Discovery of a high affinity and selective pyridine analog as a potential positron emission tomography imaging agent for cannabinoid type 2 receptor

Article abstract of DOI:10.1021/acs.jmedchem.5b00283

As part of our efforts to develop CB2 PET imaging agents, we investigated 2,5,6-substituted pyridines as a novel class of potential CB2 PET ligands. A total of 21 novel compounds were designed, synthesized, and evaluated for their potency and binding properties toward human and rodent CB1 and CB2. The most promising ligand 6a was radiolabeled with carbon-11 to yield 16 ([¹¹C]RSR-056). Specific binding of 16 to CB2-positive spleen tissue of rats and mice was demonstrated by in vitro autogadiography and verified in vivo in PET and biodistribution experiments. Furthermore, 16 was evaluated in a lipopolysaccharid (LPS) induced murine model of neuroinflammation. Brain radioactivity was strikingly higher in the LPS-treated mice than the control mice. Compound 16 is a promising radiotracer for imaging CB2 in rodents. It might serve as a tool for the investigation of CB2 receptor expression levels in healthy tissues and different neuroinflammatory disorders in humans.

Full text of DOI:10.1021/acs.jmedchem.5b00283

Article
pubs.acs.org/jmc
Discovery of a High Affinity and Selective Pyridine Analog as a
Potential Positron Emission Tomography Imaging Agent for
Cannabinoid Type 2 Receptor
Roger Slavik,† Uwe Grether,*,‡ Adrienne Muller Herde,† Luca Gobbi,‡ Jurgen Fingerle,‡
̈
̈
Christoph Ullmer,‡ Stefanie D. Kramer,† Roger Schibli,†,§ Linjing Mu,†,§ and Simon M. Ametamey*,†
̈
†Center for Radiopharmaceutical Sciences ETH, PSI and USZ, Institute of Pharmaceutical Sciences, ETH Zurich,
Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland
‡Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd.,
Grenzacherstrasse 124, 4070 Basel, Switzerland
§Department of Nuclear Medicine, University Hospital Zurich, CH-8091 Zurich, Switzerland
S
* Supporting Information
ABSTRACT: As part of our efforts to develop CB2 PET imaging agents, we investigated 2,5,6-substituted pyridines as a novel
class of potential CB2 PET ligands. A total of 21 novel compounds were designed, synthesized, and evaluated for their potency
and binding properties toward human and rodent CB1 and CB2. The most promising ligand 6a was radiolabeled with carbon-11
to yield 16 ([11C]RSR-056). Specific binding of 16 to CB2-positive spleen tissue of rats and mice was demonstrated by in vitro
autogadiography and verified in vivo in PET and biodistribution experiments. Furthermore, 16 was evaluated in a
lipopolysaccharid (LPS) induced murine model of neuroinflammation. Brain radioactivity was strikingly higher in the LPS-
treated mice than the control mice. Compound 16 is a promising radiotracer for imaging CB2 in rodents. It might serve as a tool
for the investigation of CB2 receptor expression levels in healthy tissues and different neuroinflammatory disorders in humans.
in neuroinflammatory diseases and is therefore a very promising
target for therapeutic approaches as well as for PET imaging in
Parkinson’s and Alzheimer disease, multiple sclerosis (MS), and
amyotrophic lateral sclerosis (ALS).13−16
INTRODUCTION
■
The endocannabinoid system (ES) is a rather complex and
tightly regulated signaling system and a key player in several
physiological and pathological mechanisms.1 It has prohomeo-
static effects on cannabinoid receptor activation, exerting a
general protective function. This system was discovered in the
early 90s through investigations on potential targets for
cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the
two pharmacologically most active ingredients of Cannabis
sativa L.2,3 Besides several putative cannabinoid receptors such
as GPR55,4 GPR119,5 GPR186 and TRPV1,7 two main
subtypes of cannabinoid receptors are known, designated as
CB1 and CB2. Both belong to the class A G-protein-coupled
receptor family (GPCR) containing seven transmembrane
spanning domains. CB1 is highly expressed in the central
nervous system (CNS) with highest density in cerebellum,
hippocampus, and cortex.8 CB2 on the other hand is mainly
expressed on cells related to the immune system9 but also
present in the CNS; however, its expression level is very
low.10−12 It plays an important (yet not fully understood) role
Several CB2 PET ligands from a diverse class of compounds
including quinolines,17,18 thiazoles,19 indoles,20,21 oxadiazoles,22
triazines,23 and bis-sulfones24 have been reported, but to date
no optimal ligand exists for the imaging of CB2 in human
subjects. As part of our efforts to develop a PET radioligand
targeting CB2, we have prepared and evaluated the in vitro
binding profiles of a series of 2,5,6-substituted pyridines. The
most promising compound 6a was labeled with carbon-11 to
obtain ethyl 2-(6-(cyclopropylmethoxy)-5-(3-(methoxy-11C)-
azetidin-1-yl)picolinamido)-2-ethylbutanoate ([11C]RSR056)
and evaluated in rats and in a murine model of neuro-
inflammation.
Received: February 17, 2015
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of 5-Methoxyazetidinepyridine Derivatives 6a−k
a
Reagents and conditions: (a) m-CPBA, CH2Cl2, reflux, 20 h; (b) POCl3, 95 °C, 1 h; (c) (1) cyclopropylmethanol, NaH, 95 °C, 3 h; (2) H2O, rt, 10
min; (d) methoxyazetidine, Cs2CO3, [Pd2(dba)3], BINAP, 110 °C, toluene, 16 h; (e) DIPEA, DMTMM, H2NR1, rt, CH2Cl2, 16 h.
g
Table 1. Lipophilicity (log D), Binding Affinity (Ki), and Potency (EC50) Values of Compounds 6a−k
a
b
c
d
e
f
g
n = 6. n = 8. n = 3. n = 2. n = 4. nd = not determined. The clogP and polar surface area (PSA) are calculated values. If not stated otherwise,
values represent data from a single experiment in triplicate. The asterisk (∗) indicates log D was determined with shake-flask method using
radiolabeled compound.
pot procedure afforded 5-bromo-6-(cyclopropylmethoxy)-
picolinic acid 4 in 92% excellent yield. Compound 4 was
converted in a Buchwald−Hartwig amination with 3-methox-
yazetidine to yield the desired key intermediate 5, which was
coupled to appropriate amine building blocks using DMTMM
to produce the target compounds 6a−k (Table 1) in 18−85%
yield.
RESULTS
■
Chemistry. Commercially available methyl-5-bromopicoli-
nate 1 was treated with m-chloroperoxybenzoic acid to afford
N-oxide 2 in 75% yield (Scheme 1). Reaction of the N-oxide
with POCl3 yielded chlorinated compound 3 in 59% yield via
an addition−elimination reaction. Treatment with the sodium
salt of cyclopropylmethanol and subsequent hydrolysis in a one
B
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a
Scheme 2. Synthesis of 5-Difluoroazetidinepyridine Derivatives 10a−f
a
Reagents and conditions: (a) Na2CO3, CH3I, DMF, rt, 16 h; (b) difluoroazetidine HCl, Cs2CO3, [Pd2(dba)3], BINAP, 70 °C, dioxane, 16 h; (c)
LiOH, THF/H2O, rt, 3 h; (d) DIPEA, DMTMM, H2NR2, rt, CH2Cl2, 16 h.
a
Table 2. Lipophilicity (logD), binding affinity (Ki) and potency (EC50) values of compounds 10a−f
a
Superscript c in the table indicates n = 3.
a
Scheme 3. Synthesis of 5-Cyclopentylpyridine Derivatives 13a−e
a
Reagents and conditions: (a) [Pd(dppf)Cl2], Na2CO3, DMF, 100 °C, 16 h; (b) Pd/C, H2, EtOH, rt, 4 h; (c) DIPEA, DMTMM, H2NR3, CH2Cl2,
rt, 16 h.
As part of our systematic structure−activity relationship
investigation, we replaced the 3-methoxyazetidine substituent
in position 5 of the pyridine with difluoroazetidine, applying the
reaction sequence described in Scheme 1. Scheme 2 illustrates
the synthetic pathway leading to 5-difluoroazetidinylpyridine
derivatives 10a−f starting from picolinic acid 4. Esterification of
the carboxylic functionality was accomplished by reacting the
sodium salt of picolinic acid 4 with methyl iodide at room
temperature. Methyl ester 7 was reacted with difluoroazetidine
using Buchwald−Hartwig reaction conditions to give com-
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a
Table 3. Lipophilicity (log D), Binding Affinity (Ki), and Potency (EC50) Values of Compounds 13a−e
a
Superscript d in the table indicates n = 2.
a
Scheme 4. Synthesis of Precursor 15 and Carbon-11 Labeling of 16
a
Reagents and conditions: (a) DIPEA, HBTU, DMF, rt, 16 h; (b) 3-methoxyazetidine, Cs2CO3, [Pd2(OAc)2], toluene, 110 °C, 16 h; (c) [11C]CH3I,
NaH, DMF, 90 °C, 3 min.
pound 8, which was hydrolyzed under basic conditions to
provide carboxylic acid 9. Coupling with the appropriate
amines afforded target compounds 10a−f (Table 2) in
chemical yields ranging from 22% to 85%.
In a further effort, we replaced the azetidine moiety with a
cyclopentyl group as outlined in Scheme 3. A palladium
catalyzed Suzuki cross-coupling of compound 4 afforded the
cyclopentenyl intermediate 11, which underwent reduction
with hydrogen using 10% palladium on carbon to afford
cyclopentyl analog 12. Coupling of 12 using similar conditions
as described in Scheme 1 or Scheme 2 gave the target
compounds 13a−e (Table 3) in 25−68% yield.
In Vitro Pharmacology. The selectivity and functional
activity of the synthesized compounds toward human CB1 and
CB2 were determined in a cAMP assay according to Ullmer et
al.25 Binding affinity was determined in a competitive binding
assay with membranes prepared from cells expressing human or
mouse CB2 receptors (PerkinElmer). EC50 or Ki values were
calculated from a single experiment using triplicates of 10
different concentrations of compound.
which both displayed 4.4 nM affinity toward hCB2. The
selectivity profile of hCB2 over hCB1 for these compounds was
high and greater than 1000. The binding affinities (Ki values) of
compound 6a to mouse and human CB2 were also high and
comparable to the EC50 value obtained using hCB2. The
replacement of the ethyl ester group in 6a with the N-methyl
group as in compound 6b or N-fluoroalkyl as in compounds 6c
and 6d led to a several-fold decrease in potency. A
fluoroazetidine moiety as in derivative 6e exhibited nanomolar
activity but was also shown to be a substrate for P-glycoprotein
(data not shown). Introducing an oxetane moiety to reduce
lipophilicity as in compound 6f showed a promising effect in
the cAMP assay with an EC50 value of 67 nM, but this value did
not correlate with high binding affinity.
Compounds of the 5-difluoroazetidine series shown in Table
2 exhibited EC50 values of 4 nM to 2.9 μM. Two compounds,
10e and 10f, from this series of compounds were nonbinders.
The N-methylamide analogue 10a exhibited the highest
potency with an EC50 value of 4.3 nM, which, however, did
not translate in high binding affinity to human or mouse CB2.
Compared to the 5-methoxyazetidine series, the N-fluoroethyl
side chain as in compound 10b resulted in a more than 10-fold
increase in potency (EC50 = 27 nM). Oxadiazole compounds
The 5-methoxyazetidine substituted derivatives 6a−k ex-
hibited EC50 values in the range of 4 nM to 4 μM (Table 1).
The most potent compounds in this series were 6a and 6e,
D
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10c−e were among the weakest CB2 ligands tested in this
work.
(4-morpholinyl)ethyl]-1H-indole (GW405833, 17),28 indicat-
ing specific binding of 16 to CB2.
In Vivo Characterization of 16 in Healthy Rats. Baseline
dynamic PET acquisitions were performed with 16 in rats for
60 min. For displacement studies, 17 (1.5 mg/kg) was injected
iv 10 min after injection of the radiotracer. Figure 2A shows
coronal PET images of the spleen (white arrow) under baseline
and displacement conditions. The spleen could be clearly
detected during the first 10 min after injection of 16.
Radioactivity cleared after 10 min under baseline conditions
and more rapidly after displacement with 17. Figure 2B shows
the corresponding time−activity curves (TACs) comparing
tracer uptake in spleen and background (muscle) under
baseline and displacement conditions. Spleen-TACs reached
background levels 15 min after injection of displacer. Tracer
clearance from spleen was considerably enhanced after injection
of 17, indicating competitive displacement of 16 from CB2
binding sites.
To confirm the PET data, biodistribution of 16 was studied
in rats 15 min after iv injection of 6−10 MBq (0.03−0.05
nmol) of the tracer under baseline and blocking conditions
(Figure 3). For blocking conditions, 1.5 mg/kg 17 was injected
iv 1 min prior to tracer (9−10 MBq, 0.04−0.05 nmol, n = 3)
administration. High uptake of 16 was observed in the spleen,
which was reduced by 79% upon blockade. Radioactivity uptake
in the brain was rather low, which is in agreement with low
CB2 levels under normal conditions. Highest activity was
detected in the small intestine and liver as is expected of a
rather lipophilic xenobiotic.
The metabolic stability of 16 was studied in a male Wistar rat
(n = 1). Blood samples were collected at different time points
and after 20 min, brain and urine were collected and analyzed
by radio-TLC. In brain tissue, no activity was detected after 20
min. The percentage of intact parent tracer in plasma decreased
with time to 51% after 5 min, 37% after 10 min, and 21% after
20 min. Radioactivity in urine was fully attributed to
hydrophilic radiometabolites.
In Vivo Characterization of 16 in a Neuroinflamma-
tory Mouse Model. To prove uptake of 16 in brain with
elevated CB2 levels, we used a lipopolysaccharide (LPS) mouse
model of neuroinflammation described by Qin et al.29 but with
a dose of 10 mg/kg LPS based on mRNA and autoradiographic
studies performed in our laboratories.30 Mice were injected
with 10 mg/kg LPS 5 days prior to brain PET scan. Increased
accumulation of 16 was found in the whole brain after LPS
treatment compared to vehicle group (0 mg/kg LPS) (Figure
4). Additionally, 2 mg/kg 17 was subcutaneously injected 30
min prior PET acquisition. Accumulation of 16 in the brain was
remarkably reduced under these blockade conditions but did
not reach baseline levels.
Table 3 summarizes the results obtained for the 5-substituted
cyclopentylpyridine derivatives. The potency of compounds
13a−e on hCB2 was in the range of 2.9 nM to 2.8 μM with
compound 13a as the most potent derivative. The carboxylate
ester 13b was highly active on hCB2, exhibiting an EC50 value
of 9.6 nM, but showed insufficient selectivity of ∼100-fold over
CB1. Introducing small PEG chains as in isoxazol derivatives
13c and 13d resulted in rather low potency with EC50 values of
175 and 176 nM, respectively.
From the series of compounds tested in vitro, compound 6a
from the group of 5-methoxyazetidine substituted pyridine
derivatives emerged as the most promising candidate and was
therefore selected for further evaluation. Important properties
that prompted our decision included high binding affinity
toward mouse and human CB2 receptor, high selectivity, low
polar surface area, stability in buffer, optimal membrane
permeability, non-Pgp substrate, and amenability to radio-
labeling with carbon-11.
Synthesis of Precursor and Radiolabeling. In Scheme 4
are depicted the synthesis of the desmethyl precursor 15 using
Buchwald−Hartwig amination reaction and the radiosynthesis
of [11C]RSR-056 (16). Compound 16 was successfully
obtained by reacting the sodium salt of 15 with [11C]MeI in
>99% radiochemical purity after HPLC purification. Total
synthesis time was ∼35 min from end of bombardment, and
total radioactivity obtained was 4.1
0.7 GBq (n = 22).
Specific activity at end of synthesis amounted to 194
139
GBq/μmol.
In Vitro Characterization of 16. Radiolabeled compound
16 was stable in formulated solution over a time course of 2 h.
Lipophilicity (log D) of 16 was measured in n-octanol/
phosphate buffer at pH 7.4 using the shake-flask method. The
log D7.4 value obtained was 1.94 0.08 (n = 5), which is within
the optimal range of brain penetrating compounds of 1−3.26
The stability of 16 in mouse, rat, and human plasma was
measured at 37 °C over a time course of 40 min. In rat and
mouse plasma, 86% and 84%, respectively, of intact compound
was found. No radioactive degradation products of 16 were
observed in human plasma after 40 min. Sections of rat and
mouse spleen, an organ with high basal CB2 levels,27 were used
for the in vitro autoradiography experiments (Figure 1).
Compound 16 markedly bound to spleen tissue, and the
binding was reduced by co-incubation with 5 μM CB2-selective
compound 1-(2,3-dichlorobenzoyl)-5-methoxy-2-methyl-3-[2-
DISCUSSION
■
CB2 plays a crucial (yet not fully understood) role in
neuroinflammatory diseases. To gain deeper insight into the
pathophysiology on a molecular basis, a specific CB2 PET
tracer would be very useful.
Considering the structure−activity relationship results
described in the patent literature,31 we designed a series of
2,5,6-substituted pyridines as novel potential CB2 PET tracers
based on the fact that various sets of 2-, 5-, and 6-substituted
pyridines show high binding affinity and selectivity toward CB2.
At position 2 of the pyridine ring, carboxamides are well
tolerated, whereas position 5 tolerates smaller lipophilic
Figure 1. Rat and mouse spleen autoradiograms after incubation with
16 (0.2 nM) under baseline and blocking (5 μM 17) conditions.
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Figure 2. In vivo uptake of 16 in rat spleen under baseline and displacement (1.5 mg/kg 17 injected 10 min after radiotracer) conditions. (A)
Coronal 16 PET/CT images. PET images were averaged to three 10 min sequences. White arrow indicates spleen. Color bar indicates standardized
uptake value (detected kBq/cm3 normalized by injected kBq/g body weight, SUV). (B) 16-TACs of spleen and background tissue (muscle) from
baseline and displacement experiments.
always correlated well, and species differences between mouse
and human were observed in some examples. Carboxylic acid
building block 5 was shown to be particularly useful, as its
derivatives possess high binding affinity to CB2 and high
selectivity over CB1. Combination with a fluoroazetidine amine
building block afforded compound 6e with excellent single digit
nanomolar potency on hCB2 and optimal lipophilicity but
suffered from interactions with both human and mouse P-gp
efflux protein. From all the novel compounds evaluated, 6a
exhibited the most promising profile and was therefore selected
for radiolabeling with carbon-11.
Radiolabeling of 16 was accomplished starting from hydroxyl
precursor 15 using [11C]methyl iodide. High chemical purity of
Figure 3. Biodistribution study in rats. Animals were sacrificed 15 min
after injection of 16: 6−10 MBq (0.03−0.05 nmol, n = 2) for baseline
and 9−10 MBq (0.04−0.05 nmol, n = 3) for blocking conditions.
the precursor compound was essential for reliable and high
radiochemical yields of the radiolabeled product. The binding
of 16 was further evaluated in CB2-positive spleen tissues in
vitro and in vivo. Blocking and displacement experiments with
the CB2-selective agonist 17 successfully demonstrated the
specificity of 16 binding to CB2.
substituents such as halogens, methyl, cyclic alkyls, pyrrolidine,
or substituted azetidines. Cycloalkyl or cycloalkylalkoxy are
particularly useful moieties in position 6. On the basis of these
facts, three carboxylic acid key intermediates 5, 9, and 12 were
synthesized. The synthetic pathways toward the three
intermediates were similar. The cyclopropylmethoxy substitu-
ent in position 6 of the pyridine ring was introduced by
chlorination followed by SNAr etherification. In a second step,
palladium catalyzed Suzuki cross-coupling or Buchwald−
Hartwig amination reactions were used to install the
substituents at position 5. Cyclopropyl derivative 12 required
an additional reduction step under hydrogen atmosphere. The
final products were obtained by amide coupling reactions.
Because of the rather low yields of compound 5 in the first
Buchwald reaction (Scheme 1), the carboxylic acid functionality
in compound 4 was protected as methyl ester to afford
compound 7 (Scheme 2), which was used for the amination
reaction. The yield for the amination of 7 was significantly
higher with 70%. The overall yields for the key intermediate
compounds 5, 9, and 12 were 11%, 20%, and 19%, respectively.
Target compounds 6a−k, 10a−f, and 13a−e were synthesized
in 18−85% yields and screened in competition binding assays
with hCB1 and hCB2.
Brain radioactivity was strikingly higher in the LPS-treated
mice than the control mice and was highest in an LPS-treated
mouse (data not shown) that died during the scan, possibly
because of the induced neuroinflammation. The increased
uptake in the neuroinflammation model may result from
elevated CB2 in the inflamed brain or from an increased tracer
influx into the brain due to the disruption of the blood−brain
barrier under neuroinflammation32 or from a combination of
both. As blood−brain barrier disruption is a common
observation in neuroinflammation, tightly linked to the
inflammatory processes, an ideal PET tracer for neuro-
inflammation should be insensitive to the integrity of the
blood−brain barrier. This is best achieved with a tracer with
maximal extraction at the blood−brain barrier, i.e., with a K1
equal to the cerebral plasma flow. Brain accumulation is in this
case dependent on cerebral blood flow but will not be increased
by blood−brain barrier disruption. We are currently investigat-
ing the kinetics of our CB2-targeting tracers regarding these
characteristics.
17 may not be the ideal blocker for the PET experiments, as
it not only blocks tracer binding to the receptor but also affects
inflammatory processes by its function as a CB2 agonist. In
addition, we cannot exclude that under the applied protocol,
CB2 levels were not fully occupied by 17. One hour after
With the exception of compound 13b, all the newly
synthesized compounds exhibited high selectivity over CB1.
In general, potency and binding affinity to hCB2 were not
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Figure 4. Brain uptake of 16 in LPS mouse model of neuroinflammation. (A−C) Coronal brain PET slices (colored, averaged 0.5−60 min pi)
superimposed on an MRI T2 template (gray). (A) Vehicle + vehicle. (B) 10 mg/kg LPS + vehicle. (C) 10 mg/kg LPS + 2 mg/kg 17. (D) Time−
activity curves of the whole brain at the three depicted conditions (M = mouse, n = 2).
intraperitoneal injection, levels in rat brain were about 5-fold
higher than in plasma.33 Extrapolating from the plasma levels of
the same study, this would result in a brain concentration of
>0.1 μM 1 h after intraperitoneal injection. This concentration
should be high enough for blockade despite the possibly lower
affinity of 17 compared to 16 (Ki values of 3.9,33 12,28 or 35
nM 21 to hCB2 compared to 2.5 nM for 16 and a 150-fold
selectivity over hCB1). It should be mentioned here, however,
that the affinity of 17 to murine CB2 is not known and remains
to be tested.
vitro using rodent spleen sections. In vivo studies with PET and
biodistribution studies confirmed also the specificity of 16 to
spleen tissue. To which extent binding to CB2 and disruption
of blood−brain barrier are involved in 16 accumulation in
experimental neuroinflammation remains to be elucidated.
In summary, the novel carbon-11 labeled 2,5,6-substituted
pyridine derivative 16 is a promising radiotracer for imaging
CB2 in rodents and might serve as a tool for the investigation
of CB2 receptor levels in healthy tissues and different brain
disorders in humans.
EXPERIMENTAL SECTION
CONCLUSIONS
■
■
General. All reagents and solvents were purchased from Sigma-
Aldrich Chemie GmbH (Germany), Merck (Germany), Acros
Organics (Belgium), ABCR GmbH (Germany), or Fluka (Switzer-
land). Purity of synthesized compounds was greater than 95% as
determined by analytical HPLC or LC−MS. 1H spectra were obtained
on a Bruker Avance FT-NMR spectrometer. Chemical shifts are given
in δ units, in ppm relative to tetramethylsilane (TMS) (0 ppm).
Multiplicities in the 1H NMR spectra are described as s = singlet, d =
We have successfully designed and synthesized a series of 2,5,6-
substituted pyridines as CB2 ligands with high selectivity over
human CB1. For the most promising compound 6a, a robust
radiosynthesis was established to afford 16 in high yields of
3.0−4.9 GBq, high specific activity of 194 139 GBq/μmol,
and excellent chemical and radiochemical purity of >99%.
Specific binding to CB2-positive tissue was demonstrated in
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doublet, t = triplet, q = quartet, m = multiplet, br = broad peak.
Coupling constants (J) are reported in Hz. Preparative HPLC was
performed with a Merck-Hitachi L7150 pump system and an Ultimate
XB-C18 column (21.2 mm × 150 mm, 5 μm) with an isocratic solvent
system 0.1% TFA in H2O and MeCN at a flow rate of 15 mL/min.
Radiolabeled product was purified with semipreparative HPLC system
using an ACE Symmetry column (C8 5 μm; 7.8 mm × 50 mm) with
an isocratic solvent system 0.1% H3PO4 in H2O (30%) and MeCN
(70%) at a flow rate of 4 mL/min. Radiolabeled product was analyzed
with an Agilent 1100 series HPLC system, equipped with UV
multiwavelength detector and a GabiStar radiodetector (Raytest) using
an ACE C18-AR column (50 mm × 4.6 mm, 3 μm) with the following
conditions: 0.1% TFA in H2O (solvent A), MeCN (solvent B); 0.0−
3.0 min, 30% B; 3.1−13.0 min, 30−95% B; 13.1−15 min, 95% B; flow
rate, 1 mL/min.
Animals. Animal care and experiments were conducted in
accordance with Swiss Animal Welfare legislation and were approved
by the Veterinary Office of Canton Zurich, Switzerland. Five-week-old
male Wistar rats and five-week-old male CD-1 mice were supplied by
Charles River (Sulzberg, Germany). We modified a previously
described model of neuroinflammation,28 by injecting 10 mg/kg
lipopolysaccharide (LPS), Escherichia coli strain O111:B4, or vehicle
(saline) ip into CD1 male mice 5 days prior to PET experiments. This
protocol resulted in increased CB2 mRNA levels in brain with the
identical batch of LPS used in this study (see Discussion).
Chemistry. 5-Bromo-2-(methoxycarbonyl)pyridine 1-Oxide
(2). A mixture of methyl 5-bromopicolinate 1 (50 g, 0.23 mol) and m-
CPBA (80 g, 0.46 mol) in CH2Cl2 (400 mL) was heated to reflux for
20 h. After cooling to rt, the mixture was quenched with saturated
Na2SO3. The organic layer was washed with brine (2 × 200 mL) and
evaporated to dryness. The residue was purified over silica gel with
petroleum ether/EtOAc (6:1) to obtain the product as a brown oil in a
yield of 75%. LC−MS: [MH+] 231.9, tR = 1.10 min.
Methyl 5-Bromo-6-chloropicolinate (3). Compound 2 (30 g,
0.13 mol) was added slowly to POCl3 (100 g) at 0 °C over 1 h, then
the mixture was heated to 95 °C for 1 h. After cooling to rt, the
mixture was evaporated and the residue was dissolved in water (50
mL). The resulting solution was extracted with EtOAc (3 × 50 mL)
and the combined organic layers were removed under reduced
pressure to obtain the product as white solid in 59% yield without
further purification. LC−MS: [MH+] 249.9, tR = 1.48 min.
5-Bromo-6-(cyclopropylmethoxy)picolinic Acid (4). Sodium
hydride (70%, 0.43 g, 13 mmol) was added to cyclopropylmethanol
(11 mL) at 0 °C and the mixture was stirred for 1 h at rt. Compound 3
(0.2 g, 1 mmol) was added, and the solution was heated to 95 °C for 3
h. The mixture was evaporated to dryness, and the residue was
dissolved in water (10 mL). The resulting solution was adjusted to pH
≈ 6 with 3 M HCl and the resulting precipitates were filtered to afford
the desired compound in 92% yield without further purification as
white powder. LC−MS: [MH+] 272.0, tR = 1.51 min.
6-(Cyclopropylmethoxy)-5-(3-methoxyazetidin-1-yl)-
picolinic Acid (5). 3-Methoxyazetidine (38 mg, 0.44 mmol), BINAP
(23 mg, 0.037 mmol), [Pd2(dba)3] (17 mg, 0.02 mmol), Cs2CO3 (240
mg, 0.735 mmol) were added to a solution of 4 (100 mg, 0.37 mmol)
in toluene (4 mL). The reaction mixture was vigorously stirred and
heated to 110 °C for 16 h. Solvent was removed under reduced
pressure, and the residue was dissolved in water and washed with ethyl
acetate (30 mL). The aqueous layer was adjusted to pH ≈ 2 using 1 M
HCl, and the resulting precipitate was collected by filtration. The
crude product was purified by column chromatography over silica gel
using petroleum ether/EtOAc (1:2) to give 5 in a yield of 27% as a
yellow solid. LC−MS: 265.2 [MH+], tR = 1.32 min.
EtOAc (20:1) to give 7 in a yield of 72% as a white solid. MS (EI):
286.0 [MH+], tR = 1.75 min.
Methyl 6-(Cyclopropylmethoxy)-5-(3,3-difluoroazetidin-1-
yl)picolinate (8). A mixture of compound 7 (1 g, 3.5 mmol), 3,3-
difluoroazetidine hydrochloride (0.5 g, 3.9 mmol), [Pd2(dba)3] (64
mg, 0.07 mmol), BINAP (88 mg, 0.14 mmol), and Cs2CO3 (2.28 g, 7
mmol) in toluene (40 mL) was stirred at 110 °C for 16 h. The solvent
was removed, and the residue was dissolved in water (30 mL) and
extracted using EtOAc (3 × 20 mL). The combined organic layers
were washed with brine (20 mL), dried over Na2SO4, and removed
under reduced pressure. The residue was purified over silica gel using
petroleum ether/EtOAc (20:1) to give the target compound 8 in
quantitative yield as a white solid. LC−MS: 299.1 [MH+], tR = 1.68
min.
6-(Cyclopropylmethoxy)-5-(3,3-difluoroazetidin-1-yl)-
picolinic Acid (9). A solution of compound 7 (1.0 g, 3.4 mmol) and
LiOH monohydrate (0.85 g, 13.6 mmol) in THF/H2O (30 mL) was
stirred at rt for 3 h. After removal of the organic solvent, water (20
mL) was added to the residue and pH was adjusted to ∼2 using 6 M
HCl. The solution was extracted with EtOAc (3 × 15 mL). The
combined organic phases were washed with brine (20 mL), dried over
Na2SO4 and solvents were removed under reduced pressure to give 9
in quantitative yields without further purification as white solid. LC−
MS: 285.1 [MH+], tR = 1.56 min.
5-Bromo-6-(cyclopropylmethoxy)picolinic Acid (11). A mix-
ture of 4 (0.2 g, 0.74 mmol), 2-cyclopentenyl-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane (171 mg, 0.9 mmol), [Pd(dppf)Cl2] (30 mg, 0.037
mmol), and Na2CO3 (2 M, 4 mL) in DMF (5 mL) was heated to 100
°C for 16 h. After cooling to rt, the solution was adjusted to pH ≈ 3
using 3 M HCl and extracted with EtOAc (2 × 5 mL). The combined
organic layers were washed with water (2 × 10 mL) and brine (10
mL) and evaporated to dryness. The residue was purified over silica gel
chromatography using petroleum ether/EtOAc (6:1) to obtain the
product as a white solid in 58% yield. LC−MS: 260.2 [MH+], tR = 1.73
min.
5-Cyclopentyl-6-(cyclopropylmethoxy)picolinic Acid (12). A
mixture of 11 (0.95 g, 4 mmol) and Pd/C (10%, 0.2 g) in EtOH (30
mL) was stirred under a H2 atmosphere for 4 h at rt. The mixture was
filtered and solvents were removed under reduced pressure to give the
desired product as a white solid in a yield of 80% without further
purification. LC−MS: 262.1 [MH+], tR = 1.75 min.
Compounds 6a−k, 10a−f, and 13a−e. General Procedure.
To a solution of key intermediate 5, 9, or 12 (20 mg, 1.0 eq ) in
CH2Cl2 (1 mL) were added DIPEA (2.5 equiv) and DMTMM (1.1
equiv). The mixture was stirred for 30 min at rt before the appropriate
amine building block was added (1.0 eq uiv). The mixture was stirred
at rt for 16 h. The reaction was diluted with CH2Cl2 (5 mL) and
washed with 0.2 M aq HCl (3 × 10 mL) and brine (15 mL). The
organic layer was dried over MgSO4 and removed under reduced
pressure. The residue was purified with flash chromatography over
silica gel to give the desired compounds 6a−k, 10a−f, and 13a−e in
yields in the range of 18−85%.
Ethyl 2-(6-(Cyclopropylmethoxy)-5-(3-methoxyazetidin-1-
yl)picolinamido)-2-ethylbutanoate (6a). Purification was accom-
1
plished using heptane/EtOAc (4:1). Yield: 83% as a colorless oil. H
NMR (300 MHz, CDCl3): δ 8.62 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H),
6.59 (d, J = 7.8 Hz, 1H), 4.31−4.25 (m, 7H), 3.90−3.83 (m, 2H), 2.94
(s, 3H), 2.67−2.55 (m, 2H), 1.93−1.81 (m, 2H), 1.36−1.29 (m, 4H),
1.29−1.24 (m, 1H), 0.77 (t, J = 7.5 Hz, 6H), 0.64−0.57 8m, 2H),
0.43−0.38 (m, 2H). LC−MS (ESI): 420.7 [MH+].
6-(Cyclopropylmethoxy)-5-(3-methoxyazetidin-1-yl)-N-(3-
(methylcarbamoyl)pentan-3-yl)picolinamide (6b). Purification:
Methyl 5-Bromo-6-(cyclopropylmethoxy)picolinate (7). To a
solution of compound 4 (0.4 g, 1.5 mmol) in DMF were added
Na2CO3 (0.16 g, 1.5 mmol) and CH3I (0.42 g, 3 mmol). The mixture
was stirred at rt for 16 h. Water was poured into the reaction mixture,
and the resulting mixture was extracted with EtOAc (3 × 30 mL). The
combined organic layers were washed with water and brine, dried over
Na2SO4, and removed under reduced pressure. The residue was
purified by chromatography over silica gel using petroleum ether/
1
heptane/EtOAc (1:1). Yield: 81% as white powder. H NMR (300
MHz, CDCl3): δ 8.48 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 6.59 (d, J = 7.9
Hz, 1H), 6.50 (br s, 1H), 4.34−4.26 (m, 3H), 4.23 (d, J = 7.1 Hz, 2H),
3.94−3.83 (m, 2H), 3.34 (s, 3H), 2.87 (d, J = 4.8 Hz, 3H), 2.49−2.36
(m, 2H), 1.90−1.76 (m, 2H), 1.36−1.29 (m, 1H), 0.85 (t, J = 7.4 Hz,
6H), 0.65−0.56 (m, 2H), 0.42−0.35 (m, 2H). MS (ESI): 405.5
[MH+].
H
DOI: 10.1021/acs.jmedchem.5b00283
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
6-(Cyclopropylmethoxy)-N-(3-((2-fluoroethyl)carbamoyl)-
pentan-3-yl)-5-(3-methoxyazetidin-1-yl)picolinamide (6c). Pu-
rification: heptane/EtOAc (2:1). Yield: 54% as white powder. 1H
NMR (300 MHz, CDCl3) δ 8.45 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H),
7.64 (d, J = 8.0 Hz, 1H), 6.75−6.88 (m, 1H), 4.35−4.46 (m, 3H),
4.26−4.34 (m, 4H), 4.22 (d, J = 7.3 Hz, 2H), 4.05 (s, 3H), 3.84−3.92
(m, 3H), 3.55−3.73 (m, 2H), 2.35−2.53 (m, 2H), 1.77−1.92 (m, 2,
H), 1.11−1.18 (m, 1H), 0.85 (t, J = 7.4 Hz, 6H), 0.49−0.59 (m, 2H),
0.34−0.43 (m, 2H). LC−MS (ESI): 435.3 [M − H−].
6-(Cyclopropylmethoxy)-5-(3,3-difluoroazetidin-1-yl)-N-(3-
(methylcarbamoyl)pentan-3-yl)picolinamide (10a). Purification:
heptane/EtOAc (2:1). Yield: 82% as colorless oil. 1H NMR (300
MHz, CDCl3): δ 8.61 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 6.65 (d, J = 7.8
Hz, 1H), 6.35−6.24 (m, 1H), 4.39 (t, J = 12.0 Hz, 4H), 4.26 (d, J = 7.3
Hz, 2H), 2.89 (d, J = 4.8 Hz, 3H), 2.59−2.45 (m, 2H), 1.84−1.70 (m,
2H), 1.38−1.21 (m, 2H), 0.83 (t, J = 7.4 Hz, 6H), 0.67−0.60 (m, 2H),
0.43−0.37 (m, 2H). LC−MS (ESI): 432.5 [MNa+].
6-(Cyclopropylmethoxy)-5-(3,3-difluoroazetidin-1-yl)-N-(3-
((2-fluoroethyl)carbamoyl)pentan-3-yl)picolinamide (10b). Pu-
rification: heptane/EtOAc (2:1). Yield: 22% as a white solid. 1H NMR
(300 MHz, CDCl3): δ 8.55 (s, 1H), 7.68 (d, J = 7.9 Hz, 1H), 6.57−
6.68 (m, 2H), 4.60 (t, J = 4.8 Hz, 1H), 4.32−4.49 (m, 5H), 4.25 (d, J =
7.3 Hz, 2H), 3.66−3.73 (m, 1H), 3.57−3.65 (m, 1H), 2.46−2.60 (m,
2H), 1.73−1.88 (m, 2H), 1.27−1.34 (m, 1H), 0.84 (t, J = 7.5 Hz, 6H),
0.59−0.68 (m, 2H), 0.34−0.47 (m, 2H). LC−MS (ESI): 443.4
[MH+].
6-(Cyclopropylmethoxy)-5-(3,3-difluoroazetidin-1-yl)-N-(2-
(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)propan-2-yl)picolinamide
(10c). Purification: heptane/EtOAc (2:1). Yield: 85% as a white solid.
1H NMR (300 MHz, CDCl3): δ 9.22 (s, 1H), 8.75 (d, J = 3.6 Hz, 1H),
8.33 (dt, J1 = 1.9, J2 = 8.1 Hz, 1H), 8.16 (s, 1H), 7.64 (d, J = 7.8 Hz,
1H), 7.43 (dd, J1 = 5.1, J2 = 7.8 Hz, 1H), 6.63 (d, J = 7.8 Hz, 1H), 4.40
(t, J = 11.9 Hz, 4H), 4.24 (d, J = 7.3 Hz, 2H), 1.96 (s, 6H), 1.36−1.29
(m, 1H), 0.72−0.65 (m, 2H), 0.45−0.37 (m, 2H). MS (ESI): 470.5
[M+].
6-(Cyclopropylmethoxy)-N-(3-((3-fluoropropyl)carbamoyl)-
pentan-3-yl)-5-(3-methoxyazetidin-1-yl)picolinamide (6d). Pu-
1
rification: CH2Cl2/MeOH (140:1). Yield: 54% as white powder. H
NMR (300 MHz, CDCl3) δ 8.44 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H),
6.74 (t, J = 5.4 Hz, 1H), 6.58 (d, J = 7.9 Hz, 1H), 4.61 (t, J = 5.7 Hz,
1H), 4.46 (t, J = 5.7 Hz, 1H), 4.25−4.35 (m, 3H), 4.22 (d, J = 7.1 Hz,
2H), 3.83−3.92 (m, 2H), 3.47 (q, J = 6.5 Hz, 2H), 3.34 (s, 3H), 2.34−
2.49 (m, 2H), 1.74−2.04 (m, 4, H), 1.28−1.33 (m, 1H), 0.84 (t, J =
7.4 Hz, 6H), 0.57−0.65 (m, 2H), 0.35−0.42 (m, 2H). LC−MS (ESI):
451.5 [MH+].
6-(Cyclopropylmethoxy)-N-(3-(3-fluoroazetidine-1-
carbonyl)pentan-3-yl)-5-(3-methoxyazetidin-1-yl)picolinamide
(6e). Purification: heptane/EtOAc (2:1). Yield: 18% as white powder.
1H NMR (300 MHz, CDCl3) δ 8.66 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H),
6.58 (d, J = 7.9 Hz, 1H), 5.35−5.24 (m, 1H), 4.18−4.55 (m, 9H),
3.81−3.92 (m, 2H), 3.34 (s, 3H), 2.52−2.66 (m, 2H), 1.68−1.80 (m,
2H), 1.29−1.32 (m, 1H), 0.86−0.79 (m, 6H), 0.56−0.64 (m, 2H),
0.36−0.44 (m, 2H). LC−MS (ESI): 449.3 [MH+].
6-(Cyclopropylmethoxy)-5-(3,3-difluoroazetidin-1-yl)-N-(1-
(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-2-methylpropan-2-
yl)picolinamide (10d). Purification: heptane/EtOAc (4:1 to 1:1).
N-(3-(2-Amino-2-oxoethyl)oxetan-3-yl)-6-(cyclopropylme-
thoxy)-5-(3-methoxyazetidin-1-yl)picolinamide (6f). Purifica-
1
Yield: 53% as a colorless oil. H NMR (300 MHz, CDCl3): δ 7.83−
1
tion: CH2Cl2/MeOH (20:1). Yield: 85% as white powder. H NMR
7.79 (m, 2H), 7.73 (d, J = 7.9 Hz, 1H), 7.65 (s, 1H), 7.10−7.05 (m,
2H), 6.66 (d, J = 7.9 Hz, 1H), 4.37 (t, J = 4.4 Hz, 4H), 4.04 (d, J = 7.3
Hz, 2H), 3.56 (s, 2H), 1.60 (s, 3H), 1.21−1.13 (m, 1H), 0.61−0.54
(m, 2H), 0.32−0.27 (m, 2H). LC−MS (ESI): 502.6 [MH+].
(300 MHz, CDCl3): δ 8.17 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 6.54 (d, J
= 8.0 Hz, 1H), 5.84 (br s, 1H), 5.41 (br s, 1H), 4.88 (d, J = 7.1 Hz,
2H), 4.75 (d, J = 7.1 Hz, 2H), 4.34−4.25 (m, 3H), 4.15 (d, J = 7.3 Hz,
2H), 3.93−3.84 (m, 2H), 3.34 (s, 3H), 3.21 (s, 2H), 1.34−1.20 (m,
1H), 0.65−0.58 (m, 2H), 0.41−0.34 (m, 2H). LC−MS (ESI): 391.2
[MH+].
6-(Cyclopropylmethoxy)-5-(3,3-difluoroazetidin-1-yl)-N-(2-
(5-phenyl-1,3,4-oxadiazol-2-yl)propan-2-yl)picolinamide (10e).
1
Purification: heptane/EtOAc (4:1). Yield: 66% as a colorless oil. H
6-(Cyclopropylmethoxy)-N-(3-(1-hydroxy-2-methylpropan-
2-yl)isoxazol-5-yl)-5-(3-methoxyazetidin-1-yl)picolinamide
(6g). Purification: heptane/EtOAc (2:1). Yield: 76% as white solid. 1H
NMR (300 MHz, CDCl3): δ 7.60 (d, J = 7.1 Hz, 1H), 6.47 (d, J = 8.1
Hz, 1H), 5.20 (s, 1H), 4.36−4.29 (m, 5H), 4.28 (s, 2H), 4.25−4.20
(m, 2H), 3.99−3.87 (m, 2H), 3.34 (s, 3H), 1.39 (s, 6H), 1.31−1.27
(m, 1H), 0.62−0.54 (m, 2H), 0.38−0.32 (m, 2H). LC−MS (ESI):
417.7 [MH+].
NMR (300 MHz, CDCl3): δ 8.29 (s, 1H), 8.03 (dd, J1 = 1.7, J2 = 7.8
Hz, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.58−7.41 (m, 3H), 6.63 (d, J = 7.8
Hz, 1H), 4.40 (t, J = 12.0 Hz, 4H), 4.25 (d, J = 7.3 Hz, 2H), 1.96 (s,
6H), 1.36−1.29 (m, 1H), 0.72−0.63 (m, 2H), 0.48−0.36 (m, 2H).
LC−MS (ESI): 470.6 [MH+].
N-(2-(5-Cyclopropyl-1,3,4-oxadiazol-2-yl)propan-2-yl)-6-(cy-
clopropylmethoxy)-5-(3,3-difluoroazetidin-1-yl)picolinamide
(10f). Purification: heptane/EtOAc (1:1 to 1:2). Yield: 60% as a white
1
6-(Cyclopropylmethoxy)-N-(1-(5-(4-fluorophenyl)-1,3,4-oxa-
diazol-2-yl)-2-methylpropan-2-yl)-5-(3-methoxyazetidin-1-yl)-
picolinamide (6h). Purification: heptane/EtOAc (1:1). Yield: 62% as
solid. H NMR (300 MHz, CDCl3): δ 7.82 (s, 1H), 7.70 (d, J = 7.9
Hz, 1H), 6.62 (d, J = 7.9 Hz, 1H), 4.40 (t, J = 11.9 Hz, 4H), 4.15 (d, J
= 7.3 Hz, 2H), 1.69 (s, 6H), 1.52−1.38 (m, 1H), 1.34−1.28 (m, 1H),
1.06−0.99 (m, 2H), 0.85−0.77 (m, 2H), 0.70−0.62 (m, 2H), 0.41−
0.34 (m, 2H). MS (ESI): 434.5 [MH+].
Ethyl 2-(5-Cyclopentyl-6-(cyclopropylmethoxy)-
picolinamido)-2-ethylbutanoate (13a). Purification: heptane/
EtOAc (4:1 to 2:1). Yield: 68% as a white solid. 1H NMR (300
MHz, CDCl3): δ 8.87 (s, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.56 (d, J = 7.5
Hz, 1H), 4.34−4.25 (m, 4H), 3.30−3.19 (m, 1H), 2.69−2.55 (m, 2H),
2.12−2.00 (m, 2H), 1.96−1.56 (m, 8H), 1.36−1.31 (m, 4H), 0.78 (t, J
= 7.5 Hz, 6H), 0.66−0.55 (m, 2H), 0.46−0.39 (m, 2H). MS (ESI):
402.6 [M+].
2-(5-Aminoisoxazol-3-yl)-2-methylpropyl-5-cyclopentyl-6-
cyclopropylmethoxy)picolinate (13b). Purification: heptane/
EtOAc (4:1). Yield: 67% as a white solid. 1H NMR (300 MHz,
CDCl3): δ 7.63−7.56 (m, 1H), 7.54−7.48 (m, 1H), 5.21 (s, 1H), 4.32
(s, 4H), 4.27 (d, J = 7.1 Hz, 2H), 3.31−3.18 (m, 1H), 2.14−1.97 (m,
2H), 1.84−1.75 (m, 2H), 1.75−1.66 (m, 2H), 1.65−1.56 (m, 2H),
1.41 (s, 6H), 1.35−1.29 (m, 1H), 0.62−0.54 (m, 2H), 0.40−0.34 (m,
2H). LC−MS (ESI): 400.7 [MH+].
5-Cyclopentyl-6-(cyclopropylmethoxy)-N-(3-(1-(2-methox-
yethoxy)-2-methylpropan-2-yl)isoxazol-5-yl)picolinamide
(13c). Purification: heptane/EtOAc (2:1). Yield: 26% as a colorless oil.
1H NMR (300 MHz, CDCl3): δ 10.19 (s, 1H); 7.79 (d, J = 7.4 Hz,
1
a colorless oil. H NMR (300 MHz, CDCl3): δ 7.79−7.74 (m, 2H),
7.71 (d, J = 7.9 Hz, 1H), 7.59 (s, 1H), 7.09−1.02 (m, 2H), 6.61 (d, J =
7.9 Hz, 1H), 4.35−4.26 (m, 3H), 3.99 (d, J = 7.1 Hz, 2H), 3.87−3.84
(m, 2H), 3.58 (s, 2H), 3.35 (s, 3H), 1.58 (s, 6H), 1.19−1.11 (m, 1H),
0.56−0.50 (m, 2H), 0.29−0.24 (m, 2H). LC−MS (ESI): 496.7
[MH+].
6-(Cyclopropylmethoxy)-N-((1-hydroxycyclohexyl)methyl)-
5-(3-methoxyazetidin-1-yl)picolinamide (6i). Purification: hep-
tane/EtOAc (2:1 to 1:1). Yield: 65% as a colorless oil. 1H NMR (300
MHz, CDCl3): δ 7.86 (t, J = 6.2 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H),
6.58 (d, J = 7.9 Hz, 1H), 4.34−4.25 (m, 3H), 4.14 (d, J = 7.1 Hz, 2H),
3.92−3.83 (m, 2H), 3.46 (d, J = 6.2 Hz, 2H), 3.35 (s, 3H), 2.62 (s,
1H), 1.67−1.42 (m, 10H), 1.34−1.29 (m, 1H), 0.66−0.56 (m, 2H),
0.39−0.32 (m, 2H). LC−MS (ESI): 390.7 [MH+].
N-(1-(5-Cyclobutyl-1,3,4-oxadiazol-2-yl)-2-methylpropan-2-
yl)-6-(cyclopropylmethoxy)-5-(3-methoxyazetidin-1-yl)-
picolinamide (6k). Purification: heptane/EtOAc (1:1). Yield: 38% as
white solid. 1H NMR (300 MHz, CDCl3): δ 7.67 (d, J = 8.0 Hz, 1H),
7.61 (s, 1H), 6.59 (d, J = 8.0 Hz, 1H), 4.34−4.25 (m, 3H), 4.10 (d, J =
7.3 Hz, 2H), 3.88−3.81 (m, 2H), 3.60 (quin, J = 8.3 Hz, 1H), 3.46 (s,
2H), 3.34 (s, 3H), 2.31−2.12 (m, 4H) 2.07−1.93 (m, 1H), 1.92−1.77
(m, 1H), 1.55 (s, 6H), 0.91−0.81 (m, 1H), 0.65−0.57 (m, 2H), 0.39−
0.32 (m, 2H). LC−MS (ESI): 456.7 [MH+].
1H), 7.64 (d, J = 7.4 Hz, 1H), 6.49 (s, 1H), 4.24 (d, J = 6.9 Hz, 2H),
I
DOI: 10.1021/acs.jmedchem.5b00283
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3.62−3.51 (m, 6H), 3.37 (s, 3H), 3.32−3.25 (m, 1H), 2.14−2.04 (m,
2H), 1.84−1.57 (m, 6H), 1.48−1.41 (m, 1H), 1.36 (s, 6H), 0.69−0.63
(m, 2H), 0.44−0.39 (m, 2H). LC−MS (ESI): 458.7 [MH+].
the excitation at 355 nm and at the emission with a delay of 100 ns and
a gate of 100 ns, total exposure time of 10 s at 730 nm (bandwidth 30
nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is
calculated as follows: FRET = T730 − Alexa730 − P(T645 − B645)
with P = (Ru730 − B730)/(Ru645 − B645), where T730 is the test
well measured at 730 nM, T645 is the test well measured at 645 nm,
B730 and B645 are the buffer controls at 730 and 645 nm, respectively.
cAMP content is determined from the function of a standard curve
spanning from 10 μM to 0.13 nM cAMP. EC50 values were determined
using Activity Base analysis (ID Business Solution, Limited). The EC50
values for a wide range of cannabinoid agonists or inverse agonists
generated from this assay for reference compounds were in agreement
with the values published in the scientific literature.
Radioligand Binding Assay. The binding affinity was determined
using membrane preparations from CHO cells expressing the human
CB1 or CB2 or murine CB2 receptor in conjunction with [3H]CP-
55,940 (PerkinElmer) as radioligand. Binding was performed in
binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5%
fatty acid free BSA, pH 7.4, for CB1 receptor and 50 mM Tris, 5 mM
MgCl2, 2.5 mM EGTA, and 0.1% fatty acid free BSA, pH 7.4 for CB2
receptor) in a total volume of 0.2 mL for 1 h at 30 °C shaking. The
reaction was terminated by rapid filtration through microfiltration
plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate;
Packard). Bound radioactivity was analyzed for Ki using nonlinear
regression analysis (Activity Base, ID Business Solution, Limited), with
the Kd values for [3H]CP55,940 determined from saturation
experiments.31
5-Cyclopentyl-6-(cyclopropylmethoxy)-N-(3-(1-(2-ethoxye-
thoxy)-2-methylpropan-2-yl)isoxazol-5-yl)picolinamide (13d).
Purification: heptane/EtOAc (20:1 to 9:1). Yield: 25% as a colorless
oil. 1H NMR (300 MHz, CDCl3): δ 10.20 (s, 1H), 7.79 (d, J = 7.5 Hz,
1H), 7.64 (d, J = 7.5 Hz, 1H), 6.49 (s, 1H), 4.24 (d, J = 6.9 Hz, 2H),
3.63−3.48 (m, 8H), 3.35−3.22 (m, 1H), 2.15−2.01 (m, 2H), 1.88−
1.59 (m, 6H), 1.36 (s, 6H), 1.19 (t, J = 7.0 Hz, 3H), 0.70−0.62 (m,
2H), 0.45−0.38 (m, 2H). LC−MS (ESI): 472.8 [MH+].
5-Cyclopentyl-6-(cyclopropylmethoxy)-N-(1-(5-(4-fluoro-
phenyl)-1,3,4-oxadiazol-2-yl)-2-methylpropan-2-yl)-
picolinamide (13e). Purification: heptane/EtOAc (4:1 to 2:1). Yield:
1
49% as a white solid. H NMR (300 MHz, CDCl3): δ 7.85 (s, 1H),
7.80−7.76 (m, 2H), 7.73 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H),
7.07−7.00 (m, 2H), 4.04 (d, J = 7.0 Hz, 2H), 3.58 (s, 2H), 3.27−3.16
(m, 1H), 2.09−2.00 (m, 2H), 1.88−1.64 (m, 6H), 1.60 (s, 6H), 1.20−
1.14 (m, 1H), 0.55−0.49 (m, 2H), 0.30−0.25 (m, 2H). LC−MS
(ESI): 479.7 [MH+].
Ethyl 2-(5-Bromo-6-(cyclopropylmethoxy)picolinamido)-2-
ethylbutanoate (14). To a solution of 4 (423 mg, 1.56 mmol) in
DMF (10 mL) was added DIPEA (0.731 mL, 4.19 mmol), and the
mixture is stirred at rt for 30 min. HBTU (1043 mg, 2.75 mmol) was
added portionwise, and after 1 h, ethyl 2-amino-2-ethylbutanoate
hydrochloride (234 mg, 1.196 mmol) was added. The mixture is
stirred for 16 h, diluted with EtOAc (100 mL), and washed with 0.2 M
HCl (3 × 15 mL), water (15 mL), and then brine (20 mL). Solvents
were evaporated under reduced pressure and the residue was purified
over silica gel using hexane/EtOAc (10:1) and additionally via
preparative HPLC using isocratic MeCN/0.1% TFA in water (70:30)
to give compound 14 in a yield of 40% as a yellow oil. 1H NMR (300
MHz, CDCl3): δ 8.85 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 7.8
Hz, 1H), 4.37 (d, J = 7.0 Hz, 2H), 4.30 (q, J = 7.1 Hz, 2H), 2.61−2.55
(m, 2H), 1.94−1.85 (m, 2H), 1.42−1.31 (m, 4H), 0.77 (t, J = 7.4 Hz,
6H), 0.66−0.61 (m, 2H), 0.49−0.45 (m, 2H). MS (ESI): 414.4
[MH+].
Radiochemistry. [11C]CO2 was produced via the 14N(p,α)11C
nuclear reaction by bombardment of a nitrogen gas target fortified
with 0.5% oxygen using a Cyclone 18/9 cyclotron (18 MeV; IBA,
Belgium). After reduction over a supported nickel catalyst to
[11C]CH4 and subsequent gas phase iodination, [11C]CH3I was
bubbled through a mixture of hydroxyl precursor 15 (0.7 mg) and
sodium hydride (1 mg/mL in DMF, 0.4 mL) in DMF (0.2 mL). The
mixture was heated to 90 °C for 3 min. After dilution with water (1.4
mL), the crude product was purified using semipreparative HPLC.
The collected product was diluted with water (10 mL), trapped on a
C18 cartridge (Waters, preconditioned with 5 mL of EtOH and 10 mL
of water), washed with water (5 mL), and eluted with EtOH (0.5 mL).
For formulation of the final product 16, saline (9.5 mL) was added to
give an ethanol concentration of 5%. For quality control, an aliquot of
the formulated solution was injected into an analytical HPLC system.
The product was confirmed by co-injection of cold reference
compound 6a. Specific activity of the radiolabeled product was
calculated by comparison of UV peak intensity with a calibration curve
of the cold reference compound.
Determination of log D7.4. The partition coefficient D was
determined by the shake-flask method as previously reported.34 In
brief, n-octanol saturated with phosphate buffer, pH 7.4 (0.5 mL), and
phosphate buffer saturated with n-octanol (0.5 mL) were mixed with
formulated 16 (20 μL, 8 MBq). The samples were shaken for 15 min
and centrifuged at 5000g for 5 min. Radioactivity concentrations of
both phases were measured in a γ counter (Wizard, PerkinElmer) and
D was calculated as their ratio.
In Vitro Autoradiography. Mouse and rat spleen tissue were
embedded in TissueTek and cut into 20 μm thick sections on a
cryostat. The slices were absorbed on SuperFrost Plus slides (Menzel)
and stored at −80 °C until use. For the experiment, the slices were
thawed on ice for 10 min before conditioning in incubation buffer (50
mM Tris/HCl, 5% BSA, pH 7.4) on ice for 10 min. Excess solution
was carefully removed, and the tissue slices were dried for 10 min. The
slices were then dripped with 600 μL of incubation buffer containing
0.2 nM 16 and incubated for 15 min at rt in a humid chamber. For
blockade conditions, the slices were dripped with 600 μL of a mixture
of radioligand (0.2 nM) and 17 (5 μM). After incubation, the slices
were washed with washing buffer (50 mM Tris/HCl, 1% BSA, 5%
EtOH, pH 7.4) for 2 min (2×) and distilled water for 5 s (2×) on ice.
After drying, the slices were exposed (30 min) to appropriate
phosphor imager plates (Fuji) and the films were scanned in a
BAS5000 reader (Fuji).
Precursor Compound Ethyl 2-(6-(Cyclopropylmethoxy)-5-(3-
hydroxyazetidin-1-yl)picolinamido)-2-ethylbutanoate (15). In
a 2.5 mL vial were combined 14 (170 mg, 0.411 mmol), azetidin-3-ol
HCl (54.1 mg, 0.494 mmol), diacetoxypalladium (9.23 mg, 0.041
mmol), and Cs2CO3 (335 mg, 1.028 mmol) in toluene (2.2 mL). The
reaction mixture was vigorously stirred at 110 °C for 16 h. The
reaction mixture was filtered on a pad of Celite, and the filtrate was
diluted with EtOAc (25 mL). The resulting solution was washed with
0.2 M HCl (3 × 10 mL). Solvents were dried over MgSO4 and
removed under reduced pressure. The crude material was prepurified
over silica gel using hexane/EtOAc (4:1 to 2:1) and finally via
preparative HPLC using ACN/0.1% TFA in water (50:50) to give 15
1
in a yield of 31% as a yellowish oil. H NMR (300 MHz, CDCl3): δ
8.64 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 6.57 (d, J = 7.9 Hz, 1H), 4.78−
4.69 (m, 1H), 4.40−4.30 (m, 2H), 4.30−4.21 (m, 4H), 3.83 (m, 2H),
2.72−2.45 (m, 3H), 1.94−1.79 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H),
1.29−1.24 (m, 1H), 0.77 (t, J = 7.5 Hz, 6H), 0.65−0.56 (m, 2 H)
0.44−0.36 (m, 2H). MS (ESI): 405.5 [MH+].
Functional Adenylcyclase Assay. CHO cells expressing human
CB1 or CB2 receptors were seeded 1 day prior to the experiment in
DMEM (Invitrogen no. 31331), 1× HT supplement, with 10% fetal
calf serum and incubated at 5% CO2 and 37 °C in a humidified
incubator. The growth medium was exchanged with Krebs Ringer
bicarbonate buffer with 1 mM IBMX and incubated at 30 °C for 30
min. Compounds to test were added to a final assay volume of 100 μL
and incubated for 30 min at 30 °C. By use of the cAMP-Nano-TRF
detection kit (Roche Diagnostics), the assay was stopped by the
addition of 50 μL of lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5%
NP40, 10% NaN3) and 50 μL of detection solutions (20 μM mAb
Alexa700-cAMP 1:1, and 48 μM ruthenium-2-AHA-cAMP) and
shaken for 2 h at rt. The time-resolved energy transfer is measured
by a TRF reader (Evotec Technologies GmbH), equipped with a
ND:YAG laser as excitation source. The plate is measured twice with
J
DOI: 10.1021/acs.jmedchem.5b00283
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
In Vitro Stability. Mouse, rat, and human plasma (400 μL) were
incubated with 16 formulated solution (10 μL, 4 MBq) at 37 °C under
shaking. At 5, 10, 15, and 20 min, aliquots (100 μL) were collected and
the reaction was stopped with 100 μL of ice-cooled CH3CN. The
samples were centrifuged (3 min, 5000 rpm), and the supernatant was
collected and analyzed by radio-TLC Instant Imager (Packard,
Canberra Company) using hexane/EtOAc (1:1).
AUTHOR INFORMATION
■
Corresponding Authors
*U.G.: phone, 0041 61 688 54 46; e-mail, uwe.grether@roche.
com.
*S.M.A.: phone, 0041 44 6337463; e-mail, simon.ametamey@
pharma.ethz.ch.
In Vivo Metabolic Stability. In vivo stability was performed in a
healthy Wistar rat. Radiotracer solution (48 MBq, 0.19 nmol) was
injected via tail vein, and blood was collected at 5, 10, and 20 min.
After 20 min the rat was sacrificed, urine was collected, and brain was
removed. Plasma was separated from the blood cells by centrifugation
at 5000g for 5 min at 4 °C. Proteins of plasma and urine were
precipitated with ice-cold CH3CN and then separated by centrifuga-
tion. The brain tissue was homogenized in PBS (2 mL), and cold
CH3CN was added to precipitate proteins followed by centrifugation.
Supernatants were analyzed by radio-TLC using hexane/EtOAc (1:1).
Results are expressed as percentage of total activity.
Biodistribution Studies. For biodistribution studies in male
Wistar rats, an amount of 6−10 MBq (0.03−0.05 nmol) of radiotracer
solution was administered via tail vein injection into Wistar rats (n =
3). For blocking conditions, 17 (1.5 mg/kg) was injected iv shortly
before the tracer (n = 3). Animals were sacrificed under anesthesia
with isoflurane by decapitation at 15 min pi. Blood and organs were
collected, weighed, and radioactivity was measured in a γ-counter. The
accumulated radioactivity in the organs was expressed as percent
normalized injected dose per gram of tissue normalized to 1 kg body
weight of the animals (norm. % ID/g tissue). One of the control
animals was excluded from data analysis because the tracer was
injected paravenously, resulting in low counts in all tissues.
In Vivo PET. For spleen-PET scans, four rats (two baseline and two
displacement) were anesthetized with isoflurane, and 16 (11−18 MBq,
0.06−0.09 nmol) was injected via the tail vein. For displacement
experiments, 1.5 mg/kg 17 was iv injected 10 min after radiotracer
administration. For brain PET scans, eight mice in total (six with LPS
treatment) were anesthetized with isoflurane and an amount of 10−27
MBq (0.05−0.014 nmol) 16 was injected via the tail vein. For blocking
conditions, 2.0 mg/kg 17 or vehicle was injected sc 30 min before
tracer application (two mice each). Two mice were injected sc with 2
mg/kg 17 10 min before tracer application to evaluate the best
protocol for maximal blocking. However, one of the two mice died
during the scan.
In both species, PET acquisition was started simultaneously with
radiotracer injection and lasted for 60 min followed by CT. Depth of
anesthesia of rats or mice was monitored by measuring respiratory
frequency (SA Instruments, Inc., Stony Brook, USA). Body temper-
ature was controlled by a rectal probe connected to a thermocoupler
for regulating temperature to 37 °C via an air stream. Data were
reconstructed in user-defined time frames with a voxel size of 0.3875 ×
0.3875 × 0.775 mm3 by two-dimensional-ordered subsets expectation
maximization (2D-OSEM). Random and single but no attenuation
correction was applied. PET acquisitions were followed by a CT for
anatomical orientation. Image files were analyzed with PMOD 3.5
software (PMOD Technologies Ltd., Zurich, Switzerland). Brain
regions of interest (ROI) were defined on the mouse MRI T2
template. Tissue radioactivity of the brain ROI was expressed as SUV,
that is, the decay-corrected radioactivity per cm3 divided by the
injected radioactivity dose per gram of body weight.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Ernst Schaffter, Anja Osterwald, Elisabeth Zirwes,
and Isabella Erbsmehl for their technical assistance and Mark
Rogers-Evans and Thomas Hartung for discussions. We thank
Bruno Mancosu for his support with carbon-11 module and
Claudia Keller for performing PET/CT scans. We thank the
Swiss ALS Foundation for partly financing this project and
Prof. M. Weber for discussions.
ABBREVIATIONS USED
■
DMTMM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmor-
pholinium chloride; hCB2, human cannabinoid 2 receptor;
mCB2, murine cannabinoid receptor type 2; OSEM, ordered
subset expectation maximization; PET, positron emission
tomography
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* Supporting Information
Molecular formula strings in csv file. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.jmedchem.5b00283.
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