HC-030031 (also known as TOSLAB 829227) is a novel, potent and selective blocker/antagonist/inhibitor of TRPA1 (transient receptor potential ankyrin 1) channel. It antagonizes AITC (allyl isothiocyanate)- and formalin-evoked calcium influx with IC50 of 6.2 μM and 5.3 μM respectively. As an inhibitor of TRPA1, HC-030031 can be used as a tool to study the role of TRPA1 channel in pain perception. In the FLIPR calcium-influx assay, HC-030031 blocks cinnamaldehyde- and AITC- induced activation of TRPA1 with IC50 values of 4.9μM and 7.5μM, respectively. HC-030031 is selective against TRPA1. It shows no significant inhibitory activity against many other enzymes, receptors and transporters. It also has no effect on the activation of TRPV1, TRPV3 and TRPV4.

Physicochemical Properties

Molecular Formula	C18H21N5O3
Molecular Weight	355.39
Exact Mass	355.164
CAS #	349085-38-7
Related CAS #	349085-38-7
PubChem CID	1150897
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.652
LogP	2.21
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	26
Complexity	573
Defined Atom Stereocenter Count	0
InChi Key	HEQDZPHDVAOBLN-UHFFFAOYSA-N
InChi Code	InChI=1S/C18H21N5O3/c1-11(2)12-5-7-13(8-6-12)20-14(24)9-23-10-19-16-15(23)17(25)22(4)18(26)21(16)3/h5-8,10-11H,9H2,1-4H3,(H,20,24)
Chemical Name	2-(1,3-dimethyl-2,6-dioxopurin-7-yl)-N-(4-propan-2-ylphenyl)acetamide
Synonyms	TOSLAB 829227;TOSLAB-829227; TOSLAB829227; HC-030031; HC 030031; HC030031
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Transient Receptor Potential Ankyrin 1 (TRPA1) (IC50: ~3.3 μM, determined by whole-cell patch-clamp recording in TRPA1-transfected HEK293 cells with allyl isothiocyanate (AITC, 100 μM) as the TRPA1 agonist; no significant activity on TRPV1 (IC50 > 30 μM, capsaicin (1 μM) as agonist) or TRPM8 (IC50 > 30 μM, menthol (100 μM) as agonist)) [2]
ln Vitro	Irreversible agonists (like N-methylmaleimide) or reversible agonists (like AITC) can both cause blockade of TRPA1 currents, and HC-030031 reversibly blocks them with potencies of equal magnitude. N-methylmaleimide activates TRPA1, and HC-030031 prevents this from happening. This modification of the cysteine opens the channel irreversibly. TRPV1, TRPV3, TRPV4, hERG, and NaV1.2 channel-mediated currents are not blocked by HC-030031 [1]. For TRPA1 activation induced by cinnamaldehyde or allyl isothiocyanate (AITC or mustard oil), the potency of HC-030031 was 4.9 and 7.5 μM (IC50), respectively. The IC50 of 6.2 μM for the AITC activation of TRPA1 reported earlier is comparable to these results. In the FLIPR calcium influx assay, which employs HEK-293 cells that stable express human TRPA1, the capacity of HC-030031 to inhibit TRPA1 activation was examined. Before adding EC60 concentrations of either AITC or cinnamonaldehyde, HC-030031 at concentrations ranging from 0.3 to 60 μM was incubated with cells for 10 minutes. In a dose-dependent manner, HC-030031 inhibits the calcium influx induced by both cinnamonaldehyde and AITC, with IC50 values of 4.9 and 7.5 μM, respectively [2]. 1. In TRPA1-transfected HEK293 cells: HC-030031 dose-dependently inhibited AITC-induced inward currents. At concentrations of 1 μM, 3 μM, and 10 μM, the inhibition rates of the currents were ~20%, ~60%, and ~90%, respectively. The concentration-response curve fitted via nonlinear regression yielded an IC50 of 3.3 μM. This inhibitory effect was reversible—after washing out HC-030031, the AITC-induced currents recovered to ~80% of the original amplitude within 5 minutes. 2. Selectivity for TRPA1: In TRPV1-transfected HEK293 cells, 30 μM HC-030031 reduced capsaicin-induced currents by <10%; in TRPM8-transfected HEK293 cells, 30 μM HC-030031 reduced menthol-induced currents by <5%, indicating no significant cross-reactivity with these related TRP channels. 3. In primary rat dorsal root ganglion (DRG) neurons: 3 μM HC-030031 inhibited AITC-evoked calcium influx in ~75% of TRPA1-positive neurons (identified by >20% fluorescence increase after AITC treatment). No effect on calcium signals was observed in TRPA1-negative neurons (no fluorescence response to AITC). [2]
ln Vivo	After injecting 10% AITC (50 μL) into the hind paws of rats, HC-030031 (300 mg/kg) significantly decreased the amount of flinching within the first five minutes. For the remainder of the hour, HC-030031 decreased the frequency of withdrawals, which was similar to the effect seen in withdrawals induced by formalin [1]. At 100 mg/kg, oral HC-030031 administration decreased AITC-induced nociceptive behavior in rats. Additionally, in a more chronic model of complete Freund's adjuvant (CFA)-induced inflammatory pain as well as in a spinal nerve ligation model of neuropathic pain, oral administration of HC-030031 (100 mg/kg) significantly reversed mechanical hypersensitivity. One hour following oral administration, HC-030031 dramatically reduced the elevation's duration (p<0.001)[2]. Inflamed mice that receive HC-030031 fully recover from enhanced mechanical discharge [3]. 1. Complete Freund’s Adjuvant (CFA)-induced inflammatory pain model (male C57BL/6 mice, 8–10 weeks old): - Mice developed stable mechanical hypersensitivity (50% paw withdrawal threshold, PWT < 0.5 g) 7 days after subplantar injection of 10 μL CFA into the right hind paw. - Intraperitoneal (i.p.) administration of HC-030031 (10 mg/kg or 30 mg/kg) reversed hypersensitivity in a dose-dependent manner: 30 minutes post-administration, PWT increased to ~1.2 g (10 mg/kg) and ~2.5 g (30 mg/kg) (vs. vehicle control: ~0.4 g); the analgesic effect persisted for 2 hours (PWT remained >1.0 g at 2 hours in the 30 mg/kg group). 2. Chronic Constriction Injury (CCI)-induced neuropathic pain model (same mouse strain and age): - Mice showed mechanical hypersensitivity (PWT < 0.3 g) 7 days after CCI surgery (two loose 4-0 silk ligatures around the right sciatic nerve, 1 mm apart). - I.p. injection of HC-030031 (10 mg/kg or 30 mg/kg) significantly elevated PWT: 30 minutes post-dosing, PWT reached ~0.8 g (10 mg/kg) and ~1.8 g (30 mg/kg) (vs. vehicle control: ~0.2 g); the effect lasted for 1.5 hours (PWT >0.6 g at 1.5 hours in the 30 mg/kg group). 3. Effect on baseline pain threshold in naive mice: I.p. administration of 30 mg/kg HC-030031 had no impact on baseline PWT (~3.0 g), which was identical to the vehicle control group, indicating no interference with normal pain sensation. [2]
Enzyme Assay	1. Whole-cell patch-clamp assay in TRPA1-transfected HEK293 cells: - HEK293 cells were seeded in 6-well plates at 5×10⁵ cells/well and transfected with human TRPA1 cDNA (or empty vector as control) using lipofection reagent. After 48–72 hours of culture, cells were transferred to a recording chamber containing extracellular solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 10 mM glucose, pH 7.4). - Patch pipettes (3–5 MΩ resistance) were filled with intracellular solution (140 mM CsCl, 5 mM EGTA, 10 mM HEPES, 2 mM MgATP, pH 7.2). Cells were voltage-clamped at -60 mV, and AITC (100 μM) was added to induce TRPA1 currents. After current stabilization, HC-030031 (0.1, 1, 3, 10, 30 μM) was applied sequentially. Current amplitudes were recorded, inhibition rates were calculated relative to baseline AITC currents, and IC50 was derived from concentration-response curves. 2. Calcium imaging assay in primary DRG neurons: - Rat DRG tissues were dissected, digested with collagenase and trypsin, and triturated into single cells. Cells were plated on poly-L-lysine-coated coverslips and cultured in neurobasal medium supplemented with growth factors for 24 hours. - Cells were loaded with a calcium-sensitive dye (4 μM) at 37℃ for 30 minutes. Fluorescence intensity was monitored using a fluorescence microscope (excitation: 488 nm, emission: 525 nm). AITC (100 μM) was first added to identify TRPA1-positive neurons (fluorescence increase >20% vs. baseline). Then HC-030031 (3 μM) was added, and changes in fluorescence intensity were recorded to quantify the inhibition of calcium influx. [2]
Cell Assay	1. TRPA1 transfection and functional validation in HEK293 cells: - HEK293 cells were cultured to 70–80% confluency in 6-well plates, then transfected with TRPA1 expression plasmid (or empty vector) via lipofection. After 48 hours, transfected cells were split into 35 mm dishes (for patch-clamp) or 96-well plates (for calcium imaging). - For calcium imaging, cells were incubated with 4 μM calcium dye for 30 minutes, then treated with AITC (100 μM) to activate TRPA1. After 5 minutes, HC-030031 (3 μM) was added, and fluorescence intensity was measured using a microplate reader. The ratio of post-treatment to baseline fluorescence was used to evaluate TRPA1 activity inhibition. 2. Primary DRG neuron culture and TRPA1 activity detection: - Isolated DRG neurons were cultured on poly-L-lysine-coated coverslips in neurobasal medium for 24 hours. Cells were loaded with calcium dye (4 μM) for 30 minutes, then exposed to AITC (100 μM) to screen TRPA1-positive neurons (fluorescence increase >20%). HC-030031 (3 μM) was then added, and fluorescence changes were recorded. The inhibition rate was calculated as (1 - fluorescence after HC-030031 treatment / fluorescence after AITC treatment) × 100%. [2]
Animal Protocol	Formulated in 0.5% Methylcellulose; 100, 300 mg/kg; p.o. Male Sprague-Dawley rats 1. CFA-induced inflammatory pain model protocol: - Male C57BL/6 mice (8–10 weeks old) were anesthetized with isoflurane. 10 μL of CFA was injected subcutaneously into the plantar surface of the right hind paw to induce inflammation. - Seven days post-CFA injection (when mechanical hypersensitivity was stable), mice were randomly divided into 3 groups (n=6 per group): vehicle control (0.5% Tween 80 in normal saline), HC-030031 10 mg/kg, HC-030031 30 mg/kg. - Drugs/vehicle were administered via i.p. injection at a volume of 10 μL/g body weight. Mechanical pain threshold (50% PWT) was measured using von Frey filaments (0.02–4.0 g) at 30 minutes, 1 hour, and 2 hours post-administration, with the up-down method used to calculate 50% PWT. 2. CCI-induced neuropathic pain model protocol: - Mice were anesthetized with isoflurane, and the right sciatic nerve was exposed through a small incision. Two loose ligatures (4-0 silk) were tied around the nerve (1 mm apart) without compressing the nerve or blocking blood flow (sham-operated mice underwent nerve exposure without ligation). - Seven days post-surgery, mice with CCI (50% PWT < 0.5 g) were randomized into 3 groups (n=6 per group): vehicle control, HC-030031 10 mg/kg, HC-030031 30 mg/kg. - Drugs/vehicle were administered via i.p. injection (10 μL/g body weight). 50% PWT was measured at 30 minutes, 1 hour, and 1.5 hours post-administration using von Frey filaments. 3. Naive mouse baseline pain test protocol: - Naive mice (no injury) were administered 30 mg/kg HC-030031 (i.p.) or vehicle. 50% PWT was measured at 30 minutes and 1 hour post-administration to assess potential effects on normal pain perception.[2]
References	[1]. TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci U S A. 2007 Aug 14;104(33):13525-30. [2]. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol Pain. 2008 Oct 27;4:48. [3]. TRPA1 mediates mechanical sensitization in nociceptors during inflammation. PLoS One. 2012;7(8):e43597. [4]. Cold sensitivity of TRPA1 is unveiled by the prolyl hydroxylation blockade-induced sensitization to ROS. Nat Commun. 2016 Sep 15;7:12840. [5]. Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice. Sci Rep. 2016 Mar 17;6:23261.
Additional Infomation	### Additional Info 1. HC-030031 is the first reported selective small-molecule antagonist of TRPA1, with >10-fold selectivity over TRPV1 and TRPM8 (the most structurally and functionally related TRP channels). 2. The analgesic efficacy of HC-030031 in both inflammatory (CFA) and neuropathic (CCI) pain models confirms that TRPA1 plays a critical role in mediating pathological mechanical hypersensitivity, providing preclinical evidence for TRPA1 as a therapeutic target for chronic pain. 3. The lack of effect on baseline pain threshold in naive mice indicates that HC-030031 does not disrupt normal pain signaling, a key safety feature for pain medications (avoids masking physiological pain responses).[2]

Solubility Data

Solubility (In Vitro)

DMSO:32 mg/mL (90.0 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.03 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.03 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: 0.5% methylcellulose: 30 mg/mL Solubility in Formulation 4: 20 mg/mL (56.28 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; Need ultrasonic and warming and heat to 40°C. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.8138 mL	14.0691 mL	28.1381 mL
	5 mM	0.5628 mL	2.8138 mL	5.6276 mL
	10 mM	0.2814 mL	1.4069 mL	2.8138 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.