Discovery of 1-(2,4-Dichlorophenyl)- N -(piperidin-1-yl)-4-((pyrrolidine-1- sulfonamido)methyl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)thiophene-2-yl)-1 H -pyrazole-3-carboxamide as a novel peripherally restricted cannabinoid-1 receptor antagonist with significant weight-loss efficacy in diet-induced obese mice

Article abstract of DOI:10.1021/jm401158e

After extensive synthetic efforts, we found that many structurally diverse bioisosteres could be generated via derivatizing the C-4 alkyl chain on the pyrazole ring of compound 3 (B/P = 1/33) with different electronegative groups. Especially when a sulfonamide or sulfamide moiety was added, resulting compounds exhibited not only potent CB1R activity but also a desired tPSA value over 90 ?², a threshold considered to possess a low probability to cross BBB, leading to the identification of compound 4 (B/P = 1/64) as a peripherally restricted CB1R antagonist. Apart from its significant weight-loss efficacy in DIO mice, compound 4 also displays 163 clean off-target profiles and is currently under development for treating obesity and the related metabolic syndrome.

Full text of DOI:10.1021/jm401158e

Article
pubs.acs.org/jmc
Discovery of 1‑(2,4-Dichlorophenyl)‑N‑(piperidin-1-yl)-4-
((pyrrolidine-1-sulfonamido)methyl)-5-(5-((4-
(trifluoromethyl)phenyl)ethynyl)thiophene-2-yl)‑1H‑pyrazole-3-
carboxamide as a Novel Peripherally Restricted Cannabinoid‑1
Receptor Antagonist with Significant Weight-Loss Efficacy in Diet-
Induced Obese Mice
Chun-Ping Chang,† Chien-Huang Wu,† Jen-Shin Song, Ming-Chen Chou, Ying-Chieh Wong, Yinchiu Lin,
Teng-Kuang Yeh, Amit A. Sadani, Ming-Hung Ou, Kun-Hung Chen, Pei-Hsuan Chen, Po-Chu Kuo,
Chen-Tso Tseng, Kuei-Hua Chang, Shi-Liang Tseng, Yu-Sheng Chao, Ming-Shiu Hung,*
and Kak-Shan Shia*
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli County 35053, Taiwan, R.O.C.
S
* Supporting Information
ABSTRACT: After extensive synthetic efforts, we found that many structurally diverse bioisosteres could be generated via
derivatizing the C-4 alkyl chain on the pyrazole ring of compound 3 (B/P = 1/33) with different electronegative groups.
Especially when a sulfonamide or sulfamide moiety was added, resulting compounds exhibited not only potent CB1R activity but
also a desired tPSA value over 90 Å2, a threshold considered to possess a low probability to cross BBB, leading to the
identification of compound 4 (B/P = 1/64) as a peripherally restricted CB1R antagonist. Apart from its significant weight-loss
efficacy in DIO mice, compound 4 also displays 163 clean off-target profiles and is currently under development for treating
obesity and the related metabolic syndrome.
(CP-945598), were all terminated in the late stages of
development.
INTRODUCTION
■
Cannabinoid-1 receptor (CB1R) is one of the most abundant
neuroregulatory receptors in the brain and involved mainly in
regulating feeding and appetite.1 In addition to the brain, this
receptor is also expressed in the peripheral organs, such as
adipose tissues, muscle, and liver.2 Distinct from CB1R,
cannabinoid-2 receptor (CB2R) is mostly expressed in the
immune system and primarily associated with immune
regulation and neurodegeneration.3,4 Clinically, a CB1R-
targeted antiobesity agent known as rimonabant (1) or
SR141716A was launched in Europe in 2006; however, it was
soon withdrawn in 2008 due to severe central nervous system
(CNS) adverse effects, including depression, anxiety, and stress
disorders (Figure 1). Consequently, antiobesity agents acting
on brain CB1R, such as taranabant (MK-0364) and otenabant
On the other hand, great efforts have been made particularly
on the development of peripherally selective CB1R antagonists
in that accumulating evidence from obese animal models
indicated that peripheral CB1R antagonists could also cause
weight loss and improve related metabolic disorders without
additional CNS-mediated behavioral effects.5−7 Encouragingly,
7TM Pharma has successfully completed a phase I clinical trial
of TM38837, a peripherally restricted CB1R antagonist claimed
for the treatment of obesity and type 2 diabetes, and clearly
demonstrated this CB1R restricted antagonist is lack of CNS
Received: July 30, 2013
© XXXX American Chemical Society
A
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Figure 1. Typical brain CB1R-acting antagonists reported in the literature.
Figure 2. Compounds 2−4 progressing from mainly targeting brain to targeting peripheral CB1R.
side effects as observed with 1.8 In fact, International Diabetes
Table 1. Brain-to-Plasma Ratio (B/P) and Calculated tPSA
of Compounds 1−4
Federation clearly points out that there is an urgent medical
need for pharmacological agents suitable for treating coexistent
type 2 diabetes and obesity because some common antidiabetic
agents, including thiazolidinediones (TZDs), insulin, and
sulfonylureas (SUs), are associated with undesired weight
gain.9 Thus, peripheral CB1R antagonists, which are able to
improve hyperglycemia and induce weight loss simultaneously,
might meet with this particular purpose and is worth further
development. To date, three chronic treatment drugs, namely
orlistat, lorcaserin (belviq), and qsymia, have been approved by
FDA (US) for the treatment of obesity. Mechanistically, orlistat
serves as a pancreatic lipase inhibitor with the significant
efficacy to reduce the intestinal absorption of fat, but
unpleasant side effects have resulted in little success.10,11
Lorcaserin is a selective serotonin 5-HT2C agonist, and qsymia
is a combination of two old drugs phentermine and
topiramate.12−14 However, on the basis of side effects observed
in clinical trials, these two recently approved antiobesity drugs
might have a long-term safety issue after chronic treatment.
During the past decade, tremendous efforts have been
dedicated to seeking antiobesity agents targeting CB1R in our
laboratories, and several novel series of CB1R antagonists have
been found.15−23 Representative compounds 2−4 are high-
lighted (Figure 2) in chronological order to demonstrate the
evolution of our structure-based design from mainly targeting
brain CB1R to targeting peripheral CB1R. Experimentally,
assessing brain-to-plasma ratio (B/P) appears to be an effective
and practical approach to pursue desired peripheral CB1R
antagonists. Along this axis (Table 1), structural modifications
started with agent 1 (B/P = 3/1) through second-generation
derivatives 2 (B/P = 1/6) and 3 (B/P = 1/33), leading to the
identification of a potential drug candidate 4 (B/P = 1/64) with
a significant improvement in the B/P ratio by ∼200-fold
relative to the initial model 1.
a,d
compd
tPSA (Å2)
B/P
hCB1R Ki (nM)
b
1
2
3
4
52
52
3/1
1/6
1.4 0.4
2.0 0.6
0.3 0.1
0.3 0.1
52
1/33
1/64 (1/120)
c
112
a
Nonperfused brain samples were assayed 2 h after oral dosing (20
b
mg/kg) in C57BL/6 mice (n = 4). Nonperfused brain samples were
assayed 2 h after oral dosing (2 mg/kg) in C57BL/6 mice (n = 4).
c
Nonperfused brain samples were assayed 2 h after oral dosing (20
d
mg/kg) in DIO mice (n = 4). Vehicle formulation: DMSO/Tween
80/H2O (1:1:8, v/v/v)
ring of lead 3 resulted in a 2-fold increase in the topological
polar surface area (tPSA), and more importantly, activities
toward CB1R were retained at the nanomolar level during the
current synthetic tailoring. As demonstrated by many historical
cases,24−36 in general compounds with a tPSA value higher than
90 Å2 are considered to have a low probability to permeate the
blood−brain barrier (BBB). Indeed, this synthetic strategy is
commonly applied to develop peripheral CB1R antagonists
with low brain penetration during structural optimization.
Detailed description on the design, synthesis, structure−activity
relationships (SAR) of the newly developed compounds, and
animal studies for the potential candidate are presented as
follows.
CHEMISTRY
■
Test compounds 4, 12, and 14−32 in Table 2 were prepared
according to a general synthetic method shown in Scheme 1
using compounds 4 and 12, respectively, as a typical example.
Starting material 6 was readily synthesized based on the
modified synthetic procedures reported by our laboratories.17
Radical bromination of 6 was effected under treatment with
NBS and AIBN in CCl4 at 80 °C to afford dibromo ester 7
(92%), which in turn was treated with silver nitrate in acetone/
We serendipitously discovered that installing a sulfamide or
sulfonamide functionality at the C-4 position of the pyrazole
B
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Table 2. Biological Evaluations of 5-(5-Alkynylthiophene-2-yl)pyrazole Derivatives on CB1 and CB2 Receptors
C
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Table 2. continued
a
Binding affinity determined by inhibition of [3H]-CP-55940 binding to hCB1R or hCB2R-transfected HEK 293 membrane is expressed as Ki.
b
c
Functional activity determined by inhibition of Eu-GTP binding to hCB1R-transfected HEK 293 membrane is expressed as EC50. Data are
d
e
expressed as the mean SEM of at least three independent experiments. IP: intrinsic property for inverse agonist efficacy. tPSA: topological polar
f
surface area. Tetrad model response (n = 4/group) (CP-55940 100%; “−”: >90% (weakly or not reversed); “+”: 50% < X ≤ 90%; “++”: 10% < X ≤
50%; “+++”: ≤10% (strongly reversed)). Test compound (50 mg/kg) was orally dosed 1 h prior to ip treatment with agonist CP-55940 (1 mg/kg)
in the CB1R agonist-induced hypothermia or analgesia model.
water (1:1) to give hydroxyl bromide 8 in good yield (76%).
We also attempted to brominate both the thiazole ring and 4-
methyl group of the precursor of bromide 6 in one step using
the reaction conditions of Scheme 1. However, a complex
mixture was formed, and product 7 was isolated in a yield much
lower than that of the current two-step process. Under catalysis
with aluminum chloride, compound 8 was reacted with 1-
aminopiperidine efficiently to furnish the corresponding amide
9 in quantitative yield (97%). Amide 9 thus obtained was
coupled with 1-ethynyl-4-(trifluoromethyl)benzene in the
presence of Pd(PPh3)2Cl2 and CuI as catalyst to give
compound 10 in 87% yield, which in turn underwent
bromination with PBr3 to furnish bromide 11 in quantitative
yield (98%). Compound 11 was sequentially subjected to SN2
substitution (NaN3) and Staudinger reduction (PPh3/H2O) to
afford the corresponding primary amine 13 in 66% over two
steps. Finally, amine 13 was reacted with pyrrolidine-1-sulfonyl
chloride in the presence of triethylamine to accomplish the
desired target 4 in 87% yield. Similarly, intermediate 13 was
allowed to couple individually with different sulfonyl and
sulfamoyl chlorides to give the corresponding sulfonamide and
sulfamide derivatives 23−32 in moderate to good yields (45−
89%). In addition, bromide 11 directly underwent SN2
displacement with 3-hydroxyazetidine to afford target 12 in
68% yield. Thus, following the similar synthetic operation,
intermediate 11 was coupled with selected primary and
secondary amines, respectively, to afford another series of
analogues 14−22 in moderate to good yields (56−88%). As
evidenced by HPLC analysis in the Experimental Section, all
test compounds exhibited more than 95% purity prior to in
vitro assays and animal studies.
RESULTS AND DISCUSSION
■
It is widely accepted that installing an electronegative group
could dramatically increase the tPSA value of a molecule. In
general, compounds with a tPSA over 90 Å2 are able to
significantly lower the probability to penetrate BBB. However,
prior to this strategy-oriented optimization, it is necessary to
identify the synthetically tolerated position in the initial lead,
thus allowing us to generate structurally diverse analogues
without compromising the potency and activity toward the
target of interest. Compound 3, having a low brain-to-plasma
partition (B/P = 1/33), was considered an ideal lead for
seeking novel analogues with further enhancement in peripheral
D
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a
Scheme 1. Synthesis of Sulfamide and Sulfonamide Derivatives Using Compounds 4 and 12 as an Example
a
Reagents and conditions: (a) NBS, AIBN, CCl4, 80 °C, 16 h, 92%; (b) AgNO3, acetone/water = 1/1, 60 °C, 16 h, 76%; (c) AlCl3, 1-
aminopiperidine, DCE, rt, 16 h, 97%; (d) PdCl2(PPh3)2, CuI, 1-ethynyl-4-(trifluoromethyl)benzene, 2-ethanolamine, and THF in a sealed pressure
vessel, 80 °C, 12 h, 87%; (e) PBr3, CH2Cl2, 0 °C to rt, 2 h; 98%; (f) 3-hydroxyazetidine hydrochloride, Et3N, DMF, 60 °C, 3 h, 68%; (g) NaN3,
DMF, rt, 3 h; (h) PPh3, THF/H2O = 1/1, rt, 16 h, 66% over two steps; (i) pyrrolidine-1-sulfonyl chloride, Et3N, DMF, 0 °C to rt, 16 h, 87%.
selectivity over brain. After extensive structural modifications
on its C-3, C-4, and C-5 positions on the pyrazole ring,
respectively, the C-4 alkyl group was found to be a well
tolerated position for structural variation. As listed in Table 2,
many bioisosteres with a tPSA value higher than that of parent
hit 3 (52 Å2) were obtained through appending a variety of
heteroatom-containing moieties on the C-4 side chain.
compounds potentially with limited BBB penetration, and
meanwhile their tPSA values were all greater than 90 Å2.
Compounds 28−30, though possessing a high tPSA value
(112−122 Å2) and potent CB1R activity (Ki = 0.4−1.4 nM),
were found to have positive effects on tetrad responses,
presumably due to the fact that under physiological conditions,
many other factors, including solubility, absorption, and
metabolic issues, might dominate the final consequences.
Compounds 12, 15, 17, and 20−22 with tPSA values (54−
79 Å2) less than 90 Å2 were also selected for testing. As
expected, they all failed to give a dual negative response in both
hypothermia and analgesia tests (Table 2). As such, compounds
14, 16, 18, and 19 with tPSA falling in between 54 Å2 and 78 Å2
were not elected for further tetrad-response tests. Substitution
of the conventional alkyl group with an electronegative moiety
Structurally, these potential peripheral-acting CB1R antago-
nists can be divided into two main groups as represented by
sulfamide 4 (CB1R: Ki = 0.3 0.1 nM; EC50 = 3.0 0.2 nM;
tPSA = 112 Å2) and sulfonamide 26 (CB1R: Ki = 0.8 0.1
nM; EC50 = 7.4
2.5 nM; tPSA = 109 Å2), respectively.
Compounds 4, 25−27, 31, and 32 showed a dual negative
response on CB1R agonist-induced hypothermic and analgesic
effects at a dose of 50 mg/kg, an in-house threshold to select
E
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at C-4 position could dramatically increase the tPSA value, but
this structural alteration somehow resulted in a loss of CB2/1
selectivity as demonstrated with compounds 24 and 28−30
(CB2/1 = 2−6) relative to parent hit 3 (CB2/1 = 167).19
Intriguingly, though compounds 4 and 29−32 are structurally
closely related analogues, there is no clear relationship linking
their CB2/1 selectivity to the ring size at the C-4 side chain.
Further structural design resulted in compound 32 with an
agonist, was found to cross the BBB easily and counteract
significantly both temperature-lowering and analgesic effects at
an oral dose as low as 2 mg/kg.
Compound 4 appears to be a promising peripherally
restricted CB1R inverse agonist. A long-term treatment of
compound 4 was then carried out to evaluate the weight-loss
efficacy in DIO mice. As depicted in Figure 5A, reduction of
excellent biological activity (CB1R: Ki = 0.4 0.1 nM; EC50
=
4.2 0.4 nM) and a high tPSA (126 Å2). Apparently, inserting
an extra oxygen in the terminal C-4 ring is responsible for such
a substantial increase in tPSA as compared to the
corresponding compounds 4, 29, 30, and 31 (tPSA = 112
Å2). However, compound 32 shows poorer water solubility
(<0.005 ng/mL) than 4 (0.014 ng/mL) though it has an
additional oxygen. In terms of intrinsic properties, all test
compounds induced a significant decrease in Eu-GTP binding
ranging from −14% to −51%, suggesting that they should
behave as the CB1R inverse agonist as with parent 3 (Table
2).19
In light of the lowest B/P ratios observed in both normal and
diet-induced obese (DIO) mice after 2 h acute treatment
(Table 1), compound 4 was chosen for further in vivo studies.
As illustrated in Figures 3 and 4, compound 4 did not reverse
Figure 5. Efficacy in the DIO mouse model following oral
administration of compound 4 (10 and 20 mg/kg, po qd) compared
to that in ref 1 (10 mg/kg, po qd) in the 22-day chronic study (n = 6/
group). (A) Body weight change was measured daily during
compound treatment. Data are expressed as mean standard error.
(B) The hepatic triglyceride level was measured after 22-day chronic
treatment of compounds. Statistical significance is analyzed by one-
way ANOVA followed by Dunnett’s post test (**P < 0.01).
Figure 3. Compound 4 (30 and 100 mg/kg, po) and control 1 (2 mg/
kg, po) were administrated, respectively, 1 h prior to the treatment
with agonist CP-55940 (1 mg/kg, ip) in the CB1R agonist-induced
hypothermia model. Body temperature was measured at a time point
of 30 and 65 min, respectively, after agonist dosing. (P < 0.05, #vs CP-
55940 group; P < 0.01, **vs vehicle control, ##vs CP-55940 group).
body weight occurred gradually and persistently in a dose-
dependent manner throughout the 22-day period with a relative
weight-loss rate of 26.4% and 32.8% for 10 and 20 mg/kg
groups, respectively, indicating that this compound is as
effective as its brain-acting counterpart 1 (10 mg/kg; 29.1%
weight loss) in weight-reduction efficacy, again supporting that
instead of brain CB1R, peripheral CB1R could be a potential
target for treating obesity. In addition, a significant decrease in
hepatic triglyceride level was observed in a dose-dependent
manner after the above chronic treatment (Figure 5B),
suggesting that compound 4 might have great potential to
ameliorate this related metabolic syndrome.37
More encouragingly, after 22-day repeated dosing, only trace
amounts of compound 4 were found in the mouse brain (4.3
0.4 and 7.2 0.3 ng/g for 10 and 20 mg/kg groups). These
amounts were even lower than the amount detected in brain,
15.9 3.2 ng/g, after 2 h acute treatment at an oral dose of 20
mg/kg, indicating that no severe drug accumulation occurred in
the CNS (Table 3). In sharp contrast, the positive control 1
was detected at a high brain concentration of 125.6 4.2 ng/g
(vs 4.3 0.4 ng/g of 4) over the same period (22 days) at an
oral dose of 10 mg/kg. However, whether such a low
concentration in brain is owing to P-glycoprotein (P-gp) efflux
needs to be addressed. The P-gp ATPase assay was then
performed to clarify this issue and indicated that compound 4
was not a P-gp substrate, in that ATPase activity resulted in a
Figure 4. Compound 4 (30 and 100 mg/kg, po) and control 1 (2 mg/
kg, po) were administrated, respectively, 1 h prior to the treatment
with agonist CP-55940 (1 mg/kg, ip) in the CB1R agonist-induced
analgesia model. Tail flick response was measured at a time point of 35
min after agonist dosing. (P < 0.01, *vs vehicle control, #vs CP-55940
group).
the tetrad responses at an oral dose up to 100 mg/kg, whereas
the positive control 1, a typical brain CB1R-acting inverse
F
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Mercury-300 (300 MHz) and a Varian Mercury-400 (400 MHz).
Chloroform-d or dimethyl sulfoxide-d6 was used as the solvent and
TMS (δ 0.00 ppm) as an internal standard. Chemical shift values are
reported in ppm relative to the TMS in delta (δ) units. Multiplicities
are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet),
q (quartet), dd (doublet of doublets), dt (doublet of triplets), and m
(multiplet). Coupling constants (J) are expressed in hertz. Electro-
spray mass spectra (ESMS) were recorded as m/z values using an
Agilent 1100 MSD mass spectrometer. All test compounds displayed
more than 95% purity as determined by a Hitachi 2000 series HPLC
system using a phenyl column (Waters XBridge 5 μm, 4.6 mm × 150
mm). Mobile phase A: acetonitrile; mobile phase B: 10 mM
ammonium acetate aqueous solution containing 0.1% formic acid.
The gradient system started from A:B (10%:90%) to A:B (90%:10%)
with a flow rate of 0.5 mL/min, and the injection volume was 5 μL.
The system was operated at 25 °C. Peaks were detected at 254 nm.
IUPAC nomenclature of compounds was determined with ACD/
Name Pro software.
Table 3. Determination of Brain Concentration of
Compounds 1 and 4
a
compound
dose (mg/kg)
brain (ng/g)
125.6 4.2
1
4
4
10
10
20
b
4.3 0.4
b
7.2 0.3
a
Compound 4 (10 and 20 mg/kg, po qd) compared to ref 1 (10 mg/
kg, po qd) after chronic treatment for 22 days in DIO mice (n = 6/
group). Data are presented as mean
Versus ref 1.
standard error; P < 0.01.
b
significant decrease in the single-to-noise ratio from 3.34
(verapamil-treated, positive control) to 0.87.38
The title compound, 1-(2,4-dichlorophenyl)-N-(piperidin-1-
yl)-4-((pyrrolidine-1-sulfonamido)methyl)-5-(5-((4-
(trifluoromethyl)phenyl)ethynyl)thiophene-2-yl)-1H-pyrazole-
3-carboxamide (4), is currently under preclinical development
for treating obesity and related metabolic syndrome in the
model of type 2 diabetes. The 163 off-target standard assays
were conducted, and the clean off-target profiles are displayed
in Supporting Information.39 Accordingly, besides GABAA (Ki
= 2.99 μM), sodium channel, vanilloid, and angiotensin AT1
(50−57% inhibition at 10 μM), compound 4 showed low
affinity (<50% inhibition at 10 μM) toward the other 159 off-
targets. As well, no hERG liability was observed at a
concentration up to 30 μM in the patch-clamp assay.
In conclusion, a combination of the calculated tPSA value
and experimental B/P ratio appears to be a practical approach
to modulate biopharmaceutical properties of a molecule, such
as BBB permeability, in the development of peripherally acting
derivatives. The incorporation of an electronegative fragment is
usually an effective way to significantly increase the overall tPSA
of a molecule and thereof might result in low BBB penetration.
Nevertheless, an appropriate location must be identified in the
lead molecule during synthetic tailoring to avoid compromising
any fundamental elements, such as potency and activity toward
the target of interest. Current results demonstrate that
compound 4 is not a P-gp substrate and is considered a
peripherally restricted CB1R inverse agonist as evidenced by its
negative tetrad responses, low B/P partition, and trace amount
in brain after chronic treatment.
1-(2,4-Dichlorophenyl)-N-(piperidin-1-yl)-4-((pyrrolidine-1-
sulfonamido)methyl)-5-(5-((4-(trifluoromethyl)phenyl)-
ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide (4). To a
magnetically stirred solution of 13 (1.15 g, 1.86 mmol) and Et3N (0.38
g, 3.72 mmol) in DMF (10 mL) at 0 °C was added pyrrolidine-1-
sulfonyl chloride (0.47 g, 2.79 mmol) dropwise. The resulting mixture
was allowed to warm to room temperature for 16 h and then quenched
with water. The aqueous phase was extracted with ethyl acetate (2 ×
20 mL), and the combined organic extracts were washed with water
and brine, dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure to give the crude residue,
which was purified by flash chromatography with n-hexane/ethyl
acetate (1:1) as an eluting solvent to afford the desired product 4 (1.21
1
g, 87%) as a white solid: mp 163.5−164.5 °C; H NMR (CDCl3) δ
7.71 (br s, 1H), 7.60−7.58 (m, 4H), 7.53 (d, J = 2.0 Hz, 1H), 7.38 (dd,
J = 8.4 and 2.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 3.6 Hz,
1H), 7.22 (d, J = 3.6 Hz, 1H), 6.50 (t, J = 6.8 Hz, 1H), 4.34 (d, J = 6.8
Hz, 2H), 3.28−3.25 (m, 4H), 2.87−2.81 (m, 4H), 1.87−1.85 (m, 4H),
1.79−1.77 (m, 4H), 1.48−1.42 (m, 2H); 13C NMR (CDCl3) δ 159.2,
144.3, 137.4, 136.7, 134.8, 133.3, 132.7, 131.3, 130.6, 130.2, 130.1,
130.0 (q, JC−F = 33.0 Hz), 128.5, 127.9, 125.9, 125.6, 125.0 (q, JC−F
=
4.0 Hz), 123.5 (q, JC−F = 270.5 Hz), 119.7, 93.4, 83.7, 56.9, 47.7, 37.2,
25.3, 25.0, 22.9; ESMS m/z: 751.0 (M + 1); HPLC purity = 99.21%, tR
= 46.15 min.
Ethyl 4-(Bromomethyl)-5-(5-bromothiophene-2-yl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxylate (7). To a magneti-
cally stirred solution of bromo ester 6 (2.20 g, 4.78 mmol) in CCl4 (22
mL) were sequentially added NBS (1.1 g, 6.21 mmol) and AIBN (0.05
g, 0.33 mmol) in one portion. The resulting mixture was heated under
reflux for 16 h and then cooled to room temperature. The reaction was
quenched with saturated aqueous sodium thiosulfate and extracted
with CH2Cl2 (2 × 40 mL). The organic layers were combined, washed
with water, brine, dried over Na2SO4, filtered, and concentrated to give
the crude product, which was passed through a layer of silica gel in a
sintered-glass funnel and washed with CH2Cl2. The filtrate was
concentrated to give dibromide 7 (2.52 g, 92%) as a pale yellow solid:
EXPERIMENTAL SECTION
■
General. Unless otherwise stated, all materials used were
commercially available and used as supplied. Reactions requiring
anhydrous conditions were performed in flame-dried glassware and
cooled under an argon or nitrogen atmosphere. Unless otherwise
stated, reactions were carried out under argon or nitrogen and
monitored by analytical thin layer chromatography performed on
glass-backed plates (5 × 10 cm) precoated with silica gel 60 F254 as
supplied by Merck. Visualization of the resulting chromatograms was
performed by looking under an ultraviolet lamp (λ = 254 nm)
followed by dipping in an ethanol solution of vanillin (5% w/v)
containing sulfuric acid (3% v/v) or phosphomolybdic acid (2.5% w/
v) and charring with a heat gun. Solvents for reactions were dried and
distilled under an argon or nitrogen atmosphere prior to use as
follows: THF, diethyl ether (ether), and DMF from a dark blue
solution of sodium benzophenone ketyl; toluene, dichromethane, and
pyridine from calcium hydride. Flash chromatography was used
routinely for purification and separation of product mixtures using
silica gel 60 of 230−400 mesh size as supplied by Merck. Eluent
systems are given in volume/volume concentrations. Melting points
were determined using an Electrothermal IA9000 digital melting point
apparatus. 1H and 13C NMR spectra were recorded on a Varian
1
mp 128.1−129.7 °C; H NMR (CDCl3) δ 7.48 (d, J = 2.0 Hz, 1H),
7.38−7.32 (m, 2H), 7.01 (d, J = 3.6 Hz, 1H), 6.99 (d, J = 3.6 Hz, 1H),
4.77 (s, 2H), 4.48 (q, J = 7.2 Hz, 2H), 1.43 (t, J = 7.2 Hz, 3H); 13C
NMR (CDCl3) δ 161.5, 142.2, 138.4, 137.1, 135.0, 133.8, 130.8, 130.5,
130.3, 130.2, 128.2, 128.0, 120.6, 116.4, 61.6, 22.4, 14.3; ESMS m/z:
540.8 (M + 1).
Ethyl 5-(5-Bromothiophene-2-yl)-1-(2,4-dichlorophenyl)-4-
(hydroxymethyl)-1H-pyrazole-3-carboxylate (8). To a vigorously
stirred solution of silver nitrate (3.25 g, 19.12 mmol) in 100 mL of
aqueous acetone (1:1) was added crude compound 7 (2.52 g) in one
portion. The resulting mixture was heated at 60 °C for 16 h and then
cooled to room temperature. The suspension solution was filtered, and
the filtrate was concentrated under reduced pressure to remove
acetone. The residue was extracted with CH2Cl2 (2 × 50 mL), and the
combined organic extracts were washed with brine, dried over Na2SO4,
filtered, and concentrated to give a crude residue, which was purified
G
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by flash chromatography eluting with n-hexane/ethyl acetate (2:1) to
afford hydroxy ester 8 (1.67 g, 76%) as a white solid: mp 99.2−100.4
°C; 1H NMR (CDCl3) δ 7.46 (d, J = 1.2 Hz, 1H), 7.33−7.32 (m, 2H),
6.96 (d, J = 3.6 Hz, 1H), 6.76 (d, J = 3.6 Hz, 1H), 4.71 (d, J = 7.2 Hz,
2H), 4.48 (q, J = 7.2 Hz, 2H), 3.76 (t, J = 7.2 Hz, 1H), 1.42 (t, J = 7.2
Hz, 3H); 13C NMR (CDCl3) δ 163.3, 142.9, 137.3, 136.8, 135.0,
133.7, 130.7, 130.4, 130.3, 130.1, 128.4, 127.8, 124.2, 115.8, 61.9, 54.5,
14.2; ESMS m/z: 498.9 (M + 23).
5-(5-Bromothiophene-2-yl)-1-(2,4-dichlorophenyl)-4-(hy-
droxymethyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide
(9). To a mixture of hydroxy ester 8 (4.40 g, 9.24 mmol) and
aluminum trichloride (2.46 g, 18.48 mmol) in dichloroethane (88 mL)
was added 1-aminopiperidine (3.70 g, 36.96 mmol) slowly at 0 °C
under an atmosphere of argon. The resulting mixture was allowed to
warm to room temperature, was stirred overnight, and then was
quenched with ice−water. The aqueous layer was extracted with
CH2Cl2 (2 × 40 mL). The combined organic extracts were washed
with water and brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to give a crude residue, which
was purified by flash chromatography with n-hexane/ethyl acetate
(1:1) as eluant to afford compound 9 (4.75 g, 97%) as a white solid:
mp 113.5−115.2 °C; 1H NMR (CDCl3) δ 7.73 (br s, 1H), 7.49 (d, J =
2.4 Hz, 1H), 7.35 (dd, J = 8.4, 2.4 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H),
6.95 (d, J = 4.0 Hz, 1H), 6.69 (d, J = 4.0 Hz, 1H), 5.17 (t, J = 7.2 Hz,
1H), 4.67 (d, J = 7.2 Hz, 2H), 2.83 (m, 4H), 1.78−1.73 (m, 4H),
1.43−1.41 (m, 2H); 13C NMR (CDCl3) δ 160.0, 144.7, 136.9, 136.6,
135.0, 133.6, 130.7, 130.4, 130.3, 130.2, 128.6, 128.1, 124.0, 115.8,
57.1, 54.7, 25.3, 23.2; ESMS m/z: 529.1 (M + 1).
1-(2,4-Dichlorophenyl)-5-(5-(2-(4-(trifluoromethyl)phenyl)-
ethynyl)thiophene-2-yl)-4-(hydroxymethyl)-N-(piperidin-1-yl)-
1H-pyrazole-3-carboxamide (10). A mixture of bromothiophene 9
(1.20 g, 2.26 mmol), PdCl2(PPh3)2 (0.16 g, 0.23 mmol), CuI (0.06 g,
0.28 mmol), and 2-ethanolamine (0.5 M(aq), 14 mL, 6.78 mmol) in
THF (50 mL) was stirred in a pressure vessel and degassed with argon
for 10 min, at which time 1-ethynyl-4-(trifluoromethyl)benzene (0.58
g, 3.39 mmol) was added in one portion. The resulting mixture was
heated at 80 °C in an oil bath for 12 h. After cooling to room
temperature, the reaction mixture was poured into water (20 mL) and
the aqueous layer was extracted with ethyl acetate (2 × 40 mL). The
combined organic extracts were washed with water and brine, dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure to give a crude residue, which was purified by flash
chromatography with n-hexane/ethyl acetate (1:1) to afford
compound 10 (1.22 g, 87%) as a white solid: mp 154.3−155.6 °C;
1H NMR (CDCl3) δ 7.75 (br s, 1H), 7.58−7.56 (m, 4H), 7.50 (d, J =
2.0 Hz, 1H), 7.36 (dd, J = 8.4, 2.0 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H),
7.18 (d, J = 4.0 Hz, 1H), 6.84 (d, J = 4.0 Hz, 1H), 5.21 (t, J = 7.2 Hz,
1H), 4.73 (d, J = 7.2 Hz, 2H), 2.85−2.81 (m, 4H), 1.77−1.75 (m,
4H), 1.44−1.42 (m, 2H); 13C NMR (CDCl3) δ 160.0, 144.8, 136.8,
136.6, 135.1, 133.6, 132.6, 131.5, 130.7, 130.3, 130.2 (q, JC−F = 32.6
Hz), 129.7, 129.1, 128.0, 126.1, 125.7, 125.3 (q, JC−F = 3.6 Hz), 124.0,
123.7 (q, JC−F = 270.9 Hz), 93.6, 83.7, 57.1, 54.8, 25.3, 23.1; ESMS m/
z: 619.1 (M + 1).
126.0, 125.8, 125.2 (q, JC−F = 3.7 Hz), 123.5 (q, JC−F = 270.2 Hz),
119.9, 93.8, 83.7, 56.9, 25.3, 23.1, 22.4; ESMS m/z: 681.1 (M + 1).
1-(2,4-Dichlorophenyl)-4-((3-hydroxyazetidin-1-yl)methyl)-
N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (12). To a magneti-
cally stirred solution of bromide 11 (0.33 g, 0.48 mmol) in DMF (3
mL) at room temperature were sequentially added 3-hydroxyazetidine
hydrochloride (0.08 g, 0.72 mmol) and diisopropylethylamine (0.12 g,
0.92 mmol) in one portion. The resulting mixture was heated to 60 °C
for 16 h, and then cooled to room temperature and quenched with
water. The aqueous phase was extracted with ethyl acetate (2 × 10
mL). The combined organic extracts were washed with water and
brine, dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to give a crude residue, which was subjected to
purification by flash chromatography on silica gel with n-hexane/ethyl
acetate (1:1) to afford the desired product 12 (0.22 g, 68%) as a white
1
solid: mp 174.0−175.3 °C; H NMR (CDCl3) δ 9.38 (br s, 1H),
7.59−7.57 (m, 4H), 7.46 (d, J = 2.4 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H),
7.33 (dd, J = 8.4 and 2.4 Hz, 1H), 7.18 (d, J = 4.0 Hz, 1H), 7.04 (d, J =
4.0 Hz, 1H), 4.41 (br s, 1H), 3.83 (s, 2H), 2.86−2.82 (m, 4H), 1.77−
1.73 (m, 4H), 1.43−1.41 (m, 2H); 13C NMR (CDCl3) δ 159.1, 146.1,
138.0, 136.5, 135.4, 133.6, 132.7, 131.6, 130.8, 130.3 (q, JC−F = 32.2
Hz), 130.2, 130.1, 129.5, 127.9, 126.1, 125.6, 125.3 (q, JC−F = 3.6 Hz),
123.7 (q, JC−F = 270.8 Hz), 117.1, 93.6, 83.8, 62.9, 62.0, 57.2, 50.5,
25.2, 23.3; ESMS m/z: 674.1 (M + 1); HPLC purity = 97.78%, tR =
37.81 min.
4-(Aminomethyl)-1-(2,4-dichlorophenyl)-5-(5-(2-(4-
(trifluoromethyl)phenyl)ethynyl)thiophene-2-yl)-N-(piperidin-
1-yl)-1H-pyrazole-3-carboxamide (13). To a magnetically stirred
solution of compound 11 (1.1 g, 1.62 mmol) in DMF (10 mL) was
added NaN3 (0.53 g, 8.08 mmol) at room temperature for 3 h. The
reaction mixture was poured into water (10 mL), and the aqueous
layer was extracted with ethyl acetate (2 × 20 mL). The combined
organic extracts were washed with water and brine, dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure to yield the corresponding azide. This azido compound,
without purification, was further treated with PPh3 (0.51 g, 1.94
mmol) in THF/H2O (1/1) (20 mL) at room temperature for 16 h to
afford compound 13 (0.66 g, 66% over two steps) as a pale yellow
1
solid after chromatographic purification (CH2Cl2:MeOH = 9:1): H
NMR (CDCl3) δ 7.85 (br s, 1H), 7.58−7.56 (m, 4H), 7.51 (d, J = 1.8
Hz, 1H), 7.38−7.32 (m, 2H), 7.19 (d, J = 3.9 Hz, 1H), 6.91 (d, J = 3.9
Hz, 1H), 3.99 (s, 2H), 2.87−2.79 (m, 4H), 2.01 (s, 1H), 1.74−1.76
(m, 4H), 1.49−1.38 (m, 2H); 13C NMR (CDCl3) δ 159.2, 144.6,
136.9, 136.6, 135.2, 133.5, 132.7, 131.5, 130.7, 130.2 (q, JC−F = 32.4
Hz), 130.1, 129.5, 129.4, 127.9, 126.0, 125.4, 125.2 (q, JC−F = 4.0 Hz),
124.1, 123.7 (q, JC−F = 270.4 Hz), 93.5, 83.8, 57.0, 35.4, 25.2, 23.2;
ESMS m/z: 618.0 (M + 1).
1-(2,4-Dichlorophenyl)-4-((3-fluoroazetidin-1-yl)methyl)-N-
(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (14). Following the
similar coupling-reaction and workup procedures for 12, a mixture of
bromide 11 (0.10 g, 0.15 mmol), 3-fluoroazetidine hydrochloride
(0.03 g, 0.23 mmol), and diisopropylethylamine (0.05 mL, 0.3 mmol)
in DMF (3 mL) was heated at 60 °C for 16 h to afford product 14 (82
4-(Bromomethyl)-1-(2,4-dichlorophenyl)-N-(piperidin-1-yl)-
5-(5-((4-(trifluoromethyl)phenyl)ethynyl)thiophene-2-yl)-1H-
pyrazole-3-carboxamide (11). To a magnetically stirred solution of
compound 10 (0.30 g, 0.48 mmol) in CH2Cl2 (5 mL) at 0 °C was
added PBr3 (0.26 g, 0.92 mmol) dropwise. The resulting mixture was
warmed to room temperature for 2 h and then quenched with ice−
water. The aqueous phase was extracted with CH2Cl2 (2 × 10 mL).
The combined organic extracts were washed with water and brine,
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to give the crude residue, which was purified with
flash chromatography with n-hexane/ethyl acetate (4:1) to give
bromide 11 (0.32 g, 98%) as a pale yellow solid: mp 207.3−208.6 °C ;
1H NMR (CDCl3) δ 7.59−7.57 (m, 4H), 7.52 (m, 1H), 7.38−7.37 (m,
1
mg, 83%) as a white solid: mp 186.2−187.9 °C; H NMR (CDCl3) δ
8.83 (br s, 1H), 7.61−7.58 (m, 4H), 7.48 (d, J = 2.1 Hz, 1H), 7.39 (d,
J = 8.4 Hz, 1H), 7.35 (dd, J = 8.4, 2.1 Hz, 1H), 7.19 (d, J = 3.9 Hz,
1H), 7.07 (d, J = 3.9 Hz, 1H), 5.10 (dtt, J = 57.3, 5.1, 4.8 Hz, 1H),
3.89 (s, 2H), 3.65 (m, 2H), 3.44−3.32 (m, 2H), 2.86−2.84 (m, 4H),
1.84−1.71 (m, 4H), 1.46−1.44 (m, 2H); 13C NMR (CDCl3) δ 158.9,
145.5, 138.3, 136.5, 135.3, 133.6, 132.6, 131.5, 130.7, 130.2 (q, JC−F
34.8 Hz), 130.1, 130.0, 129.6, 127.9, 126.0, 125.4, 125.2 (q, JC−F = 4.0
Hz), 123.6 (q, JC−F = 270.8 Hz), 117.6, 93.5, 83.8, 82.3 (d, JC−F
=
=
203.8 Hz), 60.2 (d, JC−F = 20.9 Hz), 57.1, 50.1, 25.1, 23.2; ES-MS (M
+ 1): 676.1; HPLC purity = 96.76%, tR = 40.11 min.
1-(2,4-Dichlorophenyl)-4-((oxetan-3-ylamino)methyl)-N-(pi-
peridin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (15). Following the
similar coupling-reaction and workup procedures for 12, a mixture of
bromide 11 (0.15 g, 0.22 mmol) and oxetan-3-amine (0.02 g, 0.33
2H), 7.23 (d, J = 4.0 Hz, 1H), 7.17 (d, J = 4.0 Hz, 1H), 4.92 (s, 2H),
2.86−2.84 (m, 4H), 1.77−1.71 (m, 4H), 1.43−1.41 (m, 2H); 13C
NMR (CDCl3) δ 158.5, 143.5, 138.4, 137.0, 134.9, 133.6, 132.8, 131.5,
130.7, 130.4, 130.3, 130.2 (q, JC−F = 37.0 Hz), 129.4, 128.8, 128.1,
H
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mmol) in DMF (3 mL) was heated at 60 °C for 16 h to afford product
15 (113 mg, 76%) as a white solid: mp 115.2−116.3 °C; H NMR
(m, 2H), 7.18 (d, J = 4.0 Hz, 1H), 7.11 (d, J = 4.0 Hz, 1H), 3.78 (s,
2H), 3.12 (d, J = 10.0 Hz, 2H), 2.93−2.83 (m, 6H), 2.13 (d, J = 12.8
Hz, 2H), 1.76−1.73 (m, 4H), 1.43−1.41 (m, 2H); 13C NMR (CDCl3)
δ 159.1, 145.7, 138.3, 136.6, 135.4, 133.7, 132.7, 131.5, 130.8, 130.5,
130.2 (q, JC−F = 32.6 Hz), 130.1, 129.7, 127.9, 126.1, 125.4, 125.3 (q,
1
(CDCl3) δ 7.73 (br s, 1H), 7.63−7.61 (m, 4H), 7.52 (d, J = 2.4 Hz,
1H), 7.36 (dd, J = 8.4 and 2.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.20
(d, J = 4.0 Hz, 1H), 6.91 (d, J = 4.0 Hz, 1H), 4.69 (t, J = 6.8 Hz, 2H),
4.49 (t, J = 6.8 Hz, 2H), 4.00 (quintet, J = 6.8 Hz, 1H), 3.86 (s, 2H),
2.85−2.83 (m, 4H), 1.81−1.76 (m, 4H), 1.68−1.59 (m, 2H); 13C
NMR (CDCl3) δ 159.1, 144.6, 137.5, 136.7, 135.1, 133.5, 132.6, 131.5,
130.6, 130.3, 130.2 (q, JC−F = 32.0 Hz), 129.6, 129.3, 128.0, 126.0,
125.6, 125.3 (q, JC−F = 4.0 Hz), 123.7 (q, JC−F = 272.0 Hz), 121.9,
93.7, 83.7, 79.5, 57.2, 53.4, 40.6, 25.2, 23.9; ES-MS (M + 1): 674.1;
HPLC purity = 97.84%, tR = 38.15 min.
JC−F = 3.7 Hz), 123.7 (q, JC−F = 270.5 Hz), 118.5, 115.4 (dd, JC−F
=
270.5 and 302.4 Hz), 93.5, 83.9, 56.6, 51.4, 46.7, 26.9 (t, JC−F = 12.4
Hz), 25.3, 23.2; ES-MS (M + 1): 720.2; HPLC purity = 98.35%, tR =
43.21 min.
1-(2,4-Dichlorophenyl)-4-((3,3-difluoropyrrolidin-1-yl)-
methyl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)-
ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide (20). Fol-
lowing the similar coupling-reaction and workup procedures for 12, a
mixture of bromide 11 (0.10 g, 0.15 mmol), 3,3-difluoropyrrolidine
hydrochloride (0.03 g, 0.23 mmol), and diisopropylethylamine (0.05
mL, 0.3 mmol) in DMF (3 mL) was heated at 60 °C for 16 h to afford
1-(2,4-Dichlorophenyl)-4-((oxetan-3-ylmethylamino)-
methyl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)-
ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide (16). Fol-
lowing the similar coupling-reaction and workup procedures for 12, a
mixture of bromide 11 (0.12 g, 0.18 mmol) and (oxetan-3-
yl)methanamine (0.03 g, 0.27 mmol) in DMF (3 mL) was heated at
60 °C for 16 h to afford product 16 (90 mg, 73%) as a white solid: mp
1
product 20 (89 mg, 86%) as a white solid: mp 75.5−76.9 °C; H
NMR (CDCl3) δ 8.55 (br s, 1H), 7.59−7.57 (m, 4H), 7.48 (d, J = 2.4
Hz, 1H), 7.41−7.34 (m, 2H), 7.18 (d, J = 4.0 Hz, 1H), 7.07 (d, J = 4.0
Hz, 1H), 3.86 (s, 2H), 3.01 (t, J = 12.8 Hz, 2H), 2.86−2.78 (m, 6H),
2.27 (tt, J = 14.4 and 7.2 Hz, 2H), 1.77−1.71 (m, 4H), 1.46−1.38 (m,
2H); 13C NMR (CDCl3) δ 159.0, 145.4, 138.7, 136.7, 135.4, 133.6,
132.6, 131.5, 130.7, 130.3 (q, JC−F = 32.6 Hz), 130.2, 130.1, 129.7,
129.6 (t, JC−F = 247.5 Hz), 128.0, 126.1, 125.6, 125.3 (q, JC−F = 4.1
Hz), 123.7 (q, JC−F = 270.8 Hz), 118.0, 93.6, 83.9, 60.6 (t, JC−F = 28.9
Hz), 57.1, 50.8, 46.7, 35.8 (t, JC−F = 24.6 Hz), 25.3, 23.2; ES-MS (M +
1): 708.1; HPLC purity = 97.39%, tR = 45.04 min.
1
96.3−97.9 °C; H NMR (CDCl3) δ 7.81 (br s, 1H), 7.59−7.57 (m,
4H), 7.50 (d, J = 2.0 Hz, 1H), 7.37−7.31 (m, 2H), 7.19 (d, J = 4.0 Hz,
1H), 6.94 (d, J = 4.0 Hz, 1H), 4.79 (dd, J = 7.6, 6.4 Hz, 2H), 4.41 (dd,
J = 6.4, 6.0 Hz, 2H), 3.87 (s, 2H), 3.12 (m, 1H), 2.97 (d, J = 7.6 Hz,
2H), 2.82−2.80 (m, 4H), 1.77−1.72 (m, 4H), 1.41−1.39 (m, 2H); 13C
NMR (CDCl3) δ 159.2, 144.8, 137.9, 136.7, 135.2, 133.6, 132.7, 131.6,
130.6, 130.3, 130.2 (q, JC−F = 32.6 Hz), 129.6, 129.5, 128.0, 126.0,
125.6, 125.3 (q, JC−F = 4.0 Hz), 123.7 (q, JC−F = 270.9 Hz), 121.4,
93.6, 83.8, 76.1, 57.2, 52.6, 42.9, 35.1, 25.3, 23.2; ES-MS (M + 1):
688.1; HPLC purity = 97.23%, tR = 37.94 min.
1-(2,4-Dichlorophenyl)-4-(((S)-2-(hydroxymethyl)pyrrolidin-
1-yl)methyl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)-
phenyl)ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide
(21). Following the similar coupling-reaction and workup procedures
for 12, a mixture of bromide 11 (0.16 g, 0.24 mmol) and ((S)-
pyrrolidin-2-yl)methanol (0.12 g, 1.2 mmol) in DMF (3 mL) was
heated at 60 °C for 16 h to afford product 21 (95 mg, 56%) as a pale
4-((Cyclopropylamino)methyl)-1-(2,4-dichlorophenyl)-N-(pi-
peridin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (17). Following the
similar coupling-reaction and workup procedures for 12, a mixture of
bromide 11 (0.16 g, 0.23 mmol) and cyclopropanamine (0.05 g, 0.92
mmol) in THF (3 mL) was stirred at room temperature for 16 h to
afford product 17 (132 mg, 84%) as a pale yellow solid: mp 85.5−87.0
°C; 1H NMR (CDCl3) δ 7.59−7.56 (m, 5H), 7.48 (d, J = 1.6 Hz, 1H),
7.36 (dd, J = 8.4 Hz, 1H), 7.33 (dd, J = 8.4, 1.6 Hz, 1H), 7.18 (d, J =
4.0 Hz, 1H), 7.01 (d, J = 4.0 Hz, 1H), 3.94 (s, 2H), 2.83−2.80 (m,
4H), 2.32−2.29 (m, 1H), 1.75−1.72 (m, 4H), 1.47−1.37 (m, 2H),
0.78−0.48 (m, 4H); 13C NMR (CDCl3) δ 159.5, 144.5, 138.3, 136.7,
135.0, 133.4, 132.7, 131.5, 130.7, 130.2 (q, JC−F = 32.6 Hz), 130.1 (q,
1
yellow solid: mp 125.8−126.0 °C; H NMR (CDCl3) δ 9.96 (br s,
1H), 7.60−7.55 (m, 4H), 7.45 (d, J = 2.0 Hz, 1H), 7.36 (d, J = 8.4 Hz,
1H), 7.32 (dd, J = 8.4, 2.1 Hz, 1H), 7.16 (d, J = 4.0 Hz, 1H), 6.84 (d, J
= 4.0 Hz, 1H), 4.18 (d, J = 12.8 Hz, 1H), 3.74 (dd, J = 11.6, 2.4 Hz,
1H), 3.55 (d, J = 12.8 Hz, 1H), 3.40 (dd, J = 11.6, 2.4 Hz, 1H), 3.12−
3.03 (m, 4H), 2.86 (m, 1H), 2.70 (m, 1H), 2.22 (m, 1H), 1.89−1.83
(m, 2H), 1.75−1.60 (m, 6H), 1.43−1.41 (m, 2H); 13C NMR (CDCl3)
δ 160.0, 145.8, 137.7, 136.5, 135.4, 133.6, 132.5, 131.5, 130.7, 130.3 (q,
JC−F = 32.6 Hz), 130.1, 129.9, 129.7, 127.9, 126.1, 125.6, 125.3 (q, JC−F
= 3.7 Hz), 123.7 (q, JC−F = 270.9 Hz), 119.7, 93.6, 83.8, 65.3, 62.1,
55.8, 54.0, 46.9, 27.1, 25.7, 23.6, 23.1; ES-MS (M + 1): 702.1; HPLC
purity = 95.00%, tR = 39.70 min.
JC−F = 4.0 Hz), 128.8, 127.9, 126.0, 125.7, 125.2, 123.6 (q, JC−F
=
272.0 Hz), 119.1, 93.5, 83.7, 57.0, 42.0, 29.7, 25.1, 23.0, 5.1; ES-MS
(M + 1): 658.1; HPLC purity = 99.29%, tR = 40.71 min.
1-(2,4-Dichlorophenyl)-4-((isopropylamino)methyl)-N-(pi-
peridin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (18). Following the
similar coupling-reaction and workup procedures for 12, a mixture of
bromide 11 (0.27 g, 0.40 mmol) and propan-2-amine (0.12 g, 2.0
mmol) in DMF (3 mL) was heated at 60 °C for 16 h to afford product
4-((1H-1,2,4-Triazol-1-yl)methyl)-1-(2,4-dichlorophenyl)-N-
(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (22). Following the
similar coupling-reaction and workup procedures for 12, a mixture of
bromide 11 (0.16 g, 0.24 mmol) and 1H-1,2,4-triazole, sodium salt
(0.11 g, 1.2 mmol) in DMF (5 mL) was heated at 60 °C for 16 h to
afford product 21 (142 mg, 88%) as a pale yellow solid: mp 218.0−
218.5 °C; 1H NMR (CDCl3) δ 8.68 (s, 1H), 7.91 (s, 1H), 7.74 (d, J =
4.0 Hz, 1H), 7.61 (s, 1H), 7.58 (m, 4H), 7.83 (d, J = 2.0 Hz, 1H), 7.37
(dd, J = 8.4 and 2.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 4.0
Hz, 1H), 5.59 (s, 2H), 2.81−2.79 (m, 4H), 1.77−1.72 (m, 4H), 1.43−
1.41 (m, 2H); 13C NMR (CDCl3) δ 158.8, 151.3, 144.7, 143.8, 139.8,
1
18 (189 mg, 72%) as a pale yellow solid: mp 110.9−112.2 °C; H
NMR (CDCl3) δ 8.15 (br s, 1H), 7.59−7.57 (m, 4H), 7.49 (d, J = 2.0
Hz, 1H), 7.34 (dd, J = 8.4, 2.0 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.19
(d, J = 3.6 Hz, 1H), 6.99 (d, J = 3.6 Hz, 1H), 3.88 (s, 2H), 2.90−2.78
(m, 5H), 1.77−1.73 (m, 4H), 1.45−1.39 (m, 2H), 1.10 (d, J = 6.0 Hz,
6H); 13C NMR (CDCl3) δ 159.2, 145.1, 137.6, 136.6, 135.4, 133.7,
132.7, 131.6, 131.5, 130.8, 130.3 (q, JC−F = 32.2 Hz), 130.2, 129.7,
128.0, 126.1, 125.4, 125.3 (q, JC−F = 3.6 Hz), 123.7 (q, JC−F = 270.9
Hz), 121.4, 93.5, 83.9, 57.3, 48.4, 40.4, 29.6, 25.3, 22.6; ES-MS (M +
1): 660.1; HPLC purity = 97.14%, tR = 40.22 min.
137.1, 134.9, 133.7, 132.9, 131.6, 131.4, 130.6, 130.4, 130.3 (q, JC−F
=
32.2 Hz), 128.2, 128.1, 126.4, 126.0, 125.3 (q, JC−F = 4.0 Hz), 123.7
(q, JC−F = 270.8 Hz), 116.8, 93.9, 83.7, 57.3, 42.7, 25.1, 23.1; ES-MS
(M + 1): 670.1; HPLC purity = 98.27%, tR = 50.79 min.
1-(2,4-Dichlorophenyl)-4-((6,6-difluoro-3-azabicyclo[3.1.0]-
h e x a n - 3 - y l ) m e t h y l ) - N - ( p i p e r i d i n - 1 - y l ) - 5 - ( 5 - ( ( 4 -
(trifluoromethyl)phenyl)ethynyl)thiophene-2-yl)-1H-pyrazole-
3-carboxamide (19). Following the similar coupling-reaction and
workup procedures for 12, a mixture of bromide 11 (0.10 g, 0.15
mmol), 6,6-difluoro-3-azabicyclo[3.1.0]hexane hydrochloride (0.03 g,
0.23 mmol), and diisopropylethylamine (0.05 mL, 0.3 mmol) in DMF
(3 mL) was heated at 60 °C for 16 h to afford product 19 (88 mg,
83%) as a white solid: mp 130.0−131.2 °C; 1H NMR (CDCl3) δ 8.78
(br s, 1H), 7.58−7.56 (m, 4H), 7.46 (d, J = 2.0 Hz, 1H), 7.40−7.32
1-(2,4-Dichlorophenyl)-4-(methylsulfonamidomethyl)-N-(pi-
peridin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (23). Following the
similar coupling-reaction and workup procedures for 4, a mixture of
amine 13 (0.10 g, 0.16 mmol), triethylamine (0.04 mL, 0.24 mmol),
and methanesulfonyl chloride (0.02 mL, 0.24 mmol) in CH2Cl2 (3
mL) at 0 °C was allowed to react for 1 h, leading to product 23 (95
1
mg, 84%) as a white solid: mp 110.2−111.9 °C; H NMR (CDCl3) δ
7.72 (br s, 1H), 7.59−7.57 (m, 4H), 7.54 (d, J = 2.0 Hz, 1H), 7.38 (dd,
I
dx.doi.org/10.1021/jm401158e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
J = 8.4 and 2.0 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 3.9 Hz,
1H), 7.20 (d, J = 3.9 Hz, 1H), 6.49 (t, J = 6.3 Hz, 1H), 4.41 (d, J = 6.3
Hz, 2H), 2.94 (s, 3H), 2.84 (m, 4H), 1.78−1.75 (m, 4H), 1.45−1.43
(m, 2H); 13C NMR (CDCl3) δ 159.5, 144.1, 138.0, 137.1, 134.9,
133.6, 133.0, 131.6, 130.7, 130.5, 130.4, 130.3 (q, JC−F = 32.5 Hz),
4H), 2.74 (s, 6H), 1.80−1.78 (m, 4H), 1.46−1.44 (m, 2H); 13C NMR
(CDCl3) δ 159.5, 144.2, 137.7, 136.9, 135.0, 133.6, 132.9, 131.6, 130.7,
130.5, 130.4, 130.3 (q, JC−F = 33.0 Hz), 129.1, 128.6, 126.1, 126.0,
125.3 (q, JC−F = 3.5 Hz), 123.7 (q, JC−F = 272.0 Hz), 119.9, 93.7, 83.8,
57.1, 38.0, 37.5, 25.1, 22.9; ES-MS (M + 1): 725.1; HPLC purity =
99.66%, tR = 45.43 min.
128.1, 128.2, 126.2, 126.0, 125.3 (q, JC−F = 3.4 Hz), 123.7 (q, JC−F
=
270.4 Hz), 119.1, 93.8, 83.7, 57.1, 40.3, 37.5, 25.1, 22.9; ES-MS (M +
1): 696.0; HPLC purity = 98.84%, tR = 43.93 min.
1-(2,4-Dichlorophenyl)-4-((N-isopropylsulfamoylamino)-
methyl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)-
ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide (28). Fol-
lowing the similar coupling-reaction and workup procedures for 4, a
mixture of amine 13 (0.10 g, 0.16 mmol), triethylamine (0.04 mL, 0.32
mmol), and isopropylsulfamoyl chloride (0.04 g, 0.24 mmol) in DMF
(3 mL) at room temperature was allowed to react for 16 h, leading to
1-(2,4-Dichlorophenyl)-4-((1-methylethylsulfonamido)-
methyl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)-
ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide (24). Fol-
lowing the similar coupling-reaction and workup procedures for 4, a
mixture of amine 13 (0.10 g, 0.16 mmol), triethylamine (0.04 mL, 0.24
mmol), and propane-2-sulfonyl chloride (0.04 g, 0.24 mmol) in
CH2Cl2 (3 mL) at 0 °C was allowed to react for 1 h, leading to
1
product 28 (54 mg, 45%) as a white solid: mp 104.1−105.2 °C; H
NMR (CDCl3) δ 8.85 (br s, 1H), 7.63−7.61 (m, 4H), 7.58 (d, J = 2.1
Hz, 1H), 7.40−7.34 (m, 2H), 7.22 (d, J = 3.9 Hz, 1H), 7.11 (d, J = 3.9
Hz, 1H), 6.10 (br s, 1H), 4.30 (s, 2H), 4.10 (m, 1H), 3.55−3.37 (m,
5H), 2.06−1.97 (m, 4H), 1.67−1.58 (m, 2H), 1.18 (d, J = 6.3 Hz,
6H); 13C NMR (CDCl3) δ 159.4, 144.2, 137.7, 136.9, 134.9, 133.5,
132.9, 131.5, 130.7, 130.4, 130.3, 130.2 (q, JC−F = 32.6 Hz), 128.6,
128.1, 126.1, 125.9, 125.3 (q, JC−F = 3.5 Hz), 123.7 (q, JC−F = 272.0
Hz), 119.8, 93.7, 83.8, 57.1, 46.0, 37.2, 25.1, 23.7, 22.9; ES-MS (M +
1): 739.1; HPLC purity = 99.18%, tR = 45.57 min.
1
product 24 (60 mg, 52%) as a white solid: mp 105.2−106.9 °C; H
NMR (CDCl3) δ 7.59−7.57 (m, 4H), 7.52 (d, J = 2.0 Hz, 1H), 7.39
(dd, J = 8.4 and 2.0 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.24−7.21 (m,
2H), 6.33 (br s, 1H), 4.41 (s, 2H), 3.20−3.01 (m, 5H), 1.94−1.88 (m,
4H), 1.59−1.44 (m, 2H), 1.36 (d, J = 6.6 Hz, 6H); 13C NMR (CDCl3)
δ 159.7, 143.8, 137.7, 137.1, 134.9, 133.6, 133.0, 131.6, 130.8, 130.6,
130.4 (q, JC−F = 32.6 Hz), 130.3, 128.3, 128.2, 126.2, 126.1, 125.3 (q,
JC−F = 4.0 Hz), 123.7 (q, JC−F = 270.4 Hz), 119.9, 93.8, 83.8, 56.9,
53.5, 37.4, 24.8, 22.6, 17.2; ES-MS (M + 1): 724.1; HPLC purity =
98.63%, tR = 45.66 min.
4-((Azetidine-1-sulfonamido)methyl)-1-(2,4-dichlorophen-
yl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (29). Following the
similar coupling-reaction and workup procedures for 4, a mixture of
amine 13 (0.07 g, 0.11 mmol), triethylamine (0.03 mL, 0.22 mmol),
and 1-azetidinesulfonyl chloride (0.03 g, 0.17 mmol) in DMF (3 mL)
at room temperature was allowed to react for 16 h, leading to product
29 (53 mg, 65%) as a white solid: mp 97.3−98.3 °C; 1H NMR
(CDCl3) δ 7.71 (br s, 1H), 7.60−7.58 (m, 4H), 7.52 (d, J = 1.8 Hz,
1H), 7.39 (dd, J = 8.4, 1.8 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.26 (d, J
= 3.9 Hz, 1H), 7.23 (d, J = 3.9 Hz, 1H), 6.58 (t, J = 6.6 Hz, 1H), 4.33
(d, J = 6.6 Hz, 2H), 3.80 (t, J = 7.8 Hz, 4H), 2.85−2.83 (m, 4H), 2.12
(quint, J = 7.8 Hz, 2H), 1.80−1.76 (m, 4H), 1.50−1.40 (m, 2H); 13C
NMR (CDCl3) δ 159.4, 144.4, 137.7, 136.9, 135.0, 133.5, 133.0, 131.5,
130.7, 130.4, 130.3, 130.2 (q, JC−F = 32.0 Hz), 128.7, 128.1, 127.3 (q,
JC−F = 270.2 Hz), 126.1, 125.9, 125.3 (q, JC−F = 3.4 Hz), 123.7 (q, JC−F
= 271.5 Hz), 120.1, 93.6, 83.9, 57.2, 50.3, 37.4, 25.2, 23.1, 22.6; ES-MS
(M + 1): 737.0; HPLC purity = 99.06%, tR = 46.22 min.
4-(Cyclopropanesulfonamidomethyl)-1-(2,4-dichlorophen-
yl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (25). Following the
similar coupling-reaction and workup procedures for 4, a mixture of
amine 13 (0.07 g, 0.11 mmol), triethylamine (0.03 mL, 0.23 mmol),
and cyclopropanesulfonyl chloride (0.03 g, 0.17 mmol) in CH2Cl2 (3
mL) at −30 °C was allowed to react for 1 h, leading to product 25 (58
mg, 71%) as a white solid: mp 105.5−106.5 °C; 1H NMR (CDCl3) δ
7.73 (br s, 1H), 7.60−7.58 (m, 4H), 7.53 (d, J = 1.5 Hz, 1H), 7.39 (dd,
J = 8.4 and 1.5 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.26−7.24 (m, 2H),
6.58 (t, J = 6.6 Hz, 1H), 4.45 (d, J = 6.6 Hz, 2H), 2.86−2.84 (m, 4H),
2.35 (m, 1H), 1.81−1.75 (m, 4H), 1.48−1.46 (m, 2H), 1.15 (m, 2H),
0.91 (m, 2H); 13C NMR (CDCl3) δ 159.3, 144.5, 138.5, 137.7, 134.9,
133.6, 133.0, 131.6, 130.7, 130.4, 130.3, 130.2 (q, JC−F = 33.0 Hz),
128.4, 128.1, 126.1, 125.5, 125.3 (q, JC−F = 3.5 Hz), 123.7 (q, JC−F
=
272.0 Hz), 119.7, 93.7, 83.8, 57.2, 37.6, 30.2, 25.2, 22.6, 5.3; ES-MS
(M + 1): 722.1; HPLC purity = 98.54%, tR = 46.01 min.
1-(2,4-Dichlorophenyl)-N-(piperidin-1-yl)-4-((piperidine-1-
sulfonamido)methyl)-5-(5-((4-(trifluoromethyl)phenyl)-
ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide (30). Fol-
lowing the similar coupling-reaction and workup procedures for 4, a
mixture of amine 13 (0.07 g, 0.11 mmol), triethylamine (0.03 mL, 0.22
mmol), and 1-piperidinesulfonyl chloride (0.03 g, 0.17 mmol) in DMF
(3 mL) at room temperature was allowed to react for 16 h, leading to
4-(Cyclopentanesulfonamidomethyl)-1-(2,4-dichlorophen-
yl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (26). Following the
similar coupling-reaction and workup procedures for 4, a mixture of
amine 13 (0.09 g, 0.15 mmol), triethylamine (0.04 mL, 0.30 mmol),
and cyclopentanesulfonyl chloride (0.04 g, 0.23 mmol)in CH2Cl2 (3
mL) at −30 °C was allowed to react for 1 h, leading to product 26 (86
mg, 76%) as a white solid: mp 111.5−112.5 °C; 1H NMR (CDCl3) δ
7.72 (br s, 1H), 7.63−7.61 (m, 4H), 7.53 (d, J = 1.8 Hz, 1H), 7.40 (dd,
J = 8.4 and 1.8 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.30−7.23 (m, 2H),
6.50 (t, J = 6.6 Hz, 1H), 4.42 (d, J = 6.6 Hz, 2H), 3.37 (quintet, J = 7.5
Hz, 1H), 2.85−2.83 (m, 4H), 2.01−1.99 (m, 4H), 1.80−1.76 (m, 6H),
1.58−1.54 (m, 2H), 1.47−1.42 (m, 2H); 13C NMR (CDCl3) δ 159.4,
144.5, 137.6, 137.0, 135.0, 133.6, 133.1, 131.6, 130.7, 130.6, 130.5 (q,
JC−F = 32.0 Hz), 130.4, 128.4, 128.2, 126.1, 126.0, 125.3 (q, JC−F = 4.6
Hz), 123.8 (q, JC−F = 272.0 Hz), 120.0, 93.7, 83.7, 62.0, 57.3, 37.4,
28.0, 25.9, 25.2, 23.2; ES-MS (M + 1): 750.1; HPLC purity = 97.72%,
tR = 47.70 min.
1-(2,4-Dichlorophenyl)-4-((N,N-dimethylsulfamoylamino)-
methyl)-N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)-
ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide (27). Fol-
lowing the similar coupling-reaction and workup procedures for 4, a
mixture of amine 13 (0.10 g, 0.16 mmol), triethylamine (0.04 mL, 0.32
mmol), and dimethylsulfamoyl chloride (0.03 mL, 0.24 mmol) in
DMF (3 mL) at room temperature was allowed to react for 16 h,
leading to product 27 (90 mg, 78%) as a white solid: mp 104.6−105.0
°C; 1H NMR (CDCl3) δ 7.99 (br s, 1H), 7.63−7.61 (m, 4H), 7.50 (m,
1H), 7.40−7.32 (m, 2H), 7.22 (d, J = 3.9 Hz, 1H), 7.16 (d, J = 3.9 Hz,
1H), 6.48 (t, J = 6.3 Hz, 1H), 4.31 (d, J = 6.3 Hz, 2H), 2.94−2.92 (m,
1
product 30 (63 mg, 73%) as a white solid: mp 113.5−114.6 °C; H
NMR (CDCl3) δ 7.72 (br s, 1H), 7.62−7.60 (m, 4H), 7.53 (d, J = 2.0
Hz, 1H), 7.38 (dd, J = 8.4 and 2.0 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H),
7.74 (d, J = 4.0 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 6.54 (t, J = 6.8 Hz,
1H), 4.32 (d, J = 6.8 Hz, 2H), 3.16−3.13 (m, 4H), 2.86−2.84 (m,
4H), 1.80−1.78 (m, 4H), 1.65−1.60 (m, 2H), 1.58−1.56 (m, 4H),
1.49−1.42 (m, 2H); 13C NMR (CDCl3) δ 159.4, 144.5, 137.6, 136.9,
135.1, 133.7, 133.0, 131.6, 130.7, 130.5, 130.4, 130.3 (q, JC−F = 32.0
Hz), 128.7, 128.1, 126.1, 126.0, 125.3 (q, JC−F = 4.0 Hz), 123.8 (q, JC−F
= 270.0 Hz), 120.1, 93.7, 83.9, 57.3, 46.9, 37.6, 25.3, 25.2, 23.7, 23.2;
ES-MS (M + 1): 765.1; HPLC purity = 98.65%, tR = 48.29 min.
4-((Azepane-1-sulfonamido)methyl)-1-(2,4-dichlorophenyl)-
N-(piperidin-1-yl)-5-(5-((4-(trifluoromethyl)phenyl)ethynyl)-
thiophene-2-yl)-1H-pyrazole-3-carboxamide (31). Following the
similar coupling-reaction and workup procedures for 4, a mixture of
amine 13 (0.07 g, 0.11 mmol), triethylamine (0.03 mL, 0.22 mmol),
and azepane-1-sulfonyl chloride (0.03 g, 0.17 mmol) in DMF (3 mL)
at room temperature was allowed to react for 16 h, leading to product
1
31 (70 mg, 80%) as a white solid: mp 110.2−110.7 °C; H NMR
(CDCl3) δ 7.85 (br s, 1H), 7.59−7.57 (m, 4H), 7.55 (d, J = 1.8 Hz,
1H), 7.38 (dd, J = 8.4, 1.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.22 (d, J
= 4.0 Hz, 1H), 7.18 (d, J = 4.0 Hz, 1H), 6.40 (t, J = 6.6 Hz, 1H), 4.27
(d, J = 6.6 Hz, 2H), 3.33−3.25 (m, 4H), 2.89−2.87 (m, 4H), 1.81−
J
dx.doi.org/10.1021/jm401158e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.77 (m, 4H), 1.76−1.67 (m, 4H), 1.66−1.55 (m, 4H), 1,54−1.40 (m,
2H); 13C NMR (CDCl3) δ 159.4, 144.3, 137.7, 137.0, 135.0, 133.7,
133.0, 131.6, 130.7, 130.5, 130.4, 130.3 (q, JC−F = 32.2 Hz), 128.7,
128.1, 126.1, 126.0, 125.3 (q, JC−F = 3.5 Hz), 123.8 (q, JC−F = 272.0
Hz), 120.0, 93.7, 83.8, 57.2, 48.3, 37.2, 29.0, 27.0, 25.2, 23.0; ES-MS
(M + 1): 798.8; HPLC purity = 98.86%, tR = 48.40 min.
increasing concentrations of test compounds after activation with 25
nM of CP-55940 and were determined by nonlinear regression
analysis using the GraphPad Prism program (GraphPad Software, San
Diego, CA). The intrinsic property for inverse agonist efficacy was
assessed by using test compounds alone in the absence of agonist CP-
55940; the percentage change of the induced Eu-GTP binding is
defined as [(Eu-GTP binding in the presence of test compound at 10
μM − basal Eu-GTP binding)/basal Eu-GTP binding] × 100% and
expressed as % efficacy.
Measurement of Body Temperature and Tail-Flick Re-
sponse. Core body temperature was measured using a TH-5
thermometer and a lubricated RET-3 rectal probe (Physitemp, Clifton,
NJ) inserted into the rectum to a constant depth of 2 cm of C57BL/6J
mice. The tail-flick response was measured by an analgesia meter
RS232 (Columbus Instruments, Columbus, OH), and the intensity of
the heat source was adjusted to produce tail-flick latency of 2 to 3 s
with a cutoff at 10 s to avoid possible tissue damage. In the CB1R
agonist-induced hypothermia and analgesia model, the body temper-
ature and the tail-flick baseline latency were measured before drug
administration. One hour after drug administration, agonist CP-55940
(1 mg/kg, Tocris Cookson, UK) was injected intraperitoneally (ip).
Body temperatures were recorded at a time point of 30 and 65 min,
respectively, after agonist dosing. The tail-flick response was measured
at a time point of 35 min after agonist dosing. Data were analyzed by
one-way ANOVA with Dunnett’s post test. A P value (*) less than
0.05 was considered significant.
Quantitation of Brain and Plasma Concentration. Com-
pounds dissolved in DMSO/Tween 80/H2O (1:1:8, v/v/v) were
administered orally (10 mL/kg) to 9- to 13-week-old C57BL/6 mice
fed ad libitum (n = 3−4/group). At defined time points, mice were
decapitated after they were sacrificed. Blood samples and brain tissues
with cerebellum removed were collected and frozen at −80 °C before
analysis. Brain tissues were weighed and homogenized with two
volumes (v/w) of acetonitrile containing 500 ng/mL of the internal
standard on ice bath and vortexed for 30 s. After centrifugation at 14
000g for 20 min at room temperature, the supernatant was transferred
to a clean tube for LC-MS/MS analysis. The chromatographic system
was composed of an Agilent 1100 series LC system (Palo Alto, CA,
USA) and an Agilent ZORBAX Eclipse XDB-C8 reverse-phase column
(5 μm, 3.0 mm × 150 mm) interfaced with an AB Sciex API 3000
tandem mass spectrometer with an ESI in the positive scanning mode
(Applied Biosystems, Foster City, CA). Mobile phase A: water
containing 0.1% formic acid; mobile phase B: acetonitrile. The
gradient system started from A:B (30%:70%) to A:B (97%:3%) with a
flow rate of 0.5 mL/min. Data were analyzed by one-way ANOVA
with Dunnett’s post test. A P value (*) less than 0.05 was considered
significant.
Diet-Induced Obese (DIO) Mouse Model. Six-week-old
C57BL/6 mice were given high-fat diet of 4.73 kcal/g energy density
(Research Diet D 12451; 45% fat, 20% protein, and 35%
carbohydrate) for 16 weeks before the drug treatment. Weight-
matched mice were assigned to different groups and orally gavaged
once daily with vehicle (10% DMSO/10% Tween 80/80% H2O) or
compounds at defined dosage for 22 days. Body weight was measured
daily. At the end of the study, blood samples after animals had fasted
for 5 h were collected and brain tissues were dissected and frozen at
−80 °C before being analyzed. The B/P partition in DIO mice was
determined using the same procedure described previously except that
DIO mice, instead of C57BL/6 mice, were used.43
1-(2,4-Dichlorophenyl)-4-((2,6-dimethylmorpholine-4-
sulfonamido)methyl)-N-(piperidin-1-yl)-5-(5-((4-
(trifluoromethyl)phenyl)ethynyl)thiophene-2-yl)-1H-pyrazole-
3-carboxamide (32). Following the similar coupling-reaction and
workup procedures for 4, a mixture of amine 13 (0.07 g, 0.11 mmol),
triethylamine (0.03 mL, 0.22 mmol), and 2,6-dimethyl-4-morpholine-
sulfonyl chloride in DMF (3 mL) at room temperature was allowed to
react for 16 h, leading to product 32 (80 mg, 89%) as a white solid: mp
169.8−170.6 °C; 1H NMR (CDCl3) δ 7.73 (br s, 1H), 7.59−7.57 (m,
4H), 7.51 (d, J = 2.1 Hz, 1H), 7.37 (dd, J = 8.4, 2.1 Hz, 1H), 7.33 (d, J
= 8.4 Hz, 1H), 7.22 (d, J = 4.0 Hz, 1H), 7.13 (d, J = 4.0 Hz, 1H), 6.68
(t, J = 6.6 Hz, 1H), 4.33 (d, J = 6.6 Hz, 2H), 3.57−3.50 (m, 2H), 3.40
(d, J = 11.1 Hz, 2H), 2.84 (m, 4H), 2.40 (t, J = 11.4 Hz, 2H), 1.79−
1.75 (m, 4H), 1.45−1.43 (m, 2H), 1.14 (d, J = 6.3 Hz, 6H); 13C NMR
(CDCl3) δ 159.4, 144.4, 137.5, 137.0, 135.0, 133.6, 132.9, 131.6, 130.7,
130.4, 130.3 (q, JC−F = 32.0 Hz), 130.2, 128.6, 128.1, 126.1, 125.5,
125.3 (q, JC−F = 4.0 Hz), 123.7 (q, JC−F = 272.0 Hz), 120.0, 93.8, 83.7,
71.3, 57.2, 51.0, 37.7, 25.3, 23.1, 18.7; ES-MS (M + 1): 795.0; HPLC
purity = 97.95%, tR = 47.19 min.
Establishing Human CB1R (hCB1R) and CB2R (hCB2R) Stable
Cell Lines and Membrane Purification. The hCB1R cDNA tagged
with Flag at the N-terminus or hCB2R cDNA was subcloned into the
pIRES2-EGFP vector (Clontech Laboratories, Inc., Mountain View,
CA). After transfection to HEK 293 cells, clones that stably expressed
either hCB1 or hCB2 were selected by GFP and G418 sulfate and
maintained in DMEM supplemented with 10% fetal bovine serum and
0.5 mg/mL of G418 sulfate under 5% CO2 at 37 °C. For membrane
purification, cells were homogenized in ice-cold buffer A (50 mM Tris,
5 mM MgCl2, 2.5 mM EDTA, pH 7.4, 10% sucrose) with 1 mM
PMSF. The homogenate was centrifuged for 15 min at 2000g at 4 °C.
The resulting supernatant was centrifuged for another 30 min at 43
000g at 4 °C. The final pellet was resuspended in buffer A and stored
at −80 °C.
Radioligand Binding Assay. The radioligand binding assay was
performed according to Felder et al.40 with minor modification. An
amount of 0.2−8 μg of the purified membrane was incubated with 0.75
nM [3H]-CP-55940 and compounds of interest in the incubation
buffer (50 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, 0.3% BSA, pH
7.4). The nonspecific binding was defined in the presence of 1 μM of
CP-55940. The reactions were incubated for 1.5 h at 30 °C in
Multiscreen microplates (Millipore Corp., Billerica, MA). The
reactions were terminated by manifold filtration and washed with
ice-cold wash buffer (50 mM Tris, pH 7.4, 0.25% BSA) four times.
The radioactivity of [3H]-CP-55940 bound to the filters was measured
by Topcount (PerkinElmer Inc., Waltham, MA). For estimation of Kd
and Bmax of hCB1R and hCB2R, the nonspecific binding was defined
in the presence of 50 μM SR141716A and 30 μM Win 552122,
respectively. Ki values were determined by the concentration of
compounds required to inhibit 50% of the specific binding of [3H]-
CP-55940 and calculated by nonlinear regression (GraphPad software,
San Diego, CA).
Eu-GTP Binding Assay. The Eu-GTP binding assay, an in vitro
functional assay, was performed using the DELFIA Eu-GTP binding
kit (Perkin-Elmer Inc., Waltham, MA) based on methods developed
by Frang et al.41,42 with minor modifications as described in the
following: 1−4 μg of purified membrane was incubated with
compounds of interest and 25 nM of CP-55940 in assay buffer (50
mM HEPES, pH 7.4, 100 mM NaCl, 100 μg/mL saponin, 5 mM
MgCl2, 2 μM GDP, 0.5% BSA) at 30 °C for 60 min in acroplates (Pall
Life Sciences, Ann Arbor, MI). Following the addition of Eu-GTP and
incubation for 30 min at 30 °C, the assay was terminated by washing
four times in washing buffer provided in the kit. The fluorescence
signal of Eu-GTP was determined by Victor 2 multilabel reader
(Perkin-Elmer Inc., Waltham, MA). EC50 values were analyzed by
Aqueous Solubility. Solubility of compound 4 and 32 were
determined by suspending 10 mg of test article in double distilled
water (1 mL), followed by shaking the mixture at room temperature
for 24 h. After 24 h shaking, the mixture was filtered to remove
undissolved test article, and the amount of test article dissolved in the
filtrate was determined by HPLC. Calibration standard curves were
prepared by dissolving known concentrations of the test article in
DMSO and then diluting with acetonitrile to conduct peaks areas vs
concentrations obtained from HPLC analysis. HPLC conditions were
the same as those used for the purity determination of the test
compounds.
K
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P-gp ATPase Assay. The P-gp ATPase assay procedures were
conducted according to the manufacturer’s protocol. The assay is
based on the determination of ATPase activity by measuring the
release of inorganic phosphate resulting from substrate-stimulated
ATP using a colorimetric method. In brief, human P-gp/MDR1
membrane or P-gp/MDR1-negative control membrane was preincu-
bated at 37 °C for 5 min in the reaction buffer containing compound 4
in the presence or absence of 10 mM Na3VO4 in a 96-well microplate.
The ATPase reaction was initiated by the addition of 20 μL of MgATP
solution. The reaction was stopped after 30 min by addition of 30 μL
of 10% (w/v) sodium dodecyl sulfate solution, and the amount of
phosphate was determined immediately by adding 200 μL of detection
color regent solution and incubated at 37 °C for 20 min in the dark.
The amount of inorganic phosphate complex was determined by
measuring the absorbance value at 800 nm wavelength. Verapamil was
used as the positive control in these experiments.
(5) Kunos, G.; Tam, J. The case for peripheral CB1 receptor blockade
in the treatment of visceral obesity and its cardiometabolic
complications. Br. J. Pharmacol. 2011, 163, 1423−1431.
(6) Janero, D. R.; Lindsley, L.; Vemuri, V. K.; Makriyannis, A.
Cannabinoid 1 G protein-coupled receptor (periphero-)neutral
antagonists: emerging therapeutics for treating obesity-driven
metabolic disease and reducing cardiovascular risk. Expert Opin.
Drug Discovery 2011, 6, 995−1025.
(7) (a) Jourdan, T.; Godlewski, G.; Cinar, R.; Bertola, A.; Szanda, G.;
Liu, J.; Tam, J.; Han, T.; Mukhopadhyay, B.; Skarulis, M. C.; Ju, C.;
Aouadi, M.; Czech, M. P.; Kunos, G. Activation of the Nlrp3
inflammasome in infiltrating macrophages by endocannabinoids
mediates beta cell loss in type 2 diabetes. Nat. Med. 2013, 1132−
́
1140. (b) Tam, J.; Vemuri, V. K.; Liu, J.; Batkai, S.; Mukhopadhyay, B.;
Godlewski, G.; Osei-Hyiaman, D.; Ohnuma, S.; Ambudkar, S. V.;
Pickel, J.; Makriyannis, A.; Kunos, G. Peripheral CB1 cannabinoid
receptor blockade improves cardiometabolic risk in mouse models of
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ASSOCIATED CONTENT
* Supporting Information
■
(8) (a) 7TM Pharma completed a phase I clinical trial and confirmed
that TM38837 is restricted to the periphery in humans (http://www.
7tm.com/News.aspx?M=News&PID=42&NewsID=58, news release
November 11, 2010). (b) Klumpers, L. E.; Fridberg, M.; de Kam,
M. L.; Little, P. B.; Jensen, N. O.; Kleinloog, H. D.; Elling, C. E.; van
Gerven, J. M. Peripheral selectivity of the novel cannabinoid receptor
antagonist TM38837 in healthy subjects. Br. J. Clin. Pharmacol. 2013,
doi: 10.1111/bcp.12141. (c) Chorvat, R. J. Peripherally restricted CB1
receptor blockers. Bioorg. Med. Chem. Lett. 2013, 23, 4751−4760.
(9) Kenkre, J.; Tan, T.; Bloom, S. Treating the obese diabetic. Expert
Rev. Clin. Pharmacol. 2013, 6, 171−183.
S
Results of 163 off-target and patch-clamp assays, conducted by
Ricerca Biosciences, for compound 4, which was renumbered as
BPRCB1184 for further development purposes. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*Tel: +886-37-246-166 ext. 35709. Fax: + 886-37-586-456. E-
mail: ksshia@nhri.org.tw.
(10) Bays, H.; Dujovne, C. Pharmacotherapy of obesity: Currently
marketed and upcoming agents. Am. J. Cardiovasc. Drugs 2002, 2,
245−253.
*Tel: +886-37-246-166 ext. 35721. Fax: + 886-37-586-456. E-
mail: mhung@nhri.org.tw.
(11) Kaya, A.; Aydin, N.; Topseverm, P.; Filiz, M.; Ozturk, A.; Dag
̆
̈
Author Contributions
†These authors contributed equally.
A.; Kilinc,
̧
combination therapy on short-term weight management in obese
patients. Biomed. Pharmacother. 2004, 58, 582−587.
Notes
The authors declare no competing financial interest.
(12) Smith, B. M.; Smith, J. M.; Tsai, J. H.; Schultz, J. A.; Gilson, C.
A.; Estrada, S. A.; Chen, R. R.; Park, D. M.; Prieto, E. B.; Gallardo, C.
S.; Sengupta, D.; Dosa, P. I.; Covel, J. A.; Ren, A.; Webb, R. R.; Beeley,
N. R. A.; Martin, M.; Morgan, M.; Espitia, S.; Saldana, H. R.; Bjenning,
C.; Whelan, K. T.; Grottick, A. J.; Menzaghi, F.; Thomsen, W. J.
Discovery and structure−activity relationship of (1R)-8-Chloro-
2,3,4,5-tetrahydro-1-methyl-1H-3-benzazepine (Lorcaserin), a selective
serotonin 5-HT2C receptor agonist for the treatment of obesity. J. Med.
Chem. 2008, 51, 305−313.
(13) Wong, D.; Sullivan, K.; Heap, G. The pharmaceutical market for
obesity therapies. Nat. Rev. Drug Discovery 2012, 11, 669−670.
(14) Colman, E.; Golden, J.; Roberts, M.; Egan, A.; Weaver, J.;
Rosebraugh, C. The FDA’s assessment of two drugs for chronic weight
management. N. Engl. J. Med. 2012, 367, 1577−1579.
(15) Tai, C. L.; Hung, M. S.; Pawar, V. D.; Tseng, S. L.; Song, J. S.;
Hsieh, W. P.; Chiu, H. H.; Wu, H. C.; Hsieh, M. T.; Kuo, C. W.;
Hsieh, C. C.; Tsao, J. P.; Chao, Y. S.; Shia, K. S. Design, synthesis, and
biological evaluation of novel alkenylthiophenes as potent and
selective CB1 cannabinoid receptor antagonists. Org. Biomol. Chem.
2008, 6, 447−450.
(16) Chu, C. M.; Hung, M. S.; Hsieh, M. T.; Kuo, C. W.; Suja, T. D.;
Song, J. S.; Chiu, H. H.; Chao, Y. S.; Shia, K. S. Bioisosteric
replacement of the pyrazole 3-carboxamide moiety of rimonabant. A
novel series of oxadiazoles as CB1 cannabinoid receptor antagonists.
Org. Biomol. Chem. 2008, 6, 3399−3407.
ACKNOWLEDGMENTS
■
We are grateful to the National Health Research Institutes and
National Science Council of the Republic of China (NSC 101-
2113-M-400-003-MY2) for financial support.
ABBREVIATIONS USED
■
CB, cannabinoid; CNS, central nervous system; B/P, brain-to-
plasma ratio; BBB, blood−brain barrier; tPSA, topological polar
surface area; SAR, structure−activity relationships; DIO, diet-
induced obese; AIBN, azobis(isobutyronitrile); P-gp, P-
glycoprotein; Eu-GTP, europium guanosine 5′-triphosphate;
ESMS, electrospray mass spectra
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