Pyrazolopyrimidines as Potent Stimulators for Transient Receptor Potential Canonical 3/6/7 Channels

Article abstract of DOI:10.1021/acs.jmedchem.7b00304

Transient receptor potential canonical 3/6/7 (TRPC3/6/7) are highly homologous receptor-operated nonselective cation channels. Despite their physiological significance, very few selective and potent agonists are available for functional examination of these channels. Using a cell-based high throughput screening approach, a lead compound with the pyrazolopyrimidine skeleton was identified as a TRPC6 agonist. Synthetic schemes for the lead and its analogues were established, and structural-activity relationship studies were carried out. A series of potent and direct agonists of TRPC3/6/7 channels were identified, and among them, 4m-4p have a potency order of TRPC3 > C7 > C6, with 4n being the most potent with an EC₅₀ of <20 nM on TRPC3. Importantly, these compounds exhibited no stimulatory activity on related TRP channels. The potent and selective compounds described here should be suitable for evaluation of the roles of TRPC channels in the physiology and pathogenesis of diseases, including glomerulosclerosis and cancer.

Full text of DOI:10.1021/acs.jmedchem.7b00304

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Article
Pyrazolopyrimidines as Potent Stimulators for
Transient Receptor Potential Canonical 3/6/7 Channels
Chunrong Qu, Mingmin Ding, Yingmin Zhu, Yungang Lu, Juan Du, Melissa R. Miller, Jinbin Tian, Jinmei
Zhu, Jian Xu, Meng Wen, AGA Er-Bu, Jule Wang, Yuling Xiao, Meng Wu, Owen B. McManus, Min Li, Jilin
Wu, Huai-Rong Luo, Zheng-Yu Cao, Bing Shen, Hongbo Wang, Michael X. Zhu, and Xuechuan Hong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00304 • Publication Date (Web): 10 Apr 2017
Downloaded from http://pubs.acs.org on April 11, 2017
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Pyrazolopyrimidines as Potent Stimulators for Tran-
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sient Receptor Potential Canonical 3/6/7 Channels
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Chunrong Qu†,※, Mingmin Ding†,※, Yingmin Zhu‡,※, Yungang Lu‡, Juan Du§, Melissa Miller∥, &, Jinbin
∥
⊥, #
Tian‡, Jinmei Zhu†, Jian Xu , Meng Wen†, AGA Er-Bu∇, Jule Wang∇, Yuling Xiao†, Meng Wu , &, Ow-
∥
⊥
∥, &
en B. McManus , Min Li , &, Jilin Wu#,¶, Huai-Rong LuoΨ,&, Zhengyu Cao , Bing Shen§, Hongbo
Wangξ,*, Michael X. Zhu‡,#,*, and Xuechuan Hong†,∇,Π,*
†State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery
(MOE) and Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuti-
cals, Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China.
‡Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of
Texas Health Science Center at Houston, Houston, TX, 77030, USA
§School of Basic Medical Sciences, Anhui Medical University, Anhui 230032, Hefei, China
∥Department of Neuroscience, High Throughput Biology Center and Johns Hopkins Ion Channel Center,
Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA
⊥State Key Laboratory of Natural Medicines, Jiangsu Provincial Key laboratory for TCM Evaluation
and Translational Development, China Pharmaceutical University, Nanjing, 211198, China
#The International Scientist Working Station of Neuropharmacology, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai, 201203, China
¶University of Chinese Academy of Sciences, Beijing, 100049, China
∇Medical College, Tibet University, Lasa, 850000, China
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ΨKey Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming, 650201, China
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ξSchool of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai Uni-
versity), Ministry of Education, Yantai University, Yantai, 264005, China
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KEYWORDS: Pyrazolopyrimidines, agonists, cation channels
ABSTRACT: Transient receptor potential canonical 3/6/7 (TRPC3/6/7) are highly homologous recep-
tor-operated non-selective cation channels. Despite their physiological significance, very few selective
and potent agonists are available for functional examination of these channels. Using a cell-based high
throughput screening approach, a lead compound with the pyrazolopyrimidine skeleton was identified
as a TRPC6 agonist. Synthetic schemes for the lead and its analogs were established and structural-
activity relationship studies were carried out. A series of potent and direct agonists of TRPC3/6/7 chan-
nels were identified and among them, 4m-4p have a potency order of TRPC3>C7>C6, with 4n being
the most potent with an EC50 of <20 nM on TRPC3. Importantly, these compounds exhibited no stimu-
latory activity on related TRP channels. The potent and selective compounds described here should be
suitable for evaluation of the roles of TRPC channels in the physiology and pathogenesis of diseases,
including glomerulosclerosis and cancer.
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■ INTRODUCTION
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Transient receptor potential canonical (TRPC) channels constitute a group of receptor-operated calcium-
permeable non-selective cation channels of the TRP superfamily. The 7 mammalian TRPC members can
be further divided into four subgroups (TRPC1; TRPC2; TRPC4/5; and TRPC3/6/7) based on their ami-
no acid sequences and functional similarities1. Native TRPC channels may be formed of either homo- or
hetero-tetramers from the same subgroup or from different subgroups; they also interact with a variety
of other proteins, which exert modulatory roles on their assembly, trafficking and/or function2. So far,
the majority of studies on TRPC channels have focused on their roles in mediating Ca2+ and Na+ influx
after the stimulation of phospholipase C (PLC) either by G protein–coupled receptors that signal
through the Gq/11 subgroup of heterotrimeric G proteins or by receptor tyrosine kinases3. These activities
cause membrane depolarization and a sustained increase in intracellular Ca2+ concentration ([Ca2+]i),
which are important for a plethora of cellular functions in many physiological systems4. On the other
hand, a few studies also implicated TRPC channel expression and function in intracellular membranes5,
suggesting that very diverse cellular activities may be regulated by these channels.
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Intensive investigations have been instrumental in defining the physiological roles of TRPC channels in
different tissues and organs, including excitation-contraction coupling in smooth muscles6, 7, protein fil-
tration in the kidney8, 9, synaptic formation and transmission10, 11, myofibroblast transdifferentiation and
wound healing12, regulation of vascular tone, cell growth and proliferation13- 15. Particularly, TRPC6 has
been implicated in the physiological regulation of glomerular hemodynamics in kidney. Genetic muta-
tions of TRPC6, including both gain-of-function and loss-of-function ones, have been linked to glomer-
ular dysfunction in the kidney, where the channel is expressed in podocytes and involved in the regula-
tion of slit diaphragm for protein filtration8,9,16. Elevated expression of TRPC6 was found in glomeruli
from patients with proteinuria, which is a common feature of kidney dysfunction of glomerular origin
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and is by itself a risk factor for both renal and extrarenal diseases17. Additionally, TRPC6 and its closely
related TRPC3, are both suggested to be involved in the development of cardiac hypertrophy and hyper-
tension14, 18- 20. The roles for TRPC3/6 in cancer have also been suggested 21,22. However, the exact
roles played by TRPC channels in kidney and cardiovascular functions and the underlying mechanisms
concerning how they contribute to these functions and/or pathogenesis have proven difficult to elucidate
due to the lack of specific compounds that modulate these channels.
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TRPC3, TRPC6, and TRPC7 are closely related, sharing 65–78% sequence identity, and form channels
that are generally activated downstream from receptors that activate PLC. These channels are also di-
rectly activated by diacylglycerols (DAG), one of the products of phosphatidylinositol 4,5-bisphosphate
(PIP2) hydrolysis by PLC23-24. Analogues of DAG, such as 1-oleoyl-2-acetyl-sn-glycerol 1 (OAG) and 1,
2-dioctanoyl-sn-glycerol 2 (DOG) have been widely used to activate TRPC3 and TRPC6 channels (Fig-
ure 1). However, not only these stimulators are non-specific for TRPC3/6/7 channels, but also they are
highly hydrophobic and unstable in the aqueous solution. Recently, small molecular probes, including
both natural products and synthetic compounds, have been reported for TRPC channels. For example, a
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novel synthetic agonist for TRPC3/6 named GSK1702934A (3) was used at 1 µM to study cardiac
contractility and arrhythmogenesis in heart26. A few small-molecule compounds including Pyr3 and
analogs27, cationic, N’-substituted 1-benzylpiperidines7, anilino-thiazole28, norgestimate29, 4-({(1R,2R)-
2-[(3R)-3-aminopiperidin-1-yl]-2,3-dihydro-1H-inden-1-yl}oxy)-3- chlorobenzonitrile30 and larixyl ace-
tate31 have been identified as inhibitors of TRPC3/6/7 channels. Englerin A32, riluzole33, clemizole hy-
drochloride34, 4-methyl-2-(1-piperidinyl)-quinoline (ML204)35, and 2-aminobenzimidazole derivatives36
have been described as TRPC4 and/or TRPC5 agonists or antagonists. Often, these probes cross-react
within the subgroup of TRPC3/6/7 or TRPC4/5, but not between the two subgroups.
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While selectivity remains lacking for most compounds, identification of their pharmacophores through
systematic structure-activity approaches can be expected to lead to development of subtype-selective
TRPC agonists or antagonists, and may perhaps lead to development of novel drug therapies, such as for
the treatment of kidney and cardiovascular diseases or cancer. For such a reason, we have carried out a
high-throughput screening (HTS) effort to identify small-molecule probes of TRPC6 and related chan-
nels as a starting point for lead optimization and target validation. Here we report the identification of a
novel pyrazolopyrimidine-based TRPC6 lead agonist, its resynthesis, and the structure-activity relation
(SAR) analysis of the initial set of its analogs on TRPC3, TRPC6, and TRPC7. With minor modifica-
tions, we obtained an agonist with relatively strong selectivity for TRPC3, exhibiting apparent affinity at
the low nanomolar range.
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Figure 1. Examples of reported TRPC3/6/7 agonists.
■ RESULTS AND DISCUSSION
Chemistry. The scarcity of TRPC3/6/7 probes has severely hampered the characterization of these
channels in their assembly, function and pathophysiological roles. To functionally screen for TRPC3/6/7
small molecular probes, we created a stable HEK293 cell line that co-expressed mouse TRPC6 and the
Gq/11-PLC coupled M5 muscarinic receptor (TRPC6 cells). These cells responded to the muscarinic
agonist, carbachol (CCh), with a strong sustained membrane depolarization, which can be detected by
the FLIPR membrane potential (FMP) dye as an increase in fluorescence (Fig. 2A, gray dashed line).
Using this cell-based assay, we screened 305,000 compounds from the Molecular Libraries Small Mole-
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cule Repository (MLSMR) supported by the Molecular Libraries Probe Production Centers Network
(MLPCN) and identified an activator with the pyrazolopyrimidine skeleton (PubChem CID: 5308428,
Table 1, compound 4o) as the primary hit. As shown in Fig. 2A, the original lead compound elicited
membrane potential depolarization in TRPC6 cells in a concentration-dependent manner, but not in the
parental HEK293 cells (Fig. 2B). It also evoked [Ca2+]i rise in the TRPC6 cells (Fig. 2C), but not paren-
tal HEK293 cells (Fig. 2D). We then confirmed that the compound concentration-dependently elicited
imal stimulation (EC50) were estimated to be 1.4 ± 0.1 µM by the FMP assay (n = 6), 2.5 ± 0.2 µM (at
+80 mV) by whole-cell recordings (n = 5-7), and 7.2 ± 0.5 µM by the Ca2+ assay (n = 12) (Fig. 2F).
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Figure 2. Lead compound 4o Identified from HTS Activated TRPC6 Expressed in HEK293 Cells. (A)
Representative traces of membrane potential changes evoked by carbachol (CCh, 1 µM, gray dashed
line) or varying concentrations of compound 4o obtained from the compound library (colored lines) in
the TRPC6 cells. The fluorescence increases indicate depolarization. (B) Compound 4o did not induce
membrane depolarization in parental HEK293 cells. (C) Representative traces of Fluo4 fluorescence
changes, indicative of [Ca2+]i increase, evoked by varying concentrations of compound 4o in the TRPC6
cells. (D) CCh, but not compound 4o, evoked [Ca2+]i increase in the parental HEK293 cells. (E) Repre-
sentative traces of whole-cell currents at +100 mV (red trace) and -100 mV (blue trace) elicited by var-
ying concentrations of compound 4o in a TRPC6 cell. The bath contained 2 mM Ca2+ and the pipette
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solution had 400 nM free Ca2+ buffered by BAPTA. Voltage ramps from +100 mV to -100 mV were
applied every second. Current-voltage (I-V) relationships obtained from the voltage ramps for peak cur-
rents elicited by selected compound concentrations are shown at right. These I-V curves are typical to
TRPC6 currents evoked through receptor stimulation37. (F) Concentration response curves for com-
pound 4o activation of TRPC6, as determined by the membrane potential assay (FMP), Ca2+ assay
(Fluo4), and electrophysiology recording shown in (A), (C) and (E), respectively. For fluorescence as-
says, area under the curve, and for whole-cell recordings, peak current changes at +80 mV were used for
quantification. Solid lines represent fits by the Hill equation, which yielded the EC50 values described in
the text.
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To further evaluate this compound and the SAR on TRPC6 activation, we first synthesized compounds
4a-4p (Scheme 1, Table 1). Starting from the commercially available piperidine-4-carboxylic acid 5, our
initial attempt to introduce a methyl group or alkyl group into the R2 position did not work until a pub-
lished protocol38 was adapted to protect the carboxyl group before adding methyl at position R2. Using
this strategy, compounds 6a-6c were prepared with high yields of 90~95% by N-acylation with ethyl
chloroformate, di-tert-butyl dicarbonate or benzyl chloroformate groups at position R2, respectively
(Scheme 1). Deprotonation of piperidine of 6a and 6b with 1.2 equiv of n-butyllithium followed by re-
action with methyl iodide afforded amides 7a and 7b in 87% and 85% yields, respectively. The ethyl
ester portion of 7a and 7b was converted to 3-oxopropanoate group using a modified Masamune proce-
dure39, which furnished β-keto esters 8a and 8b in 82% and 86% yields, respectively. Compounds 8c
and 8d were obtained with high yields from 6c and 6b, respectively, using a similar procedure. Conden-
sation of nitrile intermediate 10 with 1, 1-dimethoxy-N,N-dimethylethan-1-amine 9 using dichloro-
methane as the solvent under microwave irradiation (MWI), followed by the cyclization reaction with
hydrazine hydrochloride in EtOH generated the 5-aminopyrazoles 11a−d in excellent yields (81−88%)
over two steps. We then carried out a cyclization study of compounds 8 and 11 using a variety of acids,
such as HCl, trifluoroacetic acid (TFA), and H2SO4, in order to test the viability of our design. Unfortu-
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nately, only a trace amount of product 12 was formed in TFA at reflux for 18 h. However, mixing 8a-8c
with 11a-11d in acetic acid in a microwave reactor at 120°C overnight produced the desired products 12
in good yields. Deprotection of the Boc group of compounds 12 (R2 = Boc) with TFA/dichloromethane
(DCM) at 0°C resulted in the formation of compounds 13 in high yield. Finally, amidation of com-
pounds 13 gave rise to compounds 4 (4a-4p, Table 1) successfully (Scheme 1).
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Scheme 1. The Synthetic Route for Compound 4a and its Derivativesa.
aReagents and conditions: (a) R1O2CCl, NaOH, H2O, rt, 5h, or (Boc)2O, K2CO3, THF/H2O, rt, 7h. (b) i.
ClCO2Et, NEt3, rt, 12h; ii. n-BuLi, i-Pr2NH, THF, -78°C , 1h, MeI, THF, 5h. (c) i. 2M NaOH; ii. CDI,
THF, 4h; KO2CCH2CO2Et, MgCl2, DMAP, THF/CH3CN, overnight. (d) i. DCM, microwave, 100°C; ii.
N2H4.HCl, H2O, EtOH, 80°C, 8h. (e1) AcOH, reflux, 120°C , overnight. (e2) EtOH, TFA, reflux, 85°C ,
48h. (f) TFA, DCM, 0°C , 2h. (g) DMF, HOBt, HBTU, DIPEA, rt, overnight.
Biological Evaluation. The effect of the newly synthesized compounds (Table 1) on TRPC6 channel
function was evaluated using the fluorescence Ca2+ assay. As the original lead, the re-synthesized com-
pound 4o (R1 = H, R3 = F, R4 = CO2Et) evoked [Ca2+]i rise in the TRPC6 cells in a concentration de-
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pendent manner, with a mean EC50 value of 4.66 ± 0.03 µM (Fig. 2C and 2E, Table 1, entry 15),
demonstrating a comparable activity of the re-synthesized compound on TRPC6 as the original lead.
Compound 4o was also tested on HEK293 cells that co-expressed µ opioid receptor (MOR) and
TRPC4β35, 36, which belongs to a different subgroup of TRPC channels than TRPC3/6/7. Upon stimula-
tion by a µ agonist, (D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO, 1 µM), these cells show a robust
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36
increase in [Ca2+]i . However, compound 4o did not elicit a Ca2+ response in the MOR/TRPC4β cells;
nor did it alter the DMAGO-evoked response, demonstrating its selectivity for TRPC6 over TRPC4.
Table 1. Structures of Compounds 4a-4p and Their Effects on TRPC6 and TRPC4.
Compound
R1
R3
R4
TRPC6 EC50 (µM)a
TRPC4β+MOR IC50 (µM) b
H
F
F
12.3±1.2
NA
~10
NA
4a
4b
Me
Me
Me
F
F
NA
NA
NA
NA
4c
4d
Me
NO2
NA
NA
4e
Me
Me
Me
H
NO2
NO2
F
NA
~8c
NA
~2c
NA
NA
NA
NA
4f
4g
4h
4i
CO2Et
Boc
NO2
CO2Et
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H
F
28.8±8.3
~35
4j
5
6
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H
H
H
H
H
H
F
Cl
Cl
CF3
F
NA
NA
NA
NA
NA
NA
NA
4k
4l
Boc
Cbz
NA
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7.79±0.05
1.39±0.01
4.66±0.03
3.91±0.02
4m
4n
4o
4p
CO2Et
CO2Et
Cbz
F
aEC50 determined based on Ca2+ assay.
bIC50 against that activated by 1 µM DAMGO, estimated based on one experiment with triplicates; es-
c
timated value because of the self fluorescence of the compound; NA: No activity.
We next investigated SAR between substituents in R1 and R4 of ring A and R3 of ring B using the Ca2+
assay on the TRPC6 cells (Table 1). For most compounds, the MOR/TRPC4β cells were used as a con-
trol for subtype selectivity. First, the hydrogen is required in R1, as the substitution by a methyl group
rendered all compounds (4b-4h) inactive at stimulating TRPC6 (Table 1, entries 2-8), except for 4g,
which however, showed high fluorescence by the compound itself (not shown). This could be due in
part to the ability of methyl at position R1 to bias a particular A ring conformer. Second, the substitution
of F at R3 by Cl moderately increased the EC50 (4m, Fig. 3A and 3E, Table 1, entry 13) whereas that by
NO2 or CF3 decreased the EC50 by ~60-70% to < 2 µM (4i and 4n, Fig. 3B and 3E, Table 1, entries 9
and 14), suggesting a possible position for further modifications to improve the agonist potency. How-
ever, like 4g, the introduction of NO2 at R3 rendered the compound (4i) to become self-fluorescent (not
shown). Third, substitutions at R4 of the A ring revealed that the carbamate group, such as ethyl carba-
mate, is optimal. To identify suitable carbamate group replacements for R4, we used 6-bromo-nicotine
(4a), ethyl carbamoyl (4j), tert-butyloxycarbonyl (Boc, 4l) and carboxybenzyl (Cbz, 4m, 4p). Intri-
guingly, compound 4l did not exhibit stimulatory activity on TRPC6, likely because of the steric hin-
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drance blockade by the Boc group introduced at the R4 position. The benzyl carbamate or Cbz group in
R4 (4p) displayed comparable stimulatory activity on TRPC6 as the corresponding ethyl carbamate (4o),
giving an EC50 value of 3.91 ± 0.02 µM (Fig. 3D and 3E, Table 1, entry 16).
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Figure 3. Compounds 4m-4p Activated TRPC6 Expressed in HEK293 Cells. (A)-(E) Compounds 4m-
4p were synthesized as described in the text and tested in the Ca2+ assay using Fluo8-loaded TRPC6
cells. (A)-(D) Representative traces of [Ca2+]i rise, as indicated by the Fluo8 fluorescence increase,
evoked by varying concentrations of 4m (A), 4n (B), 4o (C), or 4p (D). (E) Summary data for (A)-(D)
and the fits by the Hill equation. (F)-(J) Compounds 4m-4p were tested on TRPC6 cells in whole-cell
recordings. The bath contained 0.1 mM Ca2+ and the pipette solution had 400 nM free Ca2+ buffered by
BAPTA. (F)-(I) Representative current traces at +100 mV (red traces) and -100 mV (blue traces) in
response to consecutive applications of increasing concentrations of 4m (F), 4n (G), 4o (H), or 4p (I)
(upper panels) and I-V curves for selected compound concentrations (lower panels). (J) Summary data
for (F)-(I) and the fits by the Hill equation.
Functional characterization of 4m-4p. To validate the agonistic action of pyrazolopyrimidine com-
pounds on TRPC6 channels, we focused on the last four compounds listed in Table 1 (4m-4p), which
evoked robust [Ca2+]i increases in TRPC6-expressing cells in the Ca2+ assay with EC50 values < 8 µM
(Fig. 3A-3E) and no self-fluorescence. In whole-cell voltage clamp recordings, we used a low Ca2+ bath
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solution (0.1 mM Ca2+) in order to minimize current rundown. Under these conditions, increasing con-
centrations (0.1 – 100 µM) of the test compound were applied consecutively and in each case the com-
pound evoked stepwise current increases in individual cells at both negative and positive potentials (Fig.
3F-3I). Noticeably, the outward currents at positive potentials developed earlier at low compound con-
centrations than the inward currents at negative potentials, indicative of a shift of voltage dependence as
the compound concentration increased. Typically, the currents did not desensitize even at highest con-
centrations (20-100 µM) used. Based on the currents at +80 mV, the EC50 values for monovalent cation
currents of TRPC6 evoked by 4m-4p were determined to be 2.05 ± 0.02 µM, 0.89 ± 0.01 µM, 6.28 ±
0.02 µM and 3.24 ± 0.01 µM (n = 6 - 8), respectively (Fig. 3J). These values are comparable to that ob-
tained from the Ca2+ assay (Table 1).
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We then examined whether the compounds directly activate closely related TRPC3 and TRPC7 channels.
Although our initial attempts using the FMP and Ca2+ assays did not reveal obvious activation of human
TRPC3 stably expressed in HEK293 cells by compound 4o (Fig. 4A1, 4A2), lowering the Ca2+ concen-
tration in the extracellular solution from the standard 2 mM to 0.5 mM allowed 4o to evoke membrane
depolarization in these cells in a concentration dependent manner (Fig. 4B). These results suggest that
the activation of TRPC3 by 4o is sensitive to inhibition by extracellular Ca2+, consistent with the previ-
ous finding that physiological concentrations of extracellular Ca2+ suppressed receptor-operated TRPC3
currents40. Supporting the FMP assay data, 4o also evoked whole-cell currents in HEK293 cells that ex-
pressed human TRPC3 (Fig. 4E). Using the FMP assay with 0.5 mM extracellular Ca2+, we determined
the EC50 values of compounds 4m-4p on stimulating TRPC3 to be 138 ± 23 nM, 19.2 ± 2.3 nM, 450 ±
87 nM, and 236 ± 47 nM (Fig. 4B-4D, n = 6 for each), respectively. Among the four analogs, 4n really
stands out, exhibiting >7-fold increase in the potency on TRPC3 as compared to other compounds
and >70-fold increase as compared to the same compound on TRPC6.
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TRPC3 (0.5 mM Ca2+
)
TRPC3 (0.5 mM Ca2+
)
TRPC3 (2 mM Ca2+
)
B
C
D
A1
1.0
0.5
0.0
1.0
4o
1.0
4o
4n
100
75
50
25
0
(µM)
30
10
3.3
1.1
0.37
0.12
0.04
0
(nM)
1111
370
123
41
4m
4n
4o
4p
0.5
0.0
0.5
0.0
14
0
9
10
10-9
10-8
10-7
10-6
10-5
0
0.5
1.0
1.5
2.0
2.5
0
0.5
1.0
1.5
2.0
2.5
0
0.5
1.0
1.5
2.0
2.5
[Compound] (M)
Time (min)
Time (min)
Time (min)
11
A2
12
TRPC3 (2 mM Ca2+
4o
)
E
4o (10 µM)
1.0
0.5
0.0
13
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21
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
nA
4o
+100 mV
-100 mV
Basal
-100 -50
50 100
mV
-0.2
-0.2
0
20
40
60
80
0
0.5
1.0
1.5
2.0
2.5
Time (sec)
Time (min)
Figure 4. Compounds 4m-4p Activated TRPC3 Expressed in HEK293 Cells when Extracellular Solu-
tion Contained 0.5 mM Ca2+. (A) Compound 4o failed to elicit membrane depolarization (A1) or [Ca2+]i
rise (A2) in TRPC3 cells in normal extracellular solution that contained 2 mM Ca2+. (B) and (C) Repre-
sentative traces of membrane potential depolarization induced by varying concentrations of compounds
4o (B) and 4n (C) in TRPC3 cells in an extracellular solution that contained 0.5 mM Ca2+. (D) Sum-
mary data for membrane depolarization induced by compounds 4m-4p in TRPC3 cells bathed in the 0.5
mM Ca2+ solution. The data points were fitted by the Hill equation. (E) Compound 4o (10 µM) elicited
TRPC3 currents in the bath that contained 0.5 mM Ca2+. The I-V curves under basal and 4o-stimulated
conditions are shown at right.
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Among the three members of the TRPC3/6/7 subgroup, the sequence of TRPC7 is more similar to
TRPC3 than to TRPC6. Interestingly, compound 4o was able to elicit membrane depolarization and
[Ca2+]i elevation in HEK293 cells that stably expressed human TRPC7 in the normal extracellular solu-
tion that contained 2 mM Ca2+ (Fig. 5A, 5B), although the extents of the response, even when they
reached the maximum appeared to be markedly lower than that seen in the TRPC6 cells. Previously, it
was shown that extracellular Ca2+ potentiated TRPC6 but inhibited TRPC7 currents41. Thus, the lower
response of TRPC7 to 4o than TRPC6 reflected at least in part a block by the 2 mM Ca2+ included in the
extracellular solution, although such a block may be much weaker than TRPC3, which was almost com-
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pletely inhibited by the same concentration of Ca2+ (see above and Fig. 4A). In whole-cell voltage
clamp recordings, 4o elicited concentration dependent current increases in the TRPC7 cells (Fig. 5D).
Different from TRPC6, the currents showed some desensitization in the high 4o concentrations (10 and
30 µM) at both negative and positive potentials even though the low Ca2+ (0.1 mM) bath solution was
used. Nonetheless, a stepwise increase in the TRPC7 currents was still detected at <10 μM 4o. Based on
the whole-cell recordings performed in the low Ca2+ bath solution, 4o had an estimated EC50 of 1.13 ±
0.03 μM (n = 6) on TRPC7. This value is only slightly higher than that obtained from the Ca2+ assay.
Based on the Ca2+ assay, the EC50 values of 4m-4p were estimated to be 213 ± 36 nM, 90 ± 12 nM, 497
± 91 nM, and 314 ± 59 nM (Fig. 5C, n = 6 for each), respectively. For each of the compounds, the EC50
value for TRPC7 is in between those for TRPC3 and TRPC6 (Table 2).
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Figure 5. Compounds 4m-4p Activated TRPC7 Expressed in HEK293 Cells. (A) and (B) Representa-
tive traces of membrane potential depolarization (A) and [Ca2+]i increase (B) induced by varying con-
centrations of compounds 4o in TRPC7 cells in normal extracellular solution that contained 2 mM Ca2+.
(C) Summary data for [Ca2+]i rise induced by compounds 4m-4p in TRPC7 cells bathed in the 2 mM
Ca2+ solution, as exemplified in (B). The data points were fitted by the Hill equation. (D) Activation of
TRPC7 currents by increasing concentrations of compound 4o in the bath solution that contained 0.1
mM Ca2+. The I-V curves under basal and selected concentrations of 4o are shown below.
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The above data clearly demonstrate the agonistic activity of the pyrazolopyrimidine compounds 4m-4p
on the TRPC3/6/7 subgroup of TRPC channels. To verify the selectivity, we tested the effects of 4m-4p
on a number of related TRP channels, including TRPC4, TRPC5, TRPA1, TRPV1, TRPV3, TRPV4,
TRPM5 and TRPM8, using either the FMP or Ca2+ assay. All compounds were applied at 33 µM when
tested for stimulatory activity, which revealed no obvious effect (data not shown). Then the channels
were stimulated with their corresponding agonists in the absence or presence of 22 µM 4m-4p. For most
channels, the compounds did not significantly alter the agonist-evoked responses, except for TRPC4,
where 4n and 4p suppressed the MOR agonist DAMGO-evoked membrane depolarization by ~33% and
13%, respectively, and for TRPM8, where 4n, 4o and 4p suppressed the menthol-evoked [Ca2+]i in-
crease by ~17%, 22% and 35%, respectively (Fig. 6). A small (<20%) inhibition by 4n was also detected
for TRPA1, TRPV1 and TRPM5 (Fig. 6).
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Table 2. The EC50 Values (µM) for Compounds 4m-4p to TRPC3/6/7 Channels.
Compound
TRPC3a
TRPC7b
TRPC6b
TRPC6c
0.213 ± 0.036
7.79±0.05
2.05±0.02
4m
4n
4o
4p
0.138 ± 0.023
0.019 ± 0.002
0.450 ± 0.087
0.236 ± 0.047
0.090 ± 0.012
0.497 ± 0.091
0.314 ± 0.059
1.39±0.01
4.66±0.03
3.91±0.02
0.89±0.01
6.28±0.02
3.24±0.01
a Cells were loaded with the FLIPR membrane potential (FMP) blue dye; the [Ca2+] in the bath was
0.5 mM.
b Cells were loaded with Fluo8-AM; the [Ca2+] in the bath was 2 mM.
c Determined based on currents at +80 mV by whole-cell patch clamp recordings using a bath solu-
tion that contained 0.1 mM Ca2+ and a peptide solution that contained 400 nM free Ca2+ buffered
with BAPTA.
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4m
4n
4o
4p
1.0
0.8
0.6
0.4
0.2
0.0
*
*
*
*
*
*
*
*
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TRPC4 TRPC5 TRPA1 TRPV1 TRPV3 TRPV4 TRPM5 TRPM8
Figure 6. Compounds 4m-4p had Little or no Effect on Related TRP Channels. Stable HEK293 cell
lines expressing MOR/TRPC4β, TRPC5, TRPA1, TRPV1, TRPV3, TRPV4, TRPM5 and TRPM8 were
seeded in wells of 96-well plates and loaded with either FMP dye (TRPC4, C5, and M5) or Fluo8-AM
(TRPA1, V1, V3, V4, and M8). Changes in membrane potential or [Ca2+]i were read in a microplate
reader while compound 4m, 4n, 4o, or 4p (33 µM) was applied for 2 min before the corresponding ago-
nist known to activate the channel (C4, 1 µM DAMGO; C5, 100 µM CCh; A1, 100 µM flufenamic acid;
V1, 1 µM capsaicin; V3, 200 µM 2-APB; V4, 30 µM RN-1747; M5, 100 µM CCh; M8, 100 µM men-
thol) was added and measurement continued for 2.5 min. After the agonist addition, the test compound
was diluted to 22 µM. Shown are summary data for the agonist-evoked response (area under the curve)
in the presence of the test compound normalized to that in the absence of the test compound. n = 4-10
measurements. * p < 0.05 compared to no test compound by Student’s t test.
Finally, we evaluated the ability of compound 4p to elicit [Ca2+]i rise in rat primary glomerular mesan-
gial cells, which endogenously express functional TRPC3/6 channels42,43. 4p (1 µM) evoked marked
increases in [Ca2+]i, as revealed by the elevation in Fluo-4 fluorescence (Fig. 7A). These activities were
abolished by 2-aminoethoxydiphenyl borate (2-APB, 30 µM), a non-specific TRP modulator that inhib-
its TRPC channels7,37
.
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4p (1 µM)
4p (1 µM)
A
B
C
3
8
6
4
2
0
8
6
4
2
0
Control
30 µM 2-APB
2
1
0
*
0
2
4
6
8
10
0
2
4
6
8
10
Control
2-APB
Time (min)
Time (min)
Figure 7. Compound 4p Induced Ca2+ Transients in Rat Glomerular Mesangial Cells. (A) Representa-
tive traces of Fluo4-loaded mesangial cells exposed to 1 µM compound 4p. Each trace represents a sin-
gle cell and the thick gray line is the average of all 36 cells in the same coverslip. (B) Similar to (A) but
the cells were pretreated for 2 min with 30 µM 2-APB, which was also present throughout the period
when compound 4p was applied. Each trace represents a single cell and the thick gray line is the aver-
age of all 30 cells in the same coverslip. (C) Summary of four experiments quantified by the mean peak
fluorescence changes induced by compound 4p for the individual experiments. * p < 0.05 compared to
no 2-APB control by Student’s t test.
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■ CONCLUSIONS
Pharmacological tool compounds play a vital role in drug development and functional characterization
of TRPC channels. Using HTS and SAR studies, we have identified a series of potent and direct ago-
nists of TRPC3/6/7 channels. Four of them, compounds 4m-4p were further characterized and shown a
potency order of TRPC3>C7>C6, with 4n being the most potent with an EC50 value of <20 nM on
TRPC3. Importantly, these compounds exhibited no stimulatory activity on related TRPC channels, in-
cluding the subfamily members TRPC4 and C5. Although not directly tested, it is unlikely that com-
pounds 4m-4p would activate other Ca2+ permeable channels endogenously expressed in HEK293 cells,
such as inositol 1,4,5-trisphosphate receptors and Orai1 or inhibit the sarco/endoplasmic reticulum Ca2+-
ATPase as they did not elicit detectable [Ca2+]i in parental HKE293 cells and a number of stable cell
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lines. A few compounds exhibited weak inhibition on TRPC4 and TRPM8 at high concentrations.
Therefore, they are quite selective for the TRPC3/6/7 subgroup of TRPCs. It remains to be determined if
the compounds affect ryanodine receptors or voltage-gated Ca2+ channels, which are mainly present in
excitable cells. Nevertheless, given the current general lack of good pharmacological agonists for the
TRPC channels and our demonstration that they activated native TRPC3/6 channels in rat glomerular
mesangial cells, the compounds described here should find excellent use in TRPC channel research. Our
results may lead to more potent and specific agonists or antagonists of targeting TRPC channels and fa-
cilitate the development of related disease therapeutics.
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■ EXPERIMENTAL SECTION
General Methods. Unless noted otherwise, all reagents and solvents were purchased from commercial
suppliers and used without further purification. Melting points were determined on a YUHUA X-5 melt-
ing point apparatus. All reactions were performed under an argon atmosphere unless otherwise specified.
Reaction progress was monitored using analytical thin-layer chromatography (TLC). The purification of
all compounds was processed by silica gel column chromatography. 1H and 13C NMR spectra were rec-
orded on a Bruker AV400 spectrometer (400 MHz, 1H NMR; 101 MHz, 13C NMR) at room temperature
(rt). NMR spectra were calibrated to the solvent signals of CDCl3 (δ 7.26 and 77.16 ppm), CD3OD (δ
3.31 and 49.00ppm), or d6-DMSO (δ 2.50 and 39.52 ppm). The chemical shifts are provided in ppm,
and the coupling constants, in Hz. The following abbreviations for multiplicities are used: s, singlet; d,
doublet; dd, double doublet; ddd, triple doublet; t, triplet; dt, double triplet; q, quadruplet; m, multiplet;
and br, broad. High-resolution MS was carried out with a Thermo LTQ XL Orbitrap instrument. The
yield of all compounds for biological testing was determined by HPLC analysis, confirming >95% puri-
ty.
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General procedure for the synthesis of 4a, 4c, 4d, 4f, 4i, 4j, 4k. To a solution of 13 (1eq) in DMF was
added appropriate carboxylic acid (1.2 eq), HOBT (3eq), HBTU (3eq). Diisopropyl ethyl amine (9eq)
was added 15 min later. The reaction was stirred for 5-24 h, quenched with water, diluted with EtOAc,
and then washed with water. The organic layer was dried over anhydrous MgSO4 and the solvent evapo-
rated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:
MeOH= 100: 1- 40: 1) to obtain the title compound.
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General procedure44 for the synthesis of 4b, 4e, 4g, 4h. To a solution of 13 (1eq) in CH2Cl2 was add-
ed appropriate acyl chloride or anhydride. Et3N (1.2eq) was added and the reaction was stirred at rt. for
4-8 h. After removing the solvent, the title compound was purified by silica gel column chromatography
(CH2Cl2: MeOH = 100: 1- 40: 1).
General procedure for the synthesis of 4l-4p. Compound 4l-4p were synthesized according to the
general procedure for the synthesis of 12 (see later).
(6-Bromopyridin-3-yl)(4-(3-(4-fluorophenyl)-7-hydroxy-2-methyl-pyrazolo[1,5-a]pyrimidin-5-
yl)piperidin-1-yl) methanone (4a)44. The title compound was isolated as a white solid in 83% yield. 1H
NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H), 8.48 (d, J = 2.3 Hz, 1H), 7.84 (dd, J = 8.2, 2.2 Hz, 1H),
7.75 (d, J = 8.1 Hz, 1H), 7.47-7.44 (m, 2H), 7.30 (t, J = 8.8 Hz, 2H), 5.67 (s, 1H), 4.61 (d, J = 10.8 Hz,
1H), 3.62 (d, J = 11.1 Hz, 1H), 3.13 (s, 1H), 2.91 (t, J = 11.4 Hz, 1H), 2.78 (s, 1H), 2.25 (s, 3H), 1.98 (d,
J = 5.2 Hz, 1H), 1.79 (s, 1H), 1.69 (d, J = 9.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.7, 162.4,
160.0, 156.2, 150.0, 148.4, 142.1, 138.0, 131.7, 131.6, 131.5, 128.1, 115.6, 115.4, 102.2, 92.8, 47.3,
41.7, 30.6, 13.0. HRMS (ESI) calcd for C24H22BrFN5O2 [M + H]+ 510.0941, found 510.0960.
3-(4-Fluorophenyl)-5-(1-((4-methoxyphenyl)sulfonyl)-4-methyl-piperidin-4-yl)-2-
methylpyrazolo[1,5-a]pyrimidin-7-ol (4b) 44. The title compound was isolated as a white solid in 60%
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yield. mp decomposition; 1H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.38
(m, 2H), 7.27 (dd, J = 8.7, 6.7 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 5.62 (s, 1H), 3.82 (s, 3H), 2.97 (d, J =
10.8 Hz, 4H), 2.19 (m, 5H), 1.77 (d, J = 4.5 Hz, 2H), 1.17 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
162.6, 158.1, 155.8, 142.8, 131.7, 131.7, 129.6, 127.4, 127.4, 127.3, 115.4, 115.2, 114.5, 93.7, 55.7,
42.2, 36.5, 33.6, 24.7, 13.0. HRMS (ESI) calcd for C26H28FN4O4S [M + H]+ 511.1815, found 511.1803
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1-(4-(3-(4-Fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a] pyrimidin-5-yl)-4-methylpiperidin-1-
yl)propan-1-one (4c) 44. The title compound was isolated as a grey solid in 73% yield. mp decomposi-
tion. 1H NMR (400 MHz, CD3OD) δ 7.45 (s, 2H), 7.21 (s, 2H), 5.36-5.33 (m, 1H), 4.65 (s, 2H), 3.60 (s,
2H), 2.43-2.39 (m, 2H), 2.32 (s, 3H), 2.04 (s, 2H), 1.76 (s, 2H), 1.39 (s, 3H), 1.11 (t, J = 7.5 Hz, 3H).
13C NMR (101 MHz, DMSO-d6) δ 171.2, 162.5, 160.1, 158.9, 156.1, 150.7, 138.9, 131.8, 127.5, 115.5,
115.3, 102.6, 93.6, 41.1, 37.4, 34.8, 34.1, 25.6, 24.3, 13.1, 9.5. HRMS (ESI) calcd for C22H26FN4O2 [M
+ H]+ 397.2040, found 397.2053.
1-(4-(3-(4-Fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a] pyrimidin-5-yl)-4-methylpiperidin-1-
yl)-2,2-dimethyl-propan-1-one (4d) 44. The title compound was isolated as a yellow solid in 55% yield.
mp 203-206 o C. 1H NMR (400 MHz, CD3OD) δ 7.42 (s, 2H), 7.20 (s, 2H), 5.87 (s, 0.5H), 5.34 (s, 0.5H),
4.65 (s, 1H), 3.71 (s, 3H), 2.30 (s, 3H), 2.10 (s, 2H), 1.78 (d, J = 12.7 Hz, 2H), 1.40 (s, 3H), 1.27 (s, 9H).
HRMS (ESI) calcd for C24H30FN4O2 [M + H]+ 425.2353, found 425.2351.
5-(1-((2,6-Difluorophenyl)sulfonyl)-4-methylpiperidin-4-yl)-2-methyl-3-(4-
nitrophenyl)pyrazolo[1,5-a]pyrimidin-7-ol (4e) 44. The title compound was isolated as an orange solid
in 67% yield. mp 224-227 o C. 1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.23 (d, J = 8.9 Hz, 3H),
7.65-7.57 (m, 2H), 7.22 (t, J = 9.1 Hz, 2H), 5.74 (d, J = 28.9 Hz, 1H), 2.96 (t, J = 9.7 Hz, 2H), 2.56 (s,
2H), 2.41-2.34 (m, 3H), 1.70 (s, 2H), 1.23 (s, 2H), 1.18 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
169.6, 160.0, 157.5, 145.1, 143.7, 140.7, 137.8, 135.9, 128.4, 123.7, 113.8, 113.8, 113.5, 113.5, 112.1,
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112.1, 93.2, 45.7, 42.9, 35.2, 22.7, 10.8, 9.0. HRMS (ESI) calcd for C25H24F2N5O5S [M + H]+ 544.1466,
found 544.1461.
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2-(3,4-Dimethoxyphenyl)-1-(4-(7-hydroxy-2-methyl-3-(4-nitro-phenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)-4-methyl-piperidin-1-yl)ethan-1-one (4f) 44. The title compound was isolated as an orange solid in
58% yield. mp 113-116 o C. 1H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H), 8.20 (s, 4H), 6.82-6.80 (m,
2H), 6.71 (d, J = 8.1 Hz, 1H), 5.85 (d, J = 8.1 Hz, 1H), 3.78 (d, J = 6.5 Hz, 1H), 3.66 (d, J = 5.7 Hz, 6H),
3.60 (s, 2H), 3.30 (d, J = 10.5 Hz, 2H), 3.17 (d, J = 9.5 Hz, 1H), 2.54 (s, 2H), 2.16 (s, 2H), 1.45 (d, J =
39.3 Hz, 2H), 1.21 (s, 1H), 1.18 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.8, 158.0, 149.3, 148.5,
147.3, 129.7, 128.4, 123.7, 120.7, 112.7, 111.8, 55.5, 55.4, 43.0, 40.1, 38.6, 36.3, 35.7, 22.1, 14.0.
HRMS (ESI) calcd for C29H32N5O6 [M + H]+ 546.2353, found 546.2322.
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Ethyl
4-(7-hydroxy-2-methyl-3-(4-nitrophenyl)
pyrazolo[1,5-a]
pyrimidin-5-yl)-4-
methylpiperidine-1-carboxylate (4g) 44. The title compound was isolated as an orange solid in 76%
yield. mp 174-178 o C. 1H NMR (400 MHz, DMSO-d6) δ 8.20 (s, 4H), 5.73 (s, 1H), 3.97 (q, J = 6.9 Hz,
2H), 3.57 (d, J = 12.4 Hz, 2H), 3.16 (s, 2H), 2.54 (s, 3H), 2.19 (s, 2H), 1.51 (s, 2H), 1.18 (s, 3H), 1.13 (t,
J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 154.7, 123.7, 60.5, 40.8, 35.7, 28.8, 14.7. HRMS
(ESI) calcd for C22H26N5O5 [M + H]+ 440.1934, found 440.1931.
Tert-butyl
4-(3-(4-fluorophenyl)-7-hydroxy-2-methylpyrazolo
[1,
5-a]pyrimidin-5-yl)-4-
methylpiperidine-1-carbo-xylate (4h) 44. The title compound was isolated as a white solid in 62%
1
yield. mp decomposition. H NMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 7.21 (dd, J = 8.0, 5.5 Hz, 2H),
7.04 (t, J = 8.5 Hz, 2H), 5.75 (s, 1H), 3.60-3.55 (m, 2H), 3.42-3.37 (m, 2H), 2.26 (s, 3H), 2.02-1.98 (m,
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2H), 1.73 (s, 2H), 1.44 (s, 9H), 1.41 (s, 3H). C NMR (101 MHz, CDCl3) δ163.1, 160.7, 157.7, 157.0,
154.8, 151.7, 138.2, 130.7, 130.6, 127.1, 116.3, 116.1, 105.3, 103.1, 95.1, 80.2, 37.4, 28.5, 25.4, 13.1,
1.2, 0.13. HRMS (ESI) calcd for C24H30FN4O3 [M + H]+ 441.2302, found 441.2310.
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Ethyl
4-(7-hydroxy-2-methyl-3-(4-nitrophenyl)pyrazolo[1,5-a]
pyrimidin-5-yl)piperidine-1-
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carboxylate (4i) 44. The title compound was isolated as a yellow solid in 61% yield. mp decomposition.
1H NMR (400 MHz, CDCl3) δ 11.37 (s, 1H), 8.05 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 5.65 (s,
1H), 4.30 (d, J = 10.9 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 2.95 (t, J = 12.1 Hz, 1H), 2.80 (s, 2H), 2.31 (s,
3H), 2.06-2.04 (m, 2H), 1.67 (ddd, J = 25.3, 12.7, 4.3 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 157.9, 157.3, 155.4, 151.0, 145.9, 139.3, 138.4, 130.1, 123.3, 102.6, 94.1, 61.7, 43.9,
40.3, 30.7, 14.8, 13.2. HRMS (ESI) calcd for C21H24N5O5 [M + H]+ 426.1777, found 426.1771.
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1-(4-(3-(4-Fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a]
pyrimidin-5-yl)piperidin-1-
yl)propan-1-one (4j) 44. The title compound was isolated as a white solid in 85% yield. mp 164-166 o C.
1H NMR (400 MHz, CD3OD) δ 7.51 (s, 2H), 7.17 (s, 2H), 5.80 (s, 1H), 4.67 (d, J = 15.8 Hz, 2H), 4.08
(d, J = 11.4 Hz, 1H), 3.16 (t, J = 12.2 Hz, 1H), 2.87 (s, 1H), 2.67 (d, J = 11.0 Hz, 1H), 2.45 (d, J = 7.0
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Hz, 2H), 2.32 (s, 3H), 1.99 (t, J = 15.4 Hz, 2H), 1.75-1.63 (m, 2H), 1.14 (t, J = 7.2 Hz, 3H). C NMR
(101 MHz, CD3OD) δ 174.7, 164.8, 162.3, 159.2, 153.0, 140.3, 133.0, 132.9, 127.9, 116.6, 116.4, 104.7,
93.9, 46.6, 42.8, 41.1, 32.3, 31.9, 27.3, 12.9, 10.0. HRMS (ESI) calcd for C21H24FN4O2 [M + H]+
383.1883, found 383.1876.
1-(4-(3-(4-Fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a] pyrimidin-5-yl)piperidin-1-yl)-2,2-
dimethylpropan-1-one (4k) 44. The title compound was isolated as a white solid in 85% yield. mp 267-
270 o C. 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 7.43 (dd, J = 8.5, 5.6 Hz, 2H), 7.30 (t, J = 8.8
Hz, 2H), 5.59 (s, 1H), 4.41 (d, J = 13.3 Hz, 2H), 2.90-2.84 (m, 1H), 2.75 (dd, J = 20.8, 8.0 Hz, 2H), 2.23
(s, 3H), 1.89 (d, J = 12.2 Hz, 2H), 1.50 (qd, J = 12.5, 3.3 Hz, 2H), 1.19 (s, 9H). 13C NMR (101 MHz,
DMSO-d6) δ 175.1, 162.6, 160.2, 157.3, 156.3, 150.3, 138.8, 131.8, 131.7, 127.2, 127.1, 115.8, 115.5,
102.3, 92.8, 44.8, 38.3, 30.9, 30.8, 28.3, 13.1. HRMS (ESI) calcd for C23H28FN4O2 [M + H]+ 411.2196,
found 411.2170.
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Tert-butyl 4-(3-(4-chlorophenyl)-7-hydroxy-2-methylpyrazolo [1, 5-a]pyrimidin-5-yl)pi -peridine-
1-carboxylate (4l) 44. The title compound was isolated as a white solid in 82% yield. mp 119-123 o C.
1H NMR (400 MHz, CDCl3) δ 11.35 (s, 1H), 7.17 (d, J = 8.2 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H), 5.51 (s,
1H), 4.22 (s, 2H), 3.07 (t, J = 11.7 Hz, 1H), 2.74 (s, 2H), 2.16 (s, 3H), 2.09 (d, J = 12.0 Hz, 2H), 1.62
(dt, J = 12.2, 8.7 Hz, 2H), 1.45 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 157.6, 156.5, 154.2, 150.4,
139.1, 132.0, 131.8, 130.1, 129.1, 102.4, 93.3, 79.2, 30.7, 28.5, 13.4. HRMS (ESI) calcd for
C23H28ClN4O3 [M + H]+ 443.1850, found 443.1832.
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Benzyl 4-(3-(4-chlorophenyl)-7-hydroxy-2-methylpyrazolo[1,5 -a]pyrimidin-5-yl)piper -idine-1-
carboxylate (4m) 44. The title compound was isolated as a white solid in 85% yield. mp 236-239 o C. 1H
NMR (400 MHz, CD3OD) δ 7.69 – 7.66 (m, 2H), 7.39 – 7.31 (m, 7H), 5.68 (s, 1H), 5.15 (s, 2H), 4.27 (d,
J = 13.3 Hz, 2H), 2.94 (s, 2H), 2.70 – 2.67 (m, 1H), 2.48 (s, 3H), 1.93 (d, J = 9.3 Hz, 2H), 1.71 (tt, J =
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12.5, 6.3 Hz, 2H). C NMR (101 MHz, CD3OD) δ 168.0, 161.4, 156.9, 150.5, 150.2, 138.2, 134.6,
131.4, 131.1, 129.5, 129.1, 129.0, 128.8, 105.0, 91.2, 68.2, 45.5, 45.2, 32.7, 14.5. HRMS (ESI) calcd for
C26H26ClN4O3 [M + H]+ 477.1693, found 477.1691.
Ethyl
4-(7-hydroxy-2-methyl-3-(4-(trifluoromethyl)
phenyl)
pyrazolo[1,5-a]pyrimidin-5-
yl)piperidine-1-carboxylate (4n) 44. The title compound was isolated as a white solid in 78% yield. mp
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258-261 o C. H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 7.85-7.81 (m, 2H), 7.68-7.66 (m, 2H),
5.64 (s, 1H), 4.06 (dt, J = 14.1, 9.2 Hz, 4H), 2.78 (t, J = 11.8 Hz, 3H), 2.30 (s, 3H), 1.85 (d, J = 12.4 Hz,
2H), 1.56 (tt, J = 12.4, 6.3 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 157.2,
156.0, 154.5, 150.0, 139.0, 135.3, 130.2, 125.5, 125.4, 101.9, 93.3, 60.7, 43.4, 30.2, 14.6, 13.1. HRMS
(ESI) calcd for C22H24F3N4O3 [M + H]+ 449.1801, found 449.1792.
Ethyl 4-(3-(4-fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a] pyrimidin-5-yl) piperi- dine-1-
carboxylate (4o) 44. The title compound was isolated as a white solid in 87% yield. mp 241-244 o C. 1H
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NMR (400 MHz, CDCl3) δ 12.12 (s, 1H), 7.14 (dd, J = 8.5, 5.4 Hz, 2H), 6.74 (t, J = 8.7 Hz, 2H), 5.48 (s,
1H), 4.26 (s, 2H), 4.09 (q, J = 7.1 Hz, 3H), 3.20 (t, J = 11.9 Hz, 1H), 2.78 (s, 2H), 2.12 (d, J = 9.7 Hz,
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6H), 1.64 (dt, J = 12.1, 8.7 Hz, 3H), 1.24 (t, J = 7.1 Hz, 4H). C NMR (101 MHz, CDCl3) δ 160.3,
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158.0, 157.7, 155.6, 150.9, 139.0, 131.2, 126.8, 115.0, 114.8, 103.17, 92.9, 61.6, 43.9, 39.8, 31.0, 14.8,
12.8. HRMS (ESI) calcd for C21H24FN4O3 [M + H]+ 399.1832, found 399.1813.
Benzyl 4-(3-(4-fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a] pyrimidin-5-yl)piperi -dine-1-
carboxylate (4p) 44. The title compound was isolated as a white solid in 82% yield. mp 206-209 o C. 1H
NMR (400 MHz, DMSO-d6) δ 7.79 (dd, J = 8.5, 5.8 Hz, 2H), 7.28 (d, J = 4.3 Hz, 4H), 7.23 (dd, J = 8.4,
4.0 Hz, 1H), 7.09 (t, J = 8.9 Hz, 2H), 5.34 (s, 1H), 5.01 (s, 2H), 4.04 (t, J = 12.0 Hz, 2H), 3.09 (d, J =
4.9 Hz, 1H), 2.82 (s, 2H), 2.37 (s, 3H), 1.74 (d, J = 11.9 Hz, 2H), 1.52 (qd, J = 12.6, 3.8 Hz, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 165.5, 158.6, 158.2, 154.6, 149.0, 147.5, 137.2, 131.7, 128.6, 128.6,
128.5, 127.9, 127.6, 114.8, 114.6, 102.1, 90.3, 66.2, 48.7, 44.0, 43.5, 15.2. HRMS (ESI) calcd for
C26H26FN4O3 [M + H]+ 461.1989, found 461.1984.
General procedure for the synthesis of 6a-6c. To a solution of 5 (1eq) in water was added NaOH (6a,
6c) (1.2eq) or K2CO3 (6b) (1.2eq), appropriate acyl chloride (6a, 6c) (1.2eq) or (Boc)2O (6b) (1.2eq)
dissolved in THF. The mixture was stirred at rt. for 5-7 h. The solvent was removed under reduced pres-
sure and the aqueous phase was adjusted to pH 2 with 1M HCl. The sample was then extracted with
CH2Cl2 and dried over anhydrous MgSO4. The solvent was evaporated under reduced pressure. The res-
idue was used in the next step without further purification.
1-(Tert-butoxycarbonyl)piperidine-4-carboxylic acid (6a)45. The title compound was isolated as a
white solid in 90% yield. 1H NMR (400 MHz, CDCl3) δ 4.01 (s, 2H), 2.85 (t, J = 11.7 Hz, 2H), 2.48 (tt,
J = 10.9, 3.9 Hz, 1H), 1.90 (d, J = 10.9 Hz, 2H), 1.68-1.58 (m, 2H), 1.45 (s, 9H).
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1-(Ethoxycarbonyl)piperidine-4-carboxylic acid (6b)46. The title compound was isolated as a white
solid in 91% yield. 1H NMR (400 MHz, CDCl3) δ 11.01 (s, 1H), 4.14-4.05 (m, 4H), 2.90 (t, J = 10.8 Hz,
2H), 2.49 (tt, J = 10.9, 3.8 Hz, 1H), 1.91 (d, J = 11.9 Hz, 2H), 1.64 (qd, J = 11.5, 4.1 Hz, 2H), 1.24 (t, J
= 7.1 Hz, 3H).
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1-((Benzyloxy)carbonyl)piperidine-4-carboxylic acid (6c)47. The title compound was isolated as a
white solid in 95% yield. 1H NMR (400 MHz, CDCl3) δ 10.72 (s, 1H), 7.39-7.29 (m, 5H), 5.13 (s, 2H),
4.10 (s, 2H), 2.95 (s, 2H), 2.51 (tt, J = 10.7, 3.8 Hz, 1H), 1.93 (s, 2H), 1.67 (d, J = 10.3 Hz, 2H).
General procedure for the synthesis of 7a-7b. To a solution of diisopropyl amine (6.73 mL, 48 mmol)
in THF (145 mL) was added n-BuLi (2M in hexane, 24mL, 48 mmol) at 0°C. The reaction was stopped
to -78°C 15 min later. Compound 6 (40mmol) in THF (60 mL) was added to the above solution and
stirred for 1h. Iodomethane (3.75 mL, 60 mmol) in THF (60 mL) was then added. The reaction was
warmed to rt. and stirred overnight, quenched with saturated NH4Cl aqueous solution at 0 °C and the
solvent removed. The sample was extracted with EtOAc (3 × 100 mL). The combined organic layer was
washed with brine (100 mL), dried over anhydrous MgSO4 and solvent evaporated under reduced pres-
sure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc, 150: 1-
50: 1) to obtain the title compound.
Diethyl 4-methylpiperidine-1, 4-dicarboxylate (7a)46. The title compound was isolated as a light yel-
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low oil in 87% yield. H NMR (400 MHz, CDCl3) δ 4.13-4.04 (m, 4H), 3.79 (s, 2H), 2.98 (t, J = 11.4
Hz, 2H), 2.03 (d, J = 13.5 Hz, 2H), 1.35-1.28 (m, 2H), 1.21 (td, J = 7.1, 5.5 Hz, 6H), 1.16 (s, 3H).
1-(Tert-butyl) 4-ethyl 4-methylpiperidine-1,4-dicarboxylate (7b)48 The title compound was isolated
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as a light yellow oil in 85% yield. H NMR (400 MHz, CDCl3) δ 4.17-4.09 (m, 2H), 3.76-3.71 (m, 2H),
2.96 (t, J = 11.5 Hz, 2H), 2.04 (d, J = 13.1 Hz, 2H), 1.43 (s, 9H), 1.37-1.30 (m, 2H), 1.24 (t, J = 7.1 Hz,
3H), 1.18 (s, 3H).
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General procedure for the synthesis of 8a-8d. To a solution of compound 7 (40 mmol) in THF (10
mL) was added 5M NaOH (40 mL, 200 mmol) at 0 °C, the resulting solution was then reacted at 70 °C
overnight. After removing the solvent, the sample was diluted with water and extracted with EtOAc (3
×15 mL). The resulting aqueous phase was adjusted to pH 3 with 1M HCl and extracted with CH2Cl2
for three times, which was then combined with the organic phase and dried over anhydrous MgSO4. The
solvent was removed under reduced pressure. The corresponding carboxylic acid was obtained and used
directly in the next step without further purification. Then, to a round bottom flask with the above car-
boxylic acid (1eq) dissolved in THF was added N, N'-carbonyldiimidazole (1.3 eq) at 0°C. The resulting
solution was stirred at rt. for 4 h. To another solution of potassium 3-ethoxy-3-oxopropanoate (1 eq) in
MeCN: THF (1: 2) was added MgCl2 (2 eq) and 4-dimethylaminopyridine (DMAP, 0.1 eq) at 0 °C. The
resulting solution was stirred at rt for 5 h. The former solution was then added to the latter solution at
0 °C. The resulting mixture was stirred at rt. overnight. After removing the solvent and adjusting pH to
4 with 1 M HCl at 0 °C , the product was extracted with EtOAc for three times. The combined organic
layer was washed with 1M HCl, 1M NaOH and brine separately, dried over anhydrous MgSO4 and the
solvent removed under reduced pressure to obtain 8 as a yellow oil, which was used directly in the next
step without further purification.
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Ethyl 4-(3-ethoxy-3-oxo)propanoyl)-4-methylpiperidine-1-carbo-xylate (8a)46. Yield of 82%. H
NMR (400 MHz, CDCl3) δ 4.21-4.15 (m, 2H), 4.10 (q, 2H), 3.62 (s, 2H), 3.51 (s, 2H), 3.35-3.20 (m,
2H), 1.97 (m, 2H), 1.44 (d, J = 3.7 Hz, 2H), 1.26 (tt, J = 10.5, 7.1 Hz, 6H), 1.18 (s, 3H).
Tert-butyl 4-(3-ethoxy-3-oxopropanoyl)-4-methylpiperidine-1-carboxylate (8b) 46. Yield of 86%. 1H
NMR (400 MHz, CDCl3) δ 4.17 (qd, J = 7.1, 1.3 Hz, 2H), 3.53-3.46 (m, 4H), 3.29-3.16 (m, 2H), 1.96-
1.91 (m, 2H), 1.42 (s, 11H), 1.29-1.23 (m, 3H), 1.16 (s, 3H).
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Benzyl 4-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate (8c) 46. Yield of 87%. H NMR (400
MHz, CDCl3) δ 7.39-7.36 (m, 5H), 5.14 (s, 2H), 4.21 (q, J = 7.1 Hz, 4H), 3.51 (s, 2H), 2.89 (d, J = 10.8
Hz, 2H), 2.67 (tt, J = 11.2, 3.7 Hz, 1H), 1.88 (s, 2H), 1.59 (m, 2H), 1.30 (td, J = 7.1, 5.6 Hz, 3H).
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Tert-butyl 4-(3-ethoxy-3-oxopropanoyl) piperidine-1-carboxylate (8d)44. Yield of 91%. H NMR
(400 MHz, CDCl3) δ 4.12 (dd, J = 14.1, 7.0 Hz, 2H), 4.04 (s, 2H), 3.43 (s, 2H), 2.72 (t, J = 11.4 Hz, 2H),
2.59-2.54 (m, 1H), 1.77 (t, J = 13.6 Hz, 2H), 1.52-1.43 (m, 2H), 1.38 (s, 9H), 1.22-1.19 (m, 3H).
General procedure for the synthesis of 11a-11d. To a sealing tube was added 9 (14.4 mL, 100 mmol),
10 (14.6 mL, 120 mmol), dry CH2Cl2 (10 mL) under N2 atmosphere. The mixture was heated at 100 °C
for 24 h. After cooling to rt., the resulting solution was added to a solution of hydrazine hydrochloride
(10.3 g, 150 mmol) in EtOH (20 mL) and H2O (20 mL) in a 250 mL round-bottom flask. The mixture
was then heated at 80 °C for 12 h. The resulting solution was concentrated and neutralized with saturat-
ed solution of NaHCO3 to pH 9. The mixture was extracted with EtOAc (3×100 mL). The combined or-
ganic layer was washed with brine (100mL), dried over anhydrous MgSO4 and the solvent removed un-
der reduced pressure. The product was recrystallized with EtOAc to obtain the title compound.
4-(4-Fluorophenyl)-3-methyl-1H-pyrazol-5-amine (11a)44. The title compound was isolated as a
white solid in 85% yield. 1H NMR (400 MHz, CDCl3) δ 7.28-7.24 (m, 2H), 7.07 (t, J = 8.7 Hz, 2H),
2.22 (s, 3H).
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4-(4-Chlorophenyl)-3-methyl-1H-pyrazol-5-amine (11b) . The title compound was isolated as a
white solid in 81% yield. 1H NMR (400 MHz, CDCl3) δ 7.35 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.2 Hz,
2H), 2.24 (s, 3H).
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3-Methyl-4-(4-(trifluoromethyl) phenyl)-1H-pyrazol-5-amine (11c) 44. The title compound was iso-
lated as a yellow solid in 88% yield. 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 8.1 Hz, 2H), 7.47 (d, J =
8.0 Hz, 2H), 2.31 (d, J = 5.5 Hz, 3H).
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3-Methyl-4-(4-nitrophenyl)-1H-pyrazol-5-amine (11d) 44. The title compound was isolated as a yel-
low solid in 82% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 8.21-8.19 (m, 2H), 7.63 (d, J =
8.7 Hz, 2H), 4.95 (s, 2H), 2.26 (s, 3H).
General procedure for the synthesis of 12. To a solution of 8 (1 eq) in AcOH was added 11 (1.1 eq),
the resulting solution was refluxed overnight. After removing the solvent and adjusting pH of the aque-
ous phase to 8 with a saturated NaHCO3 solution, the product was extracted with CH2Cl2 and dried over
anhydrous MgSO4. The solvent was evaporated under reduced pressure and the residue was purified by
silica gel column chromatography (CH2Cl2 / MeOH, 100 : 1- 40: 1) to obtain compound 12.
General procedure for the synthesis of 13a. To a solution of 12 (R2 = Boc) in CH2Cl2 (3V/V) was
added TFA (V/V) and the mixture stirred at 0 °C for 2 h, After removing the solvent and adjusting pH
of the aqueous phase to 8 with a saturated NaHCO3 solution, the resulting precipitation was filtrated,
washed with water, and dried over vacuum. 13a was obtained and used in the next step without further
purification.
General procedure for the synthesis of 13b. To a solution of 8 (1 eq) in EtOH: TFA = (50 mL : 10 mL)
was added 11a (1.2 eq). The resulting solution was stirred at 85°C when another 6.5 mL, 8.5 mL, and 25
mL of TFA was added after 10 h, 20 h, and 30 h separately. The reaction was quenched with water at 48
h. After removing the solvent and adjusting pH of the aqueous phase to 8 with a saturated NaHCO3 so-
lution, the product was filtered, the residue washed with water, CH3OH, and CH2Cl2 separately, and
then dried over vacuum to obtain 13b as a white solid, which was used directly in the next step without
further purification.
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Cell Lines and Cell Culture. HEK293 cells were grown in DMEM (high glucose) supplemented with
10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin at
37°C , 5% CO2. Stable cell lines that express human TRPC3, mouse TRPC4, mouse TRPC6, mouse
TRPV3, or mouse TRPM8 were established as described previously and maintained in the medium sup-
plemented with G418 (400 µg/mL; Invitrogen)49. For those that co-expressed MOR or M5 muscarinic
receptor, 100 µg/ml hygromycin B (Calbiochem) was also included in the culture medium. Stable cell
lines that inducibly express human TRPC5, TRPC7, TRPA1, TRPV1, TRPV4, or TRPM5, were estab-
lished as described previously and maintained in the medium supplemented with 100 μg/mL hygromy-
cin B and 5 μg/mL blasticidin50. The expression of TRP channels was induced by the addition of 0.1 µM
doxycycline to the culture medium 24 hrs prior to the assay.
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The use of animals was approved by the Animal Experimentation Ethics Committee of Anhui Medical
University and in accordance with the guidelines for ethical conduct in the care and use of animals.
Glomerular mesangial cells were prepared according to previous report43, 51. Briefly, glomeruli were iso-
lated from male Sprague-Dawley rats (260–280 g) using the graded sieving technique. Glomeruli were
collected by centrifugation and digested by collagenase (2 mg/mL, Sigma-Aldrich) for 45 min at 37°C .
Following the wash-off of the enzymes, mesangial cells were cultured in RPMI 1640 medium supple-
mented with 17% FBS, 100 units/mL penicillin G, and 100 µg/mL streptomycin at 37°C in a 5% CO2
humidified incubator.
Fluorescence Ca2+ and Membrane Potential Assays. Cells were seeded in wells of 96-well plates pre-
coated with polyornithine at ~100,000 cells/well. Cells were loaded with Fluo4-AM (Invitrogen) or
Fluo8-AM (TEFLabs) to monitor intracellular Ca2+ changes or loaded with the FLIPR Membrane Po-
tential dye (FMP, Molecular Devices) to monitor membrane potential changes with the use of a FlexSta-
tion microplate reader (Molecular Devices). The detailed protocols for the FlexStation FMP and Ca2+
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