N -(4-cyanotetrahydro-2 H -pyran-4-yl) and N -(1-cyanocyclohexyl) derivatives of 1,5-diarylpyrazole-3-carboxamides showing high affinity for 18 kDa translocator protein and/or cannabinoid receptors

Article abstract of DOI:10.1021/jm2000536

In order to develop improved radioligands for imaging brain CB₁ receptors with positron emission tomography (PET) based on rimonabant (5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl) -1H-pyrazole-3-carboxamide, 1), we synt

Full text of DOI:10.1021/jm2000536

ARTICLE
pubs.acs.org/jmc
N-(4-Cyanotetrahydro-2H-pyran-4-yl) and N-(1-Cyanocyclohexyl)
Derivatives of 1,5-Diarylpyrazole-3-carboxamides Showing High
Affinity for 18 kDa Translocator Protein and/or Cannabinoid
Receptors
Sean R. Donohue,†,‡,§ Robert F. Dannals,§ Christer Halldin,‡ and Victor W. Pike*,†
†Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, 10 Center Drive, Bethesda, Maryland
20892-1003, United States
‡Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institutet, Karolinska Hospital, S-17176, Stockholm, Sweden
§Division of Nuclear Medicine, The Johns Hopkins PET Center, Baltimore, Maryland 21287, United States
ABSTRACT: In order to develop improved radioligands for
imaging brain CB1 receptors with positron emission tomography
(PET) based on rimonabant (5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-car-
boxamide, 1), we synthesized compounds 9aꢀs in which the N-
piperidinyl ring was replaced with a 4-(4-cyanotetrahydro-2H-
pyranyl) or 1-cyanocyclohexyl ring. Such changes were expected
to be almost isosteric with 1, confer greater metabolic resistance,
and in the case of the 4-(4-cyanotetrahydro-2H-pyranyl) com-
pounds, substantially reduce lipophilicity. One derivative, 1-(2-bromophenyl)-N-(1-cyanocyclohexyl)-5-(4-methoxyphenyl)-4-
methylpyrazole-3-carboxamide (9n), showed high affinity (Ki = 15.7 nM) and selectivity for binding to CB1 receptors. The
corresponding 4-(4-cyanotetrahydro-2H-pyranyl) derivative (9m) also showed quite high affinity for CB1 receptors (Ki = 62 nM)
but was found to have even higher affinity (Ki = 29 nM) for the structurally unrelated 18 kDa translocator protein (TSPO). Some
other minor structural changes among 9aꢀs were also found to switch binding selectivity from CB1 receptors to TSPO or vice versa.
These unexpected findings and their implications for the development of selective ligands or PET radioligands for CB1 receptors or
TSPO are discussed in relation to current pharmacophore models of CB1 receptor and TSPO binding sites.
’ INTRODUCTION
In 1994 Sanofi Recherche, presently Sanofi-Aventis, intro-
duced the first high-affinity and selective CB1 receptor “antago-
nist”, rimonabant (5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-
4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide,
Marijuana (Cannabis sativa) is one of the oldest known plant-
derived substances used medicinally or as a drug of abuse.1
Marijuana’s principle psychoactive phytocannabinoid, Δ9-tetrahy-
drocannabinol (Δ9-THC),2 binds with high affinity to two known
receptor subtypes, cannabinoid type-1 (CB1)3 and cannabinoid
type-2 (CB2).4 Both the CB1 and CB2 receptor subtypes are
G-protein-coupled receptors (GPCRs) with seven-transmembrane
domains. They have 48% protein sequence homology.5 CB1
receptors have high density in brain and are also located in some
peripheral tissues, whereas CB2 receptors are mostly located in
peripheral tissues.5 Brain CB1 receptors are believed to play a role in
the regulation of neurotransmitter (e.g., glutamate and γ-amino-
butyric acid (GABA)) release through retrograde endocannabi-
noid-mediated depolarization-induced suppression of inhibition.6
Alterations in brain CB1 receptor densities and function have
been implicated in several psychiatric disorders, such as drug
dependence,7 obesity,8 depression,9 and schizophrenia.10 There-
fore, there is keen interest to study brain CB1 receptors in relation
to such disorders in living human subjects with molecular imaging
techniques, such as positron emission tomography (PET), and
suitably selective radioligands.11
SR141716A 1, Chart 1).12 This ligand is now considered to be an
inverse agonist. The key 1,5- diarylpyrazole pharmacophore of 1
has been modified extensively to evaluate substituent effects on
receptor affinity, selectivity, and physiochemical properties.13
Additionally, 1 has been modified to allow labeling with a short-
lived imaging radionuclide (e.g., carbon-11 or fluorine-18) in the
development of PET radioligands.11 Compound 1 is highly
lipophilic with a computed cLogD value of 6.32 (Table 1), and
hence, many candidate PET radioligands derived from 1 retain
high lipophilicity.11 This is usually detrimental to the success of a
PET radioligand14ꢀ18 because high lipophilicity generally pro-
motes low and poorly measurable free radioligand fraction in
plasma, low radioligand penetration from blood to brain, and
high nonspecific binding to brain tissue. Therefore, success in the
development of PET radioligands from 1 has been quite limited.
Received: January 19, 2011
Published: March 23, 2011
r
2011 American Chemical Society
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Chart 1. Structures of 1 and Some PET CB1 Receptor Radioligands in Human Use
PET CB1 receptor radioligands developed from other structural
platforms also tend to be quite lipophilic, although some are gaining
application in human subjects.11 Examples are [11C]OMAR19
PET, nearly all of the new ligands were designed to be amenable
to labeling with cyclotron-produced carbon-11 (t1/2 = 20.4 min)
by commonly used procedures such as C-11C-methylation
(9bꢀf, 9hꢀo, 9r, and 9s), O-11C-methylation (9lꢀq), N-11C-
methylation (9d), or 11C-cyanation (9g and 9p).28
[11C]2, [11C]MePPEP20,21 [11C]3, [18F]FMPEP-d2 [18F]4,
22
and [18F]MK-947023 [18F]5 (Chart 1).
Compounds were synthesized as shown in Scheme 2. Sub-
stituted anilines were first converted into diazonium salts and
then treated with ethyl 2-chloroacetoacetate under basic condi-
tions to give 6aꢀg in good yields. A series of aryl ketones (7aꢀf)
were converted into enamines with morpholine, TiCl4, and
DIPEA (diisopropylethylamine) in toluene solution using a
modified general procedure.29 Compounds 6aꢀg were then
treated with the morpholine enamines under basic conditions
to give the 1,5-diarylpyrazoles 8aꢀk, 8m, and 8n in low but
useful yields by a regioselective 1,3-dipolar cycloaddition
reaction.30 Treatment of the acylacetonitrile 7h with 6c gave
the diarylpyrazole 8p in low yield. Diarylpyrazole 8l was
prepared in high yield by bromination of 8k with NBS
(N-bromosuccinimide) using acetonitrile as a polar solvent to
favor the formation of bromine. Each of the diarylpyrazoles
8aꢀq were hydrolyzed to the acids, converted into acyl chlorides,
and treated with either 4-aminotetrahydro-2H-pyran-4-carboni-
trile or 1-aminocyclohexanecarbonitrile HCl to give the desired
carboxamides (9aꢀc and 9eꢀs). Treatment of 9c with NaH and
MeI gave the tertiary amide 9d in high yield.
Computed Ligand Lipophilicities. The most common struc-
tural change made to the scaffold of 1, namely, replacement of the
N-(piperidin-1-yl)-4H-pyrazole-3-carboxamide moiety with an
N-(4-cyanotetrahydro-2H-pyran-4-yl)-4H-pyrazole-3-carboxa-
mide moiety, was computed to reduce lipophilicity by about 0.65
cLogD units. Consequently, the majority of the prepared ligands
have much lower cLogD values than 1 (Table 1). These cLogD
values approach values in the range that is generally preferred
for PET radioligands (∼1.5ꢀ3.5).14ꢀ18 Replacement of the
4-(4-cyanotetrahydro-2H-pyranyl) group with a 1-cyanocyclo-
hexyl group increases the computed lipophilicity substantially
(Table 1). Nevertheless, the prepared examples still have sub-
stantially lower lipophilicity than 1.
The possible efficacious roles of the nitrile group in pharma-
cophore development have recently been highlighted24 and
include its ability to act as a carbonyl or halogen isostere, to act
as a hydroxyl or carboxyl surrogate, and to reduce lipophilicity
without adverse steric effect. In our search for leads to improved
PET radioligands for CB1 receptors, we considered that the
replacement of the N-piperidinyl ring in 1 with a 1-cyanocyclo-
hexyl or 4-(4-cyanotetrahydro-2H-pyranyl) ring (Scheme 1)
would be a minor steric perturbation and would confer lower
lipophilicity. Moreover, these rings, because of their nitrile
substitution, may show greater resistance to metabolism than
the replaced piperidinyl ring, which has been shown to be the
exclusive region of metabolism of 1 in vitro25 and in vivo.25,26 We
therefore set out to synthesize a set of compounds with replaced
N-piperidinyl rings and to evaluate their structureꢀactivity
relationships at CB1 receptors. Selective high-affinity CB1 ligands
were discovered and also very unexpectedly selective high-
affinity ligands for a structurally and functionally distinct site,
the 18 kDa translocator protein, previously known as the
peripheral benzodiazepine receptor (PBR) and now known as
TSPO.27 These findings and their implications for ligand and
PET radioligand development are discussed and rationalized in
the context of an identified level of congruence between pre-
viously proposed pharmacophore models of the CB1 receptor
and TSPO.
’ RESULTS AND DISCUSSION
Chemistry. N-(4-Cyanotetrahydro-2H-pyran-4-yl) and N-(1-
cyanocyclohexyl) derivatives of 1,5-diarylpyrazole 3-carboxa-
mides constituted the two core scaffolds of all newly synthesized
compounds. Substituents in the 2-, 3-, and 4-positions of the
pyrazole-N1 and -C5 aryl rings and at the pyrazole-3-carboxamide
and 4-positions of the core scaffolds were also varied. Because of
our strong interest in developing radioligands for imaging with
StructureꢀAffinity Relationships. The sequence homolo-
gies and structures of TSPO and CB1 and CB2 receptors are each
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Table 1. cLogD and Ki at CB1 and CB2 Receptors and at TSPO for Known CB1 Receptor Ligands 1, 3, 5, New Ligands 9aꢀs, and
Known TSPO Ligands 10 and 11
Ki (nM)b
ligand
R1
R2
R3
R4
R5
cLogD a
CB1
CB2
TSPO
NA
1
6.32
4.76
4.98
3.61
3.55
3.80
3.77
3.81
5.85
3.22
3.81
3.81
3.92
4.63
3.66
3.94
5.87
3.94
3.86
4.00
4.08
4.08
4.89
4.29
1.4 ( 0.2
0.472c
0.70d
927 ( 66
>1980c
3
NA
5
44d
NA
9a
9b
9c
9d
9e
9f
2-Br
2-F
H
H
H
H
H
H
H
H
H
H
H
H
O
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
297 ( 31
>10000
>10000
>10000
>10000
>10000
437 ( 44
>10000
259 ( 26
>10000
500 ( 58
115 ( 11
62.2 ( 12.8
15.7 ( 2.3
680 ( 108
>10000
356 ( 40
55 ( 6
>10000
3110 ( 572
394 ( 62.4
436 ( 40.5
880 ( 36.2
576 ( 27.3
75.2 ( 2.51
3690 ( 519
115 ( 4.9
420 ( 20.5
762 ( 51.0
2800 ( 116
132 ( 4.91
29.0 ( 3.0
279 ( 52.3
61.8 ( 2.2
1920 ( 116
155 ( 6.7
492 ( 28.2
693 ( 28.1
1.0 ( 0.0
5.70e
Me
Me
Me
Me
Me
CN
Me
Me
Me
Me
Me
Me
Me
Me
CN
Br
O
2180 ( 224
>10000
2-Cl
2-Cl
2-Br
2-Br
2-Br
3-Br
4-Br
2-I
O
O
>10000
O
>10000
CH2
O
>10000
9g
9h
9i
>10000
O
226 ( 9.5
655 ( 36.5
>10000
O
9j
O
9k
9l
2-Ph
2-F
O
>10000
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-F
O
2460 ( 214
1890 ( 350
2750 ( 183
234 ( 13.8
2370 ( 168
175 ( 10.4
>10000
9m
9n
9o
9p
9q
9r
9s
10
11
2-Br
2-Br
3-Br
2-I
O
CH2
O
O
3-Br
2-Br
2-Br
O
Me
Me
O
3-F
O
>10000
NA
>10000
NA
NA
NA
a Computed with Pallas 3.70 software (Compudrug, South San Francisco, CA) for distribution of compound between n-octanol and pH 7.4 buffer.
b Values represent the mean ( SD of three determinations. NA = not available. c Kb value from ref 20. d IC50 value from ref 23. e From ref 36.
highly conserved between species.5,27 Therefore, the binding
affinities of ligands determined for each of these sites in tissue
from one species in vitro are expected to be quite representative
of those in other animal species. Prompted by the serendipitous
and unexpected discovery of the high affinity of 9m for TSPO in a
broad receptor screen of this compound (see later), each new ligand
was assayed at TSPO, CB1, CB2, and GABAA-Bz sites (Table 1).
All compounds exhibited low affinity (Ki > 10 μM, n = 4)
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for the GABAA-Bz site. Across the synthesized series of com-
pounds, the Ki values for CB1 and CB2 receptors and TSPO
ranged widely, namely, from 15.7 to >10000 nM, from 175 to
>10000 nM, and from 29.0 to >10000 nM, respectively.
compound, the relatively lipophilic 1-cyanocyclohexyl com-
pound 9f, showed relatively higher TSPO affinity (Ki = 75
nM). Remarkably, this compound was wholly selective for TSPO
versus CB1 or CB2 receptors. Generally, high-affinity TSPO
ligands are tertiary amides and their secondary amide counter-
parts have lower affinity.31,32 However, methylation of the amido
nitrogen in the ligand 9c led to compound 9d with a marked
2-fold reduction in TSPO receptor affinity (to Ki = 880 nM) but
with retained selectivity versus CB1 or CB2 receptors. Introduc-
tion of the large phenyl substituent into the 2-position of the N1-
aryl ring in place of halogen (Cl, Br, or I), as in 9k, switched the
selectivity from TSPO to CB1 receptors. A 4-bromo substituent,
as in 9i, also induced selectivity for CB1 receptors. In this group
of compounds, replacement of the 4-methyl group (R3, Table 1)
on the pyrazole ring with hydrogen (9a) or nitrile (9g) conferred
CB1 selectivity.
The substituent in the N1-aryl ring (R1, Table 1) was mainly
varied with respect to halogen (F, Cl, Br, and I) and position. In
compounds having a pyrazole-C5 phenyl substituent (R2,
Table 1) (9bꢀf, 9hꢀk), such variations failed to generate
compounds with affinity for CB1 or CB2 receptors in the Ki <
100 nM range. All these compounds had measurable affinity for
TSPO, with the majority of 4-(4-cyanotetrahydro-2H-pyranyl)
compounds showing less than micromolar affinity. One
Scheme 1. Main Structural Changes Made to 1 in the Newly
Synthesized Set of Ligands
Introduction of a substituent into the C5-aryl ring (R2,
Table 1) had a major influence on receptor affinity and selec-
tivity. Compounds with a 4-methoxy substituent in the C5-aryl
ring plus a 2-fluoro or 2-bromo substituent on the N1-aryl ring
(9lꢀn) showed quite high affinity for both CB1 receptors and
TSPO, with very low affinity for CB2 receptors. Remarkably, the
4-(4-cyanotetrahydro-2H-pyranyl) compound (9m) with a
2-bromo substituent in the C5-aryl ring showed high affinity
Scheme 2. Syntheses of 9aꢀsa
a Reagents, conditions, and yields: (a) HCl (aq), NaNO2, AcONa, ethyl 2-chloroacetoacetate, 52-83%; (b) for 7aꢀf, morpholine, DIPEA, TiCl4,
toluene, MgSO4; (c) DIPEA, MeCN or EtOH, 18ꢀ26%; (d) ref 35; )e) DIPEA, t-BuOH or MeCN, reflux, 16 h, 6ꢀ8%, ref 36; (f) NBS, MeCN, 91%;
(g) (1) LiOH (aq)ꢀTHF; (2) oxalyl chloride, DMF (cat.), DCM; (3) 4-aminotetrahydro-2H-pyran-4-carbonitrile or 1-aminocyclohexanecarbonitrile
HCl, DIPEA, DCM, 34ꢀ71% (for 9aꢀe, 9gꢀn, and 9pꢀs), 36ꢀ68% for 9f and 9o; (h) NaH (60% dispersion in oil), DMF, MeI, 64%.
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Scheme 3. Compatibility of Some New Ligands with Previously Proposed Pharmacophore Modelsa
a (A) Compatibility of 9n with a proposed13 CB1 receptor pharmacophore model. (B) Compatibility of 9m with a proposed31ꢀ33 TSPO receptor
pharmacophore model. (C) Common general features of proposed CB1 receptor and TSPO pharmacophore models. Known CB1-selective ligands such
as 1ꢀ5 (Chart 1) and known TSPO-selective ligands 10 and 11 (Table 1) are examples of compounds from different structural classes that are
compatible with the pharmacophore motif shown in panel C.
(Ki = 29 nM) for binding to TSPO with 2-fold preference over
binding to CB1 receptors. Even more remarkably, replacement of
the tetrahydro-2H-pyranyl ring oxygen atom with a methylene
group, as in the 1-cyanocyclohexyl analogue (9n), caused
selectivity to be reversed and affinity for CB1 receptors to be
enhanced (Ki = 15.7 nM). In the whole group of pyrazole-C5
4-methoxyphenyl compounds (9lꢀq), binding site affinity and
selectivity were sensitive to position and type of substituent in the
pyrazole-C5 aryl ring. Thus, a compound with a 3-bromo
substituent (9o) showed quite a high affinity (Ki = 62 nM)
and selectivity for binding to TSPO versus CB1 receptors. This
compound also showed very low affinity for CB2 receptors, as did
the other N1-3-bromophenyl compounds (9h, 9q). A 2-iodo
substituent in the N1-aryl ring and a 4-nitrile group in the
pyrazole ring (9p) led to very low affinity at CB1 and CB2
receptors and at TSPO. Introduction of a 4-fluoro substituent
into the C5-aryl ring, as in 9r, in place of a 4-methoxy substituent
enhanced selectivity of binding to CB1 receptors versus TSPO.
However, a shift of this substituent to the 3-position, as in 9s,
abolished high-affinity CB1 receptor binding.
Receptor and Binding Site Screening of 9m and 9n.
Ligands 9m and 9n at 10 μM showed <50% inhibition (n = 4)
for radioligand binding to the following receptors and bind-
ing sites: 5-HT1A,E, 5-HT1D (CycMEP), 5-HT2AꢀC, 5-HT3,
5-HT5A, 5-HT6, 5-HT7, R1A,B, R2AꢀC, β1,3, D1ꢀ4, DAT, DOR,
H1ꢀ4, M1ꢀ5, MOR, NET, SERT, σ1,2, and V1A,1B,2. The Ki values
(n = 3) of 9m at 5-HT1A, 5-HT1D, D5, and KOR were 6280 (
1100, >10000, >10000, and 798 ( 84 nM, respectively. The
corresponding Ki values for 9n were 1130 ( 96, >10000, >10000,
and 597 ( 67 nM. Thus, 9m is highly selective for TSPO and
CB1 receptors versus other tested binding sites and 9n is highly
selective for CB1 receptors versus all other tested binding sites
and receptors.
Relationship of Ligand Structures to Previously Proposed
CB1 Receptor and TSPO Pharmacophore Models. CB1 recep-
tors and TSPO are structurally distinct proteins. The CB1
receptor belongs to the family of GPCRs having seven-trans-
membrane domains, and considerable information has been
obtained on the likely topography and makeup of the binding
site for ligands structurally resembling 1. This information has
been summarized and a pharmacophore model proposed.13 The
model proposes (i) a hydrogen bond interaction between the
amide oxygen of the ligand and the Asp366-Lys192 salt bridge in
the receptor, (ii) a pocket formed by Val 196, Phe 170, Leu 387,
and Met 384 that can accommodate a lipophilic alicyclic group,
and (iii) two distal pockets, one formed by Trp255, Tyr 275, and
Phe 278 and the other by Trp279, Trp356, and Phe 200 which
can each accept a substituted phenyl ring, as depicted in panel A,
Scheme 3. It may be hypothesized that the highest affinity and
most CB1-selective ligand found in this study, 9n, fits into the
proposed pharmacophore in the same manner as the prototypical
selective CB1 receptor ligand 1 (panel A, Scheme 3). TSPO
protein is not a GPCR but a tryptophan-rich 18 kDa protein that
is a component of a trimeric complex with the 32 kDa voltage-
dependent anion channel and the 30 kDa adenine nucleotide
translocase to constitute the mitochodrial permeability transition
pore.27 Relatively less is known about the amino acids composing
the binding site for high-affinity TSPO ligands from a plethora of
structural classes. Nevertheless, a pharmacophore model has
been proposed that comprises three lipophilic pockets (dubbed
L1, L3, and L4) and an H-bond donor group (panel B,
Scheme 3).33ꢀ35 This pharmacophore model accommodates
classical TSPO ligands such as the isoquinoline-3-carboxamide
10 (see Table 1 for structure) and TSPO ligands from some
other structural classes, including the recently described N1-
methyl-2-phenylindol-3-ylglyoxylamides, of which 11 (Table 1)
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ARTICLE
is a recent example.36 The model also accommodates the highest
affinity TSPO ligand found in this study, namely, 9m (panel B,
Scheme 3).
to a previously described procedure.30,37 The 1,5-diarylpyrazoles 8o and
8q were prepared as previously described.38,39
1
General Methods. H (400 MHz) and 13C (100 MHz) NMR
spectra were recorded at room temperature on a Varian-400 spectro-
meter (Varian, Walnut Creek, CA) or Avance-400 spectrometer
(Bruker; Billerica, MA). Chemical shifts are reported in δ units (ppm)
downfield relative to the chemical shift of tetramethylsilane. Signals are
quoted as s (singlet), d (doublet), dd (double doublet), dt (double
triplet), t (triplet), q (quartet), or m (multiplet). High-resolution mass
spectra (HRMS) were determined at the University of Illinois at Urbana
(Champaign, IL, U.S.) or Notre Dame Mass Spectrometry Facility
(Notre Dame, IN, U.S.). Melting points were determined using a Mel-
Temp II (Laboratory Devices, Holliston, MA, U.S.) or Mel-Temp
apparatus (Electrothermal, Fisher Scientific, U.S.) and are uncorrected.
Flash column chromatography was performed on silica gel (230ꢀ400
mesh, 60 Å) (Sigma-Aldrich). Elemental analyses were acquired from
Midwest Microlab, LLC (Indianapolis, IN, U.S.).
We observe that the previously described pharmacophore
models for CB1 receptors and TSPO are strikingly similar in
the arrangement of key domains of binding siteꢀligand interac-
tion; that is, the pharmacophores appear quite congruent at this
macro level of description. Ligands with the general structural
motif shown in panel C (Scheme 3) therefore have potential to
bind to either or both pharmacophores. This provides a rational
basis for our unexpected findings on binding selectivity. Clearly,
our CB1 and TSPO ligand binding data also reveal marked
sensitivities to subtle differences in ligand structure, as exempli-
fied by 9m and 9n (Table 1). At a deeper level of description the
pharmacophores would be expected to be noticeably different,
and this would account for the severe alterations or even reversals
of ligand selectivity with minor structural changes.
Chemistry. Chloro[(2-phenylphenyl)hydrazono]ethyl Acet-
ate (6e). A solution of2-aminobiphenyl (7.6 g, 45 mmol) in concentrated
HCl (12 M, 5.4 mL) plus H2O (200 mL) was stirred in a 1 L round-
bottomed flask for 30 min. The reaction mixture was cooled to about 5 °C
in an iceꢀwater bath. A solution of sodiumnitrite (3.2 g, 46 mmol) in H2O
(10 mL) was slowly added to the reaction mixture so that the reaction
temperature remained below 15 °C. The reaction mixture was then stirred
for another 30 min. A separate solution of sodium acetate (3.5 g, 43 mmol)
and ethyl 2-chloroacetoacetate (6.2 mL, 45 mmol) in EtOHꢀH2O
(400 mL, 4:1 v/v) was added dropwise. The reaction mixture was allowed
to warm to room temperature and stirred overnight (16 h). The precipitate
was filtered off, washed with water, and dried to give 6e (11.3 g, 83%) as a
light yellow solid. Mp 82ꢀ84 °C; 1H NMR δ 8.60 (s, 1H), 7.69 (d, J = 8.4
Hz, 1H), 7.51ꢀ7.36 (m, 6H), 7.26ꢀ7.22 (m, 1H), 7.12 (t, J = 6.8 Hz, 1H),
4.40 (q, J = 7.2 Hz, 2H), 1.41 (t, J = 6.8 Hz, 3H).
Implications for PET Radioligand Development. As for CB1
receptors, the development of PET radioligands for imaging
brain and peripheral TSPO currently attracts immense effort.32
This effort on TSPO radioligands is driven by their potential to
become important biomarkers of inflammation in a variety of
neuropsychiatric and peripheral conditions. Most of the radi-
oligands being developed for CB1 receptors or TSPO share the
general structural motif depicted in panel C (Scheme 3). PET
radioligands are required to bind selectively to their target
proteins. Therefore, a message from this study is to screen
candidate PET radioligands for CB1 receptors for high affinity
binding at TSPO and vice versa in order to confirm adequate
target selectivity. Screens against CB2 receptors are also advised.
The ligand 9m possesses too low affinity and selectivity to be
developed as a PET radioligand for TSPO but could be a lead to
better candidates. [11C]2 (Chart 1) has been developed as a
promising PET radioligand for brain CB1 receptors and is now in
use in human subjects. This radioligand is selective with moder-
ately high affinity (Kb = 30 nM) and a cLogD of 5.3. By
comparison, ligand 9n also shows excellent selectivity for CB1
receptors, high affinity (Ki = 16 nM), and only somewhat higher
cLogD (5.87, Table 1). The labeling of 9n with carbon-11, which
is feasible in its 4-methoxy group, for evaluation in vivo may
therefore be warranted.
Chloro[(3-bromophenyl)hydrazono]ethyl Acetate (6f).
The procedure described for the synthesis of 5e was applied to
3-bromoaniline to give 6f (52%) as a light brown solid. Mp
1
84ꢀ86 °C; H NMR δ 8.60 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.53
(t, J = 6.8, 2H), 7.45ꢀ7.36 (m, 4H), 7.26 (t, J = 6.8 Hz, 1H), 7.12 (t, J =
7.6 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.41 (t, J = 6.8 Hz, 3H).
Chloro[(4-bromophenyl)hydrazono]ethyl Acetate (6g).
The procedure described for the synthesis of 5e was applied to
4-bromoaniline to give 6g (78%) as a pale white solid. Mp
84ꢀ86 °C; 1H NMR δ 8.30 (s, 1H), 7.44 (t, J = 1.6, 1H),
7.21ꢀ7.11 (m, 3H), 4.43 (q, J = 7.2 Hz, 2H), 1.43 (t, J = 6.8 Hz, 3H).
Ethyl 1-(2-Fluorophenyl)-4-methyl-5-phenyl-1H-pyrazole-
3-carboxylate (8a). Morpholine (33 mL, 358 mmol) was added to a
suspension of MgSO4 (4 g) in toluene 60 mL) and was stirred at room
temperature. TiCl4 (47 mL, 1 M in toluene) was slowly added to give a
dark green suspension. Propiophenone (7a, 8.0 g, 60 mmol) and DIPEA
(52 mL, 298 mmol) were dissolved separately in toluene (20 mL). This
mixture was slowly added to the solution containing TiCl4 and morpholine.
The mixture was heated to 60 °C for 16 h, cooled to room temperature,
and then filtered. The cake was washed with toluene, which was then added
to the filtrate. The combined filtrate was concentrated in vacuo to give the
crude morpholine enamine (8.2 g, 68%) as a pale yellow oil.
’ CONCLUSION
Modification of the prototypical CB1 receptor ligand 1,
primarily by replacing the N-piperidinyl ring with a 4-(4-cyano-
tetrahydro-2H-pyranyl) or 1-cyanocyclohexyl ring, led respec-
tively to one high-affinity CB1 selective ligand (9n) and also
surprisingly to a ligand with a preference to bind with high affinity
to TSPO (9m). Many of the prepared ligands showed quite high
affinity for both CB1 and TSPO. These unexpected findings may
be rationalized on the basis of the observed congruence between
previously proposed phamacophore models for CB1 receptors
and TSPO.
DIPEA (7.2 mL, 41.4 mmol) wasaddedtoa stirredsolution of6a (3.3 g,
13.7 mmol) and the morpholine enamine (2.8 g, 13.7 mmol) in EtOH
(100 mL). The mixture was stirred at room temperature for 16 h and then
concentratedin vacuo. The resulting crude productwas purified by column
chromatography on silica gel (20% EtOAc in hexanes) to give 8a (1.1 g,
25%) as a white solid. Mp 66ꢀ68 °C; 1H NMR δ 7.47 (td, J = 1.6 and 7.6
Hz, 1H), 7.32ꢀ7.29 (m, 4H), 7.18ꢀ7.15 (m, 3H), 7.02 (td, J = 1.6 and 7.6
Hz), 4.49 (q, J = 7.2 Hz, 2H), 2.35 (s, 3H), 1.43 (t, J = 6.8 Hz, 3H).
Ethyl 1-(2-Chlorophenyl)-4-methyl-5-phenyl-1H-pyrazole-
3-carboxylate (8b). The procedure described for the synthesis of 8a
’ EXPERIMENTAL SECTION
Materials. All reagents and solvents (ACS or HPLC grade) were
purchased from commercial sources and used as supplied. 1-(2-
Chlorophenyl)-N-methyl-N-sec-butylisoquinoline-3-carboxamide (PK
11195, 10) was purchased from Tocris Biosciences. Chloro[-
(phenyl)hydrazono]ethyl acetates 6aꢀd were synthesized according
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Journal of Medicinal Chemistry
ARTICLE
was applied to 6b and 7a to yield 8b (21%) as a solid. Mp 116ꢀ118 °C; 1H
NMR δ 7.42 (dd, J = 1.6 and 7.2 Hz, 1H), 7.36ꢀ7.26 (m, 6H), 7.17ꢀ7.14
(m, 2H), 4.49 (q, J = 7.2 Hz, 2H), 2.36 (s, 3H), 1.45 (t, 6.8 Hz, 3H).
Ethyl 1-(2-Bromophenyl)-4-methyl-5-phenyl-1H-pyrazole-
3-carboxylate (8c). The procedure described for the synthesis of 8a
was applied to 6c and 7a to yield 8c (23%) as a solid. Mp 116ꢀ118 °C; 1H
NMR δ7.54 (dd, J = 1.6 and 7.2 Hz, 1H), 7.39 (dd, J= 1.6 and 7.2 Hz, 1H),
7.33ꢀ7.16 (m, 7H), 4.49 (q, J = 7.2 Hz, 2H), 2.37 (s, 3H), 1.45 (t, J = 6.8,
3H).
Ethyl 1-(3-Bromophenyl)-4-methyl-5-phenyl-1H-pyrazole-
3-carboxylate (8d). The procedure described for the synthesis of 8a
was applied to 6f and 7a to yield 8d (15%) as a semisolid. 1H NMR δ 7.56
(t, J = 2.0 Hz, 1H), 7.45ꢀ7.39 (m, 4H), 7.17ꢀ7.00 (m, 4H), 4.49 (q, J =
7.2 Hz, 2H), 2.39 (s, 3H), 1.43 (t, J = 6.8, 3H).
Ethyl 1-(2-Bromophenyl)-5-(4-fluorophenyl)-4-methyl-
1H-pyrazole-3-carboxylate (8m). The procedure described for
the synthesis of 8a was applied to 6c and 7d to yield 8m (20%) as a white
solid. Mp 109ꢀ110 °C; 1H NMR δ 7.56 (d, J = 7.6 Hz, 1H), 7.39ꢀ7.32
(m, 2H), 7.25ꢀ7.23 (m, 1H), 7.17 (dd, J = 5.6 and 3.2 Hz, 2H), 7.01 (t, J
= 8.4 Hz, 2H), 4.49 (q, J = 7.2 Hz, 2H), 2.34 (s, 3H), 1.45 (t, J = 6.8 Hz,
3H).
Ethyl 1-(2-Bromophenyl)-5-(3-fluorophenyl)-4-methyl-
1H-pyrazole-3-carboxylate (8n). The procedure described for
the synthesis of 8a was applied to 6c and 7e to yield 8n (19%) as a
1
white solid. Mp 106ꢀ108 °C; H NMR δ 7.57 (d, J = 76. Hz, 1H),
7.40ꢀ7.32 (m, 2H), 7.29ꢀ7.24 (m, 2H), 7.02 (t, J = 6.4 Hz, 1H), 6.96
(d, J = 8.0 Hz, 1H), 6.90 (d, J = 9.6 Hz, 1H), 4.49 (q, J = 7.2 Hz, 2H), 2.34
(s, 3H), 1.45 (t, J = 6.8 Hz, 3H).
Ethyl 1-(4-Bromophenyl)-4-methyl-5-phenyl-1H-pyrazole-
3-carboxylate (8e). The procedure described for the synthesis of 8a
was applied to 6g and 7a to yield 8e (25%) as a solid. Mp 76ꢀ78 °C; 1H
NMR δ 7.42ꢀ7.37 (m, 5H), 7.16ꢀ7.17 (m, 4H), 4.49 (q, J = 7.2 Hz, 2H),
2.39 (s, 3H), 1.43 (t, J = 6.8, 3H).
Ethyl 1-(2-Iodophenyl)-4-methyl-5-phenyl-1H-pyrazole-
3-carboxylate (8f). The procedure described for the synthesis of 8a
was applied to 6d and 7a to yield 8f (22%) as a solid. Mp 122ꢀ124 °C;
1H NMR δ 7.80 (d, J = 8.0 Hz, 1H), 7.34ꢀ7.27 (m, 5H), 7.20ꢀ7.18 (m,
2H), 7.08ꢀ7.04 (m, 1H), 4.49 (q, J = 7.2 Hz, 2H), 2.37 (s, 3H), 1.45 (t, J
= 6.8 Hz, 3H).
Ethyl 1-(2-Bromophenyl)-4-cyano-5-phenyl-1H-pyrazole-
3-carboxylate (8p). A solution of 6c (10.5 g, 34.4 mmol), benzoy-
lacetonitrile (7h, 5.0 g, 34.4 mmol), and DIPEA (13.3 g, 103.2 mmol)
was refluxed in MeCN (100 mL) for 16 h. The reaction mixture was then
concentrated in vacuo and the crude product extracted with ether (3 ꢁ
100 mL). The ethereal extracts were combined and concentrated in
vacuo. The crude was purified with flash chromatography
(hexanesꢀEtOAc, 7:3 v/v) to give 8p (1.1 g, 8%) as a pale white solid.
Mp 106ꢀ108 °C; 1H NMR δ 7.63 (d, J = 8.0 Hz, 1H), 7.46ꢀ7.35 (m,
8H), 4.56 (q, J = 7.2 Hz, 2H), 1.49 (t, J = 6.8 Hz, 3H).
1-(2-Bromophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
5-phenyl-1H-pyrazole-3-carboxamide (9a). An aqueous solution
(6 mL) of LiOH (819 mg, 34.2 mmol) was added to a stirred solution of
8c (500 mg, 966 μmol) in THF (60 mL) and heated to reflux for 6 h. The
mixture was cooled to room temperature, and volatile compounds were
removed in vacuo. The residue was dissolved in water (50 mL) and
neutralized with aqueous HCl. The resulting precipitate was filtered off
anddried invacuo, yielding crudeacid(242 mg, 52%). DMF (1 drop) and
oxalyl chloride (66 μL, 777 μmol) were added to a stirred solution of the
acid (200 mg, 518 μmol) in dry dichloromethane (DCM, 10 mL). After
bubbling had ceased, the mixture was concentrated in vacuo. A solution of
4-aminotetrahydro-2H-pyran-4-carbonitrile (101 mg, 673 μmol) and
DIPEA (90 μL, 777 μmol) in DCM (10 mL) was added to the acid
chloride and stirred at room temperature for 2 h. The volatile compounds
were removed in vacuo and the crude residue was purified with silica gel
chromatography (hexanesꢀEtOAc, 60:40 v/v) to yield 9a (58% from
acid) as a white solid. Mp 226ꢀ228 °C; 1H NMR δ 7.68 (d, J = 8.0 Hz,
1H), 7.44ꢀ7.39 (m, 2H), 7.39ꢀ7.35 (m, 1H), 7.35ꢀ7.25 (m, 3H), 7.12
(s, 1H), 7.06 (s, 1H), 4.01 (dt, J = 3.2 and 12.8 Hz, 2H), 3.88 (td, J = 2.0
and12.4Hz, 2H), 2.57 (d, J = 12.8Hz, 2H), 2.11 (td, J = 4.4 and 10.0 Hz);
HRMS (tof) [M þ Na]þ, calcd for C22H1979BrN4NaO2, 473.0584;
found, 473.0562; error, 4.5 ppm. Anal. Calcd for C22H19BrN4O2: C 58.5,
H 4.2, N 12.4. Found: C 58.4, H 4.3, N 12.3.
Ethyl 1-(2-Phenylphenyl)-4-methyl-5-phenyl-1H-pyrazole-
3-carboxylate (8g). The procedure described for the synthesis of 8a
was applied to 6e and 7a to yield 8g (17%) as a semisolid. 1H NMR δ 7.73
(d, J = 7.2 Hz, 1H), 7.21 (dd, J = 1.6 and 8.0 Hz, 1H), 7.17ꢀ7.12 (m, 2H),
7.08 (p, J = 8.4 Hz, 4H), 6.51 (d, J = 7.6 Hz, 2H), 6.42 (d, J = 7.6 Hz, 2H),
4.45 (q, J = 7.2 Hz, 2H), 2.17 (s, 3H), 1.48 (t, J = 7.2 Hz).
Ethyl 1-(2-Bromophenyl)-5-phenyl-1H-pyrazole-3-carboxy-
late (8h). The procedure described for the synthesis of 8a was applied to
6c and 7b to yield 8h (24%) as a solid. Mp 90ꢀ92 °C;1HNMRδ7.62(d, J
= 8.4 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.34ꢀ7.20
(m, 6H), 7.10 (s, 1H), 4.49 (q, J = 7.2 Hz, 2 Hz), 1.45 (t, J = 6.8 Hz).
Ethyl 1-(2-Fluorophenyl)-5-(4-methoxyphenyl)-4-methyl-
1H-pyrazole-3-carboxylate (8i). The procedure described for the
synthesis of 8a was applied to 6a and 7c to yield 8i (19%) as a white solid.
Mp 86ꢀ88 °C; 1H NMR δ 7.46 (td, J = 1.6 and 7.6 Hz, 1H), 7.35ꢀ7.29
(m, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.09 (d, J = 11.2 Hz, 2H), 7.04 (t, J = 9.2
Hz), 6.85 (d, J = 11.2 Hz, 2H), 4.48 (q, J = 7.2 Hz, 2H), 3.79 (s, 3H), 2.33
(s, 3H), 1.45 (t, J = 6.8 Hz, 3H).
Ethyl 1-(3-Bromophenyl)-5-(4-methoxyphenyl)-4-methyl-
pyrazole-3-carboxylate (8j). The procedure described for the synth-
esis of 8a was applied to 6f and 7g to yield 8j (12%) as a semisolid. 1H
NMR δ 7.57 (t, J = 1.9 Hz, 1H) 7.46ꢀ7.44 (m, 1H), 7.22ꢀ7.11 (m, 3H),
7.06ꢀ7.04 (m, 2H), 6.93 (d, J = 8.8), 4.47 (q, J = 7.2 Hz, 2H), 3.84 (s, 3H),
2.29 (s, 3H), 1.45 (t, J = 6.8 Hz, 3H).
N-(4-Cyanotetrahydro-2H-pyran-4-yl)-1-(2-fluorophenyl)-
4-methyl-5-phenyl-1H-pyrazole-3-carboxamide (9b). The
procedure described for the synthesis of 9a was applied to 8a and
4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9b (68% from acid)
Ethyl 1-(3-Bromophenyl)-5-(4-methoxyphenyl)-1H-pyrazole-
3-carboxylate (8k). The procedure described for the synthesis of 8a was
applied to 6f and 7c to yield 8k (26%) as a solid. Mp 64ꢀ68 °C; 1H NMR δ
7.61 (d, J= 8.4 Hz, 1H), 7.48 (d, J=8.0Hz,1H),7.42(t,J= 7.6 Hz, 1H), 7.33
(t, J = 7.6 Hz, 1H), 7.14 (d, J = 8.0 Hz, 2H), 7.03 (s, 1H), 6.80 (d, J = 7.2 Hz,
2H), 4.49 (q, J = 7.2 Hz, 2H), 3.78 (s, 3H), 1.45 (t, J = 6.8 Hz, 3H).
Ethyl 4-Bromo-1-(3-bromophenyl)-5-(4-methoxyphenyl)-
1H-pyrazole-3-carboxylate (8l). NBS (334 mg, 1.9 mmol) was
added to a stirred solution of 8k (500 mg, 1.2 mmol) in MeCN (25 mL).
The reaction mixture was heated to reflux for 8 h and then concentrated in
vacuo. The crude was purified with flash chromatography (hexanesꢀEtOAc,
8:2 v/v) to give 8l (543 mg, 91%). Mp 118ꢀ120 °C; 1H NMR δ 7.61 (t, J =
2.0 Hz, 1H), 7.46 (dd, J = 6.8 Hz, 1H), 7.21 (d, J = 9.3 Hz, 2H), 7.16 (t, J =
8.4 Hz, 1H), 7.06 (d, J= 7.6 Hz, 1H), 6.93 (d, J= 8.4 Hz, 2H), 4.50 (q, J=7.2
Hz, 2H), 3.84 (s, 3H), 1.47 (t, J = 6.8 Hz, 3H).
1
as a white solid. Mp 212ꢀ214 °C; H NMR δ 7.40ꢀ7.32 (m, 5H),
7.21ꢀ7.05 (m, 5H), 4.00 (dt, J = 3.2 and 12.8 Hz, 2H), 3.87 (td, J = 2.0
and 12.4 Hz, 2H), 2.56 (d, J = 12.8 Hz, 2H), 2.40 (s, 3H), 2.07ꢀ2.04 (m,
2H); HRMS (tof) [M þ H]þ, calcd for C23H22FN4O2, 405.1721; found,
3
405.1711; error, 2.5 ppm. Anal. Calcd for C23H21FN4O2 0.3 H2O: C
67.4, H 5.3, N 13.7. Found: C 67.4, H 5.3, N 13.7.
1-(2-Chlorophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
4-methyl-5-phenyl-1H-pyrazole-3-carboxamide (9c). The pro-
cedure described for the synthesis of 9a was applied to 8b and
4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9c (56% from acid)
1
as a white solid. Mp 208ꢀ210 °C; H NMR δ 7.43ꢀ7.28 (m, 7H),
7.15ꢀ7.12 (m, 2H), 7.10 (s, 1H), 4.01 (dt, J = 3.2 and 12.8 Hz, 2H), 3.86
(td, J = 2.0 and 12.4 Hz, 2H), 2.55 (d, J = 12.8 Hz, 2H), 2.40 (s, 3H),
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2.06ꢀ2.04 (m, 2H); HRMS (tof) [M
þ
Na]þ, calcd for
C23H21ClN4NaO2, 443.1245; found, 443.1231; error, 3.2 ppm. Anal.
Calcd for C23H21ClN4O2: C 65.6, H 5.0, N 13.3. Found: C 65.7, H 5.0,
N 13.0.
Hz, 2H), 2.36 (s, 3H), 2.08ꢀ2.04 (m, 2H); HRMS (tof) [M þ Na]þ,
calcd for C23H2179BrN4NaO2, 487.0740; found, 487.0721; error, 3.9
ppm. Anal. Calcd for C23H21BrN4O2: C 59.4, H 4.5, N 12.0. Found: C
59.2, H 4.6, N 11.9.
1-(2-Chlorophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
N,4-dimethyl-5-phenyl-1H-pyrazole-3-carboxamide (9d).
NaH (60% in dispersion) (17 mg, 428 μmol) was added to a stirred
solution of 9c (90 mg, 223 μmol) and methyl iodide (61 mg, 428
μmol) in DMF (5 mL). The mixture was stirred for 1 h and then con-
centrated in vacuo. The crude was purified with flash chromatography
(hexanesꢀEtOAc, 8:2 v/v) to give 9d (62 mg, 64%) as a pale white
solid. Mp 182ꢀ184 °C; 1H NMR δ 7.45 (d, J = 9.2 Hz, 1H), 7.35ꢀ7.20
(m, 6H), 7.18ꢀ7.14 (m, 2H), 4.09 (dd, J = 2.8 and 12.8 Hz, 2H), 3.45
(s, 3H), 2.58 (d, J = 2.8 Hz, 2H), 2.28 (s, 3H), 2.17 (td, J = 4.0 and 12.0
Hz, 2H); HRMS (tof) [M þ Na]þ, calcd for C24H23ClN4NaO2,
457.1402; found, 457.1386; error, 3.4 ppm. Anal. Calcd for
C24H23ClN4O2: C 66.3, H 5.3, N 12.9. Found: C 65.9, H 5.3, N 12.6.
1-(2-Bromophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
4-methyl-5-phenyl-1H-pyrazole-3-carboxamide (9e). The pro-
cedure described for the synthesis of 9a was applied to 8c and
4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9e (58% from acid)
N-(4-Cyanotetrahydro-2H-pyran-4-yl)-1-(2-iodophenyl)-
4-methyl-5-phenyl-1H-pyrazole-3-carboxamide (9j). The
procedure described for the synthesis of 9a was applied to 8f and
4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9j (61% from
1
acid) as a solid. Mp 200ꢀ202 °C; H NMR δ 7.88 (dd, J = 1.2 and
7.6 Hz, 1H), 7.39 (td, J = 1.2 and 7.6 Hz, 1H), 7.31ꢀ7.29 (m, 3H), 7.27
(dd, J = 1.6 and 7.6, 1H), 7.18ꢀ7.16 (m, 2H), 7.14ꢀ7.09 (m, 2H), 4.01
(dt, J = 3.2 and 12.8 Hz, 2H), 3.87 (td, J = 2.0 and 12.4 Hz, 2H), 2.56 (d,
J = 12.8 Hz, 2H), 2.41 (s, 3H), 2.08ꢀ2.04 (m, 2H); HRMS (tof) [M þ
H]þ, calcd for C23H22IN4O2, 513.0728; found, 513.0780; error, 0.5
ppm. Anal. Calcd for C23H21IN4O2: C 53.9, H 4.1, N 10.9. Found: C
54.0, H 4.1, N 10.8.
N-(4-Cyanotetrahydro-2H-pyran-4-yl)-1-(2-phenylphenyl)-
4-methyl-5-phenyl-1H-pyrazole-3-carboxamide (9k). The pro-
cedure described for the synthesis of 9a was applied to 8g and 4-aminote-
trahydro-2H-pyran-4-carbonitrile to yield 9k (44% from acid) as a white
solid. Mp 98ꢀ100 °C; 1H NMR δ 7.62 (d, J = 7.6, 1H), 7.51ꢀ7.47 (m,
2H), 7.29ꢀ7.02 (m, 8H), 6.51 (d, J = 7.6 Hz, 2H), 6.47 (d, J = 7.6 Hz,
2H), 4.01 (dt, J = 3.2 and 12.8 Hz, 2H), 3.87 (td, J = 2.0 and 12.4 Hz, 2H),
2.56 (d, J = 12.8 Hz, 2H), 2.41 (s, 3H), 2.08ꢀ2.04 (m, 2H); HRMS (tof)
[M þ Na]þ, calcd for C29H26N4NaO2, 463.2129; found, 463.2107; error,
4.7 ppm. Anal. Calcd for C29H26N4O2: C 75.3, H 5.7, N 12.1. Found: C
74.9, H 5.8, N 11.7.
1
as a white solid. Mp 188ꢀ190 °C; H NMR δ 7.62 (d, J = 8.0, 1H),
7.36ꢀ7.28 (m, 6H), 7.18ꢀ7.14 (m, 2H), 7.10 (s, 1H), 4.01 (td, J = 2.0
and 12.4 Hz, 2H), 3.87 (td, J = 2.0 and 12.4 Hz, 2H), 2.56 (d, J = 12.8 Hz,
2H), 2.41 (s, 3H), 2.07ꢀ2.04 (m, 2H); HRMS (tof) [M þ H]þ, calcd for
C23H2279BrN4O2, 465.0921; found, 465.0908; error, 2.8 ppm. Anal.
Calcd for C23H21BrN4O2: C 59.4, H 4.5, N 12.0. Found: C 59.5, H
4.6, N 11.7.
N-(4-Cyanotetrahydro-2H-pyran-4-yl)-1-(2-fluorophenyl)-
5-(4-methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxamide
(9l). The procedure described for the synthesis of 9a was applied to 8i
and 4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9l (59% from
acid) as a white solid. Mp 210ꢀ212 °C; 1H NMR δ 7.41ꢀ7.34 (m, 2H),
7.22(t, J = 7.2Hz, 1H), 7.10ꢀ7.05 (m, 4H), 6.85 (d, J = 8.4Hz, 2H), 4.01
(dt, J = 3.2 and 12.8 Hz, 2H), 3.88 (td, J = 2.0 and 12.4 Hz, 2H), 3.79 (s,
3H), 2.57 (d, J = 12.8 Hz, 2H), 2.35 (s, 3H), 2.11ꢀ2.04 (m, 2H); HRMS
(tof) [M þ Na]þ calcd for C24H23FN4NaO3, 457.1646; found,
1-(2-Bromophenyl)-N-(1-cyanocyclohexyl)-4-methyl-5-phen-
yl-1H-pyrazole-3-carboxamide (9f). The procedure described for the
synthesis of 9a was applied to 8c and 1-aminocyclohexanecarbonitrile
HCl to yield 9f (62% from acid) as a white solid. Mp 212ꢀ214 °C; 1H
NMR δ 7.61 (d, J = 8.0Hz, 7.37ꢀ7.25 (m, 6H), 7.16ꢀ7.14 (m, 2H), 7.04
(s, 1H), 2.51 (bs, 2H), 2.41 (s, 3H), 1.78ꢀ1.69 (m, 7H), 1.32 (bs, 1H).
HRMS (tof) [M þ H]þ, calcd for C24H2379BrN4NaO, 485.0953; found,
485.0947; error, 3.8 ppm. Anal. Calcd for C24H23BrN4O: C 62.2, H 5.0,
N 12.1. Found: C 62.1, H 5.2, N 12.3.
457.1831; error, 3.4 ppm. Anal. Calcd for C24H23FN4O3 0.3H2O: C
3
65.5, H 5.4, N 12.7. Found: C 65.5, H 5.4, N 12.7.
1-(2-Bromophenyl)-4-cyano-N-(4-cyanotetrahydro-2H-pyran-
4-yl)-5-phenyl-1H-pyrazole-3-carboxamide (9g). The procedure
described for the synthesis of 9a was applied to 8p and 4-aminotetrahydro-
2H-pyran-4-carbonitrile to yield 9g (71% from acid) as a solid. Mp
224ꢀ226 °C; 1H NMR δ 7.69 (d, J = 8.4 Hz, 1H), 7.49ꢀ7.32 (m, 8H),
6.95 (s, 1H), 4.02 (dt, J = 3.2 and 12.8 Hz, 2H), 3.86 (td, J = 2.0 and 12.4 Hz,
2H),2.58(d,J= 12.8 Hz, 2H), 2.09ꢀ2.04(m, 2H);HRMS(tof) [Mþ H]þ,
calcd for C23H1979BrN5O2, 476.0717; found, 476.0700; error, 3.6 ppm; Anal.
for C23H18BrN5O2: C 58.0, H 3.8, N 14.7. Found: C 57.6, H 3.9, N 14.5.
1-(3-Bromophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
4-methyl-5-phenyl-1H-pyrazole-3-carboxamide (9h). Thepro-
cedure described for the synthesis of 9a was applied to 8d and
4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9h (53% from acid)
as a white solid. Mp 138ꢀ140 °C; 1H NMR δ 7.56 (t, J = 2 Hz, 1H), 7.45
(m, 4H), 7.17ꢀ7.12 (m, 3H), 7.10 (s, 1H), 7.00 (d, J = 7.2 Hz, 1H), 4.02
(dt, J = 3.2 and 12.8 Hz, 2H), 3.88 (td, J = 2.0 and 12.4 Hz, 2H), 2.59 (d, J
= 12.8 Hz, 2H), 2.36 (s, 3H), 2.10ꢀ2.04 (m, 2H); HRMS (tof) [M þ
Na]þ, calcd for C23H2179BrN4NaO2, 487.0740; found, 487.0726; error,
2.8 ppm. Anal. Calcd for C23H21BrN4O2: C 59.4, H 4.5, N, 12.0. Found:
C 59.3, H 4.5, N 12.0.
1-(2-Bromophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
5-(4-methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxamide
(9m). The procedure described for the synthesis of 9a was applied to 8o
and 4-aminotetrahydro-2H-pyran-4-carbonitrile to give 9m (87 mg, 34%
from acid) as a white solid. Mp 208ꢀ210 °C; 1H NMR δ 7.62 (dd, J = 6.7
and 1.0 Hz, 1H), 7.37ꢀ7.23 (m, 3H), 7.09 (s, 1H), 7.08 (dt, J = 8.8 and
1.6 Hz, 2H), 6.82 (dt, J = 8.8 and 1.6 Hz, 2H), 3.99 (d, J = 8.1 Hz, 2H),
3.85 (t, J = 10.6 Hz, 2H), 3.77 (s, 3H), 2.54 (d, J = 13.4 Hz, 2H), 2.38 (s,
3H), 2.06 (t, J = 10.0 Hz, 3H); 13C NMR δ 162.41, 159.70, 144.30,
143.16, 133.60, 131.07, 130.81, 130.02, 128.08, 122.10, 120.88, 119.03,
113.89, 63.81, 55.21, 49.15, 35.65, 9.48; HRMS (m/z) [M þ H]þ, calcd
for C24H2479Br N4O3, 495.1032; found, 495.1029; error, ꢀ0.6 ppm.
Anal. Calcd for C24H23BrN4O3: C 58.2, H 4.7, N 11.3. Found: C 58.2, N
4.8, H 11.2.
1-(2-Bromophenyl)-N-(1-cyanocyclohexyl)-5-(4-methoxy-
phenyl)-4-methyl-1H-pyrazole-3-carboxamide (9n). The pro-
cedure described for the synthesis of 9a was applied to 8o to afford 9n
(36% from acid) as a white solid. Mp 220ꢀ222 °C; 1H NMR δ 7.61 (dd, J
= 6.7 and 1.0 Hz, 1H), 7.35ꢀ7.24 (m, 3H), 7.07 (dt, J = 8.8 and 1.6 Hz,
2H), 7.01 (s, 1H), 3.77 (s. 3H), 2.51 (bs, 2H), 2.39 (s, 3H), 1.77ꢀ1.71
(m, 8H), 1.15 (bs, 1H); 13C NMR δ 162.22, 158.67, 144.12, 143.49,
138.98, 133.56, 132.84, 131.06, 130.70, 130.06, 128.05, 122.14, 119.90,
117.99,, 114.56, 68.74, 51.39, 35.64, 24.84, 22.15, 9.55; HRMS (m/z) [M
þ H]þ, calcd for C25H2679Br N4O2, 493.1239; found, 493.1235; error,
ꢀ0.8ppm. Anal. Calcd forC25H25BrN4O2: C 60.9, H 5.1, N 11.4. Found:
C 60.8, H 5.4, N 11.2.
1-(4-Bromophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
4-methyl-5-phenyl-1H-pyrazole-3-carboxamide (9i). The pro-
cedure described for the synthesis of 9a was applied to 8e and 4-aminote-
trahydro-2H-pyran-4-carbonitrile to yield 9i (34% from acid) as a solid.
Mp176ꢀ178 °C;1H NMRδ 7.46(dt, J =2.4 and9.2 Hz, 2H), 7.41ꢀ7.38
(m, 3H), 7.16ꢀ7.13 (m,3H), 7.11 (dt, J = 2.4 and 9.2 Hz, 2H), 4.02 (dt, J
= 3.2 and 12.8Hz, 2H), 3.88 (td, J = 2.0 and 12.4Hz, 2H), 2.57 (d, J = 12.8
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Journal of Medicinal Chemistry
ARTICLE
1-(3-Bromophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
5-(4-methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxamide
(9o). The procedure described for the synthesis of 9a was applied to 8j
and 4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9o (53% from
acid) as a white solid. Mp 160ꢀ162 °C; 1H NMR δ 7.58 (bs, 1H), 7.45
(d, J = 8.4 Hz, 1H), 7.15ꢀ7.11 (m, 2H), 7.09 (d, J = 8.8 Hz, 2H), 7.00 (d, J
= 8.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 2H), 4.01 (dt, J = 3.2 and 12.8 Hz, 2H),
3.88ꢀ3.83 (m, 5H), 3.79 (s, 3H), 2.57 (d, J = 12.8 Hz, 2H), 2.35 (s, 3H),
2.10ꢀ2.04 (m, 2H); HRMS (tof) [M þ H]þ, calcd for C24H2479BrN4O3,
495.1028; found, 495.1020; error, 1.4 ppm. Anal. Calcd for
C24H23BrN4O3: C 58.2, H 4.7, N 11.3. Found: C 58.2, H 4.8, N 11.2.
4-Cyano-N-(4-cyanotetrahydro-2H-pyran-4-yl)-1-(2-iodo-
phenyl)-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxamide
(9p). The procedure described for the synthesis of 9a was applied to 8q
and 4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9p (68% from
acid) as a white solid. Mp 232ꢀ214 °C; 1H NMR δ 7.95 (dd, J = 1.2 and
8.0 Hz, 1H), 7.52 (td, J = 1.2 and 7.2 Hz, 1H), 7.38 (dd, J = 1.6 and 7.6
Hz, 1H), 7.29ꢀ7.22 (m, 3H), 6.95 (s, 1H), 6.88 (dt, J = 2.8 and 9.2 Hz),
4.02 (dt, J = 3.2 and 12.8 Hz, 2H), 3.86 (td, J = 2.0 and 12.4 Hz, 2H), 2.58
(d, J = 12.8 Hz, 2H), 2.09ꢀ2.04 (m, 2H); HRMS (tof) [M þ Na]þ,
calcd for C24H20IN5NaO3, 576.0503; found, 576.0483; error, 3.5 ppm.
Anal. Calcd for C24H20IN5O3: C 52.1, H 3.6, N 12.7. Found: C 52.0, H
3.7, N 12.7.
4-Bromo-1-(3-bromophenyl)-N-(4-cyanotetrahydro-2H-pyr-
an-4-yl)-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxamide (9q).
The procedure described for the synthesis of 9a was applied to 8l and
4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9q (56% from acid) as a
solid. Mp 182ꢀ184 °C; 1H NMR δ 7.58 (t, J = 1.6 Hz, 1H), 7.50 (d, J = 8.0
Hz, 1H), 7.21ꢀ7.15(m,3H),7.07(s,1H),7.02(d,J= 8.0 Hz, 1H), 6.94 (dt, J
= 2.8 and 8.8 Hz, 2H), 4.01 (dt, J= 3.2 and 12.8 Hz, 2H), 3.88ꢀ3.83 (m, 5H),
3.79 (s, 3H), 2.57 (d, J = 12.8 Hz, 2H), 2.35 (s, 3H), 2.10ꢀ2.04 (m, 2H);
HRMS (tof) [M þ Na]þ, calcd for C23H2079Br2N4NaO3, 580.9794; found,
580.9774; error, 3.5 ppm. Anal. Calcd for C23H20Br2N4O3: C 49.3, H 3.6, N
10.0. Found: C 49.2, H 3.6, N 9.8.
1-(2-Bromophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
5-(4-fluorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (9r).
The procedure described for the synthesis of 9a was applied to 8m and
4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9r (54%) as asolid. Mp
188ꢀ190 °C; 1H NMR δ 7.62 (d, J = 6.4 Hz, 1H), 7.40ꢀ7.28 (m, 3H),
7.15ꢀ7.11 (m, 2H), 7.09 (s, 1H), 7.02 (t, J = 9.6 Hz, 2H), 4.01 (dt, J = 3.2
and 12.8 Hz, 2H), 3.88ꢀ3.83 (m, 5H), 3.79 (s, 3H), 2.56 (d, J = 12.8 Hz,
2H), 2.38 (s, 3H), 2.07ꢀ2.00 (m, 2H); HRMS (tof) [M þ H]þ, calcd for
C23H2179BrFN4O2, 483.0826; found, 483.0821; error, 1.2 ppm. Anal. Calcd
for C23H20BrFN4O2) C 57.2, H 4.2, N 11.6. Found: C 57.2, H 4.2, N 11.4.
1-(2-Bromophenyl)-N-(4-cyanotetrahydro-2H-pyran-4-yl)-
5-(3-fluorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (9s).
The procedure described for the synthesis of 9a was applied to 8n and
4-aminotetrahydro-2H-pyran-4-carbonitrile to yield 9s (62%fromacid) asa
solid. Mp 204ꢀ206 °C; 1HNMRδ7.64 (d, J= 8.0 Hz, 1H), 7.41ꢀ7.25 (m,
4H), 7.09 (s, 1H), 7.04 (td, J= 2.4 and 8.4 Hz, 1H), 6.95 (d, J= 8.0 Hz, 1H),
6.88 (dt, J = 2.0 and 9.6 Hz, 1H), 4.01 (dt, J = 3.2 and 12.8 Hz, 2H),
3.88ꢀ3.83 (m, 5H), 3.79 (s, 3H), 2.56 (d, J = 12.8 Hz, 2H), 2.38 (s, 3H),
2.07ꢀ2.00 (m, 2H); HRMS (tof) [M þ Na]þ, calcd for C23H2079BrFNa-
N4O2, 505.0646; found, 505.0623; error, 4.6 ppm. Anal. Calcd for
C23H20BrFN4O2: C 57.2, H 4.2, N 11.6. Found: C 57.3, H 4.3, N 11.6.
Computation of cLogD. The lipophilicity of each synthesized
ligand, expressed as the partition of the compound between n-octanol
and buffer at pH 7.4 (cLogD), was computed with Pallas 3.70 software
(CompuDrug, South San Francisco, CA).
Receptor Screening. Ligands 9m and 9n were screened for
binding to a wide range of receptors and transporters by National
Institute of Mental Health Psychoactive Drug Screening Program.
Detailed protocols are available online for all binding assays at NIMH-
PDSP Web site (http://pdsp.med.unc.edu).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 301 594 5986. Fax: 301 480 5112. E-mail: pikev@mail.
nih.gov.
’ ACKNOWLEDGMENT
V.W.P. and S.R.D. were supported by the Intramural Research
Program of NIH (NIMH) (Project No. Z01-MH-002795);
specifically S.R.D. was initially supported by the National In-
stitute of Mental Health (NIMH) through a studentship to S.R.
D. under the NIH-Karolinska Institutet Graduate Partnership in
Neuroscience. S.R.D. was subsequently supported by Hoffmann-
La Roche and The Johns Hopkins PET Center through a
postdoctoral fellowship. We are grateful to the NIMH Psychoac-
tive Drug Screening Program (PDSP) for pharmacological assays
and screens. The PDSP is directed by Bryan L. Roth, Ph.D., with
project officer Jamie Driscol (NIMH), at the University of North
Carolina at Chapel Hill (Contract No. NO1MH32004).
’ ABBREVIATIONS USED
CB1, cannabinoid type 1; CB2, cannabinoid type 2; D, dopamine;
DAT, dopamine transporter; DCM, dichloromethane; DIPEA,
diisopropylethylamine; DMF, dimethylformamide; DOR, δ opi-
ate receptor; GABAA-Bz, γ-aminobutyric acidA-benzodiazepine;
GPCR, G-protein-coupled receptor; H, histamine; 5-HT, 5-hy-
droxytryptamine; KOR, κ opiate receptor; MeCN, acetonitrile;
MOR, μ opiate receptor; NBS, N-bromosuccinimide; NCA, no
carrier added; NET, noradrenaline transoporter; PET, positron
emission tomography; PBR, peripheral benzodiazepine receptor;
SERT, serotonin transporter; THF, tetrahydrofuran; Δ9-THC,
Δ9-tetrahydrocannabinol; TSPO, 18 kDa translocator protein
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