N-type calcium channel (Cav 2.2)

These compounds all displayed relatively high potency in the FDSS assay, with the unsubstituted tetrahydropyran 7 being the least potent (IC50 = 0.030 µM) and the tetramethyl-substituted analog 9/Cav 2.2 blocker 1 (compound 9) being the most potent (IC50 = 0.001 µM). The compounds were further evaluated in a high-resolution whole-cell automated patch clamp electrophysiology QPatch assay. This assay also allowed for the examination of the frequency dependence of inhibition, wherein high-frequency activation of the channel might better represent its functional state during certain chronic pain conditions and low-frequency activation more normal physiologic conditions. In this assay as well, addition of methyl groups increased the potency of the series, with Cav 2.2 blocker 1 (compound 9) having an IC50 value of 0.016 µM at the higher frequency, and 0.024 µM at the lower frequency. The trans-dimethyl compound 11 was also relatively potent in blocking the channel in this assay, demonstrating appreciable separation between the two activation states of the channel (i.e., approximately two-fold greater inhibition at high vs. low frequency), although its RLM stability was insufficient for in vivo testing in rat pain models. Having identified the tetramethylpyran Cav 2.2 blocker 1 (compound 9) as an optimal substituent for the 3-position of the pyrazole scaffold, the SAR of the 1- and 5-positions of the pyrazole scaffold was investigated. These compounds were prepared as shown in Scheme 2. The 4-ketopyran 16 was treated with TosMIC to give the nitrile that was then hydrolyzed to the carboxylic acid, followed by conversion to the benzotriazole amide 17. The benzotriazole was reacted with acylthiophenol under soft-enolization conditions to give the β-ketothioester 18. The R2 group was then introduced regioselectively via a condensation with R2-substituted hydrazines to give the 5-ketopyrazole 19. The ketopyrazole was converted to the triflate 20 and the R3-group was introduced via a Suzuki-Miyaura reaction. The FDSS and QPatch data for the 1,5-substituted pyrazoles are shown in Table 2. Regarding the R2 group, it was observed that the ortho-methoxy was important for maintaining potency in the QPatch assay. The -NEt (28) was equally potent in the FDSS assay, but was 6-fold and 25-fold less potent than -OMe (Cav 2.2 blocker 1 (compound 9)) in the QPatch assay at high and low frequency, respectively. For the R3 group, it was found that the 4-chlorophenyl could be replaced with the 2-chlorothiophene (22), 4-ethoxyphenyl (23), 4-cyanophenyl (24) or 2-ethoxypyridinyl (25). Compounds 22 and 25 showed, respectively, an approximately 2- and 4.5-fold difference in potency between high- and low-frequency activation states. Compound 25 was one of the few compounds wherein a heterocycle could be incorporated to help lower the logP of the series. Other attempts to incorporate polar functionality led to loss of potency. Cav 2.2 blocker 1 (compound 9) and 22 were selected for pharmacokinetic profiling in rats, based on in vitro potency and metabolic stability (Table 3). The two compounds exhibited long half-lives of 15.4 hr and 10.2 hr and oral bioavailabilities of 36% and 49%, respectively. The volume of distribution of both compounds was relatively high, presumably due to the lipophilic nature of the compounds, suggesting high tissue distribution. The selectivity of Cav 2.2 blocker 1 (compound 9) and 22 over the L-type calcium channel and the hERG potassium channel are shown in Table 4. Compound 9 did not inhibit the L-type at 1 uM and compound 22 showed modest inhibition of 12% at 1 uM. Both compounds had a relatively weak hERG inhibition profile. An ex vivo assay measuring the release of the neurotransmitter calcitonin gene-related peptide (CGRP) from rat spinal cord slices was used to determine the activity of Cav 2.2 blocker 1 (compound 9) and 22 in a physiologically relevant setting.In this assay, ω-conotoxin-GVIA (1 µM) was used as a positive control. At a concentration of 1 µM, compound 9 and compound 22 exhibited, respectively, 100% and 81% block of CGRP release compared with the same concentration of ω-conotoxin-GVIA.

Cav 2.2 blocker 1 (compound 9) and 22 were evaluated in the rat complete Freunds adjuvant (CFA) model of inflammatory pain.14 This test predicts the analgesic effect of numerous efficacious clinical agents, including the N-type calcium channel blocker Prialt®.15 Compounds 9 and 22 showed reversal of thermal hyperalgesia at 30 mg/kg (Fig. 2). Maximum reversal was observed 30 min post-dose with both compounds. After 180 min, latencies were similar to those in vehicle-treated animals. In separate studies, tissue concentrations of the compounds were determined. Compound 22 exhibited Cplasma = 1.1 µM, Cbrain = 2.2 µM and Cspinal cord = 1.9 µM at 1 hr following a 30 mg/kg p.o. dose; and compound 9 exhibited Cplasma = 0.7 µM, and Cbrain = 1.2 µM at 2 hr following a 30 mg/kg p.o. dose. [1]

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Cav 2.2 blocker 1 (compound 9) was also evaluated in the rat chronic constriction injury (CCI) model of neuropathic pain16 (Fig. 3). At 1–3 h following a dose of 30 mg/kg p.o. of compound 9, there was a 51% inhibition of cold allodynia, as assessed by nocifensive responses to acetone application to the affected hindpaw [1].

Pharmacokinetic studies:
Cav 2.2 blocker 1 (compound 9) was administered at 10.0/2.0 mg/kg po/iv; compound 22 was administered 6.0/1.2 mg/kg po/iv. Compounds were formulated in 20% hydroxypropyl-β-cyclodextran.

Cav 2.2 blocker 1 (compound 9) and 22 were selected for pharmacokinetic profiling in rats, based on in vitro potency and metabolic stability (Table 3). The two compounds exhibited long half-lives of 15.4 hr and 10.2 hr and oral bioavailabilities of 36% and 49%, respectively. The volume of distribution of both compounds was relatively high, presumably due to the lipophilic nature of the compounds, suggesting high tissue distribution. [1]

[1]. Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain. Bioorg Med Chem Lett. 2018 Dec 15;28(23-24):3780-3783.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain. [1]
In summary, we have identified a novel and potent series of pyrazolyltetrahydropyran N-type calcium channel blockers. Addition of methyl groups to the tetrahydropyran ring stabilized the series to HLM and RLM, leading to pharmacologically relevant exposures in plasma and spinal cord. In particular, compound 22 exhibited favorable in vitro and in vivo profiles. Further investigations will be aimed at identifying orally available N-type calcium channel blockers that may be useful in treating severe chronic pain. [1]

C25H29CLN2O2

424.962965726852

424.191

1567335-29-8

73292924

Off-white to light yellow solid powder

5.8

0

3

4

30

560

0

ClC1C=CC(=CC=1)C1=CC(C2CC(C)(C)OC(C)(C)C2)=NN1C1C=CC=CC=1OC

LDURKOGJFOYENC-UHFFFAOYSA-N

InChI=1S/C25H29ClN2O2/c1-24(2)15-18(16-25(3,4)30-24)20-14-22(17-10-12-19(26)13-11-17)28(27-20)21-8-6-7-9-23(21)29-5/h6-14,18H,15-16H2,1-5H3

5-(4-chlorophenyl)-1-(2-methoxyphenyl)-3-(2,2,6,6-tetramethyloxan-4-yl)pyrazole

Cav 2.2 blocker 1; Cav 2.2 blocker 1; 1567335-29-8; 5-(4-chlorophenyl)-1-(2-methoxyphenyl)-3-(2,2,6,6-tetramethyloxan-4-yl)pyrazole; CHEMBL4279774; SCHEMBL15568211; LDURKOGJFOYENC-UHFFFAOYSA-N; EX-A3692; BDBM50465792; compound 9

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~20.83 mg/mL (~49.02 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.89 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.89 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)