Discovery of a novel series of orally active nociceptin/orphanin FQ (NOP) receptor antagonists based on a dihydrospiro(piperidine-4,7′-thieno[2,3- C ]pyran) scaffold

Article abstract of DOI:10.1021/jm500117r

Nociceptin/OFQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the ORL1/NOP receptor. Nociceptin appears to regulate a host of physiological functions such as biological reactions to stress, anxiety, mood, and drug abuse, in addition

Full text of DOI:10.1021/jm500117r

Article
pubs.acs.org/jmc
Discovery of a Novel Series of Orally Active Nociceptin/Orphanin FQ
(NOP) Receptor Antagonists Based on a Dihydrospiro(piperidine-
4,7′-thieno[2,3‑c]pyran) Scaffold
Miguel A. Toledo,*,† Concepcion Pedregal,†,§ Celia Lafuente,† Nuria Diaz,†
́
Maria Angeles Martinez-Grau,† Alma Jimenez,† Ana Benito,† Alicia Torrado,† Carlos Mateos,†
́
Elizabeth M. Joshi,‡ Steven D. Kahl,‡ Karen S. Rash,‡ Daniel R. Mudra,‡ Vanessa N. Barth,‡
David B. Shaw,‡ David McKinzie,‡ Jeffrey M. Witkin,‡ and Michael A. Statnick‡
†Centro de Investigacion
́
Lilly, Avenida de la Industria 30, 28108-Alcobendas, Madrid, Spain
‡Eli Lilly & Co., Lilly Research Laboratories, Indianapolis, Indiana 46285, United States
S
* Supporting Information
ABSTRACT: Nociceptin/OFQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the
ORL1/NOP receptor. Nociceptin appears to regulate a host of physiological functions such as biological
reactions to stress, anxiety, mood, and drug abuse, in addition to feeding behaviors. To develop tools to study
the function of nociceptin and NOP receptor, our research effort sought to identify orally available NOP
antagonists. Our effort led to the discovery of a novel chemical series based on the dihydrospiro(piperidine-4,7′-
thieno[2,3-c]pyran) scaffold. Herein we show that dihydrospiro(piperidine-4,7′-thieno[2,3-c]pyran)-derived
compounds are potent NOP antagonists with high selectivity versus classical opioid receptors (μ, δ, and κ).
Moreover, these compounds exhibit sufficient bioavailability to produce a high level of NOP receptor
occupancy in the brain following oral administration in rats.
receptor antagonist with potency and selectivity.8 In fact this
INTRODUCTION
■
molecule shows high affinity for NOP receptor with greater
than 600-fold selectivity over the other opioid receptors.
Compound 1 has become an important pharmacological tool
for the elucidation of the different physiological functions
related to NOP receptor. Further optimization of this scaffold
delivered 2, disclosed by Banyu as the first clinical candidate for
multiple indications using an experimental medicine approach.9
3 (JTC-801) is the only NOP antagonist with potent
antinociceptive effects in animal models of acute pain.10
Although 3 has entered clinical trials as a potentially novel
analgesic,4,6 data have not been released. Phenylpiperidine 4
(SB-612111) was reported as a potent and selective NOP
receptor antagonist.11a,b It was found to inhibit food intake and
reduce immobility in forced swim and tail suspension assays.11c
4 was also recently proposed for the treatment of Parkinson’s
disease.11d Spiropiperidines (5−8) represent a very common
structural motif for NOP antagonists.12 Compounds 9,13 10,14
and 1115 are additional representative scaffolds reported in the
literature. Finally, 12 (MK-1925) characterizes a new chemical
scaffold and was selected by Banyu as the second clinical
candidate.16 When dosed orally in mice, 12 exhibited dose-
dependent antagonist activity against the reduction in
locomotor activity induced by a NOP receptor agonist, thus
reflecting target engagement.16
ORL-1 or NOP receptor is the most recently discovered
member of the opioid receptor family.1 It belongs to the class A
GPCR, and the 17 amino acid peptide nociceptin/Orphanin
FQ (N/OFQ) is the endogenous ligand.2 Activation of NOP
receptor is coupled to inhibition of adenylate cyclase as well as
to voltage-gated Ca2+ and K+ channels.3 Although the four
opioid receptors are structurally related, N/OFQ does not have
significant affinity for the classical opioid receptors, and the
opioid peptides (enkephalins, endorphin, and dynorphin) do
not bind to NOP receptor. This receptor is widely expressed in
the CNS, the peripheral nervous system, and some non-neural
tissues including gastrointestinal tract, smooth muscle, and
immune system. The activation of NOP receptor by its
endogenous ligand modulates several physiological functions
and behaviors such as anxiety, depression, stress, feeding,
bradycardia, locomotor activity, body temperature, substance
abuse, diuresis, memory, pain, and inflammatory bowel
diseases.4−6 As a result, NOP receptor represents a very
promising target to study different physiological mechanisms,
and the identification of both agonists and antagonists is an area
of interest in the discovery of novel drug therapies.4
Many pharmaceutical companies have devoted research
programs to identify small molecules as potent and selective
NOP agonists and antagonists.7 As a consequence, several NOP
antagonists have been reported in the literature (Figure 1) with
Banyu as one of the most active companies in the field. In 1999
they reported 1 (J-113397) as the first small molecule NOP
Received: January 22, 2014
Published: March 28, 2014
© 2014 American Chemical Society
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Figure 1. Reported NOP receptor antagonists.
In a paper published in 2012, Stevens reported the crystal
structure of the human nociceptin/orphanin FQ receptor in
complex with the peptide mimetic antagonist compound 5.17
The X-ray structure showed substantial conformational differ-
ences in the pocket regions between NOP and the classical
opioid receptors and provides new structural avenues for the
design of novel NOP receptor antagonists.
Early in our medicinal chemistry effort we discovered a new
series of high-affinity small molecule NOP receptor antagonists
based on a 3-[2′-halo-4′,5′-dihydrospiro(piperidine-4,7′-thieno-
[2,3-c]pyran)-1-yl]-2-(2-halobenzyl)-N-alkylpropanamide scaf-
fold (Figure 2A).18 As previously reported, this chemotype
provided very high NOP receptor binding affinity, selectivity,
rapid brain penetration, and favorable kinetics for imaging and
11C-labeling. The spiropiperidine moiety was part of our initial
hit, and the medicinal chemistry effort was developed in parallel
to the discovery of NOP receptor PET tracer B (Figure 2).
Herein, we describe the discovery of a new series of small
molecule NOP receptor antagonists based on the 2′-halo-4′,5′-
dihydrospiro(piperidine-4,7′-thieno[2,3-c]pyran) scaffold.
These derivatives exhibit high NOP receptor affinity and
antagonist potency, high selectivity versus the classic opioid
receptors, optimal oral bioavailability and PK parameters, and
very high levels of receptor occupancy maintained up to 24 h.
Figure 2. 3-(2′-Halo-4′,5′-dihydrospiro[piperidine-4,7′-thieno[2,3-c]-
pyran]-1-yl)-2-(2-halobenzyl)-N-alkylpropanamide scaffold (A) and
PET tracer for NOP receptor (B).
CHEMISTRY
■
The synthesis of the 2-chloro- and 2-fluorothienospiropiper-
idine ligands 15−21 is outlined in Scheme 1. Starting from the
previously reported 2-chloro- and 2-fluorospiro(4,5-
dihydrothieno[2,3-c]pyran-7,4′-piperidine) intermediates 13
and 14,18 the corresponding final compounds 15−21 were
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16 as a very potent and selective NOP receptor antagonist
(Table 1).
Scheme 1. Synthesis of 2-Chloro- and 2-
Fluorothienospiropiperidines as NOP Receptor
a
Table 1 shows the compounds that were prepared and tested
for NOP receptor binding and antagonist functional activity.
Compounds 15−21 were found to have very high binding
affinity, represented by subnanomolar Ki values, to human
recombinant NOP receptor using [3H]-nociceptin as radio-
ligand. In a functional assay of receptor-mediated G-protein
activation using [35S]-GTPγS binding, these ligands were found
to be very potent antagonists of the NOP receptor with
subnanomolar Kb values.
Antagonists
4-Pyrazole with a methyl substituent at position 3 was the
optimal heterocycle to maintain in vitro potency. After
extensive SAR in the distal aromatic ring, it was found that
2,6-disubstituted phenyl and 6-substituted 2-pyridine were
preferred for high in vitro potency. As it was shown with the
initial carboxamide series, the [2,3]-fused thiophene with 2-
chloro or 2-fluoro substituents produced equally high potent
NOP receptor antagonists (Table 1).
Compounds containing a thiophene functionality have been
reported to potentially form reactive intermediates due to its π-
exceeding character.20 Given this concern, in vitro trapping
assays were conducted with GSH before progressing further the
SAR with this chemical series. Compounds 17 and 20 were
incubated with rat hepatic microsomes, and metabolite profiling
by LC/MS demonstrated the presence of GSH conjugates
(results not shown). Based on these results, modifications to
the SAR were incorporated with two goals in mind: (1) block
potential benzylic oxidation; (2) decrease the electron density
of the thiophene to avoid the formation of reactive
intermediates. To this end, we decided to introduce a gem-
difluoro group at the benzylic position of the spiro moiety. This
approach yielded compound 37 which did not produce GSH-
related conjugates when incubated in the GSH trapping assay
within rat hepatic microsomes. Based on the positive results,
additional gem-difluoro substituted analogues were prepared
and evaluated.
Table 2 contains NOP receptor binding and functional
antagonism data on compounds with the gem-difluoro group.
Ligands 27−32 and 35−37 were found to have very high
binding affinitiy to human recombinant NOP receptor as
represented by subnanomolar Ki values. These compounds
were also very potent antagonists at NOP receptor with
subnanomolar Kb potencies. Selectivity versus mu, kappa, and
delta opioid receptors was also excellent (Ki > 375 nM).
These novel NOP antagonists exhibited excellent in vitro
activity and selectivity at NOP receptor, and they were tested in
the receptor occupancy assay, previously published, to
demonstrate in vivo target engagement.18 Table 3 shows RO
data for spiropiperidines 27−32 and 35−37 measured 24 h
following oral administration at 10 mg/kg in rats. All
compounds showed sustained receptor occupancy, higher
than 60% for most cases, proving brain penetration not to be
an issue.
a
Reagents and conditions: (a) Appropriate pyrazole-4-carbaldehyde,
NaBH(OAc)3, 1,2-dichloroethane or THF, rt.
prepared by reductive amination with the appropriate pyrazole-
4-carbaldehyde using classical conditions.
Scheme 2 describes the preparation of the NOP receptor
antagonists containing a gem-difluoro group at the benzylic
position of the 2-chlorospiro(4,5-dihydrothieno[2,3-c]pyran-
7,4′-piperidine). These derivatives were all prepared from the
key intermediate 25. Benzylic bromination of the previously
reported intermediate 2218 with N-bromosuccinimide followed
by oxidation under Kornblum conditions19 with sodium
bicarbonate gave a mixture of secondary alcohol and ketone
23. This mixture was subjected to a TEMPO/NaClO oxidation
to convert the secondary alcohol to the ketone 23. Fluorination
with bis(2-methoxyethyl)aminosulfur trifluoride yielded the
gem-difluoro derivative 24. This intermediate was deprotected
under standard acidic conditions to give the common
intermediate 25. Reductive amination with the corresponding
pyrazole-4-carbaldehyde took place using classical experimental
conditions to deliver key intermediates 26 and 33 and final
compounds 27−30. Reduction of the ester 26 with lithium
aluminiun hydride gave the benzylic alcohol 31 that was further
converted to the secondary amine 32 in a two-step sequence
involving oxidation with manganese dioxide and reductive
amination with methylamine. Copper-catalyzed reaction of the
pyrazole 33 with the appropriate bromopyridine gave rise to
the final product 37 and the intermediate aldehyde 34.
Intermediate 34 was further transformed to the imidazole 35
or the benzylic alcohol 36 using glyoxal with ammonia or
sodium borohydride, respectively.
RESULTS AND DISCUSSION
■
Two pairs of phenyl/pyridine analogues (27/28 and 31/36)
were selected to evaluate the pharmacokinetic profile in rats
(Table 4). All compounds show good oral bioavailability.
However, as we expected, the lipophilicity reduction provided
by the pyridine had significant impact in both the iv and the po
PK parameters. Clearance and volume of distribution were
reduced for pyridines 28 and 36 in comparison with 27 and 31,
respectively. AUC after oral administration was also signifi-
cantly higher for pyridines 28 and 36. Pyridine compounds 28
Starting from our initial hit series (A, Figure 2), the medicinal
chemistry effort focused first on modifying substitution on the
piperidine nitrogen to optimize the overall performance
characteristics of molecules. We envisioned the replacement
of the carboxamide by five-membered heterocycles to eliminate
the chiral center and reduce the number of rotatable bonds.
After the preparation of a few series of compounds with
different heterocycles and several patterns of substitution, we
identified the 2,6-difluorophenyl substituted 3-methylpyrazole
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a
Scheme 2. Synthesis of gem-Difluorothienospiropiperidines as NOP Receptor Antagonists
a
Reagents and conditions: (a) (1) NBS, ℏν, chlorobenzene, 45 °C, (2) DMSO, NaHCO3, rt, (3) KBr, 2,2,6,6-tetramethylpiperidine N-oxide, 6%
NaClO (adjusted at pH = 9 with NaHCO3), DCM, 5 °C to rt; (b) (bis(2-methoxyethyl)amino)sulfur trifluoride, THF, 70 °C; (c) (1) 37% HCl, i-
PrOH, 45 °C, (2) 6 N NaOH; (d) appropriate aldehyde, NaBH(OAc)3, 1,2-dichloroethane or THF, rt; (e) LiAlH4, THF, −20 °C; (f) (1) MnO2,
DCM, reflux, (2) 40% MeNH2 in water, NaBH4, EtOH, rt; (g) ArBr, CuI, K2CO3, trans-N,N′-dimethyl-1,2-cyclohexanediamine, toluene, 110 °C; (h)
40% glyoxal in water, 2 N ammonia in MeOH, MeOH, 0 to 40 °C; (i) NaBH4, DCM/MeOH, 0 °C to rt.
and 36 improved the overall pharmacokinetic profile due to the
reduced lipophilicity (Table 4).
NOP receptor binding and functional activity, high selectivity
over classical opioid receptors, oral bioavailability, and excellent
brain penetration to guarantee high receptor occupancy that
was maintained at 10 mg/kg up to 24 h in rats. Consistent with
the in vivo target engagement at NOP receptor, a dose-
dependently pharmacodynamic action preventing agonist-
induced hypothermia was demontrated with compound 36.
To demonstrate a pharmacodynamic action of the current
series, reversal of the hypothermic effect induced by the NOP
agonist Ro64-619821 was studied in vivo in rat. The
hypothermic effects of NOP receptor activation are well
documented in the literature.22 Consistent with its in vitro
antagonist properties, 36 reversed the NOP agonist-induced
hypothermia in a dose-dependent manner, as demonstrated by
a significant reversal of NOP-induced hypothermia at 0.1 and
0.3 mg/kg [F(4,35) = 48.03, p < 0.001; Dunnett posthoc
comparisons with alpha set at p < 0.05]. Moreover, 36 had no
effect on basal body temperature as measured prior to Ro64-
6198 administration [F(3,34) = 0.12, p > 0.95; data not
shown]. Therefore, 36 is a potent and selective NOP receptor
antagonist with CNS activity in rats following oral admin-
istration.
EXPERIMENTAL SECTION
■
Materials and Methods. All reagents used were obtained from
commercial sources (Sigma-Aldrich, unless otherwise stated). All
solvents were of an analytical grade. The NOP agonist ligand Ro64-
6198 was synthesized as reported.21 [35S]-GTPγS was obtained
commercially. Flash chromatography was performed in ISCO
automated systems using SiO2 cartridges. All final compounds
1
analyzed were >95% pure unless otherwise indicated. H NMR (300
MHz) spectra were acquired at 20 °C on a Bruker Avance DPX 300
MHz spectrometer. Abbreviations s, d, dd, ddd, m, and br denote
singlet, doublet, double doublet, double double doublet, multiplet, and
broad, respectively. High-resolution mass spectrometry (HRMS) was
performed on an Agilent 6200 Series ToF with Agilent MassHunter
Software (TOF Method, 2 μL injection, 0.2 mL/min isocratic flow
rate, 70/30 water +0.1% formic acid/acetonitrile +0.1% formic acid,
and 1.5 min run time). LC-MS was performed with an 1100 Series LC-
SUMMARY
■
We have identified orally bioavailable NOP receptor antago-
nists based on the dihydrospiro(piperidine-4,7′-thieno[2,3-
c]pyran) scaffold. These compounds display very potent
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a
Table 1. Binding Affinity and Functional Activity of the Novel NOP Receptor Antagonists 15−21
b
c
d
compound
R
X
Y
NOP binding Ki (nM)
NOP antagonism Kb (nM)
e
f
nociceptin
0.214 (0.037, n = 9)
2.59 (0.26, n = 9)
15
16
17
18
19
20
21
F
N
F
F
0.139 (0.045, n = 3)
0.136 (0.011, n = 3)
0.0494 (0.0034, n = 3)
0.0954 (0.0354, n = 3)
0.0865 (0.0183, n = 3)
0.104 (0.010, n = 3)
0.0805 (0.0080, n = 3)
0.0815 (0.0025, n = 3)
F
CF
CF
CF
CF
N
0.0711 (0.0080, n = 3)
0.0954 (0.0240, n = 4)
0.0648 (0.0433, n = 3)
0.122 (0.016, n = 3)
0.0908 (0.0246, n = 3)
F
CONH2
F
CH2NMe2
CH2NMe2
CH2OMe
Cl
Cl
F
N
CH2OMe
0.0939 (0.0207, n = 3)
a
b
c
Shown are geometric means (SEM, n) for Ki and Kb values. (L)-Tartaric acid salt. [3H]-Nociceptin was used as a reference NOP radioligand in a
d
binding assay with human recombinant NOP receptor overexpressed in CHO cells. Functional activity was determined in an assay of receptor-
mediated G-protein activation using [35S]-GTPγS and membranes from CHO cells overexpressing the human NOP receptor. See Materials and
e
Methods for assay details. Number of replicates for nociceptin control correspond to the same individual binding assays where compounds were
tested. Nociceptin response in agonist mode of assay (EC50); number of replicates correspond to the same invidual functional assays where
f
compounds were tested.
MSD single quadrupole instrument (Agilent; Santa Clara, CA) with an
ESI interface. The samples were analyzed by the following described
methods:
Method 1. Used a heated (50 °C) XBridge C18 column (3.5 μm;
2.1 mm × 50 mm) eluted at 1.0 mL/min with a gradient of A (aq 10
mM (NH4)HCO3; pH = 9) and B (acetonitrile), with B increased
linearly from 10% to 100% over 7 min. Ions in the range of m/z 100−
800 were captured after electrospray ionization of the eluted test
sample.
In Vitro Functional Blockade of NOP Receptor Agonist-
Mediated G-Protein Activation: GTPγ-[35S] Binding. Experimen-
tal details were previously disclosed.18a
In Vivo Receptor Occupancy (RO) Assays. Male Sprague−
Dawley rats weighing between 220 and 300 g were housed on a 12 h
light:dark cycle (testing during light phase) and received free access to
normal rat chow and water. Animals received either oral vehicle or a
single dose of a NOP receptor compound (10 mg/kg). Twenty-four
hours later animals received a lateral iv tail vein bolus injection of the
nonlabeled tracer (3 μg/kg). Forty minutes after tracer delivery,
animals were sacrificed by cervical dislocation and brain tissues
(hypothalamus and striatum) were harvested. Tissue samples were
weighed and placed in conical centrifuge tubes on ice. Samples were
then homogenized in four volumes (w/v) of acetonitrile (ACN)
containing 0.1% formic acid and centrifuged at 14 000 rpm for 16 min.
Supernatant liquid was diluted by adding sterile water to samples for
subsequent LC-MS/MS analysis.18a
Rat Pharmacokinetics. Male Sprague−Dawley rats (n = 3 per
treatment), fasted for a period between 4 and 24 h and water provided
ad libitum, were dosed by intravenous (iv) bolus administration (1
mL/kg) at 1 mg/kg or per oral (po) administration (5 or 10 mL/kg)
at 3 or 10 mg/kg of compound 27, 28, 31, or 36. Both iv and po doses
were formulated in 20% (w/w) Captisol (Ligand Pharmaceuticals, La
Jolla, CA) in phosphate buffer (25 mM, pH 2). Blood samples
(between 0.15 and 0.20 mL) were drawn via an arterial catheter into
disodium EDTA-containing tubes (BD, Franklin Lakes, NJ) at 0, 0.08,
0.25, 0.50, 1, 2, 4, 8, 12, and 16 or 24 h postdose. Whole blood samples
were stored on wet-ice until centrifuged (3000 rpm, 10 min, 4 °C),
and plasma supernatant was collected and stored (−20 to −70 °C)
until processed for bioanalysis. Plasma proteins were precipitated by
mixing with an internal standard (IS) solution (10 ng/mL in
methanol:acetonitrile, 1:1) and centrifuged (4000 rpm 10 min, and
4 °C) and the resulting supernatants analyzed for compounds 27, 28,
31, and 36. Analytes were separated by reverse-phase chromatography
with biphasic gradient elution (1 M NH4HCO3 mobile phase A;
MeOH/1 M NH4HCO3 (200:1, v/v) mobile phase B) on a Betasil
C18 column (2.1 × 20 mm, 5 μm; Thermo Fisher Scientific, Waltham,
MA) and detected by selective reaction monitoring in positive ion
mode (Sciex API 4000; Applied Biosystems/MDS, Foster City, CA).
Compounds 27, 28, 31, and 36 were quantified against analyte-in-
Method 2. Used a heated (50 °C) XTerra-C18 column (3.5 μm; 2.1
mm × 50 mm), eluted at 1.0 mL/min with a gradient of A (aq 10 mM
(NH4)HCO3; pH = 9) and B (acetonitrile), with B increased linearly
from 10% to 100% over 3 min. Ions in the range of m/z 100−800 were
captured after electrospray ionization of the eluted test sample.
Statistics. Values are given as means the standard deviation of
the mean, unless otherwise stated. For the NOP agonist-induced
hypothermia experiment, a between-group analysis of variance
(ANOVA) with Dunnett posthoc analyses was conducted using
vehicle and NOP agonist groups as controls.
Reversal of Nociceptin Agonist-Induced Hypothermia.
Subjects were male Sprague−Dawley rats weighing 225 g and housed
2 per cage. Rats were maintained on a 12 h light:dark cycle (testing
during light phase) and received free access to normal rat chow and
water. On test day, rats were weighed, returned to their home cage,
and allowed to acclimate in the procedure room for at least 90 min
before onset of the study. For each rat, an initial body temperature was
taken by rectal probe followed immediately by oral gavage with vehicle
or compound 36 (0.03, 0.1, or 0.3 mg/kg). Sixty minutes later, a
second body temperature was taken and followed immediately by a 6
mg/kg subcutaneous injection of vehicle or the NOP agonist Ro64-
6198. Finally, a third body temperature was taken 120 min later, a time
period in which pilot studies determined that robust hypothermic
effects of Ro64-6198 were present. All experiments with rats were
performed in accord with the National Research Council Guide under
protocols approved by the Animal Care and Use Committee of Eli
Lilly and Company.
In Vitro NOP Receptor Binding. Experimental details were
previously disclosed.18a
In Vitro Opioid Receptor Binding. Experimental details were
previously disclosed.18a
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clearance, volume of distribution, T1/2, and bioavailability calculated by
the ratio of dose normalized (dn) plasma exposures, dnAUCpo/
dnAUCiv.
Table 2. Binding affinity and functional activity of the novel
NOP receptor antagonists 27-32 and 35-37
a
Chemistry. General Procedure for Reductive Amination (Method
A). A solution of the corresponding spiropiperidine derivative and the
appropriate aldehyde was stirred at room temperature in the
appropriate solvent (0.15−0.55 M tetrahydrofuran or 1,2-dichloro-
ethane) for 10−60 min (t1). Then sodium triacetoxyborohydride
(1.2−2.0 equiv) was added and the reaction mixture was stirred at
room temperature until it was completed (2−16 h, t2). The reaction
mixture was treated with ice/saturated solution of sodium bicarbonate,
and the organic phase was extracted with ethyl acetate, dichloro-
methane, or tert-butyl ethyl ether (S2). The organic layer was
separated, washed with brine, dried over magnesium sulfate, filtered,
and concentrated under vacuum. The resulting crude product was
purified by chromatography.
NOP binding Ki
NOP antagonism
b
c
d
compound
X
R
(nM)
Kb (nM)
General Procedure for (L)-Tartrate Salt Formation (Method B).
The corresponding spiropiperidine compound (1 equiv) was dissolved
in methanol (0.2 M), and a solution of (L)-tartaric acid (1 equiv) in
methanol (0.2 M) was added. The mixture was stirred for 15 min at
room temperature. The solvent was evaporated, and the residue was
dried in vacuo overnight to give the final product in high yield (>90%).
2-Fluoro-1′-[[1-(3-fluoro-2-pyridyl)-3-methylpyrazol-4-yl]methyl]-
spiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperidine] (L)-Tartrate
(15). The free base of the title compound was essentially prepared
following the general method A using 13 (225 mg, 0.99 mmol), 1-(3-
fluoro-2-pyridyl)-3-methylpyrazole-4-carbaldehyde (284 mg, 1.38
mmol), and sodium triacetoxyborohydride (420 mg, 1.98 mmol)
using 6.6 mL of tetrahydrofuran (t1 = 10 min, t2 = 2 h, S = ethyl
acetate). The crude product was purified by reverse phase HPLC
(XBridge C18:5 μm, 19 × 100 mm. Phase A: ammonium bicarbonate
20 mM in water, pH 8; phase B: acetonitrile. Gradient: 0 → 2 min
40% solvent B isocratic, 2 → 9 min, 40% to 60% solvent B gradient, 9
→ 10 min, 60% to 99% solvent B gradient, 10 → 12 min, 99% solvent
B isocratic. Flow rate: first 2 min: 15 → 25 mL/min, from minute 2 to
end: 25 mL/min) to get 157.2 mg of the free base of the title
compound (38%). LC-MS (ESI) m/z: 417 (M + H)+, tR = 3.51 min,
method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.32 (dt, J = 4.8, 1.5 Hz,
1H), 8.23 (broad s, 1H), 7.95 (ddd, J = 11.5, 8.2, 1.5 Hz, 1H), 7.47−
7.41 (m, 1H), 6.45 (d, J = 1.8 Hz, 1H), 3.85 (t, J = 5.5 Hz, 2H), 3.33
(s, 2H), 2.67−2.60 (m, 5H), 2.35−2.25 (m, 4H), 1.95 (d, J = 12.4 Hz,
2H), 1.65 (td, J = 12.8, 4.1 Hz, 2H). The title compound (tartrate salt)
was essentially prepared following general method B with 99% yield.
LC-MS (ESI) m/z: 417 (M + H)+, tR = 3.51 min, method 1. 1H NMR
(300 MHz, DMSO-d6) δ: 8.35−8.30 (m, 2H), 7.97 (ddd, J = 11.6, 8.3,
1.4 Hz, 1H), 7.49−7.43 (m, 1H), 6.48−6.46 (m, 1H), 4.18 (s, 2H),
3.86 (t, J = 5.3 Hz, 2H), 3.64 (s, 2H), 3.57−3.51 (m, 2H), 2.84−2.79
(m, 2H), 2.54−2.48 (m, 4H), 2.27 (s, 3H), 2.06−1.98 (m, 2H), 1.78−
1.66 (m, 2H). HRMS (ESI): calcd for C21H22F2N4OS, 416.1482;
found, 416.1492.
27
CF
F
F
0.172
0.198
(0.034, n = 3)
(0.080, n = 3)
28
29
30
31
32
35
36
37
N
0.176
(0.025, n = 4)
0.273
(0.061, n = 4)
CF
N
CH2NMe2
CH2NMe2
CH2OH
0.145
(0.033, n = 3)
0.221
(0.019, n = 3)
0.371
(0.030, n = 3)
1.20 (0.32, n = 3)
CF
CF
N
0.114
(0.017, n = 3)
0.0840
(0.0177, n = 3)
CH2NHMe 0.0970
(0.0421, n = 3)
0.190
(0.019, n = 3)
2-imidazol
0.0916
(0.0122, n = 4)
0.202
(0.031, n = 4)
N
CH2OH
0.105
(0.036, n = 3)
0.166
(0.035, n = 3)
N
CH2OMe
0.185
(0.007, n = 3)
0.252
(0.070, n = 2)
a
Shown are geometric means (SEM, n) for Ki and Kb values.
Radioligand binding with the nonselective opioid antagonist, [3H]-
diprenorphine, using membranes prepared from CHO cells over-
expressing the recombinant human kappa, mu, or delta opioid
receptors gave Ki values >375 nM. Human NOP EC50 agonist activity
was >10000 nM for molecules tested (compounds 30, 32−37). See
b
Materials and Methods for assay details. (L)-Tartaric acid salt except
c
free base for compound 35. [3H]-Nociceptin was used as a reference
NOP radioligand in a binding assay with human recombinant NOP
receptor overexpressed in CHO cells. Functional activity was
determined in an assay of receptor-mediated G-protein activation
using [35S]-GTPγS and membranes from CHO cells overexpressing
the human NOP receptor.
d
Table 3. Receptor Occupancy (RO) of NOP Antagonists in
Rats after 24 h of Oral Administration
1′-[[1-(2,6-Difluorophenyl)-3-methylpyrazol-4-yl]methyl]-2-
fluorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperidine] (L)-Tar-
trate (16). Triethylamine (0.064 mL, 0.46 mmol) was added to a
suspension of 2-fluorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piper-
idine] hydrochloride (13·HCl) (121 mg, 0.46 mmol) in 1,2-
dichloroethane (1.15 mL). To the resulting solution was added 1-
(2,6-difluorophenyl)-3-methyl-1H-pyrazole-4-carbaldehyde (153 mg,
0.69 mmol) followed by sodium triacetoxyborohydride (420 mg, 1.98
mmol). The resulting reaction mixture was stirred at room
temperature overnight. The solvent was evaporated, and the residue
was purified by SCX (methanol and 2 M ammonia in methanol). The
fractions containing the product were collected and evaporated. This
crude product was further purified by reverse phase HPLC (XTerra
MS C18:5 μm, 10 × 100 mm. pH = 8. Phase A: ammonium
bicarbonate 20 mM in water, pH 8; phase B: acetonitrile. Gradient: 0
→ 2 min 50% solvent B isocratic, 2 → 9 min, 50% to 70% solvent B
gradient, 9 → 10 min, 70% to 88% solvent B gradient, 10 → 12 min,
99% solvent B isocratic. Flow rate: first 2 min: 15 → 25 mL/min, from
minute 2 to end: 25 mL/min) to get 92.2 mg of the free base of the
title compound (46%) as an oil. LC-MS (ESI) m/z: 434 (M + H)+, tR
a
compound
RO (%) 10 mg/kg
27
28
29
30
31
32
35
36
37
49
55
97
72
87
100
88
62
90
a
(L)-Tartaric acid salt.
plasma standard curve (prepared between 1 and 50 000 ng/mL)
prepared under the same conditions as the test samples, and total
plasma concentration was determined by analyte:IS chromatographic
peak area ratios. Concentration−time data were analyzed by
noncompartmental pharmacokinetic methods to determine plasma
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Article
a
Table 4. In Vivo Pharmacokinetic Data for NOP Receptor Antagonists in Rats after Oral and Intraveous Administration
intravenous administration
oral administration
compd
clogP
dose (mg/kg)
Cl (mL/min/kg)
Vd (L/kg)
t1/2 (h)
dose (mg/kg)
po AUC0‑inf (ng·h/mL)
F (%)
27
28
31
36
5.1
4.4
4.3
3.5
1
1
1
1
52.9 0.3
19.8 5.8
60.5 8.4
23.9 5.9
28.2 3.2
13.6 3.9
12.7 0.9
5.3 0.8
10.8
1
12.1 1.8
3.9 0.2
3.8 0.5
3
438 110
1815 1213
1279 469
4103 1418
49
62
51
57
3
10
10
a
Cl, clearance; Vd, volume of distribution; t1/2, plasma elimination half-life; AUC, area under the curve; F, bioavailability.
Figure 3. NOP receptor antagonist 36 prevents NOP receptor agonist-induced hypothermia in rats. Ro64-6198 (6 mg/kg, sc) was the NOP receptor
agonist used in this experiment. V = Vehicle treatment. #p < 0.05 versus V/NOP agonist group; *p < 0.05 versus V/V group.
1
= 4.10 min, method 1. H NMR (300 MHz, DMSO-d6) δ: 7.86 (s,
1H), 7.60−7.52 (m, 1H), 7.36−7.29 (m, 2H), 6.45 (d, J = 1.8 Hz,
1H), 3.86 (t, J = 5.3 Hz, 2H), 3.34−3.32 (m, 4H), 2.7−2.6 (m, 2H),
2.35−2.20 (m, 5H), 1.95 (d, J = 12.4 Hz, 2H), 1.65 (td, J = 12.9, 4.1
Hz, 2H). The title compound (tartrate salt) was essentially prepared
following general method B with 97% yield. LC-MS (ESI) m/z: 434
(M + H)+, tR = 4.13 min, method 1. 1H NMR (300 MHz, DMSO-d6)
δ: 7.94 (s, 1H), 7.61−7.51 (m, 1H), 7.37−7.30 (m, 2H), 6.47 (d, J =
1.8 Hz, 1H), 4.19 (s, 2H), 3.87 (t, J = 5.5 Hz, 2H), 3.64−3.54 (m,
2H), 2.83−2.76 (m, 2H), 2.52−2.45 (m, 4H), 2.24 (s, 3H), 2.00 (d, J
= 13.2 Hz, 2H), 1.73 (td, J = 13.0, 3.8 Hz, 2H). HRMS (ESI): calcd
for C22H22F3N3OS, 433.1436; found, 433.1488.
mmol) was added to a solution of 13 (186 mg, 0.82 mmol) in 1,2-
dichloroethane (4 mL). Then sodium triacetoxyborohydride (348 mg,
1.64 mmol) was added. The mixture was stirred at room temperature
overnight. The solvent was evaporated, and the crude material was
purified using a 5 g SCX column (methanol and 2 M ammonia in
methanol). The basic fraction was evaporated, and the material was
further purified by reserve phase HPLC (XBridge C18:5 μm, 19 × 100
mm. Phase A: ammonium bicarbonate 20 mM in water, pH 8; phase
B: acetonitrile. Gradient: 0 → 2 min 40% solvent B isocratic, 2 → 9
min, 40% to 65% solvent B gradient, 9 → 10 min, 65% to 99% solvent
B gradient, 10 → 12 min, 99% solvent B isocratic. Flow rate: first 2
min: 15 → 25 mL/min, from minute 2 to end: 25 mL/min) to give
246 mg (64%) of the free base of the title compound. LC-MS (ESI)
3-Fluoro-2-[4-[(2-fluorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-
piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]benzamide (L)-Tar-
trate (17). The free base of the title compound was essentially
prepared following the general method A using 13 (5.26 g, 23.14
mmol), 3-fluoro-2-(4-formyl-3-methylpyrazol-1-yl)benzamide (5.20 g,
21.03 mmol), and sodium triacetoxyborohydride (5.35 g, 25.24 mmol)
using 52 mL of tetrahydrofuran (t1 = 10 min, t2 = 2 h, S = ethyl
acetate). The crude product was purified by silica gel eluting with
CH2Cl2/MeOH (5%) to get 8.5 g (87%) of the free base of the title
compound as a white foam. LC-MS (ESI) m/z: 459 (M + H)+, tR =
1
m/z: 473 (M + H)+, tR = 3.96 min, method 1. H NMR (300 MHz,
DMSO-d6) δ: 7.73 (s, 1H), 7.53−7.43 (m, 1H), 7.40−7.25 (m, 2H),
6.45 (d, J = 1.8 Hz,1H), 3.85 (t, J = 5.1 Hz, 2H), 3.30 (s, 2H), 3.14 (s,
2H), 2.71−2.59 (m, 2H), 2.57−2.50 (m, 2H), 2.32−2.21 (m, 2H),
2.20 (s, 3H), 2.01 (s, 6H), 1.99−1.89 (m, 2H), 1.72−1.57 (m, 2H).
The title compound (tartrate salt) was essentially prepared following
general method B with 100% yield. LC-MS (ESI) m/z: 473 (M + H)+,
tR = 3.82 min, method 1. 1H NMR (300 MHz, MeOD) δ: 8.38 (d, J =
2.2 Hz, 1H), 7.81−7.69 (m, 3H), 6.47 (d, J = 1.8 Hz, 1H), 4.53 (s,
2H), 4.24 (d, J = 12.1 Hz, 3H), 4.16 (t, J = 5.5 Hz, 2H), 3.54 (s, 2H),
3.46 (d, J = 1.5 Hz, 1H), 3.31−3.21 (m, 2H), 2.92 (s, 6H), 2.78 (t, J =
5.3 Hz, 2H), 2.62 (s, 3H), 2.40−2.25 (m, 4H). HRMS (ESI): calcd for
C25H30F2N4OS, 472.2108; found, 472.212.
1-[2-[4-[(2-Chlorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperi-
dine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorophenyl]-N,N-di-
methylmethanamine (L)-Tartrate (19). The free base of the title
compound was essentially prepared following general method A using
14 (200 mg, 0.82 mmol), 1-[2-(dimethylaminomethyl)-6-fluorophen-
yl]-3-methylpyrazole-4-carbaldehyde (257 mg, 0.98 mmol), and
sodium triacetoxyborohydride (348 mg, 1.64 mmol) using 2 mL of
1,2-dichloroethane (t1 = 60 min, t2 = 16 h, S = dichloromethane). The
crude product was purified by reverse phase HPLC (XBridge C18:5
μm, 19 × 100 mm. Phase A: ammonium bicarbonate 20 mM in water,
pH 8; phase B: acetonitrile. Gradient: 0 → 2 min 60% solvent B
isocratic, 2 → 9 min, 60% to 80% solvent B gradient, 9 → 10 min, 80%
to 99% solvent B gradient, 10 → 12 min, 99% solvent B isocratic. Flow
1
2.73 min, method 1. H NMR (300 MHz, CDCl3) δ: 7.70 (d, J = 8.0
Hz, 1H), 7.52−7.45 (m, 2H), 7.35−7.28 (m, 1H), 6.50 (broad s, 1H),
6.11 (d, J = 1.4 Hz, 1H), 5.80 (broad s, 1H), 3.91 (t, J = 5.5 Hz, 2H),
3.47 (s, 2H), 2.70 (d, J = 11.0 Hz, 2H), 2.54 (t, J = 5.4 Hz, 2H), 2.37−
2.33 (m, 5H), 2.00 (d, J = 12.6 Hz, 2H), 1.80 (td, J = 12.9, 4.1 Hz,
2H). The title compound (tartrate salt) was essentially prepared
following general method B with 99% yield. LC-MS (ESI) m/z: 459
(M + H)+, tR = 2.78 min, method 1. 1H NMR (300 MHz, DMSO-d6)
δ: 7.83 (s, 1H), 7.64 (s, 1H), 7.57−7.46 (m, 2H), 7.41−7.36 (m, 2H),
6.47 (d, J = 1.9 Hz, 1H), 4.16 (s, 2H), 3.87 (t, J = 5.1 Hz, 2H), 3.65
(broad s, 2H), 2.93−2.82 (m, 2H), 2.60−2.46 (m, 4H), 2.21 (s, 3H),
2.02 (d, J = 13.2 Hz, 2H), 1.81−1.73 (m, 2H). HRMS (ESI): calcd for
C23H24F2N4O2S, 458.1588; found, 458.1596.
1-[3-Fluoro-2-[4-[(2-fluorospiro[4,5-dihydrothieno[2,3-c]pyran-
7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]phenyl]-N,N-di-
methylmethanamine (L)-Tartrate (18). 1-[2-(Dimethylaminometh-
yl)-6-fluorophenyl]-3-methylpyrazole-4-carbaldehyde (214 mg, 0.82
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Journal of Medicinal Chemistry
Article
rate: first 2 min: 15 → 25 mL/min, from minute 2 to end: 25 mL/
mmol) was added to a solution of tert-butyl 2′-chloro-4′,5′-
dihydrospiro[piperidine-4,7′-thieno[2,3-c]pyran]-1-carboxylate (22)
(200 g, 581.61 mmol) in chlorobenzene (1.6 L) at rt. The resulting
suspension was irradiated with three 100 W bulb lamps situated almost
in contact with the external reactor wall, and reactor T was set to 45
°C. After 4 h, N-bromosuccinimide (26.14 g, 146.87 mmol) was added
and T was maintained at 40 °C for 15 h. The reaction mixture was
cooled to 0 °C and MTBE (500 mL) was added. The solid was
filtered. The solution was concentrated to about 1000 mL solution in
chlorobenzene. Then MTBE (1000 mL) was added, the solids were
filtered, and the filtrate was concentrated to afford a 600 mL
chlorobenzene solution of the bromide. DMSO (806.47 mL, 11.35
mol) was added at room temperature, and then sodium bicarbonate
(95.38 g, 1.14 mol) was added. After stirring 24 h at room
temperature, H2O/ice (1000 mL) was added and the phases were
separated. The organic phase was washed with H2O (2 × 1 L) and
concentrated to afford a solution in chlorobenzene. Then dichloro-
methane (1.2 L) was added, and the mixture was cooled to 5 °C (ice/
H2O bath). Potassium bromide (20.27 g, 170.31 mmol) and 2,2,6,6-
tetramethylpiperidine N-oxide (4.43 g, 28.38 mmol) were added. Then
a solution of 6% sodium hypochlorite in H2O (644.40 mL, 567.68
mmol) adjusted to pH = 9 with NaHCO3 (s) was added to the
reaction mixture at 5 °C, and the resulting mixture was stirred 1 h
from 5 °C to rt. H2O (1 L) was added, and the phases were separated.
The organic phase was washed with H2O (2 × 0.5 L) and was cooled
with ice/H2O bath. Then potassium bromide (2.03 g, 17.03 mmol),
2,2,6,6-tetramethylpiperidine N-oxide (50 mg; 0.32 mmol), and a
solution of 6% sodium hypochlorite in H2O (128.88 mL, 113.54
mmol) adjusted to pH = 9 with NaHCO3 (s) was added to the
reaction mixture at 5 °C. The resulting mixture was stirred 1 h from 5
°C to room temperature. Then H2O (1L) was added, and the phases
were separated. The organic phase was washed with H2O (2 × 1 L)
dried and concentrated to afford a dark brown solid. The solid was
triturated with hexane (500 mL), methyl tert-butyl ether/hexane 5%
(250 mL), and methyl tert-butyl ether/hexane 10% (250 mL) to
obtain the title compound as a light brown solid (135 g, 65% yield).
min) to give 204 mg (51%) of the free base of the title compound. LC-
1
MS (ESI) m/z: 489 (M + H)+, tR = 4.28 min, method 1. H NMR
(300 MHz, DMSO-d6) δ: 7.81 (s, 1H), 7.52−7.44 (m, 1H), 7.37 (d, J
= 7.8 Hz, 1H), 7.30 (dt, J = 8.1, 1.5 Hz, 1H), 6.85 (s, 1H), 3.84 (t, J =
5.4 Hz, 2H), 3.33 (s, 2H), 3.14 (s, 2H), 2.70−2.60 (m, 2H), 2.57−2.50
(m, 2H), 2.34−2.22 (m, 2H), 2.21 (s, 3H), 2.01 (s, 6H), 1.99−1.90
(m, 2H), 1.75−1.61 (m, 2H). The title compound (tartrate salt) was
essentially prepared following general method B with 100% yield. LC-
1
MS (ESI) m/z: 489 (M + H)+, tR = 4.14 min, method 1. H NMR
(300 MHz, DMSO-d6) δ: 7.85 (s, 1H), 7.55−7.45 (m, 1H), 7.38−7.30
(m, 2H), 6.87 (s, 1H), 4.15 (s, 2H), 3.85 (t, J = 5.2 Hz, 2H), 3.65−
3.58 (m, 2H), 3.25 (s, 2H), 3.16 (s, 2H), 2.87−2.77 (m, 2H), 2.58−
2.48 (m, 2H), 2.24 (s, 3H), 2.08 (s, 6H), 2.06−1.96 (m, 2H), 1.86−
1.70 (m, 2H). HRMS (ESI): calcd for C25H30ClFN4OS, 488.1813;
found, 488.1821.
2-Chloro-1′-[[1-[3-(methoxymethyl)-2-pyridyl]-3-methylpyrazol-
4-yl]methyl]spiro[4,5-dihydrothieno [2,3-c]pyran-7,4′-piperidine]
(L)-Tartrate (20). The free base of the title compound was essentially
prepared following the general method A using 14 (200 mg, 0.82
mmol), 1-(3-methoxymethy1−2-pyridy1)-3-methy1-pyrazole-4-carbal-
dehyde (0.228 g, 0.984 mmol), and sodium triacetoxyborohydride
(348 mg, 1.64 mmol) using 3 mL of 1,2-dichloroethane (t1 = 60 min,
t2 = 16 h, S = dichloromethane). The crude product was purified by
reverse phase HPLC (XBridge C18:5 μm, 19 × 100 mm. Phase A:
ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile.
Gradient: 0 → 2 min 40% solvent B isocratic, 2 → 9 min, 40% to 60%
solvent B gradient, 9 → 10 min, 60% to 99% solvent B gradient, 10 →
12 min, 99% solvent B isocratic. Flow rate: first 2 min: 15 → 25 mL/
min, from minute 2 to end: 25 mL/min) to get the free base of the
title compound (286 mg, 76% yield). LC-MS (ESI) m/z: 459 (M +
H)+, tR = 4.31 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.38
(dd, J = 4.5, 1.8 Hz, 1H), 8.27 (s, 1H), 8.02 (m, 1H), 7.38 (dd, J = 7.8,
4.8 Hz, 1H), 6.85 (s, 1H), 4.79 (s, 2H), 3.82 (m, 2H), 3.41 (s, 2H),
3.33 (s, 3H), 2.66 (m, 2H), 2.54 (m, 2H), 2.28 (m, 2H), 2.25 (s, 3H),
1.96 (m, 2H), 1.68 (m, 2H). The title compound (tartrate salt) was
essentially prepared following general method B with 98% yield. LC-
t
LC-MS (ESI) m/z: 258 (M+ − 101 (− CO2 Bu)). tR = 2.54 min,
1
MS (ESI) m/z: 459 (M + H)+; tR = 4.35 min, method 1. H NMR
method 2. 1H NMR (300 MHz, CDCl3) δ: 7.18 (s, 1H), 4.32 (s, 2H),
4.19−3.90 (m, 2H), 3.12 (t, J = 11.5 Hz, 2H), 2.17−2.03 (m, 2H),
1.83−1.69 (m, 2H), 1.47 (s, 9H).
(300 MHz, DMSO-d6) δ: 8.39 (m, 1H), 8.36 (s, 1H), 8.04 (m, 1H),
7.40 (dd, J = 7.5, 4.8 Hz, 1H), 6.87 (s, 1H), 4.80 (s, 2H), 4.19 (s, 2H),
3.85 (m, 2H), 3.63 (s, 2H), 3.35 (s, 3H), 2.83 (m, 2H), 2.58−2.45 (m,
4H), 2.28 (br s, 3H), 2.02 (m, 2H), 1.76 (m, 2H). HRMS (ESI): calcd
for C23H27ClN4O2S, 458.1543; found, 458.1547.
tert-Butyl 2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-
piperidine]-1′-carboxylate (24). In a 500 mL PFA flask was added
(bis(2-methoxyethyl)amino)sulfur trifluoride (183.62 g, 829.94 mmol)
to tetrahydrofuran (81.00 mL), and then 23 (135 g, 377.24 mmol) was
added. The resulting suspension was stirred at 70 °C for 24 h. Then it
was cooled to room temperature and slowly poured over a mixture of
ice and a saturated solution of sodium bicarbonate (4 L) with stirring
(gas evolution). Methyl tert-butyl ether was used to transfer the
remaining material from flasks. After gas evolution ceased, sodium
bicarbonate (solid) was added with stirring until pH = 8 was reached.
The resulting mixture was extracted with methyl tert-butyl ether (3 ×
500 mL) until no product was detected by TLC in the aqueous phase.
Combined organics were washed with H2O (3 × 500 mL) and brine
(500 mL), dried over Na2S04, and concentrated to afford a dark thick
oil (250 g). Crude material was filtered though a SiO2 plug (dissolved
in dichloromethane), eluting with methyl tert-butyl ether/hexane 10%
(6 L) and methyl tert-butyl ether/hexane 20% (4 L). Fractions were
collected until no product was detected by TLC (20% methyl tert-
butyl ether/hexane, UV, Rf = 0.5). The filtrate was concentrated to get
the title compound as a light brown solid (93g, 65% yield). LC-MS
2-Fluoro-1′-[[1-[3-(methoxymethyl)-2-pyridyl]-3-methylpyrazol-
4-yl]methyl]spiro[4,5-dihydrothieno[2,3-c]pyran-7,4′-piperidine] (L)-
Tartrate (21). The free base of the title compound was essentially
prepared following general method A using 13 (225 mg, 0.99 mmol),
1-[3-(methoxymethyl)-2-pyridyl]-3-methylpyrazole-4-carbaldehyde
(320 mg, 1.48 mmol), and sodium triacetoxyborohydride (420 mg,
1.98 mmol) using 6 mL of tetrahydrofuran (t1 = 10 min, t2 = 2 h, S =
ethyl acetate). The crude product was purified by reverse phase HPLC
in basic conditions (XBridge C18:5 μm, 19 × 100 mm. Phase A:
ammonium bicarbonate 20 mM in water, pH 8; phase B: acetonitrile.
Gradient 40−70% B in 8 min) to get 203.4 mg of the free base of the
title compound (43%). LC-MS (ESI) m/z: 443 (M + H)+, tR = 3.94
min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.38 (dd, J = 1.5,
4.8 Hz, 1H), 8.26 (s, 1H), 8.03−8.01 (m, 1H), 7.38 (dd, J = 4.8, 7.7
Hz, 1H), 6.44 (d, J = 1.5 Hz, 1H), 4.79 (s, 2H), 3.85 (t, J = 5.3 Hz,
2H), 3.40 (s, 3H), 2.65 (d, J = 10.6 Hz, 2H), 2.50 (dd, J = 1.8, 3.7 Hz,
11H), 2.27−2.25 (m, 5H), 1.94 (d, J = 12.8 Hz, 2H), 1.65 (td, J =
12.8, 3.8 Hz, 2H). The title compound (tartrate salt) was essentially
prepared following general method B with 99% yield. LC-MS (ESI)
1
(ESI) m/z: 324 (M+ − 57 (− tBu)). tR = 2.83 min, method 2. H
NMR (300 MHz, CDCl3) δ: 6.98 (s, 1H), 4.09−3.99 (m, 4H), 3.11 (t,
J = 12.6 Hz, 2H), 2.06 (d, J = 12.9 Hz, 2H), 1.72 (td, J = 13.3, 4.8 Hz,
2H), 1.48 (s, 9H). HRMS (ESI): calcd for C16H20ClF2NO3S 379.082,
found 379.0828.
1
m/z: 443 (M + H)+, tR = 3.94 min, method 1. H NMR (300 MHz,
DMSO-d6) δ: 8.40−8.37 (m, 2H), 8.04 (d, J = 7.5 Hz, 1H), 7.40 (dd, J
= 4.7, 7.7 Hz, 1H), 6.46 (d, J = 1.8 Hz, 1H), 4.79 (s, 2H), 4.17 (s, 1H),
3.86 (t, J = 5.3 Hz, 2H), 3.66 (s, 1H), 3.35 (s, 3H), 2.85 (d, J = 11.1
Hz, 1H), 2.60−2.57 (m, 2H), 2.53−2.50 (m, 3H), 2.28 (s, 3H), 2.04−
2.00 (m, 1H), 1.80−1.74 (m, 1H).
2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-piperidine
(25). Hydrochloric acid (37%) in H2O (74.12 mL, 789.78 mmol) was
added to a solution of 24 (60 g, 157.96 mmol) in isopropyl alcohol
(420 mL) at 45 °C. The resulting solution was stirred at 45 °C 15 h.
The reaction mixture was concentrated to 1/4 volume to afford a
tert-Butyl 2′-Chloro-4′-oxospiro[piperidine-4,7′-thieno[2,3-c]-
pyran]-1-carboxylate (23). N-Bromosuccinimide (115.02 g, 646.23
3425
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Journal of Medicinal Chemistry
Article
white suspension. Then H2O (100 mL) was added, and the suspension
was basified with 6 N sodium hydroxide to obtain a two-layer mixture.
It was extracted with methyl tert-butyl ether (3 × 100 mL). Combined
organics were washed with brine (50 mL), dried over Na2SO4, and
concentrated to afford the title compound as a light brown solid (42 g,
95% yield). LC-MS (ESI) m/z: 280 (M + H)+; tR = 1.92 min, method
2. 1H NMR (300 MHz, CDCl3) δ: 6.97 (s, 1H), 4.06 (t, J = 10.2 Hz,
2H), 3.10−2.97 (m, 4H), 2.14−2.07 (m, 2H), 1.86−1.76 (m, 2H).
HRMS (ESI): calcd for C11H12ClF2NOS 279.0296, found 279.0305.
Methyl 2-[4-[(2-chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-
7,4′-piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluoroben-
zoate (26). The title compound was essentially prepared following
general method A using 25 (19.95 g, 71.31 mmol), methyl 3-fluoro-2-
(4-formyl-3-methylpyrazol-1-yl)benzoate (17 g, 64.83 mmol) and
sodium triacetoxyborohydride (16.49 g, 77.79 mmol) using 170 mL of
tetrahydrofuran (t1 = 60 min, t2 = 16 h, S = ethyl acetate). The crude
product was dissolved in methyl tert-butyl ether, and a beige solid
precipitated (excess of spiropiperidine). The solid was filtered, and the
solution was concentrated and purified by ISCO (330 g cartridge),
eluting with hexane/ethyl acetate (80:20 to 0:100 ratio) to obtain the
title compound as a beige solid (25 g, 73% yield). LC-MS (ESI) m/z:
526 (M + H)+; tR = 2.60 min, method 2. 1H NMR (300 MHz, CDCl3)
δ: 7.58 (m, 2H), 7.43−7.31 (m, 2H), 6.96 (s, 1H), 4.04 (t, J = 10.2 Hz,
2H), 3.70 (s, 3H), 3.49 (s, 2H), 2.81−2.77 (m, 2H), 2.43−2.35 (m,
2H), 2.30 (s, 3H), 2.16−2.04 (m, 2H), 1.89 (m, 2H). HRMS (ESI):
calcd for C24H23ClF3N3O3S 525.1101, found 525.1098.
2.27 (s, 3H), 2.18−2.11 (m, 2H), 1.80−1.77 (m, 2H). HRMS (ESI):
calcd for C21H20ClF3N4OS, 468.0998; found, 468.0997.
1-[2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-
piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorophenyl]-
N,N-dimethylmethanamine (L)-Tartrate (29). The free base of the
title compound was essentially prepared following general method A
using 25 (151 mg, 0.54 mmol), 1-[2-(dimethylaminomethyl)-6-
fluorophenyl]-3-methylpyrazole-4-carbaldehyde (128 mg, 0.49
mmol), and sodium triacetoxyborohydride (208 mg, 0.98 mmol)
using 3 mL of tetrahydrofuran (t1 = 60 min, t2 = 16 h, S = ethyl
acetate). The crude product was purified by Isco chromatography,
eluting with dichloromethane/2 M ammonia in methanol from 3% to
7% to get the free base of the title compound (256 mg, 100%). LC-MS
(ESI) m/z: 525 (M + H)+; tR = 2.67 min, method 2. The title
compound (tartrate salt) was essentially prepared following general
method B with 94% yield. LC-MS (ESI) m/z: 525 (M + H)+; tR = 4.47
min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 7.81 (s, 1H), 7.51
(td, J = 7.9, 5.7 Hz, 1H), 7.40−7.33 (m, 3H), 4.19−4.14 (m, 3H),
3.49−3.40 (m, 5H), 2.81−2.71 (m, 2H), 2.52−2.50 (m, 1H), 2.25−
2.33 (m, 1H), 2.23 (s, 3H), 2.18−2.07 (m, 8H), 1.86−1.70 (m, 2H).
1-[2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-
piperidine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-pyridyl]-N,N-di-
methylmethanamine (L)-Tartrate (30). The free base of the title
compound was essentially prepared following general method A using
25 (140 mg, 0.51 mmol), 1-(3-dimethylaminomethylpyridin-2-yl)-3-
methyl-1H-pyrazole-4-carbaldehyde (0.12 g, 0.51 mmol), and sodium
triacetoxyborohydride (220 mg, 1.02 mmol) using 2.5 mL of
tetrahydrofuran (t1 = 60 min, t2 = 6 h, S = ethyl acetate). The
crude product was purified by reverse phase HPLC (XBridge C18:5
μm, 19 × 100 mm. Phase A: ammonium bicarbonate 20 mM in water,
pH 8; phase B: acetonitrile. gradient 40−70% B in 8 min) to give 106
mg (40%) of the title compound as the free base. LC-MS (ESI) m/z:
508 (M + H)+. tR = 4.08 min, method 1. 1H NMR (300 MHz, DMSO-
d6) δ: 8.37 (dd, J = 1.8, 4.8 Hz, 1H), 8.16 (s, 1H), 8.03 (dd, J = 1.8, 7.7
Hz, 1H), 7.41−7.34 (m, 1H), 7.34 (s, 1H), 4.20−4.13 (m, 2H), 3.72
(s, 2H), 3.43−3.40 (m, 2H), 2.71 (d, J = 11.0 Hz, 2H), 2.28−2.25 (m,
2H), 2.25 (s, 3H), 2.10−2.08 (m, 2H), 2.08 (s, 6H), 1.83−1.72 (m,
2H). The title compound (tartrate salt) was essentially prepared
following general method B (97% yield). LC-MS (ESI) m/z: 508 (M +
H)+. tR = 3.97 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.46
(dd, J = 1.6, 4.8 Hz, 1H), 8.31 (s, 1H), 8.04 (dd, J = 1.6, 7.7 Hz, 1H),
7.42 (dd, J = 4.8, 7.7 Hz, 1H), 7.34 (s, 1H), 4.21−4.03 (m, 5H), 3.47
(s, 2H), 3.16 (s, 2H), 2.74−2.71 (m, 2H), 2.40−2.30 (m, 5H), 2.29
(m, 8H), 2.18−2.09 (m, 2H), 1.83−1.73 (m, 2H).
[2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-pi-
peridine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorophenyl]-
methanol (L)-Tartrate (31). A solution of 1 M lithium aluminum
hydride in tetrahydrofuran (45.63 mL, 45.63 mmol) was added to a
solution of 26 (30 g, 57.04 mmol) in tetrahydrofuran (240 mL) under
nitrogen at −20 °C. The cold bath was removed, allowing the reaction
mixture to reach 0 °C in 30 min. Water (2 mL) was added dropwise
carefully, followed by 2 N sodium hydroxide (2 mL) and water (6
mL). The resulting suspension was stirred at room temperature for 30
min, filtered over Celite, and solid washed with ethyl acetate (20 mL).
The filtrate was dried over sodium sulfate, concentrated, and purified
by Isco (330 g cartridge), eluting with 2-propanol/dichloromethane
(3:97 to 6:94 ratio) to get the free base of the title compound as a
colorless oil (23.75 g, 84% yield). LC-MS (ESI) m/z: 498 (M + H)+;
tR = 4.10 min, method 1. 1H NMR (300 MHz, CDCl3) δ: 7.65 (d, J =
3.9 Hz, 1H), 7.30 (m, 1H), 7.29 (s, 1H), 7.17 (m, 1H), 6.96 (s, 1H),
5.29 (br s, 1H), 4.37 (br s, 2H), 4.04 (t, J = 10.2 Hz, 2H), 3.47 (s, 2H),
2.78 (m, 2H), 2.39 (m, 2H), 2.33 (s, 3H), 2.1 (m, 2H), 1.86 (m, 2H).
HRMS (ESI): calcd for C23H23ClF3N3O2S 497.1152, found 497.1151.
The title compound (tartrate salt) was essentially prepared following
general method B with 95% yield. LC-MS (ESI) m/z: 498 (M + H)+;
tR = 4.14 min, method 1. 1H NMR (500 MHz, acetone-d6) δ: 8.04 (br
s, 1H), 7.54−7.47 (m, 2H), 7.27 (m, 1H), 7.18 (s, 1H), 4.44 (s, 2H),
4.40 (s, 2H), 4.22 (t, J = 10.2 Hz, 2H), 4.06 (s, 2H), 3.35−2.96 (m,
4H), 2.37 (s, 3H), 2.32 (m, 4H). HRMS (ESI): calcd for
C23H23ClF3N3O2S 497.1152, found 497.1148.
2-Chloro-1′-[[1-(2,6-difluorophenyl)-3-methylpyrazol-4-yl]-
methyl]-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-piperidine (L)-
Tartrate (27). The free base of the title compound was essentially
prepared following general method A using 25 (7.14 g, 25.52 mmol),
1-(2,6-difluorophenyl)-3-methyl-1H-pyrazole-4-carbaldehyde (5.40 g,
24.30 mmol), and sodium triacetoxyborohydride (9.27 g, 43.75 mmol)
using 54 mL of 1,2-dichloroethane (t1 = 30 min, t2 = 2 h, S = tert-
butyl methyl ether). The crude product was purified by silica gel
chromatography eluting with dichloromethane/methanol (97:3) to
give 7.1 g (60% yield) of the free base of the title compound. LC-MS
1
(ESI) m/z: 486 (M + H)+; tR = 4.66 min, method 1. H NMR (300
MHz, CDCl3) δ: 7.54 (s, 1H), 7.37−7.28 (m, 1H), 7.08−7.01 (m,
2H), 6.96 (s, 1H), 4.05 (t, J = 10.3 Hz, 2H), 3.49 (s, 2H), 2.84−2.80
(m, 2H), 2.41 (m, 2H), 2.35 (s, 3H), 2.13−2.06 (m, 2H), 1.98−1.81
(m, 2H). The title compound (tartrate salt) was essentially prepared
following general method B with 96% yield. LC-MS (ESI) m/z: 486
(M + H)+. tR = 4.67 min, method 1. 1H NMR (300 MHz, DMSO-d6)
δ: 7.92 (s, 1H), 7.61−7.51 (m, 1H), 7.36−7.30 (m, 3H), 4.24 (s, 2H),
4.18 (t, J = 10.7 Hz, 1H), 3.54 (s, 2H), 2.80 (d, J = 11.1 Hz, 2H),
2.47−2.37 (m, 2H), 2.23 (s, 3H), 2.19−2.12 (m, 2H), 1.88−1.76 (m,
2H). HRMS (ESI): calcd for C22H20ClF4N3OS, 485.0952; found,
485.0963.
2-Chloro-4,4-difluoro-1′-[[1-(3-fluoro-2-pyridyl)-3-methylpyrazol-
4-yl]methyl]spiro[5H-thieno[2,3-c]pyran-7,4′-piperidine] (L)-Tar-
trate (28). The free base of the title compound was essentially
prepared following general method A using 25 (90% purity) (19.99 g,
64.33 mmol), 1-(3-fluoro-2-pyridyl)-3-methylpyrazole-4-carbaldehyde
(12 g, 58.48 mmol), and sodium triacetoxyborohydride (18.59 g, 87.72
mmol) using 120 mL of 1,2-dichloroethane (t1 = 15 min, t2 = 15 h, S
= tert-butyl methyl ether). The crude product was purified by silica gel
chromatography, eluting with mixtures of dichloromethane/methanol
to give 18 g of a thick oil which was triturated with hexane and 10%
tert-butyl methyl ether/hexane to afford 16.5 g (61% yield) of the free
base of the title compound. LC-MS (ESI) m/z: 469 (M + H)+; tR =
1
4.27 min, method 1. H NMR (300 MHz, DMSO-d6) δ: 8.33−8.31
(m, 1H), 8.24 (s, 1H), 7.95 (ddd, J = 11.5, 8.2, 1.5 Hz, 1H), 7.47−7.41
(m, 1H), 7.33 (s, 1H), 4.16 (t, J = 10.8 Hz, 2H), 3.44 (s, 2H), 2.70 (d,
J = 11.7 Hz, 2H), 2.28 (m, 2H), 2.26 (s, 3H), 2.12−2.04 (m, 2H), 1.76
(td, J = 12.9, 3.8 Hz, 2H). The title compound (tartrate salt) was
essentially prepared following general method B with 97% yield. LC-
1
MS (ESI) m/z: 469 (M + H)+. tR = 4.28 min, method 1. H NMR
(300 MHz, DMSO-d6) δ: 8.33 (d, J = 4.3 Hz, 1H), 8.28 (s, 1H), 7.97−
7.93 (m, 1H), 7.48−7.43 (m, 1H), 7.35 (s, 1H), 4.24 (s, 2H), 4.18 (t, J
= 10.9 Hz, 2H), 3.55 (s, 2H), 2.81−2.77 (m, 2H), 2.42−2.39 (m, 2H),
3426
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Article
[2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-pi-
peridine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-fluorophenyl]-N-
methylmethanamine (L)-Tartrate (32). Manganese(IV) oxide (13.89
g, 159.77 mmol) was added to a solution of 31 (28 g, 56.23 mmol) in
dichloromethane (224 mL) at room temperature, and the resulting
suspension was stirred at reflux for 2.5 h. Additional manganese(IV)
oxide (33.33 g, 383.38 mmol) was added, and the mixture was stirred
at reflux for 4 h and then at room temperature for 15 h. More
manganese(IV) oxide (8.33 g, 95.82 mmol) was added, and stirring
was continued for 7 h at room temperature. The reaction mixture was
left stand for 1 h without stirring and filtered through Celite eluting
with dichloromethane. The filtrate was concentrated to provide the
intermediate aldehyde (17 g) that was used without further
purification. Methylamine (40%) in water (3.25 mL, 37.71 mmol)
was added to a solution of the intermediate aldehyde (17 g, 34.28
mmol) in ethanol (170 mL) at 0 °C. The resulting mixture was stirred
at room temperature for 15 h and then cooled with an ice/water bath.
Sodium borohydride (0.778 g, 20.57 mmol) was added, and the
mixture was stirred at room temperature for 3 h. An aqueous solution
of 5% HCl was added dropwise at 0 °C until no gas evolution was
observed (pH = 6, around 20 mL), and the mixture was concentrated
to 1/4 of the volume. A saturated aqueous solution of sodium
bicarbonate was added (100 mL), and the resulting suspension was
extracted with ethyl acetate (3 × 100 mL). Combined organic layers
were dried over sodium sulfate and concentrated to afford a crude
mixture that was purified by Isco (330 g cartridge), eluting with 7 N
ammonia in methanol/dichloromethane (3:97 to 5:95 ratio) to get the
free base of the title compound (14 g, 80% yield). LC-MS (ESI) m/z:
511 (M + H)+; tR = 4.05 min, method 1. 1H NMR (300 MHz, CDCl3)
δ: 7.49 (d, J = 2.1 Hz, 1H), 7.33 (m, 1H), 7.25 (m, 1H), 7.11 (m, 1H),
6.96 (s, 1H), 4.04 (t, J = 10.2 Hz, 2H), 3.53 (s, 2H), 3.48 (s, 2H), 2.78
(m, 2H), 2.39 (m, 2H), 2.34 (s, 3H), 2.32 (s, 3H), 2.1 (m, 2H), 1.86
(m, 2H). HRMS (ESI): calcd for C24H26ClF3N4OS 510.1468, found
510.1469. The title compound (tartrate salt) was essentially prepared
following general method B with 95% yield. LC-MS (ESI) m/z: 511
(M + H)+; tR = 4.09 min, method 1. 1H NMR (300 MHz, DMSO-d6)
δ: 7.92 (d, J = 2.1 Hz, 1H), 7.58−7.43 (m, 3H), 7.35 (s, 1H), 4.18 (t, J
= 10.8 Hz, 2H), 3.98 (s, 2H), 3.75 (s, 2H), 3.46 (s, 2H), 2.74 (m, 2H),
2.42 (s, 3H), 2.32 (m, 2H), 2.26 (s, 3H), 2.1 (m, 2H), 1.79 (m, 2H).
HRMS (ESI): calcd for C24H26ClF3N4OS 510.1468, found 510.1466.
2-Chloro-4,4-difluoro-1′-[(3-methyl-1H-pyrazol-4-yl)methyl]-
spiro[5H-thieno[2,3-c]pyran-7,4′-piperidine] (33). The title com-
pound was essentially prepared following general method A using 25
(105 g, 375 mmol), 3-methyl-1H-pyrazole-4-carbaldehyde (43.40 g,
394.12 mmol), and sodium triacetoxyborohydride (95.46 g, 450.42
mmol) using 1.58 L of tetrahydrofuran (t1 = 60 min, t2 = 15 h, S =
ethyl acetate). A 140 g amount of the title compound was obtained
(100% yield). LC-MS (ESI) m/z: 374 (M + H)+. tR = 2.09 min,
method 2. 1H NMR (300 MHz, CDCl3) δ: 7.50 (s, 1H), 6.95 (s, 1H),
4.03 (t, J = 10.3 Hz, 2H), 3.47 (s, 2H), 2.86−2.78 (m, 2H), 2.40 (t, J =
11.4 Hz, 2H), 2.30 (s, 3H), 2.11−2.04 (m, 2H), 1.96−1.84 (m, 2H).
HRMS (ESI): calcd for C16H18ClF2N3OS 373.0827, found 373.0834.
2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-pi-
peridine]-1′-yl)methyl]-3-methylpyrazol-1-yl]pyridine-3-carbalde-
hyde (34). To a 250 mL flask were added copper(I) iodide (1.91 g,
10.03 mmol), 33 (25 g, 66.87 mmol), potassium carbonate (19.60 g,
140.43 mmol), toluene (50 mL), and a stir bar. The reaction mixture
was degassed by five vacuum/refill cycles. Then 2-bromo-3-
formylpyridine (18.66 g, 100.31 mmol) and ( )-trans-N,N′-
dimethyl-1,2-cyclohexanediamine (3.16 mL, 20.06 mmol) were
added. The reaction was stirred at room temperature for 5 min.
Then it was immersed in a preheated oil bath at 115 °C and stirred for
15 h at that temperature. The reaction mixture was cooled to room
temperature, diluted with 300 mL of ethyl acetate, and filtered through
Celite. It was washed with ethyl acetate (100 mL) and aqueous
solution of ammonium hydroxide (3%) (4 × 100 mL) to remove
copper traces, and then it was washed with water (50 mL) and brine
(50 mL). The solution was dried over sodium sulfate. The solvent was
evaporated in vacuo to give a tan solid that was filtered through a pad
of silica gel, eluting with 2-propanol/dichloromethane (3% to 5% of 2-
propanol) to give 22 g of the title compound (69% yield). LC-MS
1
(ESI) m/z: 479 (M + H)+. tR = 2.72 min, 88% purity, method 2. H
NMR (300 MHz, CDCl3) δ: 10.75 (s, 1H), 8.55 (dd, J = 1.8, 4.8 Hz,
1H), 8.42 (s, 1H), 8.24 (dd, J = 1.9, 7.7 Hz, 1H), 7.30−7.28 (m, 1H),
6.96 (s, 1H), 4.04 (t, J = 10.3 Hz, 2H), 3.48 (s, 2H), 2.80 (d, J = 11.3
Hz, 2H), 2.46−2.37 (m, 2H), 2.34 (s, 3H), 2.10 (dd, J = 2.3, 14.4 Hz,
2H), 1.92−1.82 (m, 2H).
2-Chloro-4,4-difluoro-1′-[[1-[3-(1H-imidazol-2-yl)-2-pyridyl]-3-
methylpyrazol-4-yl]methyl]spiro[5H-thieno[2,3-c]pyran-7,4′-piperi-
dine] (35). A solution of 40% in water of ethanedial (1.69 mL, 14.70
mmol) and ammonium hydroxide (32% in water) (1.79 mL, 14.70
mmol) was added to a solution of 34 (1.76 g, 3.67 mmol) in methanol
(37 mL) at 0 °C. The reaction mixture was stirred for 30 min at 0 °C
and then heated at 50 °C with stirring for 16 h. The solvent was
removed under reduced pressure. The crude mixture was purified by
column chromatography with silica gel, eluting with 2 N ammonia in
methanol/dichloromethane (1:99 to 10:90 ratio) to get the title
compound (821 mg, 43% yield). LC-MS (ESI) m/z: 518 (M + H)+; tR
1
= 3.75 min, method 1. H NMR (300 MHz, DMSO-d6) δ: 12.08−
12.04 (m, 1H), 8.52 (dd, J = 1.8, 4.8 Hz, 1H), 8.22 (dd, J = 1.8, 7.7 Hz,
1H), 7.96 (s, 1H), 7.49 (dd, J = 4.8, 7.7 Hz, 1H), 7.34 (s, 1H), 7.19−
7.15 (m, 1H), 6.99 (s, 1H), 4.21−4.14 (m, 2H), 3.37 (m, 2H), 2.69 (d,
J = 10.6 Hz, 2H), 2.28−2.18 (m, 2H), 2.14−2.08 (m, 5H), 1.79−1.69
(m, 2H). HRMS (ESI): calcd for C24H23ClF2N6OS, 516.1311; found,
516.1325.
[2-[4-[(2-Chloro-4,4-difluorospiro[5H-thieno[2,3-c]pyran-7,4′-pi-
peridine]-1′-yl)methyl]-3-methylpyrazol-1-yl]-3-pyridyl]methanol
(L)-Tartrate (36). To a solution of 34 (12.5 g, 26.10 mmol) in
dichloromethane (125 mL) at 0 °C were added sodium tetrahy-
droborate (395 mg, 10.44 mmol) and methanol (37.50 mL). The ice
bath was removed, and the reaction was stirred at room temperature
for 30 min. Water (50 mL) was added, and the mixture was
concentrated to half volume to precipitate a white sticky solid.
Dichloromethane (100 mL) was added, and the biphasic mixture was
separated. The organic layer was washed with water (50 mL), dried
over sodium sulfate, and concentrated to half volume (around 100
mL). Methyl tert-butyl ether (100 mL) was added, and when the
solution was concentrated under vacuum, a solid precipitated that was
filtered and dried under vacuum to give 12 g (96% yield) of the title
compound as the free base. LC-MS (ESI) m/z: 481 (M + H)+. tR =
1
4.13 min, method 1. H NMR (300 MHz, CDCl3) δ: 8.41 (s, 1H),
8.37 (dd, J = 1.5, 4.8 Hz, 1H), 7.76 (dd, J = 1.6, 7.5 Hz, 1H), 7.18 (dd,
J = 4.8, 7.3 Hz, 1H), 6.96 (s, 1H), 6.06 (t, J = 7.7 Hz, 1H), 4.66 (d, J =
7.7 Hz, 2H), 4.04 (t, J = 10.2 Hz, 2H), 3.48 (s, 2H), 2.79 (d, J = 11.7
Hz, 2H), 2.41−2.36 (m, 5H), 2.10 (d, J = 12.1 Hz, 2H), 1.91−1.81
(m, 2H). The title compound (tartrate salt) was essentially prepared
following general method B with 100% yield. LC-MS (ESI) m/z: 481
(M + H)+. tR = 4.14 min, method 1. 1H NMR (300 MHz, DMSO-d6)
δ: 8.36−8.35 (m, 2H), 8.13 (d, J = 7.4 Hz, 1H), 7.42−7.35 (m, 2H),
4.79 (s, 2H), 4.24−4.14 (m, 4H), 3.55 (s, 2H), 2.82−2.75 (m, 2H),
2.50−2.35 (m, 2H), 2.27 (s, 3H), 2.14 (d, J = 12.9 Hz, 2H), 1.84−1.75
(m, 2H).
2-Chloro-4,4-difluoro-1′-[[1-[3-(methoxymethyl)-2-pyridyl]-3-
methylpyrazol-4-yl]methyl]spiro[5H-thieno[2,3-c]pyran-7,4′-piperi-
dine] (L)-Tartrate (37). To a screw-cap test tube were added copper(I)
iodide (1.15 g, 6.02 mmol), 33 (15 g, 40.12 mmol), potassium
carbonate (11.76 g, 85.09 mmol), 15 mL of toluene (previously
bubbled with nitrogen for 20 min), and a stir bar. The reaction mixture
was bubbled with nitrogen for additional 10 min, and then 2-bromo-3-
methoxymethylpyridine (10.54 g, 52.16 mmol) and ( )-trans-N,N′-
dimethyl-1,2-cyclohexanediamine (1.9 mL, 12.04 mmol) were added.
The reaction tube was quickly sealed (caution: buildup of pressure
possible; use a safety shield), stirred at room temperature for 5 min,
and immersed in a preheated oil bath at 115 °C for 24 h. The sample
was cooled down to room temperature, diluted with ethyl acetate, and
filtered through Celite. The solvent was evaporated in vacuo. The
residue was purified by normal phase Isco chromatography using
hexanes/ethyl acetate (30−70%). The desired fractions were collected
and evaporated. Some impure fractions (<2g) were repurified by
normal phase Isco chromatography, eluting with hexanes/ethyl acetate
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(20−60% in ethyl acetate). All the fractions containing final
compound were combined and evaporated to dryness in vacuo. The
residue was dissolved in dichloromethane and washed with 10%
aqueous solution of ammonium hydroxide to remove copper residues.
The organic solvent was evaporated, and the compound was dried
overnight under vacuum to yield 13 g (65% yield) of the free base of
the title compound. LC-MS (ESI) m/z: 495 (M + H)+. tR = 4.65 min,
J. R.; Grandy, D. K.; Langen, H.; Monsma, F., Jr.; Civelli, O.;
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1
method 1. H NMR (300 MHz, CDCl3) δ: 8.34 (d, J = 4.4 Hz, 1H),
8.22 (s, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.26−7.21 (m, 1H), 6.95 (s,
1H), 4.86 (s, 2H), 4.07−4.00 (m, 2H), 3.47 (bs, 5H), 2.79 (d, J = 11.7
Hz, 2H), 2.39−2.33 (m, 5H), 2.09 (d, J = 13.0 Hz, 2H), 1.91−1.81
(m, 2H). The title compound (tartrate salt) was essentially prepared
following general method B (96%). LC-MS (ESI) m/z: 495 (M + H)+.
tR = 4.60 min, method 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.39 (d, J
= 3.8 Hz, 1H), 8.34 (s, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.42−7.35 (m,
2H), 4.80 (s, 2H), 4.24−4.14 (m, 4H), 3.57(s,2H), 3.35 (s, 3H), 2.82
(d, J = 10.4 Hz, 2H), 2.50−2.39 (m, 2H), 2.27 (s, 3H), 2.14 (d, J =
13.4 Hz, 2H), 1.87−1.78 (m, 2H).
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ASSOCIATED CONTENT
* Supporting Information
■
S
Additional experimental details on the synthesis and character-
ization of all intermediates. This material is available free of
charge via the Internet at http://pubs.acs.org.
(6) (a) Bignan, G. C.; Connolly, P. J.; Middleton, S. A. Recent
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antagonists. Expert Opin. Ther. Pat. 2005, 15, 357−388. (b) Largent-
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Nakashima, H.; Kato, T.; Ohta, H.; Iwasawa, Y. Discovery of the
first potent and selective small molecule opioid receptor-like (ORL-1)
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(9) Satoh, A.; Sagara, T.; Sakoh, H.; Hashimoto, M.; Nakashima, H.;
Kato, T.; Goto, Y.; Mizutani, S.; Azuma-Kanoh, T.; Tani, T.; Okuda,
S.; Okamoto, O.; Ozaki, S.; Iwasawa, Y.; Ohta, H.; Kawamoto, H.
Identification of an orally active opioid receptor-like 1 (ORL-1)
receptor antagonist 4-{3-[(2R)-2,3-dihydroxypropyl]-2-oxo-2,3-dihy-
dro-1H-benzimidazol-1-yl}-1-[(1S,3S,4R)-spiro[bicyclo[2.2.1]heptane-
2,1′-cyclopropan]-3-ylmethyl]piperidine as clinical candidate. J. Med.
Chem. 2009, 52, 4091−4094.
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I. 4-Aminoquinolines: Novel nociceptin antagonists with analgesic
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AUTHOR INFORMATION
Corresponding Author
*Tel: 34-91-6233582. Fax: 34-91-6633411. E-mail: toledoma@
lilly.com.
■
Notes
The authors declare no competing financial interest.
§Deceased November 1, 2013.
ACKNOWLEDGMENTS
■
We thank the Analytical Technologies group of Lilly Spain for
the purification of compounds. We also acknowledge Anju
Lewis, Keyue Chen, and Lorrel Burison for the generation of
the in vitro NOP binding and functional data, and the binding
data for the classical opioid receptors. We dedicate this paper to
the memory of Dr. Concepcion Pedregal, our friend and
́
colleague, for her scientific talent, creativity, and enthusiasm.
ABBREVIATIONS USED
■
GSH, glutathione; N/OFQ, nociceptin/orphanin FQ peptide;
NOP, nociceptin receptor; ORL, opioid receptor-like; RO,
receptor occupancy; PFA, perfluoroalkoxy polymer; SCX,
strong cationic exchange
(11) (a) Zaratin, P. F.; Petrone, G.; Sbacchi, M.; Garnier, M.; Fossati,
C.; Petrillo, P.; Ronzoni, S.; Giardina, G. A. M.; Scheideler, M. A.
Modification of nociception and morphine tolerance by the selective
opiate receptor-like orphan receptor antagonist (−)-cis-1-methyl-7-
[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-
benzocyclohepten-5-ol (SB-612111). J. Pharmacol. Exp. Ther. 2004,
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