenyl group
of the HZ compounds may allow for larger substituents directed out from the binding
pocket opening towards the surrounding solvent (Figure 3). Thus, compounds 53 and
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4 were synthesized, both with sterically demanding substituents in the meta position
2
of the Ar phenyl group. Compound 53, containing an ester substituent on the aryl, was
a potent DHODH inhibitor, illustrating the versatility of the co-crystal structure model
as a guide for modifications of the HZ compounds. However, compound 54 (Supporting
Information Figure S2a), containing a carboxylic acid substituent, showed low potency.
2
These results imply that the meta position of the Ar phenyl group may be used to
prepare, for example, photoactive or fluorescent tagged compounds, useful in future
cell studies of this compound series.
Solubility and In vitro Metabolic Stability. Compounds 30, 38, 46 and 51, among the
most potent inhibitors from the HZ00 and HZ05 series, were further tested for their
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solubility and metabolic stability in human (HLM) and mouse liver microsomes
(MLM). The results are summarized in Table 3.
Of the HZ compounds tested, compound 38 and 51 exhibited high solubility in the
Prima HT aqueous solubility test solution (Table 3). Compounds 46 and 30 were
markedly less soluble, and would demand more elaborate formulations for use in vivo.
Metabolic stability studies in liver microsomes were conducted with all four HZ
compounds and brequinar, which was used as a reference. Compound 30 showed high
stability in MLM, however, the result must be interpreted cautiously since the
compound demonstrated low aqueous solubility. Compounds 38 and 51 both had a half-
life of more than 60 min in HLM and compound 51 demonstrated the highest stability
in MLM of the two with a half-life of 36 min. Compound 46 showed the lowest stability
in both HLM and MLM. These results, in combination with the measured aqueous
solubilities, suggest that compound 51 has the best properties to allow preclinical
mouse studies in the future. Additionally, compound 51 was tested against a panel of
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68 kinases at a concentration of 1 µM. The results from this screen (Supporting
Information Table S2) support that the compound might be a specific inhibitor of
DHODH.
Activation of the transcription factor function of p53 in cells. (R)-HZ00 and its
analogue HZ05, were identified as inducers of the transcription factor activity of p53
in the ARN8 p53-reporter melanoma cell line (Supporting Information Figure S3).30
Therefore, the ability of selected HZ analogues and brequinar (used as a reference) to
activate p53-induced transcription, was tested in this reporter cell line in the p53
reporter assay (CPRG assay). As shown in Figure 4, compounds 30, 38, 43, 45, and 51,
the most active analogues in the enzymatic assay from the HZ00 series and the HZ05
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series, were all potent activators of p53-dependent transcription in ARN8 cells.
Furthermore, supplementation with high concentrations of uridine (100 M) prevented
the activation of p53-dependent transcription, suggesting that the increased activity of
p53 after treatment with the HZ compounds is due to inhibition of the de novo
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pyrimidine ribonucleotide synthesis pathway. At higher concentrations, however,
some of the compounds, such as 43 and 45 (Figure 4), caused a decrease in the detected
p53 reporter activity. Compound 46 (Supporting Information Figure S3), the (R)-
enantiomer of 43, showed similar activity to the racemic analogue, including a p53
activity decrease at higher concentrations. The reduction observed with the strongest
inhibitors from the series might be explained by a decrease in cell growth or viability,
thus leading to a lower level of p53 transcription factor function.
The analogues, which had low activity in the in vitro enzymatic assay did not induce
p53-dependent transcription (Supporting Information Figure S3). This is also
exemplified by the enatiomeric pairs of R- and S- HZ00 and HZ05, where only the
active R-enatiomers had an effect on p53-dependent transcription.
Effect of HZ analogues on cell growth/viability. Following the activity studies of the
compounds on isolated DHODH enzyme and testing their ability to activate the
transcription factor function of p53 in cells, we analyzed the effect of HZ analogues
with an IC < 20 nM against purified DHODH, on the viability of two cell types: ARN8
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melanoma cells (Table 4 and Figure 5a) and human normal dermal fibroblasts (HNDFs;
Table 4 and Figure 5b). Brequinar (Figure 5),16, 34 was again used as a reference
compound (Table 4). Compounds with low or no activity in the enzymatic assay were
also tested for their effect on the viability of ARN8 and HNDFs (Supporting
Information Figure S4).
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The HZ00 analogues with highest activity in the enzymatic assay, compounds 30 and
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8, were more potent than (R)-HZ00 (Supporting Information Figure S4a) at reducing
growth/viability of ARN8 cells as measured by a sulforhodamine B (SRB) assay.
Furthermore, these analogues had little or no effect on the growth/viability of HNDFs
at the tested concentrations within 72 h of treatment. Compounds 36, 37, and (S)-HZ00,
which had low inhibitory activity in the in vitro enzymatic assay, had little or no effect
on both cell lines at the tested concentrations (Supporting Information Figure S4).
Most of the tested HZ05 analogues showed improved or similar growth/viability
inhibitory activities in ARN8 cells when compared to their racemic and enantiomeric
parent counterparts HZ05 and (R)-HZ05 (Table 4, Figure 5, Supporting Information
Figure S4). In contrast, when the new analogues were tested for growth/viability
inhibition in HNDF cells, only weak effects were observed at the higher concentrations.
These results show that the compounds are more toxic for ARN8 melanoma cells than
for normal fibroblasts. Compounds 42 and (S)-HZ05, which had low inhibitory activity
in the in vitro anzymatic assay, had little or no effect on both cell lines at the tested
concentrations (Supporting Information Figure S4).
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To establish whether the main effect of the compounds on cell growth/viability is due
to DHODH inhibition, we tested whether their effect was prevented by addition of a
high concentration of uridine to the medium. Similar to the result obtained in the CPRG
assay (Figure 4, Supporting Information Figure S3), we observed that supplementation
with uridine allows cells to overcome the effect on cell growth/viability of the selected
HZ00 and HZ05 analogues, except at high concentrations of some compounds, such as
43, 45, and 46 (Figure 5; Supporting Information Figure S4). The results indicate that
depletion of pyrimidine ribonucleotides, caused by inhibition of DHODH by the HZ
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compounds, leads to the increased activity of p53 and also to reduced growth/viability
of ARN8 melanoma cells.
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Effects on p53 levels and -H2AX phosphorylation. Some of the HZ analogues with
the strongest effect on cell growth/viability of ARN8 cells, were selected for further
analysis. Compound 51, with the overall most favourable potency and ADME-
properties, and the racemic compounds 43 and 45 were used for analysis of p53 protein
levels. In agreement with their ability to increase p53-dependent transcription in ARN8
melanoma cells, compounds 43, 45, 51 also increased p53 protein levels, and this was
prevented by addition of high concentrations of uridine (Figure 6a). A similar result
was observed with brequinar. In contrast, the increase in p53 levels by compounds that
have mechanisms of action other than DHODH inhibition, such as the mdm2 antagonist
nutlin-3a (N3a) 35, 36 or the topoisomerase II inhibitor etoposide (ETP) , was not
prevented by the supplementation with uridine (Figure 6a).
37
An important consideration regarding the use of DHODH inhibitors in the clinic is
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whether they can cause damage to the genome. In the study of Ladds et al. an increase
of DNA damage markers was not detected after treatment with HZ00. As the new HZ
analogues are more potent, we re-examined several proteins associated with the DNA
damage response. We checked whether cells treated with compounds 43, 45, 51, or
brequinar affected the levels of p53 phosphorylated at Ser15 (p-Ser15 p53), a marker
for the activation and stabilization of p53 as a result of DNA damage,38-40 as well as
H2AX phosphorylated at Ser139 (-H2AX), a marker for double strand DNA breaks.41,
42
As shown in Figure 6a, the levels of both markers were increased after 24 h treatment
with all DHODH inhibitors. These effects, however, were prevented by
supplementation with high concentrations of uridine, suggesting that they result from
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inhibition of DHODH, and that the compounds themselves most likely do not exert
direct effects on DNA through mechanisms such as covalent modification of DNA
bases or by drug-DNA intercalation. Treatment with etoposide, which induces double
strand DNA breaks, increased p-Ser15 p53 and -H2AX levels regardless of uridine
supplementation (Figure 6a).
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Phosphorylation of p53 at Serine-15 has also been associated with the transcription
43
factor function of p53. An increase in p-Ser15 p53 was observed not only in the
DHODH inhibitor treated samples, but also after treatment with the non-genotoxic p53
activator nutlin-3a (Figure 6).35, 44 This increase in p-Ser15 p53, although lower in the
mdm2 inhibitor treated samples, was accompanied by substantial rise in total p53 levels
(Figure 6). Therefore, the increased p-Ser15 p53 observed after inhibition of DHODH
is likely due to basal level of phosphorylation of p53. In contrast, the DNA damaging
agent etoposide (Figure 6), as well as staurosporine (Figure 6b), caused a more
pronounced rise in p-Ser15 p53 levels compared to levels of total p53.
Although the uridine supplementation demonstrated that the HZ compounds analyzed
here are unlikely to directly damage DNA, this does not preclude the idea that targeting
the de novo pyrimidine pathway and the subsequent pyrimidine ribonucleotide
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depletion affects DNA synthesis. A recent study by Mathur et al., shows that Chk-1
is phosphorylated at Ser345 (p-Ser435) and -H2AX increases after treatment of PTEN-
mutant cancer cells with DHODH inhibitors. The study, in spite of using high
concentrations (100–150 M) of leflunomide or teriflunomide (A771726), compounds
with known off-target effects at high doses,14, 15 suggested that inhibition of DHODH
leads to cell death caused by chromosome breaks and DNA damage at replication
forks.45 Therefore, we examined the levels of p-Ser345 Chk-1 and Chk-2
phosphorylated at Thr68 (p-Thr68 Chk-2) after 24 h treatment with the HZ compounds
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or brequinar (Supporting Information Figure S5). In our study, induction of p-Ser345
Chk-1 or p-Thr68 Chk-2 in response to inhibition of DHODH was not detected.
An increase of -H2AX levels can be due to DNA damage41, 42 or replication fork
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stalling, but it can also be a consequence of the induction of apoptosis.
Therefore,
we tested whether the increased phosphorylation of H2AX in response to treatment
with the HZ compounds is due to DNA fragmentation caused by activation of apoptosis.
49
For this purpose, the pan caspase inhibitor Z-VAD-FMK was used. As shown in
Figure 6b, the levels of -H2AX were reduced considerably in all DHODH inhibitor
treated cells after addition of Z-VAD-FMK. This pan caspase inhibitor, however, did
not lower the levels of -H2AX in the presence of etoposide. Additionally, co-treatment
with staurosporine (STP), a compound that can induce caspase dependent and caspase
independent apoptosis,50 and Z-VAD-FMK resulted in a small decrease of
phosphorylated H2AX. These results show that the appearance of -H2AX after
treatment with DHODH inhibitors correlates with the induction of caspase-dependent
apoptosis.
Supporting the notion that DHODH inhibitors cause apoptosis in ARN8 cells we
measured the levels of other apoptosis related proteins in response to treatment with
DHODH inhibitors. As shown in figure 6b, the levels of caspase cleaved PARP,51
increased after treatment with DHODH inhibitors, and the cleavage was prevented by
the addition of Z-VAD-FMK.
To confirm the activation of the caspase cascade in DHODH inhibitor treated cells, we
performed live cell imaging using a caspase-3/7 substrate that emits a fluorescent signal
upon binding to DNA following caspase 3 or 7 cleavage (Figure 7a) and the cell-
impermeant nuclear dye YOYO-3 (Figure 7b) that fluoresces upon binding to nucleic
acids. All tested compounds increased the signal of the caspase 3/7 substrate, and co-
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treatment with Z-VAD-FMK led to its reduction. In agreement with the previously
discussed results, supplementation with uridine ablated the effect of DHODH inhibitors
on the marker for caspase 3/7 activation, while the samples treated with staurosporine
were not affected. These results support our hypothesis that inhibition of DHODH
induces caspase-dependent apoptosis.
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Treatment with DHODH inhibitors increased the fraction of cells positive for YOYO-
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as well, although to a lesser extent than treatment with staurosporine (Figure 7b).
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Furthermore, the number of YOYO-3 positive cells/mm /confluence % was lower
compared to caspase 3/7 substrate. Supplementation with uridine prevented the effect
of the DHODH inhibitors and co-treatment with Z-VAD-FMK also reduced the
proportion of YOYO-3 positive cells in all samples.
The levels of several proteins located upstream of the caspases were also analyzed by
western blotting. The pro-apoptotic Bcl family member Bax, a downstream effector of
p53, was increased after 24-hour treatment with DHODH inhibitors, while the levels of
the anti-apoptotic Bcl-2 protein were decreased (Supporting Information Figure S6).
The levels of Bax and Bcl-2 (Supplementary Figure 3), as well as p53/p-Ser15 p53
(Figure 6b), which are upstream of the effector caspases, were not affected by Z-VAD-
FMK in response to any of the treatments.
Conclusion
Several of the new HZ analogues presented in this study are potent inhibitors of
DHODH in an enzymatic assay and are active on cultured cells. Furthermore, the rescue
of cell growth/viability by supplementation with high concentrations of uridine for the
selected new HZ compounds suggests that the observed effects are indeed due to
inhibition of the de novo pyrimidine synthesis pathway. Out of the new HZ00 and HZ05
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analogues, compound 51 is the most favorable one, based on its potency, solubility and
metabolic stability in both HLM and MLM. This makes the compound a suitable
candidate for future testing in in vivo pharmacokinetic, toxicity and efficacy studies.
Finally, we present evidence suggesting that the appearance of p-Ser15 p53 and -
H2AX upon DHODH inhibition does not necessarily indicate activation of the DNA
damage response but rather induction of apoptosis. Assessing the potential genotoxicity
arising from depletion of pyrimidine ribonucleotide pools is of interest from a
mechanistic perspective as well as from a clinical safety perspective.
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Experimental Section
Chemistry. Brequinar was purchased from Sigma-Aldrich (#SML0113), methyl 6-
(([1-(2-fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-yl]amino)carbonyl)nicotinate
(35) was purchased from Chembridge (#34540860). The following starting materials
were commercially available or synthesized as described in literature: 1-Phenyl-
4
,5,6,7-tetrahydro-1H-indazol-4-one, (4) was purchased from Enamine; 1-(2-
Fluorophenyl)-6,7-dihydro-1H -indazol-4(5H )-one (5), 1-(2-fluorophenyl)-4,5,6,7-
tetrahydro-1H-indazol-4-amine (12), (S)-N-((R)-1-(2-Fluorophenyl)-4,5,6,7-
tetrahydro-1H-indazol-4-yl)-2-methylpropane-2-sulfinamide (17), and (R)-1-(2-
Fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-amine hydrochloride (21) were
30
synthesized as described in Ladds et al. ; 2-bromo-4,5-dimethyl-pyridine (25) was
synthesized from 3,4-dimethylpyridine as described in T. Kaminski et al.52
Additional chemicals and reagents were obtained from commercial suppliers and were
used as received unless otherwise stated. Tetrahydrofuran (THF) and dichloromethane
(DCM) were obtained dry from solvent purification systems. Flash column
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chromatography was performed using silica gel (40–63 m) and automated flash
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chromatography was performed using prepacked silica columns. H NMR and C
1
NMR spectra were recorded in CDCl (internal standard: 7.26 ppm, H; 77.16 ppm,
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C), DMSO-d (internal standard: 2.50 ppm, H; 39.52 ppm, C) or methanol-d
6
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1
(
internal standard: 3.31 ppm, H; 49.00 ppm, 13C) on 300, 400 or 500 MHz
spectrometers. Coupling constants (J) are quoted in Hz and are recorded to the nearest
.1 Hz. The following abbreviations are used: s, singlet; d, doublet; dd, doublet of
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doublets; t, triplet; m, multiplet; q, quartet; and br, broad.
Analytical RPLC-MS was performed using a HPLC-MS Dionex Ultimate 3000 with a
Bruker amaZon SL, under the following conditions: Column, Kinetex C18 (2.6 μm, 50
mm  3.0 mm); Mobile phases, MeCN/water gradients (0.05% HCOOH); Flowrate, 1.5
mL/min; Detection, UV (214, 254, 280 nm) and MS (ESI, pos, neg or alternate polarity)
or Agilent/HP 1200 system 6110 mass spectrometer with electrospray ionization
(ESI+). HPLC−MS methods were the following: Method 1: Waters XBridge C18 3.5
μm column (3.0 mm × 50 mm), 3.5 min gradient mobile phase [MeCN] / [10 mM
NH HCO /H O]; Method 2: MeCN C18 3.5 μm column (3.0 mm × 50 mm), mobile
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phase [0.1% TFA/MeCN] / [0.1% TFA/H O] on a Water micro mass ZQ 2000 using
2
positive and negative electrospray ionization.
High resolution molecular masses (HRMS) were determined on a mass spectrometer
equipped with an ESI source and a 7-T hybrid linear ion trap (LTQ).
Preparative RP-LC was performed using either a Zorbax SB-C8, a VP 250/21
Nucleodur C-18, HTec, or an Ace C-8 column with a MeCN/water eluent system with
either 0.1% TFA or 0.05% HCOOH as additive. Chiral Separations were performed
using a SFC Waters Investigator system with Waters 2998 PDA detector. The column
temperature was set to 45 °C.
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The purity of the tested compounds 29-54 is at least 95% as assessed by HPLC-UV at
2
14, 254 and 280 nm or by NMR. Exceptions are stated in the synthesis description.
1-(2-Methylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-one (6). The title compound
0
1
2
3
4
5
6
7
8
9
0
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2
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8
9
0
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0
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4
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32
1
was prepared using the method described by Guo et al. Yield: 1.11 g, 70%. H NMR
(
400 MHz, CDCl ) δ 8.07 (s, 1H), 7.45–7.29 (m, 3H), 7.29–7.25 (solvent), 7.24 (dd, J
3
=
7.8, 1.4 Hz, 1H), 2.64 (t, J = 6.2 Hz, 2H), 2.58–2.50 (m, 2H), 2.21–2.11 (m, 2H), 2.11
+
+
(
s, 3H); m/z (ES ) 227 [M+H] .
1
-(2-Fluoro-5-methylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-one (7). The title
3
2
compound was prepared using the method described by Guo et al. Yield: 1.07 g, 73%.
1
H NMR (400 MHz, CDCl ) δ 8.09 (s, 1H), 7.33 (dd, J = 7.0, 1.9 Hz, 1H), 7.27–7.21
3
(m, 1H), 7.14 (dd, J = 10.1, 8.5 Hz, 1H), 2.80 (td, J = 6.3, 1.4 Hz, 2H), 2.55 (dd, J =
+
+
7
.3, 5.6 Hz, 2H), 2.39 (s, 3H), 2.21–2.10 (m, 2H). m/z (ES ) 245 [M+H] .
1-(3-Methylphenyl)-1,5,6,7-tetrahydro-4H-indazol-4-one (8). The title compound
32
1
was prepared using the method described by Guo et al. Yield: 0.405 g, 89.4%. H
NMR (400 MHz, CDCl ) δ 8.08 (s, 1H), 7.42–7.36 (m, 2H), 7.30–7.24 (m, 2H), 2.98
3
+
+
(
t, J = 6.2 Hz, 2H), 2.55 (m, 2H), 2.45 (s, 3H), 2.18 (m, 2H); m/z (ES ) 227 [M+H] .
1
-(2,3-Dimethylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-one (9). The title
3
2
compound was prepared using the method described by Guo et al. Yield: 0.425 g,
1
8
8.4%. H NMR (400 MHz, CDCl ) δ 8.07 (s, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.21 (t, J
3
= 7.6 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 2.62 (t, J = 6.0 Hz, 2H), 2.52–2.55 (m, 2H),
+
+
2
.35 (s, 3H), 2.13–2.18 (m, 2H), 1.94 (s, 3H); m/z (ES ) 241 [M+H] .
Ethyl 3-(4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzoate
(10).
2-
Dimethylaminomethylenecyclohexane-1,3-dione (0.3344 g, 2.0 mmol) and 3-
hydrazinylbenzoic acid (0.3043 g, 2.0 mmol) were dissolved in methanol (12.0 mL).
Water and NaOH (aq, 1.0 mL, 2.0 M) were added. The reaction mixture was heated at
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0 ºC for 2 h and then concentrated under vacuum. To the residue acetic acid (12.0 mL)
and water (6.0 mL) were added and the mixture was heated at 110 ºC for 1.5 h. The
solution was concentrated under vacuum. Ethyl acetate (20 mL) was added to the
residue and the mixture was washed with brine (20 mL), dried over Na SO and
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concentrated. To the residue ethanol (10 mL) and concentrated sulfuric acid (0.15 mL)
were added. The mixture was refluxed overnight. The solvents were removed under
vacuum and ethyl acetate (20 mL) was added to the residue. The mixture was washed
with NaHCO and brine and was dried over Na SO . The residue was purified by silica
3
2
4
1
gel chromatography (petroleum ether/ethyl acetate, 2:1) to yield 0.240 g, 42%. H NMR
(400 MHz, CDCl ) δ 8.15–8.17 (m, 1H), 8.09–8.11 (m, 2H), 7.73–7.76 (m, 1H), 7.58–
3
7
2
.62 (m, 1H), 4.41 (q, J = 7.1 Hz, 2H), 3.01 (t, J = 6.2 Hz, 2H), 2.55–2.58 (m, 2H),
+
+
.16–2.22 (m, 2H), 1.41 (t, J = 7.1 Hz, 3H); m/z (ES ) 285 [M+H] .
1-Phenyl-4,5,6,7-tetrahydroindazol-4-amine hydrochloride (11). The compound
32
was synthesized using the method described by Guo et al. The hydrochloric salt was
obtained by treatment with HCl in EtOH and EtOAc, and evaporation to give 141 mg,
1
5
4%. H NMR (400 MHz, DMSO-d ) δ ppm 8.48 (br. s., 3H), 7.90 (s, 1H), 7.61–7.48
6
(m, 4H), 7.45–7.33 (m, 1H), 4.47–4.29 (m, 1H), 2.77 (t, J = 5.69 Hz, 2H), 2.16–1.88
+
+
(
m, 2H), 1.86–1.67 (m, 2H); m/z (ES ) 214 [M+H] .
-(2-Methylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-amine (13). The compound
1
3
2
was synthesized using the method described by Guo et al. to yield the crude product
+
+
as a solid (47 mg, 100%). m/z (ES ) 228 [M+H] .
-(3-Methylphenyl)-4,5,6,7-tetrahydro-H-indazol-4-amine
compound was synthesized using the method described by Guo et al. The crude
1
(14).
The
title
3
2
+
+
product (0.504 g) was used without further purification. m/z (ES ) 228 [M+H] .
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1
-(2,3-Dimethylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-amine (15). The title
3
2
compound was synthesized using the method described by Guo et al. The crude
+
+
product (0.585 g) was used without further purification. m/z (ES ) 242 [M+H] .
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Ethyl 3-(4-amino-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzoate (16). The title
3
2
compound was synthesized using the method described by Guo et al. by using ethyl
-(4,5,6,7-tetrahydro-4-oxo-1H-indazol-1-yl)benzoate as a starting material, with the
modification that the heating time was 7 h at 70 ºC, to give a crude product (0.342 g).
3
+
+
m/z (ES ) 286 [M+H] .
S)-2-Methyl-N-[(4R)-1-(2-methylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-
yl]propane-2-sulfinamide (18). The compound was synthesized according to the
(
30
1
method described in Ladds et al. to give a solid (1.6 g, 70%). H NMR (400 MHz,
DMSO-d ) δ 7.51 (s, 1H), 7.45–7.36 (m, 2H), 7.36–7.24 (m, 1H), 7.21 (m, 1H), 5.40
6
(d, J = 8.4 Hz, 1H), 4.40–4.30 (m, 1H), 2.41–2.20 (m, 2H), 2.07–1.88 (m, 5H), 1.83–
+
+
1
.63 (m, 2H), 1.16 (s, 9H); m/z (ES ) 332 [M+H] .
S)-N-[(4R)-1-(2-Fluoro-5-methylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-yl]-2-
methylpropane-2-sulfinamide (19). The compound was synthesized according to the
(
3
0
1
method described in Ladds et al. to give the title compound (0.94 g, 61%). H NMR
(400 MHz, CDCl ) δ 7.64 (s, 1H), 7.29–7.23 (m, 1H, overlapping with the solvent
3
signal), 7.21–7.14 (m, 1H), 7.13–7.05 (m, 1H), 4.55–4.45 (m, 1H), 3.34 (d, J = 9.5 Hz,
1H), 2.62–2.42 (m, 2H), 2.36 (s, 3H), 2.33–2.24 (m, 1H), 2.00–1.73 (m, 3H), 1.26 (s,
+
+
9
H); m/z (ES ) 350 [M+H] .
Propan-2-yl-3-[(4R)-4-([(R)-2-methylpropane-2-sulfinyl]amino)-4,5,6,7-
tetrahydro-1H-indazo-1-yl]benzoate (20). The compound was synthesized from 10
30
using the method described in Ladds et al. with the modification that Ti(O-i-Pr) was
4
1
used instead of Ti(OEt) , to give the title compound as a solid (0.70 g, 52%). H NMR
4
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(
400 MHz, DMSO-d ) δ ppm 8.07 (t, J = 1.8 Hz, 1H), 7.90–7.95 (m, 1H), 7.83 (ddd, J
6
=
8.1, 2.3, 1.0 Hz, 1H), 7.66 (t, J = 7.90 Hz, 1H), 7.61 (s, 1H), 5.46 (d, J = 8.7 Hz, 1H),
5.17 (spt, J = 6.3 Hz, 1H), 4.29–4.39 (m, 1H), 2.65–2.84 (m, 2H), 1.91–2.08 (m, 2H),
1
1
1
1
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2
2
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2
2
2
2
2
2
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
1
.65–1.86 (m, 2H), 1.34 (d, J = 6.2 Hz, 6H), 1.16 (s, 9H). m/z (ES ) 404 [M+H] .
4R)-1-(2-Methylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-amine
Concentrated hydrochloric acid (4 mL) was added dropwise to a solution of 18 (1.11 g,
.32 mmol) in methanol (40 mL). The mixture was stirred at rt for 2 h. Saturated
aqueous NaHCO was added to pH 7 followed by water. The mixture was extracted
(
(22).
3
3
with DCM. The organic layer was washed with saturated aqueous NaHCO and brine.
3
The organic layer was concentrated under vacuum to afford the title compound (0.52
+
+
g, 68%) which was used without further purification. m/z (ES ) 228 [M+H] .
4R)-1-(2-Fluoro-5-methylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-amine (23).
(
19 (300 mg, 0.858 mmol) was dissolved in methanol (2 mL) and concentrated
hydrochloric acid (196 µL, 2.42 mmol) was added. The mixture was stirred at rt for 30
min. NaHCO (sat, aq.) and DCM were added. The phases were separated and the
3
organic phase was washed with NaHCO (sat. aq.) and brine, dried over MgSO and
3
4
1
concentrated to give the title compound (171 mg, 81%). H NMR (400 MHz, CDCl ) δ
3
7
4
.69 (s, 1H), 7.28–7.22 (m, 1H), 7.18–7.12 (m, 1H), 7.07 (dd, J = 10.1, 8.5 Hz, 1H),
.07–3.98 (m, 1H), 2.60–2.42 (m, 2H), 2.35 (s, 3H), 2.10–1.88 (m, 2H), 1.80–1.68 (m,
+
+
1H), 1.56–1.46 (m, 1H); m/z (ES ) 246 [M+H] .
Propan-2-yl-3-[(4R)-4-amino-4,5,6,7-tetrahydro-1H-indazo-1-yl]benzoate (24). 20
(172 mg, 0.341 mmol) was dissolved in methanol (4.5 mL) and HCl (conc. 0.5 mL)
was added dropwise at 0 C. The mixture was stirred for 30 min at 0 C and then let to
rt over 90 min. The mixture was neutralized with NaHCO , extracted with EtOAC and
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dried (Na SO ). The solvents were evaporated to give the title compound as an oil (85
2
4
mg, 83%) which was used directly in the next step.
Methyl 4,5-dimethylpyridine-2-carboxylate
tolylphosphino)benzyl]dipalladium(II) (44
butylphosphonium tetrafluoroborate (55 mg, 0.19 mmol), molybdenumhexacarbonyl
248 mg, 0.940 mmol) and 2-bromo-4,5-dimethyl-pyridine (175 mg, 0.940 mmol) were
(26).
Bis(acetato)bis[o-(di-o-
mmol), tri-tert-
0
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2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
mg, 0.047
(
dissolved in methanol (4 mL). 1,8-Diazabicyclo[5.4.0]undec-7-ene (0.42 mL, 2.8
mmol) was added and the vial was capped and heated in a microwave reactor at 120 °C
for 15 min. After cooling, the reaction mixture was filtered through a silica plug and
the product was eluted with EtOAc. The solution was concentrated and the title
compound was purified by column chromatography on silica eluting with gradients of
1
EtOAc in pentane (5–50%) to give the title compound (40 mg, 26%). H NMR (400
MHz, CDCl ) δ 8.45 (s, 1H), 7.92 (s, 1H), 4.00 (s, 3H), 2.34 (s, 3H), 2.32 (s, 3H). m/z
3
+
+
(
ES ) 166 [M+H] .
Lithium 4,5-dimethylpyridine-2-carboxylate (27). 26 (39 mg, 0.24 mmol) was
dissolved in THF (0.6 mL) and methanol (0.6 mL). Lithium hydroxide (2 M, 0.24 mL)
was added and the reaction mixture was stirred at rt overnight. The solvent was
removed at reduced pressure and the residue co-evaporated with toluene to dryness.
+
+
The mixture was used without further purification. m/z (ES ) 152 [M+H] .
N-[(4R)-1-(2-Fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-yl]-5-
methylpyridine-2-carboxamide (28). 21 (27 mg, 0.101 mmol), 5-(methyl)pyridine-2-
carboxylic acid (14 mg, 0.106 mmol), HATU (42 mg, 0.111 mmol) and DIPEA (70
µL, 0.37 mmol) were dissolved in DCM (1.5 mL) and DMF (0.2 mL) and was stirred
at rt overnight. The reaction mixture was purified by column chromatography (silica
column, 12 g) eluting with a gradient of EtOAc in hexanes (20–70%). The fractions
2
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containing product were evaporated and dried under vacuum to give the title
1
compound (15 mg, 42%). H NMR (400 MHz, DMSO-d ) δ 8.55 (d, J = 8.3 Hz, 1H),
6
8.49–8.42 (m, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.86–7.78 (m, 1H), 7.60–7.44 (m, 4H),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
7
.41–7.33 (m, 1H), 5.19–5.10 (m, 1H), 2.38 (s, 3H), 2.05–1.68 (m, 4H). C NMR (101
MHz, DMSO) δ 163.4, 148.7, 147.4, 141.1, 138.7, 138.0, 136.5, 130.5z, 128.7, 125.2
(d, J = 3.4) 121.6, 118.4, 116.8 (d, 20 Hz), 41.89, 29.30, 21.0 (d, 4.6 Hz), 20.0, 18.0;
+
ESI-HRMS (m/z): [M+H] calcd for C H FN O 351.1621; obsd, 351.1614.
2
0
19
4
(R)-N-(1-(2-Fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-yl)-4-
methylpicolinamide (29). 21 (18 mg, 0.07 mmol), 4-(methyl)pyridine-2-carboxylic
acid (11 mg, 0.08 mmol), HATU (28 mg, 0.07 mmol) in DMF (0.1 mL) and DIPEA
(0.05 mL, 0.28 mmol) were dissolved in DCM (1.0 mL) and DMF (0.1 mL) and was
stirred at rt overnight. The reaction mixture was purified by column chromatography
(silica column, 4 g) eluting with a gradient of EtOAc in hexanes (20–60%). The
solvents were evaporated to give the title compound as a dry film (11 mg, 47%, purity
1
9
4%). H NMR (400 MHz, methanol-d ) δ 8.46 (dd, J = 5.0, 0.8 Hz, 1H), 8.03–7.96
4
(m, 1H), 7.65 (s, 1H), 7.60–7.46 (m, 2H), 7.43–7.32 (m, 3H), 5.27 (t, J = 5.8 Hz, 1H),
1
3
2
1
1
.69–2.49 (m, 1H), 2.47 (s, 3H), 2.23–1.83 (m, 5H). C NMR (101 MHz, CDCl ) δ
3
64.1, 156.5 (d, J = 255.8 Hz), 149.8, 149.0, 148.0, 141.9, 139.6, 130.1 (d, J = 7.6 Hz),
28.8, 127.1, 124.9 (d, J = 4.0 Hz), 123.3, 118.2, 116.8 (d, J = 19.8 Hz), 42.5, 30.3,
+
21.7 (J = 4.8 Hz), 21.3, 20.3. HRMS (m/z): [M+H] C calcd for C H FN O, 351.1621;
2
0
19
4
obsd, 351.1611.
N-[(4R)-1-(2-Fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-yl]-4-
(trifluoromethyl)pyridine-2-carboxamide (30). 21 (230 mg, used as crude, max 0.7
mmol), 4-(trifluoromethyl)pyridine-2-carboxylic acid (149 mg, 0.78 mmol), HATU
(312 mg, 0.82 mmol) and DIPEA (0.52 mL, 2.99 mmol) were dissolved in DCM (4
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mL) and DMF (0.4 mL) and was stirred at rt overnight. The mixture was washed with
brine and dried (MgSO ). The residue was purified by column chromatography (silica
4
column, 24 g) eluting with a gradient of EtOAc in hexanes (20–70%). The fractions
0
1
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containing product were evaporated and dried under vacuum to give the title compound
1
as a white solid (200 mg, 66%). H NMR (400 MHz, DMSO-d ) δ 8.92 (dd, J = 16.9,
6
6
7
.7 Hz, 2H), 8.33–8.27 (m, 1H), 8.07–8.00 (m, 1H), 7.60 (s, 1H), 7.58–7.43 (m, 3H),
1
.42–7.33 (m, 1H), 5.25–5.17 (m, 1H), 2.04–1.83 (m, 4H), 1.83–1.68 (m, 2H). H NMR
(
400 MHz, CDCl ) δ 8.73 (d, J = 5.0 Hz, 1H), 8.55–8.45 (m, 1H), 8.18 (d, J = 8.1 Hz,
3
1
H), 7.73–7.60 (m, 2H), 7.54–7.35 (m, 2H), 7.32–7.17 (m, 2H, overlapping with
solvent), 5.42–5.27 (m, 1H), 2.60 (qt, J = 16.5, 6.1 Hz, 2H), 2.19 (m, 1H), 2.03–1.81
13
(
m, 3H). C NMR (101 MHz, CDCl ) δ 162.5, 156.8 (d, J = 255.5 Hz), 151.68, 149.3,
3
1
42.0, 140.2 (q*, J = 34.2 Hz), 139.4, 130.2 (d, J = 7.6 Hz), 128.8, 127.5 (d, J = 12.0
Hz), 125.0 (d, J = 4.0 Hz), 122.7 (q*, 273.6 Hz), 121.9 (q, 3.6 Hz), 118.6 (q, 3.6 Hz),
17.8, 116.8 (d, 20.0 Hz), 42.8, 30.3, 21.7 (d, 4.8 Hz), 20.3. HRMS (m/z): [M+H]+
1
calcd for C H F N O, 405.1339; obsd, 405.1328. *Due to low signal to noise, only
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0
16
4
4
two out of four peaks in expected quartet detected.
‐Chloro‐N‐[(4R)‐1‐(2‐fluorophenyl)‐4,5,6,7‐tetrahydro‐1H‐indazol‐4‐yl]pyridine
4
‐2‐carboxamide (31). 21 (20 mg, 0.0750 mmol), 4-chloropyridine-2-carboxylic acid
(12 mg, 0.0750 mmol), 1,3,5-tripropyl-1,3,5-triphosphinane-2,4,6-trione (93 µL, 0.162
mmol) and triethylamine (62 µL, 0.440 mmol) were mixed in THF (0.5 mL). The
mixture was stirred at rt for 1 h. The solvents were evaporated and the residue was
1
purified by preparative HPLC to give the title compound (12 mg, 43%). H NMR (400
MHz, methanol- d ) δ ppm 8.54 (br. s., 1H), 8.15 (br. s., 1H), 7.65 (s, 1H), 7.62 (d, J =
4
3
.5 Hz, 1H), 7.57–7.52 (m, 1H), 7.51–7.46 (m, 1H), 7.39–7.33 (m, 2H), 5.27 (t, J = 5.4
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Hz, 1H), 2.67–2.46 (m, 2H), 2.19–1.97 (m, 2H), 1.95–1.77 (m, 2H). HRMS (m/z):
+
[
M+H] calcd for C H ClFN O, 371.1075; obsd, 371.1084.
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16
4
4‐Bromo‐N‐[(4R)‐1‐(2‐fluorophenyl)‐4,5,6,7‐tetrahydro‐1H‐indazol‐4‐yl]pyridine
1
1
1
1
1
1
1
1
1
1
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0
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‐2‐carboxamide (32). 21 (20 mg, 0.0750 mmol), 4-bromopyridine-2-carboxylic acid
(15 mg, 0.0750 mmol), 1,3,5-tripropyl-1,3,5-triphosphinane-2,4,6-trione (93 µL, 0.162
mmol) and triethylamine (62 µL, 0.440 mmol) were mixed in THF (0.5 mL). The
mixture was stirred at rt for 1 h. The solvents were evaporated and the residue was
1
purified by preparative HPLC to give the title compound (9 mg, 29%). H NMR (400
MHz, methanol-d ) δ ppm 8.46 (br. s., 1H), 8.31 (br. s., 1H), 7.78 (d, J = 3.8 Hz, 1H),
4
7.65 (s, 1H), 7.57–7.52 (m, 1H), 7.52–7.46 (m, 1H), 7.40–7.31 (m, 2H), 5.27 (t, J = 5.5
Hz, 1H), 2.67–2.46 (m, 2H), 2.19–1.97 (m, 2H), 1.96–1.82 (m, 2H). HRMS (m/z):
+
[
M+H] calcd for C H BrFN O, 415.0570; obsd, 415.0567.
1
9
16
4
4-Fluoro-N-[(4R)-1-(2-fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-
yl]pyridine-2-carboxamide (33). 21 (25 mg, 0.093 mmol), 4-fluoropyridine-2-
carboxylic acid (16 mg, 0.11 mmol), HATU (39 mg, 0.10 mmol), and DIPEA (0.07
mL, 0.37 mmol) were dissolved in DCM (1.0 mL) and DMF (0.1 mL) and the mixture
was stirred at rt overnight. The mixture was purified by column chromatography (silica
column, 12 g) eluting with a gradient of EtOAc in hexanes (20–70%). The solvents
1
were evaporated to give the title compound as a white solid (28 mg, 85%). H NMR
(400 MHz, methanol-d ) δ 8.64 (dd, J = 8.0, 5.6 Hz, 1H), 7.90 (dd, J = 9.5, 2.4 Hz, 1H),
4
7
1
.65 (s, 1H), 7.58–7.45 (m, 2H), 7.42–7.32 (m, 3H), 5.28 (t, J = 5.7 Hz, 1H), 4.62 (s,
1
3
H), 2.59 (m, 2H), 2.22–1.83 (m, 5H). C NMR (101 MHz, methanol-d ) δ 171.2 (d,
4
J = 263 Hz), 164.9 (d, J = 3.7 Hz), 158.1 (d, J = 251), 154.6 (d, J = 6.6 Hz), 152.7 (d,
J = 7.1 Hz) 143.6, 140.0, 132.1 (d, 7.8 Hz), 130.0, 128.0 (d, 12 Hz), 126.2 (d, 4.0 Hz),
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1
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19.3, 117.8 (d, 20 Hz), 115.2 (d, 17 Hz), 111.1 (d, 19 Hz), 30.8, 22.30 (d, 3.4 Hz),
+
1.3. HRMS (m/z): [M+H] calcd for C H F N O, 355.1370; obsd, 355.1373.
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N-[(4R)-1-(2-Fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-yl]-4,5-
0
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dimethylpyridine-2-carboxamide (34). 27 (19 mg, 0.12 mmol) was suspended in
DMF (2 mL). HBTU (59 mg, 0.16 mmol) was added and the mixture was left to stir at
rt for 1 h. 21 (45 mg, 0.17 mmol) was added and the reaction mixture was stirred at rt
overnight. DIPEA (81 µL, 0.47 mmol) and HBTU (59 mg, 0.16 mmol) was added and
the reaction was stirred at rt overnight. The solvent was removed under a stream of
nitrogen and the product was purified by column chromatography eluting with
gradients of EtOAc in pentane (20–70%) to give the title compound (34 mg, 79%,
1
purity 90%), yield over two steps from 26. H NMR (400 MHz, CDCl ) δ 8.22 (s, 1H),
3
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.18 (d, J = 8.2 Hz, 1H), 8.02 (s, 1H), 7.68 (s, 1H), 7.47 (td, J = 7.7, 1.8 Hz, 1H), 7.44–
7.36 (m, 1H), 7.29–7.20 (m, 2H, overlapping with the solvent peak), 5.39–5.28 (m, 1H),
2
.68–2.48 (m, 2H), 2.35 (s, 3H), 2.30 (s, 3H), 2.23–2.09 (m, 1H), 2.02–1.81 (m, 3H).
1
3
C NMR (101 MHz, CDCl ) 164.3, 156.4 (d, J = 251 Hz), 148.6, 147.9, 147.3, 141.8,
3
1
1
39.6,135.5, 130.0 (d, J = 7.9 Hz), 128.7, 127.5 (d, J = 12 Hz), 124.9 (d, J = 3.8 Hz),
23.3, 118.3, 116.8 (d, J = 20 Hz), 42.4, 30.3, 21.7 (d, J = 4.7 Hz), 20.3, 19.5, 16.7.
+
HRMS (m/z): [M+H] calcd for C H FN O, 365.1778; obsd, 365.1780.
2
1
21
4
N-[(4R)-1-(2-Fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-yl]pyrimidine-2-
carboxamide (36). 21 (40 mg, 0.15 mmol), pyrimidine-2-carboxylic acid (27 mg, 0.16
mmol), HATU (62 mg, 0.16 mmol) and DIPEA (78 µL, 0.45 mmol) were dissolved in
DCM (1.5 mL) and DMF (0.2 mL) and the mixture was stirred at rt overnight. The
solvents were removed at reduced pressure and the residue was purified by column
chromatography (silica column, 12 g), first eluting with a gradient of EtOAc in hexanes
(20–80%) followed by a gradient of MeOH in DCM (2–10%). Fractions were pooled
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and evaporated. The residue was dissolved in DCM and the organic phase was washed
with sat NH Cl. The organic phase was evaporated to give the title compound (6 mg,
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1
12%). H NMR (400 MHz, CDCl ) δ 8.89 (d, J = 4.9 Hz, 2H), 8.22 (d, J = 8.1 Hz, 1H),
3
1
1
1
1
1
1
1
1
1
1
2
2
2
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2
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2
2
2
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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7
5
.71 (s, 1H), 7.55–7.36 (m, 3H), 7.32–7.19 (m, 2H, overlapping with solvent peak),
1
3
.48–5.37 (m, 1H), 2.70–2.47 (m, 2H), 2.27–2.11 (m, 1H), 2.02–1.80 (m, 3H); C
NMR (101 MHz, CDCl ) δ 161.7, 157.9, 157.6, 156.8 (d, J = 252 Hz), 142.0, 139.5,
3
1
1
30.2 (d, J = 7.7 Hz), 128.7, 127.5 (d, J = 11.7 Hz), 124.9 (d, J = 4.0 Hz), 122.7, 117.8,
+
16.8 (d, J = 20 Hz), 43.0, 30.2, 21.7 (d, J = 4.8 Hz), 20.3. HRMS (m/z): [M+H] calcd
for C H FN O, 338.1417; obsd, 338.1412.
18 16
5
N-[1-(2-Fluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-yl]benzamide (37). To a
mixture of 12 (10 mg, 0.0432 mmol), benzoic acid (11 mg, 0.0868 mmol), and DIPEA
(22 µL, 0.13 mmol) in DCM (2 mL) was added HATU (18 mg, 0.0476 mmol) and the
reaction was stirred at rt for 16 h. The organic layer was washed with brine (3  5 mL),
and concentrated. The residue was dissolved in MeOH (1 mL) and purified by HPLC.
Fractions were pooled and extracted with DCM to give the title compound (4 mg, 28%).
1
H NMR (400 MHz, CDCl ) δ 7.84–7.77 (m, 2H), 7.70 (s, 1H), 7.56–7.37 (m, 5H),
3
7
.32–7.20 (m, 2H), 6.29 (d, J = 7.8 Hz, 1H), 5.41–5.32 (m, 1H), 2.58 (m, 2H), 2.22–
+
2
.12 (m, 1H), 1.91 (m, 3H). HRMS (m/z): [M+H] calcd for C H FN O, 336.1512;
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0
18
3
obsd, 336.1526.
4
-Methyl-N-[(4R)-1-(2-methylphenyl)-4,5,6,7-tetrahydro-1H-indazol-4-
yl]pyridine-2-carboxamide (38). 22 (30 mg, 0.13 mmol), 4-methylpyridine-2-
carboxylic acid (19 mg, 0.14 mmol), HATU (55 mg, 0.145 mmol) and DIPEA (69 µL,
0.40 mmol) were dissolved in DCM (2 mL) and DMF (0.2 mL) and was stirred at rt for
4
h. Water (1 mL) was added and the phases were separated. The aqueous phase was
extracted with DCM (2  1 mL). The organic phases were combined and concentrated.
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