3H,4H,5H,6H,7H-Pyrimido[4,5-B][1,4]oxazine-4,6-dione derivatives as TRPA1 inhibitors

Abstract

The present disclosure provides certain 3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione derivatives that are inhibitors of transient receptor potential ankyrin 1 (TRPA1) and are therefore useful in treating diseases treatable by inhibition of TRPA1, as well as pharmaceutical compositions containing same and methods for preparing said compounds. TIFF2024511480000052.tif36114

Description

[Technical field]

[0001] The present disclosure provides certain 3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione derivatives that are inhibitors of transient receptor potential ankyrin 1 (TRPA1) and are therefore useful in treating diseases treatable by inhibition of TRPA1, as well as pharmaceutical compositions containing same and methods for preparing said compounds. [Background technology]

[0002] Transient receptor potential channels (TRP channels) are a group of voltage-gated ion channels that are mostly located on the plasma membrane of many mammalian cell types. There are approximately 30 structurally related TRP channels, which are classified into the following groups: TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, and TRPV. Transient receptor potential cation channel subfamily A, member 1 (TRPA1), also known as transient receptor potential ankyrin 1, is the only member of the TRPA gene subfamily. Structurally, TRPA channels are characterized by multiple N-terminal ankyrin repeats (approximately 14 in the N-terminus of human TRPA1), which leads to the "A" in the ankyrin name (Montell, 2005).

[0003] TRPA1 is highly expressed in the plasma membrane of sensory neurons in the dorsal root and nodose ganglia that serve both the skin and lungs, as well as in the small intestine, colon, pancreas, skeletal muscle, heart, brain, bladder, and lymphocytes (https: / / www.proteinatlas.org / ), and in human lung fibroblasts.

[0004] TRPA1 is best known as a sensor for environmental stimuli that result in somatosensory modalities, such as pain, cold, and itch. TRPA1 is activated by a number of reactive electrophilic stimuli (e.g., allylisothiocyanate, reactive oxygen species) as well as non-reactive compounds (e.g., icilin) ​​and is involved in asthma-related cough, chronic pulmonary obstructive disease (COPD), idiopathic pulmonary fibrosis (IPF), or cough after viral infection, or in chronic idiopathic cough, as well as cough in susceptible patients (Song and Chang, 2015; Grace and Belvisi, 2011). Based on studies showing cough-induced elevation of TGF-β, TRPA1 inhibitors may be useful in the treatment of IPF, where coughing is highly prevalent due to the association between coughing and lung injury (Xie et al., 2009; Froese et al., 2016; Tschumperlin et al., 2003; Yamamoto et al., 2002; Ahamed et al., 2008). Acute lung injury as a result of SARS-Cov-2 infection is mediated at least in part by reactive oxygen species (ROS). ROS are direct activators of TRPA1. Furthermore, desensitization of TRPA1 via the consumption of spicy foods has been hypothesized to modulate the Nrf2 pathway and reduce oxidative stress (Bousquet et al., 2020; Bousquet et al., 2021). Thus, TRPA1 inhibitors have potential in the treatment of Covid-19 / SARS-Cov-2-induced lung injury. TRPA1 antagonists inhibit calcium signaling triggered by cough triggers, e.g., cigarette smoke extract (CSE), oxidative stress, inflammatory mediator release, and downregulated antioxidant gene expression (Lin et al., 2015; Wang et al., 2019). TRPA1 antagonists are effective in studies of atopic dermatitis (Oh et al., 2013; Wilson et al., 2013), contact dermatitis (Liu et al., 2013), psoriasis-associated itch (Wilson et al., 2013), and IL-31-dependent itch (Cevikbas et al., 2014). Human TRPA1 gain of function is associated with familial episodic pain syndromes (Kremeyer et al., 2010). TRPA1 antagonists were effective in a behavioral model of migraine-associated allodynia (Edelmayer et al., 2012).TRPA1 is selectively increased in trigeminal ganglia innervating injured teeth when compared with TRPA1 expression in trigeminal ganglia innervating healthy teeth (Haas et al., 2011). Several anesthetics, including isoflurane, are known to be TRPA1 agonists (Matta et al., 2008), providing a rationale for TRPA1 inhibitors to reduce pain after surgery. TRPA1 knockout mice and wild-type mice treated with TRPA1 antagonists exhibited anxiolytic- and antidepressant-like phenotypes (de Moura et al., 2014). TRPA1 inhibitors are expected to have benefits in the treatment of diabetic neuropathy based on studies showing a mechanistic link between AMPK and counterregulation of TRPA1 (Hiyama et al., 2018; Koivisto and Pertovaara, 2013; Wang et al., 2018). TRPA1 knockout mice exhibit smaller myocardial infarct size compared to wild-type mice (Conklin et al., 2019). TRPA1 knockout and pharmacological intervention inhibit TNBS-induced colitis in mice (Engel et al., 2011). TRPA1 knockout and TRPA1 antagonists reduce myelin damage in a mouse cerebral ischemia model (Hamilton et al., 2016). Uric acid crystals and arthritis are reduced in TRPA1 knockout mice in the monosodium urate mouse model of gout (Moilanen et al., 2015). TRPA1 deletion in rats reversed arthritis and hyperalgesia in a rat model of acute gout flare (Trevisan et al., 2014). TRPA1 activation causes an inflammatory response in osteoarthritic chondrocytes (Nummenmaa et al., 2016). TRPA1 inhibition and genetic deletion reduce inflammatory mediators in osteoarthritic mouse chondrocytes and murine cartilage (Nummenmaa et al., 2016). Finally, TRPA1 knockout mice showed improved weight bearing on osteoarthritic limbs in a MIA-induced knee swelling model (Horvath et al., 2016).TRPA1 is differentially expressed in the bladder epithelium of rats (Du et al., 2007) and patients with bladder outlet obstruction (Du et al., 2008).TRPA1 receptor modulation attenuates bladder overactivity in a rat model of spinal cord injury ( Andrade et al., 2011 ), and intrathecal administration of a TRPA1 antagonist attenuates cyclophosphamide-induced cystitis in rats with hyperreflexic voiding ( Chen et al., 2016 ). It would therefore be desirable to provide potent TRPA1 inhibitors.

[0005] TRPA1 inhibitors of various structural classes are reviewed in S. Skerratt, Progress in Medicinal Chemistry, 2017, Volume 56, 81-115, and D. Preti, G. Saponaro, A. Szallasi, Pharm. Pat. Anal. (2015) 4 (2), 75-94, and H. Chen, Transient receptor potential ankyrin 1 (TRPA1) antagonists: a patent review (2015-2019), Expert Opin Ther Pat., 2020.

[0006] WO2017 / 060488 discloses a compound having the general structural formula: [ka] Compounds having the formula:

[0007] Among them, the TRPA1 activity of Examples 53, 72, 73, 86, and 90 was measured by IC 50 It is disclosed that the concentration is less than 100 nM. In L. Schenkel, et al., J. Med. Chem. 2016, 59, 2794-2809, the general structure is

[0008] [ka] Among them, compound 31, in which R is OH, showed an IC 50 has been disclosed as having an antagonistic TRPA1 activity of 58 nM and an intrinsic clearance of less than 14 μL / min / kg in human liver microsomes. Summary of the Invention

[0009] Detailed Description of the Invention The present invention discloses novel 3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione derivatives that are inhibitors of transient receptor potential ankyrin 1 (TRPA1) and have suitable pharmacological and pharmacokinetic properties that enable their use as pharmaceuticals for treating conditions and / or diseases treatable by inhibition of TRPA1. The compounds of the present invention may offer several advantages, such as enhanced potency, high metabolic and / or chemical stability, high selectivity, safety and tolerability potential, enhanced solubility, enhanced permeability, favorable plasma protein binding, enhanced bioavailability, favorable pharmacokinetic profiles, and the potential to form stable salts.

[0010] Compounds of the Invention The present invention is surprisingly a potent inhibitor of TRPA1 (Assay A), - Improved stability in human liver microsomes (Assay B) - Improved stability in human hepatocytes (Assay C) The present invention provides a 3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione derivative, further characterized by: The compounds of the present invention are structurally distinct from Examples 53, 72, 73, 86, and 90 in WO2017 / 060488 and Example 31 in L. Schenkel, et al., J. Med. Chem. 2016, 59, 2794-2809 in that they contain a substituted 3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione core and a substituent adjacent to a secondary aliphatic alcohol. These structural differences unexpectedly result in a favorable combination of (i) inhibition of TRPA1, (ii) stability in human liver microsomes, and (iii) stability in human hepatocytes.

[0011] Stability in human liver microsomes refers to the susceptibility of a compound to biotransformation in the context of selecting and / or designing drugs with pharmacokinetic properties as a preferred first screening step. The main metabolic site for many drugs is the liver. Human liver microsomes contain cytochrome P450 (CYP) and therefore represent a model system for studying phase I drug metabolism in vitro. Enhanced stability in human liver microsomes is associated with several advantages, including high bioavailability and suitable half-life, which can allow for reduced dosage and frequency of administration to patients. Therefore, enhanced stability in human liver microsomes is a preferred feature for compounds to be used in drugs. Thus, in addition to being able to inhibit TRPA1, the compounds of the present invention are expected to have favorable in vivo clearance and therefore a desired duration of action in humans. Stability in human hepatocytes refers to the susceptibility of a compound to biotransformation in the context of selecting and / or designing drugs with favorable pharmacokinetic properties. The main metabolic site for many drugs is the liver. Human hepatocytes contain cytochrome P450 (CYP) and other drug metabolizing enzymes and therefore represent a model system for studying drug metabolism in vitro (importantly, in contrast to liver microsomal assays, hepatocyte assays also cover phase II biotransformation as well as liver-specific transporter-mediated processes and therefore represent a more complete system for drug metabolism studies). Enhanced stability in human hepatocytes is associated with several advantages, including high bioavailability and suitable half-life, which can allow for reduced dosage and dosing frequency to patients. Therefore, enhanced stability in human hepatocytes is a favorable feature for compounds to be used in drugs. The present invention provides novel compounds according to formula (I).

[0012] [ka] (I) (In the formula, A is selected from the group consisting of phenyl, thiophenyl, benzofuranyl, and benzothiophenyl, A is unsubstituted or selected from the group consisting of halogen and C 1-4 R consisting of alkyl 1 substituted with one or two members of the group

[0013] Another embodiment of the present invention relates to compounds of formula (I), wherein A is selected from the group consisting of phenyl, thiophenyl, benzofuranyl, and benzothiophenyl, and A is unsubstituted or R is selected from the group consisting of F, Cl, I, and CH3. 1 substituted with one or two members of the group Another embodiment of the present invention relates to compounds of formula (I), wherein A is selected from the group consisting of phenyl, benzofuranyl, and benzothiophenyl, and A is unsubstituted or R is selected from the group consisting of F, Cl, I, and CH3.1 substituted with one or two members of the group Another embodiment of the present invention relates to compounds of formula (I), wherein A is

[0014] [ka] is selected from the group consisting of: wherein A is unsubstituted or R 1 is substituted with one or two members of the R 1 is defined as in any of the preceding embodiments.

[0015] [ka] Preferred are compounds according to formula (I) selected from the group consisting of: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Terms and definitions used Terms not specifically defined herein should be given the meaning that would be given to them by one of ordinary skill in the art in light of the present disclosure and the context. However, as used herein, unless otherwise specified, the following terms have the meanings indicated and the following rules are to be observed:

[0017] In the groups, radicals, or moieties defined below, the number of carbon atoms is often specified before the group, e.g., C 1-6 Alkyl means an alkyl group or radical having 1 to 6 carbon atoms. Generally, in groups such as HO, H2N, (O)S, (O)2S, NC (cyano), HOOC, F3C, etc., one skilled in the art can recognize the point of radical attachment to the molecule from the free valence of the group itself. In the case of linking groups containing two or more subgroups, the last named subgroup is the point of radical attachment, e.g., "aryl-C 1-3 The alkyl substituent is C 1-3It refers to an aryl group bonded to an alkyl group, where the latter moiety is bonded to a core or group to which the substituents are bonded.

[0018] When compounds of the invention are depicted in the form of a chemical name and as a formula, the formula shall prevail in the event of a discrepancy. An asterisk may be used in a sub-formula to indicate a bond that is attached to a defined core molecule. Numbering of substituent atoms begins with the atom closest to the core or group to which the substituent is attached. For example, the term "3-carboxypropyl group" represents the following substituent:

[0019] [ka] (wherein the carboxy group is attached to the third carbon atom of the propyl group). The term "1-methylpropyl", "2,2-dimethylpropyl", or "cyclopropylmethyl" group represents the following group:

[0020] [ka] An asterisk may be used in a sub-formula to indicate a bond that is attached to a defined core molecule. "C 1-n The term "alkyl" (wherein n is an integer selected from 2, 3, 4, or 5), alone or in combination with other groups, denotes an acyclic, saturated, branched or linear hydrocarbon group containing 1 to n C atoms. For example, 1-5The term alkyl encompasses HC-, HC-CH-, HC-CH-CH-, HC-CH(CH)-, HC-CH-CH-CH-, HC-CH(CH)-, HC-CH-CH-CH-, HC-CH(CH)-, HC-CH(CH)-CH-, HC-C(CH)-, HC-CH-CH-CH-CH-, HC-CH-CH-CH-CH(CH)-, HC-CH-CH(CH)-CH-, HC-CH-CH(CH)-CH-, HC-CH-CH-CH-CH-, HC-CH-C(CH)-, HC-C(CH)-CH-, HC-CH(CH)-CH-CH-, HC-CH-CH(CH)-CH-, and HC-CH-CH(CHCH)- radicals. The term "fluoro" appended to an "alkyl," "alkylene," or "cycloalkyl" group (saturated or unsaturated) means an alkyl or cycloalkyl group in which one or more hydrogen atoms have been replaced with fluorine atoms. Examples include, but are not limited to, H2FC-, HF2C-, and F3C-. The term phenyl refers to the following ring radical: [ka] The term thiophenyl refers to the following ring radical:

[0021] [ka] The term benzofuranyl refers to the following ring radical:

[0022] [ka] The term benzothiophenyl refers to the following ring radical:

[0023] [ka] The term 3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione refers to the following core radical:

[0024] [ka] The term "substituted," as used herein, means that either one or more hydrogens of the specified atom are replaced with a group selected from the indicated groups, provided that the normal valence of the specified atom is not exceeded, and further, that the substitution results in a stable compound. Unless otherwise indicated, throughout this specification and the appended claims, a given chemical formula or name is intended to encompass tautomers and all stereo, optical, and geometric isomers (e.g., enantiomers, diastereomers, E / Z isomers, etc.), as well as racemates thereof, and, where isomers and enantiomers exist, mixtures of the individual enantiomers in different ratios, mixtures of diastereomers, or mixtures of any of the foregoing forms, as well as salts, including pharma- ceutically acceptable salts thereof, and solvates thereof, such as hydrates, including solvates of the free compounds, or solvates of salts of the compounds.

[0025] In general, substantially pure stereoisomers can be obtained according to synthetic principles known to those skilled in the art, for example, by separating the corresponding mixtures, by using stereochemically pure starting materials, and / or by stereoselective synthesis. Methods for preparing optically active forms, for example, by resolving racemic forms, or by synthesis, for example, starting from optically active starting materials, and / or by using chiral reagents, are known in the art. Enantiomerically pure compounds or intermediates of the invention may, for example, be prepared via asymmetric synthesis by preparing and then separating the appropriate diastereomeric compounds or intermediates, which may be separated by known methods (e.g., by chromatographic separation or crystallization), and / or by the use of chiral reagents, e.g., chiral starting materials, chiral catalysts, or chiral auxiliaries. Furthermore, methods are known to those skilled in the art for preparing enantiomerically pure compounds from the corresponding racemic mixtures, for example by chromatographic separation on a chiral stationary phase; or by resolution of the racemic mixture using a suitable resolving agent, for example by forming a diastereomeric salt of the racemate with an optically active acid or base and then resolving the salt and releasing the desired compound from the salt; or by derivatizing the corresponding racemate with an optically active chiral auxiliary reagent and then diastereomeric separation and removal of the chiral auxiliary; or by kinetic resolution of the racemate (for example by enzymatic resolution); by enantioselective crystallization from a consortium of enantiomorphous crystals under suitable conditions; or by (partial) crystallization from a suitable solvent in the presence of an optically active chiral auxiliary.

[0026] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, and are commensurate with a reasonable benefit / risk ratio. As used herein, "pharmaceutical acceptable salt" refers to derivatives of the disclosed compounds in which the parent compound forms a salt or complex with an acid or base. Examples of acids that form pharmaceutical acceptable salts with parent compounds containing a basic moiety include inorganic or organic acids, such as benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid, gentisic acid, hydrobromic acid, hydrochloric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, 4-methylbenzenesulfonic acid, phosphoric acid, salicylic acid, succinic acid, sulfuric acid, and tartaric acid.

[0027] Examples of cations and bases that form pharma- ceutically acceptable salts with the parent compound that contains an acidic moiety include Na + , K + , Ca 2+ , Mg 2+ , NH4 +, L-arginine, 2,2'-iminobisethanol, L-lysine, N-methyl-D-glucamine, or tris(hydroxymethyl)aminomethane. The pharma- ceutically acceptable salts of the present invention may be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. In general, such salts may be prepared by reacting the free acid or base form of these compounds with a sufficient amount of an appropriate base or acid in water or an organic diluent such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or a mixture thereof. For example, salts of acids other than those mentioned above that are useful for purifying or isolating the compounds of this invention (eg, trifluoroacetates) are also included as part of this invention.

[0028] Biological assays Assessment of TRPA1 activity Assay A: TRPA1 assay The activity of the compounds of the invention may be determined using the following in vitro TRPA1 cellular assay: method: Human HEK293 cell line (Perkin Elmer, product number AX-004-PCL) overexpressing human TRPA1 ion channel is used as a test system for compound efficacy and potency. Compound activity is determined by measuring the effect of compound on intracellular calcium concentration induced by AITC (Allylisothiocyanat) agonism in FLIPRtetra system (Molecular Devices).

[0029] Cell culture: Cells are obtained as frozen cells in cryovials and stored at -150°C until use. Cells are grown in culture medium (MEM / EBSS medium with 10% FCS and 0.4 mg / ML Geneticin). It is important that the density does not exceed 90% confluence. For subculture, cells are detached from flasks by Versene. The day before the assay, cells are detached, washed twice with medium (MEM / EBSS medium with 10% FCS) and seeded at 20 000 cells in 20 μL / well into Corning poly-D-lysine biocoated 384-well plates (black, clear bottom, catalog 356697). Plates are incubated at 37°C / 5% CO2 for 24 hours before use in the assay.

[0030] Preparation of compounds Test compounds are dissolved in 100% DMSO at a concentration of 10 mM, diluted in DMSO to a concentration of 5 mM in a first step, followed by serial dilution steps in 100% DMSO. The dilution factor and the number of dilution steps can be different as required. Typically, 8 different concentrations are prepared by dilution of 1:5, and further intermediate dilutions (1:20) of the substance are performed using HBSS / HEPES buffer (1×HEPES, catalog 14065, Gibco; 20 mM HEPES, catalog 83264, SIGMA; 0.1% BSA, catalog 11926, Invitrogen, pH 7.4).

[0031] FLIPR Assay: On the day of the assay, cells are washed three times with assay buffer (puffer), leaving 20 μL of buffer in the well after washing. 10 μL of Ca6 kit (cat. R8191 Molecular Devices) loading buffer in HBSS / HEPES is added to the cells, the plate is covered and incubated for 120 min at 37°C / 5% CO2. 10 μL of compound or control in HBSS / HEPES buffer / 5% DMSO from the intermediate dilution plate is carefully added to the well. Luminescence (indicating calcium influx or release) is read for 10 min on the FLIPRtetra device to monitor compound-induced effects (e.g. agonism). Finally, 10 μL of 50 μM agonist AITC dissolved in HBSS / HEPES buffer / 0.05% DMSO (final concentration 10 μM) is added to the well, followed by an additional reading for 10 min on the FLIPRtetra device. The area under the signal curve (AUC) after AITC addition is used to calculate IC50 / % inhibition.

[0032] Data evaluation and calculations: Each assay microtiter plate contains wells containing a vehicle (1% DMSO) control instead of compound as a control for AITC-induced luminescence (100% CTL; high control), and wells containing a vehicle control without AITC as a control for non-specific changes in luminescence (0% CTL; low control). The data is analyzed by calculating the area under the signal curve for each individual well. Based on this value, the % values ​​for the measurement of each substance concentration are calculated using the MegaLab software (in-house development) (AUC(sample)-AUC(low))*100 / (AUC(high)-AUC(low)). The IC50 values ​​are calculated from the % control values ​​using the MegaLab software. Calculation: [y=(ad) / (1+(x / c)^b)+d], a=low value, d=high value; x=concentration M; c=IC50M; b=Hill; y=Ctrl%

[0033] [Table 1]

[0034] [Table 2]

[0035] [Table 3]

[0036] Assessment of microsomal clearance Assay B: Microsomal Clearance: Metabolic degradation of test compounds is assayed using pooled liver microsomes at 37° C. A final incubation volume of 100 μl per time point contains room temperature Tris buffer pH 7.6 (0.1 M), magnesium chloride (5 mM), microsomal protein (1 mg / ml), and test compound at a final concentration of 1 μM. After a short preincubation period at 37°C, the reaction is started by adding reduced beta-nicotinamide adenine dinucleotide phosphate (NADPH, 1 mM) and terminated by transferring aliquots into the solvent after different time points (0, 5, 15, 30, 60 min). In addition, NADPH-independent degradation is monitored in incubations without NADPH and terminated at the last time point. [%] remaining test compound after NADPH-independent incubation is reflected by the parameter c (control) (metabolic stability). The quenched incubations are pelleted by centrifugation (10000g, 5 min).

[0037] Aliquots of the supernatant are assayed for the amount of parent compound by LC-MS / MS. Half-lives (t1 / 2 INVITRO) are determined by the slope of a semi-log plot of the concentration-time profile. The intrinsic clearance (CL_INTRINSIC) is calculated taking into account the amount of protein in the incubation: CL_INTRINSIC[μl / min / mg protein]=(Ln2 / (half-life [min]*protein content [mg / ml]))*1000 CL_INTRINSIC_INVIVO[ml / min / kg]=(CL_INTRINSIC[μL / min / mg protein]×MPPGL[mg protein / g liver]×liver factor[g / kg body weight]) / 1000 Qh[%]=CL[ml / min / kg] / hepatic blood flow [ml / min / kg]) Hepatocellular, human: 120 x 10e6 cells / g liver Liver factor, human: 25.7 g / kg body weight Blood flow rate, human: 21 ml / (min x kg)

[0038] [Table 4]

[0039] [Table 5]

[0040] [Table 6] Assessment of hepatocyte clearance Assay C: Hepatocyte Clearance The metabolic degradation of the test compounds is assayed in hepatocyte suspensions: Hepatocytes (cryopreserved) are incubated in Dulbecco's modified Eagle's medium containing 5% seed serum (supplemented with 3.5 μg glucagon / 500 mL, 2.5 mg insulin / 500 mL, and 3.75 mg / 500 mL hydrocortisone).

[0041] After 30 min preincubation in an incubator (37 °C, 10% CO), add 5 µl of test compound solution (80 µM; from 2 mM in DMSO stock solution diluted 1:25 with culture medium) into 395 µl of hepatocyte suspension (cell density ranging from 0.25 to 5 Mio cells / mL depending on species, typically 1 Mio cells / mL; final concentration of test compound is 1 µM, final DMSO concentration is 0.05%). Cells are incubated for 6 hours (incubator, orbital shaker) and samples (25 μl) are taken at 0, 0.5, 1, 2, 4 and 6 hours. Samples are transferred into acetonitrile and pelleted by centrifugation (5 min). Supernatants are transferred to a new 96 deep-well plate, evaporated under nitrogen and resuspended. The loss of parent compound is analyzed by HPLC-MS / MS.

[0042] CLint is calculated as follows: CL_INTRINSIC = dose / AUC = (C0 / CD) / (AUD+clast / k) x 1000 / 60. CO: initial concentration in incubation [μM], CD: cell density of differentially dependent cells [10e6 cells / mL], AUD: area under the data [μM x time], clast: concentration of last data point [μM], k: slope of regression line for parental reduction [h-1] The calculated in vitro hepatic intrinsic clearance may be scaled up to the intrinsic in vivo hepatic clearance and used to predict the hepatic in vivo blood clearance (CL) by using a liver model (well-stirred model). CL_INTRINSIC_INVIVO[ml / min / kg]=(CL_INTRINSIC[μL / min / 10e6 cells] x hepatocellularity [10e6 cells / g liver] x liver factor [g / kg body weight]) / 1000 CL[ml / min / kg]=CL_INTRINSIC_INVIVO[ml / min / kg]×hepatic blood flow [ml / min / kg] / (CL_INTRINSIC_INVIVO[ml / min / kg]+hepatic blood flow [ml / min / kg]) Qh[%]=CL[ml / min / kg] / hepatic blood flow [ml / min / kg]) Hepatocellular, human: 120 x 10e6 cells / g liver Liver factor, human: 25.7 g / kg body weight Blood flow rate, human: 21 ml / (min x kg)

[0043] [Table 7]

[0044] [Table 8]

[0045] [Table 9]

[0046] Permeability assessment Caco-2 cells (1~2×10 5 cells / 1cm 2 Cells (100 μM area) are seeded onto filter inserts (Costar transwell polycarbonate or PET filters, 0.4 pm pore size) and cultured for 10-25 days (DMEM). Compounds are dissolved in an appropriate solvent (e.g., DMSO, 1-20 mM stock solution). Stock solutions are diluted with HTP-4 buffer (128.13 mM NaCl, 5.36 mM KCl, 1 mM MgSO4, 1.8 mM CaCl2, 4.17 mM NaHCO3, 1.19 mM Na2HPO4 x 7H20, 0.41 mM NaH2PO4 x H2O, 15 mM HEPES, 20 mM glucose, 0.25% BSA, pH 7.2) to prepare transport solution (0.1-300 μM compound, final DMSO 0.5% or less). Transport solution (TL) is applied to the apical or basolateral donor side to measure AB or BA permeability (reproduced on three filters), respectively. Samples are collected from the donor at the beginning and end of the experiment and from the receiver side at various time intervals for up to 2 h to measure concentrations by HPLC-MS / MS or scintillation counting. The sampled receiver volume is replaced with fresh receiver solution.

[0047] Assessment of plasma protein binding This equilibrium dialysis (ED) technique is used to determine the approximate in vitro partial binding of test compounds to plasma proteins. Dianorm Teflon dialysis cells (0.2 micro) are used. Each cell consists of a donor and an acceptor chamber, separated by an ultra-thin semi-permeable membrane with a molecular weight cut-off of 5 kDa. Stock solutions of each test compound are prepared in 1 mM DMSO and diluted to a final concentration of 1.0 μM. Subsequent dialysis solutions are prepared in pooled human or rat plasma (containing NaEDTA) from male and female donors. An aliquot of 200 μL of dialysis buffer (100 mM potassium phosphate, pH 7.4) is dispensed into the buffer chamber. An aliquot of 200 μL of test compound dialysis solution is dispensed into the plasma chamber. Incubation is performed for 2 hours at 37°C with rotation. At the end of the dialysis period, the dialysate is transferred into the reaction tube. The tube for the buffer fractionation contains 0.2 mL ACN / water (80 / 20). An aliquot of 25 μL of plasma dialysate is transferred to a deep-well plate and mixed with 25 μL ACN / water (80 / 20), 25 μL buffer, 25 μL calibration solution, and 25 μL internal standard solution. Protein precipitation is performed by adding 200 μL ACN. An aliquot of 50 μL of buffer dialysate is transferred to a deep-well plate and mixed with 25 μL blank plasma, 25 μL internal standard solution, and 200 μL ACN. The samples are measured in an HPLC-MS / MS system and evaluated in the Analyst software. The binding percentage is calculated by the formula: % bound = (plasma concentration - buffer concentration / plasma 30 concentration) x 100.

[0048] Solubility evaluation Saturated solutions are prepared in well plates (format depends on the robot) by adding an appropriate volume (typically in the range of 0.25-1.5 ml) of the selected aqueous medium to each well containing a known amount of solid drug substance (typically in the range of 0.5-5.0 mg). The wells are shaken or agitated for a predefined time (typically in the range of 2-24 hours) and then filtered using an appropriate filter membrane (typically a PTFE filter with 0.45 μm pore size). Filter absorption is avoided by discarding the first few drops of the filtrate. The amount of dissolved drug substance is determined by UV spectroscopy. Additionally, the pH of the aqueous saturated solution is measured using a glass electrode pH meter.

[0049] Pharmacokinetic characterization Test compounds are administered to individual test species either intravenously or orally. After application of the test compound, blood samples are taken at several time points, anticoagulated, and centrifuged. Analyte concentrations - administered compounds and / or metabolites - are quantified in plasma samples. PK parameters are calculated using non-compartmental methods. AUC and Cmax are normalized to a dose of 1 μmol / kg.

[0050] Assessment of metabolism in human hepatocytes in vitro The metabolic pathway of the test compound is investigated using primary human hepatocytes in suspension. After recovery from cryopreservation, the human hepatocytes are incubated in Dulbecco's modified Eagle's medium containing 5% human serum and supplemented with 3.5 μg glucagon / 500 ml, 2.5 mg insulin / 500 ml, and 3.75 mg / 500 ml hydrocortisone. After pre-incubation for 30 minutes in a cell culture incubator (37°C, 10% CO2), the test compound solution was added to the hepatocyte suspension to make the final cell density 1.0*10 6 ~4.0*10 6 cells / mL (according to compound turnover rates observed in primary human hepatocytes), final test compound concentration of 10 μM, and final DMSO concentration of 0.05%. Cells are incubated for 6 hours in a cell culture incubator on a horizontal shaker and samples are removed from incubation after 0, 0.5, 1, 2, 4, or 6 hours depending on turnover rate. Samples are quenched with acetonitrile and pelleted by centrifugation. Supernatants are transferred to 96 deep-well plates, evaporated under nitrogen, resuspended, and then bioanalyzed by liquid chromatography-high resolution mass spectrometry to identify putative metabolites. The structure was analyzed by Fourier transform-MS n Assignments are tentative based on the data. Metabolites are reported as percentage of the parent in human hepatocyte incubations with a threshold above 4%.

[0051] Treatment The present invention is directed to compounds of general formula 1 useful in the prevention and / or treatment of diseases and / or conditions associated with or modulated by TRPA1 activity, including, but not limited to, fibrotic diseases, inflammatory and immunoregulatory disorders, respiratory or gastrointestinal diseases or complaints, ophthalmic diseases, inflammatory diseases of the joints, and inflammatory diseases of the nasopharynx, eyes, and skin, as well as the treatment and / or prevention of pain and neurological disorders, including cough, idiopathic pulmonary fibrosis, other pulmonary interstitial diseases, and other fibrotic, asthmatic, or allergic diseases, eosinophilic diseases, chronic obstructive pulmonary disease, and inflammatory and immunoregulatory disorders, such as rheumatoid arthritis and atherosclerosis, as well as pain and neurological disorders, such as acute pain, postoperative pain, chronic pain, and depression, and bladder disorders. The compounds of general formula 1 are useful for the prevention and / or treatment of (1) Cough, e.g., chronic idiopathic cough or chronic refractory cough, cough associated with asthma, COPD, lung cancer, post-viral and idiopathic pulmonary fibrosis, and other pulmonary interstitial diseases. (2) Pulmonary fibrotic diseases, such as collagen diseases, such as lupus erythematosus erythematodes), pneumonitis or interstitial pneumonitis associated with systemic sclerosis, rheumatoid arthritis, polymyositis, and dermatomyositis, idiopathic interstitial pneumonias such as pulmonary fibrosis (IPF), nonspecific interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, desquamative interstitial pneumonia, idiopathic stromal pneumonia, acute interstitial pneumonia, and lymphocytic interstitial pneumonia, lymphangioleiomyomatosis, pulmonary alveolar proteinosis, Langerhans cell histiocytosis, pleuroparenchymal fibroblastosis, interstitial lung diseases of known etiology such as those resulting from occupational exposures such as asbestosis, silicosis, miner's lung (coal dust), farmer's lung (hay and mold), pigeon breeder's lung (birds), or other occupational airborne triggers such as metal dust or mycobacteria, or resulting from treatments such as radiation, methotrexate, Interstitial pneumonitis resulting from bronchitis, amiodarone, nitrofurantoin, or chemotherapy, or resulting from granulomatous diseases, e.g. polyangiitis, Churg-Strauss syndrome, sarcoidosis, granulomatosis presenting as hypersensitivity pneumonitis, or interstitial pneumonitis caused by different causes, e.g. inhalation of toxic gases, vapors, inhalation, heart failure, x-rays, radiation, chemotherapy, M. boeck, or sarcoidosis, granulomatosis, cystic fibrosis, or cystic fibrosis, alpha-1-antitrypsin deficiency, acute lung injury resulting from Covid-19 / SARS-Cov-2 infection, or pulmonary fibrosis secondary to Covid-19 / SARS-Cov-2 infection.

[0052] (3) Other fibrotic diseases, e.g., hepatic bridging fibrosis, liver cirrhosis, nonalcoholic steatohepatitis (NASH), atrial fibrosis, endomyocardial fibrosis, old myocardial infarction, glial scar, atherosclerosis, arthrofibrosis, Dupuytren's contracture, keloid, scleroderma / systemic sclerosis, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, retroperitoneal fibrosis, and adhesive capsulitis.

[0053] (4) Inflammatory autoimmune or allergic diseases and conditions, such as allergic or non-allergic rhinitis or sinusitis, chronic sinusitis or rhinitis, nasal polyposis, chronic rhinosinusitis, acute rhinosinusitis, asthma, childhood asthma, allergic bronchitis, alveolitis, hyperreactive airways, airways), allergic conjunctivitis, bronchiectasis, adult respiratory distress syndrome, bronchial and pulmonary edema, bronchitis or pneumonitis, eosinophilic cellulitis (e.g. Wells' syndrome), eosinophilic pneumonia (e.g. Löffler's syndrome, chronic eosinophilic pneumonia), eosinophilic fasciitis (e.g. Shulman's syndrome), delayed hypersensitivity, non-allergic asthma; exercise-induced bronchoconstriction; chronic obstructive pulmonary disease (COPD), acute bronchitis, chronic bronchitis, cough, emphysema; systemic anaphylaxis or hypersensitivity response, drug allergy (e.g. to penicillin, cephalosporins), eosinophilic myalgia syndrome to ingestion of contaminated tryptophan, insect bite allergy; autoimmune diseases, e.g. rheumatoid arthritis, Graves' disease, Sjögren's syndrome, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, immune-mediated Thrombocytopenia (adult ITP, neonatal thrombocytopenia, pediatric ITP), immune hemolytic anemia (autoimmune and drug-induced), Evans syndrome (platelet and red blood cell immune cytopenia), neonatal Rh disease, Goodpasture syndrome (anti-GBM disease), celiac disease, autoimmune cardiomyopathy, juvenile onset diabetes; glomerulonephritis, autoimmune thyroiditis, Behcet's disease; graft rejection, including allograft rejection or graft-versus-host disease inflammatory bowel disease, e.g., Crohn's disease and ulcerative colitis; spondyloarthropathy; scleroderma; psoriasis (including T-cell mediated psoriasis), and inflammatory skin diseases, e.g., dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous, and hypersensitivity vasculitis); erythema nodosum; eosinophilic myositis, eosinophilic fasciitis, cancers presenting with leukocyte infiltration of the skin or organs;Ophthalmic diseases such as age-related macular degeneration, diabetic retinopathy and diabetic macular edema, keratitis, eosinophilic keratitis, keratoconjunctivitis, vernal conjunctivitis, scarring, anterior segment scarring, blepharitis, blepharoconjunctivitis, bullous disorders, cicatricial pemphigoid, conjunctival melanoma, papillary conjunctivitis, dry eye, episcleritis, glaucoma, gliosis, granuloma annulare, Graves' ophthalmopathy, intraocular melanoma, pinguecula, proliferative vitreoretinopathy, pterygium, scleritis, uveitis, acute gout flare-up, gout, or osteoarthritis; (5) Pain, e.g. chronic idiopathic pain syndromes, neuropathic pain, dysesthesias, allodynia, migraine, dental pain, and post-surgical pain. (6) depression, anxiety, diabetic neuropathy, and bladder disorders, e.g., bladder outlet obstruction, overactive bladder, cystitis; myocardial reperfusion injury, or cerebral ischemic injury. Thus, the present invention relates to compounds of general formula 1 for use as medicines. Furthermore, the present invention relates to the use of compounds of general formula 1 for treating and / or preventing diseases and / or conditions which are associated with or modulated by TRPA1 activity. Furthermore, the present invention relates to the use of compounds of general formula 1 for treating and / or preventing fibrotic diseases, inflammatory and immunoregulatory disorders, diseases or complaints of the respiratory or gastrointestinal tract, ophthalmic diseases, inflammatory diseases of the joints, and inflammatory diseases of the nasopharynx, eyes, and skin, pain and neurological disorders, such as cough, idiopathic pulmonary fibrosis, other pulmonary interstitial diseases, and other fibrotic, asthmatic, or allergic diseases, eosinophilic diseases, chronic obstructive pulmonary disease, and inflammatory and immunoregulatory disorders, such as rheumatoid arthritis, and atherosclerosis, as well as pain and neurological disorders, such as acute pain, postoperative pain, chronic pain, and depression, and bladder disorders.

[0054] Furthermore, the present invention relates to the use of compounds of general formula 1 for the treatment and / or prevention of: (1) Cough, e.g., chronic idiopathic cough or chronic refractory cough, cough associated with asthma, COPD, lung cancer, post-viral and idiopathic pulmonary fibrosis, and other pulmonary interstitial diseases.

[0055] (2) Pulmonary fibrotic diseases, such as collagen diseases, e.g., lupus erythematosus erythematodes), pneumonitis or interstitial pneumonitis associated with systemic sclerosis, rheumatoid arthritis, polymyositis, and dermatomyositis, idiopathic interstitial pneumonias, e.g., pulmonary fibrosis (IPF), nonspecific interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, desquamative interstitial pneumonia, idiopathic stromal pneumonia, acute interstitial pneumonia, and lymphocytic interstitial pneumonia, lymphangioleiomyomatosis, pulmonary alveolar proteinosis, Langerhans cell histiocytosis, pleuroparenchymal fibroblastosis, interstitial lung diseases of known etiology, e.g., asbestosis, silicosis, miner's lung (coal dust), farmer's lung (hay and mold), pigeon breeder's lung (birds), or other occupational airborne triggers, e.g., metal dust or mycobacteria, or resulting from treatments, e.g., radiation, methotrexate, Interstitial pneumonitis resulting from bronchitis, amiodarone, nitrofurantoin, or chemotherapy, or resulting from granulomatous diseases, e.g. polyangiitis, Churg-Strauss syndrome, sarcoidosis, granulomatosis presenting as hypersensitivity pneumonitis, or interstitial pneumonitis caused by different causes, e.g. inhalation of toxic gases, vapors, inhalation, heart failure, x-rays, radiation, chemotherapy, M. boeck, or sarcoidosis, granulomatosis, cystic fibrosis, or cystic fibrosis, alpha-1-antitrypsin deficiency, acute lung injury resulting from Covid-19 / SARS-Cov-2 infection, or pulmonary fibrosis secondary to Covid-19 / SARS-Cov-2 infection.

[0056] (3) Other fibrotic diseases, e.g., hepatic bridging fibrosis, liver cirrhosis, nonalcoholic steatohepatitis (NASH), atrial fibrosis, endomyocardial fibrosis, old myocardial infarction, glial scar, atherosclerosis, arthrofibrosis, Dupuytren's contracture, keloid, scleroderma / systemic sclerosis, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, retroperitoneal fibrosis, and adhesive capsulitis.

[0057] (4) Inflammatory autoimmune or allergic diseases and conditions, e.g., allergic or nonallergic rhinitis or sinusitis, chronic sinusitis or rhinitis, nasal polyposis, chronic rhinosinusitis, acute rhinosinusitis, asthma, childhood asthma, allergic bronchitis, alveolitis, reactive airway hyperactivity, allergic conjunctivitis, bronchiectasis, adult respiratory distress syndrome, bronchial and pulmonary edema, bronchitis or pneumonitis, eosinophilic cellulitis (e.g., Wells' syndrome), eosinophilic pneumonia (e.g., Loeffler's syndrome), group, chronic eosinophilic pneumonia), eosinophilic fasciitis (e.g., Shulman's syndrome), delayed hypersensitivity, nonallergic asthma; exercise-induced bronchoconstriction; chronic obstructive pulmonary disease (COPD), acute bronchitis, chronic bronchitis, cough, emphysema; systemic anaphylaxis or hypersensitivity reactions, drug allergies (e.g., to penicillin, cephalosporins), eosinophilic myalgia syndrome to ingestion of contaminated tryptophan, insect bite allergy; autoimmune diseases, e.g., rheumatoid arthritis , Graves' disease, Sjögren's syndrome, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, immune thrombocytopenia (adult ITP, neonatal thrombocytopenia, pediatric ITP), immune hemolytic anemia (autoimmune and drug-induced), Evans syndrome (platelet and red blood cell immune cytopenia), neonatal Rh disease, Goodpasture's syndrome (anti-GBM disease), celiac disease, autoimmune cardiomyopathy, juvenile onset diabetes mellitus; glomerulonephritis, autoimmune thyroiditis, Behcet's disease Diseases; graft rejection (e.g. in transplants), including allograft rejection or graft-versus-host disease; inflammatory bowel diseases, e.g., Crohn's disease and ulcerative colitis; spondyloarthropathy; scleroderma; psoriasis (including T-cell mediated psoriasis), and inflammatory skin diseases, e.g., dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous, and hypersensitivity vasculitis); erythema nodosum; eosinophilic myositis, eosinophilic fasciitis, cancers presenting with leukocyte infiltration of the skin or organs;Ophthalmological diseases such as age-related macular degeneration, diabetic retinopathy and diabetic macular edema, keratitis, eosinophilic keratitis, keratoconjunctivitis, vernal conjunctivitis, scars, abdominal scars, blepharitis, blepharoconjunctivitis, bullous disorders, cicatricial pemphigoid, conjunctival melanoma, papillary conjunctivitis, dry eye, episcleritis, glaucoma, gliosis, granuloma annulare, Graves' ophthalmopathy, intraocular melanoma, pinguecula, proliferative vitreoretinopathy, pterygium, scleritis, uveitis, acute gout flare-ups, gout, or osteoarthritis;

[0058] (5) Pain, e.g. chronic idiopathic pain syndromes, neuropathic pain, dysesthesias, allodynia, migraine, dental pain, and post-surgical pain. (6) depression, anxiety, diabetic neuropathy, and bladder disorders, e.g., bladder outlet obstruction, overactive bladder, cystitis; myocardial reperfusion injury, or cerebral ischemic injury. In a further aspect, the present invention relates to compounds of general formula 1 for use in the treatment and / or prevention of the aforementioned diseases and conditions. In a further aspect, the present invention relates to the use of compounds of general formula 1 for preparing a medicament for the treatment and / or prevention of the aforementioned diseases and conditions. In a further aspect, the present invention relates to a method for treating or preventing the above mentioned diseases and conditions, comprising administering to a human an effective amount of a compound of general formula 1.

[0059] Combination Therapy The compound of the present invention may further be used in combination with one or more therapeutic agents, preferably one additional therapeutic agent.According to one embodiment, the additional therapeutic agent is selected from the group of therapeutic agents useful for treating fibrotic diseases, inflammatory and immunoregulatory disorders, respiratory or gastrointestinal diseases or complaints, inflammatory diseases or conditions of the joints, or of the nasopharynx, eyes, and skin, such as cough, idiopathic pulmonary fibrosis, other pulmonary interstitial diseases, asthmatic or allergic diseases, eosinophilic diseases, chronic obstructive pulmonary disease, atopic dermatitis, and autoimmune conditions such as rheumatoid arthritis and atherosclerosis, or therapeutic agents useful in treating ophthalmological diseases, pain, and depression. Suitable additional therapeutic agents for such combinations are in particular those which allow the dosing of one or more active substances to enhance and / or reduce the therapeutic effect of the active substance or substances, for example, with respect to the mentioned indications.

[0060] Thus, the compounds of the present invention may be used in combination with one or more additional therapeutic agents selected from the group consisting of antifibrotic agents, antitussives, anti-inflammatory agents, anti-atopic dermatitis agents, analgesics, anticonvulsants, anxiolytics, sedatives, skeletal muscle relaxants, or antidepressants.

[0061] Antifibrotic agents include, for example, nintedanib, pirfenidone, phosphodiesterase-IV (PDE4) inhibitors, such as roflumilast, autotaxin inhibitors, such as GLPG-1690 or BBT-877; connective tissue growth factor (CTGF) blocking antibodies, such as pamrevlumab; B-cell activating factor receptor (BAFF-R) blocking antibodies, such as lanalumab; alpha-V / beta-6 blocking inhibitors, such as BG-0001l / STX-100, recombinant pentraxin-2 (PTX-2), such as P RM-151; c-Jun N-terminal kinase (JNK) inhibitors, e.g., CC-90001; galectin-3 inhibitors, e.g., TD-139; G protein-coupled receptor 84 (GPR84) inhibitors, e.g., GLPG-1205; G protein-coupled receptor 84 / G protein-coupled receptor 40 dual inhibitors, e.g., PBI-4050; Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) inhibitors, e.g., KD-025; heat shock protein 47 (HSP47) small interfering RNA, e.g., BMS-986263 / ND-L02-S020 1; Wnt pathway inhibitors, e.g., SM-04646; LD4 / PDE3 / 4 inhibitors, e.g., Tipelukast; recombinant immunomodulating domain of histidyl-tRNA synthetase (HARS), e.g., ATYR-1923; prostaglandin synthase inhibitors, e.g., ZL-2102 / SAR-191801; 15-hydroxy-eicosapentaenoic acid (15-HEPE, e.g., DS-102); lysyl oxidase-like 2 (LOXL2) inhibitors, e.g., PAT-1251, PXS-5382 / PXS-5338; phosphoinositide 3-carboxylates, e.g., ... enzyme (PI3K) / mammalian target of rapamycin (mTOR) dual inhibitors, e.g., HEC-68498; calpain inhibitors, e.g., BLD-2660; mitogen-activated protein kinase kinase kinase (MAP3K19) inhibitors, e.g., MG-S-2525; chitinase inhibitors, e.g., OATD-01; mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) inhibitors, e.g., MMI-0100; transforming growth factor beta 1 (TGF-beta 1) small interfering RNA, e.g., TRK250 / BNC-1021;or a lysophosphatidic acid receptor antagonist, such as BMS-986278;

[0062] Antitussives include, for example, purinergic receptor 3 (P2X3) receptor antagonists, such as gefapixant, S-600918, BAY-1817080, or BLU-5937; neurokinin 1 (NK-1) receptor antagonists, such as orbepitant, aprepitant; nicotinic acetylcholine receptor alpha 7 subunit agonists, such as ATA-101 / bradanicline; codeine, gabapentin, pregabalin, or azithromycin. Anti-inflammatory agents are, for example, corticosteroids such as prednisolone or dexamethasone; cyclooxygenase-2 (COX2) inhibitors such as celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib; prostaglandin E2 antagonists; leukotriene B4 antagonists; leukotriene D4 antagonists such as montelukast; 5-lipoxygenase inhibitors; or other nonsteroidal anti-inflammatory agents (NSAIDs), such as aspirin, diclofenac, diflunisal, etodolac, ibuprofen, or indomethacin.

[0063] Anti-atopic dermatitis agents include, for example, cyclosporine, methotrexate, mycophenolate mofetil, azathioprine, phosphodiesterase inhibitors (e.g., apremilast, crisaborole), Janus-associated kinase (JAK) inhibitors (e.g., tofacitinib), neutralizing antibodies against IL-4 / IL-13 (e.g., dupilumab), IL-13 (e.g., lebrikizumab, tralokinumab), and IL-31 (nemolizumab).

[0064] The analgesic may be, for example, of the opioid type, such as morphine, oxymorphine, levopane, oxycodone, propoxyphene, nalmefene, fentanyl, hydrocondone, hydromorphone, mellipidine, methadone, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, pentazocine; or of the non-opioid type, such as acetophenamine.

[0065] Antidepressants include, for example, tricyclic antidepressants, such as amitriptyline, clomipramine, despramine, doxepin, desipramine, imipramine, nortriptyline; selective serotonin reuptake inhibitor antidepressants (SSRIs), such as fluoxetine, paroxetine, sertraline, citalopram, escitalopram; norepinephrine reuptake inhibitor antidepressants (SNRIs), such as maprotiline, lofepramine, mirtazapine, oxaprotiline, fezolamine, tomoxetine, mianserin, serotonin reuptake inhibitors (SNRIs), such as ... a dual serotonin norepinephrine reuptake inhibitor antidepressant (SNRI), such as duloxetine, venlafaxine, desvenlafaxine, levomilnacipran; an atypical antidepressant, such as trazodone, mirtazapine, vortioxetine, vilazodone, bupropion; or a monoamine oxidase inhibitor antidepressant (MAOI), such as tranylcypromine, phenelzine, or isocarboxazid.

[0066] The anti-anxiety drugs are, for example, benzodiazepines, such as alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, or tofisopam; or they are non-benzodiazepine hypnotics, such as eszopiclone, zaleplon, zolpidem, or zopiclone; or they are carbamates, such as meprobamate, carisoprodol, tibamate, or lorbamate; or they are antihistamines, such as hydroxyzine, chlorpheniramine, or diphenhydramine. The sedatives may be, for example, barbiturate sedatives, such as amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, secobarbital, talbutal, theamylal, or thiopental; or they may be non-barbiturate sedatives, such as glutethimide, meprobamate, methaqualone, or dichloralphenazone. Skeletal muscle relaxants are, for example, baclofen, meprobamate, carisoprodol, cyclobenzaprine, metaxalone, methocarbamol, tizanidine, chlorzoxazone, or orphenadrine.

[0067] Other suitable combination partners are inhibitors of acetylcholinesterase inhibitors, such as donepezil; 5-HT-3 antagonists, such as ondansetron; metabotropic glutamate receptor antagonists; antiarrhythmics, such as mexiletine or phenytoin; or NMDA receptor antagonists. Further suitable combination partners are incontinence drugs, such as anticholinergics, such as oxybutynin, tolterodine, darifenacin, fesoterodine, solifenacin or trospium; or they are bladder muscle relaxants, such as mirabegron; or they are alpha blockers, such as tamsulosin, alfuzosin, silodosin, doxazosin or terazosin. The dosages for the above mentioned combination partners are in most cases 1 / 5 of the lowest normally recommended dose to 1 / 1 of the normally recommended dose. Therefore, in another aspect, the invention relates to the use of a compound according to the invention in combination with one or more additional therapeutic agents as described above or below for the treatment of diseases or conditions which may be affected or mediated by TRPA1, in particular the diseases or conditions mentioned above and below.

[0068] In a further aspect, the present invention relates to a method for treating a disease or condition that may be affected by inhibiting TRPA1 in a patient, comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula (I), or a pharma- ceutically acceptable salt thereof, in combination with a therapeutically effective amount of one or more additional therapeutic agents. In a further aspect, the invention relates to the use of a compound of formula (I) or a pharma- ceutically acceptable salt thereof in combination with one or more additional therapeutic agents in a patient in need thereof for treating a disease or condition that may be affected by inhibiting TRPA1. In yet another aspect, the invention relates to a method for treating a disease or condition mediated by TRPA1 activity in a patient, comprising administering to a patient, preferably a human, in need of such treatment a therapeutically effective amount of a compound of the invention in combination with a therapeutically effective amount of one or more additional therapeutic agents as described above and below.

[0069] The use of the compounds according to the invention in combination with an additional therapeutic agent may be simultaneous or staggered. The compound according to the invention and the one or more additional therapeutic agents may both be present together in one formulation, e.g. a tablet or capsule, or separately in two identical or different formulations, e.g. as a so-called kit of parts. Consequently, in another aspect, the invention relates to a pharmaceutical composition comprising a compound according to the invention and one or more additional therapeutic agents as described above and below, optionally together with one or more inert carriers and / or diluents. In yet another aspect the present invention relates to the use of a compound according to the invention in a cough measuring device. Other features and advantages of the present invention will become apparent from the following more detailed embodiments which illustrate, by way of example, the principles of the invention.

[0070] preparation The compounds according to the invention and intermediates thereof may be obtained using synthetic methods known to those skilled in the art and described in the organic synthesis literature.Preferably, the compounds are obtained in the same manner as the preparation methods described more fully below, especially in the experimental section.In some cases, the order of carrying out the reaction steps may be changed.Various reaction methods known to those skilled in the art but not detailed here may also be used. General methods for preparing compounds according to the invention will be apparent to one skilled in the art upon studying the following schemes. Any functional groups in the starting materials or intermediates may be protected using conventional protecting groups. These protecting groups may be cleaved again at a suitable stage in the reaction sequence using methods well known to those skilled in the art.

[0071] The compounds according to the present invention are prepared by the synthetic methods described below, in which the substituents of the general formula have the meanings given hereinabove.These methods are intended to illustrate the present invention, without limiting the scope of its subject matter and compounds claimed in these examples.If the preparation of starting compounds is not described, they may be commercially available or may be prepared analogously to known compounds or methods described herein.Substances described in the literature are prepared according to published synthetic methods.Abbreviations are as defined in the Examples section. Scheme 1:

[0072] [ka]

[0073] In Scheme 1, compounds of formula I may be synthesized via N-alkylation of intermediate (A) with chloromethyleneoxadiazole (B) in the presence of a base, such as potassium carbonate. Scheme 2:

[0074] [ka]

[0075] In Scheme 2, 5-aminopyrimidine-4,6-diol is amidated with an activated acetic acid (e.g., chloroacetyl chloride) bearing a leaving group at the alpha position, followed by cyclization in the presence of a base (e.g., DIPEA) to give (C). N-alkylation of pyrimidones (C) with a protecting group, e.g., para-methoxybenzyl, may be achieved by treating (C) with a suitable reagent bearing a leaving group (e.g., para-methoxybenzyl chloride, PMB-Cl) in the presence of a base (e.g., K2CO3). This allows for the subsequent N-methylation (e.g., with MeI) of (D) in the presence of a base (e.g., K2CO3) to give (E). Finally, intermediate (A) is obtained by cleaving the protecting group of (E) under suitable conditions (e.g., PMB:trifluoroacetic acid, 100°C). Scheme 3:

[0076] [ka] In Scheme 3, alpha-cyanoketones (E) synthesized from carboxylic esters (D) are selectively reduced to alcohols (F) by using an appropriate catalytic system using a transition metal complex (e.g., of Ru or Ir) in combination with a chiral ligand (e.g., [(1S,2S)-2-amino-1,2-di-phenylethyl](4-toluenesulfonyl)amide) and a hydrogen source, e.g., triethylamine formate complex. Addition of hydroxylamine to these alcohols (F) gives dihydroxypropaneimidamides (G). Ring closure to chloromethyleneoxadiazoles (B) may be achieved by stirring the reaction mixture with chloroacetyl chloride in the presence of a base, e.g., DIPEA. EXAMPLES

[0077] preparation The compounds according to the invention and intermediates thereto may be obtained using synthetic methods known to those skilled in the art and described in the literature of organic synthesis, for example, using the methods described in "Comprehensive Organic Transformations", 2nd Edition, Richard C. Larock, John Wiley & Sons, 2010, and "March's Advanced Organic Chemistry", 7th Edition, Michael B. Smith, John Wiley & Sons, 2013. Preferably, the compounds may be obtained analogously to the preparation methods more fully described below, especially as described in the experimental section. In some cases, the order adopted in carrying out the reaction schemes may be modified. Variants of these reactions known to those skilled in the art and not detailed herein may also be used. General methods for preparing the compounds according to the invention will be clear to the skilled person studying the following schemes. The starting compounds are commercially available or may be prepared by methods described in the literature or herein, or in a similar or similar manner. Before carrying out the reaction, any corresponding functional groups in the starting compounds may be protected using conventional protecting groups. These protecting groups may be cleaved again at a suitable stage in the reaction sequence using methods well known to those skilled in the art and described in the literature, e.g., in “Protecting Groups”, 3rd Edition, Philip J. Kocienski, Thieme, 2005, and “Protective Groups in Organic Synthesis”, 4th Edition, Peter GM Wuts, Theodora W. Greene, John Wiley & Sons, 2006. The terms “room temperature” and “room temperature” are used interchangeably and denote a temperature of about 20° C., e.g. between 19 and 24° C.

[0078] [Table 10]

[0079] Preparation of intermediates Intermediate I Intermediate I.1 (general route) (3S)-3-(4-chlorophenyl)-3-hydroxypropanenitrile

[0080] [ka] 10.0 g (55.7 mmol) of 4-chlorobenzoylacetonitrile are added to 100 mL of ACN under an inert atmosphere. 142 mg (0.23 mmol) of chloro([(1S,2S)-2-amino-1,2-diphenylethyl](4-toluenesulfonyl)amido)(mesitylene)ruthenium(II) (CAS174813-81-1) are added, followed by dropwise addition of 8.30 mL (19.8 mmol) of formate triethylamine complex (5:2). After stirring for 3 h at room temperature, the solvent is removed in vacuo. Water is added to the remaining crude mixture, and the mixture is extracted twice with EtOAc. The organic layers are combined, dried over MgSO4, filtered, and the solvent is removed in vacuo to give intermediate I.1. C9H8ClNO (M=181.6g / mol) ESI-MS: 226[M+HCOO] - R t (HPLC): 0.81 min (Method B)

[0081] The following compounds are prepared using procedures similar to those described for intermediate I.1 using the appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations of the general reaction conditions.

[0082] [Table 11]

[0083] Intermediate II Intermediate II.1 (general route) (3S)-3-(4-chlorophenyl)-N,3-dihydroxypropanimidamide

[0084] [ka] To 9.82 g (54.1 mmol) of (3S)-3-(4-chlorophenyl)-3-hydroxypropanenitrile (Intermediate I.1) in 100 mL of MeOH, 8.00 mL (136 mmol) of hydroxylamine (50%, in water) is added and the mixture is stirred for 1.5 h at 75° C. After cooling to room temperature, all volatiles are removed in vacuo to give the crude product, which is used without further purification. C9H 11 ClN2O2 (M = 214.6 g / mol) ESI-MS: 215[M+H] + R t (HPLC): 0.60 min (Method B) The following compounds are prepared using procedures similar to those described for intermediate II.1 using the appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations of the general reaction conditions.

[0085] [Table 12]

[0086] Intermediate III Intermediate III.1 (general route) (1S)-2-[5-(chloromethyl)-1,2,4-oxadiazol-3-yl]-1-(4-chlorophenyl)ethan-1-ol

[0087] [ka] To 11.2 g (52.4 mmol) of intermediate II.1 in 55 mL of NMP is added 10.0 mL (57.8 mmol) of DIPEA. The mixture is cooled to 0° C., then 4.60 mL (57.7 mmol) of chloroacetyl chloride dissolved in 5 mL of NMP is slowly added and the mixture is stirred at 0° C. for 45 min. The mixture is then heated to 95° C. and stirring is continued for 4 h. After cooling to room temperature, 200 mL of water is added and the resulting mixture is extracted three times with EtOAc. The organic layers are combined, dried over MgSO4, filtered and the solvent is removed in vacuum. The residue is purified by column chromatography (silica gel; PE / EtOAc, 7 / 3). C 11 H 10 Cl2N2O2 (M = 273.1 g / mol) ESI-MS: 271[MH] - R t (HPLC): 0.93 min (Method B) The following compounds are prepared using procedures similar to those described for intermediate III.1 using the appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations of the general reaction conditions.

[0088] [Table 13]

[0089] Intermediate IV Intermediate IV.1 (general route) 3-(6-fluoro-1-benzothiophene-2-yl-3-oxopropanenitrile

[0090] [ka] To 0.63 g (3.00 mmol) of methyl 6-fluoro-1-benzothiophene-2-carboxylate in 9.0 mL of dry toluene and 0.78 mL of dry ACN is added 0.36 g (9.00 mmol) of NaH (60%, in oil) at room temperature under an inert atmosphere. The mixture is heated to reflux and stirred for 16 h, cooled to room temperature, poured into ice / water (30 mL) and treated with 2 M HCl to pH=1. EtOAc (20 mL) is added and the phases are separated. The aqueous phase is extracted again with EtOAc (20 mL), the combined organic phase is washed with brine (20 mL) and the solvent is removed under reduced pressure. The crude product is purified by silica gel column chromatography using a gradient of EtOAc / hexanes (30%-40%). C 11 H6FNOS (M=219.23g / mol) ESI-MS: 218[MH] - R t (HPLC): 3.31 minutes (E)

[0091] The following compounds are prepared using procedures similar to those described for intermediate IV.1 using the appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations of the general reaction conditions.

[0092] [Table 14] Intermediate V 3H,4H,5H,6H,7H-Pyrimido[4,5-b][1,4]oxazine-4,6-dione

[0093] [ka] To 10.0 g (127.10 mmol) of 5-aminopyrimidine-4,6-diol in 300 mL of DMF, 7.5 mL (94.41 mmol) of chloroacetyl chloride is slowly added with stirring at 65° C. After stirring at 65° C. for 1.5 h, 36.1 mL (129.94 mmol) of DIPEA is slowly added to the reaction mixture and stirring at 65° C. is continued for 45 min. The reaction mixture is concentrated under reduced pressure, treated with water, and the precipitated product is filtered, washed with a small amount of EtOH and dried. C6H5N3O3 (M = 167.1 g / mol) ESI-MS: 168[M+H] + R t (HPLC): 0.20 min (Method G)

[0094] Intermediate VI 3-[(4-Methoxyphenyl)methyl]-5-methyl-3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione [ka] 680 mg (4.07 mmol) of 3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione (Intermediate V) in 15 mL of DMF is treated with 843 mg (6.10 mmol) of potassium carbonate and stirred at room temperature for 15 min. 0.58 mL (4.27 mmol) of 1-(chloromethyl)-4-methoxybenzene is added and the reaction mixture is stirred for 20 h. 843 mg (6.10 mmol) of potassium carbonate and 0.30 mL (4.88 mmol) of methyl iodide are added and the reaction mixture is stirred at room temperature for 20 h. 0.26 mL (4.07 mmol) of methyl iodide is added and the reaction mixture is stirred at room temperature for 20 h, filtered, concentrated under reduced pressure and purified by reverse phase HPLC (ACN / H2O gradient, 0.1% TFA). C 15 H 15 N3O4 (M = 301.3 g / mol) ESI-MS: 302[M+H] + R t(HPLC): 0.95 min (Method B)

[0095] Intermediate VII 5-Methyl-3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione

[0096] [ka] 5.30 g (14.07 mmol) of 3-[(4-methoxyphenyl)methyl]-5-methyl-3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione (intermediate VI) is treated with 16 ml of TFA and stirred at 100° C. for 1.5 h. The reaction mixture is then stirred in a microwave at 120° C. for 15 min, poured into ice-cold water and filtered. The filtrate is lyophilized and used directly without further purification. C7H7N3O3 (M = 181.2 g / mol) ESI-MS: 182[M+H] + R t (HPLC): 0.37 min (Method B)

[0097] Preparation of final compounds Example 1 (General Procedure) 3-({3-[(2S)-2-(4-chlorophenyl)-2-hydroxyethyl]-1,2,4-oxadiazol-5-yl}methyl)-5-methyl-3H,4H,5H,6H,7H-pyrimido[4,5-b][1,4]oxazine-4,6-dione

[0098] [ka] A mixture of 30 mg (0.11 mmol) of intermediate VII, 20 mg (0.11 mmol) of intermediate III.1, and 30 mg (0.22 mmol) of KCO in 1.0 mL of DMF is stirred at room temperature for 1 h. The reaction mixture is filtered and the filtrate is purified by reverse-phase HPLC (ACN / HO gradient, 0.1% TFA) to give the desired product. C18 H 16 ClNO4 (M = 417.80 g / mol) ESI-MS: 418[M+H] + R t (HPLC): 0.44 min (Method A) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 2.90 - 3.10 (m, 2 H), 3.33 (s, 3 H), 4.82 (s, 2 H), 4.96 (dd, J=7.9, 5.7 Hz, 1 H), 5.48 (s, 2 H), 7.32-7.39 (m, 4 H), 8.44 (s, 1 H) The following compounds are prepared using the appropriate starting materials and procedures similar to those described in the general procedure of Example 1. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0099] [Table 15] Analytical data for the compounds listed in the above table:

[0100] [Table 16] Analytical HPLC method

[0101] [Table 17]

[0102] [Table 18] [Table 19]

[0103]

Table 20

[0104]

Table 21

[0105]

Table 22

[0106]

Table 23

[0107]

Table 24

Claims

1. A compound of formula (I). 【Chemistry 1】 (I) (In the formula, A is selected from the group consisting of phenyl, thiophenyl, benzofuranyl, and benzothiophenyl, A is unsubstituted or selected from the group consisting of halogen and C. 1-4 R consisting of alkyl 1 substituted with one or two members of the group

2. R 1 is F, Cl, I, and CH 3 2. The compound of formula (I) according to claim 1, selected from:

3. A, 【Chemistry 2】 2. The compound of formula (I) according to claim 1, selected from the group consisting of: wherein A is unsubstituted or R 1 substituted with one or two members of the group 【Request 4】 【Chemical 3】 2. The compound according to formula (I) of claim 1, selected from the group consisting of:

5. A salt of the compound according to any one of claims 1 to 4.

6. A pharmaceutical composition comprising at least one compound of formula (I) according to any one of claims 1 to 4, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable excipients.

7. A composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 4 for use as a medicine.

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 or a pharma- ceutically acceptable salt thereof for treating or preventing an inflammatory airway disease or a fibrotic disease or cough.

9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 or a pharma- ceutically acceptable salt thereof for treating or preventing idiopathic pulmonary disease (IPF) or cough.

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