The pentafluorosulfanyl group in cannabinoid receptor ligands: Synthesis and comparison with trifluoromethyl and tert-butyl analogues

Article abstract of DOI:10.1039/c4ra01212g

An array of cannabinoid ligands, bearing meta- and para-substituted pentafluorosulfanyl (SF₅) aniline groups in position 3 of the pyrazole ring, was efficiently synthesised and compared with the exact trifluoromethyl and tert-butyl analogues. In general, the SF₅ substituted ligands showed higher lipophilicity (i.e. logP values) than the CF₃ counterparts and lower lipophilicity than the tert-butyl ones. In terms of pharmacological activity, SF₅ pyrazoles generally showed slightly higher or equivalent CB₁ receptor affinity (K_i), always in the nanomolar range, and selectivity towards the CB₂ relative to both CF₃ and tert-butyl analogues. Functional β-arrestin recruitment assays were used to determine equilibrium dissociation constants (K_b) and showed that all of the tested SF ₅ and CF₃ compounds are CB₁ neutral antagonists. These results confirm the possibility of successfully using an aromatic SF₅ group as a stable, synthetically accessible and effective bioisosteric analogue of the electron-withdrawing CF₃ group, and possibly also of bulky aliphatic groups, for drug discovery and development applications. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra01212g

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PAPER
The pentafluorosulfanyl group in cannabinoid
receptor ligands: synthesis and comparison with
trifluoromethyl and tert-butyl analogues†
Cite this: RSC Adv., 2014, 4, 20164
Stefano Altomonte,a Gemma L. Baillie,b Ruth A. Ross,b Jennifer Rileyc
ad
*
and Matteo Zanda
An array of cannabinoid ligands, bearing meta- and para-substituted pentafluorosulfanyl (SF5) aniline groups
in position 3 of the pyrazole ring, was efficiently synthesised and compared with the exact trifluoromethyl
and tert-butyl analogues. In general, the SF5 substituted ligands showed higher lipophilicity (i.e. log P values)
than the CF3 counterparts and lower lipophilicity than the tert-butyl ones. In terms of pharmacological
activity, SF5 pyrazoles generally showed slightly higher or equivalent CB1 receptor affinity (Ki), always in
the nanomolar range, and selectivity towards the CB2 relative to both CF3 and tert-butyl analogues.
Functional b-arrestin recruitment assays were used to determine equilibrium dissociation constants (Kb)
and showed that all of the tested SF5 and CF3 compounds are CB1 neutral antagonists. These results
confirm the possibility of successfully using an aromatic SF5 group as a stable, synthetically accessible
and effective bioisosteric analogue of the electron-withdrawing CF3 group, and possibly also of bulky
aliphatic groups, for drug discovery and development applications.
Received 11th February 2014
Accepted 9th April 2014
DOI: 10.1039/c4ra01212g
www.rsc.org/advances
in drug discovery and materials science came in the late 90s,
with the improvement of their synthesis.
The synthesis of pentauorosulfanyl compounds, their bio-
logical applications and properties have been reviewed.6–9
Importantly, the pentauorosulfanyl group is oen compared
to the triuoromethyl group, and because of its higher lip-
ophilicity,10 electronegativity,11 chemical stability and greater
steric demand, which is only slightly lower than that of the tert-
butyl group,12 the SF5 group is oen referred to as a “super-
triuoromethyl” group (Fig. 1).13–15
Introduction
Pentauorosulfanyl group
Although only a few uorinated natural compounds have been
isolated,1 it is well known that introduction of one or more
uorine atoms into a molecule can have profound effects on the
binding to a receptor, and can improve both its metabolic
stability and bioavailability.2 Fluorine could be incorporated by
uorination or alternatively via a building-block approach.
Among the uorinated motifs, the triuoromethyl group
occupies a prominent role in drug discovery,3 and several
blockbuster drugs display a CF3 substituent.4
In 1960 Sheppard reported for the rst time the synthesis of
an aromatic compound bearing a pentauorosulfanyl group,
SF5.5 However, due to of the inconvenient synthetic access to
pentauorosulfanyl arenes, the breakthrough in the commer-
cialization rst and then in the application of SF5-compounds
Cannabinoid receptors
Cannabinoid receptors belong to the G-protein coupled recep-
tors family (GPCRs).16,17 At least two cannabinoid receptor
subtypes have been identied: CB1 and CB2,18 furthermore, the
CB1 type has two splice variants, denominated CB1A and
19,20
CB1B
.
The distribution of CB1 receptors is localised
predominantly in the brain21 whereas the CB2 are present in the
peripheral nervous system (PNS) cells.22 However, recent studies
have demonstrated the presence of CB1 in the PNS23 and, on the
aKosterlitz Centre for Therapeutics, Institute of Medical Sciences and “John Mallard”
Scottish PET Centre, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD,
Scotland, UK. E-mail: m.zanda@abdn.ac.uk
bMedical Sciences Building, University of Toronto, 1 King's College Circle, Toronto,
Ontario, M5S 1A8, Canada
cDrug Discovery Unit, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland,
UK
dC.N.R.-Istituto di Chimica del Riconoscimento Molecolare, via Mancinelli 7, 20131
Milano, Italy. Fax: +44 01224 437465
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra01212g
Fig. 1 Steric demand of the three groups compared in this work: tBu >
SF5 [ CF3.
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SF5-benzene was treated with iron-powder in a reuxing HCl–
ethanol solution,34 affording the corresponding 3-(pentauoro-
l6-sulfanyl)aniline in good yield (83%). The SF5 group remained
unreactive under these conditions, conrming the high chem-
ical stability of this group.
Thiophenyl-compounds 5a–f and 9a–d were prepared
according to the general synthetic methods shown in Schemes 2
and 3, respectively.35 4-Methyl-pyrazole-substituted compounds
5 were synthesised starting with a reaction of 2-propionyl-thio-
phene with diethyl oxalate using LHMDS as a base which
provided the stable lithium salt 1 in moderate yields. The latter
was allowed to react rst with 2,4-dihalo-phenylhydrazine
hydrochloride in ethanol, followed by intramolecular cycliza-
tion in reuxing acetic acid to provide the pyrazole ester 2.
Treatment of 2 with NBS afforded the 2-bromothiophene 3 in
90% yield via regioselective bromination in position 5 of the
thiophene ring. The ester 3 was hydrolysed under basic condi-
tions to give the carboxylic acid 4 in very good yield. The acid 4
was rst converted into the corresponding acyl chloride with
Fig. 2 Chemical structure of Rimonabant (SR141716).
other hand, of the CB2 in the central nervous system, albeit in
low density.24 Since CB1 receptors are associated with several
disorders, such as depression,25 anxiety,26 stress,27 schizo-
phrenia,28 chronic pain29 and obesity,30 several cannabinoid
ligands were developed. Among these ligands, the most studied
is probably SR141716 (Rimonabant),31 a pyrazole-core inverse
agonist which was discovered by Sano-Synthelabo (now Sano-
Aventis) in 1994 (Fig. 2), marketed in Europe as anti-obesity
drug and subsequently withdrawn from the market owing to its
side-effects.
The scientic question we wanted to address in this work
was about the position occupied by the SF5 group relative to its
closest bioisosteric substituents, namely CF3 and tert-butyl
groups, in terms of its effect on key pharmacological and
physico-chemical properties, such as lipophilicity and solu-
bility. To answer, we decided to use a Rimonabant-type scaffold
as a model bioactive structure for incorporating an SF5-group,
as well as CF3 and the tert-butyl groups, and directly compare
the SF5 derivatives with their CF3 and tert-Bu counterparts from
the pharmacological viewpoint. SF5, CF3 or tert-butyl groups
were incorporated on a carboxy-aniline residue in position 3 of
the pyrazole ring, since (1) 3-carboxy-aniline Rimonabant
analogues were shown to have excellent CB1-affinity and selec-
32
tivity vs. the CB2 and (2) SF5-substituted anilines or nitro-
anilines are accessible starting materials (see below).
Since, to the best of our knowledge, SF5-substituted canna-
binoid receptor ligands have never been described in the
literature, we decided to synthesise two different classes of
pyrazole-core CB1 receptor ligands: the former based on a
Rimonabant-type structure and the latter based on ligands
described in a Pharmaness' patent,33 where the 4-chloro-phenyl
ring is replaced by a 2-bromo-thiophene.
Results and discussion
Chemistry
The starting para-SF5-substituted aniline is commercially
available whereas meta-SF5-aniline was synthesised from the
commercially available 3-nitro-SF5 benzene (Scheme 1). 3-Nitro-
Scheme 2 Reagents and conditions: (a) diethyl oxalate, LiHMDS, THF/
Et2O (2/1), ꢀ78 ꢁC to room temp., 16 h; (b) EtOH, room temp., 24 h; (c)
AcOH, 120 ꢁC, 16 h; (d) NBS, CH3CN, 0 ꢁC to r.t., 16 h; (e) KOH, MeOH,
reflux, 3 h; (f) thionyl chloride, toluene, reflux, 3 h; (g) Et3N, DCM, 0 ꢁC
to r.t., 16 h.
Scheme 1 Reagents and conditions: Fe, HCl–EtOH, reflux, 2 h.
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Scheme 3 Reagents and conditions: (a) diethyl oxalate, LiHMDS, THF/
Et2O (2/1), ꢀ78 ꢁC to room temp, 16 h; (b) EtOH, room temp, 24 h; (c)
AcOH, 120 ꢁC, 16 h; (d) KOH, MeOH, reflux, 3 h; (e) thionyl chloride,
toluene, reflux, 3 h; (f) Et3N, DCM, 0 ꢁC to room temp, 16 h.
Scheme 4 Reagents and conditions: (a) diethyl oxalate, LiHMDS, THF/
Et2O (2/1), ꢀ78 ꢁC to room temp, 16 h; (b) EtOH, room temp, 24 h; (c)
AcOH, 120 ꢁC, 16 h; (d) KOH, MeOH, reflux, 3 h; (e) thionyl chloride,
toluene, reflux, 3 h; (f) Et3N, DCM, 0 ꢁC to room temp, 16 h.
thionyl chloride and then reacted with several anilines to afford
the desired products 5.
signicant discrepancies among the calculated values. We
therefore decided to set up an experimental method for
determining the log P of these CB1 ligands via reverse phase-
HPLC analysis.
Similar procedure was used for the synthesis of the pyrazoles
9 (Scheme 3), having no substitution in position 4. In this case,
however, the overall synthesis was one-step shorter thanks to
the commercial availability of 2-bromo-5-acetyl-thiophene,
which allowed us to skip the bromination reaction.
The synthesis of the Rimonabant-like derivatives 13, shown
in Scheme 4, was analogously accomplished following the
strategy described by Lan et al.32
The retention times obtained (which are proportional to the
lipophilic character of the molecules) conrmed that molecules
bearing the SF5 moiety are more lipophilic than the CF3 coun-
terparts and, in general, a substituent in para position inu-
ences the hydrophobicity of the entire molecule to a greater
extent (Dlog P(SF –CF )p ¼ 0.3) than in meta (Dlog P(SF –CF )m ¼ 0.2)
5
3
5
3
log P measurement
(Tables 1 and 2). Not surprisingly, replacement of these uori-
Streich et al. showed that an SF5 substituent on phenoxyacetic nated functions with a strongly lipophilic tert-butyl group
acid imparts higher lipophilicity than a CF3 group.10 We there- further increased the log P of the molecules.
fore investigated whether this was the case for our pyrazole
cannabinoid ligands too.
Replacement of the 4-methyl group on the pyrazole, as in
compounds 5 and 13, with a hydrogen, as in compounds 9,
Several soware packages allow the prediction of physico- reduced the lipophilicity of both SF5 and CF3 derivatives by
chemical molecular properties, including the log P (octanol/ Dlog P(CH –H)SF ¼ 0.7 and Dlog P(CH –H)CF ¼ 0.6 units respec-
3
5
3
3
water partition coefficient). However, there are oen tively. A further hydrophilicity enhancement was observed by
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Table 1
Compound
R1
R2
log Pa (ꢂSEM)
4.67 (ꢂ0.20)
5.60 (ꢂ0.27)
CB1 Ki (nM) (ꢂSEM)
817.8 (ꢂ247.2)
CB2 Ki (nM) (ꢂSEM)
2991.0 (ꢂ988.2)
528.4 (ꢂ204.8)
9a
9b
137.7 (ꢂ36.9)
9c
4.97 (ꢂ0.22)
5.89 (ꢂ0.30)
302.7 (ꢂ72.1)
58.8 (ꢂ13.4)
1009.0 (ꢂ349.2)
588.2 (ꢂ229.9)
9d
a
Determined experimentally by means of RP-HPLC (see experimental section for details).
Table 2
Compound
R1
R2
log Pa (ꢂSEM)
CB1 Ki (nM) (ꢂSEM)
CB2 Ki (nM) (ꢂSEM)
5a
5d
6.19 (ꢂ0.32)
6.34 (ꢂ0.34)
50.9 (ꢂ6.1)
362.2 (ꢂ67.6)
706.6 (ꢂ309.7)
65.3 (ꢂ13.0)
5b
5e
6.42 (ꢂ0.35)
6.61 (ꢂ0.37)
66.1 (ꢂ15.5)
7.9 (ꢂ3.3)
991.2 (ꢂ231.7)
306.4 (ꢂ140.3)
5c
5f
7.09 (ꢂ0.40)
7.04 (ꢂ0.41)
19.9 (ꢂ4.6)
102.1 (ꢂ25.0)
906.2 (ꢂ361.0)
106.9 (ꢂ30.1)
13a
13d
13b
13e
5.97 (ꢂ0.30)
6.14 (ꢂ0.32)
6.21 (ꢂ0.33)
6.41 (ꢂ0.35)
26.5 (ꢂ6.4)
31.3 (ꢂ9.8)
11.2 (ꢂ2.4)
17.1 (ꢂ3.4)
609.6 (ꢂ195.5)
392.8 (ꢂ91.4)
637.3 (ꢂ205.2)
537.2 (ꢂ210.3)
13c
6.95 (ꢂ0.38)
6.81 (ꢂ0.40)
8.5 (ꢂ1.5)
1950.0 (ꢂ852.6)
2053.0 (ꢂ949.8)
13f
47.9 (ꢂ19.8)
a
Determined experimentally by means of RP-HPLC (see Experimental section for details).
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substitution of the 2,4 diuoro aryl group in 9a,c with the methyl group in position 4 of the pyrazole ring results in an
chlorinated analogue in 9b,d (Dlog P(F–Cl) ¼ 0.9). affinity increase versus CB1 and, on the other hand, decreased
When 4-chloro-phenyl moiety (compounds 13) was the Emax measured by the displacement assay on CB2 CHO cells
replaced by a 5-bromo-thiophene group (compounds 5), the (see Table S1, ESI†).
a
log P decreased by Dlog P(Thy–Ph) ¼ 0.2, despite the benzene
In the para-phenyl substituted series of compounds 5d–f, the
group (log P benzene ¼ 2.15)36 is reported to be more hydro- SF5-compound 5e showed the highest CB1 affinity, whereas the
phobic than the thiophene group (log P thiophene ¼ 1.81).36 presence of the tert-butyl group in 5f caused a substantial drop
These results are presumably inuenced by the presence on the both in affinity and CB1/CB2 selectivity. Interestingly, the CB1/
thiophene ring of the bromine atom, which is soer and more CB2 selectivity was higher for the SF5 derivative 5e relative to the
hydrophobic than chlorine.
CF3 analogue 5d. In the meta-substituted series of compounds
Most of the tabulated log P values for the CB1 inverse agonist 5a–c, the tert-butyl derivative 5c featured the highest CB1
Rimonabant (SR141716) were determined in silico, and the two affinity, whereas the SF5 compound 5b and the CF3-analogue 5a
experimental values reported in literature, obtained through the showed comparable CB1 affinities.
ask-shake technique, are quite different (log D7.4 ¼ 4.6 ꢂ 0.837
For the 4-chlorophenyl Rimonabant-type series of
and log D7.4 ¼ 3.838). In order to obtain a new experimental value compounds 13a–f (Table 2), we also observed nanomolar
we decided to test SR141716 using the previously described RP- affinities for the CB1, in the same range of compounds 5a–f, but
HPLC method, which provided a log P value of 4.73 ꢂ 0.20 for the CB1/CB2 selectivity was generally higher. In the meta-
Rimonabant. This conrmed that all the new ligands presented substituted series of compounds 13a–c, the SF5 derivative 13b
in this article exhibit higher hydrophobicity than SR141716.
and the tert-butyl 13c displayed the highest CB1 affinity, and 13b
showed 2-fold higher affinity than the CF3 analogue. The situ-
ation was somewhat reversed for the para-substituted
compounds 13d–f where the SF5 compound 13e displayed the
Binding affinity and SAR
Equilibrium binding assays. The binding affinities for the lowest apparent CB1 Ki, whereas 13d and 13f showed similar
cannabinoid receptors of all the compounds 5, 9 and 13 were affinities.
determined by radio-receptor binding assays using the protocol
However, importantly, all of the 4-methyl-substituted pyr-
t
previously described.39 In this assay compounds 5, 9 and 13 azoles bearing the SF5 or Bu groups in para position, namely
were subjected to equilibrium binding studies with the 5e, 5f, 9d, 9c and 13f, displaced [3H]CP55940 only partially at
orthosteric agonist probe [3H]CP55940, and the ligands were the maximum concentration of 10ꢀ5 M with Emax values
assayed for their capacity to displace [3H]CP55940 from mouse ranging from 35.3 to 43.2 (see Fig. 3 and Table S1, ESI†).
brain membranes which express high levels of CB1 receptors Considering that the 4-triuomethyl arene analogue 5d
and from hCB2 transfected CHO cells.
produced a nearly full displacement of [3H]CP55940, we
Compounds 9a–d lack a substituent in position 4 of the initially hypothesised that this behaviour could be explained
pyrazole ring, incorporate a (5-bromo-thiophene) residue in by the binding of the compound to a topographically distinct
position 5 and carry different aryl residues in position 1 (2,4- site on CB1 (an ‘allosteric binding site’). We therefore inves-
diuorophenyl for 9a,c and 2,4-dichlorophenyl for 9b,d). In this tigated the effect of thiophenyl 5e on the dissociation of [3H]
series, we compared the para-phenyl substituted compounds 9a CP55940 from mouse brain membranes; as this is the gold
and 9b, bearing a CF3 group, with, respectively, 9c and 9d, standard method of assessing an allosteric interaction.41
carrying an SF5 group (Table 1). In both cases, the SF5 However, compound 5e had no signicant effects on CB1
compounds 9c,d showed higher affinity (lower Ki) than the CF3 agonist dissociation indicating that it is not an allosteric
counterparts. In terms of CB1/CB2 selectivity, while 9a,c were modulator.
comparable, 9d showed modest but higher CB1/CB2 selectivity
(10-fold) than 9b (4-fold).
It has been previously demonstrated that an aliphatic
substituent in position 4 of the pyrazole ring imparts higher CB1
selectivity in pyrazole-based ligands; in particular Chen et al.
performed a 3D quantitative structure–activity relationship
(QSAR) of 5-aryl pyrazole structures using the comparative
molecular eld analysis (CoMFA),40 highlighting the impor-
tance of the 4-methyl group on pyrazole ring to achieve a better
CB1/CB2 ratio.
We therefore investigated also the effect of SF5, CF3 and tert-
butyl groups as 3-phenyl-carboxamide substituents in two series
of 4-methyl-pyrazole cannabinoid ligands: 5-(5-bromo-thio-
phene)-pyrazoles 5a–f and Rimonabant-type 5-(4-chlorophenyl)-
Fig. 3 Effect of compounds 5d,e on equilibrium binding of [3H]
pyrazoles 13a–f.
CP55940 to mouse brain membranes. Data shown are mean ꢂ SEM
As expected, the lower apparent Kis of all compounds 5 and
3–4 independent experiments. Data were best fitted by a sigmoidal
13 (Table 2) relative to 9a–d conrmed that introduction of a concentration–response curve.
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At that point, we hypothesised that the partial displacement produce severe adverse effects that limit their clinical applica-
might be due to a solubility issue at the highest concentrations tions, whereas CB1 neutral antagonists might have improved
tested, i.e. 10ꢀ5 M. In this context, Jackson et al. had previously clinical utility.45 Furthermore SF5-compounds showed nano-
shown that, although the S–F bond is longer and more polar- molar inhibition (Ki) and equilibrium dissociation constants
izable than C–F one, the entropic cost that derives from dis- (Kb) for the CB1 receptor, with moderate CB1/CB2 selectivity.
solving in water a larger group played a major role, resulting in
Both affinity and selectivity of SF5-compounds were generally
lower Sw (water solubility) values for most of the penta- slightly superior or comparable to that of CF3 and tert-butyl
uorosulfanyl analytes relative to their CF3 analogues.42 Laser analogues. Experimental lipophilicity (log P) was found to
nephelometry has become the method of choice for measuring follow the trend tert-butyl > SF5 > CF3. Functional assays showed
solubility of molecules in a drug discovery setting.43
that all of the tested compounds, belonging to the SF5 and CF3
We therefore submitted compounds 5e, 5d and 5b to a laser series, are CB1 neutral antagonists. It is worth noting that some
nephelometry assay, for assessing the solubility of each of the compounds, incorporating para-SF5- or tert-butyl-aryl
compound at different concentrations. However, these experi- groups on the C-3 pyrazole ring, displayed an apparent partial
ments showed that all of the compounds above were soluble at displacement of [3H]CP55940 in the functional assays (Emax
the highest concentration of 10ꢀ5 M (99.9% aqueous buffer, values ranging from 35.3 to 43.2), while ligands binding to the
0.1% DMSO) (see Table S2, ESI,† for details).
orthosteric binding pocket would be expected to fully displace
Functional assays. b-Arrestin recruitment assays, such as the the radioligand. One explanation for this observation could be
PathHunter® b-arrestin assay, can be used for the identication that the compounds are binding to an allosteric pocket.
of compounds with an agonistic, antagonistic, inverse However, this seems unlikely as we observed no change in
agonistic, or allosteric modulation prole for GPCR ligands.44 agonist dissociation. Solubility problems were also excluded by
Increasing concentrations of an agonist binding to the orthos- means of nephelometry assays, so we are currently unclear as to
teric binding site on the receptor will result in a dose response the explanation for this observation. Overall, the data collected
curve where the EC50 value is determined as the half maximal in this work conrm that (1) an aromatic SF5 group is an
response. The addition of a competitive antagonist will result in effective bioisosteric analogue of the CF3 group, and possibly
a signicant rightward shi in this dose response curve. This is also of bulky aliphatic groups like the tert-butyl, (2) it can be
due to the antagonist competing for the same site on the successfully used as a substitutent in biologically active
receptor as the agonist; therefore a higher concentration of compounds and drug candidates.
agonist is required to reach the same maximal response. This
will result in a signicant increase in the EC50 value obtained.
Experimental section
Chemistry
Using the PathHunter® b-arrestin assay in hCB1 cells, [3H]
CP55940 stimulated b-arrestin recruitment with an EC50 of 16.9
nM (11–27 nM) (95% condence limits for all of the b-arrestin
Solvents, reagents and apparatus. Reagent-grade commer-
experiments herein described). The uorinated para- cially available solvents and reagents were used without further
substituted CB1 receptor ligands, 5e, 5d, 13e and 13d (Table 2) purication.
1
caused a signicant rightward shi in the dose response curve
NMR data were recorded on Bruker ADVANCE III for H at
of [3H]CP55940, with an EC50 of 111.0 nM (82–151 nM), 235.8 400.13 MHz, for 13C at 100.58 MHz and for 19F at 376.45 MHz.
nM (157–355 nM), 245.8 nM (194–311 nM) and 439.6 nM (298– 1H NMR chemical shis are reported relative to TMS, and the
649 nM) respectively (see ESI† for the graphics). Analogously, solvent resonance was employed as the internal standard
with the uorinated meta-substituted CB1 receptor ligands 13b, (CDCl3 d ¼ 7.26). 13C NMR spectra were recorded with complete
13a, 5b, and 5a, [3H]CP55940 stimulated beta-arrestin recruit- proton decoupling, and the chemical shis are reported relative
ment with an EC50 of 17.2 nM (14–22 nM) and the SF5 to TMS with the solvent resonance as internal standard (CDCl3
compounds again, caused a signicant rightward shi in the d ¼ 77.0). 19F NMR spectra were referenced to CFCl3 as the
dose response curve of [3H]CP55940 with EC50 values of external standard. All chemical shi (d) are reported in parts per
932.6 nM (451–1927 nM), 1528 nM (336–6953 nM), 158.6 nM million (ppm) downeld from TMS and coupling constant (J) in
(116–218 nM) and 57.0 nM (42–77 nM), respectively.
Hertz. Splitting patterns are reported as follows: s, singlet; d,
All of the tested compounds produced a signicant increase doublet; dd, doublet of doublets; ddd, doublet of doublet of
in the EC50 values with a rank order of efficacy of 13a, 13b, 13d, doublets; t, triplet; td, triplet of doublets; appt, apparent triplet;
13e, 5d, 5b, 5e, and 5a, and should be therefore considered q, quadruplet; qd, quadruplet of doublets; m, multiplet; br,
competitive antagonists of [3H]CP55940 for the CB1 receptor.
broad signal.
Mass Analysis was performed using an Agilent 1200 HPLC
system coupled to an Agilent G6120 single quadrupole detector
equipped with electrospray ionization (ESI) source in direct
Summary and conclusions
We have shown that the pentauorosulfanyl group can effec- infusion modality.
tively replace a triuoromethyl group in pyrazole-type canna-
Lipophilicities were determined using a Reverse Phase (RP)-
binoid ligands. The resulting SF5-compounds behaved as HPLC with an Agilent 1200 HPLC system equipped with a DAD,
competitive CB1 receptor antagonists, which is an interesting analytical Phenomenex Luna C-18 column (250 ꢃ 4.60 mm L ꢃ
property since CB1 inverse agonists have been reported to ID, particle size: 5 m) and an ESI-MS detector.
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HRMS analysis were performed using an LTQ Orbitrap XL chromatography on silica gel with DCM gave ester 2 (5.33 g,
1
MS spectrometer.
All reactions were carried out in oven- or ame-dried glass- spectra matched to those reported by Tseng et al.35
ware under nitrogen atmosphere, unless stated otherwise, and 5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
were magnetically stirred and monitored by TLC on silica gel (60 pyrazole-3-carboxylic acid ethyl ester (3). To a magnetically stir-
F254 pre-coated glass plates, 0.25 mm thickness). red solution of 2 (2.60 g, 6.61 mmol) in acetonitrile (23 mL) was
42% over two steps) as a white solid: the obtained H, 12C and
Visualization was accomplished using irradiation with a UV added NBS (1.43 g, 7.94 mmol) in small portions under nitrogen
lamp (l ¼ 254 nm or l ¼ 365 nm), and/or staining with potas- at 0 ꢁC. The resulting mixture was then warmed to room
sium permanganate or ceric ammonium molybdate solution.
temperature and stirred for 16 h. The reaction was quenched
Purication of reaction products was performed using ash with saturated aqueous sodium thiosulfate and concentrated
˚
chromatography on silica gel (60 A, particle size 40–63 mm) under reduced pressure to remove acetonitrile. The aqueous
according to the procedure of Still and co-workers.46
layer was extracted with ethyl acetate (2 ꢃ 40 mL). The organic
Yields refer to chromatographically and spectroscopically layers were combined, washed with water, brine, dried over
pure compounds, unless stated otherwise. anhydrous sodium sulfate, and concentrated to give the crude
3-(Pentauoro-l6-sulfanyl)aniline. To a stirred acidic warm residue, which was subjected to purication by ash chroma-
solution (50 ꢁC) of pentauoro(3-nitrophenyl)-l6-sulfane (1.00 g, tography on silica gel with n-hexane–ethyl acetate (8 : 2) to
3.93 mmol) in ethanol–HCl (11 mL:0.8 mL, 37% v/v), iron afford bromo ester 3 (2.70 g, 90%) as a white solid: 1H NMR (400
powder (1.22 g, 21.63 mmol) was added portionwise. The reac- MHz, chloroform-d) d 7.46 (d, J ¼ 2.0 Hz, 1H), 7.36 (d, J ¼ 8.5 Hz,
tion was reuxed for 2 h. Aer cooling, the solid was removed by 1H), 7.33 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.95 (dd, J ¼ 3.9, 0.8 Hz, 1H),
means of ltration; the ltered was diluted with dichloro- 6.64 (dd, J ¼ 3.9, 0.8 Hz, 1H), 4.44 (q, J ¼ 7.1 Hz, 2H), 2.42 (s,
methane. The organic phase was acidied with HCl 2N. The 3H), 1.42 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 162.5,
aqueous phase was separated, basied and extracted with 142.9, 136.9, 136.6, 135.7, 133.8, 130.9, 130.2, 130.1, 130.1,
dichloromethane (3 ꢃ 50 mL). The combined extracts were 129.3, 127.9, 120.4, 115.0, 61.1, 14.5, 10.0. ESMS, calculated m/z
washed with brine, dried over anhydrous sodium sulfate,
C
17H13BrCl2N2O2S 459.92 [M]+, found m/z (relative intensity)
ltered, and evaporated to give the crude aniline as a yellow oil 460.9 [M + H]+ (100), 482.9 [M + Na]+ (100).
1
(0.72 g, 83% yield): the obtained H, 12C and 19F NMR spectra
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid (4). To a solution of bromo ester 3
matched to those reported by Bowden et al.34
Lithium salt of ethyl 3-methyl-2,4-dioxo-4-(thiophen-2-yl)buta- (2.64 g, 5.62 mmol) in methanol (26 mL) was added potassium
noate (1). To a magnetically stirred solution of lithium bis(tri- hydroxide (0.73 g, 11.24 mmol) in methanol (8 mL) dropwise at
methylsilyl)amide (61 mL, 60.60 mmol, 1.0 M in THF) in diethyl room temperature. The resulting mixture was heated to reux
ether (110 mL) at ꢀ78 ꢁC, 1-(2-thienyl)-1-propanone (7 mL, 55.10 for 3 h. Aer hydrolysis was complete, the reaction mixture was
mmol) in diethyl ether (42 mL) was added dropwise under a cooled to room temperature, poured into ice-water, and acidi-
nitrogen atmosphere. Aer the mixture was stirred at the same ed with 2N hydrochloric acid. The precipitate was ltered,
temperature for an additional 45 min, diethyl oxalate (9 mL, washed with water, and dried under vacuum to give the thio-
66.11 mmol) was added dropwise. The reaction mixture was phene carboxylic acid 4 (2.19 g, 90%) as a white solid: 1H NMR
allowed to warm to room temperature and stirred for another 16 (400 MHz, DMSO-d6) d 13.05 (s, 1H), 7.89 (d, J ¼ 2.3 Hz, 1H),
h. The reaction precipitate was ltered, washed with diethyl 7.73 (d, J ¼ 8.5 Hz, 1H), 7.63 (dd, J ¼ 8.5, 2.3 Hz, 1H), 7.23 (d, J ¼
ether, and dried under vacuum to afford the crude lithium salt 1 3.9 Hz, 1H), 6.88 (d, J ¼ 3.9 Hz, 1H), 2.32 (s, 3H); 13C NMR (101
(9.50 g, 70%) as a pale-yellow solid. The product was used MHz, DMSO) d 163.9, 143.3, 136.7, 136.2, 135.9, 133.1, 132.3,
without further purication. ESMS, calculated m/z C11H11LiO4S 131.5, 130.8, 130.2, 130.0, 129.0, 119.6, 114.6, 10.2. ESMS,
246.05 [M]+, found m/z (relative intensity) 247.0 [M + H]+ (100), calculated m/z C15H9BrCl2N2O2S 431.9 [M]+, found m/z (relative
265.0 [M + H + Na ꢀ Li]+ (100), 279.0 [M + H + K ꢀ Li]+ (100).
intensity) 470.9 [M + K]+ (100).
General procedure for the synthesis of compounds 5a–5f. The
1-(2,4-Dichlorophenyl)-4-methyl-5-thiophen-2-yl-1H-pyrazole-3-
carboxylic acid ethyl ester (2). To a solution of lithium salt 1 general procedure is illustrated below for compound 5a.
(8.50 g, 33.83 mmol) in ethanol (26 mL) was added 2,4- 5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
dichlorophenylhydrazine hydrochloride (8.85 g, 40.60 mmol) in pyrazole-3-carbonyl chloride. A solution of the crude acid 4 (0.80
one portion at room temperature under nitrogen. The resulting g, 1.81 mmol) and thionyl chloride (399 mL, 5.44 mmol) in
mixture was stirred at the same temperature for 24 h. Aer the toluene (12 mL) was reuxed for 3 h. Solvent was evaporated
reaction was complete, the precipitate was ltered, washed with under reduced pressure, and the residue was then re-dissolved
ethanol and diethyl ether, and dried under vacuum to give a in toluene (8 mL) rst and then in hexane (5 mL); aer evapo-
light-yellow solid (7.47 g). This crude solid, without purication, ration the crude acyl chloride (0.80 g, 98% yield) was obtained
was dissolved in acetic acid (68 mL) and heated to reux for as a white solid.
16 h. The reaction mixture was poured into ice-water and
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-N-(3-
extracted with ethyl acetate (3 ꢃ 50 mL). The combined extracts (triuoromethyl)phenyl)-1H-pyrazole-3-carboxamide (5a). A solu-
were washed with water, saturated aqueous sodium bicar- tion in dichloromethane of the carboxylic chloride (1 mL,
bonate, and brine, dried over anhydrous sodium sulfate, 0.36 M) obtained previously from 4, was added dropwise to a
ltered, and evaporated. Purication by ash column solution of 3-(triuomethyl)aniline (50 mL, 0.40 mmol) and
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triethylamine (50 mL, 0.36 mmol) in dichloromethane (0.5 mL) 130.4, 130.2, 129.8, 129.4, 128.2, 126.25 (q, J ¼ 3.6 Hz), 125.57 (q,
at 0 ꢁC. Aer stirring at room temperature for 16 h, to the J ¼ 32.5 Hz, ꢃ2), 124.13 (q, J ¼ 272.2), 119.6, 119.2 (ꢃ2), 115.2,
reaction mixture was added water and the organic phase was 9.7; 19F NMR (376 MHz, chloroform-d) d ꢀ62.07. ESMS, calcu-
extracted with dichloromethane (3 ꢃ 3 mL). The combined lated m/z C22H13BrCl2F3N3OS 574.93 [M]+, found m/z (relative
extracts were washed with brine, dried over anhydrous sodium intensity) 575.8 [M + H]+ (100). HRMS m/z M+H+ calcd for
sulfate, ltered, and evaporated. Flash column chromatography
C
22H13BrCl2F3N3OS: 573.9370; found: 573.9361%.
on silica gel with n-hexane–ethyl acetate (8 : 2) gave carbox-
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-N-(4-
1
amide 5a (150 mg, 66% yield) as a white solid: H NMR (400 (pentauoro-l6-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (5e).
1
MHz, chloroform-d) d 8.87 (s, 1H), 8.01 (s, 1H), 7.85 (d, J ¼ 7.8 5e (100 mg, 42% yield) was obtained as a white solid: H NMR
Hz, 1H), 7.54 (d, J ¼ 2.1 Hz, 1H), 7.46 (t, J ¼ 8.0 Hz, 1H), 7.39 (dd, (400 MHz, chloroform-d) d 8.92 (s, 1H), 7.81–7.68 (m, 4H), 7.53
J ¼ 8.5, 2.1 Hz, 1H), 7.36 (d, J ¼ 8.4 Hz, 2H), 6.98 (d, J ¼ 3.9 Hz, (d, J ¼ 2.1 Hz, 1H), 7.42–7.31 (m, 2H), 6.98 (d, J ¼ 3.9 Hz, 1H),
1H), 6.68 (d, J ¼ 3.9 Hz, 1H), 2.52 (s, 3H); 13C NMR (101 MHz, 6.68 (d, J ¼ 3.9 Hz, 1H), 2.51 (s, 3H); 13C NMR (101 MHz, CDCl3)
CDCl3) d 160.4, 144.3, 138.3, 137.7, 136.8, 135.5, 133.7, 131.42 d 160.4, 149.03 (appt, J ¼ 17.6 Hz), 144.1, 140.5, 137.8, 136.9,
(q, J ¼ 32.4 Hz), 130.6, 130.4, 130.2, 129.9, 129.5, 129.4, 128.1, 135.4, 133.7, 130.6, 130.4, 130.2, 129.7, 129.4, 128.1, 126.99 (p, J
125.2, 123.87 (q, J ¼ 272.6 Hz), 122.57, 120.55 (q, J ¼ 3.9 Hz), ¼ 4.7 Hz, ꢃ2), 119.7, 118.8 (ꢃ2), 115.2, 9.7; 19F NMR (377 MHz,
119.6, 116.34 (q, J ¼ 4.0 Hz), 115.2; 19F NMR (376 MHz, chlo- chloroform-d) d 85.35 (p, J ¼ 150.1, 149.4 Hz), 63.55 (d, J ¼ 150.0
roform-d) d ꢀ62.72. ESMS, calculated m/z C22H13BrCl2F3N3OS Hz). ESMS, calculated m/z C21H13BrCl2F5N3OS2 632.90 [M]+,
574.93 [M]+, found m/z (relative intensity) 575.9 [M + H]+ (100). found m/z (relative intensity) 631.8 [M ꢀ H]ꢀ. HRMS m/z M+H+
HRMS m/z M+H+ calcd for C22H13BrCl2F3N3OS: 573.9370; found: calcd for C21H13BrCl2F5N3OS2: 631.9059; found: 631.9048%.
573.9361%.
5-(5-Bromothiophen-2-yl)-N-(4-(tert-butyl)phenyl)-1-(2,4-dichlor-
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-N-(3- ophenyl)-4-methyl-1H-pyrazole-3-carboxamide (5f). 5f (84 mg,
1
(pentauoro-l6-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (5b). 40% yield) was obtained as a white solid: H NMR (400 MHz,
1
5b (163 mg, 68% yield) was obtained as a white solid: H NMR chloroform-d) d 8.67 (s, 1H), 7.58 (d, J ¼ 8.7 Hz, 2H), 7.53 (dd, J
(400 MHz, chloroform-d) d 8.87 (s, 1H), 8.13 (t, J ¼ 2.0 Hz, 1H), ¼ 1.9, 0.6 Hz, 1H), 7.41–7.32 (m, 4H), 6.97 (d, J ¼ 3.9 Hz, 1H),
7.87 (dd, J ¼ 8.1, 1.9 Hz, 1H), 7.54 (d, J ¼ 2.1 Hz, 1H), 7.50 (d, J ¼ 6.67 (d, J ¼ 3.8 Hz, 1H), 2.52 (s, 3H), 1.32 (s, 9H); 13C NMR (101
8.3 Hz, 1H), 7.44 (d, J ¼ 8.1 Hz, 1H), 7.40 (dd, J ¼ 8.5, 2.1 Hz, MHz, CDCl3) d 160.2, 147.0, 144.9, 137.4, 136.7, 135.6, 135.2
1H), 7.36 (d, J ¼ 8.4 Hz, 1H), 6.98 (d, J ¼ 3.9 Hz, 1H), 6.68 (d, J ¼ (ꢃ2), 133.8, 130.7, 130.4, 130.2, 129.2, 128.0, 125.8 (ꢃ2), 119.6
3.9 Hz, 1H), 2.52 (s, 3H); 13C NMR (101 MHz, CDCl3) d 160.4, (ꢃ2), 119.4, 115.0, 34.4, 31.4 (ꢃ3) 9.8. ESMS, calculated m/z
154.25 (appt, J ¼ 17.1 Hz), 144.2, 138.2, 137.8, 136.9, 135.4,
C
25H22BrCl2N3OS 563.0 [M]+, found m/z (relative intensity) 564.0
133.8, 130.6, 130.4, 130.3, 129.8, 129.4, 129.2, 128.1, 122.5, [M + H]+ (100). HRMS m/z M+H+ calcd for C25H22BrCl2N3OS:
121.28 (p, J ¼ 4.7 Hz), 119.6, 117.25 (p, J ¼ 4.0 Hz), 115.2, 9.7; 19
F
562.0122; found: 562.0113%.
Lithium salt of ethyl 4-(5-bromothiophen-2-yl)-2,4-dioxobuta-
NMR (377 MHz, chloroform-d) d 84.17 (p, J ¼ 149.2 Hz), 62.73
(d, J ¼ 150.0 Hz). ESMS, calculated m/z C21H13BrCl2F5N3OS2 noate (6). To a magnetically stirred solution of lithium bis(tri-
632.90 [M]+, found m/z (relative intensity) 631.9 [M ꢀ H]ꢀ (100). methylsilyl)amide (23 mL, 22.56 mmol, 1.0 M in THF) in diethyl
HRMS m/z M+H+ calcd for C21H13BrCl2F5N3OS2: 631.9059; ether (41 mL) at ꢀ78 ꢁC, 1-(5-bromothiophen-2-yl)ethanone
found: 631.9052%.
(4.25 g, 20.51 mmol) in diethyl ether (16 mL) was added drop-
5-(5-Bromothiophen-2-yl)-N-(3-(tert-butyl)phenyl)-1-(2,4-dichlor- wise under a nitrogen atmosphere. Aer the mixture was stirred
ophenyl)-4-methyl-1H-pyrazole-3-carboxamide (5c). 5c (87 mg, at the same temperature for an additional 45 min, diethyl
1
63% yield) was obtained as a white solid H NMR (400 MHz, oxalate (3.4 mL, 24.62 mmol) was added dropwise. The reaction
chloroform-d) d 8.71 (s, 1H), 7.64–7.57 (m, 1H), 7.59 (d, J ¼ 2.0 mixture was allowed to warm to room temperature and stirred
Hz, 1H), 7.54 (dd, J ¼ 1.9, 0.7 Hz, 1H), 7.38 (dd, J ¼ 1.9, 1.3 Hz, for another 16 h. The reaction precipitate was ltered, washed
2H), 7.30 (d, J ¼ 8.6 Hz, 1H), 7.18–7.12 (m, 1H), 6.97 (d, J ¼ 3.9 with diethyl ether, and dried under vacuum to afford the crude
Hz, 1H), 6.67 (d, J ¼ 3.8 Hz, 1H), 2.52 (s, 3H), 1.33 (s, 9H); 13C lithium salt 6 (6.31 g, 99%) as a pale-yellow solid. The product
NMR (101 MHz, CDCl3) d 160.2, 152.2, 144.9, 137.5, 137.4, 136.7, was used without further purication. ESMS, calculated m/z
135.6, 133.8, 130.7, 130.4, 130.2, 130.1, 129.2, 128.6, 128.0,
C
10H8BrLiO4S 309.9 [M]+, found m/z (relative intensity) 326.9 [M
121.2, 119.4, 117.0, 116.9, 115.0, 34.8, 31.3 (x 3), 9.8. ESMS, ꢀ Li + H + Na]+ (63), 343 [M ꢀ Li + H + K]+ (31).
calculated m/z C25H22BrCl2N3OS 563.00 [M]+, found m/z (relative
General procedure for the synthesis of compounds 7a,7b. The
intensity) 564.0 [M + H]+ (100). HRMS m/z M+H+ calcd for general procedure is illustrated below for compound 7a.
25H22BrCl2N3OS: 562.0122; found: 562.0111%. Ethyl 5-(5-bromothiophen-2-yl)-1-(2,4-diuorophenyl)-1H-pyr-
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-N-(4- azole-3-carboxylate (7a). To a solution of lithium salt 6 (3 g, 9.55
C
(triuoromethyl)phenyl)-1H-pyrazole-3-carboxamide (5d). 5d (97 mmol) in ethanol (26 mL) was added 2,4-diuorophenylhy-
mg, 44% yield) was obtained as a white solid: 1H NMR (400 drazine hydrochloride (2.143 g, 11.46 mmol) in one portion at
MHz, chloroform-d) d 8.89 (s, 1H), 7.80 (d, J ¼ 8.5 Hz, 2H), 7.60 room temperature under nitrogen. The resulting mixture was
(d, J ¼ 8.6 Hz, 2H), 7.54 (d, J ¼ 2.1 Hz, 1H), 7.40 (dd, J ¼ 8.5, 2.1 stirred at the same temperature for 24 h. Aer reaction was
Hz, 1H), 7.36 (d, J ¼ 8.4 Hz, 1H), 6.98 (d, J ¼ 3.9 Hz, 1H), 6.68 (d, complete, the precipitate was ltered, washed with ethanol and
J ¼ 3.9 Hz, 1H), 2.52 (d, J ¼ 0.5 Hz, 3H); 13C NMR (101 MHz, diethyl ether, and dried under vacuum to give a light-yellow
CDCl3) d 160.4, 144.3, 140.9, 137.7, 136.9, 135.4, 133.7, 130.6, solid (2.66 g). This crude solid, without purication, was
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dissolved in acetic acid (20 mL) and heated to reux for 16 h. ESMS, calculated m/z C14H7BrCl2N2O2S 417.9 [M]+, found m/z
The reaction mixture was poured into ice-water and extracted (relative intensity) 456.9 [M + K]+ (100).
with ethyl acetate (3 ꢃ 70 mL). The combined extracts were
washed with water, saturated aqueous sodium bicarbonate, and general procedure is illustrated below for compound 9a.
brine, dried over anhydrous sodium sulfate, ltered, and 5-(5-Bromothiophen-2-yl)-1-(2,4-diuorophenyl)-1H-pyrazole-
General procedure for the synthesis of compounds 9a–9d. The
evaporated. Purication by ash column chromatography on 3-carbonyl chloride. A solution of the crude acid 8 (0.20 g, 0.51
silica gel with DCM gave ester 7a (2.08 g, 58% over two steps) as mmol) and thionyl chloride (112 mL, 1.53 mmol) in toluene (3
1
a white solid. H NMR (400 MHz, chloroform-d) d 7.51 (td, J ¼ mL) was reuxed for 3 h. The solvent was evaporated under
8.5, 5.7 Hz, 1H), 7.08 (s, 1H), 7.08–6.92 (m, 2H), 6.93 (d, J ¼ 3.9 reduced pressure, and the residue was then re-dissolved in
Hz, 1H), 6.69 (d, J ¼ 3.9 Hz, 1H), 4.44 (q, J ¼ 7.1 Hz, 2H), 1.41 (t, J toluene (3 mL) rst and then in hexane (4 mL); aer evaporation
¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 163.54 (dd, J ¼ the crude acyl chloride (0.19 g, 95% yield) was obtained as a
254.1, 11.0 Hz), 161.6, 157.88 (dd, J ¼ 256.9, 12.8 Hz), 145.3, white solid.
139.5, 130.83 (d, J ¼ 10.1 Hz), 130.8, 130.4, 127.4, 123.29 (dd, J ¼
5-(5-Bromothiophen-2-yl)-1-(2,4-diuorophenyl)-N-(4-(triuoromethyl)-
12.7, 4.1 Hz), 114.4, 112.25 (dd, J ¼ 22.7, 3.8 Hz), 108.4, 105.29 phenyl)-1H-pyrazole-3-carboxamide (9a). A solution in dichloro-
(dd, J ¼ 26.5, 23.0 Hz), 61.3, 14.3; 19F NMR (376 MHz, chloro- methane of the acyl chloride obtained previously (0.9 mL, 0.24
form-d) d ꢀ104.59 (qd, J ¼ 8.4, 5.7 Hz), ꢀ115.10 to ꢀ115.23 (m). M), was added dropwise to a solution of 4-(triuomethyl)aniline
ESMS, calculated m/z C16H11BrF2N2O2S 414.0 [M]+, found m/z (30 mL, 0.23 mmol) and triethylamine (33 mL, 0.23 mmol) in
(relative intensity) 415.0 [M + H]+ (100), 435.0 [M + Na]+ (100).
dichloromethane (0.2 mL) at 0 ꢁC. Aer stirring at room
Ethyl 5-(5-bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-1H-pyr- temperature for 16 h, to the reaction mixture was added water
azole-3-carboxylate (7b). 7b (2.36 g, 60% over two steps) was and the organic phase was extracted with dichloromethane (3 ꢃ
1
obtained as a white solid: H NMR (400 MHz, chloroform-d) d 3 mL). The combined extracts were washed with brine, dried
7.54 (d, J ¼ 2.1 Hz, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H), 7.41 (dd, J ¼ 8.5, over anhydrous sodium sulfate, ltered, and evaporated. Flash
2.1 Hz, 1H), 7.08 (s, 1H), 6.92 (d, J ¼ 3.9 Hz, 1H), 6.66 (d, J ¼ 3.9 column chromatography on silica gel with n-hexane–ethyl
Hz, 1H), 4.44 (q, J ¼ 7.1 Hz, 2H), 1.41 (t, J ¼ 7.1 Hz, 3H); 13C NMR acetate (9 : 1) gave carboxamide 9a (88 mg, 72% yield) as a white
(101 MHz, CDCl3) d 161.7, 145.2, 139.5, 137.2, 135.3, 134.0, solid: 1H NMR (400 MHz, chloroform-d) d 8.81 (s, 1H), 7.81 (d, J
131.0, 130.8, 130.5, 130.4, 128.2, 127.3, 114.5, 108.1, 61.4, 14.4. ¼ 8.5 Hz, 2H), 7.61 (d, J ¼ 8.6 Hz, 2H), 7.51 (td, J ¼ 8.5, 5.7 Hz,
ESMS, calculated m/z 16H11BrCl2N2O2S 445.9 [M]+, found m/z 1H), 7.17 (s, 1H), 7.12–7.00 (m, 2H), 6.95 (d, J ¼ 3.9 Hz, 1H), 6.74
(relative intensity) 446.9 [M + H]+ (100).
(d, J ¼ 3.9 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 163.74 (dd, J ¼
General procedure for the synthesis of compounds 8a,8b. The 254.8, 10.9 Hz), 159.1, 158.03 (dd, J ¼ 257.3, 12.8 Hz), 147.8,
general procedure is illustrated below for compound 8a.
140.6, 130.7, 130.64 (d, J ¼ 10.2 Hz), 130.5, 127.7, 126.32 (q, J ¼
5-(5-Bromothiophen-2-yl)-1-(2,4-diuorophenyl)-1H-pyrazole-3- 3.8 Hz, ꢃ2), 125.99 (q, J ¼ 32.6 Hz), 124.09 (q, J ¼ 271.8 Hz),
carboxylic acid (8a). To a solution of bromo ester 7a (1.72 g, 4.07 123.15 (dd, J ¼ 13.0, 4.2 Hz), 122.74, 119.3 (ꢃ2), 114.8, 112.48
mmol) in methanol (19 mL) was added potassium hydroxide (dd, J ¼ 22.8, 3.9 Hz), 107.0, 105.66 (dd, J ¼ 26.5, 23.0 Hz); 19F
(0.80 g, 12.21 mmol) in methanol (9 mL) dropwise at room NMR (376 MHz, chloroform-d) d ꢀ62.11, ꢀ104.02 (qd, J ¼ 8.3,
temperature. The resulting mixture was heated to reux for 3 h. 5.7 Hz), ꢀ115.07 to ꢀ115.16 (m). ESMS, calculated m/z
Aer hydrolysis was complete, the reaction mixture was cooled
C
21H11BrF5N3OS 529 [M]+, found m/z (relative intensity) 530 [M
to room temperature, poured into ice-water, and acidied with + H]+ (100). HRMS m/z M+H+ calcd for C21H11BrF5N3OS:
2N hydrochloric acid. The precipitate was ltered, washed with 527.9805; found: 527.9794%.
water, and dried under vacuum to give thiophene carboxylic
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-N-(4-(triuoromethyl)-
acid 8a (1.34 g, 85%) as a white solid: 1H NMR (400 MHz, DMSO- phenyl)-1H-pyrazole-3-carboxamide (9b). 9b (69 mg, 53%) was
1
d6) d 13.18 (bs, 1H), 7.83 (td, J ¼ 8.8, 5.9 Hz, 1H), 7.64 (ddd, J ¼ obtained as a white solid: H NMR (400 MHz, chloroform-d) d
10.3, 9.0, 2.8 Hz, 1H), 7.42–7.32 (m, 1H), 7.28 (s, 1H), 7.23 (d, J ¼ 8.81 (s, 1H), 7.80 (d, J ¼ 8.5 Hz, 2H), 7.65–7.56 (m, 3H), 7.46 (d, J
4.0 Hz, 1H), 7.11 (d, J ¼ 4.0 Hz, 1H); 13C NMR (101 MHz, DMSO) ¼ 1.9 Hz, 2H), 7.18 (s, 1H), 6.94 (d, J ¼ 3.9 Hz, 1H), 6.71 (d, J ¼
d 163.66 (dd, J ¼ 251.4, 11.7 Hz), 163.0, 157.92 (dd, J ¼ 253.6, 3.9 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 159.1, 147.7, 140.6,
13.7 Hz), 146.0, 139.7, 132.28 (d, J ¼ 10.7 Hz), 131.6, 130.8, 140.5, 137.5, 135.1, 134.1, 130.7, 130.7, 130.7, 130.5, 128.4,
129.3, 123.43 (dd, J ¼ 12.6, 3.8 Hz), 114.0, 113.45 (dd, J ¼ 22.9, 127.6, 126.33 (q, J ¼ 3.7 Hz, ꢃ2), 125.98 (q, J ¼ 32.8 Hz), 124.08
3.6 Hz), 108.6, 106.16 (dd, J ¼ 27.4, 23.6 Hz); 19F NMR (376 MHz, (q, J ¼ 271.3 Hz), 119.3 (ꢃ2), 114.8, 106.6; 19F NMR (376 MHz,
DMSO-d6) d ꢀ104.80 (qd, J ¼ 8.7, 5.8 Hz), ꢀ117.33 (q, J ¼ 9.4 chloroform-d)
d
ꢀ62.10.
21H11BrCl2F3N3OS 560.9 [M]+, found m/z (relative intensity)
583.8 [M
Na]+ (100). HRMS m/z M+H+ calcd for
21H11BrCl2F3N3OS: 559.9214; found: 559.9203%.
ESMS,
calculated
m/z
Hz). ESMS, calculated m/z C14H7BrF2N2O2S 383.9 [M]+, found m/
z (relative intensity) 422.9 [M + K]+ (100).
C
+
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-1H-pyrazole-3-
carboxylic acid (8b). 8b (1.42 g, 79%) was obtained as a white
C
5-(5-Bromothiophen-2-yl)-1-(2,4-diuorophenyl)-N-(4-(pentauoro-
solid: 1H NMR (400 MHz, DMSO-d6) d 13.18 (s, 1H), 8.00 (d, J ¼ l6-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (9c). 9c (110 mg,
1
2.3 Hz, 1H), 7.82 (d, J ¼ 8.5 Hz, 1H), 7.71 (dd, J ¼ 8.5, 2.3 Hz, 70%) was obtained as a white solid: H NMR (400 MHz, chlo-
1H), 7.31 (s, 1H), 7.22 (d, J ¼ 4.0 Hz, 1H), 7.09 (d, J ¼ 4.0 Hz, 1H); roform-d) d 8.81 (s, 1H), 7.81–7.72 (m, 4H), 7.51 (td, J ¼ 8.5, 5.7
13C NMR (101 MHz, DMSO) d 163.0, 145.8, 139.5, 136.9, 135.5, Hz, 1H), 7.18 (s, 1H), 7.13–7.01 (m, 2H), 6.96 (d, J ¼ 3.9 Hz, 1H),
133.6, 132.5, 131.6, 130.8, 130.6, 129.4, 129.1, 114.0, 108.3. 6.74 (d, J ¼ 3.9 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 163.76 (dd,
20172 | RSC Adv., 2014, 4, 20164–20176
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J ¼ 254.8, 11.0 Hz), 159.1, 158.02 (dd, J ¼ 257.2, 12.8 Hz), 149.14 129.3 (ꢃ2), 128.2, 127.5, 119.6, 61.4, 14.9, 10.1. ESMS, calculated
(appt, J ¼ 17.5 Hz), 147.6, 140.7, 140.3, 130.63 (d, J ¼ 10.4 Hz), m/z C19H15Cl3N2O2 408.0 [M]+, found m/z (relative intensity)
130.5, 127.7, 127.04 (p, J ¼ 4.6 Hz, ꢃ2), 123.11 (dd, J ¼ 12.6, 4.2 409.0 [M + H]+ (100).
Hz), 118.9 (ꢃ2), 114.9, 112.50 (dd, J ¼ 22.8, 3.9 Hz), 107.0,
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-
105.67 (dd, J ¼ 26.4, 22.9 Hz), 100; 19F NMR (377 MHz, chlo- carboxylic acid (12). To a solution of bromo ester 3 (2.32 g, 5.54
roform-d) d 85.18 (p, J ¼ 150.6, 149.7 Hz), 63.49 (d, J ¼ 150.0 Hz), mmol) in methanol (26 mL) was added potassium hydroxide
ꢀ103.91 (qd, J ¼ 8.3, 5.7 Hz), ꢀ115.12 (q, J ¼ 8.9 Hz). ESMS, (0.72 g, 11.09 mmol) in methanol (8 mL) dropwise at room
calculated m/z C20H11BrF7N3OS2 586.9 [M]+, found m/z (relative temperature. The resulting mixture was heated to reux for 3 h.
intensity) 585.9 [M ꢀ H]ꢀ (100). HRMS m/z M+H+ calcd for Aer hydrolysis was complete, the reaction mixture was cooled
C20H11BrF7N3OS2: 585.9493; found: 585.9484%.
to room temperature, poured into ice-water, and acidied with
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-N-(4-(pentauoro- 2N hydrochloric acid. The precipitate was ltered, washed with
l6-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (9d). 9d (60 mg, water, and dried under vacuum to give the carboxylic acid 12
1
1
40%) was obtained as a white solid: H NMR (400 MHz, chlo- (2.10 g, 98%) as a white solid: H NMR (400 MHz, DMSO-d6) d
roform-d) d 8.82 (s, 1H), 7.82–7.70 (m, 4H), 7.61 (d, J ¼ 1.9 Hz, 7.73 (d, J ¼ 2.3 Hz, 1H), 7.67 (d, J ¼ 8.5 Hz, 1H), 7.54 (dd, J ¼ 8.5,
1H), 7.52–7.40 (m, 2H), 7.19 (s, 1H), 6.95 (d, J ¼ 3.9 Hz, 1H), 6.71 2.3 Hz, 1H), 7.42 (d, J ¼ 8.4 Hz, 2H), 7.21 (d, J ¼ 8.4 Hz, 2H), 2.20
(d, J ¼ 3.9 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 159.1, 149.13 (s, 3H); 13C NMR (101 MHz, DMSO) d 164.1, 143.4, 142.9, 136.1,
(p, J ¼ 17.9 Hz), 147.5, 140.6, 140.2, 137.6, 135.1, 134.1, 130.7, 135.6, 134.3, 132.3, 132.1, 131.8 (ꢃ2), 130.0, 129.2 (ꢃ2), 128.8,
130.7, 130.6, 130.5, 128.4, 127.6, 127.07 (p, J ¼ 4.3 Hz, ꢃ2), 118.9 127.4, 118.4, 9.9. ESMS, calculated m/z C17H11Cl3N2O2 380.0
(ꢃ2), 114.9, 106.6; 19F NMR (377 MHz, chloroform-d) d 85.17 (p, [M]+, found m/z (relative intensity) 381.0 [M + H]+ (100).
J ¼ 150.7, 149.7 Hz), 63.48 (d, J ¼ 150.0 Hz). ESMS, calculated m/
General procedure for the synthesis of compounds 13a–13g. The
z C20H11BrCl2F5N3OS2 618.9 [M]+, found m/z (relative intensity) general procedure is illustrated below for compound 13a.
617.8 [M
ꢀ
H]ꢀ (100). HRMS m/z M+H+ calcd for
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-
3-carbonyl chloride. A solution of the crude acid 12 (1 g, 2.57
C
20H11BrCl2F5N3OS2: 617.8902; found: 617.8894%.
Lithium salt of ethyl 4-(4-chlorophenyl)-3-methyl-2,4-dioxobu- mmol) and thionyl chloride (565 mL, 7.70 mmol) in toluene (17.1
tanoate (10). To a magnetically stirred solution of lithium bis(tri- mL) was reuxed for 3 h. Solvent was evaporated under reduced
methylsilyl)amide (64 mL, 63.93 mmol, 1.0 M in THF) in diethyl pressure, and the residue was then re-dissolved in toluene (10
ꢁ
ether (63 mL) at ꢀ78 C, 1-(2-thienyl)-1-propanone (10 g, 58.12 mL) rst and then in hexane (10 mL); aer evaporation the
mmol) in diethyl ether (73 mL) was added dropwise under a crude acyl chloride (0.98 g, 95% yield) was obtained as a white
nitrogen atmosphere. Aer the mixture was stirred at the same solid.
temperature for an additional 45 min, diethyl oxalate (9 mL,
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(3-(tri-
63.93 mmol) was added dropwise. The reaction mixture was uoromethyl)phenyl)-1H-pyrazole-3-carboxamide (13a). A solu-
allowed to warm to room temperature and stirred for another 16 tion in dichloromethane of the acyl chloride obtained
h. The reaction precipitate was ltered, washed with diethyl previously (0.96 mL, 0.41 M), was added dropwise to a solution
ether, and dried under vacuum to afford the crude lithium salt of 3-(triuomethyl)aniline (55 mL, 0.44 mmol) and triethylamine
ꢁ
10 (8.05 g, 50%) as a pale-yellow solid. The product was used (61 mL, 0.44 mmol) in dichloromethane (0.4 mL) at 0 C. Aer
without further purication.
stirring at room temperature for 16 h, to the reaction mixture
Ethyl 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H- was added water and the organic phase was extracted with
pyrazole-3-carboxylate (11). To a solution of lithium salt 10 (8.05 dichloromethane (3 ꢃ 3 mL). The combined extracts were
g, 28.73 mmol) in ethanol (99 mL) was added 2,4- dichlor- washed with brine, dried over anhydrous sodium sulfate,
ophenylhydrazine hydrochloride (6.88 g, 31.60 mmol) in one ltered, and evaporated. Flash column chromatography on
portion at room temperature under nitrogen. The resulting silica gel with n-hexane–ethyl acetate (8 : 2) gave carboxamide
1
mixture was stirred at the same temperature for 24 h. Aer 13a (160 mg, 70% yield) as a white solid: H NMR (400 MHz,
reaction was complete, the precipitate was ltered, washed with chloroform-d) d 8.91 (s, 1H), 8.03 (t, J ¼ 1.9 Hz, 1H), 7.86 (d, J ¼
ethanol and diethyl ether, and dried under vacuum to give a 8.3 Hz, 1H), 7.51–7.41 (m, 2H), 7.40–7.27 (m, 5H), 7.12–7.07 (d, J
light-yellow solid (6.1 g). This crude solid, without purication, ¼ 8.5 Hz, 2H), 2.43 (s, 3H); 13C NMR (101 MHz, CDCl3) d 160.6,
was dissolved in acetic acid (45 mL) and heated to reux for 16 144.4, 143.7, 138.4, 136.3, 135.7, 135.2, 133.0, 131.40 (q, J ¼ 32.3
h. The reaction mixture was poured into ice-water and extracted Hz), 130.8 (ꢃ2), 130.5, 130.4, 129.5, 129.0 (ꢃ2), 128.0, 126.8,
with ethyl acetate (3 ꢃ 50 mL). The combined extracts were 122.55, 122.53 (q, J ¼ 272.0 Hz), 120.50 (q, J ¼ 3.9 Hz), 118.4,
washed with water, saturated aqueous sodium bicarbonate, and 116.32 (q, J ¼ 4.0 Hz), 9.5; 19F NMR (376 MHz, chloroform-d) d
brine, dried over anhydrous sodium sulfate, ltered, and ꢀ62.74. ESMS, calculated m/z C24H15Cl3F3N3O 523 [M]+, found
evaporated. Purication by ash column chromatography on m/z (relative intensity) 546.0 [M + Na]+ (100). HRMS m/z M+H+
silica gel with hexane–acetate (8 : 2) gave ester 11 (3.44 g, 30% calcd for C24H15Cl3F3N3O: 524.0311; found: 524.0301%.
over two steps) as a white solid: 1H NMR (400 MHz, chloroform-
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(3-(penta-
d) d 7.38 (d, J ¼ 2.2 Hz, 1H), 7.34 (d, J ¼ 8.4 Hz, 1H), 7.32–7.27 uoro-l6-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (13b). 13b
(m, 3H), 7.07 (d, J ¼ 8.6 Hz, 2H), 4.45 (q, J ¼ 7.1 Hz, 2H), 2.33 (s, (182 mg, 70% yield) was obtained as a white solid: 1H NMR (400
3H), 1.43 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 163.2, MHz, chloroform-d) d 8.92 (s, 1H), 8.16 (t, J ¼ 2.1 Hz, 1H), 7.91
143.4, 143.3, 136.5, 136.3, 135.4, 133.5, 131.3 (ꢃ2), 131.2, 130.5, (d, J ¼ 9.0 Hz, 1H), 7.55–7.43 (m, 3H), 7.39–7.31 (m, 4H), 7.12 (d,
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J ¼ 8.5 Hz, 2H), 2.45 (s, 3H); 13C NMR (101 MHz, CDCl3) d 160.6, [M + Na]+ (100). HRMS m/z M+H+ calcd for C27H24Cl3N3O:
154.25 (appt, J ¼ 17.4 Hz), 144.2, 143.7, 138.3, 136.3, 135.7, 512.1063; found: 512.1053%.
135.2, 133.0, 130.8 (ꢃ2), 130.5, 130.4, 129.2, 129.0 (ꢃ2), 128.0,
Determination of the partition coefficients (log P). Measurement
126.8, 122.4, 121.24 (p, J ¼ 3.9, 3.4 Hz), 118.4, 117.22 (p, J ¼ 4.9 of chromatographic capacity factor (k0) by C-18 HPLC method
Hz), 9.5; 19F NMR (377 MHz, chloroform-d) d 84.17 (p, J ¼ 150.4 was performed using the equation k0 ¼ (tr ꢀ t0)/t0,47,48 where t0 is
Hz), 62.72 (d,
C
J
¼
150.0 Hz). ESMS, calculated m/z the retention time of unretained substance and tr is the com-
23H15Cl3F5N3OS 583.0 [M]+, found m/z (relative intensity) 582.0 pound's retention time. The mobile phase was prepared mixing
[M ꢀ H]ꢀ (56). HRMS m/z M+H+ calcd for C23H15Cl3F5N3OS: methanol with water in proportions of 85 : 15 and the ow rate
582.0000; found: 581.9991%.
was 1 mL minꢀ1. A solution of urea in a methanol–water solvent
N-(3-(tert-Butyl)phenyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)- (85 : 15) was used for measurement of the column dead time (t0
4-methyl-1H-pyrazole-3-carboxamide (13c). 13c (88 mg, 63% yield) ¼ 2.673 ꢂ 0.002, n ¼ 3). Seven compounds having known log P
was obtained as a white solid: 1H NMR (400 MHz, chloroform-d) values, tabulated and in the range from 1.10 to 5.70 (benzyl
d 8.76 (s, 1H), 7.64–7.59 (m, 2H), 7.46 (t, J ¼ 1.3 Hz, 1H), 7.35– alcohol, log P 1.10; benzene, log P 2.10; toluene, log P 2.70;
7.25 (m, 5H), 7.18–7.13 (m, 1H), 7.10 (d, J ¼ 8.5 Hz, 2H), 2.44 (s, naphthalene, log P 3.60; biphenyl, log P 4; phenanthrene, log P
3H), 1.33 (s, 9H); 13C NMR (101 MHz, CDCl3) d 160.4, 152.2, 4.50; triphenylamine, log P 5.70) were chosen as a “standard”
145.0, 143.4, 137.6, 136.2, 135.8, 135.0, 133.1, 130.9 (ꢃ2), 130.6, calibration mixture for the determination of retention times (tr).
130.4, 128.9 (ꢃ2), 128.6, 128.0, 127.1, 121.1, 118.2, 117.1, 116.9, Every measure was obtained in triplicate and at controlled
34.8, 31.4 (ꢃ3), 9.6. ESMS, calculated m/z C27H24Cl3N3O 511.1 temperature of 25 ꢁC. Capacity factors (k0) were calculated. The
[M]+, found m/z (relative intensity) 512.1 [M + H]+ (100). log k' value for each of seven compounds was plotted against its
HRMS m/z M+H+ calcd for C27H24Cl3N3O: 512.1063; found: relative lipophilicity value reported in literature, based on the
512.1052%.
established linear relationship (log P ¼ 3.64 log k0 + 3.39,
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(4-(tri- correlation coefficient ¼ 0.95). The capacity factor of each
uoromethyl)phenyl)-1H-pyrazole-3-carboxamide (13d). 13d (141 compound was determined (the value was obtained on average
mg, 58% yield) was obtained as a white solid: 1H NMR (400 of three experiments) and the relative log P value was obtained
MHz, chloroform-d) d 8.93 (s, 1H), 7.82 (d, J ¼ 8.3 Hz, 2H), 7.60 by extrapolation.
(d, J ¼ 8.3 Hz, 2H), 7.47 (s, 1H), 7.37–7.28 (m, 4H), 7.09 (d, J ¼ 8.5
Hz, 2H), 2.43 (s, 3H); 13C NMR (101 MHz, CDCl3) d 160.6, 144.4,
In vitro assays
143.7, 141.0, 136.3, 135.7, 135.2, 133.0, 130.8 (ꢃ2), 130.5, 129.0
Equilibrium binding assays. Binding assays were performed
(ꢃ2), 128.2, 128.0, 126.8, 126.27 (q, J ¼ 3.7 Hz, ꢃ2), 125.55 (q, J
¼ 31.9 Hz), 124.93 (q, J ¼ 272.0 Hz), 119.2 (ꢃ2), 118.5, 9.5.
ESMS, calculated m/z C24H15Cl3F3N3O 523 [M]+, found m/z
(relative intensity) 524.0 [M + H]+ (100). HRMS m/z M+H+ calcd
for C24H15Cl3F3N3O: 524.0311; found: 524.0300%.
with the CB1 receptor agonist, [3H]CP55940 (0.7 nM), 1 mg mLꢀ1
bovine serum albumin (BSA) and 50 mM Tris buffer containing
0.1 mM EDTA and 0.5 mM MgCl2 (pH 7.4), total assay volume 500
mL. Binding was initiated by the addition of mouse brain
membranes (30 mg) or CB2 transfected CHO cells (5 mg). Assays
were carried out at 37 ꢁC for 60 minutes before termination by
addition of ice-cold wash buffer (50 mM Tris buffer, 1 mg mLꢀ1
BSA) and vacuum ltration using a 24-well sampling manifold
(Brandel Cell Harvester) and Whatman GF/B glass-bre lters
that had been soaked in wash buffer at 4 ꢁC for 24 h. Each
reaction tube was washed ve times with a 4 mL aliquot of buffer.
The lters were oven-dried for 60 min and then placed in 5 mL of
scintillation uid (Ultima Gold XR, Packard), and radioactivity
quantitated by liquid scintillation spectrometry. Specic
binding was dened as the difference between the binding that
occurred in the presence and absence of 1 mM of the corre-
sponding unlabelled ligandand was 70–80% of the total binding.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(4-(penta-
uoro-l6-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (13e). 13e
(177 mg, 68% yield) was obtained as a white solid: 1H NMR (400
MHz, chloroform-d) d 8.93 (s, 1H), 7.79 (d, J ¼ 9.0 Hz, 2H), 7.77–
7.69 (m, 2H), 7.47 (d, J ¼ 2.1 Hz, 1H), 7.37–7.28 (m, 4H), 7.09 (d, J
¼ 8.4 Hz, 2H), 2.43 (s, 3H); 13C NMR (101 MHz, CDCl3) d 160.6,
148.97 (appt, J ¼ 17.0 Hz), 144.2, 143.8, 140.6, 136.4, 135.6,
135.2, 133.0, 130.8 (ꢃ2), 130.43, 130.42, 129.0 (ꢃ2), 128.0,
126.97 (p, J ¼ 4.6 Hz, ꢃ2), 126.72, 118.8 (ꢃ2), 118.5, 9.5; 19F
NMR (377 MHz, chloroform-d) d 85.34 (p, J ¼ 150.1, 149.1 Hz),
63.53 (d, J ¼ 149.9 Hz). ESMS, calculated m/z C23H15Cl3F5N3OS
5823.0 [M]+, found m/z (relative intensity) 582.0 [M ꢀ H]ꢀ (64).
HRMS m/z M+H+ calcd for C23H15Cl3F5N3OS: 582.0000; found:
581.9990%.
b-Arrestin assays
N-(4-(tert-Butyl)phenyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-
4-methyl-1H-pyrazole-3-carboxamide (13f). 13f (162 mg, 78% PathHunter® HEK293 CB1 beta-arrestin cells were plated 48
1
ꢁ
yield) was obtained as a white solid: H NMR (400 MHz, chlo- hours before use and incubated at 37 C, 5% CO2 in a humid-
roform-d) d 8.71 (s, 1H), 7.60 (d, J ¼ 8.7 Hz, 2H), 7.46 (dd, J ¼ 1.8, ied incubator. Compounds were dissolved in DMSO and
0.8 Hz, 1H), 7.36 (d, J ¼ 8.7 Hz, 2H), 7.33–7.29 (m, 4H), 7.09 (d, J diluted in OCC media to the required concentrations. 5 mL of
¼ 8.5 Hz, 2H), 2.43 (s, 3H), 1.32 (s, 9H); 13C NMR (101 MHz, compound or vehicle solution was added to each well and
CDCl3) d 160.4, 147.0, 145.0, 143.4, 136.1, 135.9, 135.2, 135.0, incubated for 60 minutes at 37 ꢁC, 5% CO2 in a humidied
133.0, 130.9 (ꢃ2), 130.6, 130.4, 128.9 (ꢃ2), 127.9, 127.1, 125.8 incubator. 5 mL of increasing concentrations of CP55940 was
(ꢃ2), 119.5 (ꢃ2), 118.2, 34.4, 31.4 (ꢃ3), 9.5. ESMS, calculated m/ added to each well followed by a 90 minute incubation at 37 ꢁC,
z C27H24Cl3N3O 511.1 [M]+, found m/z (relative intensity) 534.1 5% CO2 in a humidied incubator. 55 mL of detection reagent is
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then added followed by a further 90 minute incubation at room 15 L. J. Sæthre, N. Berrah, J. D. Bozek, K. J. Børve, T. X. Carroll,
temperature in the dark. Chemiluminescence, indicated as
RLU, was measured on a standard luminescence plate reader.
E. Kukk, G. L. Gard, R. Winter and T. D. Thomas, J. Am.
Chem. Soc., 2001, 123, 10729–10737.
16 A. Kapur, P. Samaniego, G. A. Thakur, A. Makriyannis
and M. E. Abood, J. Pharmacol. Exp. Ther., 2008, 325,
341–348.
Solubility tests
The aqueous solubility was measured using laser nephelometry
(BMG Labtech Nephelometer) following serial dilution of DMSO
stocks into Tris Buffer (50 mM Tris–HCL, 50 mM Tris base and
0.1% BSA) to give nal concentrations of 0.01, 0.1,1 and 10 mM
and a nal DMSO concentration of 0.1%. The amount of laser
scatter caused by insoluble particulates (relative nephelometry
units) was measured. RFU values 3-fold greater than control (0
mM) indicate insolubility.
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Acknowledgements
¨
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579, 259–264.
We thank the European Commission for nancial support
(Industry Academia Partnerships and Pathways project “PET
BRAIN”, Contract no. 251482) and Miteni S.p.A. (Italy) for a kind
gi of the SF5 precursors. The EPSRC National Mass Spec-
trometry Service Centre (Swansea, UK) is gratefully acknowl-
edged for performing HRMS analyses. We thank Dr Kevin Read
(University of Dundee) for support and advice with the nephe-
lometry assays. S.A. thanks the Northern Research Partnership
(http://www.northscotland-research.ac.uk/) and Pharmaness
(http://www.pharmaness.it) for co-funding a PhD studentship.
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