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

Abstract

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 (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 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.

---

Introduction

Pentafluorosulfanyl group

Although only a few fluorinated natural compounds have been isolated,¹ it is well known that introduction of one or more fluorine 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 fluorination or alternatively via a building-block approach. Among the fluorinated motifs, the trifluoromethyl group occupies a prominent role in drug discovery,³ and several blockbuster drugs display a CF₃ substituent.⁴

In 1960 Sheppard reported for the first time the synthesis of an aromatic compound bearing a pentafluorosulfanyl group, SF₅.⁵ However, due to of the inconvenient synthetic access to pentafluorosulfanyl arenes, the breakthrough in the commercialization first and then in the application of SF₅-compounds in drug discovery and materials science came in the late 90s, with the improvement of their synthesis.

The synthesis of pentafluorosulfanyl compounds, their biological applications and properties have been reviewed.^6–9 Importantly, the pentafluorosulfanyl group is often compared to the trifluoromethyl group, and because of its higher lipophilicity,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 often referred to as a “super-trifluoromethyl” group (Fig. 1).^13–15

Fig. 1 Steric demand of the three groups compared in this work: ^tBu > SF₅ ≫ CF₃.

Cannabinoid receptors

Cannabinoid receptors belong to the G-protein coupled receptors family (GPCRs).^16,17 At least two cannabinoid receptor subtypes have been identified: CB₁ and CB₂,¹⁸ furthermore, the CB₁ type has two splice variants, denominated CB_1A and CB_1B.^19,20 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 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 schizophrenia,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 Sanofi-Synthelabo (now Sanofi-Aventis) in 1994 (Fig. 2), marketed in Europe as anti-obesity drug and subsequently withdrawn from the market owing to its side-effects.

Fig. 2 Chemical structure of Rimonabant (SR141716).

The scientific 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 solubility. 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 selectivity vs. the CB₂³² and (2) SF₅-substituted anilines or nitro-anilines are accessible starting materials (see below).

Since, to the best of our knowledge, SF₅-substituted cannabinoid 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-SF₅-benzene was treated with iron-powder in a refluxing HCl–ethanol solution,³⁴ affording the corresponding 3-(pentafluoro-λ⁶-sulfanyl)aniline in good yield (83%). The SF₅ group remained unreactive under these conditions, confirming the high chemical stability of this group.

Scheme 1 Reagents and conditions: Fe, HCl–EtOH, reflux, 2 h.

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-thiophene with diethyl oxalate using LHMDS as a base which provided the stable lithium salt 1 in moderate yields. The latter was allowed to react first with 2,4-dihalo-phenylhydrazine hydrochloride in ethanol, followed by intramolecular cyclization in refluxing 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 conditions to give the carboxylic acid 4 in very good yield. The acid 4 was first converted into the corresponding acyl chloride with thionyl chloride and then reacted with several anilines to afford the desired products 5.

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 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.

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

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.

log P measurement

Streich et al. showed that an SF₅ substituent on phenoxyacetic acid imparts higher lipophilicity than a CF₃ group.¹⁰ We therefore investigated whether this was the case for our pyrazole cannabinoid ligands too.

Several software packages allow the prediction of physicochemical molecular properties, including the log P (octanol/water partition coefficient). However, there are often significant 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.

The retention times obtained (which are proportional to the lipophilic character of the molecules) confirmed that molecules bearing the SF₅ moiety are more lipophilic than the CF₃ counterparts and, in general, a substituent in para position influences the hydrophobicity of the entire molecule to a greater extent (Δlog P_(SF5–CF3)p = 0.3) than in meta (Δlog P_(SF5–CF3)m = 0.2) (Tables 1 and 2). Not surprisingly, replacement of these fluorinated functions with a strongly lipophilic tert-butyl group further increased the log P of the molecules.

Table 1

Compound	R^1	R^2	log P^a (±SEM)	CB1 Ki (nM) (±SEM)	CB2 Ki (nM) (±SEM)
a Determined experimentally by means of RP-HPLC (see experimental section for details).
9a			4.67 (±0.20)	817.8 (±247.2)	2991.0 (±988.2)
9b			5.60 (±0.27)	137.7 (±36.9)	528.4 (±204.8)
9c			4.97 (±0.22)	302.7 (±72.1)	1009.0 (±349.2)
9d			5.89 (±0.30)	58.8 (±13.4)	588.2 (±229.9)

---

Table 2

Compound	R^1	R^2	log P^a (±SEM)	CB1 Ki (nM) (±SEM)	CB2 Ki (nM) (±SEM)
a Determined experimentally by means of RP-HPLC (see Experimental section for details).
5a			6.19 (±0.32)	50.9 (±6.1)	362.2 (±67.6)
5d			6.34 (±0.34)	65.3 (±13.0)	706.6 (±309.7)
5b			6.42 (±0.35)	66.1 (±15.5)	991.2 (±231.7)
5e			6.61 (±0.37)	7.9 (±3.3)	306.4 (±140.3)
5c			7.09 (±0.40)	19.9 (±4.6)	102.1 (±25.0)
5f			7.04 (±0.41)	106.9 (±30.1)	906.2 (±361.0)
13a			5.97 (±0.30)	26.5 (±6.4)	609.6 (±195.5)
13d			6.14 (±0.32)	31.3 (±9.8)	392.8 (±91.4)
13b			6.21 (±0.33)	11.2 (±2.4)	637.3 (±205.2)
13e			6.41 (±0.35)	17.1 (±3.4)	537.2 (±210.3)
13c			6.95 (±0.38)	8.5 (±1.5)	1950.0 (±852.6)
13f			6.81 (±0.40)	47.9 (±19.8)	2053.0 (±949.8)

---

Replacement of the 4-methyl group on the pyrazole, as in compounds 5 and 13, with a hydrogen, as in compounds 9, reduced the lipophilicity of both SF₅ and CF₃ derivatives by Δlog P_(CH3–H)SF5 = 0.7 and Δlog P_(CH3–H)CF3 = 0.6 units respectively. A further hydrophilicity enhancement was observed by substitution of the 2,4 difluoro aryl group in 9a,c with the chlorinated analogue in 9b,d (Δlog P_(F–Cl) = 0.9).

When a 4-chloro-phenyl moiety (compounds 13) was replaced by a 5-bromo-thiophene group (compounds 5), the log P decreased by Δlog P_(Thy–Ph) = 0.2, despite the benzene group (log P benzene = 2.15)³⁶ is reported to be more hydrophobic than the thiophene group (log P thiophene = 1.81).³⁶ These results are presumably influenced by the presence on the thiophene ring of the bromine atom, which is softer and more hydrophobic than chlorine.

Most of the tabulated log P values for the CB₁ inverse agonist Rimonabant (SR141716) were determined in silico, and the two experimental values reported in literature, obtained through the flask-shake technique, are quite different (log D_7.4 = 4.6 ± 0.8³⁷ and log D_7.4 = 3.8³⁸). In order to obtain a new experimental value we decided to test SR141716 using the previously described RP-HPLC method, which provided a log P value of 4.73 ± 0.20 for Rimonabant. This confirmed that all the new ligands presented in this article exhibit higher hydrophobicity than SR141716.

Binding affinity and SAR

Equilibrium binding assays. The binding affinities for the cannabinoid receptors of all the compounds 5, 9 and 13 were determined by radio-receptor binding assays using the protocol previously described.³⁹ In this assay compounds 5, 9 and 13 were subjected to equilibrium binding studies with the orthosteric agonist probe [³H]CP55940, and the ligands were assayed for their capacity to displace [³H]CP55940 from mouse brain membranes which express high levels of CB₁ receptors and from hCB₂ transfected CHO cells.

Compounds 9a–d lack a substituent in position 4 of the pyrazole ring, incorporate a (5-bromo-thiophene) residue in position 5 and carry different aryl residues in position 1 (2,4-difluorophenyl for 9a,c and 2,4-dichlorophenyl for 9b,d). In this series, we compared the para-phenyl substituted compounds 9a and 9b, bearing a CF₃ group, with, respectively, 9c and 9d, carrying an SF₅ group (Table 1). In both cases, the SF₅ compounds 9c,d showed higher affinity (lower K_i) than the CF₃ counterparts. In terms of CB₁/CB₂ selectivity, while 9a,c were 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 field analysis (CoMFA),⁴⁰ highlighting the importance 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-thiophene)-pyrazoles 5a–f and Rimonabant-type 5-(4-chlorophenyl)-pyrazoles 13a–f.

As expected, the lower apparent K_is of all compounds 5 and 13 (Table 2) relative to 9a–d confirmed that introduction of a methyl group in position 4 of the pyrazole ring results in an affinity increase versus CB₁ and, on the other hand, decreased the E_max measured by the displacement assay on CB₂ CHO cells (see Table S1, ESI†).

In the para-phenyl substituted series of compounds 5d–f, the SF₅-compound 5e showed the highest CB₁ affinity, whereas the presence of the tert-butyl group in 5f caused a substantial drop both in affinity and CB₁/CB₂ selectivity. Interestingly, the CB₁/CB₂ selectivity was higher for the SF₅ derivative 5e relative to the CF₃ analogue 5d. In the meta-substituted series of compounds 5a–c, the tert-butyl derivative 5c featured the highest CB₁ affinity, whereas the SF₅ compound 5b and the CF₃-analogue 5a showed comparable CB₁ affinities.

For the 4-chlorophenyl Rimonabant-type series of compounds 13a–f (Table 2), we also observed nanomolar affinities for the CB₁, in the same range of compounds 5a–f, but the CB₁/CB₂ selectivity was generally higher. In the meta-substituted series of compounds 13a–c, the SF₅ derivative 13b and the tert-butyl 13c displayed the highest CB₁ affinity, and 13b showed 2-fold higher affinity than the CF₃ analogue. The situation was somewhat reversed for the para-substituted compounds 13d–f where the SF₅ compound 13e displayed the lowest apparent CB₁ K_i, whereas 13d and 13f showed similar affinities.

However, importantly, all of the 4-methyl-substituted pyrazoles bearing the SF₅ or ^tBu groups in para position, namely 5e, 5f, 9d, 9c and 13f, displaced [³H]CP55940 only partially at the maximum concentration of 10⁻⁵ M with E_max values ranging from 35.3 to 43.2 (see Fig. 3 and Table S1, ESI†). Considering that the 4-trifluomethyl arene analogue 5d produced a nearly full displacement of [³H]CP55940, we initially hypothesised that this behaviour could be explained by the binding of the compound to a topographically distinct site on CB₁ (an ‘allosteric binding site’). We therefore investigated the effect of thiophenyl 5e on the dissociation of [³H]CP55940 from mouse brain membranes; as this is the gold standard method of assessing an allosteric interaction.⁴¹ However, compound 5e had no significant effects on CB₁ agonist dissociation indicating that it is not an allosteric modulator.

Fig. 3 Effect of compounds 5d,e on equilibrium binding of [³H]CP55940 to mouse brain membranes. Data shown are mean ± SEM 3–4 independent experiments. Data were best fitted by a sigmoidal concentration–response curve.

At that point, we hypothesised that the partial displacement might be due to a solubility issue at the highest concentrations tested, i.e. 10⁻⁵ M. In this context, Jackson et al. had previously shown that, although the S–F bond is longer and more polarizable than C–F one, the entropic cost that derives from dissolving in water a larger group played a major role, resulting in lower S_w (water solubility) values for most of the pentafluorosulfanyl analytes relative to their CF₃ analogues.⁴² Laser nephelometry has become the method of choice for measuring solubility of molecules in a drug discovery setting.⁴³

We therefore submitted compounds 5e, 5d and 5b to a laser nephelometry assay, for assessing the solubility of each compound at different concentrations. However, these experiments showed that all of the compounds above were soluble at the highest concentration of 10⁻⁵ M (99.9% aqueous buffer, 0.1% DMSO) (see Table S2, ESI,† for details).

Functional assays. β-Arrestin recruitment assays, such as the PathHunter® β-arrestin assay, can be used for the identification of compounds with an agonistic, antagonistic, inverse agonistic, or allosteric modulation profile for GPCR ligands.⁴⁴ Increasing concentrations of an agonist binding to the orthosteric binding site on the receptor will result in a dose response curve where the EC₅₀ value is determined as the half maximal response. The addition of a competitive antagonist will result in a significant rightward shift in this dose response curve. This is due to the antagonist competing for the same site on the receptor as the agonist; therefore a higher concentration of agonist is required to reach the same maximal response. This will result in a significant increase in the EC₅₀ value obtained.

Using the PathHunter® β-arrestin assay in hCB₁ cells, [³H]CP55940 stimulated β-arrestin recruitment with an EC₅₀ of 16.9 nM (11–27 nM) (95% confidence limits for all of the β-arrestin experiments herein described). The fluorinated para-substituted CB₁ receptor ligands, 5e, 5d, 13e and 13d (Table 2) caused a significant rightward shift in the dose response curve of [³H]CP55940, with an EC₅₀ of 111.0 nM (82–151 nM), 235.8 nM (157–355 nM), 245.8 nM (194–311 nM) and 439.6 nM (298–649 nM) respectively (see ESI† for the graphics). Analogously, with the fluorinated meta-substituted CB₁ receptor ligands 13b, 13a, 5b, and 5a, [³H]CP55940 stimulated beta-arrestin recruitment with an EC₅₀ of 17.2 nM (14–22 nM) and the SF₅ compounds again, caused a significant rightward shift in the dose response curve of [³H]CP55940 with EC₅₀ values of 932.6 nM (451–1927 nM), 1528 nM (336–6953 nM), 158.6 nM (116–218 nM) and 57.0 nM (42–77 nM), respectively.

All of the tested compounds produced a significant increase in the EC₅₀ values with a rank order of efficacy of 13a, 13b, 13d, 13e, 5d, 5b, 5e, and 5a, and should be therefore considered competitive antagonists of [³H]CP55940 for the CB₁ receptor.

Summary and conclusions

We have shown that the pentafluorosulfanyl group can effectively replace a trifluoromethyl group in pyrazole-type cannabinoid ligands. The resulting SF₅-compounds behaved as competitive CB₁ receptor antagonists, which is an interesting property since CB₁ inverse agonists have been reported to produce severe adverse effects that limit their clinical applications, whereas CB₁ neutral antagonists might have improved clinical utility.⁴⁵ Furthermore SF₅-compounds showed nanomolar inhibition (K_i) and equilibrium dissociation constants (K_b) for the CB₁ receptor, with moderate CB₁/CB₂ selectivity.

Both affinity and selectivity of SF₅-compounds were generally slightly superior or comparable to that of CF₃ and tert-butyl analogues. Experimental lipophilicity (log P) was found to follow the trend tert-butyl > SF₅ > CF₃. Functional assays showed that all of the tested compounds, belonging to the SF₅ and CF₃ series, are CB₁ neutral antagonists. It is worth noting that some of the compounds, incorporating para-SF₅- or tert-butyl-aryl groups on the C-3 pyrazole ring, displayed an apparent partial displacement of [³H]CP55940 in the functional assays (E_max values ranging from 35.3 to 43.2), while ligands binding to the orthosteric binding pocket would be expected to fully displace the radioligand. One explanation for this observation could be that the compounds are binding to an allosteric pocket. However, this seems unlikely as we observed no change in agonist dissociation. Solubility problems were also excluded by means of nephelometry assays, so we are currently unclear as to the explanation for this observation. Overall, the data collected in this work confirm that (1) an aromatic SF₅ group is an effective bioisosteric analogue of the CF₃ group, and possibly also of bulky aliphatic groups like the tert-butyl, (2) it can be successfully used as a substitutent in biologically active compounds and drug candidates.

Experimental section

Chemistry

Solvents, reagents and apparatus. Reagent-grade commercially available solvents and reagents were used without further purification.

NMR data were recorded on Bruker ADVANCE III for ¹H at 400.13 MHz, for ¹³C at 100.58 MHz and for ¹⁹F at 376.45 MHz. ¹H NMR chemical shifts are reported relative to TMS, and the solvent resonance was employed as the internal standard (CDCl3 δ = 7.26). ¹³C NMR spectra were recorded with complete proton decoupling, and the chemical shifts are reported relative to TMS with the solvent resonance as internal standard (CDCl3 δ = 77.0). ¹⁹F NMR spectra were referenced to CFCl₃ as the external standard. All chemical shift (δ) are reported in parts per million (ppm) downfield from TMS and coupling constant (J) in Hertz. Splitting patterns are reported as follows: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; t, triplet; td, triplet of doublets; appt, apparent triplet; q, quadruplet; qd, quadruplet of doublets; m, multiplet; br, 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 infusion modality.

Lipophilicities were determined using a Reverse Phase (RP)-HPLC with an Agilent 1200 HPLC system equipped with a DAD, analytical Phenomenex Luna C-18 column (250 × 4.60 mm L × ID, particle size: 5 μ) and an ESI-MS detector.

HRMS analysis were performed using an LTQ Orbitrap XL MS spectrometer.

All reactions were carried out in oven- or flame-dried glassware under nitrogen atmosphere, unless stated otherwise, and were magnetically stirred and monitored by TLC on silica gel (60 F254 pre-coated glass plates, 0.25 mm thickness).

Visualization was accomplished using irradiation with a UV lamp (λ = 254 nm or λ = 365 nm), and/or staining with potassium permanganate or ceric ammonium molybdate solution.

Purification of reaction products was performed using flash chromatography on silica gel (60 Å, particle size 40–63 μm) according to the procedure of Still and co-workers.⁴⁶

Yields refer to chromatographically and spectroscopically pure compounds, unless stated otherwise.

3-(Pentafluoro-λ⁶-sulfanyl)aniline. To a stirred acidic warm solution (50 °C) of pentafluoro(3-nitrophenyl)-λ⁶-sulfane (1.00 g, 3.93 mmol) in ethanol–HCl (11 mL:0.8 mL, 37% v/v), iron powder (1.22 g, 21.63 mmol) was added portionwise. The reaction was refluxed for 2 h. After cooling, the solid was removed by means of filtration; the filtered was diluted with dichloromethane. The organic phase was acidified with HCl 2N. The aqueous phase was separated, basified and extracted with dichloromethane (3 × 50 mL). The combined extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated to give the crude aniline as a yellow oil (0.72 g, 83% yield): the obtained ¹H, ¹²C and ¹⁹F NMR spectra matched to those reported by Bowden et al.³⁴
Lithium salt of ethyl 3-methyl-2,4-dioxo-4-(thiophen-2-yl)butanoate (1). To a magnetically stirred solution of lithium bis(tri-methylsilyl)amide (61 mL, 60.60 mmol, 1.0 M in THF) in diethyl ether (110 mL) at −78 °C, 1-(2-thienyl)-1-propanone (7 mL, 55.10 mmol) in diethyl ether (42 mL) was added dropwise under a nitrogen atmosphere. After the mixture was stirred at the same temperature for an additional 45 min, diethyl oxalate (9 mL, 66.11 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for another 16 h. The reaction precipitate was filtered, washed with diethyl ether, and dried under vacuum to afford the crude lithium salt 1 (9.50 g, 70%) as a pale-yellow solid. The product was used without further purification. ESMS, calculated m/z C₁₁H₁₁LiO₄S 246.05 [M]⁺, found m/z (relative intensity) 247.0 [M + H]⁺ (100), 265.0 [M + H + Na − Li]⁺ (100), 279.0 [M + H + K − Li]⁺ (100).
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 (8.50 g, 33.83 mmol) in ethanol (26 mL) was added 2,4-dichlorophenylhydrazine hydrochloride (8.85 g, 40.60 mmol) in one portion at room temperature under nitrogen. The resulting mixture was stirred at the same temperature for 24 h. After the reaction was complete, the precipitate was filtered, washed with ethanol and diethyl ether, and dried under vacuum to give a light-yellow solid (7.47 g). This crude solid, without purification, was dissolved in acetic acid (68 mL) and heated to reflux for 16 h. The reaction mixture was poured into ice-water and extracted with ethyl acetate (3 × 50 mL). The combined extracts were washed with water, saturated aqueous sodium bicarbonate, and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. Purification by flash column chromatography on silica gel with DCM gave ester 2 (5.33 g, 42% over two steps) as a white solid: the obtained ¹H, ¹²C and spectra matched to those reported by Tseng et al.³⁵
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid ethyl ester (3). To a magnetically stirred solution of 2 (2.60 g, 6.61 mmol) in acetonitrile (23 mL) was added NBS (1.43 g, 7.94 mmol) in small portions under nitrogen at 0 °C. The resulting mixture was then warmed to room temperature and stirred for 16 h. The reaction was quenched with saturated aqueous sodium thiosulfate and concentrated under reduced pressure to remove acetonitrile. The aqueous layer was extracted with ethyl acetate (2 × 40 mL). The organic layers were combined, washed with water, brine, dried over anhydrous sodium sulfate, and concentrated to give the crude residue, which was subjected to purification by flash chromatography on silica gel with n-hexane–ethyl acetate (8 : 2) to afford bromo ester 3 (2.70 g, 90%) as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 7.46 (d, J = 2.0 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.33 (dd, J = 8.4, 2.0 Hz, 1H), 6.95 (dd, J = 3.9, 0.8 Hz, 1H), 6.64 (dd, J = 3.9, 0.8 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 2.42 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 162.5, 142.9, 136.9, 136.6, 135.7, 133.8, 130.9, 130.2, 130.1, 130.1, 129.3, 127.9, 120.4, 115.0, 61.1, 14.5, 10.0. ESMS, calculated m/z C₁₇H₁₃BrCl₂N₂O₂S 459.92 [M]⁺, found m/z (relative intensity) 460.9 [M + H]⁺ (100), 482.9 [M + Na]⁺ (100).
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid (4). To a solution of bromo ester 3 (2.64 g, 5.62 mmol) in methanol (26 mL) was added potassium hydroxide (0.73 g, 11.24 mmol) in methanol (8 mL) dropwise at room temperature. The resulting mixture was heated to reflux for 3 h. After hydrolysis was complete, the reaction mixture was cooled to room temperature, poured into ice-water, and acidified with 2N hydrochloric acid. The precipitate was filtered, washed with water, and dried under vacuum to give the thiophene carboxylic acid 4 (2.19 g, 90%) as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 13.05 (s, 1H), 7.89 (d, J = 2.3 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.63 (dd, J = 8.5, 2.3 Hz, 1H), 7.23 (d, J = 3.9 Hz, 1H), 6.88 (d, J = 3.9 Hz, 1H), 2.32 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 163.9, 143.3, 136.7, 136.2, 135.9, 133.1, 132.3, 131.5, 130.8, 130.2, 130.0, 129.0, 119.6, 114.6, 10.2. ESMS, calculated m/z C₁₅H₉BrCl₂N₂O₂S 431.9 [M]⁺, found m/z (relative intensity) 470.9 [M + K]⁺ (100).
General procedure for the synthesis of compounds 5a–5f. The general procedure is illustrated below for compound 5a.
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbonyl chloride. A solution of the crude acid 4 (0.80 g, 1.81 mmol) and thionyl chloride (399 μL, 5.44 mmol) in toluene (12 mL) was refluxed for 3 h. Solvent was evaporated under reduced pressure, and the residue was then re-dissolved in toluene (8 mL) first and then in hexane (5 mL); after evaporation the crude acyl chloride (0.80 g, 98% yield) was obtained as a white solid.
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-N-(3-(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (5a). A solution in dichloromethane of the carboxylic chloride (1 mL, 0.36 M) obtained previously from 4, was added dropwise to a solution of 3-(trifluomethyl)aniline (50 μL, 0.40 mmol) and triethylamine (50 μL, 0.36 mmol) in dichloromethane (0.5 mL) at 0 °C. After stirring at room temperature for 16 h, to the reaction mixture was added water and the organic phase was extracted with dichloromethane (3 × 3 mL). The combined extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated. Flash column chromatography on silica gel with n-hexane–ethyl acetate (8 : 2) gave carboxamide 5a (150 mg, 66% yield) as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.87 (s, 1H), 8.01 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 2.1 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.39 (dd, J = 8.5, 2.1 Hz, 1H), 7.36 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 3.9 Hz, 1H), 6.68 (d, J = 3.9 Hz, 1H), 2.52 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 144.3, 138.3, 137.7, 136.8, 135.5, 133.7, 131.42 (q, J = 32.4 Hz), 130.6, 130.4, 130.2, 129.9, 129.5, 129.4, 128.1, 125.2, 123.87 (q, J = 272.6 Hz), 122.57, 120.55 (q, J = 3.9 Hz), 119.6, 116.34 (q, J = 4.0 Hz), 115.2; ¹⁹F NMR (376 MHz, chloroform-d) δ −62.72. ESMS, calculated m/z C₂₂H₁₃BrCl₂F₃N₃OS 574.93 [M]⁺, found m/z (relative intensity) 575.9 [M + H]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₂H₁₃BrCl₂F₃N₃OS: 573.9370; found: 573.9361%.
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-N-(3-(pentafluoro-λ⁶-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (5b). 5b (163 mg, 68% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.87 (s, 1H), 8.13 (t, J = 2.0 Hz, 1H), 7.87 (dd, J = 8.1, 1.9 Hz, 1H), 7.54 (d, J = 2.1 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.40 (dd, J = 8.5, 2.1 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 3.9 Hz, 1H), 6.68 (d, J = 3.9 Hz, 1H), 2.52 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 154.25 (appt, J = 17.1 Hz), 144.2, 138.2, 137.8, 136.9, 135.4, 133.8, 130.6, 130.4, 130.3, 129.8, 129.4, 129.2, 128.1, 122.5, 121.28 (p, J = 4.7 Hz), 119.6, 117.25 (p, J = 4.0 Hz), 115.2, 9.7; ¹⁹F NMR (377 MHz, chloroform-d) δ 84.17 (p, J = 149.2 Hz), 62.73 (d, J = 150.0 Hz). ESMS, calculated m/z C₂₁H₁₃BrCl₂F₅N₃OS₂ 632.90 [M]⁺, found m/z (relative intensity) 631.9 [M − H]⁻ (100). HRMS m/z M⁺H⁺ calcd for C₂₁H₁₃BrCl₂F₅N₃OS₂: 631.9059; found: 631.9052%.
5-(5-Bromothiophen-2-yl)-N-(3-(tert-butyl)phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (5c). 5c (87 mg, 63% yield) was obtained as a white solid ¹H NMR (400 MHz, chloroform-d) δ 8.71 (s, 1H), 7.64–7.57 (m, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.54 (dd, J = 1.9, 0.7 Hz, 1H), 7.38 (dd, J = 1.9, 1.3 Hz, 2H), 7.30 (d, J = 8.6 Hz, 1H), 7.18–7.12 (m, 1H), 6.97 (d, J = 3.9 Hz, 1H), 6.67 (d, J = 3.8 Hz, 1H), 2.52 (s, 3H), 1.33 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 160.2, 152.2, 144.9, 137.5, 137.4, 136.7, 135.6, 133.8, 130.7, 130.4, 130.2, 130.1, 129.2, 128.6, 128.0, 121.2, 119.4, 117.0, 116.9, 115.0, 34.8, 31.3 (x 3), 9.8. ESMS, calculated m/z C₂₅H₂₂BrCl₂N₃OS 563.00 [M]⁺, found m/z (relative intensity) 564.0 [M + H]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₅H₂₂BrCl₂N₃OS: 562.0122; found: 562.0111%.
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-N-(4-(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (5d). 5d (97 mg, 44% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.89 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 2.1 Hz, 1H), 7.40 (dd, J = 8.5, 2.1 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 3.9 Hz, 1H), 6.68 (d, J = 3.9 Hz, 1H), 2.52 (d, J = 0.5 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 144.3, 140.9, 137.7, 136.9, 135.4, 133.7, 130.6, 130.4, 130.2, 129.8, 129.4, 128.2, 126.25 (q, J = 3.6 Hz), 125.57 (q, J = 32.5 Hz, ×2), 124.13 (q, J = 272.2), 119.6, 119.2 (×2), 115.2, 9.7; ¹⁹F NMR (376 MHz, chloroform-d) δ −62.07. ESMS, calculated m/z C₂₂H₁₃BrCl₂F₃N₃OS 574.93 [M]⁺, found m/z (relative intensity) 575.8 [M + H]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₂H₁₃BrCl₂F₃N₃OS: 573.9370; found: 573.9361%.
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-4-methyl-N-(4-(pentafluoro-λ⁶-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (5e). 5e (100 mg, 42% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.92 (s, 1H), 7.81–7.68 (m, 4H), 7.53 (d, J = 2.1 Hz, 1H), 7.42–7.31 (m, 2H), 6.98 (d, J = 3.9 Hz, 1H), 6.68 (d, J = 3.9 Hz, 1H), 2.51 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 149.03 (appt, J = 17.6 Hz), 144.1, 140.5, 137.8, 136.9, 135.4, 133.7, 130.6, 130.4, 130.2, 129.7, 129.4, 128.1, 126.99 (p, J = 4.7 Hz, ×2), 119.7, 118.8 (×2), 115.2, 9.7; ¹⁹F NMR (377 MHz, chloroform-d) δ 85.35 (p, J = 150.1, 149.4 Hz), 63.55 (d, J = 150.0 Hz). ESMS, calculated m/z C₂₁H₁₃BrCl₂F₅N₃OS₂ 632.90 [M]⁺, found m/z (relative intensity) 631.8 [M − H]⁻. HRMS m/z M⁺H⁺ calcd for C₂₁H₁₃BrCl₂F₅N₃OS₂: 631.9059; found: 631.9048%.
5-(5-Bromothiophen-2-yl)-N-(4-(tert-butyl)phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (5f). 5f (84 mg, 40% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.67 (s, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.53 (dd, J = 1.9, 0.6 Hz, 1H), 7.41–7.32 (m, 4H), 6.97 (d, J = 3.9 Hz, 1H), 6.67 (d, J = 3.8 Hz, 1H), 2.52 (s, 3H), 1.32 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 160.2, 147.0, 144.9, 137.4, 136.7, 135.6, 135.2 (×2), 133.8, 130.7, 130.4, 130.2, 129.2, 128.0, 125.8 (×2), 119.6 (×2), 119.4, 115.0, 34.4, 31.4 (×3) 9.8. ESMS, calculated m/z C₂₅H₂₂BrCl₂N₃OS 563.0 [M]⁺, found m/z (relative intensity) 564.0 [M + H]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₅H₂₂BrCl₂N₃OS: 562.0122; found: 562.0113%.
Lithium salt of ethyl 4-(5-bromothiophen-2-yl)-2,4-dioxobutanoate (6). To a magnetically stirred solution of lithium bis(tri-methylsilyl)amide (23 mL, 22.56 mmol, 1.0 M in THF) in diethyl ether (41 mL) at −78 °C, 1-(5-bromothiophen-2-yl)ethanone (4.25 g, 20.51 mmol) in diethyl ether (16 mL) was added dropwise under a nitrogen atmosphere. After the mixture was stirred at the same temperature for an additional 45 min, diethyl oxalate (3.4 mL, 24.62 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for another 16 h. The reaction precipitate was filtered, washed with diethyl ether, and dried under vacuum to afford the crude lithium salt 6 (6.31 g, 99%) as a pale-yellow solid. The product was used without further purification. ESMS, calculated m/z C₁₀H₈BrLiO₄S 309.9 [M]⁺, found m/z (relative intensity) 326.9 [M − Li + H + Na]⁺ (63), 343 [M − Li + H + K]⁺ (31).
General procedure for the synthesis of compounds 7a,7b. The general procedure is illustrated below for compound 7a.
Ethyl 5-(5-bromothiophen-2-yl)-1-(2,4-difluorophenyl)-1H-pyrazole-3-carboxylate (7a). To a solution of lithium salt 6 (3 g, 9.55 mmol) in ethanol (26 mL) was added 2,4-difluorophenylhydrazine hydrochloride (2.143 g, 11.46 mmol) in one portion at room temperature under nitrogen. The resulting mixture was stirred at the same temperature for 24 h. After reaction was complete, the precipitate was filtered, washed with ethanol and diethyl ether, and dried under vacuum to give a light-yellow solid (2.66 g). This crude solid, without purification, was dissolved in acetic acid (20 mL) and heated to reflux for 16 h. The reaction mixture was poured into ice-water and extracted with ethyl acetate (3 × 70 mL). The combined extracts were washed with water, saturated aqueous sodium bicarbonate, and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. Purification by flash column chromatography on silica gel with DCM gave ester 7a (2.08 g, 58% over two steps) as a white solid. ¹H NMR (400 MHz, chloroform-d) δ 7.51 (td, J = 8.5, 5.7 Hz, 1H), 7.08 (s, 1H), 7.08–6.92 (m, 2H), 6.93 (d, J = 3.9 Hz, 1H), 6.69 (d, J = 3.9 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 163.54 (dd, J = 254.1, 11.0 Hz), 161.6, 157.88 (dd, J = 256.9, 12.8 Hz), 145.3, 139.5, 130.83 (d, J = 10.1 Hz), 130.8, 130.4, 127.4, 123.29 (dd, J = 12.7, 4.1 Hz), 114.4, 112.25 (dd, J = 22.7, 3.8 Hz), 108.4, 105.29 (dd, J = 26.5, 23.0 Hz), 61.3, 14.3; ¹⁹F NMR (376 MHz, chloroform-d) δ −104.59 (qd, J = 8.4, 5.7 Hz), −115.10 to −115.23 (m). ESMS, calculated m/z C₁₆H₁₁BrF₂N₂O₂S 414.0 [M]⁺, found m/z (relative intensity) 415.0 [M + H]⁺ (100), 435.0 [M + Na]⁺ (100).
Ethyl 5-(5-bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylate (7b). 7b (2.36 g, 60% over two steps) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 7.54 (d, J = 2.1 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.41 (dd, J = 8.5, 2.1 Hz, 1H), 7.08 (s, 1H), 6.92 (d, J = 3.9 Hz, 1H), 6.66 (d, J = 3.9 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 161.7, 145.2, 139.5, 137.2, 135.3, 134.0, 131.0, 130.8, 130.5, 130.4, 128.2, 127.3, 114.5, 108.1, 61.4, 14.4. ESMS, calculated m/z 16H₁₁BrCl₂N₂O₂S 445.9 [M]⁺, found m/z (relative intensity) 446.9 [M + H]⁺ (100).
General procedure for the synthesis of compounds 8a,8b. The general procedure is illustrated below for compound 8a.
5-(5-Bromothiophen-2-yl)-1-(2,4-difluorophenyl)-1H-pyrazole-3-carboxylic acid (8a). To a solution of bromo ester 7a (1.72 g, 4.07 mmol) in methanol (19 mL) was added potassium hydroxide (0.80 g, 12.21 mmol) in methanol (9 mL) dropwise at room temperature. The resulting mixture was heated to reflux for 3 h. After hydrolysis was complete, the reaction mixture was cooled to room temperature, poured into ice-water, and acidified with 2N hydrochloric acid. The precipitate was filtered, washed with water, and dried under vacuum to give thiophene carboxylic acid 8a (1.34 g, 85%) as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 13.18 (bs, 1H), 7.83 (td, J = 8.8, 5.9 Hz, 1H), 7.64 (ddd, J = 10.3, 9.0, 2.8 Hz, 1H), 7.42–7.32 (m, 1H), 7.28 (s, 1H), 7.23 (d, J = 4.0 Hz, 1H), 7.11 (d, J = 4.0 Hz, 1H); ¹³C NMR (101 MHz, DMSO) δ 163.66 (dd, J = 251.4, 11.7 Hz), 163.0, 157.92 (dd, J = 253.6, 13.7 Hz), 146.0, 139.7, 132.28 (d, J = 10.7 Hz), 131.6, 130.8, 129.3, 123.43 (dd, J = 12.6, 3.8 Hz), 114.0, 113.45 (dd, J = 22.9, 3.6 Hz), 108.6, 106.16 (dd, J = 27.4, 23.6 Hz); ¹⁹F NMR (376 MHz, DMSO-d₆) δ −104.80 (qd, J = 8.7, 5.8 Hz), −117.33 (q, J = 9.4 Hz). ESMS, calculated m/z C₁₄H₇BrF₂N₂O₂S 383.9 [M]⁺, found m/z (relative intensity) 422.9 [M + K]⁺ (100).
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 solid: ¹H NMR (400 MHz, DMSO-d₆) δ 13.18 (s, 1H), 8.00 (d, J = 2.3 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.71 (dd, J = 8.5, 2.3 Hz, 1H), 7.31 (s, 1H), 7.22 (d, J = 4.0 Hz, 1H), 7.09 (d, J = 4.0 Hz, 1H); ¹³C NMR (101 MHz, DMSO) δ 163.0, 145.8, 139.5, 136.9, 135.5, 133.6, 132.5, 131.6, 130.8, 130.6, 129.4, 129.1, 114.0, 108.3. ESMS, calculated m/z C₁₄H₇BrCl₂N₂O₂S 417.9 [M]⁺, found m/z (relative intensity) 456.9 [M + K]⁺ (100).
General procedure for the synthesis of compounds 9a–9d. The general procedure is illustrated below for compound 9a.
5-(5-Bromothiophen-2-yl)-1-(2,4-difluorophenyl)-1H-pyrazole-3-carbonyl chloride. A solution of the crude acid 8 (0.20 g, 0.51 mmol) and thionyl chloride (112 μL, 1.53 mmol) in toluene (3 mL) was refluxed for 3 h. The solvent was evaporated under reduced pressure, and the residue was then re-dissolved in toluene (3 mL) first and then in hexane (4 mL); after evaporation the crude acyl chloride (0.19 g, 95% yield) was obtained as a white solid.
5-(5-Bromothiophen-2-yl)-1-(2,4-difluorophenyl)-N-(4-(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (9a). A solution in dichloromethane of the acyl chloride obtained previously (0.9 mL, 0.24 M), was added dropwise to a solution of 4-(trifluomethyl)aniline (30 μL, 0.23 mmol) and triethylamine (33 μL, 0.23 mmol) in dichloromethane (0.2 mL) at 0 °C. After stirring at room temperature for 16 h, to the reaction mixture was added water and the organic phase was extracted with dichloromethane (3 × 3 mL). The combined extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated. Flash column chromatography on silica gel with n-hexane–ethyl acetate (9 : 1) gave carboxamide 9a (88 mg, 72% yield) as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.81 (s, 1H), 7.81 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.51 (td, J = 8.5, 5.7 Hz, 1H), 7.17 (s, 1H), 7.12–7.00 (m, 2H), 6.95 (d, J = 3.9 Hz, 1H), 6.74 (d, J = 3.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl3) δ 163.74 (dd, J = 254.8, 10.9 Hz), 159.1, 158.03 (dd, J = 257.3, 12.8 Hz), 147.8, 140.6, 130.7, 130.64 (d, J = 10.2 Hz), 130.5, 127.7, 126.32 (q, J = 3.8 Hz, ×2), 125.99 (q, J = 32.6 Hz), 124.09 (q, J = 271.8 Hz), 123.15 (dd, J = 13.0, 4.2 Hz), 122.74, 119.3 (×2), 114.8, 112.48 (dd, J = 22.8, 3.9 Hz), 107.0, 105.66 (dd, J = 26.5, 23.0 Hz); ¹⁹F NMR (376 MHz, chloroform-d) δ −62.11, −104.02 (qd, J = 8.3, 5.7 Hz), −115.07 to −115.16 (m). ESMS, calculated m/z C₂₁H₁₁BrF₅N₃OS 529 [M]⁺, found m/z (relative intensity) 530 [M + H]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₁H₁₁BrF₅N₃OS: 527.9805; found: 527.9794%.
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-N-(4-(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (9b). 9b (69 mg, 53%) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.81 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.65–7.56 (m, 3H), 7.46 (d, J = 1.9 Hz, 2H), 7.18 (s, 1H), 6.94 (d, J = 3.9 Hz, 1H), 6.71 (d, J = 3.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 159.1, 147.7, 140.6, 140.5, 137.5, 135.1, 134.1, 130.7, 130.7, 130.7, 130.5, 128.4, 127.6, 126.33 (q, J = 3.7 Hz, ×2), 125.98 (q, J = 32.8 Hz), 124.08 (q, J = 271.3 Hz), 119.3 (×2), 114.8, 106.6; ¹⁹F NMR (376 MHz, chloroform-d) δ −62.10. ESMS, calculated m/z C₂₁H₁₁BrCl₂F₃N₃OS 560.9 [M]⁺, found m/z (relative intensity) 583.8 [M + Na]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₁H₁₁BrCl₂F₃N₃OS: 559.9214; found: 559.9203%.
5-(5-Bromothiophen-2-yl)-1-(2,4-difluorophenyl)-N-(4-(pentafluoro-λ⁶-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (9c). 9c (110 mg, 70%) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.81 (s, 1H), 7.81–7.72 (m, 4H), 7.51 (td, J = 8.5, 5.7 Hz, 1H), 7.18 (s, 1H), 7.13–7.01 (m, 2H), 6.96 (d, J = 3.9 Hz, 1H), 6.74 (d, J = 3.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 163.76 (dd, J = 254.8, 11.0 Hz), 159.1, 158.02 (dd, J = 257.2, 12.8 Hz), 149.14 (appt, J = 17.5 Hz), 147.6, 140.7, 140.3, 130.63 (d, J = 10.4 Hz), 130.5, 127.7, 127.04 (p, J = 4.6 Hz, ×2), 123.11 (dd, J = 12.6, 4.2 Hz), 118.9 (×2), 114.9, 112.50 (dd, J = 22.8, 3.9 Hz), 107.0, 105.67 (dd, J = 26.4, 22.9 Hz), 100; ¹⁹F NMR (377 MHz, chloroform-d) δ 85.18 (p, J = 150.6, 149.7 Hz), 63.49 (d, J = 150.0 Hz), −103.91 (qd, J = 8.3, 5.7 Hz), −115.12 (q, J = 8.9 Hz). ESMS, calculated m/z C₂₀H₁₁BrF₇N₃OS₂ 586.9 [M]⁺, found m/z (relative intensity) 585.9 [M − H]⁻ (100). HRMS m/z M⁺H⁺ calcd for C₂₀H₁₁BrF₇N₃OS₂: 585.9493; found: 585.9484%.
5-(5-Bromothiophen-2-yl)-1-(2,4-dichlorophenyl)-N-(4-(pentafluoro-λ⁶-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (9d). 9d (60 mg, 40%) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.82 (s, 1H), 7.82–7.70 (m, 4H), 7.61 (d, J = 1.9 Hz, 1H), 7.52–7.40 (m, 2H), 7.19 (s, 1H), 6.95 (d, J = 3.9 Hz, 1H), 6.71 (d, J = 3.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 159.1, 149.13 (p, J = 17.9 Hz), 147.5, 140.6, 140.2, 137.6, 135.1, 134.1, 130.7, 130.7, 130.6, 130.5, 128.4, 127.6, 127.07 (p, J = 4.3 Hz, ×2), 118.9 (×2), 114.9, 106.6; ¹⁹F NMR (377 MHz, chloroform-d) δ 85.17 (p, J = 150.7, 149.7 Hz), 63.48 (d, J = 150.0 Hz). ESMS, calculated m/z C₂₀H₁₁BrCl₂F₅N₃OS₂ 618.9 [M]⁺, found m/z (relative intensity) 617.8 [M − H]⁻ (100). HRMS m/z M⁺H⁺ calcd for C₂₀H₁₁BrCl₂F₅N₃OS₂: 617.8902; found: 617.8894%.
Lithium salt of ethyl 4-(4-chlorophenyl)-3-methyl-2,4-dioxobutanoate (10). To a magnetically stirred solution of lithium bis(tri-methylsilyl)amide (64 mL, 63.93 mmol, 1.0 M in THF) in diethyl ether (63 mL) at −78 °C, 1-(2-thienyl)-1-propanone (10 g, 58.12 mmol) in diethyl ether (73 mL) was added dropwise under a nitrogen atmosphere. After the mixture was stirred at the same temperature for an additional 45 min, diethyl oxalate (9 mL, 63.93 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for another 16 h. The reaction precipitate was filtered, washed with diethyl ether, and dried under vacuum to afford the crude lithium salt 10 (8.05 g, 50%) as a pale-yellow solid. The product was used without further purification.
Ethyl 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylate (11). To a solution of lithium salt 10 (8.05 g, 28.73 mmol) in ethanol (99 mL) was added 2,4- dichlorophenylhydrazine hydrochloride (6.88 g, 31.60 mmol) in one portion at room temperature under nitrogen. The resulting mixture was stirred at the same temperature for 24 h. After reaction was complete, the precipitate was filtered, washed with ethanol and diethyl ether, and dried under vacuum to give a light-yellow solid (6.1 g). This crude solid, without purification, was dissolved in acetic acid (45 mL) and heated to reflux for 16 h. The reaction mixture was poured into ice-water and extracted with ethyl acetate (3 × 50 mL). The combined extracts were washed with water, saturated aqueous sodium bicarbonate, and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. Purification by flash column chromatography on silica gel with hexane–acetate (8 : 2) gave ester 11 (3.44 g, 30% over two steps) as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 7.38 (d, J = 2.2 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.32–7.27 (m, 3H), 7.07 (d, J = 8.6 Hz, 2H), 4.45 (q, J = 7.1 Hz, 2H), 2.33 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 163.2, 143.4, 143.3, 136.5, 136.3, 135.4, 133.5, 131.3 (×2), 131.2, 130.5, 129.3 (×2), 128.2, 127.5, 119.6, 61.4, 14.9, 10.1. ESMS, calculated m/z C₁₉H₁₅Cl₃N₂O₂ 408.0 [M]⁺, found m/z (relative intensity) 409.0 [M + H]⁺ (100).
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid (12). To a solution of bromo ester 3 (2.32 g, 5.54 mmol) in methanol (26 mL) was added potassium hydroxide (0.72 g, 11.09 mmol) in methanol (8 mL) dropwise at room temperature. The resulting mixture was heated to reflux for 3 h. After hydrolysis was complete, the reaction mixture was cooled to room temperature, poured into ice-water, and acidified with 2N hydrochloric acid. The precipitate was filtered, washed with water, and dried under vacuum to give the carboxylic acid 12 (2.10 g, 98%) as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 7.73 (d, J = 2.3 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.54 (dd, J = 8.5, 2.3 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 2.20 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 164.1, 143.4, 142.9, 136.1, 135.6, 134.3, 132.3, 132.1, 131.8 (×2), 130.0, 129.2 (×2), 128.8, 127.4, 118.4, 9.9. ESMS, calculated m/z C₁₇H₁₁Cl₃N₂O₂ 380.0 [M]⁺, found m/z (relative intensity) 381.0 [M + H]⁺ (100).
General procedure for the synthesis of compounds 13a–13g. The general procedure is illustrated below for compound 13a.
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 mmol) and thionyl chloride (565 μL, 7.70 mmol) in toluene (17.1 mL) was refluxed for 3 h. Solvent was evaporated under reduced pressure, and the residue was then re-dissolved in toluene (10 mL) first and then in hexane (10 mL); after evaporation the crude acyl chloride (0.98 g, 95% yield) was obtained as a white solid.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(3-(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (13a). A solution in dichloromethane of the acyl chloride obtained previously (0.96 mL, 0.41 M), was added dropwise to a solution of 3-(trifluomethyl)aniline (55 μL, 0.44 mmol) and triethylamine (61 μL, 0.44 mmol) in dichloromethane (0.4 mL) at 0 °C. After stirring at room temperature for 16 h, to the reaction mixture was added water and the organic phase was extracted with dichloromethane (3 × 3 mL). The combined extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated. Flash column chromatography on silica gel with n-hexane–ethyl acetate (8 : 2) gave carboxamide 13a (160 mg, 70% yield) as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.91 (s, 1H), 8.03 (t, J = 1.9 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.51–7.41 (m, 2H), 7.40–7.27 (m, 5H), 7.12–7.07 (d, J = 8.5 Hz, 2H), 2.43 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 144.4, 143.7, 138.4, 136.3, 135.7, 135.2, 133.0, 131.40 (q, J = 32.3 Hz), 130.8 (×2), 130.5, 130.4, 129.5, 129.0 (×2), 128.0, 126.8, 122.55, 122.53 (q, J = 272.0 Hz), 120.50 (q, J = 3.9 Hz), 118.4, 116.32 (q, J = 4.0 Hz), 9.5; ¹⁹F NMR (376 MHz, chloroform-d) δ −62.74. ESMS, calculated m/z C₂₄H₁₅Cl₃F₃N₃O 523 [M]⁺, found m/z (relative intensity) 546.0 [M + Na]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₄H₁₅Cl₃F₃N₃O: 524.0311; found: 524.0301%.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(3-(pentafluoro-λ⁶-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (13b). 13b (182 mg, 70% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.92 (s, 1H), 8.16 (t, J = 2.1 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.55–7.43 (m, 3H), 7.39–7.31 (m, 4H), 7.12 (d, J = 8.5 Hz, 2H), 2.45 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 154.25 (appt, J = 17.4 Hz), 144.2, 143.7, 138.3, 136.3, 135.7, 135.2, 133.0, 130.8 (×2), 130.5, 130.4, 129.2, 129.0 (×2), 128.0, 126.8, 122.4, 121.24 (p, J = 3.9, 3.4 Hz), 118.4, 117.22 (p, J = 4.9 Hz), 9.5; ¹⁹F NMR (377 MHz, chloroform-d) δ 84.17 (p, J = 150.4 Hz), 62.72 (d, J = 150.0 Hz). ESMS, calculated m/z C₂₃H₁₅Cl₃F₅N₃OS 583.0 [M]⁺, found m/z (relative intensity) 582.0 [M − H]⁻ (56). HRMS m/z M⁺H⁺ calcd for C₂₃H₁₅Cl₃F₅N₃OS: 582.0000; found: 581.9991%.
N-(3-(tert-Butyl)phenyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (13c). 13c (88 mg, 63% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.76 (s, 1H), 7.64–7.59 (m, 2H), 7.46 (t, J = 1.3 Hz, 1H), 7.35–7.25 (m, 5H), 7.18–7.13 (m, 1H), 7.10 (d, J = 8.5 Hz, 2H), 2.44 (s, 3H), 1.33 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 160.4, 152.2, 145.0, 143.4, 137.6, 136.2, 135.8, 135.0, 133.1, 130.9 (×2), 130.6, 130.4, 128.9 (×2), 128.6, 128.0, 127.1, 121.1, 118.2, 117.1, 116.9, 34.8, 31.4 (×3), 9.6. ESMS, calculated m/z C₂₇H₂₄Cl₃N₃O 511.1 [M]⁺, found m/z (relative intensity) 512.1 [M + H]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₇H₂₄Cl₃N₃O: 512.1063; found: 512.1052%.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(4-(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (13d). 13d (141 mg, 58% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.93 (s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 8.3 Hz, 2H), 7.47 (s, 1H), 7.37–7.28 (m, 4H), 7.09 (d, J = 8.5 Hz, 2H), 2.43 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 144.4, 143.7, 141.0, 136.3, 135.7, 135.2, 133.0, 130.8 (×2), 130.5, 129.0 (×2), 128.2, 128.0, 126.8, 126.27 (q, J = 3.7 Hz, ×2), 125.55 (q, J = 31.9 Hz), 124.93 (q, J = 272.0 Hz), 119.2 (×2), 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%.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(4-(pentafluoro-λ⁶-sulfanyl)phenyl)-1H-pyrazole-3-carboxamide (13e). 13e (177 mg, 68% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.93 (s, 1H), 7.79 (d, J = 9.0 Hz, 2H), 7.77–7.69 (m, 2H), 7.47 (d, J = 2.1 Hz, 1H), 7.37–7.28 (m, 4H), 7.09 (d, J = 8.4 Hz, 2H), 2.43 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 160.6, 148.97 (appt, J = 17.0 Hz), 144.2, 143.8, 140.6, 136.4, 135.6, 135.2, 133.0, 130.8 (×2), 130.43, 130.42, 129.0 (×2), 128.0, 126.97 (p, J = 4.6 Hz, ×2), 126.72, 118.8 (×2), 118.5, 9.5; ¹⁹F NMR (377 MHz, chloroform-d) δ 85.34 (p, J = 150.1, 149.1 Hz), 63.53 (d, J = 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%.
N-(4-(tert-Butyl)phenyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (13f). 13f (162 mg, 78% yield) was obtained as a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.71 (s, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.46 (dd, J = 1.8, 0.8 Hz, 1H), 7.36 (d, J = 8.7 Hz, 2H), 7.33–7.29 (m, 4H), 7.09 (d, J = 8.5 Hz, 2H), 2.43 (s, 3H), 1.32 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 160.4, 147.0, 145.0, 143.4, 136.1, 135.9, 135.2, 135.0, 133.0, 130.9 (×2), 130.6, 130.4, 128.9 (×2), 127.9, 127.1, 125.8 (×2), 119.5 (×2), 118.2, 34.4, 31.4 (×3), 9.5. ESMS, calculated m/z C₂₇H₂₄Cl₃N₃O 511.1 [M]⁺, found m/z (relative intensity) 534.1 [M + Na]⁺ (100). HRMS m/z M⁺H⁺ calcd for C₂₇H₂₄Cl₃N₃O: 512.1063; found: 512.1053%.
Determination of the partition coefficients (log P). Measurement of chromatographic capacity factor (k′) by C-18 HPLC method was performed using the equation k′ = (t_r − t₀)/t₀,^47,48 where t₀ is the retention time of unretained substance and t_r is the compound's retention time. The mobile phase was prepared mixing methanol with water in proportions of 85 : 15 and the flow rate was 1 mL min⁻¹. A solution of urea in a methanol–water solvent (85 : 15) was used for measurement of the column dead time (t₀ = 2.673 ± 0.002, n = 3). Seven compounds having known log P values, tabulated and in the range from 1.10 to 5.70 (benzyl alcohol, log P 1.10; benzene, log P 2.10; toluene, log P 2.70; naphthalene, log P 3.60; biphenyl, log P 4; phenanthrene, log P 4.50; triphenylamine, log P 5.70) were chosen as a “standard” calibration mixture for the determination of retention times (t_r). Every measure was obtained in triplicate and at controlled temperature of 25 °C. Capacity factors (k′) were calculated. The log k' value for each of seven compounds was plotted against its relative lipophilicity value reported in literature, based on the established linear relationship (log P = 3.64 log k′ + 3.39, correlation coefficient = 0.95). The capacity factor of each compound was determined (the value was obtained on average of three experiments) and the relative log P value was obtained by extrapolation.

In vitro assays

Equilibrium binding assays. Binding assays were performed with the CB₁ receptor agonist, [3H]CP55940 (0.7 nM), 1 mg mL⁻¹ 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 μL. Binding was initiated by the addition of mouse brain membranes (30 μg) or CB₂ transfected CHO cells (5 μg). 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 mL⁻¹ BSA) and vacuum filtration using a 24-well sampling manifold (Brandel Cell Harvester) and Whatman GF/B glass-fibre filters that had been soaked in wash buffer at 4 °C for 24 h. Each reaction tube was washed five times with a 4 mL aliquot of buffer. The filters were oven-dried for 60 min and then placed in 5 mL of scintillation fluid (Ultima Gold XR, Packard), and radioactivity quantitated by liquid scintillation spectrometry. Specific binding was defined as the difference between the binding that occurred in the presence and absence of 1 μM of the corresponding unlabelled ligand and was 70–80% of the total binding.

β-Arrestin assays

PathHunter® HEK293 CB₁ beta-arrestin cells were plated 48 hours before use and incubated at 37 °C, 5% CO₂ in a humidified incubator. Compounds were dissolved in DMSO and diluted in OCC media to the required concentrations. 5 μL of compound or vehicle solution was added to each well and incubated for 60 minutes at 37 °C, 5% CO₂ in a humidified incubator. 5 μL of increasing concentrations of CP55940 was added to each well followed by a 90 minute incubation at 37 °C, 5% CO₂ in a humidified incubator. 55 μL of detection reagent is then added followed by a further 90 minute incubation at room temperature in the dark. Chemiluminescence, indicated as RLU, was measured on a standard luminescence plate reader.

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 final concentrations of 0.01, 0.1,1 and 10 μM and a final 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 μM) indicate insolubility.

Acknowledgements

We thank the European Commission for financial support (Industry Academia Partnerships and Pathways project “PET BRAIN”, Contract no. 251482) and Miteni S.p.A. (Italy) for a kind gift of the SF₅ precursors. The EPSRC National Mass Spectrometry Service Centre (Swansea, UK) is gratefully acknowledged for performing HRMS analyses. We thank Dr Kevin Read (University of Dundee) for support and advice with the nephelometry 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.

Notes and references

1. D. O'Hagan and D. B. Harper, J. Fluorine Chem., 1999, 100, 127–133.
2. S. Purser, P. R. Moore, S. Swallow and V. Gouverneur, Chem. Soc. Rev., 2008, 37, 320–330.
3. M. Zanda, New J. Chem., 2004, 28, 1401–1411.
4. D. O'Hagan, J. Fluorine Chem., 2010, 131, 1071–1081.
5. W. A. Sheppard, J. Am. Chem. Soc., 1960, 82, 4751–4752.
6. J. T. Welch, in Fluorine In Pharmaceutical And Medicinal Chemistry, Imperial college press, 2012, vol. 6, pp. 175–207.
7. S. Altomonte and M. Zanda, J. Fluorine Chem., 2012, 143, 57–93.
8. R. W. Winter, R. A. Dodean, and G. L. Gard, in Fluorine-Containing Synthons, ed. V. A. Soloshonok, American Chemical Society, Washington, DC, 2005, vol. 911, pp. 87–118.
9. W. A. Sheppard, J. Am. Chem. Soc., 1965, 87, 2410–2420.
10. C. Hansch, R. M. Muir, T. Fujita, P. P. Maloney, F. Geiger and M. Streich, J. Am. Chem. Soc., 1963, 85, 2817–2824.
11. W. A. Sheppard, J. Am. Chem. Soc., 1962, 84, 3072–3076.
12. J. T. Welch and D. S. Lim, Bioorg. Med. Chem., 2007, 15, 6659–6666.
13. P. Kirsch, Modern fluoroorganic chemistry synthesis, reactivity, applications, Wiley-VCH, Weinheim, 2004.
14. D. Lentz and K. Seppelt, in Chemistry of Hypervalent Compounds, ed. K. Akiba, Wiley-VCH, New York, 1999, pp. 295–323.
15. L. J. Sæthre, N. Berrah, J. D. Bozek, K. J. Børve, T. X. Carroll, 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.
17. S. Munro, K. L. Thomas and M. Abu-Shaar, Nature, 1993, 365, 61–65.
18. A. C. Howlett, F. Barth, T. I. Bonner, G. Cabral, P. Casellas, W. A. Devane, C. C. Felder, M. Herkenham, K. Mackie, B. R. Martin, R. Mechoulam and R. G. Pertwee, Pharmacol. Rev., 2002, 54, 161–202.
19. D. Shire, C. Carillon, M. Kaghad, B. Calandra, M. Rinaldi-Carmona, G. L. Fur, D. Caput and P. Ferrara, J. Biol. Chem., 1995, 270, 3726–3731.
20. E. Ryberg, H. K. Vu, N. Larsson, T. Groblewski, S. Hjorth, T. Elebring, S. Sjögren and P. J. Greasley, FEBS Lett., 2005, 579, 259–264.
21. M. Herkenham, A. B. Lynn, M. D. Little, M. R. Johnson, L. S. Melvin, B. R. de Costa and K. C. Rice, Proc. Natl. Acad. Sci. U. S. A., 1990, 87, 1932–1936.
22. G. Griffin, S. R. Fernando, R. A. Ross, N. G. McKay, M. L. J. Ashford, D. Shire, J. W. Huffman, S. Yu, J. A. H. Lainton and R. G. Pertwee, Eur. J. Pharmacol., 1997, 339, 53–61.
23. R. G. Pertwee, Life Sci., 1999, 65, 597–605.
24. J. C. Ashton, D. Friberg, C. L. Darlington and P. F. Smith, Neurosci. Lett., 2006, 396, 113–116.
25. J. Horder, M. Browning, M. Di Simplicio, P. J. Cowen and C. J. Harmer, J. Psychopharmacol., 2012, 26, 125–132.
26. G. Kunos, D. Osei-Hyiaman, S. Bátkai, K. A. Sharkey and A. Makriyannis, Trends Pharmacol. Sci., 2009, 30, 1–7.
27. E. Kirilly, X. Gonda and G. Bagdy, Acta Physiol., 2012, 205, 41–60.
28. B.-C. Ho, T. H. Wassink, S. Ziebell and N. C. Andreasen, Schizophr. Res., 2011, 128, 66–75.
29. B. Costa, A. E. Trovato, M. Colleoni, G. Giagnoni, E. Zarini and T. Croci, Pain, 2005, 116, 52–61.
30. P. Gazzerro, M. G. Caruso, M. Notarnicola, G. Misciagna, V. Guerra, C. Laezza and M. Bifulco, Int. J. Obes., 2006, 31, 908–912.
31. M. Rinaldi-Carmona, F. Barth, M. Héaulme, D. Shire, B. Calandra, C. Congy, S. Martinez, J. Maruani, G. Néliat, D. Caput, P. Ferrara, P. Soubrié, J. C. Brelière and G. Le Fur, FEBS Lett., 1994, 350, 240–244.
32. R. Lan, Q. Liu, P. Fan, S. Lin, S. R. Fernando, D. McCallion, R. Pertwee and A. Makriyannis, J. Med. Chem., 1999, 42, 769–776.
33. P. Lazzari, S. Ruiu, G. A. Pinna and G. Murineddu, US Pat., 0 261 281, 2005.
34. R. D. Bowden, P. J. Comina, M. P. Greenhall, B. M. Kariuki, A. Loveday and D. Philp, Tetrahedron, 2000, 56, 3399–3408.
35. S.-L. Tseng, M.-S. Hung, C.-P. Chang, J.-S. Song, C.-L. Tai, H.-H. Chiu, W.-P. Hsieh, Y. Lin, W.-L. Chung, C.-W. Kuo, C.-H. Wu, C.-M. Chu, Y.-S. Tung, Y.-S. Chao and K.-S. Shia, J. Med. Chem., 2008, 51, 5397–5412.
36. A. Leo, C. Hansch and D. Elkins, Chem. Rev., 1971, 71, 525–616.
37. H. Fan, H. T. Ravert, D. P. Holt, R. F. Dannals and A. G. Horti, J. Labelled Compd. Radiopharm., 2006, 49, 1021–1036.
38. S. Tobiishi, T. Sasada, Y. Nojiri, F. Yamamoto, T. Mukai, K. Ishiwata and M. Maeda, Chem. Pharm. Bull., 2007, 55, 1213–1217.
39. R. A. Ross, H. C. Brockie, L. A. Stevenson, V. L. Murphy, F. Templeton, A. Makriyannis and R. G. Pertwee, Br. J. Pharmacol., 1999, 126, 665–672.
40. J.-Z. Chen, X.-W. Han, Q. Liu, A. Makriyannis, J. Wang and X.-Q. Xie, J. Med. Chem., 2006, 49, 625–636.
41. M. R. Price, G. L. Baillie, A. Thomas, L. A. Stevenson, M. Easson, R. Goodwin, A. McLean, L. McIntosh, G. Goodwin, G. Walker, P. Westwood, J. Marrs, F. Thomson, P. Cowley, A. Christopoulos, R. G. Pertwee and R. A. Ross, Mol. Pharmacol., 2005, 68, 1484–1495.
42. D. A. Jackson and S. A. Mabury, Environ. Toxicol. Chem., 2009, 28, 1866.
43. B. Hoelke, S. Gieringer, M. Arlt and C. Saal, Anal. Chem., 2009, 81, 3165–3172.
44. M. M. C. van D. Lee, M. Bras, C. J. van Koppen and G. J. R. Zaman, J. Biomol. Screening, 2008, 13, 986–998.
45. B. D. Kangas, M. S. Delatte, V. K. Vemuri, G. A. Thakur, S. P. Nikas, K. V. Subramanian, V. G. Shukla, A. Makriyannis and J. Bergman, J. Pharmacol. Exp. Ther., 2013, 344, 561–567.
46. W. C. Still, M. Kahn and A. Mitra, J. Org. Chem., 1978, 43, 2923–2925.
47. Organisation for Economic Co-operation and Development, Test No. 117: Partition Coefficient (n-octanol/water), HPLC Method, OECD Publishing, Paris, 2004.
48. X. Fei and Q. Zheng, J. Liq. Chromatogr. Relat. Technol., 2005, 28, 939–945.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra01212g

---