Discovery, optimization, and biological evaluation of 5-(2- (trifluoromethyl)phenyl)indazoles as a novel class of transient receptor potential A1 (TRPA1) antagonists

Article abstract of DOI:10.1021/jm401986p

A high throughput screening campaign identified 5-(2-chlorophenyl)indazole compound 4 as an antagonist of the transient receptor potential A1 (TRPA1) ion channel with IC₅₀ = 1.23 μM. Hit to lead medicinal chemistry optimization established the SAR around the indazole ring system, demonstrating that a trifluoromethyl group at the 2-position of the phenyl ring in combination with various substituents at the 6-position of the indazole ring greatly contributed to improvements in vitro activity. Further lead optimization resulted in the identification of compound 31, a potent and selective antagonist of TRPA1 in vitro (IC₅₀ = 0.015 μM), which has moderate oral bioavailability in rodents and demonstrates robust activity in vivo in several rodent models of inflammatory pain.

Full text of DOI:10.1021/jm401986p

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Article
Discovery, Optimization, and Biological Evaluation of 5-
(2-(trifluoromethyl)phenyl)-indazoles as a Novel Class
of Transient Receptor Potential A1 (TRPA1) Antagonists
Lisa Rooney, Agnès Vidal, Anne-Marie D’Souza, Nick Devereux, Brian Masick, Valerie Boissel, Ryan
West, Victoria Head, Rowan Stringer, Jianmin Lao, Matt J Petrus, Ardem Patapoutian, Mark Nash, Natalie
Stoakley, Moh Panesar, J Martin Verkuyl, Andrew M Schumacher, H. Michael Petrassi, and David C. Tully
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401986p • Publication Date (Web): 02 Jun 2014
Downloaded from http://pubs.acs.org on June 10, 2014
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Discovery, Optimization, and Biological Evaluation
of 5ꢀ(2ꢀ(trifluoromethyl)phenyl)ꢀindazoles as a
Novel Class of Transient Receptor Potential A1
(TRPA1) Antagonists
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Lisa Rooney, ‡ Agnès Vidal, † Anne-Marie D’Souza,‡ Nick Devereux, ‡ Brian Masick, † Valerie
Boissel, ‡ Ryan West, ‡ Victoria Head, ‡ Rowan Stringer, ‡ Jianmin Lao, † Matt J. Petrus, † Ardem
Patapoutian, † Mark Nash, ‡ Natalie Stoakley, ‡ Moh Panesar, ‡ J. Martin Verkuyl, ‡ Andrew M.
Schumacher†, H. Michael Petrassi, † and David C. Tully*†
†Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San
Diego, California 92121, United States
‡Novartis Institutes for Biomedical Research, Wimblehurst Road, Horsham RH12 5AB, United
Kingdom
RECEIVED DATE
ABSTRACT. A high throughput screening campaign identified 5ꢀ(2ꢀchlorophenyl)ꢀindazole
Compound 4 as an antagonist of the Transient Receptor Potential A1 (TRPA1) ion channel with
IC50=1.23 µM. Hit to lead medicinal chemistry optimization established the SAR around the
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indazole ring system, demonstrating that a trifluoromethyl group at the 2ꢀposition of the phenyl
ring in combination with various substituents at the 6ꢀposition of the indazole ring greatly
contributed to improvements in vitro activity. Further lead optimization resulted in the
identification of Compound 31, a potent and selective antagonist of TRPA1 in vitro (IC50 = 0.015
µM), which has moderate oral bioavailability in rodents and demonstrates robust activity in vivo
in several rodent models of inflammatory pain.
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KEYWORDS. TRPA1 antagonist, ANKTM1, transient receptor potential ion channel,
inflammatory pain, visceral pain
INTRODUCTION
Transient Receptor Potential A1 (TRPA1) is a nonꢀselective cation channel that functions
as a polymodal sensory receptor and plays an important role in nociception, or the sensing of
noxious stimuli. The channel is expressed in sensory neurons, particularly within the dorsal root
ganglia (DRG), and in the visceral tissues such as the small intestine, colon, bladder, and
stomach.1ꢀ4 TRPA1 is activated by Ca2+,5,6 cold temperatures,3 inflammatory peptides,7 and
naturally occurring electrophilic compounds such as allyl isothiocyanate (AITC),
cinnamaldehyde, and allicin, which covalently modify the protein and result in the channel
opening.6,8,9 TRPA1 activation by these agents likely accounts for the pungent sensation elicited
by foods such as garlic, horseradish, and wasabi. In addition, TRPA1 activation in sensory
neurons results in secretion of proꢀinflammatory peptides such as substance P and calcitonin
geneꢀrelated peptide (CGRP), which both play a role in pain transmission from the DRG
neurons.3,7,10,11 TRPA1 signaling is believed to play a role in nociception involving neuropathic
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pain resulting from injury or disease, as well as visceral pain stemming from underlying
gastrointestinal disease or urinary bladder dysfunction.12
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Human genetics further support a role for TRPA1 in pain. A gain of function mutation in
TRPA1 has been linked to Familial Episodic Pain Syndrome (FEPS), and patients carrying this
mutation experience episodes of excruciating pain, mainly in the abdominal region, which are
triggered by fasting, cold, or physical exercise.13 Conversely, TRPA1 knockout mice show a
reduced pain response to TRPA1 agonists, including inflammatory mediators such as 4ꢀ
hydroxynonenal.8,14ꢀ17 This evidence suggests that TRPA1 may be responsible for transmitting
pain signals and that antagonizing its calcium channel activity may be a useful approach for the
treatment of pain.18,19
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There have been only a few classes of TRPA1 antagonists reported in the literature to date:18ꢀ20
oximes such as APꢀ18 (1)21 and Aꢀ967079 (2)22,23 and xanthines such as HCꢀ030031 (3)24,25
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although there are several additional scaffold classes which have been claimed in patents
applications and subsequently evaluated in recent review articles.19,26,27 Consistent with the
TRPA1 knockout mouse findings, 2 and 3 exhibited analgesic effects in rodent pain models in
response to sensitization induced by mustard oil.23,24 Furthermore, oral administration of 3 was
shown to reduce Complete Freund’s Adjuvant (CFA)ꢀinduced inflammatory pain, as well as
neuropathic pain caused by spinal nerve ligation in rats.24 Taken together, these genetic and
pharmacological results suggest the potential utility of antagonizing TRPA1 to treat pain.
However, the relatively low potency of 3 (IC50 = 2.3 µM) and poor in vivo pharmacokinetics of 2
(high Cl, short T1/2 in rodents) leave plenty of room for the optimization of potent TRPA1
antagonists with improved in vivo pharmacokinetics that would better suited for further
pharmaceutical development.
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Herein we report the discovery and SAR surrounding a novel scaffold class of TRPA1
antagonists derived from 5ꢀphenyl indazole. Compound 4 was originally identified in a highꢀ
throughput screening (HTS) campaign of our proprietary compound collection. We initiated
medicinal chemistry on this scaffold with the primary objectives of improving in vitro potency
and in vivo PK, and our efforts have resulted in a novel series of potent TRPA1 antagonists with
potent activity in vivo in rodent models of inflammatory pain.
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Chemistry
The 5ꢀphenyl indazoles could all be synthesized using a Suzuki palladium catalyzed crossꢀ
coupling reaction to form the biaryl scaffold (Scheme 1). The reaction of unprotected 5ꢀbromo
indazole (6a) with commercially available boronic acids proceeded to give sufficient yields of
many of the desired products shown in Table 1. Where the aryl boronic acids were not available,
the alternative coupling of tertꢀbutylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀ
indazoleꢀ1ꢀcarboxylate (9) with the aryl bromide provided the products in a single step as the
protecting group was lost under the reaction conditions.
For the investigation of the effect of substitution on the indazole ring, the 5ꢀbromo indazoles
required were synthesized from their corresponding 4ꢀbromoꢀ2ꢀmethylaniline (5). Route A
provided acetyl protected indazoles which were taken into the Suzuki crossꢀcoupling step. The
acetyl protecting group was not stable under the Suzuki conditions, and in the absence of a
protecting group on the indazole, the competing debromination reaction tended to become more
prevalent. The rate of the cross coupling versus the deacetylation varied due to the nature of the
R group, and therefore a more stable protecting group was desired. Route B provided the
unprotected indazoles in good yields and a THP group was then introduced prior to the Suzuki
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reaction. Following the completion of this work it has been noted that conditions have now been
reported to allow the Suzukiꢀtype cross coupling of unprotected indazoles and other azoles to
proceed with high yields.28
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Late stage functionalization from the common ester intermediate 10h was used to access 35,
40 and 42 (Scheme 2). Reduction to the alcohol 45 and alkylation using methyl iodide, followed
by removal of the THP protecting group provided 40. Oxidation of alcohol 45 with manganese
dioxide and treatment with DAST introduced the CF2H group. Hydrolysis of the ester 10h and
amide formation via the acid chloride allowed access to amide derivative 44.
The intermediate 10d, also used in the synthesis of 36 could be further transformed to the 6ꢀ
cyclopropyl derivative 34 (Scheme 3). Deprotection of the aryl methyl ether and formation of
the aryl triflate 51 allowed for Suzuki coupling with cyclopropyl boronic acid to introduce the
alkyl substituent.
Results and Discussion
As part of our efforts to discover novel chemical scaffolds that could inhibit TRPA1 activation,
a high throughput screen (HTS) of our compound collection was conducted on approximately 1.8
M compounds using an antagonist mode FLIPR Ca2+ imaging assay in 1536ꢀwell format.
Compound 4 was identified as a primary hit in this HTS using selection criteria of >50%
inhibition at 10 µM. Following the hitꢀpick and reconfirmation assay, compound 4 was shown to
have IC50 = 1.23 µM with 91% inhibition of TRPA1 activation by cinnamaldehyde. Indazole 4
was considered to be a particularly attractive starting point for medicinal chemistry optimization
due to its relative drugꢀlikeness based on calculated parameters such as low molecular weight
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(MW = 228 Da), number of rotatable bonds (=1), number of hydrogen bond donors (=1), and
number of hydrogen bond acceptors (=2).
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During our initial exploration of the SAR of this scaffold, we first evaluated the effect of the
phenyl substituent by directly replacing the chlorine on compound 4 with other substituents.
Replacement of the chlorine with hydrogen (compound 11) results in complete loss of activity,
while the fluoro analog 12 was a weak, partial blocker of TRPA1 activation. The SAR of the
phenyl ring demonstrates sensitivity toward the size of the alkyl substituent in that ethyl and
isopropyl groups are fairly well tolerated (compounds 14 and 15, respectively), while a methyl
group (compound 13) or a tertꢀbutyl group (compound 16) results in a significant loss of
potency. Following the SAR at the ortho position of the phenyl ring, the trifluoromethyl group
was then explored in compound 17 (IC50 = 0.164 µM). The favorable steric and unique
inductive electronꢀwithdrawing properties of the trifluoromethyl group ultimately turned out to
be the most preferred substituent as reflected by the order of magnitude improvement in potency
from the original hit compound 4. The SAR around this position proved to be quite steep as
exemplified by loss of potency by subtle changes to compound 17 such as replacing fluorine
with hydrogen, as in the difluoromethyl analog 18, or inserting oxygen, as in the
trifluoromethoxy analog 19. A similar loss in potency was also observed with the small polar
nitrile group (compound 20) despite the strong electron withdrawing nature of this substituent.
Prior to embarking on more extensive lead optimization efforts, we investigated whether this
indazole scaffold was able to block TRPA1 activation by different chemical agonists. In
addition to cinnemaldehyde, the TRPA1 agonist used in our primary FLIPR assay (at EC80
concentration), compound 17 was able to effectively block TRPA1 activation by a variety of
other agonists, including AITC and also the highly potent agonist dibenzo[b,f][1,4]oxazepineꢀ4ꢀ
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carboxamide, previously described by Gijsen, et. al.29 As shown in Table 4, IC50 values for
blocking TRPA1 activation are similar (roughly within threeꢀfold) with different chemical
agonists, each with effectively complete inhibition. This attribute was reassuring, and based on
our internal experience with this and other scaffolds suggested that this indazole series of
antagonists would likely have the ability to block TRPA1 activation regardless of the nature of
activating stimuli.
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In continuation of our lead optimization efforts from compound 17, we next chose to explore
whether additional substituents were permitted at any other position of the phenyl ring or around
the indazole ring with the optimal CF3 group in place. In consideration of the initial SAR for
this scaffold which was quite steep, we set out to introduce subtle changes by strategically
installing either fluorine or a methyl group around the phenyl indazole ring system in order to
learn which positions would tolerate or benefit from substituents (Table 2). Neither the 3ꢀ nor 4ꢀ
positions of the phenyl ring tolerated the addition of fluorine as demonstrated by compounds 21
and 22, while the 5ꢀfluoro and 6ꢀfluoro analogs (23 and 24) resulted in a loss of activity.
Substitution of the indazole ring with fluorine demonstrated subtle effects, with the 4ꢀ
fluoroindazole analog (25) losing twofold in potency over 17, while installation of a fluorine at
the indazole 6ꢀposition affords a threefold improvement in potency (compound 26). Since the
incorporation of methyl groups to either the 3ꢀ or 7ꢀpositions of the indazole resulted in a steep
loss of activity (compounds 27 and 28), we chose to focus our efforts on the indazole 6ꢀposition
for further exploration of the SAR of this scaffold.
The SAR of 6ꢀsubstituted indazole analogs is described in Table 3, with each of the analogs
bearing the requisite trifluoromethyl group at the ortho position of the phenyl ring. In general
small, hydrophobic substituents tend to improve the potency, while hydrophobic groups with
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larger steric demands tend to result in a significant loss of activity. The more optimal
substituents were found to be chloro (compound 30) and small alkyl (Me, Et) or alkyl ethers
(MeO, EtO, CF3O) resulting in potent in vitro activities with IC50 values generally below 50 nM.
Interestingly, while the 6ꢀisoꢀpropyl analog is nearly inactive (compound 33), the cyclopropyl
remains quite potent (compound 34). Likewise a similar trend is observed for isopropoxy (38,
IC50 = 0.970 µM) versus cyclopropylmethyloxy (39, IC50 = 0.026 µM). Both of these
observations further demonstrate the subtleties of the steric and electronic demands in this region
of the scaffold. Small polar substituents such as a nitrile are tolerated (43), but do not confer any
improvement in potency over the lead compound 17. In contrast, larger polar substituents are
not tolerated, particularly when bearing a hydrogen bond donor, such as simple amides
exemplified by compound 44.
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In advance of selecting compounds for evaluation in preclinical rodent models, several of the
more potent analogs were tested in vitro against rat and mouse TRPA1 in order to determine the
extent of species crossꢀreactivity (Table 5). There have been previous reports demonstrating
speciesꢀspecific differential pharmacology, whereby antagonists of human TPRA1 exhibit
agonist activities at rat and/or mouse TRPA1 homologs.6,30,31 As outlined in Table 5, most
compounds tend to exhibit on average a threeꢀfold to fiveꢀfold drop off in activity between
hTRPA1 and either rTRPA1 or mTRPA1, with some wider variances. In general, both rat and
mouse TRPA1 homologs appear to be less tolerant of the larger substituents at the 6ꢀposition of
the indazole ring, and as such the sterically less demanding hydrophobic substituents such as
methyl (31) and chloro (30) tended to be more potent in vitro against rat and mouse TRPA1.
Furthermore, as shown in Table 4, compound 31 was also highly effective at blocking TRPA1
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activation by multiple agonists, which proved to be a consistent trend with this indazole scaffold
class of TRPA1 antagonists.
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We also tested this subset of analogs against several related thermoꢀTRP channels that have
been shown to play a role in the transduction of nociceptive pain, including the vanilloid
receptors TRPV1 and TRPV3, as well as the cold and menthol receptor TRPM8. As shown in
Table 5, this series of indazoles displays only weak or partial antagonist activity towards each of
the TRP channels tested, each with greater than 100ꢀfold window of selectivity over TRPA1.
Potent, antagonist activity towards TRPA1 across multiple species and against multiple agonists,
combined with a favorable selectivity profile over related thermoꢀTRP channels provided
confidence that observed analgesic activity in vivo would be mediated through TRPA1 directly.
Additionally, as the indazole is a common hingeꢀbinding motif found in kinase inhibitors, we
tested compounds 17, 30, 31, and 36 across our inꢀhouse panel of kinases, and no inhibitory
activity was observed below 10 ꢁM.
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The in vivo pharmacokinetic properties in male Wistar rats selected indazole analogs are
summarized in Table 6. Single dose intravenous PK demonstrated that the volumes of
distribution for most analogs from this series tend to remain consistently within the moderate
range of 1.6 – 3.6 L/kg, while the nature of the substituent at the 6ꢀposition of the indazole ring
had a significant influence on the in vivo clearance rates. Analogs 17 and 30, with hydrogen and
chlorine, respectively, had relatively low clearance, while the substituents that are more prone to
oxidative metabolism such as methyl (31) and ethers (36 and 39) lead to relatively higher in vivo
clearance and shorter T1/2 in the rat. Oral pharmacokinetics were studied using a 10 mg/kg dose
administered from a 0.5% methylcelluose/0.5% Tween80 aqueous suspension formulation. The
overall trend for oral exposure reflected the relative trend from IV clearance, whereby the
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compounds with higher IV clearance had overall lower oral AUC and Cmax. Oral bioavailability
was moderate in the range of 37ꢀ63% for each of the compounds with low to moderate clearance,
whereas 39, with a fairly high clearance in rat (Cl = 50.0 mL/min/kg), exhibited low oral
bioavailability (%F = 4).
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Compound 31 was prioritized for in vivo efficacy studies due to the combination of attractive
in vivo PK properties and in vitro potency across all species of TRPA1 tested. Established
models of somatic and visceral inflammatory pain were used according published methods.32
The analgesic effect of 31 was evaluated in the Freund’s Complete Adjuvant (FCA)ꢀinduced
mechanical hyperalgesia model of inflammatory pain.33,34 Oral administration of 31 significantly
reversed intraplantar FCAꢀinduced mechanical hyperalgesia, with 58.6% reversal after 1 h
following a 10 mg/kg oral dose (D50 = 5.5 mg/kg) (Figure 2). The effect of 31 was also
evaluated in the mustard oil (MO)ꢀinduced allodynia model of visceral pain in the mouse.35 In
this model, orally administered 31 also elicited a significant reversal of MOꢀinduced allodynia
with a full effect after 1 h with a 10 mg/kg dose (Figure 3). The pharmacokinetic profile of 31 in
mouse had been established to be reasonably consistent with what was observed in the rat (Table
7) with a slightly higher clearance and lower oral bioavailability.
TRP channels in general mediate thermosensation and thermoregulation in mammals, and
specifically TRPA1 is believed to be a sensor of noxious cold and appears to be activated by cold
temperatures. In one study, TRPA1ꢀ/ꢀ knockout mice displayed a reduced sensitivity to cold, as
measured by paw withdrawal from a 0 °C plate or paw shaking in response to acetoneꢀinduced
cooling.17 We therefore evaluated the effect of an orally administered TRPA1 antagonist (31) on
thermosensation in naive mice when placed upon a cold (ꢀ5 °C) surface. As shown in Figure 4,
naïve mice administered with compound 31 demonstrated a reduced sensitivity to the cold
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surface in a doseꢀdependent manner, as measured by the latency to respond with behavioural
queues (determined by the time to the first observed hind paw flick/shake, lick, or jump) similar
in magnitude to that observed with the TRPA1 knockout phenotype.17
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Antagonists of the related sensory TRP channel, TRPV1, have been shown to elicit significant
increases in body temperature.36 This marked hyperthermia has limited the clinical utility of
TRPV1 antagonists for the treatment of pain.37 To understand whether TRPA1 antagonists
would decrease body temperature, compound 31 was orally administered to naïve rats at and
above the therapeutic dose range (Figure 5). The cannabinoid agonist WIN55,212ꢀ238 was
chosen as the positive control because historically in our laboratory this compound induces a
robust body temperature change over the course of the experiment, whereas the TRPV1 agonist
capsaicin gives a shorterꢀduration hypothermia and could elicit a nociceptive response.
Important to note for the future development potential of TRPA1 antagonists, Compound 31 did
not alter body temperature despite the effect observed on noxious cold sensation.
Conclusion
In summary, medicinal chemistry efforts initiated from a HTS hit compound 4 resulted in the
discovery of a novel series of TRPA1 antagonists derived from a 5ꢀphenyl indazole scaffold.
The SAR demonstrated a strong preference for a trifluoromethyl group at the 2ꢀposition of the
phenyl ring and small hydrophobic substituents at the 6ꢀposition of the indazole ring system, and
in combination, provided a number of potent and selective TRPA1 antagonists. In vivo rodent
PK of selected compounds with potent in vitro antagonist activities across multiple species of
TRPA1 were evaluated, and they were found to have adequate pharmacokinetic profiles with
modest oral bioavailability in rodents. Compound 31 was selected for further evaluation in
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rodent pain models, and following a single oral dose of 10 mg/kg, compound 31 significantly
reversed FCAꢀinduced inflammatory pain in the rat as well as mustard oilꢀinduced allodynia in
the mouse. An effect on the sensation of cold at ꢀ5 °C was also observed in the mouse, which is
consistent with the TRPA1 KO phenotype and TRPA1 regulation in vitro by noxious cold
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temperatures.
Compound 31 and related indazoles have provided us with useful
pharmacological tools in preꢀclinical rodent models for the evaluation of TRPA1 as a target for
the treatment of pain. Likewise, as the potential exists for the development of TRPA1
antagonists for indications beyond pain, valuable tool compounds such as 31 can be utilized in
preꢀclinical in vivo models to explore the role of TPRA1 in various disease model settings where
TRPA1 is believed to play a key role.
Experimental Section
General Methods All commercial reagents and solvents were used as received. 5ꢀBromoꢀ1Hꢀ
indazole
and
tertꢀButyl
5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀindazoleꢀ1ꢀ
carboxylate were purchased from SigmaꢀAldrich. 5ꢀbromoꢀ6ꢀmethylindazole (6c) was purchased
from Manchester Organics. The synthetic procedures for the preparation of substituted 5ꢀ
bromoindazoles 6b, 6d-j and 7a-f from commercial 2ꢀmethylanilines are provided in the
Supporting Information. 1H and 13C NMR spectra were obtained on a Bruker AVANCE 400
NMR spectrometer using ICONꢀNMR. Spectra were measured at 298K and were referenced
using the solvent peak. Mass spectra were run using electrospray ionization on either a
Micromass Platform Mass Spectrometer or SQD Mass Spectrometer. Final compounds had a
purity of ≥ 95% as assessed by analytical HPLC using either an Agilent 1100 HPLC or Waters
Acquity UPLC. High resolution mass spectrometry was obtained using LTQ Orbitrap. Melting
points were measured by DSC on a Perkin Elmer Pyris 1, heating from 25 °C to 300 °C at 20
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°C/min under nitrogen. Elemental analysis was measured on a Leco CHNSꢀ932. Microwave
reactions were performed using a Biotage Initiator Robot Sixty. All in vivo study protocols were
approved by the Institutional Animal Care and Use Committee (IACUC) and the Novartis
Animal Welfare and Ethics Committee and performed in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Animals (USA) and Home Office (United
Kingdom) guidelines.
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Typical Procedure for Suzuki Coupling of tert-Butyl 5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-indazole-1-carboxylate (9) used for the synthesis of 14, 15, 16, 20, 21
and 22.
5-(2-Isopropyl-phenyl)-1H-indazole (15)
To 1ꢀbromoꢀ2ꢀisopropylbenzene (43.4 mg, 0.218 mmol) in water (0.2 mL) and ethanol (0.7 mL)
was added tertꢀbutyl 5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀindazoleꢀ1ꢀcarboxylate
(75 mg, 0.218 mmol), sodium carbonate (69.3 mg, 0.654 mmol) and
tetrakis(triphenylphosphine)palladium(0) (50.4 mg, 0.044 mmol) and the mixture was heated to
120 ºC in the microwave for 1 h. The reaction mixture was partitioned between water and ethyl
acetate. The organic layer was separated, dried over magnesium sulfate, and the solvent was
evaporated. The crude residue was purified by column chromatography on silica (0ꢀ100%
1
EtOAc/isoꢀhexane) to give 15 as a colorless solid (40mg, 0.169 mmol, 78% yield): H NMR
(400 MHz, DMSOꢀd6) δ 13.10 (s, 1H), 8.10 (s, 1H), 7.62 (s, 1H), 7.59 (d, J = 8.5 Hz, 1H), 7.41
(d, J = 7.9 Hz, 1H), 7.35 (dd, J = 7.4, 7.4 Hz, 1H), 7.22 (m, 2H), 7.18 (m, 1H), 3.00 (sept, J = 6.8
Hz, 1H), 1.10 (s, 6H); MS(ESI) m/z 237.13 [M+H]+.
Typical Procedure for Suzuki coupling of 5-Bromo-1H-indazole used for the synthesis of 4,
12, 17, 18, 19, 23, 24, 25, 27 and 28.
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5-(2-(Trifluoromethyl)phenyl)-1H-indazole (17)
5
6
7
8
A mixture of 5ꢀbromoꢀ1Hꢀindazole (100 mg, 0.5 mmol), 2ꢀ(trifluoromethyl)phenylboronic acid
(106 mg, 0.58 mmol, 1.1 equiv), Cs2CO3 (413 mg, 1.27 mmol, 2.5 equiv), chloro(diꢀ2ꢀ
norbornylphosphino)(2'ꢀdimethylaminoꢀ1,1'ꢀbiphenylꢀ2ꢀyl)palladium (II) (28 mg, 0.05 mmol, 0.1
equiv) in water (0.1 mL) and dioxane (1 mL) was sealed and heated in the microwave for 5 min
at 160 ºC. The mixture was partitioned between water and EtOAc. The organic phase was
separated and dried over MgSO4 and then concentrated in vacuo. The residue was purified by
9
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silica gel chromatography to give 17 as a white solid. MP 120ꢀ126º C; H NMR (400 MHz,
DMSOꢀd6) δ 13.18 (s, 1H), 8.11 (s, 1H), 7.84 (d, J = 7.9 Hz 1H), 7.72 (dd, J = 7.5 Hz; 7.5 Hz,
1H), 7.68 (s, 1H), 7.61 (dd, J = 7.8; 7.8 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 7.6 Hz,
13
1H), 7.28 (d, J = 8.6 Hz, 1H); C NMR (125MHz, DMSOꢀd6) δ 141.2 (q, J = 1.8 Hz), 139.2,
133.8, 132.7, 132.1, 131.3, 127.7, 127.2, 127.0 (q, J = 32 Hz), 126.0 (q, J = 5.0 Hz), 124.2 (q, J
= 272.2 Hz), 122.4, 120.3, 109.5; 19F NMR (376 MHz, DMSOꢀd6) δ ꢀ55.35; EA: Calc for
C14H9F3N2: C, 64.12; H, 3.46; N, 10.68: Found: C, 63.99; H, 3.37, N, 10.75; HRMS m/z Calc for
C14H9F3N2: 263.0796, Found: 263.0804.
Typical procedure for Suzuki Coupling of 1-(5-Bromo-1H-indazol-1-yl)ethanones used for
the synthesis of 30, 32, 33, 37, 38 and 41.
6-Chloro-5-(2-(trifluoromethyl)phenyl)-1H-indazole (30)
A mixture of 7a (500 mg, 2.160 mmol), 2ꢀtrifluoromethylphenylboronic acid (410 mg, 2.160
mmol), cesium carbonate (2111 mg, 6.48 mmol), tetrakis(triphenylphosphine)palladium(0) (499
mg, 0.432 mmol) in water (1 mL) and dioxane (6 mL) was sealed and heated for 1 hr at 120 ºC in
the microwave. The reaction mixture was partitioned between water and ethyl acetate. The
organic phase was separated, dried over magnesium sulfate, and the solvent was evaporated.
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The residue was purified by silica gel chromatography (0ꢀ50% EtOAc/isoꢀhexane) to yield 30 as
5
6
1
a colourless solid (70mg, 0.24 mmol, 11% yield): H NMR (400 MHz, DMSOꢀd6) δ 13.35 (s,
7
8
9
1H), 8.14 (s, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.75 (m, 3H), 7.68 (m, 1H), 7.41 (d, J = 7.5 Hz, 1H);
MS(ESI) m/z 297.19 [M+H]+.
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Typical Procedure via THP Protection Route used for the synthesis of 13, 26, 31, 36, 39, 42
and 43.
6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (31)
5-bromo-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (8c)
To a stirred suspension of 5ꢀbromoꢀ6ꢀmethylꢀ1Hꢀindazole (6c) (10 g, 47.4 mmol) in chloroform
(200 ml) was added pꢀtoluenesulfonic acid monohydrate (0.901 g, 4.7 mmol) followed by 3,4ꢀ
dihydroꢀ2Hꢀpyran (8.66 mL, 95 mmol) and the mixture was stirred at room temperature
overnight. The reaction mixture was then heated at 50 °C for 5 h. The product mixture was
concentrated under vacuum, dissolved in ethyl acetate, and washed with water, saturated aqueous
sodium hydrogen carbonate and brine. The organics were dried over sodium sulfate, filtered and
concentrated in vacuo to yield the title compound (15.5 g) which was used in the next step
without further purification. Purification of a small sample by column chromatography (0ꢀ30%
EtOAc/isoꢀhexane) and recrystallization from diethyl ether provided a sample for full analysis:
1H NMR (400 MHz, DMSOꢀd6) δ 8.04 (s, 1H), 8.03 (s, 1H), 7.75 (s, 1H), 5.80 (dd, J = 9.7, 2.5
Hz, 1H), 3.91ꢀ3.84 (m, 1H), 3.79ꢀ3.68 (m, 1H), 2.44 (s, 3H), 2.46ꢀ2.32 (m, 1H), 2.09ꢀ1.99 (m,
1H), 1.99ꢀ1.91 (m, 1H), 1.81ꢀ1.66 (m, 1H), 1.62ꢀ1.54 (m, 2H), 13C NMR (100 MHz, DMSOꢀd6)
δ 138.7, 134.9, 132.6, 124.0, 123.8, 117.0, 111.6, 84.0, 66.5, 28.8, 24.7, 23.4, 22.2; EA: Calc for
C13H15BrN2O: C, 52.9; H, 5.12; N, 9.49: Found: C, 53.05; H, 5.30, N, 9.51; MS(ESI) m/z 295.23
[M+H]+.
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6-Methyl-1-(tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole (10c)
A mixture of 8c (8 g, 27.1 mmol), 2ꢀ(trifluoromethyl)phenylboronic acid (5.15 g, 27.1 mmol),
cesium carbonate (26.5 g, 81 mmol), tetrakis(triphenylphosphine)palladium(0) (3.13 g, 2.71
mmol) in water (15 mL) and dioxane (80 mL) was sealed and heated in the microwave for 1 hour
at 120ºC. The mixture was partitioned between water and ethyl acetate. The organic phase was
separated and dried over magnesium sulfate and concentrated in vacuo. The residue was purified
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by chromatography on silica to give 10c as a white solid (8.7 g, 24.14 mmol, 89% yield): H
NMR (400 MHz, DMSOꢀd6) δ 8.06 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.73 (dd, J = 7.5; 7.5 Hz,
1H), 7.63 (m, 2H), 7.49 (s, 1H), 7.37 (dd, J = 8.3, 8.3 Hz, 1H), 5.85 (m, 1H), 3.91 (m, 1H), 3.77
(m, 1H), 2.45 (m, 1H), 2.10 (s, 3H), 2.10ꢀ1.95 (m, 2H), 1.82ꢀ1.70 (m, 1H), 1.63ꢀ1.57 (m, 2H).
13C NMR (125MHz, DMSOꢀd6) δ 140.1, 139.3, 134.6, 133.4, 132.5, 132.4, 132.4, 128.0, 127.5
(q, J = 28.8 Hz), 125.8 (q, J = 5.6 Hz), 124.1 (q, J = 273.6Hz), 121.8, 120.8, 110.1, 83.9, 66.5,
28.9, 24.8, 22.3, 20.8; HRMS: Calc for C20H20F3N2O: 361.153; Found: 361.152.
6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (31)
To a solution of 10c (5 g, 13.87 mmol) in methanol (150 mL) was added 4M HCl in dioxane
(2.108 ml, 69.4 mmol) and the mixture was stirred at rt overnight then left to stand for 3 nights.
The mixture was partitioned between ethyl acetate and saturated aqueous sodium hydrogen
carbonate. The organic phase was separated and the aqueous extracted with ethyl acetate. The
combined organics were dried over magnesium sulfate and concentrated in vacuo. The residue
was purified by chromatography on silica (0ꢀ70% EtOAc/isoꢀhexane) and then was dissolved in
ethanol (~100 mL) and macroporous polystyreneꢀ2,4,6ꢀtrimercaptotriazine (4 g) was added and
mixture stirred at 40°C for 1h. The mixture was filtered and to the filtrate was added a further 2 g
of macroporous polystyreneꢀ2,4,6ꢀtrimercaptotriazine and stirred 40 °C for 1 h. This was filtered
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again and filtrate evaporated. The residue was recrystallized from methanol/water to give 31 as a
5
6
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white solid (1.52 g, 5.5 mmol, 40% yield): Mp 176ꢀ182ºC; H NMR (400 MHz, DMSOꢀd6) δ
7
8
9
13.01 (br s, 1H), 8.00 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.72 (m, 1H), 7.62 (m, 1H), 7.47 (s, 1H),
7.43 (s, 1H), 7.35 (d, J = 7.5 Hz, 1H), 2.05 (s, 3H); 13C NMR (125MHz, DMSOꢀd6) δ 140.4 (q,
J = 2.1 Hz), 139.9, 134.1, 133.4, 132.4, 132.1, 131.6, 127.9, 127.6 (q, J = 28.5 Hz), 125.9 (q, J =
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5.4 Hz), 124.1 (q, J = 274.9 Hz), 120.7, 120.4, 109.8, 20.7; F NMR (400 MHz, DMSOꢀd6) δ ꢀ
57.48; EA: Calc for C15H11F3N2: C, 65.22; H, 4.01; N, 10.14: Found: C, 65.04; H, 4.32, N,
10.28; MS(ESI) m/z 277.16 [M+H]+.
1-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (29)
17 (80 mg; 0.3 mmol) was added to a mixture of 60 % NaH (15 mg; 0.37 mmol) in DMF (1 mL).
After 15 min at room temperature, iodomethane (52 mg; 0.36 mmol) was added, the reaction was
stirred for 2 hr, and then diluted with water and extracted with EtOAc. The extracts were rinsed
with water and brine, dried over magnesium sulfate evaporated, and the product 29 isolated by
1
HPLC: H NMR (400 MHz, DMSOꢀd6) δ 8.10 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.75ꢀ7.62 (m,
3H), 7.61 (dd, J = 7.6, 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.32 (d, J = 8.9 Hz, 1H), 4.10 (s,
3H).MS(ESI) m/z 277.1 [M+H]+.
6-Cyclopropyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (34)
1-(Tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazol-6-ol (50)
To a solution of 1ꢀdodecanethiol (1.613 g, 7.97 mmol) in anhydrous Nꢀmethylꢀ2ꢀpyrrolidinone
(3 ml) under nitrogen was added sodium hydroxide (638 mg, 15.94 mmol). The resulting mixture
was stirred at room temperature for 30 minutes and treated with 10d (2.0 g, 5.31 mmol) in Nꢀ
methylꢀ2ꢀpyrrolidinone (7 mL) via syringe. The vial was subsequently sealed and heated at 130
°C. After 5 h, the mixture was acidified with 2M hydrochloric acid and extracted with ethyl
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acetate. The combined organic extracts were dried over magnesium sulfate, filtered and
concentrated to give a yellow oil. The residue was purified by chromatography on silica (0ꢀ
100% EtOAc/isoꢀhexane) to afford 50 as a foamy colorless solid (1.84 g, 5.08 mmol, 96% yield):
1H NMR (400 MHz, DMSOꢀd6) δ 9.80 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.70 (dd, J
= 7.5, 7.5 Hz, 1H), 7.60 (dd, J = 7.7, 7.7 Hz, 1H), 7.40 (s, 1H), 7.35 (dd, J = 7.7, 7.7 Hz, 1H), 7.0
(s, 1H), 5.70 (m, 1H), 3.90 (m, 1H), 3.70 (m, 1H), 2.40 (m, 1H), 2.05 (m, 2H), 1.75 (m, 1H),
1.60 (m, 2H); MS(ESI) m/z 363.3 [M+H]+.
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1-(Tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazol-6-yl
trifluoromethanesulphonate (51)
1ꢀ(Tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ5ꢀ(2ꢀ(trifluoromethyl)phenyl)ꢀ1Hꢀindazolꢀ6ꢀol (50) (100 mg,
0.276 mmol), phenyl triflimide (99 mg, 0.276 mmol) and potassium carbonate (114 mg, 0.828
mmol) in tetrahydrofuran (1 mL) were heated using microwave radiation at 120 °C for 24
minutes. After cooling, the mixture was filtered and washed with ethyl acetate. The filtrate was
evaporated to give a pink residue. The residue was purified by chromatography on silica (0ꢀ
100% EtOAc/isoꢀhexane) followed by repurification (0ꢀ30% EtOAc/isoꢀhexane) to afford 51
1
(110 mg, 0.21 mmol, 77% yield): H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.72 (d, J = 7.9
Hz, 1H), 7.64 (s, 1H), 7.58 (d, J = 5.8 Hz, 1H), 7.51 (m, 2H), 7.31 (m, 1H), 5.69 (m, 1H), 3.98
(m, 1H), 3.72 (m, 1H), 2.48 (m, 1H), 2.10 (m, 2H), 1.70 (m, 3H); 19F (376 MHz, CDCl3) δ ꢀ59, ꢀ
74; MS(ESI) m/z 495.4 [M+H]+.
6-Cyclopropyl-1-(tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole (52)
1ꢀ(Tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ5ꢀ(2ꢀ(trifluoromethyl)phenyl)ꢀ1Hꢀindazolꢀ6ꢀyl
trifluoromethanesulphonate (51) (110 mg, 0.222 mmol), cyclopropylboronic acid (287 mg, 3.34
mmol), tetrakis(triphenylphosphine)palladium(0) (51 mg, 0.044 mmol) and cesium carbonate
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(217 mg, 0.667 mmol) in dioxane (2 mL) and water (0.22 mL) were heated using microwave
radiation at 100 °C for 1 h. After cooling to room temperature, the reaction was quenched with
water and extracted with ethyl acetate. The combined organic extracts were dried over
magnesium sulfate, filtered and concentrated in vacuo to give a yellow residue. The residue was
purified by chromatography on silica (0ꢀ60% EtOAc/isoꢀhexane) to afford 52 (63mg, 0.15 mmol,
68% yield): MS(ESI) m/z 387.4 [M+H]+.
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6-Cyclopropyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (34)
To a solution of 52 (63 mg, 0.163 mmol) in methanol (1 mL) was added 4M hydrogen chloride
in dioxane (0.204 mL, 0.815 mmol). The reaction mixture was stirred at room temperature
overnight. The solvent was removed in vacuo, and the residue was diluted with ethyl acetate and
basified with saturated aqueous sodium hydrogen carbonate. The combined organic extracts were
dried over magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by
chromatography on silica (0ꢀ100% EtOAc/isoꢀhexane) to afford an oily residue. The residue was
recrystallized from 1:1 methanol/water, passed through an Isolute® PEꢀAX cartridge rinsing
with methanol before the product was eluted with ammonia in methanol. This afforded 34 as a
1
colorless solid (18.6 mg, 0.062 mmol, 38% yield): H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H),
7.81 (d, J = 7.9 Hz, 1H), 7.60 (dd, J = 7.2, 7.2 Hz, 1H), 7.56ꢀ7.50 (m, 2H), 7.39 (d, J = 7.5 Hz,
1H), 7.14 (s, 1H), 1.51ꢀ1.60 (m, 1H), 0.73ꢀ0.86 (m, 3H), 0.59ꢀ0.67 (m, 1H); 19F (376 MHz,
CDCl3) δ ꢀ58.2; MS(ESI) m/z 303.3 [M+H]+.
6-(Difluoromethyl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole (35)
Methyl 1-(tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl) phenyl)-1H-indazole-6-carboxylate
(10h)
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10h was prepared analogously to 10c: H NMR (400 MHz, DMSOꢀd6) δ 8.36 (d, J = 3.6 Hz,
5
6
7
8
1H), 8.25 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.68 (s, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.59 (dd, J =
7.6, 7.6 Hz, 1H), 7.32 (dd, J = 7.0, 7.0 Hz, 1H), 6.09ꢀ6.04 (m, 1H), 3.90ꢀ3.78 (m, 2H), 2.48ꢀ2.38
(m, 1H), 2.10ꢀ2.00 (m, 2H), 1.83ꢀ1.74 (m, 1H), 1.64ꢀ1.57 (m, 2H); MS(ESI) m/z 306.15 [M+H]+.
(1-(Tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazol-6-yl)methanol (45)
To an ice cooled solution of 10h (968 mg, 2.394 mmol) in tetrahydrofuran (5 mL) under nitrogen
was added lithium aluminum hydride (2M in THF) (4.79 ml, 9.58 mmol). The mixture was
stirred with ice cooling for 30 minutes and then allowed to warm to room temperature over 1.5 h.
To the mixture was added water (360 ꢁL), followed by 2M sodium hydroxide (360 ꢁL), water (1
mL) and 2M sodium hydroxide (1 mL). The quenched mixture was left at room temperature
overnight. The resulting suspension filtered and washed with dichloromethane. The filtrate was
diluted with water and the organic portion was separated, washed with water, dried over sodium
sulfate and concentrated in vacuo. The residue was purified by chromatography on silica (0ꢀ
100% EtOAc/isoꢀhexane) to afford 45 as a white solid (622mg, 1.65 mmol, 69% yield): 1H NMR
(400 MHz, DMSOꢀd6) δ 8.08 (s, 1H), 7.85 (m, 2H), 7.71 (dd, J = 7.5, 7.5 Hz, 1H), 7.63 (dd, J =
7.6, 7.6 Hz, 1H), 7.47 (s, 1H), 7.38 (dd, J = 8.1, 8.1 Hz, 1H), 5.92ꢀ5.87 (m, 1H), 5.30 (t, J = 5.2
Hz, 1H), 4.27 (m, 1H), 4.15 (m, 1H), 3.96ꢀ3.87 (m, 1H), 3.82ꢀ3.73 (m, 1H), 2.49ꢀ2.40 (m, 1H),
2.10ꢀ1.97 (m, 2H), 1.86ꢀ1.74 (m, 1H), 1.60 (m, 2H): MS(ESI) m/z 377.35 [M+H]+.
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1-(Tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole-6-carbaldehyde (46)
To a solution of 45 (250 mg, 0.664 mmol) in dichloromethane (10 mL) was added manganese
dioxide (289 mg, 3.32 mmol) and the mixture was stirred at room temperature for 3 days. The
resulting mixture was filtered through Celite® and washed with dichloromethane. The filtrate
was concentrated in vacuo and purification of the residue by chromatography on silica (0ꢀ50%
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EtOAc/isoꢀhexane) afforded 46 (153mg, 0.41 mmol, 62% yield): H NMR (400 MHz, DMSOꢀ
d6) δ 9.76 (s, 1H), 8.39 (d, J = 6.2 Hz, 1H), 8.29 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.75 (s, 1H),
7.73 (d, J = 7.4 Hz, 1H), 7.67 (dd, J = 7.7, 7.7 Hz, 1H), 7.45 (dd, J = 7.5, 7.5 Hz, 1H), 6.11ꢀ6.06
(m, 1H), 3.97ꢀ3.78 (m, 2H), 2.48ꢀ2.39 (m, 1H), 2.10ꢀ2.02 (m, 2H), 1.86ꢀ1.73 (m, 1H), 1.65ꢀ1.57
(m, 2H); MS(ESI) m/z 291.30 [M+H]+.
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6-(Difluoromethyl)-1-(tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole
(48)
46 (25mg, 0.067 mmol) was dissolved in dry dichloromethane (1 mL) and to this was added
diethylaminosulfur trifluoride (13ꢁl, 0.10 mmol, 1.5 equiv) and the reaction was heated to reflux
overnight. Additional diethylaminosulfur trifluoride (25ꢁl, 0.2 mmol) was added and the mixture
was heated to reflux overnight. The reaction mixture cooled, diluted with dichloromethane and
washed with saturated aqueous sodium carbonate, 1M hydrochloric acid and brine. The organic
phase was dried over magnesium sulfate and solvent removed in vacuo. The residue was purified
by column chromatography (10% EtOAc/isoꢀhexane) to yield 49 (12 mg, 0.030 mmol, 45%
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yield): H NMR (400MHz, CDCl3) δ 8.00 (s, 1H), 7.91 (s, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.50
(m, 3H), 7.25 (m, 1H), 6.29 (t, J = 55.3 Hz, 1H), 5.75 (d, J = 9.3 Hz, 1H), 4.01 (m, 1H), 3.72 (m,
1H), 2.55 (m, 1H), 2.09 (m, 2H), 1.69 (m, 3H); MS(ESI) m/z 313.2 [MHꢀTHP]+.
6-(Difluoromethyl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole (35)
49 (12mg, 0.03 mmol) was dissolved in dioxane (100 ꢁL) and to this was added 4M hydrogen
chloride in dioxane (40 ꢁL, 0.151 mmol) and methanol (100 ꢁL). This was left to stir for 4 hours
at room temperature, then heated to 50°C overnight. The reaction mixture was concentrated in
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vacuo to yield 35 (3 mg, 0.0096 mmol, 37% yield): H NMR (400MHz, CDCl3) δ 8.05 (s, 1H),
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7.81 (s, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.55 (s, 1H), 7.48 (m, 2H), 7.25 (d, J = 7.3 Hz, 1H), 6.25
(t, J = 55.4 Hz, 1H); MS(ESI) m/z 313.1 [M+H]+.
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6-(Methoxymethyl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole
(40)6-(Methoxymethyl)-1-
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(tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole (48)
To a slurry of sodium hydride (60% in mineral oil) (12.75 mg, 0.319 mmol) in N,Nꢀ
dimethylformamide (2.5 ml) cooled in an iceꢀbath was added a solution of 45 (100 mg, 0.266
mmol) in N,Nꢀdimethylformamide (1.5 mL) and the mixture was stirred at 0°C. After 1 h,
methyl iodide (0.020 ml, 0.319 mmol) was added to the mixture and it was allowed to warm to
room temperature and stirred for 1.5 h. The mixture was added to ice water, and a white
precipitate formed which was removed by filtration. The filtrate was extracted with ethyl acetate.
The organic extracts were washed with water, dried over magnesium sulfate, filtered and
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concentrated in vacuo to give 48 as an oil (97 mg, 0.25 mmol, 94% yield): H NMR (400 MHz,
CDCl3) δ 8.04 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.76 (s, 1H), 7.58 (m, 1H), 7.54 (m, 1H), 7.52 (s,
1H), 7.30 (m, 1H), 5.80 (d, J = 9.6 Hz, 1H), 4.27ꢀ4.18 (m, 1H), 4.14 (m, 1H), 3.82 (m, 1H), 3.32
(s, 3H), 2.70ꢀ2.59 (m, 1H), 2.24ꢀ2.07 (m, 1H), 1.88ꢀ1.60 (m, 1H), 1.72ꢀ1.58 (m, 1H), 1.28 (s,
2H), 0.94ꢀ0.85 (m, 1H); MS(ESI) m/z 391.37 [M+H]+.
6-(Methoxymethyl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole (40)
To a solution of 48 (97 mg, 0.248 mmol) in dioxane (0.5 mL) was added 4M hydrogen chloride
in dioxane (0.311 mL, 1.242 mmol) followed by methanol (2 mL) and the mixture was stirred at
room temperature overnight. The reaction mixture was diluted in ethyl acetate and washed with
water, saturated aqueous sodium hydrogen carbonate and brine. The organic extracts were dried
over sodium sulfate, filtered and concentrated under vacuum. The crude residue was purified by
column chromatography on silica (0ꢀ100% EtOAc/isoꢀhexane) to give 40 as a white glassy solid
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(57.7 mg, 0.19 mmol, 76% yield): H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.80 (d, J = 7.7
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Hz, 1H), 7.70 (s, 1H), 7.59 (m, 1H), 7.57 (s, 1H), 7.53 (m, 1H), 7.34 (d, J = 7.4 Hz, 1H), 4.22 (s,
1H), 3.32 (s, 3H); MS(ESI) m/z 307.29 [M+H]+.
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5-(2-Trifluoromethyl-phenyl)-1H-indazole-6-carboxylic acid methylamide (44)
1-(tetrahydro-2H-pyran-2-yl)-5-(2-(trifluoromethyl)phenyl)-1H-indazole-6-carboxylic acid (47)
To a solution of 10h (889 mg, 2.198 mmol) in tetrahydrofuran (10 mL) was added sodium
hydroxide (879 mg, 21.98 mmol), water (1.67 mL) and methanol (10 mL), and this was stirred at
room temperature overnight. The reaction mixture was concentrated under vacuum and 2M
hydrochloric acid was added until the reaction mixture was pH 2. This was extracted with ethyl
acetate and the organic extracts washed with saturated aqueous sodium hydrogen carbonate and
brine, dried over sodium sulfate, and concentrated in vacuo. The product 47 was used in the
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following step without further purification (984 mg): H NMR (400 MHz, DMSOꢀd6) δ 12.5 (s,
1H), 8.35 (d, 1H), 8.22 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.64ꢀ7.60 (m, 2H), 7.56 (dd, J = 7.5, 7.5
Hz, 1H), 7.361 (dd, J = 6.2, 6.2 Hz, 1H), 6.07ꢀ6.00 (m, 1H), 3.96ꢀ3.76 (m, 2H), 2.48ꢀ2.37 (m,
1H), 2.10ꢀ1.98 (m, 2H), 1.86ꢀ1.74 (m, 1H), 1.64ꢀ1.57 (m, 2H); MS(ESI) m/z 391.43 [M+H]+.
5-(2-Trifluoromethyl-phenyl)-1H-indazole-6-carboxylic acid methylamide (44)
To a solution of 47 (75 mg, 0.192 mmol) in toluene (0.5 mL) was added thionyl chloride (0.5
mL, 6.85 mmol) and the mixture was heated at 70 °C for 4 h after which time the thionyl
chloride was removed under vacuum. The residue was dissolved in toluene (0.5 mL) and
diisopropylethylamine (0.2 mL, 1.153 mmol) and methylamine (2M in tetrahydrofuran) (0.38
mL, 0.769 mmol) were added and the mixture was stirred at 70 °C overnight. Water was added
and the mixture was extracted with dichloromethane. The organic extracts were washed with
saturated aqueous sodium hydrogen carbonate, dried over sodium sulfate and concentrated in
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vacuo. The residue was purified by preparative reverse phase HPLC (40ꢀ80% CH3CN/water with
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0.1% TFA) to yield 44 as a white glassy solid (13.4 mg, 0.042 mmol, 21.8% yield): H NMR
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(400 MHz, DMSOꢀd6) δ 13.4 (s, 1H), 8.24 (m, 1H), 8.13 (s, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.69
(s, 1H), 7.60 (dd, J = 7.4; 7.4 Hz, 1H), 7.56 (s, 1H), 7.52 (m, 1H), 7.30 (d, J = 7.5 Hz, 1H), 2.57
(d, J = 4.6 Hz, 3H); MS(ESI) m/z 320.30 [M+H]+.
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TRP channel cellular assays
Full length human, rat, or mouse TRPA1 cDNA was subꢀcloned into the expression vector
pcDNA™4/TO/mycꢀHis (Invitrogen) and stable expression clones were created in CHOꢀTREX
cells. Stable human TRPV1 and TRPM8ꢀexpressing clones were similarly created in CHOꢀK1
using the plasmids pcDNA™3.1 (Geneticin) and pcDNA™3.1 (Hygromycin) respectively.
Human TRPV3 expression clones were created in CHOꢀK1 cells using the vector pcDNA™3.1
(Hygromycin).
Human TRPA1 cells were maintained in Hams Fꢀ12 nutrient mixture (HyClone) containing 10%
Fetal Bovine Serum (FBS, Invitrogen), 5 µg/mL Blasticidin (Invitrogen), 200 µg/mL
Hygromycin B (Invitrogen), 1% antibiotic/antimycotic solution (Invitrogen), and 1% sodium
pyruvate (Invitrogen). CHOꢀTREX cells stably expressing mouse and rat TRPA1 (CHOꢀ
mTRPA1 and CHOꢀrTRPA1) were maintained in Ham’s F12 nutrient mix, 10% FBS, 1%
antibiotics/antimycotics, 5 µg/mL blasticidin, 300 µg/mL Zeocin (Invitrogen). For compound
screening, a similar medium was used containing 0.5 µg/mL or 1 µg/mL of tetracycline (Sigma)
for human TRPA1, or mouse or rat TRPA1 cells, respectively. TRPV1 cell lines were grown in
MEM Alpha, 10% FBS, 1% antibiotic/antimycotic solution, and 500 µg/mL G418 (Invitrogen).
TRPV3 cells were grown in Ham’s F12 with 10% FBS, 1% antibiotic/antimycotic, and 300
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µg/mL hygromycin.
TRPM8 cells were grown in DMEM with 10% FBS, 1%
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Compounds were seriallyꢀdiluted in DMSO and stock solutions were then made in assay buffer
containing HBSS and 20 mM HEPES (Gibco), 2.5 mM probenecid (Sigma), and 0.1% BSA.
Cells were incubated with the calcium indicator dye Fluo4ꢀAM (Molecular Probes) at [1ug/ml
concentration] for 90 min at room temperature, washed once with assay buffer, then transferred
to the FLIPR (Molecular Devices) in assay buffer for calcium flux imaging. Antagonist
compounds were added to the cells first, followed 10 min later by addition of the agonists
cinnamaldehyde (Sigma Aldrich), AITC (Fluka), 2ꢀAminoethoxydiphenylborane (2ꢀAPB, Tocris
Bioscience), or dibenzo[b,f][1,4]oxazepineꢀ4ꢀcarboxamide, which was synthesized according to
the literature procedures.23
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Agonists are used at EC80 concentration for the respective assay:
CHOꢀhTRPA1: 30 µM cinnamaldehyde
CHOꢀhTRPA1: 2.5 µM allyl isothiocyanate (AITC)
CHOꢀhTRPA1: 5 nM dibenzo[b,f][1,4]oxazepineꢀ4ꢀcarboxamide
CHOꢀrTRPA1: 40 µM cinnamaldehyde
CHOꢀmTRPA1: 40 µM cinnamaldehyde
CHOꢀhTRPV1: 40 nM capsaicin
CHOꢀhTRPV3: 40 µM 2ꢀAminoethoxydiphenylborane (2ꢀAPB)
CHOꢀhTRPM8: 40 µM menthol
Data was exported using the FLIPR software, applying the bias subtraction and the spatial
uniformity, and calculated as the stop response (the time point when the positive control gave
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maximal response minus the minimum response. The mean of triplicate values were used to
calculate the inhibition and to plot sigmoidal dose response curves. Dose response curves were
fitted using an XLfit program to determine IC50 values.
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Freund’s complete adjuvant induced mechanical hyperalgesia.
Male wistar rats (200ꢀ250g) were used for the study. Mechanical hyperalgesia was assessed by
measuring ipsi and contralateral hindpaw withdrawal thresholds to a pressure stimulus applied by
an analgesymeter (Ugo Basile, Italy) with a cut off threshold of 180 g. The endpoint was taken
as the first sign of pain response (struggling, vocalization, or paw withdrawal). Withdrawal
thresholds of both hind paws were measured immediately before and 72 h after intraplantar
injection of 25 ml of complete Freund’s adjuvant (CFA) into one hind paw, and at 1,3,6h after
oral administration of vehicle (0.5% methylcellulose and 0.5%Tween 80) or compound 31 (3, 10
or 30 mg/kg). D50 refers to the calculated dose at which 50% reversal of mechanical
hyperalgesia would be observed.
Reversal of established hyperalgesia was calculated according to the formula:
% Reversal
Postdose threshold ꢀ Predose threshold x 100
Naive threshold ꢀ Predose threshold
Mustard oil-Induced Allodynia Model. C57Bl6 male mice (20ꢀ25g) were anesthetized with
isoflurane and mustard oil (0.25% in 70% ethanol; 50 ml per mouse) was administered by
inserting a fine cannula with a rounded tip (3 cm long) into the colon via the anus. Referred
allodynia was measured from withdrawal thresholds to the application of von Frey
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filaments(SemmesꢀWeinsten Monofilaments; Linton Instrumentation,UK) to the abdomen of
mice before (baseline) and 48 h after administration of mustard oil. Animals were placed in a
raised Perspex ventilated box with a wire mesh floor and allowed to acclimatize prior to
measurement of withdrawal thresholds. Allodynia was tested by touching the lower abdomen
with von Frey filaments in ascending order of force (0.02–15 g) for up to 6 s. The endpoint
(response) was taken as an abdominal retraction, jumping, or immediate licking or scratching of
the site of filament application and the lowest force required to elicit a response was recorded as
withdrawal threshold (in grams). Vehicle (0.5% methylcellulose and 0.5% tween 80) and
compound 31 (3, 10 and 30mg/kg) were administered orally 48h after mustard oil and referred
allodynia measured 1h later.
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Thermosensation A clear Plexiglas chamber containing a Peltierꢀcooled cold plate, temperature
controller, and heat sink (TECA) was used to assess cold responses. Mice were weighed and
treated via oral gavage with vehicle (0.5% methylcellulose and 0.5% Tween80) or compound 31
(10mg/kg or 100mg/kg) (n = 7 mice per group) and acclimated in an equivalent chamber at room
temperature for 40 min. Individual mice were placed on ꢀ5 °C cold plate and observed for up to 2
min . Latency to onset was then assessed by counting the first jump, brisk hindpaw lift or
flicking/licking of the hindpaws.
Body Temperature Measurement. Rectal temperatures in rats were measured by using a rectal
probe before and 1, 3, and 6 h after oral administration of vehicle (0.5%methylcellulose and
0.5%Tween 80), compound 31 (10, 30 and 100 mg/kg p.o.) or WIN55,212ꢀ2 (6mg/kg s.c).
ACKNOWLEDGMENT. The authors thank the Analytical Chemistry and Metabolism and
Pharmacokinetics groups for their contributions.
SUPPORTING INFORMATION
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General procedures and experimental details for the syntheses of key intermediates used for
the preparation of compounds 11ꢀ44, and analytical data for final compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Corresponding Author Eꢀmail: david.tully@novartis.com; Current Address: Novartis Institutes
for Biomedical Research, 4560 Horton St, Emeryville, California 94608.
Ancillary Information
Abbreviations and acronyms used: DAST (Diethylaminosulfur trifluoride), FLIPR®
(Fluorometric Imaging Plate Reader, Molecular Devices), HBSS (Hank's Balanced Salt
Solution), HEPES (4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid), AITC (allyl
isothiocyanate)
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Urology 2008, 72, 450ꢀ455.
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