Pyrazole antagonists of the CB1 receptor with reduced brain penetration

Article abstract of DOI:10.1016/j.bmc.2016.01.033

Type 1 cannabinoid receptor (CB1) antagonists might be useful for treating obesity, liver disease, metabolic syndrome, and dyslipidemias. Unfortunately, inhibition of CB1 in the central nervous system (CNS) produces adverse effects, including depression,

Full text of DOI:10.1016/j.bmc.2016.01.033

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrazole antagonists of the CB1 receptor with reduced brain
penetration
Alan Fulp, Yanan Zhang, Katherine Bortoff, Herbert Seltzman, Rodney Snyder, Robert Wiethe,
⇑
George Amato, Rangan Maitra
Discovery Science and Technology, RTI International, 3040 Cornwallis Rd., Research Triangle Park, NC 27709-2194, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Type 1 cannabinoid receptor (CB1) antagonists might be useful for treating obesity, liver disease, meta-
bolic syndrome, and dyslipidemias. Unfortunately, inhibition of CB1 in the central nervous system (CNS)
produces adverse effects, including depression, anxiety and suicidal ideation in some patients, which led
to withdrawal of the pyrazole inverse agonist rimonabant (SR141716A) from European markets. Efforts
are underway to produce peripherally selective CB1 antagonists to circumvent CNS-associated adverse
effects. In this study, novel analogs of rimonabant (1) were explored in which the 1-aminopiperidine
group was switched to a 4-aminopiperidine, attached at the 4-amino position (5). The piperidine nitrogen
was functionalized with carbamates, amides, and sulfonamides, providing compounds that are potent
inverse agonists of hCB1 with good selectivity for hCB1 over hCB2. Select compounds were further stud-
ied using in vitro models of brain penetration, oral absorption and metabolic stability. Several compounds
were identified with predicted minimal brain penetration and good metabolic stability. In vivo pharma-
cokinetic testing revealed that inverse agonist 8c is orally bioavailable and has vastly reduced brain pen-
etration compared to rimonabant.
Received 5 November 2015
Revised 6 January 2016
Accepted 17 January 2016
Available online xxxx
Keywords:
CB1
Cannabinoid
Peripheral
Antagonist
Rimonabant
Pyrazole
CB2
MDCK
PAMPA
Ó 2016 Published by Elsevier Ltd.
Blood brain barrier
1. Introduction
potential medications for CNS related disorders including alco-
holism and drug dependence.5,6 Additionally, through both periph-
The endocannabinoid (EC) system is comprised of endocannabi-
noids, receptors, transporters, and enzymes involved in the synthe-
sis and degradation of endocannabinoids. Cannabinoid receptors,
CB1 and CB2, are G protein-coupled receptors (GPCRs) whose pri-
mary function is to activate G proteins (Gi/o).1 While CB1 is present
throughout the body, it is primarily expressed in the central ner-
vous system (CNS).2 In contrast, CB2 is nominally expressed in
the CNS, but is highly expressed in the human immune system.3
While agonism of the CB1 receptor is most well known as a tar-
get of drug abuse,4 CB1 antagonists have received attention as
eral and CNS mechanisms, the CB1 receptor has been validated as a
target for the treatment of obesity, liver disease, metabolic syn-
drome, and dyslipidemias.7,8 Unfortunately, due to adverse CNS-
related side effects, anxiety and depression, the first clinically
approved
CB1
antagonist/inverse
agonist,
rimonabant
(SR141716A, 1) (Fig. 1) was withdrawn from European markets.
Furthermore, the adverse side effects seen with 1 precipitated
the withdrawal of other CB1 antagonists, otenabant (2), tarana-
bant, and ibipinabant, from clinical development.7
In hope of avoiding CNS-related side effects, an alternate strat-
egy has been developed which selectively targets the CB1 receptors
in the periphery for indications such as obesity, diabetes, and liver
diseases. By developing CB1 antagonists that do not cross the
blood–brain barrier (BBB), it is reasonable to assume that CNS side
effects can be mitigated. A similar strategy has been previously
used to develop peripherally selective opioids.9 This strategy is
currently being pursued for the CB1 receptor system by several
groups.10 One common approach to limit compounds to the
periphery is to considerably increase their topological polar surface
area (TPSA). Using this strategy, some pyrazole CB1 antagonists
Abbreviations: BBB, blood–brain barrier; BOP, benzotriazol-1-yl-oxytris
(dimethylamino)phosphonium hexafluorophosphate; CB, cannabinoid receptors;
CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; CHO-K1, Chinese
hamster ovary cells; CMA 80, 80% chloroform, 18% methanol, and 2% ammonium
hydroxide; CNS, central nervous system; Ke, apparent affinity constant; MDCK-
mdr1, Madin–Darby canine kidney cells transfected with the human MDR1 gene;
IP3, inositol phosphatase 3; MRM, multiple reaction monitoring; NA, not applicable;
PAMPA, parallel artificial membrane permeability assay.
⇑
Corresponding author. Tel.: +1 919 541 6795; fax: +1 919 541 8868.
E-mail address: rmaitra@rti.org (R. Maitra).
http://dx.doi.org/10.1016/j.bmc.2016.01.033
0968-0896/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Fulp, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.033

2
A. Fulp et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
nitrogen as carbamates, amides, and sulfonamides (Fig. 3). This
strategy has been proven successful at discovering potent and
selective analogs of both rimonabant (1) and otenabant (2).15–18
In the SAR studies of compound 4, one of the goals was good oral
bioavailability. A perceived advantage with compound 4 compared
to 3 was its lower molecular weight, which enabled exploration of
larger R groups while keeping the MW <600. The TPSA was gener-
ally kept between 60 and 90 Å2 to potentially minimize CNS pene-
tration while not precluding oral absorption.
Syntheses were carried out under standard conditions
(Scheme 1). The preparation of carbamates 8a–e began with react-
ing the previously reported amine 615 with 4-nitrophenyl chloro-
formate to form intermediate 7 in 75% yield. Reaction of 7 with
the appropriate alcohol afforded the desired carbamate in yields
ranging from 46% to 88%. Amides 9c–f were prepared in 79–99%
yield by coupling the appropriate carboxylic acid and 6 using (ben-
O
N
N
NH
N
N
Cl
O
N
N
N
N
NH2
Cl
Cl
NH
Cl
Cl
2 (otenabant,
CP-945,598)
1 (rimonabant,
SR141716A)
Figure 1. Examples of reported CB1 antagonists.
have been reported. These compounds include structural modifica-
tions at the 4-methyl,11,12 the 5-aryl position,13 as well as the 1-
aryl position of rimonabant.14 While further characterization and
validation of these compounds are required, such peripherally
selective CB1 antagonists hold much promise.
zotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluo-
rophosphate (BOP). Compound 9g was obtained by reacting 6
with benzoyl chloride in 82% yield. Finally, 9b was prepared in
99% yield by reacting 6 with trifluoroacetic anhydride. The synthe-
sis of sulfonamides 10a–f were accomplished by coupling 6 and
the appropriate sulfonyl chloride in yields ranging from 57% to
85%.
Our group has been focusing on introducing changes at the 3-
position of the pyrazole that increase the TPSA relative to rimona-
bant (Fig. 2).15,17 We recently reported peripherally selective CB1
antagonists based on the scaffold of rimonabant, including com-
pounds 3 and 4 (Fig. 2).15–18 Both 3 and 4 were found to have brain
to plasma ratios that were <0.04 when dosed at 10 mg/kg ip in
Sprague-Dawley rats. When un-perfused brains are used, the cere-
bral blood volume present in rodent brains is 4–6%; therefore, a
brain to plasma ratio of 60.04 represents little to no brain penetra-
tion.19–21 Unfortunately, neither 3 nor 4 are orally bioavailable.
While the SAR of 3 have been extensively studied in our earlier
work, the SAR requirement for 4 remained unclear.15 The molecu-
lar weight of compounds based on 4 also have the advantage of
being lower compared to 3. Our previous structural modifications
mainly focused on compounds with the piperidine nitrogen func-
tionalized as a urea. Herein we will describe our efforts to broaden
the SAR around 4 to include additional carbamates, amides and
sulfonamides. The in vitro pharmacological characterization of
these compounds, including predictive assays for CNS penetration
and oral absorption were conducted, leading to identification of 8c
for further testing. In vivo pharmacokinetic (PK) testing was per-
formed, thus revealing that 8c is orally bioavailable and has
reduced brain penetration.
2.2. Pharmacological characterization
All target compounds were pharmacologically evaluated in
FLIPR-based calcium mobilization assay for hCB1 (Table 1).15,16
The potency of each compound was first determined in the calcium
assay. Those that demonstrated apparent antagonist dissociation
equilibrium constant Ke <50 nM were further tested for affinity at
hCB1 and selectivity for hCB1 over hCB2 using radioligand dis-
placement of [3H]CP55940 in purified membrane fractions overex-
pressing either human CB1 or CB2.15,16 As previously reported by
our group, the parent amine 6 had a Ke >1 lM at the hCB1 receptor
in the calcium assays.15 Most target compounds reported in this
publication were found to be potent antagonists of the hCB1 recep-
tor and several had Ke values at hCB1 of 620 nM in the calcium
assays against agonist CP55940. In addition, most compounds
had high affinity for the hCB1 receptor. While the aryl carbamate
7 had significantly decreased potency, the alkyl carbamates 8a–e
were all active at the hCB1 receptor in both assays. Alkyl groups
smaller than the t-butyl in 4 were well tolerated. In particular,
the ethyl, propyl and isopropyl carbamates, 8b, 8c and 8e, respec-
tively, showed good potency, affinity and selectivity. These carba-
mates are potentially less acid labile than 4, which may be
important for oral bioavailability. In the amide series, straight
chain alkyl, branched chain alkyl, cyclic alkyl and aryl groups were
active as well. The trifluoromethyl, cyclopentyl and phenyl amides
(9b, 9f and 9g, respectively) were particularly potent and selective.
2. Results and discussion
2.1. Compound design and synthesis
The new analogs developed have been designed to broaden the
SAR around 5 by examining functionalization of the piperidine
O
R
N
O
H
N
N
O
O
O
O
NH
NH
N
NH
3
3
4
5
4
N
N
N
N
N
N
5
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
4
3
5
Figure 2. Peripherally selective CB1 antagonists.
Figure 3. General strategy for SAR studies.
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A. Fulp et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
NO2
3
O
NH
R
O
O
O
N
O
NH
N
O
O
NH
a
N
b
N
NH
Cl
N
Cl
N
N
Cl
N
Cl
Cl
Cl
Cl
8
6
Cl
7
Cl
f
c, d, or e
O
N
O
S
O
R
R
N
O
O
N
NH
NH
N
N
N
Cl
Cl
Cl
Cl
9
10
Cl
Cl
Scheme 1. Reagents and conditions: (a) 4-nitrophenyl chloroformate, NEt3, THF; (b) ROH, NaH, THF; (c) RCO2H, BOP, NEt3, THF; (d) benzoyl chloride, NEt3, THF; (e)
trifluoroacetic anhydride, NEt3, THF; (f) RSO2Cl, NEt3, THF.
Table 1
Pharmacological characteristics of hCB1 antagonists
#
R
TPSA (Å2)
Ke hCB1 (nM)
Ki hCB1a (nM)
Ki hCB2a (nM)
Selectivity CB2/CB1
51
1
Rimonabant
Unsubstituted piperidine
Me
Et
n-Pr
n-Bu
i-Pr
t-Bu
p-NO2-Ph
Me
CF3
n-Bu
i-Bu
50
59
76
76
76
76
76
76
122
67
67
67
67
1.1
5115
59
3
12
6.2
313
6b
8a
8bb
8c
9.2
8.82
3000
1545
326
175
8d
8e
88
20
6.4
2.9
1929
2510
301
878
8fb (4)
7
4.7
212
195
0.67
9.1
16.0
9ab
9b
9c
65
1.9
14.5
28.9
2240
1041
1785
1808
34
548
123
63
9d
9e
70
2198
N
c-Pen
Ph
Me
Et
9f
67
67
93
84
3.6
7.0
269
18.7
9.2
7.5
2496
1725
271
230
9g
10ab
10b
44.4
9.6
4407
4719
99
F
F
10c
84
4.3
492
F
i-Bu
c-Pr
10d
10e
10f
84
84
84
9.9
292
45.8
43.5
43.0
6213
143
Ph
>20000
>465
a
Displacement was measured using [3H]CP55940 in CHO cell membrane preparations overexpressing human CB1 or CB2 receptors.
Our previous results from Ref. 15.
b
The small, but electron withdrawing CF3 group of analog 9b pro-
vided the most potent compound of the series, demonstrating
potency and affinity better than 4 and comparable to 1. This is in
stark contrast to the result for the methyl amide 9a, which is much
less active. Compared to the potent i-butyl amide 9d, the sterically
similar dimethylamine analog 9e was much less active, indicating
low tolerance for a basic amine in this region. The sulfonamides
showed a similar trend as the amides. Both alkyl (10b, 10c, 10d)
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A. Fulp et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 2
and aryl (10f) groups provided compounds with good activity. The
size of the group is important, as demonstrated by the tenfold
increase in potency of the ethyl sulfonamide 10b compared to
the methyl sulfonamide 10a. Interestingly, the 3,3,3-trifluoro-
propyl analog (10c) was the most potent and selective of the sul-
fonamides, demonstrating similar potency and affinity as 4.
In summary, it was shown that potent & selective compounds
can be made when compound 5 is either an amide, sulfonamide
or carbamate. Straight chain alkyl, branched chain alkyl, cycloalkyl
and aryl groups are well tolerated. The small, but electron with-
drawing trifluoromethyl amide 9f was the most potent and selec-
tive of the compounds prepared. All compounds tested in the
radioligand binding assays were highly selective with at least
>50-fold selectivity for hCB1 over hCB2.
ADME properties of CB1 antagonists
#
R
MDCK-mdr1 A S9% remaining
PAMPA A to B (%)a
pH 7.4, 5.5
to Ba (%)
at 60 min
8c
8e
9b
9c
9d
9f
n-Pr
i-Pr
CF3
n-Bu
i-Bu
c-Pen
Ph
<1
<1
<1
<1
1
<1
<1
<1
66
82
74
72
72
62
>90
68
1.9, 1.4
0.1, 0
0.2, 0.4
1.8, 1.1
3.6, 3.3
0.2, 0.2
0.2, 0.3
0.05, 0.1
9g
10b Et
F
F
F
10c
<1
>90
0.1, 0.1
10d i-Bu
10f
11
<1
<1
80
>90
>90
0.2, 0.1
0.1, 0.03
70, 67
Ph
Caffeine
2.3. Compound 8c is an inverse agonist of hCB1 receptor
a
% transported from apical (A) to basal (B) side.
Compound 8c was further characterized using the calcium
assay to establish whether this compound acted as a neutral antag-
onist or inverse agonist of hCB1 receptor. Structurally related
rimonabant is an inverse agonist. As indicated by Figure 4, both
compounds are inverse agonists of hCB1 as constitutive signaling
through hCB1 was suppressed with increasing concentration of
each compound. The EC50 of 8c was 20 nM compared to 5 nM for
rimonabant.
compounds is related to efflux by P-glycoprotein or a function of
increased TPSA.
In vitro stability of the compounds was evaluated in pooled
human S9 liver fractions (Table 2). Compounds that are >50% stable
after a 30 or 60 min incubation period are generally considered
favorable in this assay. In general, these compounds were stable,
assuaging any concerns related to poor metabolic stability. The
phenyl amide 9g, the alkyl sulfonamides 10c and 10d, and the phe-
nyl sulfonamide 10f demonstrated particularly high stability with
>90% of the parent surviving the incubations.
2.4. In vitro permeability and metabolic stability testing
Select compounds were further tested in an in vitro model of
BBB penetration comprising monolayers of MDCK cells expressing
the human P-glycoprotein efflux transporter (MDCK-mdr1) cells
grown on filters.22 Permeability of the compounds across the
monolayer from the top (apical, A) to the bottom (basal, B) com-
partment approximates brain penetration. Caffeine is included as
a reference compound that has good brain penetration. The com-
pounds tested had little to no permeability (expressed as% trans-
ported A to B) across monolayers as indicated in Table 2. It is
currently unknown whether the lack of transport noted with these
PAMPA was used to predict oral absorption of these com-
pounds. Testing was performed at pH 7.4 and 5.5 to mimic the
varying pH in the intestines and allow for possible effects of com-
pound ionization (Table 2).23,24 Efficient apical to basal transport,
mimicking transcellular and paracellular transport across mem-
branes coated with a lipid mixture, indicates possibility of good
oral absorption in vivo. Caffeine was included as a reference com-
pound with good permeability. Most compounds tested demon-
strated low permeability in the PAMPA system, indicative of
limited oral absorption. Similar results were obtained at both neu-
tral and acidic pH. Three compounds, the n-propyl carbamate 8c
and the n-butyl and i-butyl amides 9c and 9d, however, showed
some permeability. Compound 8c is a close analog of the previ-
ously reported t-butyl carbamate 4. In rats, 4 has a good brain to
plasma ratio but is not orally bioavailable.15 It was hypothesized
that the lack of oral bioavailability of 4 may be due to the potential
instability of the t-butyl carbamate group in the acidic parts of the
digestive tract. In compound 8c, the t-butyl group is replaced with
a more stable n-propyl group and hence this compound was
selected for additional testing.
6000
5500
2.5. In vivo snapshot testing of compound 8c
Compound 8c was subjected to snapshot PK testing to assess
whether this compound had limited brain exposure. As such, three
12 week old male Sprague Dawley rats were administered a bolus
dose of 8c by oral gavage. Animals were sacrificed 4 h after dosing.
Samples were collected and concentration of the parent compound
8c
Rimonabant
5000
-1
0
1
2
3
4
5
Table 3
Concentration, log [nM]
Rat brain & plasma levels of 8c, 4 h after administration of 10 mg/kg po
Tissue
Mean
SD
Figure 4. Compound 8c is an inverse agonist of hCB1. CHO-CB1 cells were loaded
with calcium indicator dye for 60 min as has been described in Section 4. Cells were
then stimulated by various concentrations of each compound and fluorescence
change recorded using FLIPR Tera (Molecular Devices) instrument. Data reported as
Mean SEM from 3 independent measurements.
Brain (ng/g)
Plasma (ng/ml)
Ratio (%)
69
455
15
8
95
3
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A. Fulp et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
was determined in plasma and brain. As demonstrated in Table 3,
4.1.1. 4-Nitrophenyl-4-[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-
carboxylate (7)
compound 8c was orally bioavailable and had limited brain pene-
tration. Approximately 15% exposure was noted in un-perfused
brain. When un-perfused brains are used, cerebral blood volume
present in this tissue is 4–6% by different estimates in rodents.19,21
Based on this observation, compound 8c has >9-fold selectivity for
plasma versus brain. Generally speaking, compounds that have a
10-fold or greater selectivity for plasma over brain are considered
peripherally selective. Compound 8c, thus, represents a starting
point for further optimization.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piper-
idin-4-yl)-1H-pyrazole-3-carboxamide (61 mg, 0.132 mmol,
6
1 equiv), p-nitrophenyl chloroformate (29 mg, 0.144 mmol,
1.1 equiv), and triethylamine (0.06 mL, 0.395 mmol, 3 equiv) were
stirred for 16 h in THF (2 mL). The reaction was concentrated in
vacuo. The crude material was purified by column chromatography
0–100% ethyl acetate/hexanes to yield 62 mg (75%) of desired
product. Compound was 91% pure by HPLC analysis. 1H NMR
(300 MHz, CDCl3) d ppm 1.54–1.74 (m, 2H), 2.18 (br s, 2H), 2.42
(s, 3H), 3.04–3.38 (m, 2H), 4.21–4.43 (m, 3H), 6.95 (d, J = 7.91 Hz,
1H), 7.10 (d, J = 8.29 Hz, 2H), 7.30–7.41 (m, 5H), 7.47 (s, 1H), 8.29
(d, J = 9.04 Hz, 2H); [M+H]+ 628.7.
3. Conclusions
To avoid the psychiatric side effects associated with CB1 antag-
onists, peripherally restricted CB1 antagonists that have limited
BBB penetration have been pursued. In this study, a series of carba-
mates, amides and sulfonamides with higher TPSA than 1 were
synthesized and evaluated in a number of pharmacological assays.
Representatives of all three compound classes were found to be
potent antagonists of the hCB1 receptor with good selectivity over
hCB2. Straight chain alkyl, branched chain alkyl, cyclic alkyl and
aryl groups were tolerated for activity. These compounds have pre-
dicted good metabolic stability and low BBB penetration, as deter-
mined by incubation with human S9 liver fractions and MDCK-
MDR1 assays, respectively. In general, predicted oral absorption
was low based on results from testing in the PAMPA system. Three
compounds (8c, 9c and 9d), however, were significantly better
than the others and 8c was further studied for oral PK in rats.
The results indicate that 8c is orally bioavailable and mostly com-
partmentalized in plasma. It has vastly reduced penetration into
the CNS compared to rimonabant, which has >50% brain penetra-
tion. Compound 8c, thus, serves as a lead structure for producing
potent and selective compounds with oral bioavailability and lim-
ited CNS penetration.
4.1.2. General procedure for the conversion of 4-nitrophenyl 4-
[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-amido]piperidine-1-carboxylate (7) into other
carbamates
To 4-nitrophenyl 4-[5-(4-chlorophenyl)-1-(2,4-dichlorophe-
nyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate
(0.02 mmol, 1 equiv) and the appropriate alcohol (0.5 mL) in THF
(2 mL) was added sodium hydride 60% dispersion in mineral oil
(4 mg, 0.1 mmol, 5 equiv). The reaction was stirred for 16 h and
quenched with acetic acid. The reaction was concentrated in vacuo.
The crude material was purified by column chromatography using
0–100% ethyl acetate/hexanes to yield desired product.
4.1.3. Methyl 4-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (8a)
Reaction proceeded in 78% yield. 1H NMR (300 MHz, CDCl3) d
ppm 1.37–1.52 (m, 2H), 1.96–2.11 (m, 3H), 2.37 (s, 3H), 2.97 (t,
J = 12.15 Hz, 2H), 3.69 (s, 3H), 3.98–4.26 (m, 3H), 6.85 (d,
J = 8.01 Hz, 1H), 7.06 (d, J = 8.29 Hz, 2H), 7.27–7.34 (m, 3H), 7.43
(s, 1H); [M+H]+ 521.7.
The results of this study establishes a way to analyze and triage
compounds for good in vitro activity, good metabolic stability, low
predicted CNS penetration and high predicted oral absorption.
Future studies will further refine balancing physical properties
such as MW, H-bond donating groups and TPSA to obtain com-
pounds that are potent, selective, have low CNS penetration and
good oral bioavailability.
4.1.4. Propyl 4-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (8c)
Reaction proceeded in 88% yield. 1H NMR (300 MHz, CDCl3) d
ppm 0.90–0.99 (m, 3H), 1.40–1.53 (m, 2H), 1.60–1.72 (m, 2H),
2.02 (d, J = 14.13 Hz, 2H), 2.37 (s, 3H), 2.96 (t, J = 12.15 Hz, 2H),
4.03 (t, J = 6.64 Hz, 2H), 4.07–4.25 (m, 3H), 6.85 (d, J = 8.10 Hz,
1H), 7.06 (d, J = 8.38 Hz, 2H), 7.27–7.34 (m, 3H), 7.43 (d,
J = 1.51 Hz, 1H); [M+H]+ 549.7.
4. Experimental
4.1. Chemistry general
Purity and characterization of compounds were established by a
combination of HPLC, TLC, and NMR analytical techniques
described below. 1H spectra were recorded on a Bruker Avance
DPX-300 (300 MHz) spectrometer and were determined in chloro-
form-d (CDCl3, 7.26 ppm) or methanol-d4 (CD3OD, 3.31 ppm) with
tetramethylsilane (TMS, 0.00 ppm) or solvent peaks as the internal
reference unless otherwise noted. Chemical shifts are reported in
ppm relative to the solvent signal, and coupling constant (J) values
are reported in hertz (Hz). Thin-layer chromatography (TLC) was
performed on EMD precoated silica gel 60 F254 plates, and spots
were visualized with UV light or I2 detection. Low-resolution mass
spectra were obtained using a Waters Alliance HT/Micromass ZQ
system (ESI). Unless stated otherwise, all test compounds were at
least 95% pure as determined by HPLC on an Agilent 1100 system
4.1.5. Butyl 4-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (8d)
Reaction proceeded in 51% yield. 1H NMR (300 MHz, CDCl3) d
ppm 0.81–0.94 (m, 3H), 1.25–1.65 (m, 6H), 1.96 (d, J = 12.72 Hz,
2H), 2.30 (s, 3H), 2.89 (t, J = 12.15 Hz, 2H), 3.93–4.18 (m, 5H),
6.78 (d, J = 8.10 Hz, 1H), 6.99 (d, J = 8.38 Hz, 2H), 7.20–7.27 (m,
3H), 7.36 (d, J = 1.51 Hz, 1H); [M+H]+ 563.5.
4.1.6. Propan-2-yl-4-[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-
carboxylate (8e)
Reaction proceeded in 46% yield. 1H NMR (300 MHz, CDCl3) d
ppm 1.17 (d, J = 6.22 Hz, 6H), 1.28–1.46 (m, 2H), 1.87–2.02 (m,
2H), 2.30 (s, 3H), 2.87 (t, J = 11.87 Hz, 2H), 3.93–4.17 (m, 3H),
4.84 (dt, J = 12.43, 6.22 Hz, 1H), 6.78 (d, J = 8.10 Hz, 1H), 6.99
(d, J = 8.38 Hz, 2H), 7.20–7.27 (m, 3H), 7.35 (s, 1H); [M+H]+
549.4.
using an Agilent Zorbax SB-Phenyl, 2.1 ꢀ 150 mm, 5
lm column
with gradient elution using the mobile phases (A) H2O containing
0.05% CF3COOH and (B) Methanol. A flow rate of 1.0 mL/min was
used.
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6
A. Fulp et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.7. General procedure for making amides, using BOP, from 5-
(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-
(piperidin-4-yl)-1H-pyrazole-3-carboxamide (6)
J = 12.53 Hz, 1H), 2.37 (s, 3H), 2.72–2.85 (m, 1H), 2.90 (t,
J = 7.82 Hz, 1H), 3.18 (t, J = 11.87 Hz, 1H), 3.98 (d, J = 13.56 Hz,
1H), 4.12–4.28 (m, 1H), 4.60 (d, J = 13.28 Hz, 1H), 6.86 (d,
J = 8.01 Hz, 1H), 7.06 (d, J = 8.38 Hz, 2H), 7.21–7.36 (m, 4H), 7.43
(d, J = 1.51 Hz, 1H); [M+H]+ 559.7.
To a solution of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
(6)
(20 mg, 0.043 mmol, 1 equiv) in 2 mL of THF was added triethy-
lamine (0.02 mL, 0.13 mmol, 3 equiv), BOP (19 mg, 0.043 mmol,
1 equiv), and the appropriate carboxylic acid (1 equiv). The reac-
tion was stirred for 16 h and then concentrated in vacuo. The crude
material was purified by silica gel column chromatography using
0–100% ethyl acetate/hexane to yield compound.
4.1.13. N-(1-Benzoylpiperidin-4-yl)-5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (9g)
To a solution of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
(6)
(23 mg, 0.050 mmol, 1 equiv) in 2 mL of THF was added triethy-
lamine (0.02 mL, 0.15 mmol, 3 equiv) and benzoyl chloride
(0.01 mL, 0.075 mmol, 1.5 equiv). The reaction was stirred for
16 h and then concentrated in vacuo. The crude material was puri-
fied by silica gel column chromatography using 0–100% ethyl acet-
ate/hexane to yield 23 mg (82%) of desired compound. 1H NMR
(300 MHz, CDCl3) d ppm 1.33–1.62 (m, 2H), 1.98–2.21 (m, 2H),
2.37 (s, 3H), 2.87–3.30 (m, 2H), 3.81 (d, J = 17.71 Hz, 1H), 4.15–
4.34 (m, 1H), 4.68 (br s, 1H), 6.90 (d, J = 8.01 Hz, 1H), 7.06 (d,
J = 8.29 Hz, 2H), 7.25–7.35 (m, 4H), 7.36–7.47 (m, 5H); [M+H]+
567.4.
4.1.8. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-
[1-(trifluoroacetyl)piperidin-4-yl]-1H-pyrazole-3-carboxamide
(9b)
To a solution of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
(6)
(22 mg, 0.048 mmol, 1 equiv) in 2 mL of THF was added triethy-
lamine (0.02 mL, 0.143 mmol, 3 equiv) and trifluoromethanesul-
fonic anhydride (0.01 mL, 0.071 mmol, 1.5 equiv). The reaction
was stirred 16 h and then concentrated in vacuo. The crude mate-
rial was purified by silica gel column chromatography using 0–
100% ethyl acetate/hexane to yield 25 mg (99%) of desired com-
pound. 1H NMR (300 MHz, CDCl3) d ppm 1.95–2.08 (m, 2H),
2.12–2.28 (m, 2H), 2.30–2.44 (m, 3H), 2.67–2.86 (m, 2H), 3.09–
3.26 (m, 1H), 4.12 (d, J = 14.13 Hz, 1H), 4.64–4.74 (m, 1H), 7.07
(d, J = 8.29 Hz, 2H), 7.20–7.37 (m, 4H), 7.40 (d, J = 1.70 Hz, 1H);
[M+H]+ 559.8.
4.1.14. General procedure for making sulfonamides from 5-(4-
chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-
4-yl)-1H-pyrazole-3-carboxamide (6)
To a solution of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
(6)
(20 mg, 0.043 mmol, 1 equiv) in 2 mL of THF was added triethy-
lamine (0.02 mL, 0.13 mmol, 3 equiv) and the appropriate sulfonyl
chloride (1 equiv). The reaction was stirred for 16 h and then con-
centrated in vacuo. The crude material was purified by silica gel
column chromatography using 0–100% ethyl acetate/hexane to
yield compound.
4.1.9. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-
(1-pentanoylpiperidin-4-yl)-1H-pyrazole-3-carboxamide (9c)
Reaction proceeded in 89% yield. 1H NMR (300 MHz, CDCl3) d
ppm 0.88–0.98 (m, 3H), 1.31–1.48 (m, 4H), 1.53–1.67 (m, 2H),
2.03 (d, J = 11.77 Hz, 1H), 2.13 (d, J = 12.34 Hz, 1H), 2.27–2.41 (m,
5H), 2.70–2.86 (m, 1H), 3.18 (t, J = 11.68 Hz, 1H), 3.87 (d,
J = 13.56 Hz, 1H), 4.09–4.29 (m, 1H), 4.59 (d, J = 13.56 Hz, 1H),
6.87 (d, J = 8.01 Hz, 1H), 7.06 (d, J = 8.48 Hz, 2H), 7.23–7.35 (m,
4H), 7.43 (d, J = 1.60 Hz, 1H); [M+H]+ 547.9.
4.1.15. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-[1-(ethan-
esulfonyl)piperidin-4-yl]-4-methyl-1H-pyrazole-3-carboxamide
(10b)
Reaction proceeded in 77% yield. 1H NMR (300 MHz, CDCl3) d
ppm 1.37 (t, J = 7.39 Hz, 3H), 1.54–1.71 (m, 2H), 2.01–2.18 (m,
2H), 2.36 (s, 3H), 2.97 (q, J = 7.69 Hz, 4H), 3.83 (d, J = 12.53 Hz,
2H), 4.00–4.21 (m, 1H), 6.87 (d, J = 8.01 Hz, 1H), 7.06 (d,
J = 8.29 Hz, 2H), 7.23–7.36 (m, 4H), 7.44 (s, 1H); [M+H]+ 555.6.
4.1.10. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-
[1-(3-methylbutanoyl)piperidin-4-yl]-1H-pyrazole-3-
carboxamide (9d)
Reaction proceeded in 99% yield. 1H NMR (300 MHz, CDCl3) d
ppm 0.86–0.95 (m, 6H), 1.29–1.45 (m, 2H), 1.93–2.09 (m, 3H),
2.11–2.20 (m, 2H), 2.30 (s, 3H), 2.71 (t, J = 11.54 Hz, 1H), 3.11 (t,
J = 11.77 Hz, 1H), 3.75–3.88 (m, 1H), 4.11 (dd, J = 7.39, 3.53 Hz,
1H), 4.54 (d, J = 13.28 Hz, 1H), 6.80 (d, J = 8.01 Hz, 1H), 6.99 (d,
J = 8.38 Hz, 2H), 7.14–7.29 (m, 4H), 7.36 (d, J = 1.70 Hz, 1H); [M
+H]+ 547.7.
4.1.16. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-
{1-[(3,3,3-trifluoropropane)sulfonyl]piperidin-4-yl}-1H-
pyrazole-3-carboxamide (10c)
Reaction proceeded in 78% yield. 1H NMR (300 MHz, CDCl3) d
ppm 1.58–1.72 (m, 2H), 2.07–2.20 (m, 2H), 2.37 (s, 3H), 2.55–
2.71 (m, 2H), 2.93–3.06 (m, 2H), 3.06–3.17 (m, 2H), 3.84 (d,
J = 12.43 Hz, 2H), 4.12 (ddt, J = 11.07, 7.28, 3.69 Hz, 1H), 6.88 (d,
J = 8.01 Hz, 1H), 7.06 (d, J = 8.48 Hz, 2H), 7.23–7.37 (m, 4H), 7.43
(d, J = 1.79 Hz, 1H); [M+H]+ 625.7.
4.1.11. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-{1-[2-(di-
methylamino)acetyl]piperidin-4-yl}s-4-methyl-1H-pyrazole-3-
carboxamide (9e)
Reaction proceeded in 93% yield. 1H NMR (300 MHz, CDCl3) d
ppm 1.46 (br s, 2H), 2.02–2.16 (m, 2H), 2.27 (s, 6H), 2.37 (s, 3H),
2.73–2.88 (m, 1H), 3.07 (s, 1H), 3.10–3.23 (m, 2H), 4.04–4.28 (m,
2H), 4.47–4.63 (m, 1H), 6.85 (d, J = 8.01 Hz, 1H), 7.06 (d,
J = 8.38 Hz, 2H), 7.23–7.34 (m, 4H), 7.43 (d, J = 1.32 Hz, 1H); [M
+H]+ 548.8.
4.1.17. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-
{1-[(2-methylpropane)sulfonyl]piperidin-4-yl}-1H-pyrazole-3-
carboxamide (10d)
Reaction proceeded in 57% yield. 1H NMR (300 MHz, CDCl3) d
ppm 1.11 (d, J = 6.69 Hz, 6H), 1.58–1.71 (m, 2H), 2.12 (d,
J = 10.83 Hz, 2H), 2.22–2.32 (m, 1H), 2.32–2.40 (m, 3H), 2.76
(d, J = 6.50 Hz, 2H), 2.83–2.99 (m, 2H), 3.82 (d, J = 12.53 Hz,
2H), 3.99–4.19 (m, 1H), 6.87 (d, J = 8.01 Hz, 1H), 7.06 (d,
J = 8.48 Hz, 2H), 7.21–7.36 (m, 4H), 7.43 (d, J = 1.70 Hz, 1H);
[M+H]+ 583.9.
4.1.12. 5-(4-Chlorophenyl)-N-(1-cyclopentanecarbonylpiperidin-
4-yl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbox-
amide (9f)
Reaction proceeded in 79% yield. 1H NMR (300 MHz, CDCl3) d
ppm 1.37–1.87 (m, 10 H), 1.98–2.07 (m, 1H), 2.13 (d,
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A. Fulp et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
4.1.18. 5-(4-Chlorophenyl)-N-[1-(cyclopropanesulfonyl)piperid-
in-4-yl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbo-
xamide (10e)
ple reaction monitoring (MRM). Flow injection analysis was also
conducted to optimize for mass spectrometer parameters. Samples
from the apical and basolateral side of the MDCK cell assay were
dried under nitrogen on a Turbovap LV. The chromatography was
conducted with an Agilent 1100 binary pump with a flow rate of
0.5 mL/min. Mobile phase solvents were A, 0.1% formic acid in
water, and B, 0.1% formic acid in methanol. The initial solvent con-
ditions were 10% B for 1 min, then a gradient was used by increas-
ing to 95% B over 5 min, then returning to initial conditions. Data
reported are average values from 2 to 3 measurements.
Reaction proceeded in 85% yield. 1H NMR (300 MHz, CDCl3) d
ppm 0.95–1.05 (m, 2H), 1.13–1.23 (m, 2H), 1.60–1.73 (m, 2H),
2.13 (d, J = 10.55 Hz, 2H), 2.24–2.31 (m, 1H), 2.37 (s, 3H), 2.98 (t,
J = 11.02 Hz, 2H), 3.83 (d, J = 12.34 Hz, 2H), 4.00–4.21 (m, 1H),
6.87 (d, J = 8.01 Hz, 1H), 7.06 (d, J = 8.38 Hz, 2H), 7.22–7.37 (m,
4H), 7.43 (d, J = 1.51 Hz, 1H); [M+H]+ 567.4.
4.1.19. N-[1-(Benzenesulfonyl)piperidin-4-yl]-5-(4-chlorophen-
yl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxami-
de (10f)
4.4. In vitro stability testing
Reaction proceeded in 84% yield. 1H NMR (300 MHz, CDCl3) d
ppm 1.56–1.72 (m, 2H), 2.04–2.14 (m, 2H), 2.33 (s, 3H), 2.46 (t,
J = 11.02 Hz, 2H), 3.72–3.99 (m, 3H), 6.81 (d, J = 8.10 Hz, 1H), 7.04
(d, J = 8.38 Hz, 2H), 7.22–7.36 (m, 4H), 7.43 (d, J = 1.51 Hz, 1H),
7.49–7.68 (m, 3H), 7.78 (d, J = 7.44 Hz, 2H); [M+H]+ 605.5.
Stability of compounds to plasma and S9 fraction was per-
formed as previously described.15 In vitro testing for metabolic sta-
bility was conducted in mixed gender pooled hepatic S9 fraction
supplied by Xenotech, LLC, Lenexa, KS. Identity of the donors was
unknown.
For the hepatic S9 metabolism studies, all samples were tested
4.2. Calcium mobilization and radioligand displacement assays
at 10 lM final concentration in a 1 mL volume containing 1 mg/mL
S9. Samples were incubated in a buffer containing 50 mM potas-
sium phosphate, pH 7.4 with 3 mM MgCl2 and a NADPH regenera-
tion system comprising of NADP (1 mM), glucose-6-phosphate
(5 mM) and glucose-6-phosphate dehydrogenase (1 unit/mL). Trip-
licate samples were incubated for 60 min. Reactions were termi-
nated by addition of 3 volumes of acetonitrile and processed as
described for the MDCK-mdr1 assays, but standard curves were
prepared in blank matrix for each compound for quantitative
Each compound was pharmacologically characterized using a
functional fluorescent CB1 activated Gaq16-coupled intracellular
calcium mobilization assay in CHO-K1 cells, as has been described
in our previous publications and apparent affinity (Ke) values were
determined.15–18 Briefly, CHO-K1 cells were engineered to co-
express human CB1 and G q16. Activation of CB1 by an agonist then
a
leads to generation of inositol phosphatase 3 (IP3) and activation of
IP3 receptors, which leads to mobilization of intracellular calcium.
Calcium flux was monitored in a 96-well format using the fluores-
cent dye Calcein-4 AM in an automated plate reader (Flexstation,
Molecular Devices). The antagonism of a test compound was mea-
sured by its ability to shift the concentration response curve of the
synthetic CB1 agonist CP55940 rightwards using the equation:
assessment. Data reported are average values from
measurements.
3
4.5. Parallel artificial membrane permeability assay (PAPMA)
PAMPA was conducted using a 96-well plate based kit from BD
Biosciences (RTP, NC) and manufacturer’s instructions were fol-
lowed closely. Briefly, all samples were tested at 10 lM final con-
Ke ¼ ½Ligandꢁ=½DR ꢂ 1ꢁ
where DR is the EC50 ratio of CP55940 in the presence or absence of
a test agent.
For some assays, cells were loaded with Calcein-4 AM as
described below and directly stimulated with various concentra-
tions of a test agent for 90 s. Decrease in basal fluorescence was
used in these assays to calculate EC50 values.
Further characterization of select compounds was performed
using radioligand displacement of [3H]CP55940 and equilibrium
dissociation constant (Ki) values were determined as described
previously.15–18,25 Selectivity of these compounds at hCB1 versus
hCB2 was also determined by obtaining Ki values at either receptor
in membranes of CHO cells over-expressing either receptor. Data
reported are average values from 3 to 6 measurements typically
with ꢃ25% standard error.
centration by adding them to the apical (top) compartment in PBS
at pH 5.5 or 7.4. Samples were incubated at room temperature for
4 h. Apical to basal transport was evaluated by sampling from the
top and bottom wells of the plate and measuring concentrations of
compounds in each compartment using LC–MS. Processing and
methods for analytical evaluation were similar to those described
for MDCK-mdr1 transport assays. Data reported are average values
from 3 measurements.
4.6. Pharmacokinetic testing
Male Sprague Dawley rats were procured from Charles River
Laboratories (Durham, NC) at the age of 10 weeks and allowed to
acclimate to the facility. Animals were dosed with compound 8c
at a concentration of 10 mg/kg in a vehicle comprised of 90% corn
oil and 10% DMSO. Four hours after dosing, the animals were sac-
rificed and tissue collected for analyses essentially as described in
our previous publications.18 LC–MS/MS was used for quantification
of parent compound in a given tissue.
4.3. MDCK-mdr1 permeability assays
MDCK-mdr1 cells obtained from the Netherlands Cancer Insti-
tute were grown on Transwell type filters (Corning) for 4 days to
confluence in DMEM/F12 media containing 10% fetal bovine serum
and antibiotics as has been described previously.17 Compounds
Acknowledgements
were added to the apical side at a concentration of 10
transport buffer comprising of 1X Hank’s balanced salt solution,
25 mM -glucose and buffered with HEPES to pH 7.4. Samples were
incubated for 1 h at 37 °C and carefully collected from both the
apical and basal side of the filters. Compounds selected for
MDCK-mdr1 cell assays were infused on an Applied Biosystems
API-4000 mass spectrometer to optimize for analysis using multi-
lM in a
The authors would like to thank Ann Gilliam for performing the
binding assays, Ms. Sherry Black and Ms. Purvi Patel for help with
metabolic stability studies. We express our gratitude to the NIDA
drug supply program for providing radiolabeled probes and to Dr.
Brian Thomas for supplying the CB1 cells. This research was funded
by research Grants AA022235 and DK100414 to R.M. from NIH.
D
Please cite this article in press as: Fulp, A.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.033

8
A. Fulp et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
14. Sasmal, P. K.; Reddy, D. S.; Talwar, R.; Venkatesham, B.; Balasubrahmanyam,
Supplementary data
D.; Kannan, M.; Srinivas, P.; Kumar, K. S.; Devi, B. N.; Jadhav, V. P.; Khan, S. K.;
Mohan, P.; Chaudhury, H.; Bhuniya, D.; Iqbal, J.; Chakrabarti, R. Bioorg. Med.
Chem. Lett. 2011, 21, 562.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.01.033.
15. Fulp, A.; Bortoff, K.; Seltzman, H.; Zhang, Y.; Mathews, J.; Snyder, R.; Fennell, T.;
Maitra, R. J. Med. Chem. 2012, 55, 2820.
16. Fulp, A.; Bortoff, K.; Zhang, Y.; Seltzman, H.; Mathews, J.; Snyder, R.; Fennell, T.;
Maitra, R. J. Med. Chem. 2012, 55, 10022.
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