Preparation, Antiepileptic Activity, and Cardiovascular Safety of Dihydropyrazoles as Brain-Penetrant T-Type Calcium Channel Blockers

Article abstract of DOI:10.1021/acs.jmedchem.6b00756

A series of dihydropyrazole derivatives was developed as potent, selective, and brain-penetrating T-type calcium channel blockers. An optimized derivative, compound 6c, was advanced to in vivo studies, where it demonstrated efficacy in the WAG/Rij rat model of generalized nonconvulsive, absence-like epilepsy. Compound 6c was not efficacious in the basolateral amygdala kindling rat model of temporal lobe epilepsy, and it led to prolongation of the PR interval in ECG recordings in rodents.

Full text of DOI:10.1021/acs.jmedchem.6b00756

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Article
Preparation, antiepileptic activity and cardiovascular safety of
dihydropyrazoles as brain-penetrant T-type calcium channel blockers
Lubos Remen, Olivier Bezençon, Lloyd Simons, Rick D. Gaston, Dennis Downing,
John Gatfield, Catherine Roch, Melanie Kessler, Johannes Mosbacher, Thomas
Pfeifer, Corinna Grisostomi, Markus Rey, Eric A. Ertel, and Richard Moon
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00756 • Publication Date (Web): 31 Aug 2016
Downloaded from http://pubs.acs.org on September 1, 2016
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Preparation, antiepileptic activity and cardiovascular
safety of dihydropyrazoles as brainꢀpenetrant Tꢀtype
calcium channel blockers
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Lubos Remen , Olivier Bezençon *, Lloyd Simons , Rick Gaston , Dennis Downing , John
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Gatfield , Catherine Roch , Melanie Kessler , Johannes Mosbacher , Thomas Pfeifer , Corinna
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Grisostomi , Markus Rey, Eric A. Ertel and Richard Moon
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Drug Discovery Chemistry, Biology and Pharmacology, Actelion Pharmaceuticals Ltd.,
Gewerbestrasse 16, CHꢀ4123 Allschwil, Switzerland
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Kalexsyn, Inc., 4502 Campus Drive, Kalamazoo, Michigan 49008, USA
ABSTRACT: A series of dihydropyrazole derivatives was developed as potent, selective, brainꢀ
penetrating Tꢀtype calcium channel blockers. An optimized derivative, compound 6c, was
advanced to in vivo studies, where it demonstrated efficacy in the WAG/Rij rat model of
generalized nonꢀconvulsive, absenceꢀlike epilepsy. Compound 6c was not efficacious in the
basolateral amygdala kindling rat model of temporal lobe epilepsy, and it led to prolongation of
the PR interval in ECG recordings in rodents.
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INTRODUCTION
Epilepsy represents one of the most common chronic brain diseases. It is estimated that 0.5%
to 1% of the worldwide population will experience at least one unprovoked seizure in their
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lifespan . Epilepsy is a brain disorder characterized by an enduring predisposition to generate
epileptic seizures and by the neurobiological, cognitive, psychological, and social consequences
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of this condition . Epileptic patients experience recurrent spontaneous seizures, which may
present various phenotypes ranging from mild brief lapses of attention or muscle jerks up to
severe and prolonged convulsions. Seizures are transient events due to abnormal, excessive or
synchronous neuronal activity in the brain. They are classified either as focal seizures when they
remain restricted to networks in one hemisphere or as generalized seizures when they rapidly
engage bilaterally distributed networks. Etiologies and clinical presentations are multiple, so that
the soꢀcalled epileptic syndromes are classified in approximately sixty different types and subꢀ
types based on features such as clinical observations, electroencephalographic (EEG) pattern,
age of onset, cognitive and developmental antecedents and consequences, provoking or
triggering factors, that also includes genetic variations.
Many antiepileptic drugs (AEDs) exert their actions through direct or indirect modulation of
ion channels . Foremost pharmacological targets are GABA ꢀreceptors (targeted by diazepam
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and other benzodiazepines), voltageꢀgated sodium channels (targeted by carbamazepine ,
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lacosamide , lamotrigine , zonisamide and others), voltageꢀgated potassium channels
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retigabine ), and high voltageꢀgated calcium channels (lamotrigine , levetiracetam ,
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topiramate ). More recently an AMPA receptor blocker, perampanel, was approved as AED .
In spite of about 25 commerciallyꢀavailable AEDs, about one in three epileptic patients remains
or develops pharmacoresistant, meaning that sustained seizure freedom is not achieved following
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tryout of two tolerated and appropriately chosen and used AED schedules (whether as
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monotherapy or in combination) . This proportion, which has not decreased markedly for more
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than a century , clearly calls for new compounds with new mechanisms of action. At Actelion
Pharmaceuticals, we decided to target Tꢀtype calcium channels and to develop a new
mechanismꢀbased inhibitor that could be useful to treat epilepsy.
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Lowꢀvoltageꢀgated Tꢀtype calcium channels (TTCCs) are the products of three different genes
in mammals, CACNA1G, CACNA1H, and CACNA1I, leading to the Ca 3.1, Ca 3.2, and Ca 3.3
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channels, respectively . They are largely expressed in the brain , particularly in the thalamus
and cortex areas, suggesting an important role in thalamocortical signaling and rhythms. TTCCs
play a critical role in idiopathic generalized epilepsies (IGE), such as absence epilepsy, both in
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humans and in animals . Ethosuximide, the drug of choice for absence epilepsy , has been
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claimed to block TTCCs at pharmacologically relevant concentrations . Genetic variants of the
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human CACNA1H gene have been linked to absence epilepsies and those variants are
predicted to increase neuronal excitability either by altering the biophysical properties of the
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channel or by increasing surface expression,
and thereby to influence neuronal
excitability. In addition, Ca 3.1ꢀknockꢀdown mice are more resistant to absenceꢀtype seizures
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than their wild type littermates . More recently, TTCC antagonists were shown to attenuate or to
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suppress seizures in the WAG/Rij rat and the GAERS rat , two genetic animal models for
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absenceꢀlike epilepsy . Information on the efficacy of TTCC blockers in other types of
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epilepsies has remained sparse: a role for TTCCs has been suggested in temporal lobe epilepsy
but the effect of a selective TTCC blocker in relevant animal models has not been shown.
Brain penetrant, selective TTCC blockers have been reported only recently. As mentioned
above, ethosuximide has been claimed to block TTCCs, and zonisamide also blocks TTCCs in
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the micromolar range
. A first selective TTCC blocker 1 (MKꢀ8998, presumed structure,
Figure 1, IC ꢀvalues of 1.2 nM, 8.2 nM and 1.5 nM on the Ca 3.1ꢀ, Ca 3.2ꢀ, and Ca 3.3ꢀ
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channels respectively, in our hands) was evaluated in phase 1 and phase 2 clinical trials,
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targeting acute psychosis in patients with schizophrenia . Another compound 2 (Zꢀ944, IC ꢀ
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values of 37 nM, 73 nM and 86 nM on the Ca 3.1ꢀ, Ca 3.2ꢀ, and Ca 3.3ꢀchannels respectively, in
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our hands) was in phase 1 clinical trials and may have proceeded to phase 2 for neuropathic
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pain . Several other potent, selective TTCC blockers have been used in preclinical research to
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investigate different indications, such as pain , essential tremor , Parkinson disease , obesity ,
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addiction , or psychosis .
Our program started with the development of highꢀthroughput FLIPRꢀassays using conditions
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similar to those published previously . Since genetic variants of the CACNA1H gene had the
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strongest link to idiopathic generalized epilepsies , we selected Ca 3.2 for the highꢀthroughput
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screening (HTS) and we measured block of Ca 3.1 and Ca 3.3 in secondary assays. We
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developed a counter assay on Ca 1.2, an Lꢀtype high voltage activated calcium channel, to assess
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hypotension .
RESULTS AND DISCUSSION
From our HTS campaign, we identified dihydropyrazole 5a (Table 1) as a compound with a
promising potency on Ca 3.2, and with good selectivity toward Ca 1.2. A three step synthesis
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following known procedure gave us a rapid access to this class of compounds, presented in
Tables 1 and 2. An aldol condensation between a benzaldehyde and a methylphenyl ketone
(
Scheme 1) yielded α,βꢀunsaturated ketones of type 3. Cyclization with hydrazine led to
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dihydropyrazoles of type 4, which were converted immediately to the final, desired (racemic)
products of type 5 or 6. Nonꢀacylated dihydropyrazoles of type 4 proved to be unstable on
storage, oxidizing rapidly to the corresponding pyrazoles. Slight modifications led to amide
derivative 5i and to thiourea 5m (see supporting information for experimental details). This
approach allowed a straightforward investigation of the structure activity relationship (SAR) at
the urea moiety (Table 1). We found that an electron withdrawing, para fluoro substituent
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increased the potency on Ca 3.2 (compound 5b). A meta fluorine substituent did not contribute
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to potency (compound 5c), while an ortho fluorine substituent (compound 5d) was not tolerated.
Electron donating or electron neutral substituents at position 4 were detrimental (compounds 5e
and 5f). Replacing the phenyl substituent by a cycloalkyl or an alkyl group (compounds 5g and
5h) led to a decrease in potency and in selectivity versus Ca 1.2. Replacing the urea either by an
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amide (compound 5i), or by a thiourea (compound 5m), or elongating the urea substituent
(compound 5j) led to lower potencies as well. Finally the presence of heteroaryls, like a
pyridinyl in derivatives 5k and 5l was not tolerated.
This first SAR investigations led to potent Ca 3.2 blockers (compounds 5b, 5c). On the other
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hand, these compounds contained four aromatic rings and a urea functionality and were therefore
poorly soluble (< 1mg/L in aqueous buffer, see Table 5 for selected examples). The development
of an SAR at the other two aryl positions proved more complex, since it required the introduction
of diversity at the very first step of the synthesis. Therefore, we focused on substituents that
might improve solubility. Complete removal of an aryl substituent at either position 3 or 5 led to
a loss in potency (Table 2, compounds 6b and 6e) but suppression of the paraꢀbromo substituent
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on Ar was well tolerated (compound 6a). To our delight, introduction of a 4ꢀpyridinyl group at
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the Ar ꢀposition was tolerated (compound 6c), while the corresponding Nꢀoxide (compound 6d)
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was less potent. At the Ar ꢀposition, a paraꢀmethyl sulfone led to a potent blocker (compound
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f) but a combination of polar substituents at both Ar ꢀ and Ar ꢀpositions was not tolerated well
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compound 6g). Selectivity toward Ca 1.2 was well retained throughout the compound series.
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We also attempted to increase solubility by introducing a new substituent bearing a polar,
solubilizing group. One possibility was offered by the quaternization of the chiral center. This
quaternization should not only allow us to introduce a hydrophilic substituent (vide infra) but it
would prevent a possible aromatization, and it would increase the proportion of rotatable, nonꢀ
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aromatic bonds . As outlined in Scheme 2 , acylation of an activated alkyne led to Michael
acceptors of type 7, that upon addition of an aryl cuprate yielded compounds of type 8
Cyclization with hydrazine (compounds of type 9), formation of the urea (compounds of type
10), and deprotection led to final products 11a-l. We observed that at this position a
hydroxymethyl substituent was wellꢀtolerated (Table 3, compound 11a) and an elongated, polar
hydroxyethyl group was also tolerated (compound 11b). Based on this last substituent, we found
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that the potentially solubilizing 4ꢀmethoxyethoxy substituent on Ar (compound 11c) led to a
potent Ca 3.2 blocker, in contrast to a tertiary amine (compound 11d). Compound 11c was
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indeed slightly soluble in aqueous buffer, and replacement of a phenyl by a 4ꢀpyridinyl
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compound 11e) led to a moderate loss in potency but increased solubility from < 1mg/L to 35
mg/L in aqueous buffer at pH7 (Table 5). Further combinations of polar groups led to diverse
results; while compound 11f still retained most of its potency, and some solubility, compound
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1g proved to be less potent, most probably being too polar. Additional modifications at the
polar chain, in particular introduction of tertiary amines, were not well tolerated (Table 4,
compound 11iꢀ11l), even if these examples displayed a sufficient solubility profile (Table 5,
compounds 11k and 11l). From the solubility profiles presented in Table 5, no simple trend can
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be found between physicochemical properties and solubility, demonstrating the complexity of
the factors controlling this parameter. In general, an ionizable substituent has to be introduced
(compounds 6c, 11e, 11f, 11k and 11l) but compound 11c represent an exception. Unfortunately,
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gain in solubility often happened at the detriment of potency toward the target. Indeed, it seems
that the high potency values observed on Ca 3.2 in this series are at least partially driven by
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lipophilicity, even if a high polar surface area (PSA) is sometimes tolerated (compounds 6f, 11c,
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and 11f). The most potent derivative, compound 5b, has a PSA of 44.7 Å , and a very high clogP
value of 5.5. In terms of ligand efficiency (LE), the best blockers described here are compounds
6a (LE = 0.38), and 6c (LE = 0.37), which can be regarded as reasonable values. The lipophilic
ligand efficiencies (LLE), though, remain low. The best value is obtained for compound 6c (LLE
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= 3.6), clearly inferior to the threshold value of 5 recommended in the literature .
Since our goal was to achieve brain penetration, we monitored the central nervous system
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multiparameter optimization (CNSꢀMPO)ꢀvalues as well. When considering compounds with a
minimum of solubility, we realized that these values were rather low, in particular due to high
clogP, clogD, and molecular weight. For instance MPO values for compounds 6f, 11c, and 11f,
were of 3.8, 2.8, and 3.2, respectively. Selected compounds were measured in a multiꢀdrug
resistance gene 1 (MDR1) assay with a transfected MDCK cell line to assess their brain
permeability and their potential as Pꢀglycoprotein (Pꢀgp) substrates (Table 6). While compound
6c was permeable and was not effluxed by Pꢀgp, the more polar compounds 11f and 11g remain
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permeable but were clearly substrates for Pꢀgp. A limit with a PSA around 60 Å seemed to
emerge. At this stage it became clear that the ridge between adequate solubility and problematic
efflux substrate would be very narrow for this class of compounds, and we decided to use
compound 6c, which displayed acceptable solubility, as a model compound.
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As mentioned earlier all blockers were, up to now, prepared and tested as racemates. At this
stage, we prepared the reasonably potent compound 11h (Table 3), a very close analogue to
compound 11f, we separated its enantiomers by HPLC using a chiral solid phase, and we
crystallized one enantiomer to determine its absolute configuration by Xꢀray diffraction analysis.
This enantiomer was (S)ꢀconfigured (compound (S)-11h, Fig. 2) and this allowed us to identify
the (R)ꢀenantiomer as being the active one for compound 11h (Table 3). By analogy, we
assumed that the (R)ꢀenantiomer was responsible for the observed biological activity in all
compounds 11aꢀ11g, and that the (S)ꢀenantiomer should be the active one for compounds 5aꢀ5m
and 6aꢀ6g. Due to its importance, the enantiomers of compound 6c were separated as well (Table
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2). The discrimination between the enantiomers was not as marked in this case as for compounds
(R)-11 and (S)-11. Nevertheless, a factor ten difference in potency was observed on the cav3.2
channel.
Compound 6c proved to be a rather well balanced blocker for all three channels Ca 3.1,
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Ca 3.2, and Ca 3.3, with a somewhat lower affinity for Ca 3.3 (Table 7). The Ca ꢀFLIPR assay
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used to characterize and optimize our blockers did not control cell membrane potential, which
rests spontaneously at around ꢀ25 mV in these cells. Therefore, IC ꢀvalues measured in this
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system mostly reflect compound binding to channels in the inactivated state . In order to
measure IC ꢀvalues reflecting compound binding to channels in the closed or open states, we set
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up automated patchꢀclamp (QPatch) measurements using resting potentials at ꢀ90 mV and ꢀ60
mV. From these experiments, compound 6c displayed a moderate voltageꢀdependence on all
three TTCCs. Compound 6c proved to be selective toward other channels: weak inhibition of
Ca 1.2 was observed (Table 2) as well as weak inhibition of endogenous voltage regulated
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sodium and potassium channels in rodent neurons (Table 7). Most probably owing to its basic
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pyridinyl moiety, compound 6c blocked the hERG channel with an IC ꢀvalue of 0.8 ꢀM. It
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should be noted that this was rather an exception within this series. For instance, both compound
a and compound 5c blocked the hERG channel with IC ꢀvalues above 10 ꢀM. On the other
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hand, compound 6c inhibited CYP3A4, due to its 4ꢀpyridinyl moiety. The CYP2C9 was
inhibited as well, on the contrary to CYP2D6. It was metabolically rather stable using human, rat
and mouse microsomes, it had an unbound fraction of 2ꢀ3% in rat and human plasma (Table 7)
and a good permeability (Table 6): these data encouraged us to profile this compound in vivo.
A first pharmacokinetic (PK) study in rats (Table 8) indicated a systemic clearance equal to
liver blood flow, in spite of its low intrinsic clearance in rat microsomes (Table 7, 104
.
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L/min.mg protein, or about 10 mL/min kg, with f
= 0.5) and in rat hepatocytes (Clintr. =
u,microsomes
.
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.7 ꢀL/min 10 cells). The high in vivo clearance led to low exposure after oral administration at
a dose of 2 mg/kg. However, oral coꢀadministration of the general broadꢀband CYPꢀinhibitor
aminobenzotriazol (ABT) 2 hours before application of the iv dose led to an almost complete
suppression of clearance, indicating that oxidative clearance by the CYPꢀenzymes represented
the main metabolism pathway for this compound in rats. Under this condition, the compound
was quantitatively bioavailable, confirming excellent absorption properties. In dogs (without
ABT), an acceptable maximal concentration was observed after oral dose of 10 mg/kg. Brain
exposure was studied as well, albeit with higher doses, in order to saturate metabolism and
achieve pharmacologically relevant concentrations (Table 9). Indeed, plasma concentrations at 1
and 3 h postꢀdose indicated a nonꢀlinear behavior between 2 and 10 mg/kg (compare data in
Tables 8 and 9), most probably due to a saturation of metabolism at the higher dose. The
compound penetrated the brain very well at all doses between 10 and 100 mg/kg and the CSF
concentrations were in a fair agreement with the unbound concentrations in plasma.
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Since there is a strong rationale for a role of TTCCs in idiopathic generalized epilepsies
including absence epilepsy, we tested the efficacy of compound 6c in the WAG/Rij rat (Wistar
albino rats of Glaxo Rijswijk) model of generalized nonꢀconvulsive absenceꢀlike epilepsy. Male
WAG/Rij rats older than 6 months were implanted with radiotelemetry probes allowing
continuous recording of the electroencephalogram/electromyogram and they were administered
with a single oral dose of 100 mg/kg compound 6c (or matching vehicle) at the beginning of
their nocturnal active phase. Compound 6c decreased by 63.5 % the number of seizures over the
12 h night period compared to vehicle (p = 0.0013, paired tꢀtest; Fig. 3A), corresponding to a
decrease of 62.2 % of the total duration of the seizures during this period (or 1239 seconds; Fig.
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3B). Over the first 4 h following administration, spontaneous seizures were nearly completely
suppressed by compound 6c with a decrease of more than 99 % of the number and duration of
seizures compared to vehicle. From the brain exposure experiment (Table 9), at 1 h Cu,brain is
equal to 128 nM. This is above the IC ꢀvalues for the Ca 3.1ꢀ and Ca 3.2 channels in our FLIPR
50
v
v
assay, while the other IC ꢀvalues are above this concentration. We can hypothesize that either
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the Cav3.3 channel is less important for the generation of seizure in this animal model, or that
the IC ꢀvalues do not need to be reached to already have a full suppression of seizures. Further,
5
0
a compound blocking the channels at the inactivated state seems to be sufficient to prevent
seizures. Those data are in accordance with published studies showing strong reductions of
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seizures in this model with potent TTCC blockers
.
We also tested compound 6c at 300 mg/kg p.o. in the basolateral amygdala kindling rat model
of temporal lobe epilepsy (a type of focal epilepsies). In this model, animals are electrically
stimulated daily in the basolateral amygdala until they are fully kindled, i.e. until they reach
43
robust occurrence of stage 5 seizures on Racine's scale at each stimulation . Compound 6c, at
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the dose tested, was neither able to decrease the focal susceptibility to generate seizures, nor to
block the secondary generalization. In fully kindled rats, 2 h following administration of
compound 6c, the afterdischarge threshold, defined as the lowest current intensity inducing an
electrographic response ≥ 3 sec, was not changed compared to vehicle treated animals (Wilcoxon
test, p = 0.125, Fig. 4A). The duration of the afterdischarge on EEG at the threshold intensity and
the behavioral seizure response (on the Racine's scale: stage 0: arrest, normal behavior; stage 1:
facial twitches; stage 2: chewing, head nodding; stage 3: forelimb clonus; stage 4: rearing, falling
on forelimbs; stage 5: rearing, falling on side or back, rolling) were also unchanged compared to
vehicle treated rats (p = 0.3125 and p = 0.75, Figs. 4B and C respectively). Similar results were
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recently published with compound 2 , in line with what we observed with compound 6c.
The cardiovascular safety of compound 6c was evaluated in freely moving, conscious male
spontaneously hypertensive rats (SHRs) equipped with a telemetry system allowing noninvasive
monitoring of mean arterial pressure (MAP), heart rate (HR) and electrocardiogram. The
compound was administered as a single oral dose of 100 mg/kg in the morning at the end of the
wake phase of the animals. MAP was decreased ~12 mmHg by Compound 6c and it returned to
baseline values within 18 hours (Fig. 5A). A slight decrease in HR was observed which returned
to baseline values within 6 hours (data not shown). On the electrocardiogram, compound 6c
increased PRꢀintervals (indicative of AVꢀblock type I, Fig. 5B) and induced AVꢀblocks type II
(Wenckebach and Mobitz II). In spite of the low IC ꢀvalue of compound 6c on the hERG
5
0
channel, no QTꢀprolongation was observed. If we assume that exposure in these animals should
be similar to what we observed in the bloodꢀbrain pharmacokinetic experiments we run in Wistar
rats (Table 9), free concentration at 1h (C = 37 nM) corresponds to IC ꢀvalues on Ca 3.1 and
u
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Ca 3.2, both present in the heart, and remain much below the IC ꢀvalue on the hERG channel.
v
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CONCLUSIONS
In conclusion, we have developed a series of dihydropyrazole compounds as potent, selective
and brain penetrant TTCC blockers. The lead compound of this series, dihydropyrazole 6c, was
used as a tool compound to evaluate potential efficacy for epilepsy indications and
cardiovascular safety; it showed good efficacy and minimal safety issues. Issues related to
solubility and intellectual property led us to stop the development of this class of compounds.
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EXPERIMENTAL SECTION
Chemistry. All final compounds were analyzed by 1HꢀNMR and LCꢀMS, and were found to
be > 95% pure by these analytical methods. Description of general methods, especially of
gradients used in LCꢀMS, can be found in the supporting information.
(E)-1-(4-Bromophenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3a). To a solution of 4ꢀ
fluorobenzaldehyde (3.00 g, 24.2 mmol) in ethanol (50 ml) was added aqueous 30% KOH (10
ml) during 2 minutes, and the mixture was stirred for 5 minutes at 23 °C. A solution of 1ꢀ(4ꢀ
bromophenyl)ethanꢀ1ꢀone (4.80 g; 24.2mmol) in diethyl ether (20 ml) was added during 5
minutes at rt and the mixture was stirred for 50 minutes. The suspension was poured into ice ꢀ
water and aqueous 1N HCl (150 ml) was added to maintain the pH = 1. The precipitate was
filtered off, washed with cold water (100 ml) and stirred in ethanol (75 ml) for 1 h. The crude
1
product was filtered off and dried to afford 5.6 g (76%) of a yellowish solid. H NMR (400 MHz,
CDCl3) δ 7.90 (d, J = 8.5 Hz, 2H), 7.80 (d, J =15.7 Hz, 1H), 7.63ꢀ7.70 (m, 4H), 7.43 (d, J = 15.7
Hz, 1H), 7.14 (t, J = 8.6 Hz, 2H). LCꢀMS (method A): t = 1.13 min; [M+H]+: not visible.
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3
-(4-Bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole (4a). To a solution of 3a
(
1.43 g, 4.70 mmol) in toluene (60 ml) was added hydrazine hydrate (0.75 ml, 15.5 ml) and the
mixture was stirred at rt for 21 h. The solvent was evaporated in vacuo and crude product (1.52 g
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of brown oil) was used in the next step without purification. LCꢀMS (method A): t = 1.05 min;
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+
[M+H] : not visible.
3-(4-Bromophenyl)-5-(4-fluorophenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-
carboxamide (5a). To a solution of 4a (1.52 g, 4.76 mmol) in toluene (120 ml) was added
phenylisocyanate (568 mg; 4.77 mmol) and Et N (3 drops) and the mixture was stirred at 90 °C
3
for 40 minutes. The solvent was evaporated in vacuo and crude was purified by automated flash
chromatography (Büchi Sepacore, 50 g cartridge, solvent A: heptane, solvent B: ethyl acetate,
1
gradient in % B: 2 to 10, flow rate: 30ml/min) to afford 0.9 g of a yellowish foam. H NMR (400
MHz, CDCl3) δ 8.08 (s, 1H), 7.59ꢀ7.46 (m, 4H), 7.52 (d, J = 8.0 Hz, 2H), 7.28ꢀ7.33 (m, 4H),
7
1
1
.02ꢀ7.08 (m, 3H), 5.60 (dd, J = 12.2 Hz, J = 5.5 Hz, 1H), 3.83 (dd, J = 17.7 Hz, J = 12.2 Hz,
1 2 1 2
1
3
H), 3.19 (dd, J = 17.8 Hz, J = 5.5 Hz, 1H). C NMR (100 MHz, CDCl ) δ 163.5, 161.0,
1
2
3
51.4, 150.7, 138.3, 138.0, 132.0, 130.0, 128.9, 127.9, 127.4, 124.7, 123.1, 119.0, 115.8. LCꢀMS
+
(method A): t = 1.16 min; [M+H] : 437.67.
r
Following compounds have been synthesized from appropriate starting materials according to
the procedures described for compound 5a:
3-(4-Bromophenyl)-N,5-bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (5b).
1
H NMR (400 MHz, CDCl ) δ 8.02 (s, 1H), 7.59ꢀ7.64 (m, 4H), 7.47 (dd, J = 9.0 Hz, J = 4.8
3
1
2
Hz, 2H), 7.28ꢀ7.31 (m, 2H), 7.05 (t, J = 8.6 Hz, 2H), 7.02 (t, J = 8.6 Hz, 2H), 5.59 (dd, J = 12.2
1
Hz, J = 5.9 Hz, 1H), 3.85 (dd J = 17.7 Hz, J = 12.1 Hz, 1H), 3.20 (dd, J = 17.8 Hz, J = 5.9
2
1
2
1
2
+
Hz, 1H). LCꢀMS (method A): t = 1.16 min; [M+H] : 455.82.
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-(4-Bromophenyl)-N-(3-fluorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5c). H NMR (400 MHz, CDCl ) δ 8.14 (s, 1H), 7.49ꢀ7.55 (m, 4H), 7.46 (dt, J =
3
1
11.2 Hz, J = 2.1 Hz, 1H), 7.27ꢀ7.31 (m, 2H), 7.23 (t, J = 6.6 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H),
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.05 (t, J = 8.6 Hz, 1H), 6.75 (td, J = 8.3 Hz, J = 2.3 Hz, H), 5.59 (dd, J = 12.1 Hz, J = 5.8
1
2
1
2
Hz, 1H), 3.85 (dd, J = 17.8 Hz, J = 12.2 Hz, 1H), 3.20 (dd, J = 17.8 Hz, J = 5.8 Hz, 1H).
1
2
1
2
+
LCꢀMS (method A): t = 1.16 min; [M+H] : 455.70.
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3-(4-Bromophenyl)-N-(2-fluorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5d). H NMR (400 MHz, CDCl ) δ 8.38 (d, J = 2.6 Hz, 1H), 8.22 (t, J = 8.1 Hz,
3
1H), 7.59ꢀ7.64 (m, 4H), 7.29ꢀ7.32 (m, 2H), 6.95ꢀ7.17 (m, H), 5.60 (dd, J = 12.1 Hz, J = 5.8
1 2
Hz, 1H), 3.85 (dd, J = 17.7 Hz, J = 12.1 Hz, 1H), 3.21 (dd, J = 17.7 Hz, J = 5.8 Hz, 1H).
1
2
1
2
+
LCꢀMS (method A): t = 1.18 min; [M+H] : 455.91.
r
3-(4-Bromophenyl)-5-(4-fluorophenyl)-N-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5e). H NMR (300 MHz, CDCl ) δ 7.91 (s, 1H), 7.53ꢀ7.64 (m, 4H), 7.39 (d, J =
3
9.0 Hz, 2H), 7.23ꢀ7.29 (m, 2H), 7.02 (t, J = 8.7 Hz, 2H), 6.83 (d, J = 9.3 Hz, 2H), 5.56 (dd, J =
1
12.0 Hz, J = 5.90 Hz, 1H), 3.80 (dd, J = 17.9 Hz, J = 12.3 Hz, 1H), 3.77 (s, 3H), 3.16 (dd, J
1
2
1
2
+
= 17.6 Hz, J = 5.9 Hz, 1H). LCꢀMS (method A): t = 1.15 min; [M+H] : 467.79.
2
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3-(4-Bromophenyl)-N-(4-ethylphenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5f) . H NMR (400 MHz, CDCl ) δ 8.02 (s, 1H), 7.58ꢀ7.64 (m, 4H), 7.42 (d, J =
3
8.4 Hz, 2H), 7.27ꢀ7.31 (m, 2H), 7.14 (d, J = 8.3 Hz, 2H), 7.04 (t, J = 8.6 Hz, 2H), 5.60 (dd, J =
1
12.1 Hz J = 5.8 Hz, 1H), 3.83 (dd, J = 17.7 Hz, J = 12.2 Hz, 1H), 3.18 (dd, J = 17.7 Hz, J =
2 1 2 1 2
5.8 Hz, 1H), 2.62 (q J = 7.8 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). LCꢀMS (method A): t = 1.20 min;
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[
M+H] : 466.23.
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-(4-Bromophenyl)-N-cyclohexyl-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5g). H NMR (400 MHz, CDCl ) δ 7.54ꢀ7.59 (m, 4H), 7.28 (s, 1H), 7.24 (dd, J =
3
1
8.6 Hz, J = 5.3 Hz, 2H), 7.02 (t, J = 8.6 Hz, 2H), 5.89ꢀ5.93 (m, 1H), 5.50 (dd, J = 12.2 Hz, J
2
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2
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=
6.0 Hz, 1H), 3.75 (dd, J = 17.6 Hz, J = 12.2 Hz, 1H), 3.59ꢀ3.69 (m, 1H), 3.11 (dd, J = 17.6
1 2 1
Hz, J = 6.0 Hz, 1H), 1.88ꢀ2.03 (m, 2H), 1.70ꢀ1.77 (m, 2H), 1.28ꢀ1.40 (m, 4H). LCꢀMS (method
2
+
A): t = 1.16 min; [M+H] : 444.09.
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3-(4-Bromophenyl)-5-(4-fluorophenyl)-N-isopropyl-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5h). H NMR (400 MHz, CDCl ) δ 7.53ꢀ7.59 (m, 4H), 7.28 (s, 1H), 7.24 (dd, J
3
1
=
7.6 Hz, J = 5.6 Hz, 2H), 7.02 (t, J = 8.4 Hz, 2H), 5.86 (d, J = 7.9 Hz, 1H), 5.50 (dd, J = 12.3
2 1
Hz, J = 6.0 Hz, 1H), 4.15 (q, J = 7.1 Hz, 1H), 3.95ꢀ4.00 (m, 1H), 3.75 (dd, J = 17.8 Hz, J =
2
1
2
12.2 Hz, 1H), 3.10 (dd, J = 17.7 Hz, J2 = 5.9 Hz, 1H), 1.23 (dd, J = 6.1 Hz, J = 3.6 Hz, 6H).
1
1
2
+
LCꢀMS (method A): t = 1.10 min; [M+H] : 403.99.
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(3-(4-Bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(phenyl)methanone
1
(5i). H NMR (400 MHz, CDCl ) δ 8.01 (d, J = 7.4 Hz, 2H), 7.45ꢀ7.60 (m, 7H), 7.33 (dd, J =
3
1
8.5 Hz, J = 5.3 Hz, 2H), 7.06 (t, J = 8.6 Hz, 2H), 5.84 (dd, J = 11.8 Hz, J = 5.2 Hz, 1H), 3.80
2
1
2
(dd, J = 17.7 Hz, J = 11.9 Hz, 1H), 3.18 (dd, J = 17.7 Hz, J = 5.2 Hz, 1H). LCꢀMS (method
1
2
1
2
+
A): t = 1.14 min; [M+H] : 422.89.
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3-(4-Bromophenyl)-5-(4-fluorophenyl)-N-phenethyl-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5j). H NMR (400 MHz, CDCl ) δ 7.56 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.3 Hz,
3
2H), 7.23ꢀ7.36 (m, 7H), 7.04 (t, J = 8.3 Hz, 2H), 6.14 (t, J = 5.6 Hz, 1H), 5.51 (dd, J = 12.1 Hz,
1
J = 6.0 Hz, 1H), 3.75 (dd, J = 17.6 Hz, J = 12.2 Hz, 1H), 3.56 (m, 2H), 3.11 (dd, J = 17.6
2
1
2
1
+
Hz, J = 5.9 Hz, 1H), 2.81ꢀ2.95 (m, 2H). LCꢀMS (method A): t = 1.15 min; [M+H] : 468.66.
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-(4-Bromophenyl)-5-(4-fluorophenyl)-N-(pyridin-4-yl)-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5k). H NMR (400 MHz, CDCl ) δ 8.45 (d, J = 5.4 Hz, 2H), 8.33 (s, 1H), 7.75ꢀ
3
7
.80 (m, 2H), 7.47ꢀ7.54 (m, 5H), 7.30ꢀ7.32 (m, 2H), 7.07 (t, J = 8.6 Hz, 2H), 5.59 (dd, J = 12.0
1
0
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Hz, J = 5.7 Hz, 1H), 3.91 (dd, J = 17.9 Hz, J = 12.0 Hz, 1H), 3.28 (dd, J = 17.8 Hz, J = 5.6
2
1
2
1
2
+
Hz, 1H). LCꢀMS (method A): t = 0.87 min; [M+H] : 361.26.
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3-(4-Bromophenyl)-5-(4-fluorophenyl)-N-(pyridin-2-yl)-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (5l). H NMR (400 MHz, CDCl ) δ 8.93ꢀ9.06 (m, 1H), 8.28 (d, J = 4.5 Hz, 1H),
3
8.14 (d, J = 8.5 Hz, 1H), 7.75ꢀ7.83 (m, 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.44ꢀ7.50 (m, 3H), 7.31
(dd, J = 8.6 Hz, J = 5.3 Hz, 2H), 7.06 (t, J = 8.6 Hz, 2H), 7.00 (t, J = 6.1 Hz, 1H), (m, 1H),
1 2
6
.83 (s), 5.60 (dd, J = 12.0 Hz, J = 5.5 Hz, 1H), 3.88 (dd, J = 17.7 Hz, J = 12.0 Hz, 1H),
1 2 1 2
+
3.25 (dd, J = 17.7 Hz, J = 5.5 Hz, 1H). LCꢀMS (method A): t = 0.92 min; [M+H] : 361.32.
1
2
r
3
-(4-Bromophenyl)-N,5-bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide
1
(5m). H NMR (400 MHz, CDCl ) δ 9.12 (s, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 8.0 Hz,
3
2
H), 7.56 (dd, J = 7.7 Hz, J = 5.0 Hz, 2H), 7.26 (dd, J = 12.7 Hz, J = 5.9 Hz, 2H), 7.02ꢀ7.08
1 2 1 2
(m, 4H), 6.16 (dd, J = 3.4 Hz, J = 11.6 Hz, 1H), 3.87 (dd, J = 17.8 Hz, J = 11.7 Hz, 1H),
1 2 1 2
+
3.20 (dd, J = 17.8 Hz, J = 3.5 Hz, 1H). LCꢀMS (method A): t = 1.18 min; [M+H] : 471.92.
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2
r
1
N,5-Bis(4-fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6a). H NMR
400 MHz, CDCl ) δ 8.08 (s, 1H), 7.75ꢀ7.80 (m, 2H), 7.46ꢀ7.52 (m, 5H), 7.27ꢀ7.33 (m, 2H), 7.05
(
3
( t, J = 8.6 Hz, 2H), 7.00 ( t, J = 8.6 Hz, 2H), 5.59 (dd, J = 12.1 Hz, J2 = 5.8 Hz, 1H), 3.88 (dd,
1
J1 = 17.7 Hz, J2 = 12.1 Hz, 1H), 3.24 (dd, J1 = 17.7 Hz, J = 5.7 Hz, 1H). LCꢀMS (method B):
2
+
t = 1.00 min; [M+H] : 378.12.
r
1
N,5-Bis(4-fluorophenyl)-3-methyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6b). H NMR
400 MHz, CDCl ) δ 7.91 (s, 1H), 7.42ꢀ7.45 (m, 2H), 7.24 (dd, J = 8.6 Hz, J = 5.3 Hz, 2H),
(
3
1
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=
.04 ( t, J = 8.6 Hz, 2H), 6.97 ( t, J = 8.6 Hz, 2H), 5.40 (dd, J = 11.9 Hz, J = 5.9 Hz, 1H),
1
2
.29ꢀ3.68 (m, 1H), 2.77 (dd, J = 18.2 Hz, J = 5.9 Hz, 1H), 2.11 (s, 3H). LCꢀMS (method A): t
r
1
2
+
1.03 min; [M+H] : 316.42.
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1
N,5-Bis(4-fluorophenyl)-3-(pyridin-4-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide (6c). H
NMR (400 MHz, CDCl3) δ: 8.74 (dd, J1 = 6.1 Hz, J2 = 1.6 Hz, 2H), 8.03 (s, 1H), 7.60 (dd, J1 =
6
.1 Hz, J2 = 1.6 Hz, 2H), 7.46 (dd, J = 9.3 Hz, J = 4.8 Hz, 2H), 7.27ꢀ7.30 (m, 2H), 7.05 (t, J =
1 2
8
.5 Hz, 2H), 7.00 (t, J = 8.5 Hz, 2H), 5.61 (dd, J = 12.6 Hz, J = 6.4 Hz, 1H), 3.85 (dd, J = 18.2
1
2
1
1
3
Hz, J = 13.1 Hz, 1H), 3.21 (dd, J = 18.4 Hz, J = 6.4 Hz, 1H). C NMR (100 MHz, CDCl ) δ
2
1
2
3
1
63.3, 161.3, 159.9, 157.9, 151.2, 150.4, 149.5, 138.2, 137.7, 134.0, 127.4, 127.3, 121.0, 120.9,
+
1
20.2, 116.3, 116.1, 115.9, 115.6, 60.3, 42.2. LCꢀMS (method A): t = 0.87 min; [M+H] :
r
379.43.
R)-N,5-Bis(4-fluorophenyl)-3-(pyridin-4-yl)-4,5-dihydro-1H-pyrazole-1-carboxamide ((R)-
c) and (S)-N,5-Bis(4-fluorophenyl)-3-(pyridin-4-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide ((S)-6c). Compound 6c was separated by HPLC using a chiral stationary phase
(
6
(
Diacel ChiralPak IB 5 ꢀm column, 30 x 250 mm, EtOH / heptane 4:6 with 0.05% Et NH, 34
2
1
ml/min, 15 min). Compound (R)-6c eluted at 11.8 min, compound (S)-6c at 8.68 min. HꢀNMR
and LCꢀMS spectra for each compound was identical to 6c. Enantiomeric purity > 95% by
analytical HPLC (Diacel ChiralPak IB 5 ꢀm column, 4.6 x 250 mm, EtOH / heptane 4:6 with
0
6
4
.05% Et NH, 0.8 ml/min, 15 min). [α] (c = 1, CHCl ): +81.2 ° for (R)-6c, and ꢀ85.8 ° for (S)-
2
D
3
c. Absolute configuration determined by analogy to compounds (R)-11h and (S)-11h.
-(5-(4-Fluorophenyl)-1-((4-fluorophenyl)carbamoyl)-4,5-dihydro-1H-pyrazol-3-
yl)pyridine 1-oxide (6d). A solution of N,5ꢀbis(4ꢀfluorophenyl)ꢀ3ꢀ(pyridinꢀ4ꢀyl)ꢀ4,5ꢀdihydroꢀ
1Hꢀpyrazoleꢀ1ꢀcarboxamide (100 mg, 0.26 mmol) and MCPBA (68 mg, 0.40 mmol) in CH Cl2
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(
6 ml) was stirred at rt for 3 h. The mixture was diluted with aqueous 1N NaOH and ethyl
acetate. The layers were separated, the aqueous phase was washed with CH Cl and the
2
2
combined organic layers were dried over MgSO , filtrated off and evaporated in vacuo. The
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crude was purified by automated flash chromatography (Büchi Sepacore, 5 g cartridge, solvent
A: CH Cl , solvent B: 7M NH in MeOH, gradient in %B: 0 to 2, flow rate: 12.5 ml/min) to
2
2
3
1
afford 92 mg (89%) of an yellowish foam. H NMR (400 MHz, CDCl ) δ 8.27 (d, J = 7.2 Hz,
3
2
H), 7.96 (s, 1H), 7.62 (d, J = 7.2 Hz, 2H), 7.45 (dd, J = 9.0 Hz, J = 4.5 Hz, 2H), 7.28ꢀ7.45 (m,
1 2
2H), 7.06 ( t, J = 8.6 Hz, 2H), 7.01 ( t, J = 8.7 Hz, 2H), 5.64 (dd, J = 12.3 Hz, J = 6.0 Hz, 1H),
1
2
5
.32 (s, 1 H), 3.82 (dd, J = 17.7 Hz, J = 12.3 Hz, 1H), 3.18 (m, 1H). LCꢀMS (method A): t =
1
2
r
+
1
.02 min; [M+H] : 395.59.
1
N-(4-Fluorophenyl)-5-methyl-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6e).
H
NMR (400 MHz, CDCl ) δ 8.00 (s, 1H), 7.73ꢀ7.76 (m, 2H), 7.44ꢀ7.54 (m, 4H), 7.28 (s, 1H), 7.04
3
(t, J = 8.6 Hz, 2H), 4.71 (m, 1H), 3.54 (dd, J = 17.3 Hz, J = 11.1 Hz, 1H), 2.91 (m, 1H), 1.50
1 2
+
(d, J = 6.3 Hz, 3H). LCꢀMS (method A): t = 1.03 min; [M+H] : 298.00.
r
N-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (6f). H NMR (400 MHz, CDCl ) δ 8.08 (s, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.76
3
(dd, J = 8.0 Hz, J = 4.0 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.41ꢀ7.53 (m, 5H), 7.00 (t, J = 8.0
1 2
Hz, 2H), 5.65 (dd, J = 12.2 Hz, J = 6.0 Hz, 1H), 3.93 (dd, J = 17.8 Hz, J = 12.2 Hz, 1H), 3.22
1
2
1
2
+
(dd, J = 17.8 Hz, J = 6.2 Hz, 1H), 3.04 (s, 3H). LCꢀMS (method C): t = 4.42 min; [M+H] :
1
2
r
4
38.30.
N-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-3-(pyridin-4-yl)-4,5-dihydro-1H-
1
pyrazole-1-carboxamide (6g). H NMR (400 MHz, CDCl ) δ 9.34 (s, 1H), 8.70 (d, J = 5.8 Hz,
3
2
H), 7.92 (d, J = 8.3 Hz, 2H), 7.86 (d, J = 5.8 Hz, 2H), 7.56ꢀ7.65 (m, 2H), 7.54 (d, J = 8.3 Hz,
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H), 7.12 (t, J = 8.0 Hz, 2H), 5.70 (dd, J = 12.4 Hz, J = 6.2 Hz, 1H), 3.94 (dd, J = 18.1 Hz, J
1
2
1
2
+
=
12.3 Hz, 1H), 3.11ꢀ3.29 (m, 4H). LCꢀMS (method C): t = 2.87 min; [M+H] : 439.40.
r
1-Phenyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-yn-1-one (7a). To a solution of 2ꢀ(propꢀ2ꢀ
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ynꢀ1ꢀyloxy)tetrahydroꢀ2Hꢀpyran (2.23 g, 15.9 mmol) in THF (40 ml) was added at ꢀ78°C nꢀBuLi
(1.6 M in THF, 9.95 ml, 15.9 mmol) and the mixture was stirred at this temperature for 1 h. A
solution of NꢀmethoxyꢀNꢀmethylbenzamide (2.5 g, 15.2 mmol) in THF (10 ml) was added and
the mixture was stirred at rt for 1 h. The mixture was diluted with saturated aqueous NH Cl and
4
ethyl acetate. The layers were separated, the aqueous phase was washed with ethyl acetate, and
the combined organic layers were dried over MgSO , filtrated off and evaporated in vacuo. The
4
crude was purified by automated flash chromatography (Büchi Sepacore, 70 g cartridge, solvent
A: heptane, solvent B: ethyl acetate, gradient in %B: 1 to 3, flow rate: 50 ml/min) to afford 3.0 g
1
(
77%) of an yellowish oil. H NMR (400 MHz, CDCl ) δ 8.17 (d, J = 7.5 Hz, 2H), 7.64 (t, J =
3
7.1 Hz, 1H), 7.52 (t, J = 7.8 Hz, 2H), 4.92 (t, J = 3.3 Hz, 1H), 4.57 (s, 2H), 3.85ꢀ3.95 (m, 1H),
+
3
.56ꢀ3.64 (m, 1H), 1.51ꢀ1.95 (m, 6H). LCꢀMS (A): t = 1.04 min; [M+H] : 245.38.
r
(E/Z)-3-(4-Fluorophenyl)-1-phenyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-one (8a).
.
To a solution of CuBr Me S (1.03 g, 4.9 mmol) in THF (15 ml) was added at ꢀ78 °C (4ꢀ
2
fluorophenyl)magnesium bromide (1 M in THF, 9.8 ml, 9.8 mmol), and the mixture was stirred
at this temperature for 1.5 h. The solution of 7a (1 g, 4.1 mmol) was added in THF (5 ml) and the
reaction was stirred at ꢀ78 °C for 3 h. The mixture was diluted at 0 °C with saturated aqueous
NH Cl and ethyl acetate. The layers were separated, the aqueous phase was washed with ethyl
4
acetate, and the combined organic layers were dried over MgSO , filtrated off and evaporated in
4
vacuo. The crude was purified by automated flash chromatography (Büchi Sepacore, 20 g
cartridge, solvent A: heptane, solvent B: ethyl acetate, gradient in %B: 1 to 5, flow rate: 35
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+
ml/min) to afford 182 mg (13%) of an yellowish oil. LCꢀMS (method A): t =1.10 min; [M+H] :
r
341.32.
5-(4-Fluorophenyl)-3-phenyl-5-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-4,5-dihydro-1H-
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pyrazole (9a). A solution of 8a (177 mg, 0.52 mmol) and hydrazine monohydrate (53 mg, 1.04
mmol) in EtOH (3 ml) was stirred in microwave at 150°C for 1h. The solvent was evaporated in
vacuo and crude product (184 mg of yellowish oil) was used in the next step without
+
purification. LCꢀMS (method A): t = 1.08 min; [M+H] : 355.43.
r
N,5-Bis(4-fluorophenyl)-3-phenyl-5-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-4,5-dihydro-
1
H-pyrazole-1-carboxamide (10a). A solution of 9a (184 mg, 0.52 mmol), 1ꢀfluoroꢀ4ꢀ
isocyanatobenzene (71 mg, 0.52 mmol) and Et N (10 mg, 0.1 mmol) in toluene (5 ml) was
3
stirred at 90 °C overnight. Additional 1ꢀfluoroꢀ4ꢀisocyanatobenzene was added (28 mg, 0.20
mmol) and the reaction was stirred for 2 h. The mixture was diluted at rt with ethyl acetate, and
aqueous HCl (1 M). The layers were separated, the organic phase was washed with saturated
aqueous NaHCO . The layers were separated and the org. layer was dried over MgSO , filtrated
3
4
off and evaporated in vacuo. The crude was purified by automated flash chromatography (Büchi
Sepacore, 5 g cartridge, solvent A: heptane, solvent B: ethyl acetate, gradient in %B: 1 to 4, flow
rate: 15 ml/min) to afford 106 mg (41%) of an yellowish resin. LCꢀMS (method A): t = 1.17
r
+
min; [M+H] : not visible.
N,5-Bis(4-fluorophenyl)-5-(hydroxymethyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-
carboxamide (11a). A solution of 10a (100 mg, 0.2 mmol) and PTSA (2.9 mg, 0.01 mmol) in
Et O (1 ml) was stirred at 0 °C for 1.5 h. The mixture was diluted at rt with ethyl acetate, and
2
saturated aqueous NaHCO The layers were separated, the org. phase was washed with saturated
3
.
aqueous NaCl. The layers were separated and the organic layer was dried over MgSO , filtrated
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off and evaporated in vacuo. The crude was purified by flash chromatography (Büchi Sepacore,
2
g cartridge, solvent A: heptane, solvent B: ethyl acetate, gradient in %B: 2, flow rate: 15
1
ml/min) to afford 40 mg (48%) of an colorless foam. H NMR (400 MHz, CDCl ) δ 8.31 (s, 1H),
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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38
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40
41
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43
44
45
46
47
48
49
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53
54
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57
58
59
60
7
.68ꢀ7.75 (m, 2H), 7.43ꢀ7.53 (m, 5H), 7.32 (dd, J = 8.9 Hz, J = 5.1 Hz, 2H), 7.08 ( t, J = 8.6
1 2
Hz, 2H), 7.04 ( t, J = 8.7 Hz, 2H), 4.89ꢀ5.26 (m, 1H), 4.50 (d, J = 12.3 Hz, 1H), 4.24 (d, J =
12.3 Hz, 1H), 3.50 (d, J = 18.2 Hz, 1H), 3.38 (d, J = 17.8 Hz, 1H). LCꢀMS (method A): t =
r
+
1
.09 min; [M+H] : 407.98.
Following compounds have been synthesized from appropriate starting materials according to
the procedures described for compound 11a:
N-(4-Fluorophenyl)-5-(2-hydroxyethyl)-3,5-diphenyl-4,5-dihydro-1H-pyrazole-1-
1
carboxamide (11b). H NMR (500 MHz, CDCl ) δ 8.30 (s, 1H), 7.70ꢀ7.74 (m, 2H), 7.48 (dd, J
3
1
=
9.1 Hz, J = 4.8 Hz, 2H), 7.43ꢀ7.47 (m, 3H), 7.35ꢀ7.40 (m, 4H), 7.26ꢀ7.30 (m, 1H), 7.00 (t, J =
2
8
.5 Hz, 2H), 3.87ꢀ3.99 (m, 2H), 3.80 (d, J = 21.1 Hz, 1H), 3.48 (d, J = 17.8 Hz, 1H), 3.13 (dt, J1
+
=
14.2 Hz, J = 5.9 Hz, 1H), 2.60ꢀ2.67 (m, 1H). LCꢀMS (method B): t = 0.93 min; [M+H] :
2
r
4
04.17.
N-(4-Fluorophenyl)-5-(2-hydroxyethyl)-5-(4-(2-methoxyethoxy)phenyl)-3-phenyl-4,5-
1
dihydro-1H-pyrazole-1-carboxamide (11c). H NMR (400 MHz, CDCl ) δ 8.28 (s, 1H), 7.23
3
(dd, J = 5.2 Hz, J = 2.8 Hz, 2H), 7.43ꢀ7.50 (m, 5H), 7.25ꢀ7.31 (m, 2H), 7.00 (t, J = 8.4 Hz,
1 2
2H), 6.92 (t, J = 9.2 Hz, 2H), 4.11 (t, J = 2.2 Hz, 2H), 3.83ꢀ3.96 (m, 3H), 3.74 (t, J = 4.8 Hz,
2
H), 3.45 (s, 3H), 3.44 (d, J = 18.0 Hz, 1H), 3.05ꢀ3.12 (m, 1H), 2.56ꢀ2.63 (m, 1H). LCꢀMS
+
(method C): t = 4.61 min; [M+H] : 478.33.
r
5
-(4-(2-(Dimethylamino)ethoxy)phenyl)-N-(4-fluorophenyl)-5-(2-hydroxyethyl)-3-phenyl-
1
4
,5-dihydro-1H-pyrazole-1-carboxamide (11d). H NMR (400 MHz, CDCl ) δ 8.28 (s, 1H),
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.22 (dd, J = 6.0 Hz, J = 2.4 Hz, 2H), 7.42ꢀ7.50 (m, 5H), 7.25ꢀ7.32 (m, 2H), 7.00 (t, J = 8.8
1
2
Hz, 2H), 6.90 (t, J = 8.8 Hz, 2H), 4.07 (t, J = 5.2 Hz, 2H), 3.83ꢀ3.97 (m, 3H), 3.44 (d, J = 18.0
Hz, 1H), 3.05ꢀ3.12 (m, 1H), 2.76 (t, J = 5.2 Hz, 2H), 2.56ꢀ2.63 (m, 1H), 2.36 (s, 6H). LCꢀMS
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
(method C): t = 3.59 min; [M+H] : 491.49.
r
N-(4-Fluorophenyl)-5-(2-hydroxyethyl)-3-(pyridin-4-yl)-5-(p-tolyl)-4,5-dihydro-1H-
1
pyrazole-1-carboxamide (11e). H NMR (400 MHz, CDCl ) δ 8.69 (d, J = 4.6 Hz, 2H), 8.22 (s,
3
1
H), 7.53 (d, J = 6.0 Hz, 2H), 7.42ꢀ7.50 (m, 2H), 7.12ꢀ7.27 (m, 4H), 6.99 (t, J = 8.4 Hz, 2H),
3.79ꢀ4.03 (m, 2H), 3.91 (d, J = 17.6 Hz, 1H), 3.39 (d, J = 18.0 Hz, 1H), 3.03ꢀ3.15 (m 1H), 2.56ꢀ
+
2
.67 (m, 1H), 2.32 (s, 3H). LCꢀMS (method C): t = 3.22 min; [M+H] : 419.39.
r
(R)-N-(4-Chlorophenyl)-5-(2-hydroxyethyl)-5-(4-methoxyphenyl)-3-(pyridin-3-yl)-4,5-
1
dihydro-1H-pyrazole-1-carboxamide (11f). H NMR (400 MHz, CDCl ) δ 8.93 (s, 1H), 8.66
3
(broad d, J = 4.7 Hz, 1H), 8.30 (s, 1H), 8.03 ( broad d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.5 Hz, 2H),
7
.39 (dd, J = 7.9 Hz, J = 5.0 Hz, 1H), 7.27 (m, 4H), 6.90 (d, J = 8.6 Hz, 2H), 3.93(d, J = 17.5
1
2
Hz, 1H), 3.82ꢀ3.97 (m, 2H), 3.80 (s, 3H), 3.43 (d, J = 17.9 Hz, 1H), 3.08 (dt, J = 16.0 Hz, J =
1
2
+
6.4 Hz, 1H), 2.58ꢀ2.67 (m, 1H), 1.95 (broad s, 1H). LCꢀMS (method B): t = 0.79 min; [M+H] :
r
451.11.
N-(4-Fluorophenyl)-5-(2-hydroxyethyl)-5-(4-(methylsulfonyl)phenyl)-3-phenyl-4,5-dihydro-
1
1
H-pyrazole-1-carboxamide (11g). H NMR (400 MHz, CDCl ) δ 8.28 (s, 1H), 7.95 (d, J = 8.4
3
Hz, 2H), 7.69ꢀ7.74 (m, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.42ꢀ7.50 (m, 5H), 7.02 (t, J = 8.4 Hz,
2
H), 4.0 (d, J = 17.6 Hz, 1H), 3.86ꢀ4.02 (m, 2H), 3.39 (d, J = 18 Hz, 1H), 3.03ꢀ3.15 (m 1H),
+
3
.06 (s, 3H), 2.56ꢀ2.67 (m, 1H). LCꢀMS (method C): t = 4.13 min; [M+H] : 482.19.
r
N-(4-Bromophenyl)-5-(2-hydroxyethyl)-5-(4-methoxyphenyl)-3-(pyridin-3-yl)-4,5-dihydro-
1
1H-pyrazole-1-carboxamide (11h). H NMR (400 MHz, CDCl ) δ 8.92 (d, J = 1.8 Hz, 1H),
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.65 (dd, J = 4.8 Hz, J = 1.5 Hz, 1H), 8.29 (s, 1H), 8.02 (dt, J = 8.0 Hz, J = 1.8 Hz, 1H),
1
2
1
2
.35ꢀ7.46 (m, 5H), 7.28 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 3.95(d, J = 18.2 Hz 1H),
.81ꢀ3.96 (m, 2H), 3.80 (s, 3H), 3.43 (d, J = 17.9 Hz, 1H), 3.08 (dt, J = 14.3 Hz, J = 5.7 Hz,
1
2
10
11
12
13
14
15
16
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+
H), 2.62 (broad s, 1H). LCꢀMS (method B): t = 0.80 min; [M+H] : 495.06.
r
(R)-N-(4-Bromophenyl)-5-(2-hydroxyethyl)-5-(4-methoxyphenyl)-3-(pyridin-3-yl)-4,5-
dihydro-1H-pyrazole-1-carboxamide ((R)-11h) and (S)-N-(4-bromophenyl)-5-(2-
hydroxyethyl)-5-(4-methoxyphenyl)-3-(pyridin-3-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide ((S)-11h). Compounds 11h was separated by HPLC using a chiral stationary phase
(
Diacel ChiralPak IB 5 ꢀm column, 30 x 250 mm, EtOH / heptane 4:6 with 0.05% Et NH, 34
2
1
ml/min, 12 min). Compound (R)-11h eluted at 6.49 min, compound (S)-11h at 8.27 min. Hꢀ
NMR and LCꢀMS spectra for each compound was identical to 11h. Enantiomeric purity > 95%
by analytical HPLC (Diacel ChiralPak IS 5 ꢀm column, 4.6 x 250 mm, EtOH / heptane 4:6 with
0
.05% Et NH, 0.8 ml/min, 15 min). [α] (c = 1, methanol): ꢀ10.3 ° for (R)-11h, and +11.3 ° for
2
D
(S)-11h.
X-ray structure analysis of (S)-11h. Formula: C H BrN O , formula weight 495.4, space
2
4
23
4
3
group: P21212. The structure was deposited with Cambridge Crystallographic Data Centre, code
498622.
1
N-(4-Fluorophenyl)-5-(methoxymethyl)-3-phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-
carboxamide (11i). Obtained from a solution of the corresponding hydroxymethyl derivative (80
mg, 0.20 mmol) in THF (3 ml) at rt. NaH (60% in oil, 12 mg, 0.30 mmol) was added, and the
mixture was stirred for 15 min. MeI (14 mg, 0.10 mmol) was added, and the mixture was stirred
at 60 °C for 1 h. The mixture was diluted with water (30 ml), and was extracted ethyl acetate (50
ml). Purification by flash chromatography (ethyl acetate / hexanes 0:100 ꢀ 20:80) yielded the
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desired product. H NMR (500 MHz, CDCl ) δ 8.25 (s, 1H), 7.73ꢀ7.76 (m, 2H), 7.47 (dd, J =
3
1
9
2
1
.1 Hz, J = 4.8 Hz, 2H), 7.43ꢀ7.46 (m, 3H), 7.24 (d, J = 8.30 Hz, 2H), 7.16 (d, J = 8.00 Hz,
2
H), 6.99 (t, J = 8.60 Hz, 2H), 4.45 (d, J = 9.2 Hz, 1H), 4.23 (d, J = 9.2 Hz, 1H), 3.98 (d, J =
0
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7.6 Hz, 1H), 3.50 (s, 3H), 3.40 (d, J = 17.5 Hz, 1H), 2.33 (s, 3H). LCꢀMS (method B): t = 1.03
r
+
min; [M+H] : 418.05.
N-(4-Fluorophenyl)-5-(2-morpholinoethyl)-3-phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-
carboxamide (11j). Obtained from the corresponding hydroxyethyl derivative (1.00 eq.) that
was dissolved in DMSO/CH Cl (1:4, 10 mL/mmol) at rt. Nꢀ(3ꢀDimethylaminopropyl)ꢀN'ꢀ
2
2
ethylcarbodiimide hydrochloride (6 eq.) and dichloroacetic acid (0.5 eq.) were added after each
other. The reaction mixture was stirred at rt for 45 min. CH Cl was added, and the mixture was
2
2
washed with aq. sat. NaHCO . The organic layer was dried over MgSO , filtered, and the
3
4
solvents were removed under reduced pressure to yield the crude aldehyde, which was converted
to the desired, final product by reductive amination (1.3 eq. of the amine, 4.0 eq. of BH(OAc)3,
1
in THF at rt overnight). H NMR (500 MHz, CDCl ) δ 8.25 (s, 1H), 7.70ꢀ7.74 (m, 2H), 7.44ꢀ
3
7
.48 (m, 5H), 7.24 (d, J = 8.05 Hz, 2H), 7.17 (d, J = 8.10 Hz, 2H), 7.01 (t, J = 8.65 Hz, 2H),
3.53ꢀ3.96 (m, 4H), 3.47 (d = 17.5 Hz, 1H), 3.02ꢀ3.21 (broad s, 1H), 2.37ꢀ2.83 (m, 4H), 2.33 (s,
+
3
H). LCꢀMS (method B): t = 0.84 min; [M+H] : 487.14.
r
5
-(2-(Dimethylamino)ethyl)-N-(4-fluorophenyl)-3-phenyl-5-(p-tolyl)-4,5-dihydro-1H-
pyrazole-1-carboxamide (11k). Obtained from the corresponding hydroxyethyl derivative
1
similarly to compound 11j. H NMR (500 MHz, CDCl ) δ 8.29 (s, 1H), 7.68ꢀ7.77 (m, 2H), 7.48
3
(
dd, J = 9.1 Hz, J = 4.8 Hz, 2H), 7.44ꢀ7.47 (m, 3H), 7.23 (d, J = 8.30 Hz, 2H), 7.17 (d, J =
1 2
8
.15 Hz, 2H), 7.01 (t, J = 8.60 Hz, 2H), 3.84 (d, J = 18.0 Hz, 1H), 3.47 (d, J = 18.05 Hz, 1H),
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.10ꢀ3.21 (m, 1H), 2.50ꢀ2.85 (m, 3H), 2.45 (broad s, 6H), 2.33 (s, 3H). LCꢀMS (method B): t =
r
+
0.83 min; [M+H] : 445.16.
N-(4-Fluorophenyl)-5-(2-(4-methylpiperazin-1-yl)ethyl)-3-phenyl-5-(p-tolyl)-4,5-dihydro-
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1
H-pyrazole-1-carboxamide (11l). Obtained from the corresponding hydroxyethyl derivative
1
similarly to compound 11j. H NMR (500 MHz, CDCl ) δ 8.24 (s, 1H), 7.70ꢀ7.74 (m, 2H), 7.44ꢀ
3
7
.51 (m, 5H), 7.25 (d, J = 8.30 Hz, 2H), 7.16 (d, J = 8.05 Hz, 2H), 7.00 (t, J = 8.60 Hz, 2H),
3
.74ꢀ3.78 (m, 1H), 3.44 (d, J = 17.9 Hz, 1H), 3.02ꢀ3.16 (m, 1H), 2.40ꢀ3.01 (m, 11H), 2.32 (s,
+
3H), 2.30 (broad s, 3H). LCꢀMS (method B): t = 0.77 min; [M+H] : 500.17.
r
Biology
In vitro Methods – Measurement of calcium channel flux by means of FLIPR assays:
HEK293 cells recombinantly expressing either voltageꢀdependent Tꢀtype calcium channel
subunit alphaꢀ1H (Ca 3.2) or voltageꢀdependent Lꢀtype calcium channel subunit alphaꢀ1C
v
(Ca 1.2) were assayed for calcium flux using the calcium indicator dye Fluoꢀ4ꢀAM (Molecular
v
Devices) and FLIPR technology (Fluorometric Imaging Plate Reader, Molecular Devices) (Xie
X, Van Deusen AL, Vitko I, Babu DA, Davies LA, Huynh N, Cheng H, Yang N, Barrett PQ,
PerezꢀReyes E. Validation of high throughput screening assays against three subtypes of Ca(v)3
Tꢀtype channels using molecular and pharmacologic approaches. Assay and Drug Development
Technologies 2007, 5(2), 191ꢀ203). The HEK293 cells recombinantly expressing Ca 3.2 were
v
maintained in DMEM growth medium (Life Technologies) supplemented with 10 % Fetal
Bovine Serum (FBS), 100 U/ml penicilin (Life Technologies), 100 ꢀg/ml streptomycin (Life
Technologies) and 1 mg/ml G418 (Life Technologies). HEK293 cells recombinantly expressing
Ca 1.2 were maintained in DMEM growth medium (Life Technologies) supplemented with 10
v
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%
FBS, 0.1 mg/ml G418 (Life Technologies), 0.1 mg/ml hygromycin (Life Technologies) and
40 ꢀg/ml zeocin (Life Technologies).
Cells were washed once with PBS, then dissociated in 0.25 % trypsin/EDTA (Life Technologies)
and seeded into polyꢀDꢀlysine coated 384ꢀwell black, clear bottom plates (BD Biosciences) at a
density of 30,000 cells/well. The seeded plates were incubated overnight at 37°C.
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Immediately prior to performing the assay, medium was removed and cells were treated for 1
hour at 37˚C with loading buffer containing HBSS 1X (137 mM NaCl; 5.4 mM KCl; 0.25 mM
Na HPO ; 1.3 mM CaCl ; 0.4 mM MgSO ; 0.5 mM MgCl ; 0.4 mM KH PO , pH 7.4), 0.375
2
4
2
4
2
2
4
g/L NaHCO , 20 mM Hepes, supplemented with 3 ꢀM Fluoꢀ4ꢀAM and 0.15 % Pluronic (Life
3
Technologies). The cells are then washed three times with assay buffer (HBSS 1X; 0.375 g/L
NaHCO ; 20 mM Hepes; 1 % FBS; pH 7.4) and allowed to rest in 50 ꢀl of wash buffer for 30
3
minutes.
Stock solutions of test compounds were prepared to a concentration of 10 mM in DMSO. For the
Cav3.2 assay, serial dilutions of the compounds were prepared in TEAC buffer (100 mM
tetraethylammonium chloride; 20 mM Hepes; 2.5 mM CaCl ; 5 mM KCl; 1 mM MgCl ; 1 %
2
2
FBS; pH 7.2), for the Ca 1.2 assay serial dilutions were prepared in assay buffer. Test
v
compounds were added to the cells to give a 3ꢀfold dilution series ranging from 10 ꢀM to 0.05
2
+
nM. The compounds were incubated with the cells for 3 minutes and Ca entry was stimulated
by adding either CaCl to a final concentration of 10 mM (Ca 3.2 assay) or by adding KCl to a
2
v
final concentration of 20 mM ( Ca 1.2 assay). The kinetics of fluorescence increase were
v
recorded for every well and the area under the fluorescence trace for every compound
concentration was used to generate inhibition curves using nonꢀlinear regression sigmoidal
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concentrationꢀresponse curve analysis with inꢀhouse software. IC50 values were calculated and
represent the compound concentration required to inhibit 50% of the signal that is obtained in the
presence of vehicle instead of test compound. In analogy, antagonistic activities (IC50 values) of
all exemplified compounds have been measured for the Ca 3.1ꢀ and the Ca 3.3ꢀchannel.
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v
Antagonistic activities (IC50 values) of all exemplified compounds are in the range of 0.3 to
210 nM with respect to Ca 3.1; and in the range of 0.8 to 1280 nM with respect to Ca 3.3.
1
v
v
Electrophysiology: Patch-clamp assays. Compounds were evaluated for block of hCav3.1,
hCav3.2, and hCav3.3 channels using CHO cells stably expressing the human Cacna1h,
Cacna1g, and Cacna1i genes (from E. PerezꢀReyes, U. Virginia, USA) and the QPatch platform
2+
(
Sophion, Ballerup, Denmark). Ca tail currents were measured at ꢀ60 mV following a 3ꢀms
depolarization to +20 mV from holding voltages of either ꢀ90 mV or ꢀ60 mV. The CsꢀGlutamateꢀ
2
+
2+
based internal solution also contained 1 mM TBA, 25 mM Clꢀ, 1.8 mM Ca , 1 mM Mg , 11
mM BAPTA and 20 mM HEPES. The TEAꢀClꢀbased external solution also contained 5 mM
2
+
2+
Ca , 0.5 mM Mg and 10 mM HEPES. Compound effects were quantified 150s after
application to the cells. Compounds were evaluated for block of neuronal Na and K channels
using rat cortical neurons (cryopreserved or freshlyꢀprepared) dissociated from E18 Wistar rat
embryos and cultured for 9 to 15 days, using a manual patchꢀclamp system (HEKAꢀ10 amplifier,
+
Darmstadt, Germany). Fast and slow K currents were measured during one 50ꢀms
depolarization to 0 mV from a holding potential of ꢀ80 mV, as the peak outward current and as
+
the outward current at the endꢀofꢀpulse. Na currents were measured during a 30 Hz train of
fifteen 10ꢀms depolarizations to 0 mV at 50 Hz from a holding potential of ꢀ80 mV, and
compound effects were measured as effect on the peak inward current during the first and last
+
2+
pulses of the train. The KꢀClꢀbased internal solution also contained 10 mM Na , 11 mM Mg , 5
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mM EGTA, 0.1 mM GTP, 5 mM ATP and 10 mM HEPES. The NaꢀClꢀbased external solution
+
2+
2+
also contained 4 mM K , 1.2 mM Ca , 1 mM Mg and 10 mM HEPES. Compound effects were
quantified at least180s after application to the cells. hKV11.1 ("hERG") assay. Compounds were
evaluated for block of hKV11.1 channels using CHO cells stably expressing the hERG gene
(bSys, Witterswil, Switzerland) and the QPatch platform (Sophion, Ballerup, Denmark). K+ tail
currents were measured at ꢀ40 mV following a 500ꢀms depolarization to +20 mV from a holding
voltage of ꢀ80 mV. The external solution contained 150 mM Na+, 4 mM K+, 1 mM Mg2+ and
1.2 mM Ca2+. Compound effects were quantified 3 minutes after application to the cells.
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Pharmacology: Male Wistar rats and WAG/Rij rats were purchased from Harlan (Horst
Netherlands) and Charles River (Sulzfeld, Germany) respectively. All animals were maintained
under standard lab conditions (temperature 20 ± 2°C, relative humidity 55–70%, on a 12 h light /
12 h dark cycle from 6 am to 6 pm) with free access to normal chow and water. The
experimental procedures used in this study were approved based on international guidelines and
adherence to Swiss federal regulations on animal experimentation (license # KVꢀBL 420 for
kindling experiment, # KVꢀBL 205 for EEG experiments).
Amygdala-kindling rat model: Adult male Wistar rats (Harlan Laboratories, Netherlands; body
weight 300ꢀ350g) were stereotaxically implanted with twisted bipolar plasticꢀcoated stainless
steel electrode (MS333ꢀ2ꢀBIU 10mm, Plastics One, Roanoke, USA) into the right basolateral
amygdala under isoflurane anesthesia. To place the electrode, trepanations were made in the
skull and the electrode was lowered into the right basolateral amygdala (from bregma: anteriorꢀ
posterior (AP): ꢀ2.5mm, medioꢀlateral (ML): ꢀ3.5mm, dorsoꢀventral (DV): ꢀ8.6mm; α=10°) and
secured to the skull with screws and dental acrylate. After one week of recovery, they were
handled daily and habituated over one week to the kindling setꢀup.
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Kindling procedure: For a kindling session each rat was placed individually into a smooth acrylic
plastic, roundꢀbottomed bowl (Ø 36cm, height 36cm, BASi movementꢀresponsive caging
system; West Lafayette, USA) and its intracranial implanted electrode was connected to the
stimulator (STG4008, Multichannel Systems GmbH, Germany) and the recording devices
(PowerLab 8/35, ADInstruments Ltd, Oxford, UK) via a cable (335ꢀ340/3 (C), Plastics One,
Roanoke, USA). Before starting the daily electrical stimulation, the preꢀkindling afterdischarge
threshold (ADT) was defined by administering a series of electrical stimulations (1sꢀtrain of 50
Hz squareꢀwave biphasic pulses of 1ꢀms duration) at 1ꢀmin intervals with ascending intensity,
starting at 10µA and increasing in 10µAꢀsteps. The ADT was defined as the minimal stimulation
intensity necessary to evoke an afterdischarge (electroencephalographically measured neuronal
hyperꢀsynchronous activity with an amplitude 2ꢀtimes higher than baseline amplitude and a
frequency of ≥1 Hz) of at least 3sec duration. For the kindling procedure, each rat was exposed
once daily to an electrical stimulation and the behavioral symptoms of the evoked seizure were
observed and classified according to the modified Racine scale (stage 0, arrest, wet dog shakes,
normal behaviour; stage 1, facial twitches: nose, lips, eyes; stage 2, chewing, head nodding;
stage 3, forelimb clonus; stage 4, rearing, falling on forelimbs; stage 5, rearing, falling on side or
back, rolling). The electrical stimulus consists of a 1sꢀtrain of 50 Hz squareꢀwave biphasic pulses
of 1ꢀms duration at an intensity of 400µA (suprathreshold intensity). The stimulus was applied
daily until each rat was fully kindled, i.e. it showed seizures of severity stage 4 and 5 upon
electrical stimulation in at least ten consecutive kindling sessions. Finally, the postꢀkindling ADT
was defined using the same protocol as described for the preꢀkindling ADT. Due to the kindlingꢀ
induced increase in seizure susceptibility of the temporal lobe tissue the postꢀkindling ADT is
lower than the preꢀkindling ADT.
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