Design of Aminobenzothiazole Inhibitors of Rho Kinases 1 and 2 by Using Protein Kinase†A as a Structure Surrogate.

We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.

Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel and Selective Transient Receptor Potential Vanilloid 3 Antagonists.

Transient receptor potential vanilloid 3 (TRPV3) is a Ca(2+)- and Na(+)-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.

Discovery of (R)-1-(7-chloro-2,2-bis(fluoromethyl)chroman-4-yl)-3-(3-methylisoquinolin-5-yl)urea (A-1165442): a temperature-neutral transient receptor potential vanilloid-1 (TRPV1) antagonist with analgesic efficacy.

The synthesis and characterization of a series of selective, orally bioavailable 1-(chroman-4-yl)urea TRPV1 antagonists is described. Whereas first-generation antagonists that inhibit all modes of TRPV1 activation can elicit hyperthermia, the compounds disclosed herein do not elevate core body temperature in preclinical models and only partially block acid activation of TRPV1. Advancing the SAR of this series led to the eventual identification of (R)-1-(7-chloro-2,2-bis(fluoromethyl)chroman-4-yl)-3-(3-methylisoquinolin-5-yl)urea (A-1165442, 52), an analogue that possesses excellent pharmacological selectivity, has a favorable pharmacokinetic profile, and demonstrates good efficacy against osteoarthritis pain in rodents.

Quantification of TRPV1 protein levels in rat tissues to understand its physiological roles.

Transient receptor potential subfamily V, member 1 (TRPV1) is a nonselective cation channel expressed in both the peripheral and central nervous systems (CNS). TRPV1 protein levels in rat tissues were determined under normal and pain states using enzyme-linked immunosorbent assay. In naive rats, brain TRPV1 protein concentrations ranged from 1.5 to 4 ng/mg in hippocampus, cortex, hypothalamus, and cerebellum. Rat spinal cord TRPV1 protein levels were 40-50 ng/mg in L1-L5 of the lumbar regions, but increased to 97 ± 9.3 ng/mg toward the end of the lumbar region (L6-S1). In the complete Freund's adjuvant (CFA)-induced inflammatory pain model, TRPV1 protein level significantly increased on both the contralateral (36.5 %, p < 0.05) and ipsilateral (31.4 %, p < 0.05) L4-L6 dorsal root ganglia (DRG). TRPV1 protein levels also increased 33.3 % (p < 0.05) on the ipsilateral sciatic nerve, but no significant change in the lumbar spinal cord of CFA rats. In the monoiodoacetate-induced rat knee joint pain model, TRPV1 protein level was significantly reduced in the ipsilateral L3-L5 DRG (33.3 %, p < 0.01), no significant difference was detected in the lumbar region of the spinal cord. Quantitative determination of TRPV1 protein levels may help to elucidate the TRPV1 physiological roles and regulatory mechanisms in various pain states.

An oral TRPV1 antagonist attenuates laser radiant-heat-evoked potentials and pain ratings from UV(B)-inflamed and normal skin.

AIMS: Laser (radiant-heat) evoked potentials (LEPs) from vertex-EEG peak-to-peak (PtP) amplitude were used to determine acute antinociceptive/antihyperalgesic efficacy of ABT-102, a novel TRPV1 antagonist efficacious in preclinical pain models, compared with active controls and placebo in normal and UV(B)-inflamed skin. METHODS: This was a randomized, placebo- and active-controlled, double-blind, intra-individual, crossover trial. Twenty-four healthy subjects received six sequences of single doses of ABT-102 (0.5, 2, 6 mg), etoricoxib 90 mg, tramadol 100 mg and placebo. Painful stimuli were induced by CO(2) -laser on normal and UV(B) -inflamed skin. LEPs and visual analogue scale (VAS-pain) ratings were taken at baseline and hourly up to 8 h post-dose from both skin types. RESULTS: Compared with placebo, significant mean decreases in the primary variable of LEP PtP-amplitude from UV(B)-inflamed skin were observed with ABT-102 6 mg (P < 0.001), ABT-102 2 mg (P = 0.002), tramadol 100 mg (P < 0.001), and etoricoxib 90 mg (P = 0.001) over the 8 h period; ABT-102 0.5 mg was similar to placebo. ABT-102 6 mg was superior to active controls over the 8 h period (P < 0.05) whereas ABT-102 2 mg was comparable. Improvements in VAS scores compared with placebo were observed with ABT-102 6 mg (P < 0.001) and ABT-102 2 mg (P = 0.002). ABT-102 average plasma concentrations were 1.3, 4.4 and 9.4 ng ml(-1) for the 0.5, 2 and 6 mg doses, respectively. There were no clinically significant safety findings. CONCLUSIONS: TRPV-1 antagonism appears promising in the management of clinical pain, but requires further investigation.

Pharmacology of modality-specific transient receptor potential vanilloid-1 antagonists that do not alter body temperature.

The transient receptor potential vanilloid-1 (TRPV1) channel is involved in the development and maintenance of pain and participates in the regulation of temperature. The channel is activated by diverse agents, including capsaicin, noxious heat (≥ 43°C), acidic pH (< 6), and endogenous lipids including N-arachidonoyl dopamine (NADA). Antagonists that block all modes of TRPV1 activation elicit hyperthermia. To identify efficacious TRPV1 antagonists that do not affect temperature antagonists representing multiple TRPV1 pharmacophores were evaluated at recombinant rat and human TRPV1 channels with Ca(2+) flux assays, and two classes of antagonists were identified based on their differential ability to inhibit acid activation. Although both classes of antagonists completely blocked capsaicin- and NADA-induced activation of TRPV1, select compounds only partially inhibited activation of the channel by protons. Electrophysiology and calcitonin gene-related peptide release studies confirmed the differential pharmacology of these antagonists at native TRPV1 channels in the rat. Comparison of the in vitro pharmacological properties of these TRPV1 antagonists with their in vivo effects on core body temperature confirms and expands earlier observations that acid-sparing TRPV1 antagonists do not significantly increase core body temperature. Although both classes of compounds elicit equivalent analgesia in a rat model of knee joint pain, the acid-sparing antagonist tested is not effective in a mouse model of bone cancer pain.

Development of ELISA to measure TRPV1 protein in rat tissues.

The transient receptor potential vanilloid receptor type 1 (TRPV1) is a non-selective cation channel expressed in both the peripheral and the central nervous systems. To quantitatively determine TRPV1 protein levels in native rat tissues, novel monoclonal antibodies were raised against full-length recombinant human TRPV1 protein and utilized to develop a sandwich ELISA assay. Monoclonal antibody 10E3-1A2 specifically recognized TRPV1 protein and the recognition epitope was determined to reside in amino acids 45-58 of human and rat TRPV1. Using the TRPV1 polyclonal antibody ABRK4 as the capturing antibody and the monoclonal antibody 10E3-1A2 as the detection antibody, a sandwich ELISA that detected both human and rat TRPV1 protein was established. Recombinant human TRPV1 heterologously expressed in mammalian HEK293-F cells, which showed high ligand-binding affinity, was purified by TRPV1 monoclonal antibody affinity chromatography and used as protein standard to quantify TRPV1 protein levels. This ELISA detected TRPV1 protein as low as 1.5ng/ml (15pM), and was able to determine TRPV1 protein levels in native rat tissues such as DRG and spinal cord. This is the first TRPV1 sandwich ELISA that determines the abundance of TRPV1 protein in different tissues. It provides a powerful tool to quantify changes of TRPV1 protein levels in pathological states.

Synthesis and in vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7) antagonists.

Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X(7) antagonists. These compounds were assayed for activity at both the human and rat P2X(7) receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X(7) receptors. Compounds 12 and 38 displayed hP2X(7)pIC(50)s>7.8 with less than 2-fold difference in potency at the rP2X(7).

Oral and cutaneous thermosensory profile of selective TRPV1 inhibition by ABT-102 in a randomized healthy volunteer trial.

The capsaicin receptor (TRPV1) antagonist ABT-102 demonstrates efficacy in multiple preclinical pain models. However, evolving clinical data for this compound class suggest potentially profound drug-induced thermosensory impairment. Safety and tolerability of ABT-102 were assessed in a multiple-dose, double-blind, placebo-controlled, randomized healthy volunteer trial. Thirty-six participants were randomized in a 2:1 ratio to ABT-102:placebo in 3 dose groups (1 mg, 2 mg, and 4 mg twice a day) and confined to an inpatient research unit for a 7-day treatment period and 3 follow-up days. Outcome measures included: oral and cutaneous cold detection, warm detection (WDT), and heat pain thresholds (HPT); oral perceived heat intensity (oral liquid test); time to hand withdrawal (water bath test); and cutaneous pain intensity (long thermal stimulus). Significant dose-dependent (placebo- and baseline-adjusted) increases in HPT and reduced painfulness of suprathreshold heat were present from days 1-7. For ABT-102 4 mg twice a day, model-based mean differences from placebo (95% confidence interval) were as follows: oral HPT, day 1=2.5°C (0.6-4.4), day 5=4.4°C (2.5-6.3); cutaneous HPT, day 2=3.3°C (1.4-5.3), day 5=5.3°C (3.3-7.2); oral WDT, day 1=2.6°C (0.5-4.7), day 5=2.7°C (0.6-4.9); cutaneous WDT, day 2=1.3 (0.0-2.6), day 5=1.6 (0.3-2.8) (all P<0.05). Oral liquid test and water bath test results followed a similar pattern. There was no effect on cutaneous cold detection. All effects were fully reversed by day 10. There were no other relevant safety findings. Core body temperature remained below 39°C in all participants. In conclusion, ABT-102 potently and reversibly increased HPT and reduced painfulness of suprathreshold oral/cutaneous heat.

Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation.

Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A-967079 potently blocks human (IC(50): 51 nmol/L, electrophysiology, 67 nmol/L, Ca(2+) assay) and rat TRPA1 (IC(50): 101 nmol/L, electrophysiology, 289 nmol/L, Ca(2+) assay). It is >1000-fold selective over other TRP channels, and is >150-fold selective over 75 other ion channels, enzymes, and G-protein-coupled receptors. Oral dosing of A-967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate-induced nocifensive response and osteoarthritic pain in rats (ED(50): 23.2 mg/kg, p.o.). A-967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A-967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects.

Chroman and tetrahydroquinoline ureas as potent TRPV1 antagonists.

Novel chroman and tetrahydroquinoline ureas were synthesized and evaluated for their activity as TRPV1 antagonists. It was found that aryl substituents on the 7- or 8-position of both bicyclic scaffolds imparted the best in vitro potency at TRPV1. The most potent chroman ureas were assessed in chronic and acute pain models, and compounds with the ability to cross the blood-brain barrier were shown to be highly efficacious. The tetrahydroquinoline ureas were found to be potent CYP3A4 inhibitors, but replacement of bulky substituents at the nitrogen atom of the tetrahydroisoquinoline moiety with small groups such as methyl can minimize the inhibition.

Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Na(v)1.8 sodium channel with efficacy in a model of neuropathic pain.

Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.

Subtype-selective Na(v)1.8 sodium channel blockers: identification of potent, orally active nicotinamide derivatives.

A series of aryl-substituted nicotinamide derivatives with selective inhibitory activity against the Na(v)1.8 sodium channel is reported. Replacement of the furan nucleus and homologation of the anilide linker in subtype-selective blocker A-803467 (1) provided potent, selective derivatives with improved aqueous solubility and oral bioavailability. Representative compounds from this series displayed efficacy in rat models of inflammatory and neuropathic pain.

A-995662 [(R)-8-(4-methyl-5-(4-(trifluoromethyl)phenyl)oxazol-2-ylamino)-1,2,3,4-tetrahydronaphthalen-2-ol], a novel, selective TRPV1 receptor antagonist, reduces spinal release of glutamate and CGRP in a rat knee joint pain model.

The TRPV1 antagonist A-995662 demonstrates analgesic efficacy in monoiodoacetate-induced osteoarthritic (OA) pain in rat, and repeated dosing results in increased in vivo potency and a prolonged duration of action. To identify possible mechanism(s) underlying these observations, release of neuropeptides and the neurotransmitter glutamate from isolated spinal cord was measured. In OA rats, basal release of glutamate, bradykinin and calcitonin gene-related peptide (CGRP) was significantly elevated compared to naïve levels, whereas substance P (SP) levels were not changed. In vitro studies showed that capsaicin-evoked TRPV1-dependent CGRP release was 54.7+/-7.7% higher in OA, relative to levels measured for naïve rats, suggesting that TRPV1 activity was higher under OA conditions. The efficacy of A-995662 in OA corresponded with its ability to inhibit glutamate and CGRP release from the spinal cord. A single, fully efficacious dose of A-995662, 100 micromol/kg, reduced spinal glutamate and CGRP release, while a single sub-efficacious dose of A-995662 (25 micromol/kg) was ineffective. Multiple dosing with A-995662 increased the potency and duration of efficacy in OA rats. Changes in efficacy did not correlate with plasma concentrations of A-995662, but were accompanied with reductions in spinal glutamate release. These findings suggest that repeated dosing of TRPV1 antagonists enhances therapeutic potency and duration of action against OA pain, at least in part, by the sustained reduction in release of glutamate and CGRP from the spinal cord.

A-887826 is a structurally novel, potent and voltage-dependent Na(v)1.8 sodium channel blocker that attenuates neuropathic tactile allodynia in rats.

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na(v)1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na(v)1.8 channel blockers. A-887826 exemplifies this series and potently (IC(50)=11nM) blocked recombinant human Na(v)1.8 channels. A-887826 was approximately 3 fold less potent to block Na(v)1.2, approximately 10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na(+)) currents and was >30 fold less potent to block Na(V)1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na(+)) currents (IC(50)=8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund's adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by approximately 4mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na(v)1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na(v)1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na(v)1.8 sodium channels in pathological pain states.

Discovery of TRPV1 antagonist ABT-116.

The synthesis and SAR of a series of indazole TRPV1 antagonists leading to the discovery of 21 (ABT-116) is described. Biological studies demonstrated potent in vitro and in vivo activity for 21, as well as suitable physicochemical and pharmacokinetic properties for advancement to clinical development for pain management.

Discovery and biological evaluation of novel cyanoguanidine P2X(7) antagonists with analgesic activity in a rat model of neuropathic pain.

We disclose the design of a novel series of cyanoguanidines that are potent (IC(50) approximately 10-100 nM) and selective (> or = 100-fold) P2X(7) receptor antagonists against the other P2 receptor subtypes such as the P2Y(2), P2X(4), and P2X(3). We also found that these P2X(7) antagonists effectively reduced nociception in a rat model of neuropathic pain (Chung model). Particularly, analogue 53 proved to be effective in the Chung model, with an ED(50) of 38 micromol/kg after intraperitoneal administration. In addition compound 53 exhibited antiallodynic effects following oral administration and maintained its efficacy following repeated administration in the Chung model. These results suggest an important role of P2X(7) receptors in neuropathic pain and therefore a potential use of P2X(7) antagonists as novel therapeutic tools for the treatment of this type of pain.

Antagonism of TRPV1 receptors indirectly modulates activity of thermoregulatory neurons in the medial preoptic area of rats.

In order to enhance understanding of TRPV1 contributions to thermoregulation, we measured the effects of a TRPV1 receptor antagonist, A-889425, on thermoregulatory neurons in the medial preoptic area of the hypothalamus (mPOA) of rats while simultaneously monitoring rectal temperature (T(r)). Administration of A-889425 (4 micromol/kg, i.v.) significantly increased T(r) by 0.42+/-0.02 degrees C in anesthetized rats. Warm-sensitive (WS) neurons in the mPOA increase firing in response to body warming, and when active stimulate heat loss and inhibit heat production. WS neurons were initially inhibited by A-889425. Subsequently, WS neuronal activity diverged, differentiating WS neurons into two subgroups. One group of WS neurons continued to be inhibited during the recording period while another group of "biphasic" WS neurons increased firing as T(r) increased. Cold-sensitive (CS) neurons fire at a higher rate during cooling of the body, and when active, may contribute to heat production. Injection of A-889425 affected CS neurons in a manner opposite to the biphasic WS neurons; activity was initially increased followed by a later decrease. Direct administration of A-889425 into the mPOA (10 and 30 nmol) or spinal cord (30 nmol) did not affect T(r). Disruption of abdominal TRPV1 receptor function by injection of the TRPV1 receptor agonist, resiniferatoxin (20 microg/kg, i.p.), 9-15 days prior to experiments, blocked the effects of systemically injected A-889425 on T(r) and mPOA neuronal activity. These data demonstrate that antagonist block of abdominal TRPV1 receptors indirectly modulates activity of thermoregulatory neurons in the mPOA in a manner that is consistent with producing an acute rise in body temperature.

Repeated dosing of ABT-102, a potent and selective TRPV1 antagonist, enhances TRPV1-mediated analgesic activity in rodents, but attenuates antagonist-induced hyperthermia.

Transient receptor potential vanilloid type 1 (TRPV1) is a ligand-gated ion channel that functions as an integrator of multiple pain stimuli including heat, acid, capsaicin and a variety of putative endogenous lipid ligands. TRPV1 antagonists have been shown to decrease inflammatory pain in animal models and to produce limited hyperthermia at analgesic doses. Here, we report that ABT-102, which is a potent and selective TRPV1 antagonist, is effective in blocking nociception in rodent models of inflammatory, post-operative, osteoarthritic, and bone cancer pain. ABT-102 decreased both spontaneous pain behaviors and those evoked by thermal and mechanical stimuli in these models. Moreover, we have found that repeated administration of ABT-102 for 5-12 days increased its analgesic activity in models of post-operative, osteoarthritic, and bone cancer pain without an associated accumulation of ABT-102 concentration in plasma or brain. Similar effects were also observed with a structurally distinct TRPV1 antagonist, A-993610. Although a single dose of ABT-102 produced a self-limiting increase in core body temperature that remained in the normal range, the hyperthermic effects of ABT-102 effectively tolerated following twice-daily dosing for 2 days. Therefore, the present data demonstrate that, following repeated administration, the analgesic activity of TRPV1 receptor antagonists is enhanced, while the associated hyperthermic effects are attenuated. The analgesic efficacy of ABT-102 supports its advancement into clinical studies.

Pore dilation occurs in TRPA1 but not in TRPM8 channels.

Abundantly expressed in pain-sensing neurons, TRPV1, TRPA1 and TRPM8 are major cellular sensors of thermal, chemical and mechanical stimuli. The function of these ion channels has been attributed to their selective permeation of small cations (e.g., Ca2+, Na+ and K+), and the ion selectivity has been assumed to be an invariant fingerprint to a given channel. However, for TRPV1, the notion of invariant ion selectivity has been revised recently. When activated, TRPV1 undergoes time and agonist-dependent pore dilation, allowing permeation of large organic cations such as Yo-Pro and NMDG+. The pore dilation is of physiological importance, and has been exploited to specifically silence TRPV1-positive sensory neurons. It is unknown whether TRPA1 and TRPM8 undergo pore dilation. Here we show that TRPA1 activation by reactive or non-reactive agonists induces Yo-Pro uptake, which can be blocked by TRPA1 antagonists. In outside-out patch recordings using NMDG+ as the sole external cation and Na+ as the internal cation, TRPA1 activation results in dynamic changes in permeability to NMDG+. In contrast, TRPM8 activation does not produce either Yo-Pro uptake or significant change in ion selectivity. Hence, pore dilation occurs in TRPA1, but not in TRPM8 channels.