Discovery and optimization of 1-(4-(Pyridin-2-yl)benzyl)imidazolidine-2,4- dione derivatives as a novel class of selective cannabinoid CB2 receptor agonists

Article abstract of DOI:10.1021/jm200916p

Here, we report the identification and optimization of 1-(4-(pyridin-2-yl) benzyl)imidazolidine-2,4-dione derivatives as a novel chemotype with selective cannabinoid CB2 receptor agonist activity. 1 is a potent and selective cannabinoid CB2 receptor agonist (hCB2 pEC₅₀ = 8.6). The compound was found to be metabolically unstable, which resulted in low oral bioavailability in rat (F_po = 4%) and possessed off-target activity at the hERG ion channel (pK_i = 5.5). Systematic modification of physicochemical properties, such as lipophilicity and basicity, was used to optimize the pharmacokinetic profile and hERG affinity of this novel class of cannabinoid CB2 receptor agonists. This led to the identification of 44 as a potent, selective, and orally bioavailable cannabinoid CB2 receptor agonist (hCB2 pEC₅₀ = 8.0; hERG pK_i < 4; F_po = 100%), which was active in a rat spinal nerve ligation model of neuropathic pain.

Full text of DOI:10.1021/jm200916p

ARTICLE
pubs.acs.org/jmc
Discovery and Optimization of 1-(4-(Pyridin-2-yl)benzyl)-
imidazolidine-2,4-dione Derivatives As a Novel Class of Selective
Cannabinoid CB2 Receptor Agonists
Mario van der Stelt,*,†,# Jos Cals,† Silvia Broeders-Josten,† Jean Cottney,‡ Antoon A. van der Doelen,§
Marcel Hermkens,† Vera de Kimpe,† Angela King,|| Jan Klomp,^ Julia Oosterom,§ Ilse Pols-de Rooij,†
Jeroen de Roos,§ Martin van Tilborg,† Susan Boyce,‡ and James Baker‡
†Department of Medicinal Chemistry, Merck Research Laboratories, Oss, The Netherlands
‡Department of Pharmacology, Merck Research Laboratories, Newhouse, Scotland
§Department of Molecular Pharmacology & DMPK, Merck Research Laboratories, Oss, The Netherlands
Department of Molecular Pharmacology, Merck Research Laboratories, Newhouse, Scotland
^Department of Molecular Design & Informatics, Merck Research Laboratories, Oss,The Netherlands
ABSTRACT: Here, we report the identification and optimiza-
tion of 1-(4-(pyridin-2-yl)benzyl)imidazolidine-2,4-dione deri-
vatives as a novel chemotype with selective cannabinoid CB2
receptor agonist activity. 1 is a potent and selective cannabinoid
CB2 receptor agonist (hCB2 pEC50 = 8.6). The compound was
found to be metabolically unstable, which resulted in low oral
bioavailability in rat (Fpo = 4%) and possessed off-target activity
at the hERG ion channel (pKi = 5.5). Systematic modification of
physicochemical properties, such as lipophilicity and basicity, was used to optimize the pharmacokinetic profile and hERG affinity of
this novel class of cannabinoid CB2 receptor agonists. This led to the identification of 44 as a potent, selective, and orally bioavailable
cannabinoid CB2 receptor agonist (hCB2 pEC50 = 8.0; hERG pKi < 4; Fpo = 100%), which was active in a rat spinal nerve ligation
model of neuropathic pain.
’ INTRODUCTION
Marijuana has been used in the treatment of various kinds of
ailments for centuries.3 Emerging clinical evidence, as well as
anecdotal reports from patients self-medicating with cannabis,
suggest that cannabinoid receptor agonists may have a role in
treating pain.4,5 The antinociceptive properties of Δ9-tetra-
hydrocannabinol, the main psychoactive ingredient in cannabis,
have been well established in preclinical models, but its ther-
apeutic effect in man is limited by its undesirable psychotropic
activities and abuse potential.3,4 Δ9-Tetrahydrocannabinol acts
on two G-protein coupled receptors, namely the cannabinoid
CB1 and CB2 receptors.6 The cannabinoid CB1 receptor is
abundantly expressed in the central nervous system (CNS) and is
responsible for the psychotropic side effects.3,6,7 The cannabi-
noid CB2 receptor is mainly found in cells of the immune system
and is upregulated in the CNS under pathological conditions.8,9
For example, Anand et al. describe the expression of cannabinoid
CB2 receptor on human sensory neurons, which increased after
trauma. They demonstrated that the cannabinoid CB2 receptor
is colocalized with TRPV1 and suggest that CB2 receptor agonists
reduce Ca2+ influx by blockade of TRPV1 receptor activation (via
depletion of cAMP).10
Neuropathic pain, defined as chronic pain caused by injury,
disease, or dysfunction of the nervous system, is present in
∼1% of the population; the largest patient populations include
those with painful diabetic peripheral neuropathy and those
with neuralgia that persists after an attack of herpes zoster
(postherpetic neuralgia). It is characterized by a complex com-
bination of symptoms, including spontaneous pain that can occur
in the absence of tissue damage. Patients suffering from neuro-
pathic pain also have increased sensitivity both to stimuli
normally perceived as painful (hyperalgesia) as well as to stimuli
that do not normally provoke pain (allodynia). These symptoms
are often refractory to conventional analgesic therapies, with
most patients achieving incomplete relief of their symptoms.
Currently, antidepressants, anticonvulsants, and opioids remain
first-line treatment, with pregabalin as the gold standard. All of
these drugs have significant side effects that are dose limiting. In
addition, efficacy is a considerable problem in the neuropathic
pain market, with current treatments showing a maximum of 50%
reduction in overall pain scores from baseline. Consequently,
there remains an unmet medical need for agents that have higher
efficacy/responder rate and with reduced side-effects compared
with currently used drugs.1,2
Received: July 12, 2011
Published: September 18, 2011
r
2011 American Chemical Society
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The focus of drug discovery efforts have recently turned to the
cannabinoid CB2 receptor, because selective activation of this
receptor has been shown to elicit analgesic effects in animals
without the CNS-mediated side effects typical of a nonselective
CB receptor agonist such as THC.11ꢀ13 It has been shown that
CB2 receptor expression on monocytes, microglial, and astro-
cytic cells is of crucial importance to establish neuropathic pain in
a sciatic nerve injury model.14 In CB2 receptor KO mice,
allodynia and hyperalgesia were not restricted to the injured
limb, but mice also developed a contralateral mirror image pain
which was also associated with increased microglial and astro-
cytic activation; both the behavioral and histological changes
were attenuated in CB2 receptor overexpressing mice. The
neuropathic pain phenotype of CB2 receptor KO mice was also
manifested in irradiated wild-type mice reconstituted with bone
marrow cells from CB2 receptor KO mice, thereby demonstrat-
ing the implication of the CB2 receptor on immune cells in the
development of neuropathic pain in this model. Enhanced IFN-γ
signaling was present in CB2 receptor KO mice, but the double
KO mice (of CB2 and IFN-γ) did not show any longer the
enhanced manifestations of neuropathic pain observed in the
CB2 receptor KO mice.15
Besides evidence from genetically engineered animals, several
selective CB2 receptor agonists from different chemotypes have
also been reported to be effective in neuropathic pain models in
rodents (see Thakur et al. for a review).16,17 For example, HU-
308,18 AM1241,19 GW842166X,20 and JWH-13321 belong to the
first classes of selective cannabinoid CB2 receptor agonists that
have been used to study the action of the cannabinoid CB2
receptor in preclinical models of pain and other diseases, e.g.
osteoporosis, multiple sclerosis, melanoma, and neurodegenera-
tive diseases (ALS, Alzheimer’s, and Huntington’s diseases).8,9
Here, we report the identification and optimization of 1-
(4-(pyridin-2-yl)benzyl)imidazolidine-2,4-dione derivatives as a
novel chemotype with selective cannabinoid CB2 receptor
agonist activity. The initial hit 1 was found to be metabolically
unstable, which resulted in low oral bioavailability and possessed
off-target activity at the hERG ion channel. In this paper, we
describe the optimization of pharmacokinetic properties and
hERG affinity of this novel class of cannabinoid CB2 receptor
agonists by systematically modulating lipophilicity and basicity.
This led to the identification of 44 as a potent, selective, and
orally bioavailable cannabinoid CB2 receptor agonist, which was
active in a rat spinal nerve ligation model of neuropathic pain.
Table 1. Comparison of Rat Plasma PK for Compounds 1 and
44 a
1
44
CL (mL/min/kg)
Vss (L/kg)
100 ( 12
5.4 ( 1
0.8
8.1 ( 2.6
0.94 ( 0.08
1.7 ( 0.7
100 ( 19
3.8 ( 0.9
0.44
T1/2 (h)
F (%)
4 ( 2
0.02
oral Cmax (μM)
plasma fu
0.25
oral Cmax,u (μM)
0.006
1.7
a Compounds dosed at 1 mg/kg iv and 5 mg/kg po to fed male Wistar
rats (n = 4 per route). Both compounds were dosed orally in 0.5% (w/v)
gelatin in 5% (w/v) mannitol.
cardiotoxicity.22 To evaluate compounds for their inhibition of
hERG channels, a displacement assay was performed utilizing
hERG channels stably expressed in HEK293 cells and radioli-
gand [3H]-dofetilide, which is a class III antiarrhythmic drug with
high affinity for the hERG channel.22 1 was found to displace
[3H]-dofetilide with a pKi of 5.5.
1 has good Caco-2 permeability (210 nm/s), which was used
as an in vitro model of intestinal absorption. Low metabolic
stability was observed in human and rat liver microsomes (t1/2
=
24 and 21 min, CLint = 58 and 64 μL/min/mg, respectively),
which were used as in vitro models to estimated in vivo hepatic
clearance.
Rat pharmacokinetic studies were performed to test whether
sufficient exposure levels could be reached in the blood after oral
dosing to modulate cannabinoid CB2 receptor activity in vivo. It
was found that 1 had low oral bioavailability (4%) and a Cmax of
0.024 μM after oral dosing at 5 mg/kg in 0.5% gelatin in 5%
mannitol (Table 1). These low exposure levels after oral dosing
could be explained by high first pass extraction due to high
clearance (100 mL/min/kg).
We set out to systematically modulate the physicochemical
properties of the 1-(4-(pyridin-2-yl)benzyl)imidazolidine-2,4-
dione series to improve the ADME properties. A synthetic route
was designed which would allow the rapid and simultaneous
optimization at three variation points (i.e., R1, R2, and R3) in
the molecule. Our approach for the synthesis of the 1-(4-(pyridin-
2-yl)benzyl)imidazolidine-2,4-dione derivatives was first to pre-
pare strategic building blocks I and II (Scheme 1). Depending on
the type of substituent at R1, building block I can be prepared via
two methods, both starting from the commercially available
4-bromobenzaldehyde (Scheme 2). In method 1, 4-bromobenzal-
dehyde was converted via a reductive amination with glycinamide
hydrochloride to 2-(4-bromobenzylamino)acetamide 3. Under
basic conditions and in the presence of CDI, the resulting
intermediate was converted to 1-(4-bromobenzyl)-imidazoli-
dine-2,4-dione 4. The R1-group was introduced by alkylation of
4 with an appropriate alkylhalide in the presence of K2CO3, which
gave 5aꢀg in good yields. In case R1 = cyclopropyl, cyclobutyl,
isopropyl, or 2,2,2-trifluoro-ethyl, the substituent was introduced
according to method 2 because the corresponding alkyl halides did
not react properly. Glycine hydrochloride was used for the
reductive amination with 4-bromobenzaldehyde to form 2-(4-
bromobenzylamino)acetic acid 6. Amide coupling using PyBob
and the appropriate amine gave 7hꢀk. The corresponding
imidazolidine-2,4-dione derivatives 5hꢀk were obtained by a ring
closure reaction of 7hꢀk, as previously described.
’ RESULTS AND DISCUSSION
A focused target family library screen, with proprietary com-
pounds, followed by a small optimization campaign resulted in
the discovery of 1, which is a low molecular weight compound
(420 Da) with moderate lipophilicity (Log D = 2.8) and high
basicity (pKa = 8.9). 1 belongs to a novel chemical class of
1-(4-(pyridin-2-yl)benzyl)-imidazolidine-2,4-dione derivatives.
1 has a pEC50 = 8.6 for the cannabinoid CB2 receptor and was
more than 1000-fold selective over the cannabinoid CB1 recep-
tor in a functional assay using the inhibition of forskolin-induced
cAMP formation as a read out in CHO cells stably overexpressing
the human recombinant cannabinoid CB2 or CB1 receptors.
Because of its physicochemical properties, it was anticipated that
the compound may also have an affinity for the hERG ion
channel. Compounds that display a high affinity for hERG ion
channels are considered to have an increased probability of
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Scheme 1. Synthetic Strategy for Synthesis of 1-(4-(Pyridin-2-yl)benzyl)imidazolidine-2,4-dione Derivatives
Scheme 2. Synthesis of the 1-(4-Bromobenzyl)-imidazolidine-2,4-dione (Building Block I)a
a (a) Glycinamide or glycine hydrochloride, CH3OH/H2O, NaOH, NaBH4, 0 ꢀC or rt; (b) CDI, DMAP, CH3CN, 60 ꢀC; (c) alkylhalide, K2CO3, 50 ꢀC;
(d) alkylamine, PyBOP, Et3N, CH2Cl2, rt; (e) CDI,DMAP, CH3CN, 60 ꢀC
from 2.8 to 1.3 and led to an improved metabolic stability, i.e. the
half-life time increased from 22 to 90 min in rat liver microsomes.
This was a general trend because the metabolic half-life time of
otherbranchedalkyl 21orcycloalkyls22ꢀ24 analoguescorrelated
also with the lipophilicity (Figure 1a). Introducing fluorine at the
alkyl chain to block oxidation increased the metabolic stability as
observed with compounds 25 and 26, whereas fluorination of the
scaffold at the 5-pyridyl position 27ꢀ33 was detrimental to
metabolic stability. The most lipophilic compound 40 (log D =
4.7) was also the most labile compound with a half-life time of
3 min (CLint > 270 μL/min/mg).
Scheme 3. Synthesis of 2-Bromopyridine (Building Block
II)a
a (a) HOAc, NaBH(OAc)3,CH2Cl2, 10 ꢀC.
The potency of the compounds at the cannabinoid CB2
receptor was also dependent on the lipophilicity of the alkyl chain
(Table 2; Figure 1b). There was an inverse correlation with the
metabolic stability. Increasing the lipophilicity from 1.3 in com-
pound 20 to 2.2 increased the potency by 50-fold, giving a pEC50 =
8.9 for 18. Replacement of the hydrogen at R2 by a fluorine
increased potency in most cases (e.g., 27 vs 19; 30 vs 22; 33 vs 25).
The most potent compound identified in this series was 28 with a
pEC50 = 9.1. Interestingly, there was no apparent correlation of
lipophicity withcannabinoid CB1receptor activity. All compounds
are weak partial cannabinoid CB1 receptor agonists (Table 2).
However, lipophilicity did correlate with affinity for the hERG
channel. Molecules with smaller alkyl groups at R1 than an isobutyl
group did have lower affinity for hERG compared to 1 (Figure 2a).
Second, we investigated the influence of the basicity of the
amine substituent at the R3 position by introducing different
electron modulating groups in the piperidine ring 19ꢀ28, while
isobutyl was chosen as the R1 substituent. The cannabinoid CB2
receptor potency was not affected by the level of basicity
(Table 2). Both very basic compounds (i.e., 1 with a pKa = 8.9)
and weakly basic compounds (i.e., 42 with a pKa = 3.0) were very
Building blocks II 10aꢀk were prepared via reductive amina-
tion of 6-bromopicolinaldehyde 8a or 6-bromo-3-fluoropicoli-
naldehyde 8b with the appropriate amine (Scheme 3). For
preparation of the cyclohexyl derivative 15 (Scheme 4), 2,6-
dibromopyridine 11 was used as a starting point. After bromo/
lithium exchange of 11, it was reacted with cyclohexanecarboni-
trile to afford (6-bromopyridin-2-yl)(cyclohexyl)methanone 13.
Compound 13 was converted into the 4-methylbenzenesulfo-
nohydrazide 14, which was subsequently reduced to 15.
Final compounds were prepared by a Suzuki coupling of the two
building blocks 10 and 5using tetrakis(triphenylphosphine)palladium-
(0) as a catalyst (Scheme 5). It should be noted that building
blocks 5aꢀk were first converted to the corresponding boronic
esters and used directly in the Suzuki coupling without purifica-
tion because the boronic esters were unstable during SiO2
column chromatography. Final products 1,18ꢀ44 were obtained
in 3ꢀ80% yield.
First, the effect of reducing lipophilicity was investigated by
introducing smaller alkyl groups at R1. Replacing the isobutyl
group by a propyl 18, ethyl 19, or methyl 20 group decreased log D
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Scheme 4. Synthesis of 2-Bromo-6-cyclohexylmethyl-pyridinea
a (a) BuLi, hexane/THF, ꢀ78 ꢀC, H2SO4; (b) 4-methylbenzenesulfonyl hydrazide, EtOH, 100 ꢀC; (c) DiBAlH, CH2Cl2, rt.
Scheme 5. Synthesis of 1-(4-(Pyridin-2-yl)benzyl)imidazolidine-2,4-dione Derivativesa
a (a) Bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, DMF, 75 ꢀC; (b) K2CO3, Pd(PPh3)4, toluene/EtOH, 75 ꢀC.
Figure 1. Correlation of human liver microsomal half-life time (a) and potency on human cannabinoid CB2 receptors (b) with lipophilicity of
1-(4-(pyridin-2-yl)benzyl)imidazolidine-2,4-dione derivatives.
potent cannabinoid CB2 receptor agonists (pEC50 = 8.6 for
both). Moreover, 43, in which the nitrogen was replaced by a
carbon, did not lose potency (pEC50 = 7.7). This indicates that
the amine is not essential for the interaction with the cannabinoid
CB2 receptor. On the other hand, the hERG ion channel is well-
known for its preference of ligands with a basic amine group.23
This provided an opportunity to separate the biological activity
of this chemical series at the cannabinoid CB2 receptor from the
hERG channel. Indeed, we have found that lowering the basicity
of the amine at R3 reduced the affinity of the compounds at the
hERG channel (Figure 2b) while cannabinoid CB2 receptor
potency was maintained (Table 2). For example, 42 (pKa = 3)
had only marginal affinity (27% @ 0.1 mM) for the hERG
channel, while its potency at the cannabinoid CB2 receptor was
high (pEC50 = 8.6). Unfortunately, the metabolic stability of
these compounds was very low, which could be attributed to the
high lipophilicity (Table 2).
The next step was to combine the substituents at the R1, R2,
and R3 position. To avoid the elaborate synthesis of all possible
combinations, we have generated COMFA models for CB2
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Table 2. StructureꢀProperty Relationships of 1-(4-(Pyridin-2-yl)benzyl)imidazolidine-2,4-dione Derivatives
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Table 2 Continued
potency, metabolic stability, and hERG affinity to predict the
biological activity for these novel combinations. The generation
of these COMFA models, validation, and prediction of the
biological activity of ∼3000 in silico compounds will be de-
scribed by Klomp et al (in preparation). We have synthesized
only the compounds for which the biological activity was
potent cannabinoid CB2 receptor agonist (CB2 pEC50 = 8.0)
with more than 1000-fold selectivity over the cannabinoid CB1
receptor. It lacked any affinity for the hERG ion channel (ꢀ3% @
0.1 mM). The compound was metabolically stable (HLM/RLM
t1/2> 70 min, CLint < 20 μL/min/mg) and was highly permeable
in the Caco-2 model (Papp 173 nm/s). These favorable in vitro
ADME parameters resulted in excellent in vivo pharmacokinetic
properties as summarized in Table 1. 44 had a low clearance and
100% oral bioavailability. This compound was tested in the rat
spinal nerve ligation model of neuropathic pain.24 In this model,
mechanical allodynia is induced by tight ligation of the left L5
predicted to be within the predefined criteria (CB2 pEC50
>
7.5; RLM t1/2 > 20 min; hERG < 50% @ 10 μM). This resulted in
the identification of 44.
44 had good physicochemical properties, such as a MW = 473,
log D = 1.0, pKa = 2.8, and good solubility (89 mg/L). 44 was a
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Figure 2. Correlation of hERG channel affinity (as measured by % displacement of 3H-dofetilide with 10ꢀ4 M of test compound) with lipophilicity
(a) and basicity (b) of 1-(4-(pyridin-2-yl)benzyl)imidazolidine-2,4-dione derivatives.
cannabinoid CB2 receptor agonists is not due to residual
cannabinoid CB1 receptor activity.26,27 New series of imidazo-
pyridine and decahydroquinoline amides CB2 agonists, which
are highly selective, did not show any efficacy in a rat inflamma-
tory pain model, whereas closely related, albeit less selective,
analogues were active.26,27 44 is more than 1000-fold selective
over the cannabinoid CB1 receptor, but it does have some
residual cannabinoid CB1 receptor activity (30% @ 10 micro-
Molar). Additional pharmacological profiling of 44 and other
compounds from this novel chemical series will be described in
future papers.
Figure 3. The effect of acute oral administration of compound 44 on
’ CONCLUSION
neuropathy-induced mechanical allodynia in male wistar rats after
dosing of 2, 6, and 20 mg/kg after 0 and 2 h. The reading at 0 min
denotes the postsurgery withdrawal threshold (the difference between
the presurgery and 0 min reading denotes the development of mechan-
ical allodynia), this reading was followed by administration of 44.
Gabapentin (50 mg/kg, po) was included as a positive control. Data
are expressed as mean ( SEM.
We have disclosed the 1-(4-(pyridin-2-yl)benzyl)imidazolidine-
2,4-dione series as a novel chemotype possessing cannabinoid
CB2 receptor activity. 44 was identified after a multiparameter
optimization process in which lipophilicity and basicity were
systematically varied to study their effect on metabolic stability,
potency at the human cannabinoid CB2 receptor, and selectivity
over the human cannabinoid CB1 receptor and the hERG
channel. 44 is a potent and selective cannabinoid CB2 receptor
agonist, which is orally bioavailable and has shown in vivo activity
in a spinal nerve ligation model of neuropathic pain. The
compound has low brain penetration, therefore it can be
considered as a peripherally restricted agonist. This compound
may be useful to study cannabinoid CB2 receptor biology in
various preclinical models of human diseases such as pain,
osteoporosis, and cancer.8,9,21,28
spinal nerve. This assay has been employed successfully to
demonstrate antiallodynic effects of anticonvulsants (e.g.,
gabapentin), antidepressants (e.g., duloxetine), and opioid an-
algesics (e.g., tramadol), which are used clinically in the treat-
ment of neuropathic pain.1,2 44 induced a dose-dependent
antinociceptive effect after 2 h of dosing (Figure 3). Significant
relieve of mechanical allodynia was observed at 6 mg/kg (po).
High free plasma levels (>1900 nM) were required to observe
significant antinociception. These levels were higher than ex-
pected based on the potency of the compound at the cannabinoid
CB2 receptor, therefore we determined the brain penetration
and its CSF levels. The total brain to total plasma ratio was 0.05
and the CSF/free plasma concentration ratio was 0.14, indicating
active efflux out of the CNS. In the Caco-2 model, an A to B/B to
A efflux ratio of 3.0 was observed, further suggesting that 44 may
be subject to active transport. If the mechanism is centrally
mediated, this may explain the high dose needed for efficacy in
the spinal nerve ligation model, but other explanations cannot be
ruled out at the moment. It is noteworthy that it was recently
reported that the selective CB2 agonist GW842166 was not
efficacious in patients with acute pain following third molar tooth
extraction.25 It is questioned whether the efficacy of selective
’ EXPERIMENTAL SECTION
All commercial chemicals and solvents are reagent grade and were
used without further purification. The residues were purified by silica gel
column (63ꢀ200 μm) chromatography or recrystallized from an
appropriate solvent. The progress of reactions was monitored by thin
layer chromatography (TLC) using commercially prepared silica gel 60
F254 glass-backed plates. Compounds were visualized under ultraviolet
(UV) light. Purification performed on Gilson SemiPrep HPLC. Col-
umn: Luna 5 μm C18 (20), 100A, Axiapack, 150 mm ꢁ 21.20 mm, 5 μm.
Detection: UV at 210 nm. Eluent A: Milli-Q water 900 mL + CH3CN
100 mL + 0.05% HCOOH. Eluent B: CH3CN 900 mL + MQ-water
100 mL + 0.05% HCOOH (Table 3). Flow: 20 mL/min .
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Table 3
1-(4-Bromo-benzyl)-3-methyl-imidazolidine-2,4-dione (5a). Over-
all yield: 65%. 1H NMR (400 MHz, CDCl3): δ 7.50 (d, J = 8.61 Hz, 2H),
7.14 (d, J = 8.61 Hz, 2H), 4.52 (s, 2H), 3.73 (s, 2H), 3.06 (s, 3H).
1-(4-Bromo-benzyl)-3-ethyl-imidazolidine-2,4-dione (5b). Overall
yield: 60%. 1H NMR (400 MHz, CDCl3): δ 7.50 (d, J = 8.22 Hz, 2H),
7.13 (d, J = 8.22 Hz, 2H), 4.52 (s, 2H), 3.71 (s, 2H), 3.59 (q, J = 7.43 Hz,
2H), 1.24 (t, J = 7.43 Hz, 3H).
1-(4-Bromo-benzyl)-3-propyl-imidazolidine-2,4-dione (5c). Over-
all yield: 63%. 1H NMR (400 MHz, CDCl3): δ 7.49 (d, J = 8.22 Hz, 2H),
7.13 (d, J = 8.22 Hz, 2H), 4.51 (s, 2H), 3.72 (s, 2H), 3.50 (dd J = 7.43 and
6.26 Hz, 2H), 1.72ꢀ1.60 (m, 2H), 0.93 (t, J = 7.43 Hz, 3H).
1-(4-Bromo-benzyl)-3-isobutyl-imidazolidine-2,4-dione (5d). Over-
all yield: 67%. 1H NMR (400 MHz, CDCl3): δ 7.50 (d, J = 8.22 Hz, 2H),
7.14 (d, J = 8.22 Hz, 2H), 4.50 (s, 2H), 3.72 (s, 2H), 2.15ꢀ2.05 (m, 1H),
1.50ꢀ1.42 (m, 2H), 0.93 (d, J = 6.65 Hz, 6H).
T (min)
A (%)
B (%)
0.0
90
0
10
90
6.00
30.00
31.00
34.00
35.00
0
90
0
100
100
10
0
90
Table 4
T (min)
A (%)
B (%)
0.0
98
0
02
100
100
02
1-(4-Bromo-benzyl)-3-(2,2-difluoro-ethyl)-imidazolidine-2,4-dione
(5e). Overall yield: 67%. 1H NMR (400 MHz, CDCl3): δ 7.51 (d, J = 8.22
Hz, 2H), 7.14 (d, J = 8.22 Hz, 2H), 6.21ꢀ5.87 (tt, J = 55.95, 4.70, and 4.30
Hz, 1H), 4.53 (s, 2H), 3.90 (td, J = 13.70 and 4.30 Hz, 2H), 3.80 (s, 2H).
1-(4-Bromo-benzyl)-3-cyclopropylmethyl-imidazolidine-2,4-dione
(5f). Overall yield: 59%. 1H NMR (400 MHz, CDCl3): δ 7.50 (d, J = 8.22
Hz, 2H), 7.15 (d, J = 8.22 Hz, 2H), 4.53 (s, 2H), 3.74 (s, 2H), 3.56 (d, J =
7.44 Hz, 2H), 1.23ꢀ1.12(m, 1H),0.54ꢀ0.45 (m, 2H), 0.38ꢀ0.32 (m, 2H).
1-(4-Bromo-benzyl)-3-cyclobutylmethyl-imidazolidine-2,4-dione (5g).
Overall yield: 56%. 1H NMR (400 MHz, CDCl3):δ7.49 (d, J=8.22Hz,2H),
7.13(d,J= 8.22 Hz, 2H), 4.50 (s, 2H), 3.71 (s, 2H), 3.57 (d, J=7.43Hz,2H),
2.75ꢀ2.62 (m, 1H), 2.08ꢀ1.97 (m, 2H), 1.92ꢀ1.70 (m, 4H).
Method 2
(i) To a solution of glycine hydrochloride (108 mmol) in water
(40 mL) were added an aqueous solution of sodium hydroxide
(108 mmol) and a solution of 4-bromobenzaldehyde 2 (108
mmol) in methanol (240 mL). After 30 min of stirring at room
temperature, sodium borohydride (108 mmol) was added por-
tionwise to this suspension. After 18 h of stirring at room
temperature, the reaction mixture was concentrated under re-
duced pressure and the resulting aqueous phase was washed with
diethyl ether. The aqueous phase was neutralized by the addition
of an aqueous solution of 2 M hydrochloric acid. The resulting
precipitate was collected by filtration and washed with water and
diethyl ether. Drying the white solid afforded (4-bromo-
benzylamino)-acetic acid 6 (y = 71%). The product was used in
the following step without further purification.
5.00
5.70
5.71
7.00
0
98
98
02
The purity of all final compounds was >95% as determined with
UPLC-MS; Waters Aquity Ultra Performance. Column: Acquity UPLC
BEH C18, 2.1 mm ꢁ 100 mm, 1.7 μm. Detection: UV at 210 nm. Eluent
A: MQ-water 900 mL + CH3CN 100 mL + 0.05% HCOOH. Eluent B:
CH3CN 900 mL + MQ-water 100 mL + 0.05% HCOOH (Table 4).
Flow: 0.60 mL/min.
1H NMR was recorded on a Bruker Spectrospin 400 Ultrashield
General Procedures for Synthesis of 1-(4-Bromobenzyl)-
3-alkyl-imidazolidine-2,4-dione Building Blocks. Method 1
(i) To a solution of 4-bromobenzaldehyde 2 (0.54 mol) and glycina-
mide hydrochloride (0.48 mol) methanol/water (1500 mL,
5.5/1) was added sodium hydroxide (0.54 mol). After stirring
for 17 h at room temperature, the reaction mixture was cooled
to 0 ꢀC. Sodium borohydride (1.0 mol) was added, and the
mixture was stirred until a clear solution was obtained. The
reaction was quenched by addition of concentrated hydrochlo-
ric acid until pH = 3. After stirring for 17 h, the mixture was
neutralized with a saturated aqueous solution of sodium hy-
drogen carbonate and the product was extracted into dichlor-
omethane. The combined organic phases were washed with
water, brine, dried over sodium sulfate, and concentrated under
reduced pressure to afford 2-(4-bromo-benzylamino)-aceta-
mide 3 (92 g).
(ii) To a suspension of the product 6 obtained in the previous step
(6.96 mmol) in dichloromethane (20 mL) were added triethy-
lamine (13.9 mmol), an alkylamine (7.66 mmol), and o-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (PyBOP) (8.36 mmol). After 17 h of stirring at room
temperature, the reaction mixture was concentrated under
reduced pressure. Column chromatography afforded 2-(4-bromo-
benzylamino)-N-alkyl-acetamide 7hꢀk.
(ii) To a solution of the product 3 obtained in the previous step (0.25
mol) in acetonitrile (1500 mL) were added CDI (0.5 mol)
DMAP (0.5 mol). After stirring for 16 h at 60 ꢀC, the solution
was cooled to room temperature and poured into an aqueous
solution of 2 M hydrochloric acid. The product was extracted
into ethyl acetate and the combined organic phases were washed
with water, brine, dried over sodium sulfate, and concentrated
under reduced pressure. The remaining solid was stirred with
acetone. Filtration afforded 1-(4-bromo-benzyl)-imidazolidine-
2,4-dione 4 (45 g) as a white solid. The product was used in the
following step without further purification.
(iii) To a solution of the product 4 obtained in the previous step
(37.2 mmol) in DMF (90 mL) were added at room temperature,
potassium carbonate (111 mmol) and the alkylbromide (74.3
mmol). After 17 h stirring at 50 ꢀC under a nitrogen atmosphere,
the reaction mixture was cooled to room temperature and
filtered. The clear solution was concentrated under reduced
pressure. Column chromatography afforded the corresponding
1-(4-Bromo-benzyl)-3-alkyl-imidazolidine-2,4-dione building
block 5aꢀg as a white solid.
(iii) To a solution of the product 7hꢀk obtained in the previous step
(6.96 mmol) in acetonitrile were added (diimidazol-1-yl)ketone
(13.9 mmol) and 4-dimethylaminopyridine (13.9 mmol). After
17 h of stirring at 60 ꢀC, the reaction mixture was cooled to room
temperature and quenched by addition of a saturated aqueous
solution of sodium hydrogen carbonate. The product was extracted
into ethylacetate and the combined organic phases were washed
with water and brine and dried over sodium sulfate. Column
chromatography afforded the title compound 1-(4-bromo-
benzyl)-3-alkyl-imidazolidine-2,4-dione 5hꢀk as a light-yellow oil.
1-(4-Bromo-benzyl)-3-isopropyl-imidazolidine-2,4-dione (5h).
Overall yield: 36%. 1H NMR (400 MHz, CDCl3): δ 7.50 (d, J = 8.61
Hz, 2H), 7.13 (d, J = 8.61 Hz, 2H), 4.49 (s, 2H), 4.38ꢀ4.30 (m, 1H),
1.43 (d, J = 6.65 Hz, 6H).
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1-(4-Bromo-benzyl)-3-cyclopropyl-imidazolidine-2,4-dione (5i).
Overall yield: 46%. 1H NMR (400 MHz, CDCl3): δ 7.50 (d, J = 8.61
Hz, 2H), 7.14 (d, J = 8.61 Hz, 2H), 4.48 (s, 2H), 3.66 (s, 2H),
2.65ꢀ2.56 (m, 1H), 0.97 (d, J = 5.87 Hz, 4H).
4-(6-Bromo-pyridin-2-ylmethyl)-thiomorpholine 1,1-dioxide (10j).
Yield: 90%. 1H NMR (400 MHz, CDCl3): δ 7.61ꢀ7.54 (dd, J = 7.83 and
7.43 Hz, 1H), 7.42 (d, J = 7.83 Hz, 1H), 7.39 (d, J = 7.43 Hz, 1H), 3.81 (s,
2H), 3.14ꢀ3.04 (m, 8H).
1-(4-Bromo-benzyl)-3-cyclobutyl-imidazolidine-2,4-dione (5j).
Overall yield: 57%. 1H NMR (400 MHz, CDCl3): δ 7.49 (d, J = 8.22
Hz, 2H), 7.14 (d, J = 8.22 Hz, 2H), 4.60ꢀ4.51 (m, 1H), 4.49 (s, 2H),
3.65 (s, 2H), 2.95ꢀ2.82 (m, 2H), 2.23ꢀ2.13 (m, 2H), 1.91ꢀ1.81 (m,
1H), 1.79ꢀ1.65 (m, 1H).
4-(6-Bromo-3-fluoro-pyridin-2-ylmethyl)-thiomorpholine 1,1-di-
1
oxide (10k). Yield: 77%. H NMR (400 MHz, CDCl3): δ 7.45 (dd,
J = 8.22 and 3.52 Hz, 1H), 7.32 (d, J = 8.61 and 8.22 Hz, 1H), 3.89 (d,
J = 2.74 Hz, 2H), 3.17 (m, 8H).
2-Bromo-6-cyclohexylmethyl-pyridine (15)
1-(4-Bromo-benzyl)-3-(2,2,2-trifluoro-ethyl)-imidazolidine-2,4-
dione (5k). Overall yield: 25%. 1H NMR (400 MHz, CDCl3): δ
7.52(d, J = 8.61 Hz, 2H), 7.15 (d, J = 8.61 Hz, 2H), 4.55 (s, 2H), 4.16
(q, J = 8.61 Hz, 2H), 3.83 (s, 2H).
(i) A solution of 2,6-dibromopyridine 11 (5.8 mmol) in THF/
hexane/diethyl ether (1/1/3, 15 mL) was added dropwise, under
a nitrogen atmosphere, to a solution of a 2.5 M n-butyllithium in
hexane (2.43 mL, 6.08 mmol) at ꢀ78 ꢀC . After 10 min of stirring,
a solution of cyclohexanecarbonitrile 12 (5.8 mmol) in THF/
hexane/diethyl ether (1/1/3, 4 mL) was added and the reaction
mixture was stirred for 2.5 h at ꢀ78 ꢀC. The reaction mixture was
warmed to room temperature and stirred for another 1.5 h. The
reaction mixture was quenched by the addition of an aqueous
solution of 2 M sulfuric acid (7 mL). After vigorous stirring for
2 h, water was added and the product was extracted into diethyl
ether. The combined organic phases were washed with a saturated
aqueous solution of sodium hydrogen carbonate, brine, dried
over sodium sulfate, and concentrated under reduced pressure.
Column chromatography afforded (6-bromo-pyridin-2-yl)-cyclo-
hexyl-methanone 13 (800 mg) as a clear oil.
(ii) A suspension of the product13 obtained in the previous step (2.24
mmol) and 4-methylbenzenesulfonyl hydrazide (2.46 mmol) in
ethanol (2 mL) was heated to 100 ꢀC for 15 min in a microwave.
After cooling to room temperature, the reaction mixture concen-
trated under reduced pressure. Column chromatography afforded
N0-((6-bromopyridin-2-yl)(cyclohexyl)methylene)-4-methylben-
zenesulfonohydrazide 14 (608 mg) as a white solid.
General Procedure for Synthesis of 2-Bromo-pyridine Building
Blocks. To a solution of 6-bromo-pyridine-2-carbaldehyde 8a or 8b
(135 mmol) in dichloromethane (500 mL) was slowly added an
appropriate cyclic amine (149 mmol) at 10 ꢀC. After stirring for 15
min at 10 ꢀC, acetic acid (149 mmol) was added, followed by the
portionwise addition of sodium triacetoxyborohydride, while the tem-
perature was kept at 5ꢀ10 ꢀC. After stirring for 2 h at room temperature,
the reaction mixture was poured into a saturated aqueous solution of
sodium hydrogen carbonate. The product was extracted into dichlor-
omethane, and the combined organic phases were washed with brine,
dried over sodium sulfate, and concentrated under reduced pressure.
Column chromatography (heptane/ethylacetate 8/2) afforded the
corresponding 2-bromo-pyridine 10aꢀk as a colorless oil.
2-Bromo-6-piperidin-1-ylmethyl-pyridine (10a). Yield: 93%. 1H
NMR (400 MHz, CDCl3): δ 7.54ꢀ7.43 (m, 2H), 7.33 (d, J = 7.43
Hz, 1H), 3.60 (s, 2H), 2.48ꢀ2.38 (m, 4H), 1.63ꢀ1.54 (m, 4H),
1.48ꢀ1.39 (m, 2H).
6-Bromo-3-fluoro-2-piperidin-1-ylmethyl-pyridine (10b). Yield:
80%. 1H NMR (400 MHz, CDCl3): δ 7.38 (dd, J = 8.22 and 3.52 Hz,
1H), 7.26 (d, J = 8.61 and 8.22 Hz, 1H), 3.69 (d, J = 2.74 Hz, 2H),
2.54ꢀ2.46 (m, 4H), 1.61ꢀ1.53 (m, 4H), 1.45ꢀ1.36 (m, 2H).
2-Bromo-6-(3-methyl-piperidin-1-ylmethyl)-pyridine (10c). Yield:
45%. 1H NMR (400 MHz, CDCl3): δ 7.47ꢀ7.37 (m, 2H), 7.26 (d, J =
7.43 Hz, 1H), 3.54 (s, 2H), 2.76ꢀ2.64 (m, 2H), 1.92 (td, J = 10.96 and
3.52 Hz, 1H), 1.68ꢀ1.44 (m, 5H), 0.87ꢀ0.72 (m, 4H).
(iii) To a solution of the product 14 obtained in the previous step
(1.38 mmol) in 4 mL dichloromethane was added slowly to a
solution of 20% diisobutylaluminiumhydride in toluene (13.8
mmol). After 17 h of stirring at room temperature, the reaction
mixture was quenched by adding slowly an aqueous solution of 2
M sodium hydroxide until pH = 10. The product was extracted
into ethylacetate, and the combined organic phases were washed
with water, brine, dried over sodium sulfate, and concentrated
under reduced pressure. Column chromatography afforded the
title compound 2-bromo-6-cyclohexylmethyl-pyridine 15 as a
white solid. 1H NMR (400 MHz, CDCl3): δ 7.42ꢀ7.21 (m, 3H),
3.62 (d, J = 7.04 Hz, 2H), 1.81ꢀ0.85 (m, 11H).
6-Bromo-3-fluoro-2-(3-methyl-piperidin-1-ylmethyl)-pyridine
(10d). Yield: 38%. (m/z) = 288 (M + H)+.
2-Bromo-6-(3-fluoro-piperidin-1-ylmethyl)-pyridine (10e). Yield:
24%. 1H NMR (400 MHz, CDCl3): δ 7.53 (dd, J = 7.83 and 7.43 Hz,
1H), 7.47 (d, J = 7.43 Hz, 1H), 7.36 (d, J = 7.83 Hz, 1H), 4.76ꢀ4.56 (m,
1H), 3.68 (s, 2H), 2.82ꢀ2.71 (m, 1H), 2.60ꢀ2.47 (m, 2H), 2.43ꢀ2.36
(m, 1H), 1.93ꢀ1.78 (m, 2H), 1.73ꢀ1.50 (m, 2H).
General Method for Synthesis of 1-(4-(Pyridin-2-yl)benzyl)imidazolidine-
2,4-dion Derivatives
6-Bromo-3-fluoro-2-(3-fluoro-piperidin-1-ylmethyl)-pyridine
(10f). Yield: 55%. 1H NMR (400 MHz, CDCl3): δ 7.40 (dd, J = 8.61 and
3.52 Hz, 1H), 7.28 (d, J = 8.61 and 8.22 Hz, 1H), 4.72ꢀ4.53 (m, 1H), 3.78
(bs, 2H), 2.97ꢀ2.86 (m, 1H), 2.60ꢀ2.57 (m, 1H), 2.55ꢀ2.47 (m, 1H),
2.42ꢀ2.35 (m, 1H), 1.92ꢀ1.75 (m, 2H), 1.62ꢀ1.47 (m, 2H).
4-(6-Bromo-3-fluoro-pyridin-2-ylmethyl)-morpholine (10g).
Yield: 65%. 1H NMR (400 MHz, CDCl3): δ 7.41 (dd, J = 8.22
and 3.52 Hz, 1H), 7.28 (d, J = 8.61 and 8.22 Hz, 1H), 3.73ꢀ3.69 (m,
6H), 2.61ꢀ2.54 (m, 4H).
(i) To a solution of the 1-(4-bromobenzyl)-imidazolidine-2,4-dione deri-
vative 5aꢀk (6.2 mmol), bis(pinacolato)diboron (6.2 mmol) and
potassium acetate (18.5 mmol) in DMF (50 mL) under a nitrogen
atmosphere was added 1,10-bis(diphenylphosphino)ferrocenedichloro
palladium(II) (0.18 mmol). After 17 h of stirring at 75 ꢀC, the reaction
mixture was cooled to room temperature. Water was added, and
the product was extracted into ethyl acetate. The combined
organic phases were washed with a saturated aqueous solution
of sodium hydrogen carbonate, water, and brine, dried over
sodium sulfate, and concentrated under reduced pressure to
afford the corresponding boronic ester 16aꢀk as a black oil.
The product was used in the following step without further
purification.
2-Bromo-6-(3,3-difluoro-piperidin-1-ylmethyl)-pyridine (10h).
Yield: 45%. 1H NMR (400 MHz, CDCl3): δ 7.58ꢀ7.49 (m, 2H), 7.47
(d, J = 7.43 Hz, 1H), 3.74 (s, 2H), 2.71 (dd, J = 11.35 and 10.96 Hz,
2H), 2.54 (dd, J = 5.48 and 5.09 Hz, 2H), 1.97ꢀ1.84 (m, 2H),
1.83ꢀ1.76 (m, 2H).
6-Bromo-2-(3,3-difluoro-piperidin-1-ylmethyl)-3-fluoro-pyridine
(10i). Yield: 65%. 1H NMR (400 MHz, CDCl3): δ 7.41 (dd, J = 8.22
and 3.52 Hz, 1H), 7.29 (d, J = 8.61 and 8.22 Hz, 1H), 3.86 (d, J = 2.35
Hz, 2H), 2.79 (dd, J = 11.35 and 10.95 Hz, 2H), 2.59 (dd, J = 5.48 and
5.09 Hz, 2H), 1.91ꢀ1.72 (m, 4H).
(ii) To a solution of the product 16aꢀk obtained in the previous
step (3.52 mmol) and an appropriate 2-bromopyridine deri-
vative 10aꢀk or 15 (2.93 mmol) in toluene/ethanol (4/1,
25 mL) was added an aqueous solution of 2 M potassium
carbonate. After 15 min of stirring under a nitrogen atmosphere,
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tetrakis(triphenylphosphine)palladium(0) (0.073 mmol) was
added and this mixture was stirred for 17 h at 75 ꢀC under a
nitrogen atmosphere. After completion, the mixture was cooled
to room temperature and filtered through decalite. Water was
added to the filtrate, and the product was extracted into ethylace-
tate. The combined organic phases were washed with water and
brine, dried over sodium sulfate, and concentrated under reduced
pressure. Column chromatography on silicagel (n-heptane/ethyl-
acetate) afforded the corresponding 1-(4-(pyridin-2-yl)benzyl)-
imidazolidine-2,4-dion final products 1, 18ꢀ44.
7.83 Hz, 1H), 7.44 (d, J = 7.43 Hz, 1H), 7.35 (d, J = 8.61 Hz, 2H), 6.05
(Tt, J = 55.6 and 4.30 Hz, 1H), 4.63 (s, 2H), 3.92 (Td, J = 13.7 and 4.30
Hz, 2H), 3.81 (s, 2H), 3.72 (s, 2H), 2.54ꢀ2.40 (m, 4H), 1.67ꢀ1.56 (m,
4H), 1.50ꢀ1.41 (m, 2H).
1-(4-(6-(Piperidin-1-ylmethyl)pyridin-2-yl)-benzyl)-3-(2,2,2-trifluoro-
ethyl)-imidazolidine-2,4-dione (26). 1H NMR (400 MHz, CDCl3): δ
8.00 (d, J = 8.22 Hz, 2H), 7.73 (dd, J = 7.83 and 7.43 Hz, 1H), 7.56 (d, J =
7.83 Hz, 1H), 7.44 (d, J = 7.43 Hz, 1H), 7.35 (d, J = 8.22 Hz, 2H), 4.64 (s,
2H), 4.17 (q, J = 8.61 Hz, 2H), 3.84 (s, 2H), 3.72 (s, 2H), 2.54ꢀ2.46 (m,
4H), 1.67ꢀ1.56 (m, 4H), 1.51ꢀ1.42 (m, 2H).
3-Ethyl-1-(4-(5-fluoro-6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-
imidazolidine-2,4-dione (27). 1H NMR (400 MHz, CDCl3): δ 7.97 (d,
J = 8.22 Hz, 2H), 7.63 (dd, J = 8.61 and 3.52 Hz, 1H), 7.43 (dd, J = 9.00
and 8.61 Hz, 1H), 7.35 (d, J = 8.61 Hz, 2H), 4.61 (s, 2H), 3.85 (d, J =
2.35 Hz, 2H), 3.74 (s, 2H), 3.61 (q, J = 7.43 Hz, 2H), 2.62 (bs, 4H),
1.66ꢀ1.57 (m, 4H), 1.47ꢀ1.38 (m, 2H), 1.25 (t, J = 7.43, 3H).
1-(4-(5-Fluoro-6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-3-
propylimidazolidine-2,4-dione (28). 1H NMR (400 MHz, CDCl3): δ
7.96 (d, J = 8.61 Hz, 2H), 7.63 (dd, J = 8.61 and 3.52 Hz, 1H), 7.43 (dd,
J = 9.00 and 8.61 Hz, 1H), 7.34 (d, J = 8.61 Hz, 2H), 4.61 (s, 2H), 3.74
(s, 2H), 3.51 (t, J = 7.04 Hz, 2H), 3.49 (s, 2H), 2.70ꢀ2.51 (m, 4H),
1.76ꢀ1.55 (m, 6H), 1.49ꢀ1.35 (m, 2H), 0.94 (t, J = 7.04, 3H).
1-(4-(5-Fluoro-6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-3-
isobutylimidazolidine-2,4-dione (29). 1H NMR (400 MHz, CDCl3):
δ 7.96 (d, J = 8.61 Hz, 2H), 7.62 (dd, J = 8.21 and 3.13 Hz, 1H), 7.43 (t,
J = 8.61 Hz, 1H), 7.34 (d, J = 8.21 Hz, 2H), 4.62 (s, 2H), 3.83 (d, J =
1.96 Hz, 2H), 3.75 (s, 2H), 3.37 (d, J = 7.43 Hz, 2H), 2.62ꢀ2.54 (m,
4H), 2.15ꢀ2.04 (m, 1H), 1.65ꢀ1.57 (m, 4H), 1.46ꢀ1.37 (m, 2H),
0.93 (d, J = 6.65 Hz, 6H).
3-Isobutyl-1-(4-(6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-
1
imidazolidine-2,4-dione (1). H NMR (400 MHz, CDCl3): δ 8.00
(d, J = 8.22 Hz, 2H), 7.72 (dd, J = 8.22 and 7.83 Hz, 1H), 7.56 (d, J =
8.22 Hz, 1H), 7.43 (d, J = 7.83 Hz, 1H), 7.34 (d, J = 8.22 Hz, 2H), 4.62
(s, 2H), 3.74 (s, 2H), 3.72 (s, 2H), 3.36 (d, J = 7.43 Hz, 2H),
2.53ꢀ2.47 (m, 4H), 2.15ꢀ2.05 (m, 1H), 1.65ꢀ1.58 (m, 4H),
1.50ꢀ1.42 (m, 2H), 0.93 (d, J = 6.65 Hz, 6H).
1-(4-(6-(Piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-3-propylimi-
dazolidine-2,4-dione (18). 1H NMR (400 MHz, CDCl3): δ 7.99 (d,
J = 8.22 Hz, 2H), 7.22 (dd, J = 7.83 and 7.04 Hz, 1H), 7.56 (d, J = 7.83
Hz, 1H) 7.43 (d, J = 7.04 Hz, 1H), 7.34 (d, J = 8.22 Hz, 2H), 4.61 (s,
2H), 3.73 (s, 2H), 3.71 (s, 2H), 3.51 (t, J = 7.43 Hz, 2H), 2.53ꢀ2.47 (m,
4H), 1.73ꢀ1.56 (m, 6H), 1.50ꢀ1.42 (m, 2H), 0.94 (t, J = 7.43, 3H).
3-Ethyl-1-(4-(6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-imi-
dazolidine-2,4-dione (19). 1H NMR (400 MHz, CDCl3): δ 7.99 (d,
J = 8.22 Hz, 2H), 7.74 (dd, J = 7.83 and 7.43 Hz, 1H), 7.61 (d, J = 7.83
Hz, 1H), 7.45 (d, J = 7.43 Hz, 1H), 7.35 (d, J = 8.22 Hz, 2H), 4.61 (s,
2H), 3.72 (bs, 4H), 3.61 (q, J = 7.04 Hz, 2H), 2.53ꢀ2.47 (m, 4H),
1.65ꢀ1.57 (m, 4H), 1.50ꢀ1.43 (m, 2H), 1.25 (t, 3H).
3-Cyclopropyl-1-(4-(5-fluoro-6-(piperidin-1-ylmethyl)-pyridin-2-
yl)-benzyl)-imidazolidine-2,4-dione (30). 1H NMR (400 MHz, CDCl3):
δ 7.96 (d, J = 8.22 Hz, 2H), 7.62 (dd, J = 8.61 and 3.52 Hz, 1H), 7.42 (dd,
J = 8.61 and 8.22 Hz, 1H), 7.34 (d, J = 8.22 Hz, 2H), 4.58 (s, 2H), 3.82 (d,
J = 2.74 Hz, 2H), 3.68 (s, 2H), 2.67ꢀ2.55 (m, 5H), 1.64ꢀ1.54 (m, 4H),
1.44ꢀ1.84 (m, 2H), 1.83ꢀ1.75 (m, 2H), 1.64ꢀ1.57 (m, 4H), 1.47ꢀ1.37
(m, 2H), 1.00ꢀ0.95 (m, 4H).
3-Methyl-1-(4-(6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-
imidazolidine-2,4-dione (20). 1H NMR (400 MHz, CDCl3): δ 7.99
(d, J = 8.22 Hz, 2H), 7.72 (dd, J = 7.83 and 7.04 Hz, 1H), 7.56 (d, J =
7.83 Hz, 1H), 7.43 (d, J = 7.04 Hz, 1H), 7.35 (d, J = 8.22 Hz, 2H),
4.62 (s, 2H), 3.75 (s, 2H), 3.72 (s, 2H), 3.06 (s, 3H), 2.50 (bs, 4H),
1.65ꢀ1.57 (m, 4H), 1.50ꢀ1.43 (m, 2H).
3-Isopropyl-1-(4-(6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-
imidazolidine-2,4-dione (21). 1H NMR (400 MHz, CDCl3): δ 7.99
(d, J = 8.61 Hz, 2H), 7.72 (dd, J = 7.83 and 7.43 Hz, 1H), 7.56 (d, J =
7.83 Hz, 1H), 7.43 (d, J = 7.43 Hz, 1H), 7.34 (d, J = 8.61 Hz, 2H), 4.58
(s, 2H), 4.40ꢀ4.32 (m, 1H), 3.72 (s, 2H), 3.67 (s, 2H), 2.50 (bs, 4H),
1.66ꢀ1.57 (m, 6H), 1.44 (d, J = 7.04 Hz, 6H).
3-(Cyclopropylmethyl)-1-(4-(5-fluoro-6-(piperidin-1-ylmethyl)-
1
pyridin-2-yl)-benzyl)-imidazolidine-2,4-dione (31). H NMR (400
MHz, CDCl3): δ 7.97 (d, J = 8.22 Hz, 2H), 7.62 (dd, J = 8.61 and 3.52
Hz, 1H), 7.42 (t, J = 8.61 Hz, 1H), 7.35 (d, J = 8.22 Hz, 2H), 4.62 (s,
2H), 3.82 (d, J = 2.74 Hz, 2H), 3.76 (s, 2H), 3.41 (d, J = 7.01 Hz, 2H),
2.61ꢀ2.55 (m, 4H), 1.64ꢀ1.56 (m, 4H), 1.45ꢀ1.38 (m, 2H),
1.23ꢀ1.16 (m, 1H), 0.55ꢀ0.48 (m, 2H), 0.39ꢀ0.34 (m, 2H).
3-(Cyclobutylmethyl)-1-(4-(5-fluoro-6-(piperidin-1-ylmethyl)-
pyridin-2-yl)-benzyl)-imidazolidine-2,4-dione (32). 1H NMR (400
MHz, CDCl3): δ 7.96 (d, J = 8.22 Hz, 2H), 7.62 (dd, J = 8.61 and 3.52
Hz, 1H), 7.42 (t, J = 8.61 Hz, 1H), 7.33 (d, J = 8.22 Hz, 2H), 4.60 (s, 2H),
3.82 (d, J = 2.74 Hz, 2H), 3.73 (s, 2H), 3.58 (d, J = 7.43 Hz, 2H),
2.76ꢀ2.66 (m, 1H), 2.63ꢀ2.53 (m, 4H), 2.09ꢀ1.99 (m, 2H), 1.93ꢀ1.84
(m, 2H), 1.83ꢀ1.75 (m, 2H), 1.64ꢀ1.57 (m, 4H), 1.47ꢀ1.37 (m, 2H).
3-(2,2-Difluoroethyl)-1-(4-(5-fluoro-6-(piperidin-1-ylmethyl)-pyr-
idin-2-yl)-benzyl)-imidazolidine-2,4-dione (33). 1H NMR (400 MHz,
CDCl3): δ 7.98 (d, J = 8.61 Hz, 2H), 7.62 (dd, J = 8.61 and 3.52 Hz, 1H),
7.43 (dd, J = 9.00 and 8.61 Hz, 1H), 7.37 (d, J = 8.61 Hz, 2H), 4.05 (Tt,
J = 55.95 and 4.30 Hz, 1H), 4.63 (s, 2H), 3.92 (Td, J = 13.69 and 4.30 Hz,
2H), 3.83 (s, 2H), 3.82 (s, 2H), 2.63ꢀ2.55 (m, 4H), 1.65ꢀ1.55 (m, 4H),
1.45ꢀ1.37 (m, 2H).
3-Cyclopropyl-1-(4-(6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-
imidazolidine-2,4-dione (22). 1H NMR (400 MHz, CDCl3): δ 7.98 (d,
J = 8.22 Hz, 2H), 7.72 (dd, J = 7.83 and 7.43 Hz, 1H), 7.56 (d, J = 7.83
Hz, 1H), 7.43 (d, J = 7.43 Hz, 1H), 7.34 (d, J = 8.22 Hz, 2H), 4.59 (s,
2H), 3.72 (s, 2H), 3.67 (s, 2H), 2.66ꢀ2.60 (m, 1H), 2.54ꢀ2.46 (m, 4H),
1.65ꢀ1.57 (m, 2H), 1.50ꢀ1.42 (m, 2H), 1.00ꢀ0.96 (m, 4H).
3-Cyclobutyl-1-(4-(6-(piperidin-1-ylmethyl)-pyridin-2-yl)-benzyl)-
imidazolidine-2,4-dione (23). 1H NMR (400 MHz, CDCl3): δ 7.98 (d,
J = 8.22 Hz, 2H), 7.72 (dd, J = 7.83 and 7.43 Hz, 1H), 7.56 (d, J = 7.83
Hz, 1H), 7.43 (d, J = 7.43 Hz, 1H), 7.34 (d, J = 8.22 Hz, 2H), 4.63ꢀ4.53
(m, 3H), 3.72 (s, 2H), 3.67 (s, 2H), 2.97 ꢀ2.84 (m, 2H), 2.54ꢀ2.46 (m,
4H), 2.24ꢀ2.14 (m, 2H), 1.92ꢀ1.82 (m, 1H), 1.79ꢀ1.67 (m, 1H),
1.65ꢀ1.57 (m, 4H), 1.52ꢀ1.40 (m, 2H).
3-(Cyclopropylmethyl)-1-(4-(6-(piperidin-1-ylmethyl)-pyridin-2-
yl)-benzyl)-imidazolidine-2,4-dione (24). 1H NMR (400 MHz,
CDCl3): δ 7.99 (d, J = 8.22 Hz, 2H), 7.72 (dd, J = 8.22 and 7.83 Hz,
1H), 7.56 (d, J = 8.22 Hz, 1H), 7.43 (d, J = 7.83 Hz, 1H), 7.35 (d, J =
8.22 Hz, 2H), 4.62 (s, 2H), 3.75 (s, 2H), 3.72 (s, 2H), 3.41 (d, J = 7.04
Hz, 2H), 2.53ꢀ2.47 (m, 4H), 1.654ꢀ1.58 (m, 4H), 1.52ꢀ1.42 (m,
2H), 1.24ꢀ1.14 (m, 1H), 0.56ꢀ0.47 (m, 2H), 0.40ꢀ0.33 (m, 2H).
3-(2,2-Difluoroethyl)-1-(4-(6-(piperidin-1-ylmethyl)-pyridin-2-yl)-
benzyl)-imidazolidine-2,4-dione (25). 1H NMR (400 MHz, CDCl3): δ
8.00 (d, J = 8.61 Hz, 2H), 7.73 (dd, J = 7.83 and 7.42 Hz, 1H), 7.56 (d, J =
3-Isobutyl-1-(4-(6-((3-methylpiperidin-1-yl)-methyl)-pyridin-2-
yl)-benzyl)-imidazolidine-2,4-dione (34). 1H NMR (400 MHz,
CDCl3): δ 7.99 (d, J = 8.22 Hz, 2H), 7.73 (dd, J = 7.83 and 7.43
Hz, 1H), 7.57 (d, J = 7.83 Hz, 1H), 7.44 (d, J = 7.43 Hz, 1H), 7.34 (d,
J = 8.22 Hz, 2 H), 4.63 (s, 2H), 3.74 (s, 2H), 3.73 (s, 2H), 3.37 (d, J =
7.43 Hz, 2H), 2.93ꢀ2.82 (m, 2H), 2.16ꢀ1.99 (m, 2H), 1.79ꢀ1.52 (m,
6H), 0.93 (d, J = 7.04 Hz, 6H), 0.87 (d, J = 5.87 Hz, 3H).
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Journal of Medicinal Chemistry
ARTICLE
1-(4-(5-Fluoro-6-((3-methylpiperidin-1-yl)-methyl)-pyridin-2-yl)-
benzyl)-3-isobutylimidazolidine-2,4-dione (35). 1H NMR (400
MHz, CDCl3): δ 7.96 (d, J = 8.22 Hz, 2H), 7.62 (dd, J = 8.61 and
3.52 Hz, 1H), 7.43 (dd, J = 9.00 and 8.61 Hz, 1H), 7.34 (d, J = 8.22 Hz,
2H), 4.62 (s, 2H), 3.83 (bs, 2H), 3.76 (s, 2H), 3.37 (d, J = 7.43 Hz, 2H),
3.06ꢀ2.94 (m, 2H), 2.15ꢀ2.02 (m, 2H), 1.81ꢀ1.55 (m, 6H), 0.93 (d,
J = 7.04 Hz, 6H), 0.85 (d, J = 5.87 Hz, 3H).
1-(4-(6-((3-Fluoropiperidin-1-yl)-methyl)-pyridin-2-yl)-benzyl)-3-
isobutylimidazolidine-2,4-dione (36). 1H NMR (400 MHz, CDCl3):
δ 7.99 (d, J = 8.22 Hz, 2H), 7.74 (dd, J = 7.83 and 7.43 Hz, 1H), 7.58 (d,
J = 7.83 Hz, 1H), 7.42 (d, J = 7.43 Hz, 1H), 7.35 (d, J = 8.22 Hz, 2 H),
4.62 (s, 2H), 3.76 (s, 2H), 3.75 (s, 2H), 3.37 (d, J = 7.43 Hz, 2H),
2.79ꢀ2.68 (m, 2H), 2.54ꢀ2.43 (m, 2H), 2.15ꢀ1.84 (m, 6H), 0.93 (d,
J = 6.65 Hz, 6H).
1-(4-(5-Fluoro-6-((3-fluoropiperidin-1-yl)-methyl)-pyridin-2-yl)-
benzyl)-3-isobutylimidazolidine-2,4-dione (37). 1H NMR (400 MHz,
CDCl3): δ 7.96 (d, J = 8.22 Hz, 2H), 7.64 (dd, J = 8.61 and 3.52 Hz, 1H),
7.44 (d, J = 9.00 and 8.61 Hz, 1H), 7.35 (d, J = 8.22 Hz, 2H), 4.62 (s,
2H), 3.86 (d, J = 2.35 Hz, 2H), 3.76 (s, 2H), 3.37 (d, J = 7.43 Hz, 2H),
2.84ꢀ2.75 (m, 2H), 2.64ꢀ2.54 (m, 2H), 2.17ꢀ1.85 (m, 6H), 0.93 (d,
J = 6.65 Hz, 6H).
1-(4-(5-Fluoro-6-morpholin-4-ylmethyl-pyridin-2-yl)-benzyl)-3-
isobutylimidazolidine-2,4-dione (38). 1H NMR (400 MHz, CDCl3):
δ 7.96 (d, J = 8.22 Hz, 2H), 7.62 (dd, J = 8.61 and 3.52 Hz, 1H), 7.42 (t,
J = 8.62 Hz, 1H), 7.34 (d, J = 8.22 Hz, 2H), 4.62 (s, 2H), 3.82 (d, J =
2.74 Hz, 2H), 3.75 (s, 2H), 3.36 (d, J = 7.43 Hz, 2H), 2.62ꢀ2.55 (m,
4H), 2.14ꢀ2.05 (m, 1H), 1.64ꢀ1.57 (m, 4H), 1.46ꢀ1.37 (m, 2H),
0.93 (d, J = 6.65 Hz, 6H).
7.43 Hz, 1H), 7.63 (dd, J = 9.00 and 3.52 Hz, 1H), 7.36 (d, J = 8.22 Hz,
2H), 4.59 (s, 2H), 4.02 (s, 2H), 3.70 (s, 2H), 3.24ꢀ3.19 (m, 4H),
3.13ꢀ3.07 (m, 4H), 2.67ꢀ2.59 (m, 1H), 1.01ꢀ0.96 (m, 4H).
Determination of eLogD and pKa. The physicochemical prop-
erties eLogD and pKa were experimentally determined as described
previously29,30
Agonist-Induced cAMP Change in Human CB2 Tranfected
CHO Cells. Adenylate cyclase assays were carried out using CHO cells
stably overexpressing the human recombinant cannabinoid CB2 receptor.
Cells were cultured in DMEM/HAMF12 containing 1% (v/v) penicillin/
streptomycin (Gibco 15140ꢀ122), 10% FBS Biowitthaker (DE-14-701-F),
and 400 μg/mL Geneticin (Invitrogen 10131ꢀ027). Test compounds
and reference cannabinoid CB receptor agonist CP55,940 were dis-
solved in DMSO, and dilutions were made in serum-free medium
containing 10 μM Rolipram (Sigma R6520) and 2 μM Forskolin
(Sigma, F3917). Then 10 μL of each dilution was transferred to an
assay plate (384-well white culture plate, Perkin-Elmer). Cell suspen-
sions containing 106 cells/mL in DMEM/HAMF12 containing 1%
(v/v) penicillin/streptomycin were prepared from hCB2_C2-CHO cells
and 10 μL (10000 cells/well) thereof was transferred to the assay plate and
cells were incubated for 45 min at 37 ꢀC. Homogeneous time-resolved
fluorescence (HTRF; CisBio) was used as a read-out by sequentially
adding 10 μL of cAMP-XL665 and 10 μL of anti-cAMP(Eu) cryptate; after
1 h incubation at room temperature, fluorescence at 615 nm and 665 nm
was measured on Envision (Perkin-Elmer). Results were calculated from
the 665 nm/615 nm ratios obtained for individual test compounds and
were compared to values obtained for the reference compound.
Agonist-Induced cAMP Change in Human CB1 Tranfected
CHO Cells. Adenylate cyclase assays were carried out using CHO cells
stably overexpressing the human recombinant cannabinoid CB1 recep-
tor. Cells were cultured in DMEM/HAMF12 containing 1%(v/v)
penicillin/streptomycin (Gibco 15140-122), 10% FBS Biowitthaker
(DE-14-701-F), 400 μg/mL Geneticin (Invitrogen 10131-027), and
Zeocine 250 μg/mL (Invitrogen, 45-0430). Test compounds and
reference cannabinoid CB agonist (CP55,940) were dissolved in
DMSO, and dilutions were made in serum-free medium containing 10
μM Rolipram (Sigma R6520) and 2 μM Forskolin (Sigma, F3917).
Then 10 μL of each dilution was transferred to an assay plate (384-well
white culture plate, Perkin-Elmer). Cell suspensions containing 106
cells/mL in DMEM/HAMF12 containing 1% (v/v) penicillin/strepto-
mycin were prepared from hCB1_A2-CHO cells and 10 μL (10000
cells/well) thereof was transferred to the assay plate and cells were
incubated for 45 min at 37 ꢀC. Homogeneous time-resolved fluores-
cence (HTRF; CisBio) was used as a read-out by sequentially adding 10
μL of cAMP-XL665 and 10 μL of anti-cAMP(Eu) cryptate; after 1 h
incubation at room temperature, fluorescence at 615 nm and 665 nm
was measured on Envision (Perkin-Elmer). Results were calculated from
the 665 nm/615 nm ratios obtained for individual test compounds and
were compared to values obtained for the reference compound.
In Vitro Determination of Affinity at the hERG Channel
Expressed in HEK293 Cells. This assay is used to evaluate test
compounds for their inhibition of [3H]-dofetilide binding to hERG channels
stably expressed in HEK293 cells (HEK.HERG). The class III antiarrhyth-
mic dofetilide has a high affinity for the hERG channel. Compounds that
display a high affinity for hERG and displace [3H]-dofetilide are considered
to have an increased probability of producing cardiotoxicity. For this study,
compounds were prepared in glass vials and stored at ꢀ20 ꢀC as 10ꢀ2 M
stocks. The amount of radiolabel remaining on the filters was then
determined by scintillation counting. The highest DMSO concentration
in the assay was equal to 1%. Test compounds were assayed in a 6 pt log dose
response concentration range from 10ꢀ4 M to 10ꢀ11 M.
1-(4-(6-((3,3-Difluoropiperidin-1-yl)-methyl)-pyridin-2-yl)-benzyl)-
3-isobutylimidazolidine-2,4-dione (39). 1H NMR (400 MHz, CDCl3):
δ 7.99 (d, J = 8.61 Hz, 2H), 7.75 (dd, J = 7.83 and 7.43 Hz, 1H), 7.57 (d,
J = 7.83 Hz, 1H), 7.44 (d, J = 7.43 Hz, 1H), 7.35 (d, J = 8.61 Hz, 2 H), 4.62
(s, 2H), 3.85 (s, 2H), 3.75 (s, 2H), 3.37 (d, J = 7.43 Hz, 2H), 2.78 (t, J =
11.35 Hz, 2H), 2.63ꢀ2.54 (m, 2H), 2.17ꢀ2.04 (m, 1H), 1.97ꢀ1.77)m,
4H), 0.93 (d, J = 6.65 Hz, 6H).
1-(4-(6-((3,3-Difluoropiperidin-1-yl)-methyl)-5-fluoropyridin-2-yl)-
benzyl)-3-isobutylimidazolidine-2,4-dione (40). 1H NMR (400 MHz,
CDCl3): δ 7.96 (d, J = 8.22 Hz, 2H), 7.66 (dd, J = 8.61 and 3.52 Hz, 1H),
7.46 (dd, J = 9.00 and 8.61 Hz, 1H), 7.35 (d, J = 8.22 Hz, 2H), 4.62 (s,
2H), 3.99 (d, J = 2.35 Hz, 2H), 3.76 (s, 2H), 3.37 (d, J = 7.43 Hz, 2H),
2.88 (t, J = 11.35 Hz, 2H), 2.69ꢀ2.62 (m, 2H), 2.17ꢀ2.05 (m, 1H),
1.92ꢀ1.74)m, 4H), 0.93 (d, J = 6.65 Hz, 6H).
1-(4-(6-((1,1-Dioxo-1λ6-thiomorpholin-4-yl)-methyl)-pyridin-2-yl)-
benzyl)-3-isobutyl-imidazolidine-2,4-dione (41). 1H NMR (400
MHz, CDCl3): δ 7.98 (d, J = 8.61 Hz, 2H), 7.77 (dd, J = 7.83 and
7.43 Hz, 1H), 7.63 (d, J = 7.83 Hz, 1H), 7.36 (m, 3H), 4.63 (s, 2H), 3.92
(s, 2H), 3.76 (s, 2H), 3.37 (d, J = 7.43 Hz, 2H), 3.18ꢀ3.09 (m, 8H),
2.15ꢀ2.04 (m, 1H), 0.93 (d, J = 6.65 Hz, 6H).
1-(4-(6-(1,1-Dioxo-1λ6-thiomorpholin-4-ylmethyl)-5-fluoro-pyri-
1
din-2-yl)-benzyl)-3-isobutyl-imidazolidine-2,4-dione (42). H NMR
(400 MHz, CDCl3): δ 7.94 (d, J = 8.22 Hz, 2H), 7.68 (dd, J = 8.61 and
3.52 Hz, 1H), 7.48 (dd, J = 9.00 and 8.61 Hz, 1H), 7.36 (d, J = 8.22 Hz,
2H), 4.62 (s, 2H), 4.02 (d, J = 2.35 Hz, 2H), 3.76 (s, 2H), 3.37 (s, 2H),
3.24ꢀ3.18 (m, 4H), 3.13ꢀ3.06 (m, 4H), 2.14ꢀ2.04 (m, 1H), 0.93 (d,
J = 7.04 Hz, 6H).
1-(4-(6-(Cyclohexylmethyl)-pyridin-2-yl)-benzyl)-3-ethylimidazo-
lidine-2,4-dione (43). 1H NMR (400 MHz, CDCl3): δ 7.99 (d, J = 8.22
Hz, 2H), 7.64 (dd, J = 7.83 and 7.43 Hz, 1H), 7.50 (d, J = 7.83 Hz, 1H),
7.35 (d, J = 8.22 Hz, 2H), 7.05 (d, J = 7.43 Hz, 1H), 4.62 (s, 2H), 3.73 (s,
2H), 3.61 (q, J = 7.43 Hz, 2H), 2.72 (d, J = 7.04 Hz, 2H), 1.92ꢀ1.78 (m,
1H), 1.76ꢀ1.58 (m, 6H), 1.31ꢀ1.15 (m, 5H), 1.10ꢀ0.98 (m, 2H).
3-Cyclopropyl-1-(4-(6-(1,1-dioxo-1λ6-thiomorpholin-4-ylmethyl)-
5-fluoro-pyridin-2-yl)-benzyl)-imidazolidine-2,4-dione (44). 1H NMR
(400 MHz, CDCl3): δ 7.93 (d, J = 8.22 Hz, 2H), 7.68 (dd, J = 7.83 and
Caco-2 Permeability. Test compound was added to either apical
(A) or basolateral (B) chamber at a final concentration of 20 μM in PBS,
pH7.4. After 1 h of incubation at 37 ꢀC, an aliquot from the opposite side
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Journal of Medicinal Chemistry
ARTICLE
of the monolayer was removed and terminated in acetonitrile. The
compounds were quantified by LC-MS/MS. Monolayer integrity for
each well was evaluated by lucifer yellow permeability (as determined by
fluorescence). The efflux ratio was defined as the B to A/A to B apparent
permeability (Papp) ratio.
Present Addresses
#Netherlands Translational Research Center BV, Oss, The
Netherlands
Plasma Protein Binding. Plasma protein binding was assessed by
equilibrium dialysis using Dianorm apparatus (Germany) with plasma
and buffer (PBS, pH 7.4) separated by a 5000 Da cutoff membrane
(Harvard Apparatus, Edenbridge, UK). The assay was performed for 6 h
at 37 ꢀC with compound initially at 10 μM in plasma. The concentra-
tions in plasma and buffer at the end of the incubation were determined
by LC-MS/MS following protein precipitation.
’ ACKNOWLEDGMENT
We thank Drs. Jac Wijkmans and Stan van Boeckel for
critically reading the manuscript. Lynda Allen, Gillian Gourlay,
Frank Klaassen, and Melanie Sammons are acknowledged for
their excellent technical assistance with the in vitro assays and
in vivo pain models.
Microsomal Stability. The rate of metabolism was assessed by
incubation at 37 ꢀC at pH 7.4 with hepatic microsomes (0.5 mg protein/
mL) supplemented with 1 mM NADPH and an initial substrate
concentration of 1 μM. Substrate depletion over a time course was
measured by LC-MS/MS following protein precipitation.
Pharmacokinetics in Rat. Test compounds were administered via
jugular vein cannulae (iv) or by oral gavage (po) to groups of four
nonfasted male Wistar BRL rats. Serial blood samples were taken into
EDTA-containing tubes from each rat at various time-points to 24 h.
Plasma was harvested by centrifugation (3000 rcf) with storage at ꢀ20 ꢀC
untilbioanalysis. Sampleswere analyzedfollowingprotein precipitationby
LC-MS/MS with quantitation against a standard curve prepared with
authentic standards. Timeꢀconcentration profiles were analyzed using
WinNonlin v 4.1 (Pharsight, USA) to derive PK parameters.
Serial CSF Sampling. Male Wistar BRL rats were surgically
prepared with implantation of a cannula for CSF sampling from the
cisterna magna. Three days after surgery, four rats were dosed with test
compound at 20 mg/kg by oral gavage. Serial CSF samples (30 μL) and tail
vein blood samples were taken at 0.5, 1, 2, and 4 h after dosing. Additionally,
brains were harvested at 4 h and also at 2 h in a nonsurgically prepared
satellite group. Plasma was separated from blood and analyzed as described
for rat PK studies. Brain samples were homogenized with 3 volumes of
ice-cold PBS, pH 7.4, and were protein precipitated prior to analysis. CSF
samples were quantified against standards prepared in artificial CSF.
Analysis was by LC-MS/MS for plasma, brain and CSF samples.
’ ABBREVIATIONS USED
CB1, Cannabinoid 1 receptor; CB2, Cannabinoid 2 receptor;
hERG, Human Ether-ꢀa-go-go Related Gene; TRPV1, Transient
receptor potential vanilloid 1; IFN γ, Interferon gamma; ALS,
Amyotrophic lateral sclerosis; CHO, Chinese hamster ovary;
CLint, Intrinsic clearance; CL, Clearance; PyBO, Benzotriazol-
1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; COM-
FA, Comparative Molecular Field Analysis; MW, Molecular
weight; HLM, Human liver microsomes; RLM, Rat liver micro-
somes; ADME, Absorption, distribution, metabolism, excretion;
CI, Confidence interval; CSF, Cerebrospinal fluid; CDI, 1,10-
Carbonyldiimidazole; DMAP, 4-Dimethylaminopyridine; DMF,
Dimethylformamide;M, Molar;THF, Tetrahydrofuran;DMEM,
Dulbecco's modified Eagle's medium; FBS, Fetal bovine serum;
DMSO, Dimethyl sulfoxide; PBS, Phosphate buffered saline;
NADPH, Nicotinamide adenine dinucleotide phosphate; EDTA,
Ethylenediaminetetraacetic acid; SEM, Standard error of the mean;
Vss, Volume of distribution (steady state); F, Bioavailability; fu,
fraction unbound
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’ AUTHOR INFORMATION
(10) Anand, U; Otto, W. R.; Sanchez-Herrera, D; Facer, P; Yiangou,
Y; Korchev, Y; Birch, R; Benham, C; Bountra, C; Chessell, I. P.; Anand,
P. Cannabinoid receptor CB2 localisation and agonist-mediated inhibi-
tion of capsaicin responses in human sensory neurons. Pain 2008, 138
(3), 667–680.
Corresponding Author
*Phone: +31 412 669 368. E-mail: mario.vanderstelt@
ntrc.nl. Address: NTRC, Molenstraat 110 Oss 5342 CC, Oss,
The Netherlands.
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Journal of Medicinal Chemistry
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