Tetrazole derivatives as trpa1 inhibitors

Abstract

The present invention provides certain tetrazole derivatives which are inhibitors of Transient Receptor Potential Ankyrin 1 (TRPA1) and are therefore useful in the treatment of diseases which can be treated by inhibition of TRPA1. Also provided are pharmaceutical compositions containing the compounds and methods of preparing the compounds.

Description

Technical Field

[0001] This invention provides certain tetrazolium derivatives that are inhibitors of transient receptor potential ankylosing protein 1 (TRPA1), and are therefore suitable for treating diseases that can be treated by inhibiting TRPA1. This invention also provides pharmaceutical compositions containing said compounds and methods for preparing said compounds. Background Technology

[0002] Transient receptor potential (TRP) channels are a group of voltage-gated ion channels primarily located on the plasma membranes of numerous mammalian cell types. Approximately 30 structurally related TRP channels exist, classified into the following categories: TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, and TRPV. Member A of the transient receptor potential cation channel subfamily, TRPA1 (TRPA1), also known as transient receptor potential ankyrin 1, is the sole member of the TRPA gene subfamily. Structurally, TRPA channels are characterized by multiple N-terminal ankyrin repeat sequences (approximately 14 at the N-terminus of human TRPA1), which give rise to the "A" designation for ankyrin (Montell, 2005).

[0003] TRPA1 is highly expressed in the plasma membranes of sensory neurons in the dorsal root ganglia and tuberous ganglia that serve the skin and lungs, as well as in cells of 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 produce somatosensory modalities such as pain, cold, and itching. TRPA1 is activated by a variety of reactive, electrophilic stimuli (e.g., allyl isothiocyanate, reactive oxygen species), as well as non-reactive compounds (e.g., icilin), and is involved in coughs associated with asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), or post-viral cough, or chronic idiopathic cough and cough in sensitive patients (Song and Chang, 2015; Grace and Belvisi, 2011). Based on studies showing that cough induces elevated TGF-β (Xie et al., 2009; Froese et al., 2016; Tschumperlin et al., 2003; Yamamoto et al., 2002; Ahamed et al., 2008), TRPA1 inhibitors are indicated for the treatment of IPF where cough is very common due to the link between cough and lung damage. TRPA1 antagonists inhibit calcium signaling triggered by cough precipitates such as oxidative stress from cigarette smoke extracts (CSE), release of inflammatory mediators, and downregulation of antioxidant gene expression (Lin et al., 2015; Wang et al., 2019). TRPA1 antagonists have been effective in studies of atopic dermatitis (Oh et al., 2013; Wilson et al., 2013), contact dermatitis (Liu et al., 2013), psoriasis-related pruritus (Wilson et al., 2013), and IL-31-related pruritus (Cevikbas et al., 2014). Gain of TRPA1 function in humans is associated with familial paroxysmal pain syndrome (Kremeyer et al., 2010). TRPA1 antagonists are also effective in behavioral models of migraine-related aberrant pain (Edelmayer et al., 2012). TRPA1 expression was selectively increased in the trigeminal ganglion innervating damaged teeth compared to its expression in the ganglion innervating healthy teeth (Haas et al., 2011). Several anesthetics are known to be TRPA1 agonists, including isoflurane (Matta et al., 2008), which provides the basic mechanism for TRPA1 inhibitors to relieve postoperative pain. TRPA1 knockout mice and wild-type mice treated with TRPA1 antagonists showed anxiolytic and antidepressant-like phenotypes (de Moura et al., 2014). Based on studies showing a mechanistic link between inverse regulation of AMPK and TRPA1, TRPA1 inhibitors are expected to be beneficial in the treatment of diabetic neuropathy (Hiyama et al., 2018; Koivisto and Pertovaara, 2013; Wang et al., 2018).Compared to wild-type mice, TRPA1 knockout mice exhibited smaller myocardial infarction areas (Conklin et al., 2019). TRPA1 knockout and pharmacological intervention inhibited TNBS-induced colitis in mice (Engel et al., 2011). In a mouse model of cerebral ischemia, TRPA1 knockout and TRPA1 antagonists reduced myelin sheath damage (Hamilton et al., 2016). In a mouse model of gout with monosodium urate, TRPA1 knockout mice showed reduced urate crystals and joint inflammation (Moilanen et al., 2015). TRPA1 deficiency in rats improved joint inflammation and hyperalgesia in a rat model of acute gout attacks (Trevisan et al., 2014). TRPA1 activation induced an inflammatory response in osteoarthritis chondrocytes (Nummenmaa et al., 2016). TRPA1 inhibition and gene deletion reduced inflammatory mediators in chondrocytes of osteoarthritis mice and in murine cartilage (Nummenmaa et al., 2016). Finally, TRPA1 knockout mice showed improved weight-bearing capacity in osteoarthritis-affected limbs in a MIA-induced knee swelling model (Horvath et al., 2016). TRPA1 expression differed in the bladder epithelium of rats (Du et al., 2007) and patients with bladder outlet obstruction (Du et al., 2008). TRPA1 receptor modulation reduced overactive bladder in a rat model of spinal cord injury (Andrade et al., 2011), and intrathecal administration of TRPA1 antagonists reduced cyclophosphamide-induced cystitis in rats with excessive micturition reflex (Chen et al., 2016).

[0005] Therefore, there is a need to provide effective TRPA1 inhibitors.

[0006] A review of TRPA1 inhibitors of various structural classes is available in S. Skerratt, Progress in Medicinal Chemistry, 2017, Vol. 56, pp. 81-115 and D. Preti, G. Saponaro, A. Szallasi, Pharm. Pat. Anal. (2015) 4(2), pp. 75-94.

[0007] WO2017 / 060488 discloses a compound as a TRPA1 antagonist, which has the following general structural formula.

[0008]

[0009] However, the TRPA1 activity of Examples 28 and 29, which contain a tetrazolium ring, was not disclosed.

[0010] L. Schenkel et al., J. Med. Chem. 2016, 59, 2794-2809, disclosed quinazolinone-based TRPA1 antagonists, which include compounds with the following general structural formula.

[0011]

[0012] Compound 31, where R is OH, is disclosed to have IC50 in FLIPR assays. 50 It exhibits 58 nM of TRPA1 antagonistic activity and has an intrinsic clearance rate of <14 μL / min / kg in human liver microsomes. Detailed Implementation

[0013] This invention discloses a novel tetrazolium derivative that is an inhibitor of transient receptor potential ankylosing protein 1 (TRPA1) and has suitable pharmacological and pharmacokinetic properties, making it suitable as a medicament for treating symptoms and / or diseases that can be treated by inhibiting TRPA1.

[0014] The compounds of the present invention offer several advantages, such as enhanced potency; high metabolic and / or chemical stability; high selectivity, safety and tolerability; enhanced solubility; enhanced permeability; desired plasma protein binding; enhanced bioavailability; suitable pharmacokinetic profile; and the possibility of forming stable salts.

[0015] Compounds of the present invention

[0016] This invention provides a novel tetrazolium derivative that is a surprisingly potent inhibitor of TRPA1 (assay A), characterized further in that...

[0017] -Stability in human liver microsomes (assay B) has been improved.

[0018] -Stability in human hepatocytes (measured C) has been improved.

[0019] The compounds of the present invention differ structurally from Examples 28 and 29 of WO2017 / 060488 in that the compounds of the present invention have a substituted bicyclic core (pyrimido[4,5-b][1,4]oxazine-4,6-dione, pyrimido[4,5-b][1,4]thiazine-4,6-dione, or pyrido[3,2-d][1,4]pyrimidinone-4,6-dione) and a substituent adjacent to a secondary aliphatic alcohol. The compounds of the present invention differ structurally from Example 31 of L. Schenkel et al., J. Med. Chem. 2016, 59, 2794-2809 in that the compounds of the present invention have a tetrazolium ring. These structural differences unexpectedly lead to the following advantageous combinations: (i) inhibition of TRPA1, (ii) stability in human liver microsomes, and (iii) stability in human hepatocytes.

[0020] Therefore, the compounds of the present invention are superior to those disclosed in the prior art in terms of the combination of the following parameters:

[0021] -Efficacy as a TRPA1 inhibitor

[0022] -Stability in human liver microsomes

[0023] -Stability in human liver cells.

[0024] Stability in human liver microsomes refers to the susceptibility of a compound to biotransformation in the selection and / or design of drugs with favorable pharmacokinetic properties as a first screening step. The liver is a major site of metabolism for many drugs. 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 increased bioavailability and an appropriate half-life, which allows for reduced dosage and frequency of administration to patients. Therefore, enhanced stability in human liver microsomes is an advantageous feature of compounds intended for use as drugs. Thus, in addition to the ability to inhibit TRPA1, the compounds of the present invention are also expected to have favorable in vivo clearance and therefore the desired duration of action in humans.

[0025] 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 liver is the primary site of metabolism for many drugs. Human hepatocytes contain cytochrome P450 (CYP) and other drug-metabolizing enzymes, and therefore represent a model system for studying in vitro drug metabolism. (Importantly, compared to liver microsomal assays, hepatocyte assays also encompass phase II biotransformation and processes mediated by liver-specific transporters, and therefore represent a more complete system for drug metabolism studies). Enhanced stability in human hepatocytes is associated with several advantages, including increased bioavailability and an appropriate half-life, allowing for lower doses and frequency of administration to patients. Therefore, enhanced stability in human hepatocytes is a favorable characteristic for compounds intended for drug use.

[0026] This invention provides novel compounds according to formula (I).

[0027]

[0028] in

[0029] A is selected from phenyl, thienyl, benzothienyl, or benzofuranyl, and is unsubstituted or via halogen, CN, or C. 1-4 Alkyl, OC 1-4 Alkyl, C 1-4 Fluoroalkyl, OC 1-4 fluoroalkyl, C 3-4 cycloalkyl, OC 3-4 cycloalkyl, C 3-4 Cyclofluoroalkyl and OC 3-4 The R group composed of cyclofluoroalkyl groups 3 One, two, or three members may be replaced;

[0030] or

[0031] A is selected from:

[0032]

[0033] E is selected from O, S, SO, SO2, and CH2;

[0034] as well as

[0035] R 1 and R 2 Independently selected from H and C 1-4 Alkyl, C 1-4 Fluoroalkyl and halogens,

[0036] Or R 1 and R 2 Together with the carbon atom it is attached to, it forms a cyclopropyl or cyclobutyl ring.

[0037] Another embodiment of the present invention relates to a compound of formula (I), wherein

[0038] A is selected from phenyl, thienyl, benzothienyl, or benzofuranyl, and is unsubstituted or via halogen, C 1-4 Alkyl, -CN and -OC 1-4 alkyl groups R 3 One or two members are replaced;

[0039] or

[0040] A is

[0041]

[0042] E is selected from O, S, and CH2;

[0043] as well as

[0044] R 1 and R 2 Independently selected from H and C 1-4 Alkyl groups and halogens.

[0045] Another embodiment of the present invention relates to a compound of formula (I).

[0046] in

[0047] A is selected from phenyl, thienyl, benzothienyl, or benzofuranyl, and is unsubstituted or derived from F, Cl, Br, or C. 1-4 The R group of alkyl, CN and OCH3 3 One or two members are replaced;

[0048] or

[0049] A is

[0050]

[0051] And substituents E, R 1 and R 2 As defined in the aforementioned implementation scheme.

[0052] Another embodiment of the present invention relates to a compound of formula (I), wherein

[0053] A is selected from phenyl, thienyl, benzothienyl, or benzofuranyl, and is unsubstituted or converted by a group R selected from F, Cl, Br, CH3, CN, and OCH3. 3 One or two members are replaced.

[0054] or

[0055] A is

[0056]

[0057] And substituents E, R 1 and R 2 As defined in any of the foregoing implementation schemes.

[0058] Another embodiment of the present invention relates to a compound of formula (I), wherein

[0059] A is selected from:

[0060]

[0061] Unsubstituted or via group R 3 One or two members are replaced.

[0062] or

[0063] A is

[0064]

[0065] And substituents E, R 1 R 3 As defined in any of the foregoing embodiments. Another embodiment of the invention relates to a compound of formula (I), wherein R 3 Selected from F, Cl, Br, CH3, CN, and OCH3;

[0066] And substituents A, E, R 1 and R 2 As defined in any of the foregoing embodiments. Another embodiment of the invention relates to a compound of formula (I), wherein A is selected from:

[0067]

[0068]

[0069]

[0070] And substituents E, R 1 and R 2 As defined in any of the foregoing embodiments. Another embodiment of the invention relates to a compound of formula (I), wherein E is selected from O and S;

[0071] And substituents A and R 1 R 2 and R 3 As defined in any of the foregoing embodiments. Another embodiment of the invention relates to a compound of formula (I), wherein E is selected from O and CH2:

[0072] And substituents A and R1 R 2 and R 3 As defined in any of the foregoing embodiments. Another embodiment of the invention relates to a compound of formula (I), wherein E is O;

[0073] And substituents A and R 1 R 2 and R 3 As defined in any of the foregoing embodiments. Another embodiment of the invention relates to a compound of formula (I), wherein E is S;

[0074] And substituents A and R 1 R 2 and R 3 As defined in any of the foregoing embodiments. Another embodiment of the invention relates to a compound of formula (I), wherein E is CH2;

[0075] And substituents A and R 1 R 2 and R 3 As defined in any of the foregoing embodiments. Another embodiment of the invention relates to a compound of formula (I), wherein R 1 and R 2 Independently selected from H, CH3 and halogens;

[0076] And substituents A, E and R 3 As defined in any of the foregoing implementation schemes.

[0077] Another embodiment of the present invention relates to a compound of formula (I), wherein

[0078] R 1 and R 2 Independently selected from H, CH3, and F;

[0079] And substituents A, E and R 3 As defined in any of the foregoing implementation schemes.

[0080] Another embodiment of the invention relates to a compound of formula (I), wherein R 1 and R 2 For H;

[0081] And substituents A, E and R 3 As defined in any of the foregoing implementation schemes.

[0082] Another embodiment of the present invention relates to a compound of formula (I), wherein

[0083] R 1 and R 2 One of them is H and R1 and R 2 The other one is selected from CH3 and F;

[0084] And substituents A, E and R 3 As defined in any of the foregoing implementation schemes.

[0085] Compounds selected from the following formula (I) are preferred:

[0086] and

[0087] Furthermore, substituent A is as defined in any of the foregoing embodiments. Compounds selected from formula (I) are particularly preferred:

[0088]

[0089]

[0090]

[0091]

[0092] Terms and definitions used

[0093] Terms not specifically defined herein shall be given the meanings that a person skilled in the art would assign to them in light of the disclosure and the context. However, unless otherwise stated, the following terms shall have the specified meanings as used herein and shall be subject to the following conventions.

[0094] In the groups, radicals, or portions defined below, the number of carbon atoms is usually specified before the group, for example, C. 1-6 Alkyl refers to an alkyl group / alkyl radical having 1 to 6 carbon atoms. Generally, in groups such as HO, H2N, (O)S, (O)2S, NC (cyano), HOOC, F3C, or similar groups, those skilled in the art can see the free valence of the group itself to the group linkage point of the molecule. For composite groups containing two or more subunits, the last named subunit is the group linkage point, for example, the substituent "aryl-C". 1-3 "alkyl" refers to a compound with C 1-3 alkyl-bonded aryl group, the C 1-3 The alkyl group is bonded to the nucleus or to a substituent.

[0095] If the compounds of this invention are described by chemical name and chemical formula, in case of any discrepancy, the chemical formula shall prevail. An asterisk may be used in the formula to indicate bonds attached to the nucleus molecule as defined.

[0096] The counting of substituent atoms begins with the atom closest to the nucleus or the atom closest to the group to which the substituent is attached.

[0097] For example, the term "3-carboxypropyl" indicates the following substituents:

[0098]

[0099] The carboxyl group is attached to the third carbon atom of the propyl group. The terms "1-methylpropyl-", "2,2-dimethylpropyl-", or "cyclopropylmethyl-" represent the following groups:

[0100]

[0101] An asterisk can be used in a subform to indicate a bond attached to a nuclear molecule as defined.

[0102] Term "C" 1-n Alkyl (where n is an integer selected from 2, 3, 4, or 5) alone or in combination with another group represents a non-cyclic saturated branched or straight-chain hydrocarbon group having 1 to n carbon atoms. For example, the term "C 1-5 "Alkyl" encompasses the following groups: H3C-, H3C-CH2-, H3C-CH2-CH2-, H3C-CH(CH3)-, H3C-CH2-CH2-CH2-, H3C-CH2-CH(CH3)-, H3C-CH(CH3)-CH2-, H3C-C(CH3)2-, H3C-CH2-CH2-CH2-CH2-, H3C-CH2-CH2-CH(CH3)-, H3C-CH2-CH(CH3)-CH2-, H3C-CH(CH3)-CH2-CH2-, H3C-CH2-C(CH3)2-, H3C-C(CH3)2-CH2-, H3C-CH(CH3)-CH(CH3)- and H3C-CH2-CH(CH2CH3)-.

[0103] The term "fluorine" added to "alkyl," "alkylene," or "cycloalkyl" (saturated or unsaturated) refers to an alkyl or cycloalkyl in which one or more hydrogen atoms are replaced by fluorine atoms. Examples include (but are not limited to): H2FC-, HF2C-, and F3C-.

[0104] The term phenyl refers to a group of the following ring.

[0105]

[0106] The term thiophene group refers to a group with the following ring.

[0107]

[0108] The term benzothiophene group refers to a group with the following ring.

[0109]

[0110] The term benzofuranyl refers to a group with the following ring.

[0111]

[0112] The term tetrazolium refers to a group in the following ring.

[0113]

[0114] The term pyrimido[4,5-b][1,4]oxazine-4,6-dione refers to a group with a bicyclic core.

[0115]

[0116] The term pyrimido[4,5-b][1,4]thiazine-4,6-dione refers to a group with the following bicyclic core.

[0117]

[0118] The term pyrido[3,2-d][1,4]pyrimidinone-4,6-dione refers to a group with a bicyclic core.

[0119]

[0120] As used herein, the term “substituted” means that any one or more hydrogen atoms on a specified atom are selectively replaced by a specified group, provided that the substitution does not exceed the normal valence of the specified atom and that the substitution produces a stable compound.

[0121] Unless otherwise specified, throughout the specification and appended claims, the given chemical formula or name shall cover tautomers and all stereoisomers, optical isomers and geometric isomers (e.g., enantiomers, diastereomers, E / Z isomers, etc.) and their racemates, as well as mixtures of independent enantiomers in different proportions, mixtures of diastereomers, or mixtures containing any of such isomers and enantiomers in the aforementioned forms, and salts, including pharmaceutically acceptable salts of salts and their solvates (such as hydrates of solvates comprising the free compound or salts of the compound).

[0122] Generally, substantially pure stereoisomers can be obtained according to synthetic principles known to those skilled in the art, such as by separating the corresponding mixtures, by using stereochemically pure starting materials, and / or by stereoselective synthesis. How photoactive forms are prepared is known in the art, such as by resolution of racemic forms or by synthesis, for example, starting with photoactive starting materials and / or by using chiral reagents.

[0123] The enantiomeric pure compounds or intermediates of the present invention can be prepared via asymmetric synthesis, for example by preparation and subsequent separation of suitable diastereomeric compounds or intermediates that can be separated by known methods (e.g., by chromatographic separation or crystallization) and / or by using chiral reagents (such as chiral initiators, chiral catalysts or chiral auxiliaries).

[0124] Furthermore, those skilled in the art know how to prepare enantiomerically pure compounds from corresponding racemic mixtures, such as by chromatographic separation of the corresponding racemic mixture on a chiral stationary phase; or by resolving the racemic mixture using a suitable resolving agent, for example by forming a diastereomer salt with the racemic compound and a photoactive acid or base, followed by resolving the salt and releasing the desired compound from the salt; or by deriving the corresponding racemic compound using a photoactive chiral auxiliary agent, followed by separation of the diastereomer and removal of the chiral auxiliary group; or by kinetic resolution of the racemic mixture (e.g., by enzymatic resolution); by enantioselective crystallization from aggregates of isomorphic crystals under suitable conditions; or by (fractional) crystallization in the presence of a photoactive chiral auxiliary agent with a suitable solvent.

[0125] The phrase “pharmaceutically acceptable” is used in this article to refer to compounds, substances, compositions and / or dosage forms that are suitable for use within the bounds of reasonable medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications and that are commensurate with a reasonable benefit / risk ratio.

[0126] As used herein, a "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound in which the parent compound forms a salt or complex with an acid or base. Examples of acids that form pharmaceutically acceptable salts with a parent compound 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.

[0127] Examples of cations and bases that form pharmaceutically acceptable salts with a parent compound containing an acidic moiety include Na. + K + Ca 2+ Mg 2+ NH4 +L-arginine, 2,2'-iminodiethanol, L-lysine, N-methyl-D-reduced glucosamine, or tris(hydroxymethyl)-aminomethane. The pharmaceutically acceptable salts of this invention can be synthesized from parent compounds containing a basic or acidic moiety using conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a sufficient amount of an aqueous solution or organic diluent solution (such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile) or a mixture thereof.

[0128] In addition to the salts mentioned above, salts of other acids suitable for purifying or separating the compounds of the present invention (e.g., trifluoroacetates) also constitute part of the present invention.

[0129] Bioassay

[0130] Assessment of TRPA1 activity

[0131] Determination A: TRPA1 determination

[0132] The activity of the compounds of the present invention can be demonstrated using the following in vitro TRPA1 cell assays:

[0133] method:

[0134] The human HEK293 cell line (Perkin Elmer, product number AX-004-PCL) overexpressing the human TRPA1 ion channel was used as the assay system for compound efficacy and potency. Compound activity was determined by measuring the effect of the compound on intracellular calcium concentration induced by the agonistic effect of allyl isothiocyanate (AITC) in a FLIPRtetra system (Molecular Devices).

[0135] Cell culture:

[0136] The cell lines were obtained in frozen cell form in vials and stored at -150°C before use.

[0137] Cells were grown in MEM / EBSS medium containing 10% FCS and 0.4 mg / mL Geneticin. Importantly, the cell density should not exceed 90% confluence. For subculture, cells were removed from the flasks using Versene. One day before assay, cells were removed, washed twice with MEM / EBSS medium containing 10% FCS, and 20,000 cells were seeded at 20 μL / well into 384-well plates (black clear bottom, Cat. 356697) biocoated with Poly-D-Lysine from Corning. The plates were incubated at 37°C / 5% CO2 for 24 hours before assay.

[0138] Compound preparation

[0139] The test compound was dissolved in 100% DMSO at a concentration of 10 mM, and the first step was to dilute to a concentration of 5 mM in DMSO, followed by a series of dilution steps in 100% DMSO. The dilution factor and the number of dilution steps can be varied as needed. Typically, eight different concentrations were prepared by a 1:5 dilution, and the substance was further intermediately diluted (1:20) with HBSS / HEPES buffer (1×HEPES, Cat. 14065, from Gibco; 20 mM HEPES, Cat. 83264, from SIGMA; 0.1% BSA Cat. 11926, from Invitrogen, pH 7.4).

[0140] FLIPR measurement:

[0141] On the day of assay, cells were washed three times with assay buffer, leaving 20 μL of buffer in each well. 10 μL of Ca6 kit (Cat. R8191, Molecular Devices) loading buffer (HBSS / HEPES) was added to the cells, and the wells were incubated at 37°C / 5% CO2 for 120 minutes. 10 μL of the compound or control (HBSS / HEPES buffer / 5% DMSO) was carefully added to each well from the intermediate dilution dish. The luminescence (indicating calcium influx or release) was read on a FLIPRtetra device for 10 minutes to monitor the compound-induced effect (e.g., agonist effect). Finally, 10 μL of 50 μM AITC agonist (final concentration 10 μM) dissolved in HBSS / HEPES buffer / 0.05% DMSO was added to each well, followed by another 10 minutes of reading on a FLIPRtetra device. The area under the signal curve (AUC) after AITC addition was used to calculate IC50 / inhibition %.

[0142] Data evaluation and calculation:

[0143] Each microtitration pan contains a well with a mediator (1% DMSO) control substitute compound as a control for AITC-induced luminescence (100% CTL; high control), and a well with a mediator control without AITC as a control for non-specific changes in luminescence (0% CTL; low control).

[0144] Data analysis was performed by calculating the area under the signal curve for each individual well. Based on this value, the percentage of the measured concentration of each substance was calculated using MegaLab software (internal development): (AUC(sample) - AUC(low)) * 100 / (AUC(high) - AUC(low)). The IC50 value was then calculated from the control value percentage using MegaLab software. Calculation: [y = (ad) / (1 + (x / c)^b) + d], a = low value, d = high value; x = concentration M; c = IC50 M; b = Hill slope; y = control %

[0145] Table 1: Biological data of the compounds of the present invention obtained in assay A

[0146]

[0147]

[0148] Table 2: Biodata of the background art compounds (Examples 28 and 29 in WO2017 / 060488) obtained in determination A.

[0149] Examples in WO2017 / 060488 <![CDATA[hTRPA1 IC 50 [nM]]]> 28 366 29 1120

[0150] Table 3: Biodata of the background technology compound (Example 31 in L. Schenkel et al., J. Med. Chem. 2016, 59, 2794-2809) obtained in determination A.

[0151] Examples from Med. Chem. 2016, 59, 2794-2809 <![CDATA[hTRPA1 IC 50 [nM]]]> 31 52

[0152] Assessment of microsomal clearance rate

[0153] Determination B: Microsomal clearance rate:

[0154] The metabolic degradation of the test compound was determined using pooled liver microsomes at 37°C. A final culture volume of 100 μl at each time point contained TRIS buffer at pH 7.6 (0.1 M) at RT, magnesium chloride (5 mM), microsomal protein (1 mg / ml), and the test compound at a final concentration of 1 μM.

[0155] After a brief pre-incubation period at 37°C, the reaction was initiated by adding β-nicotinamide adenine dinucleotide phosphate in its reduced form (NADPH, 1 mM) and terminated by transferring aliquots to the solvent at different time points (0, 5, 15, 30, 60 min). NADPH-independent degradation was monitored in the NADPH-free culture, and the reaction was terminated at the last time point. The percentage of remaining test compounds after NADPH-independent incubation is reflected by parameter c (control) (metabolic stability). The quenched culture was granulated by centrifugation (10000 g, 5 min).

[0156] The amount of the parent compound in aliquots of the supernatant was determined by LC-MS / MS. The half-life (t1 / 2INVITRO) was determined by the slope of the semi-logarithmic plot of the concentration-time characteristic curve.

[0157] The intrinsic clearance rate (CL_INTRINSIC) is calculated by taking into account the amount of protein in the culture:

[0158] CL_INTRINSIC[μl / min / mg protein]=(Ln 2 / (half-life[min]*protein content[mg / ml]))*1000

[0159] CL_INTRINSIC_INVIVO[ml / min / kg]=(CL_INTRINSIC[μl / min / mg protein]×MPPGL[mg protein / g liver]×liver factor[g / kg body weight]) / 1000

[0160] Qh[%]=CL[ml / min / kg] / liver blood flow[ml / min / kg])

[0161] Hepatocyte count, human: 120 × 10⁶ cells / gram of liver

[0162] Liver factor, human: 25.7 g / kg body weight

[0163] Blood flow, human: 21 ml / (min×kg)

[0164] Table 4: Biological data of the compounds of the present invention obtained from determination B

[0165]

[0166]

[0167] Table 5: Biodata of the background art compounds (Examples 28 and 29 in WO2017 / 060488) obtained in determination B.

[0168] Examples in WO2017 / 060488 Human LM[%Qh] 28 62 29 <23

[0169] Table 6: Biodata of the background art compound (Example 31 in L. Schenkel et al., J. Med. Chem. 2016, 59, 2794-2809) obtained in determination B.

[0170] Examples from Med. Chem. 2016, 59, 2794-2809 Human LM[%Qh] 31 <23

[0171] Assessment of hepatocyte clearance rate

[0172] Measurement C: Hepatocyte clearance rate

[0173] The metabolic degradation of the test compounds was determined in hepatocyte suspensions. Hepatocytes were cultured (cryopreserved) in Dulbecco's modified eagle medium (supplemented with 3.5 μg glucagon / 500 mL, 2.5 mg insulin / 500 mL, and 3.75 mg / 500 mL dihydrocorticosterone) containing 5% or 50% species serum.

[0174] After pre-incubation in an incubator (37°C, 10% CO2) for 30 min, 5 μl of the test compound solution (80 μM; prepared by diluting 2 mM DMSO stock solution with culture medium at a ratio of 1:25) was added to 395 μl of hepatocyte suspension (cell density in the range of 0.25-5 million cells / mL, depending on the species, usually 1 million cells / mL; the final concentration of the test compound was 1 μM, and the final concentration of DMSO was 0.05%).

[0175] Cells were cultured for six hours (incubator, rotary shaker) and samples (25 μl) were collected at 0, 0.5, 1, 2, 4, and 6 hours. The samples were transferred to acetonitrile and granulated by centrifugation (5 min). The supernatant was transferred to a new 96-well plate, evaporated under nitrogen, and resuspended.

[0176] The reduction of the parent compound was analyzed by HPLC-MS / MS.

[0177] CLint is calculated as follows: CL_INTRINSIC = dose / AUC = (C0 / CD) / (AUD + clast / k) × 1000 / 60. C0: initial concentration in the culture [μM], CD: cell density of viable cells [10e6 cells / mL], AUD: area under the data [μM × h], clast: concentration of the last data point [μM], k: slope of the regression line of the parent decrease [h-1].

[0178] The calculated in vitro hepatic clearance can be scaled up to the in vivo hepatic clearance and used to predict in vivo hepatic blood clearance (CL) using a liver model (a well-mixed model).

[0179] CL_INTRINSIC_INVIVO [ml / min / kg] = (CL_INTRINSIC [μL / min / 10e6 cells] × hepatocyte count [10e6 cells / gram liver] × liver factor [g / kg body weight]) / 1000

[0180] 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])

[0181] Qh[%]=CL[ml / min / kg] / liver blood flow[ml / min / kg])

[0182] Hepatocyte count, human: 120 × 10⁶ cells / gram of liver

[0183] Liver factor, human: 25.7 g / kg body weight

[0184] Blood flow, human: 21 ml / (min×kg)

[0185] Table 7: Biological data of the compounds of the present invention obtained in determination C

[0186] Example Human hepatocytes [%Qh] 1 7 2 16 3 18 4 31 5 29 6 13 7 21 8 11 9 5 10 <4 11 29 12 8 13 10 14 5 15 29 16 <4 17 13 18 8 19 19 20 <4 21 6 22 12 23 36 24 16

[0187] Table 8: Biodata of the background art compounds (Examples 28 and 29 in WO2017 / 060488) obtained in determination C.

[0188] Examples in WO2017 / 060488 Human hepatocytes [%Qh] 28 49 29 22

[0189] Table 9: Biodata of the background art compound obtained in determination C (Example 31 in L. Schenkel et al., J. Med. Chem. 2016, 59, 2794-2809).

[0190] Examples from Med. Chem. 2016, 59, 2794-2809 Human hepatocytes [%Qh] 31 73

[0191] Penetration assessment

[0192] Caco-2 cells (1-2 × 10⁵ cells / cm²) 2The inoculated areas were placed on filter inserts (Costar transwell polycarbonate or PET filters, 0.4 μm pore size) and cultured (DMEM) for 10 to 25 days.

[0193] Dissolve the compound in a suitable solvent (e.g., DMSO, 1–20 mM stock solution). Dilute the stock solution 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 Na2HPO4×7H2O, 0.41 mM NaH2PO4×H2O, 15 mM HEPES, 20 mM glucose, 0.25% BSA, pH 7.2) to prepare transport solutions (0.1–300 μM compound, final DMSO <= 0.5%). Apply the transport solution (TL) to the donor side (top or base side) for measuring AB or BA permeability, respectively (repeated filtration 3 times). Collect samples from the donor side at the beginning and end of the experiment, and from the recipient side at various time intervals for 2 hours, to determine concentrations by HPLC-MS / MS or scintillation counting. Replace the sampled receptor volume with freshly prepared receptor solution.

[0194] Assessment of plasma protein binding

[0195] This balanced dialysis (ED) technique is used to determine the approximate in vitro fractionation binding of test compounds to plasma proteins. A Dianorm Teflon dialysis unit (0.2 ml) was used. Each unit consists of a donor chamber and a recipient chamber, separated by an ultrathin semipermeable membrane with a 5 kDa molecular weight cutoff. Stock solutions for each test compound were prepared in DMSO at 1 mM and diluted to a final concentration of 1.0 μM. Subsequent dialysis solutions were prepared from combined human or rat plasma (containing NaEDTA) from male and magnetic donors. Aliquots of 200 μL of dialysis buffer (100 mM potassium phosphate, pH 7.4) were aliquoted into the buffer chamber. Aliquots of 200 μL of the test compound dialysis solution were aliquoted into the plasma chamber. The mixture was incubated at 37°C under rotation for 2 hours.

[0196] At the end of the dialysis period, transfer the dialysis buffer to the reaction tube. The tube used for the buffer portion contains 0.2 mL ACN / water (80 / 20). Transfer an aliquot of 25 μL plasma dialysis buffer to a deep-well plate and mix 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. Transfer an aliquot of 50 μL buffer dialysis buffer to a deep-well plate and mix with 25 μL blank plasma, 25 μL internal standard solution, and 200 μL ACN. Measure the samples on an HPLC-MS / MS system and evaluate using Analyst Software. Calculate the binding percentage using the following formula: Binding % = (Plasma concentration - Buffer concentration / Plasma 30 concentration) × 100.

[0197] Solubility assessment

[0198] A saturated solution is prepared in a well disc (format depends on the robot) by adding an appropriate volume of the selected liquid medium (typically in the range of 0.25–1.5 ml) to each well containing a known amount of solid active pharmaceutical ingredient (typically in the range of 0.5–5.0 mg). Each well is shaken or stirred for a predetermined period of time (typically in the range of 2–24 h) and then filtered using a suitable filter membrane (typically a PTFE membrane with a pore size of 0.45 μm). Membrane absorption is avoided by discarding the first few drops of filtrate. The amount of dissolved active pharmaceutical ingredient is determined by UV spectroscopy. Additionally, the pH of the saturated aqueous solution is measured using a glass electrode pH meter.

[0199] Assessment of pharmacokinetic characteristics in rodents

[0200] The test compound was administered intravenously to fed rats orally to fasted rats. Blood samples were collected at several time points after administration of the test compound, and the samples were anticoagulated and centrifuged.

[0201] The concentrations of the administered compounds and / or metabolites used as analytes in plasma samples were quantified. PK parameters were calculated using non-laboratory methods. AUC and Cmax were normalized to a dose of 1 μmol / kg.

[0202] Assessment of human hepatocyte metabolism in vitro

[0203] Primary human hepatocytes in suspension form were used to investigate the metabolic pathways of the test compounds. After being reconstituted from cryopreservation, human hepatocytes were cultured in Duchenne modified Eagle medium containing 5% human serum supplemented with 3.5 μg glucagon / 500 ml, 2.5 mg insulin / 500 ml, and 3.75 mg / 500 ml DHEA.

[0204] After pre-culturing in a cell culture incubator (37℃, 10% CO2) for 30 min, the test compound solution was incorporated into the hepatocyte suspension to obtain 1.0*102 6 Up to 4.0*10 6 The final cell density was determined by the number of cells per milliliter (depending on the metabolic conversion rate of the compound observed in primary human hepatocytes), the final concentration of the test compound was 10 μM, and the final concentration of DMSO was 0.05%.

[0205] Cells were incubated in a cell culture incubator with a horizontal shaker for six hours, and samples were removed after incubation at 0, 0.5, 1, 2, 4, or 6 hours depending on the metabolic conversion rate. Samples were quenched with acetonitrile and granulated by centrifugation. The supernatant was transferred to a 96-well plate, evaporated and resuspended under nitrogen, and then subjected to bioanalysis by liquid chromatography-high resolution mass spectrometry to identify putative metabolites.

[0206] Based on Fourier-Transform-MS n Data are temporarily assigned to a specific structure. Metabolites are reported as a percentage of the parent cell in human hepatocyte culture, with a cutoff value of ≥4%.

[0207] Treatment

[0208] This invention relates to compounds of general formula 1, which are suitable for the prevention and / or treatment of diseases and / or symptoms associated with or regulated by TRPA1 activity, including (but not limited to) the treatment and / or prevention of fibrotic diseases, inflammatory and immunomodulatory conditions, respiratory or gastrointestinal diseases or discomforts, ophthalmic diseases, inflammatory diseases of the joints and inflammatory diseases of the nasopharynx, eyes and skin, as well as pain and neurological conditions. The conditions, diseases and discomforts include cough, idiopathic pulmonary fibrosis, other interstitial lung diseases and other fibrotic diseases, asthma or allergic diseases, eosinophilic diseases, chronic obstructive pulmonary disease, and inflammatory and immunomodulatory conditions (such as rheumatoid arthritis and atherosclerosis) as well as pain and neurological conditions (such as acute pain, surgical pain, chronic pain and depression, and bladder disorders).

[0209] Compounds of general formula 1 are suitable for prevention and / or treatment:

[0210] (1) Cough, such as chronic idiopathic cough or chronic refractory cough, cough associated with asthma, COPD, lung cancer, post-viral infection and idiopathic pulmonary fibrosis and other interstitial lung diseases.

[0211] (2) Pulmonary fibrotic diseases (such as collagen-related pneumonia or interstitial pneumonia), such as lupus, systemic scleroderma, rheumatoid arthritis, polymyositis and dermatomyositis, idiopathic interstitial pneumonia (such as idiopathic pulmonary fibrosis (IPF)), nonspecific interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, desquamative interstitial pneumonia, cryptogenic organizing pneumonia, acute interstitial pneumonia and lymphocytic interstitial pneumonia, lymphangioleiomyomatosis, pulmonary alveolar proteinosis, Langerhans cell histiocytosis, pleural parenchymal fibroelastosis, interstitial lung diseases of known etiology, such as those caused by occupational exposure (such as asbestos deposition, silica deposition, miner's lung (coal dust), farmer's lung (hay and mold), pigeon fanciers' lung). Interstitial pneumonia or granulomatous diseases caused by lung (birds) or other occupational airborne precipitants (such as metal dust or mycobacteria) or by treatment (such as radiation, methotrexate, amiodarone, nitrofurantoin, or chemotherapy agents), such as granulomatous polyangiitis, Church-Strauss syndrome, sarcoidosis, hypersensitivity pneumonitis, or interstitial pneumonia caused by various etiologies (e.g., inhalation of toxic gases, vapors, bronchitis, or pneumonia), or by heart failure, X-rays, radiation, chemotherapy, Burke's disease or sarcoidosis, granulomatous diseases, cystic fibrosis or viscous myxopathy, or alpha-1 antitrypsin deficiency.

[0212] (3) Other fibrotic diseases, such as liver bridging fibrosis, cirrhosis, non-alcoholic steatosis (NASH), atrial fibrosis, endocardial myocardial fibrosis, old myocardial infarction, glial scar, arterial stiffness, joint fibrosis, Dupuytren's contracture, keloid, scleroderma / systemic sclerosis, mediastinal fibrosis, myelofibrosis, Peyronie's disease, renal systemic fibrosis, retroperitoneal fibrosis, and adhesion cystitis.

[0213] (4) Inflammatory, autoimmune, or allergic diseases and symptoms, such as allergic or non-allergic rhinitis or sinusitis, chronic sinusitis or rhinitis, nasal polyps, chronic sinusitis, acute sinusitis, asthma, childhood asthma, allergic bronchitis, alveolitis, hyperresponsive trachea, allergic conjunctivitis, bronchiectasis, adult respiratory distress syndrome, bronchial and pulmonary edema, bronchitis or regional pneumonia, eosinophilic cellulitis (e.g., Wells syndrome), eosinophilic pneumonia (e.g., Loeffler's syndrome), chronic eosinophilic pneumonia, eosinophilic fasciitis (e.g., Shulman's syndrome). Hypersensitivity syndrome (HSS), delayed-type hypersensitivity, non-allergic asthma; exercise-induced bronchoconstriction; chronic obstructive pulmonary disease (COPD), acute bronchitis, chronic bronchitis, cough, emphysema; systemic allergic reactions or hypersensitivity reactions, drug allergies (e.g., to penicillin, cephalosporins), eosinophilic myalgia syndrome caused by ingestion of contaminated tryptophan, insect sting allergy; autoimmune diseases such as rheumatoid arthritis, Graves' disease, Sjogren's syndrome, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, immune thrombocytopenic purpura (ITP in adults / neonatal ITP, pediatric ITP), immune hemolytic anemia (autoimmune and drug-induced), Evans syndrome (immune thrombocytopenic purpura of platelets and red blood cells), neonatal Rh disease, Goodpasture's syndrome Syndrome (anti-GBM disease), celiac disease, autoimmune cardiomyopathy, juvenile diabetes; glomerulonephritis, autoimmune thyroiditis, Behcet's disease; transplant rejection (e.g., in transplantation), including allogeneic transplant rejection or graft-versus-host disease; inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis; spondyloarthritis; psoriasis (including T-cell mediated psoriasis) and inflammatory skin diseases (such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, rubella); vasculitis (e.g., necrotizing, cutaneous, and hypersensitivity vasculitis); erythema nodosum; eosinophilic myositis, eosinophilic fasciitis, cancers of the skin or organs with leukocyte infiltration;Ophthalmic diseases such as age-related macular degeneration, diabetic retinopathy and diabetic macular edema, keratitis, eosinophilic keratitis, keratoconjunctivitis, vernal keratoconjunctivitis, scars, anterior segment scarring, blepharitis, palpebral conjunctivitis, bullous diseases, cicatricial pemphigoid, conjunctival melanoma, papillary conjunctivitis, dry eye, episcleritis, glaucoma, glioma, annular granuloma, Graves' ophthalmopathy, intraocular melanoma, conjunctival macular degeneration, proliferative vitreoretinopathy, pterygium, scleritis, acute gout attacks, gout, or osteoarthritis.

[0214] (5) Pain, such as chronic idiopathic pain syndrome, neuralgia, hypoesthesia, tenderness, migraine, toothache and postoperative pain.

[0215] (6) Depression, anxiety, diabetic neuropathy and bladder disorders, such as bladder outlet obstruction, overactive bladder, cystitis; myocardial reperfusion injury or local cerebral ischemia injury.

[0216] Therefore, the present invention relates to a compound of general formula 1, which is used as a pharmaceutical agent.

[0217] Furthermore, the present invention relates to the use of a compound of general formula 1 for the treatment and / or prevention of diseases and / or symptoms related to or regulated by TRPA1 activity.

[0218] Furthermore, this invention relates to the use of compounds of general formula 1 for the treatment and / or prevention of fibrotic diseases, inflammatory and immunomodulatory disorders, respiratory or gastrointestinal diseases or discomforts, ophthalmic diseases, inflammatory diseases of the joints and nasopharynx, eyes and skin, pain and neurological disorders. The conditions, diseases and discomforts include cough, idiopathic pulmonary fibrosis, other interstitial lung diseases and other fibrotic disorders, asthma or allergic diseases, eosinophilic diseases, chronic obstructive pulmonary disease, and inflammatory and immunomodulatory disorders (such as rheumatoid arthritis and atherosclerosis) as well as pain and neurological disorders (such as acute pain, surgical pain, chronic pain and depression, and bladder disorders).

[0219] Furthermore, the present invention relates to the use of a compound of general formula 1 for treatment and / or prevention:

[0220] (1) Cough, such as chronic idiopathic cough or chronic refractory cough, cough associated with asthma, COPD, lung cancer, post-viral infection and idiopathic pulmonary fibrosis and other interstitial lung diseases.

[0221] (2) Pulmonary fibrotic diseases (such as collagen-related pneumonia or interstitial pneumonia), such as lupus, systemic scleroderma, rheumatoid arthritis, polymyositis and dermatomyositis, idiopathic interstitial pneumonia (such as idiopathic pulmonary fibrosis (IPF)), nonspecific interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, desquamative interstitial pneumonia, cryptogenic organizing pneumonia, acute interstitial pneumonia and lymphocytic interstitial pneumonia, lymphangioleiomyomatosis, pulmonary alveolar proteinosis, Langerhans cell histiocytosis, pleural parenchymal fibroelastosis, interstitial lung diseases of known etiology, such as those caused by occupational exposure (such as asbestos deposition, silica deposition, miners). Interstitial pneumonia or granulomatous diseases caused by lung (coal dust), farmer's lung (hay and mold), pigeon breeder's lung (birds) or other occupational airborne causes, such as metal dust or mycobacteria, or by treatment (such as radiation, methotrexate, amiodarone, nitrofurantoin or chemotherapeutic agents), such as granulomatous polyangiitis, Churchill-Schönlein syndrome, sarcoidosis, allergic pneumonia, or by various causes (e.g., inhalation of toxic gases, vapors, bronchitis or pneumonia), or by heart failure, X-rays, radiation, chemotherapy, Burke's disease or sarcoidosis, granulomatous disease, cystic fibrosis or viscous myxopathy or alpha-1 antitrypsin deficiency.

[0222] (3) Other fibrotic diseases, such as liver bridging fibrosis, cirrhosis, non-alcoholic steatotic hepatitis (NASH), atrial fibrosis, endocardial myocardial fibrosis, old myocardial infarction, glial scar, arterial stiffness, joint fibrosis, Dupuytrange contracture, keloid, scleroderma / systemic sclerosis, mediastinal fibrosis, myelofibrosis, Peroni disease, renal systemic fibrosis, retroperitoneal fibrosis, and adhesion cystitis.

[0223] (4) Inflammatory, autoimmune, or allergic diseases and symptoms, such as allergic or non-allergic rhinitis or sinusitis, chronic sinusitis or rhinitis, nasal polyps, chronic sinusitis, acute sinusitis, asthma, childhood asthma, allergic bronchitis, alveolitis, hyperresponsive trachea, allergic conjunctivitis, bronchiectasis, adult respiratory distress syndrome, bronchial and pulmonary edema, bronchitis or localized pneumonia, eosinophilic cellulitis (e.g., Wells syndrome), eosinophilic pneumonia (e.g., Löffler's syndrome, chronic eosinophilic pneumonia), eosinophilic fasciitis (e.g., Schulman syndrome), delayed-type hypersensitivity, non-allergic asthma; exercise-induced bronchoconstriction. Shrinkage; Chronic obstructive pulmonary disease (COPD), acute bronchitis, chronic bronchitis, cough, emphysema; systemic allergic reactions or hypersensitivity reactions, drug allergies (e.g., to penicillin, cephalosporins), eosinophilic myalgia syndrome caused by ingestion of contaminated tryptophan, insect sting allergy; autoimmune diseases such as rheumatoid arthritis, Graves' disease, Hughley's syndrome, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, immune thrombocytopenic purpura (adult ITP / neonatal thrombocytopenic purpura, pediatric ITP), immune hemolytic anemia (autoimmune and drug-induced), Ivan Behcet's syndrome (immune thrombocytopenic purpura), neonatal Rh disease, Cuban Stockholm syndrome (anti-GBM disease), celiac disease, autoimmune cardiomyopathy, juvenile diabetes mellitus; glomerulonephritis, autoimmune thyroiditis, Behcet's disease; transplant rejection (e.g., in transplantation), including allogeneic transplant rejection or graft-versus-host disease; inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis; spondyloarthritis; psoriasis (including T-cell mediated psoriasis) and inflammatory skin diseases (such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, rubella); vasculitis (e.g., necrotizing, cutaneous, and hypersensitive vasculitis). Inflammation); erythema nodosum; eosinophilic myositis, eosinophilic fasciitis, cancer of the skin or organs with leukocyte infiltration; ophthalmic diseases such as age-related macular degeneration, diabetic retinopathy and diabetic macular edema, keratitis, eosinophilic keratitis, keratoconjunctivitis, vernal keratoconjunctivitis, scars, anterior segment scars, blepharitis, palpebral conjunctivitis, bullous diseases, cicatricial pemphigoid, conjunctival melanoma, papillary conjunctivitis, dry eye, episcleritis, glaucoma, glioma, annular granuloma, Graves' ophthalmopathy, intraocular melanoma, conjunctival macular degeneration, proliferative vitreoretinopathy, pterygium, scleritis, acute gout attack, gout or osteoarthritis.

[0224] (5) Pain, such as chronic idiopathic pain syndrome, neuralgia, hypoesthesia, tenderness, migraine, toothache and postoperative pain.

[0225] (6) Depression, anxiety, diabetic neuropathy and bladder disorders, such as bladder outlet obstruction, overactive bladder, cystitis; myocardial reperfusion injury or local cerebral ischemia injury.

[0226] In another aspect, the present invention relates to a compound of general formula 1 for treating and / or preventing the diseases and symptoms mentioned above.

[0227] In another aspect, the present invention relates to the use of a compound of general formula 1 for the preparation of a medicament for the treatment and / or prevention of the diseases and symptoms mentioned above.

[0228] In another aspect of the invention, the invention relates to a method for treating or preventing the diseases and symptoms mentioned above, the method comprising administering an effective amount of a compound of formula 1 to a human.

[0229] Combination therapy

[0230] The compounds of the present invention may be further combined with one or more, preferably one, additional therapeutic agent. According to one embodiment, the additional therapeutic agent is selected from: agents suitable for treating the diseases or conditions described above, particularly those related to fibrotic diseases, inflammatory and immunomodulatory disorders, respiratory or gastrointestinal diseases or discomforts, inflammatory diseases or conditions of the joints or nasopharynx, eyes and skin (such as cough), idiopathic pulmonary fibrosis, other interstitial lung diseases, asthma or allergic diseases, eosinophilic diseases, chronic obstructive pulmonary disease, atopic dermatitis, and autoimmune disorders (such as rheumatoid arthritis and atherosclerosis); or agents suitable for treating ophthalmic diseases, pain, and depression.

[0231] Additional therapeutic agents suitable for such combinations include, in particular, those that enhance the therapeutic effect of one or more active substances on one of the mentioned indications and / or reduce the dosage of one or more active substances.

[0232] Therefore, the compounds of the present invention can be combined with one or more additional therapeutic agents selected from the following: antifibrotic agents, antitussive agents, anti-inflammatory agents, anti-atopic dermatitis agents, analgesics, anticonvulsants, anxiolytic agents, sedatives, skeletal muscle relaxants, or antidepressants.

[0233] Anti-fibrotic agents include, for example, nintedanib, pirfenidone, phosphodiesterase type 4 (PDE4) inhibitors (such as roflumilast), autocrine motor factor inhibitors (GLP-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); α-v / β-6 blocking inhibitors (such as BG-00011 / STX-100), recombinant pentamin-2 (PTX-2) (such as PRM-151); c-Jun N-terminal kinase (JNK) inhibitors, such as CC-90001; galactoglobulin-3 inhibitors, such as TD-139; G-protein-coupled receptor 84 (GPR84) inhibitors, such as GLPG-1205; dual inhibitors of G-protein-coupled receptor 84 / G-protein-coupled receptor 40, such as PBI-4050; Rho-associated coiled-coil protein kinase 2 (ROCK2), such as KD-025; heat shock protein 47 (HSP47) small interfering RNA / ND-L02-s0201, such as BMS-986263; Wnt pathway inhibitors, such as SM-04646; LD4 / PDE3 / 4 inhibitors, such as Tipelukast; recombinant immunomodulatory domains of histyl-tRNA synthetase (HARS), such as ATYR-1923; prostaglandin synthase inhibitors, such as ZL-2102 / SAR-191. 801; 15-hydroxy-eicosapentaenoic acid (15-HEPE, e.g., DS-102); lysyl oxidase-like 2 (LOXL2) inhibitors, such as PAT-1251, PXS-5382 / PXS-5338; phosphatidylinositol 3-kinase (PI3K) / mammalian target of rapamycin (mTOR) dual inhibitors, such as HEC-68498; calpain inhibitors, such as BLD-2660; mitogen-activated protein kinase (MAP3K19) inhibitors, such as MG-S-2525; chitinase inhibitors, such as OATD-01; mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) inhibitors, such as MMI-0100; transforming growth factor β1 (TGF-β1) small interfering RNA, such as TRK250 / BNC-1021; or lysophosphatidic acid receptor antagonists, such as BMS-986278.

[0234] For example, cough suppressants are purine receptor 3 (P2X3) receptor antagonists, such as gefapixant, S-600918, BAY-1817080, or BLU-5937; neurokinin 1 (NK-1) receptor antagonists, such as orvepitant or aprepitant; and nicotinic acetylcholine receptor α7 subunit stimulators, such as ATA-101 / bradanicline, codeine, gabapentin, pregablin, or azithromycin. For example, anti-inflammatory agents are corticosteroids, such as prednisolone or dexamethasone; cyclooxygenase-2 (COX2) inhibitors, such as celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib; pre- Prostaglandin E2 antagonists; leukotriene B4 antagonists; leukotriene D4 antagonists, such as montelukast; 5-lipoxygenase inhibitors; or other nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, diclofenac, diflunisal, etodolac, ibuprofen, or indomethacin.

[0235] For example, anti-atopic dermatitis agents include cyclosporin, 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., dupilamab), IL-13 (e.g., lebrikizumab, tralokinumab), and IL-31 (nemolizumab).

[0236] For example, analgesics are opioid-like, such as morphine, oxymorphine, levopanol, oxycodone, propoxyphene, nalmefene, fentanyl, hydrocodone, hydromorphone, meripidine, methadone, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, pentazocine; or non-opioid analgesics such as acetylene.

[0237] For example, antidepressants include tricyclic antidepressants such as amitriptyline, clomipramine, despramine, doxepin, desipramine, imipramine, and nortriptyline; selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, paroxetine, sertraline, citalopram, and escitalopram; and norepinephrine reuptake inhibitors (SNRIs) such as maprotiline, lofepramine, mirtazapine, oxaprotiline, fezolamine, and atomoxetine. Moxetine, mianserin, butylamine acetone, hydroxybuproprion, nomifensine, viloxazine; dual serotonin-norepinephrine reuptake inhibitors (SNRIs), such as duloxetine, venlafaxine, desvenlafaxine, levomilnacipran; atypical antidepressants, such as trazodone, mirtazapine, vortioxetine, vilazodone, bupropion; monoamine oxidase inhibitors (MAOIs), such as tranylcypromine, phenelzine, or isocarboxazid.

[0238] For example, anxiolytics are benzodiazepines, such as alprazolam, bromazepam, chlordiazepoxide, clonazepam, clonazepam, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, or tofisopam; or non-benzodiazepines. Diazazepine hypnotics, such as eszopiclone, zaleplon, zolpidem, or zopiclone; or carbamates, such as meprobamate, carisoprodol, tybamate, or lorbamate; or antihistamines, such as hydroxyzine, chlorpheniramine, or diphenhydramine.

[0239] For example, the sedative is a barbiturate sedative, such as amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, methabarbital, methohexital, pentobarbital, secobarbital, talbutal, theamylal, or thiopental; or a non-barbiturate sedative, such as glutethimide, meprabarbital, methaqualone, or dichloal phenazone.

[0240] For example, skeletal muscle relaxants include baclofen, mepparidine, cyclobenzaprine, metaxalone, mesocarbamol, tizanidine, chlorzoxazone, or orphenadrine.

[0241] Other suitable combination therapies include acetylcholinesterase inhibitors, such as donepezil; 5-HT-3 antagonists, such as ondansetron; metabotropic glutamate receptor antagonists; antiarrhythmic agents, such as mexiletine or phenytoin; or NMDA receptor antagonists.

[0242] Other suitable combinations include incontinence drugs, such as anticholinergic agents like oxybutynin, tolterodine, darifenacin, fesoterodine, solifenacin, or trospium; or bladder relaxants like miraberon; or alpha blockers like tamsulosin, alfuzosin, silodosin, doxazosin, or terazosin.

[0243] The dosage of the above combination of drugs is usually 1 / 5 to 1 / 1 of the normal recommended minimum dose.

[0244] Therefore, in another aspect, the present invention relates to the use of compounds according to the invention in combination with one or more additional therapeutic agents as described in the context for the treatment of diseases or conditions that may be affected or mediated by TRPA1, particularly as described in the context.

[0245] In another aspect, the present invention relates to a method for treating a disease or symptom in a patient that may be affected by TRPA1 inhibition, comprising the steps of administering to a patient requiring such treatment a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of one or more additional therapeutic agents.

[0246] In another aspect, the present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with one or more additional therapeutic agents for the treatment of a disease or condition in patients in need that may be affected by TRPA1 inhibition.

[0247] In another aspect, the present invention relates to a method for treating a patient with a disease or symptom mediated by TRPA1 activity, comprising the steps of administering to a patient (preferably human) requiring such treatment a therapeutically effective amount of the compound of the present invention combined with a therapeutically effective amount of one or more additional therapeutic agents described in the context.

[0248] The use of compounds according to the invention in combination with other therapeutic agents can be carried out simultaneously or at staggered times.

[0249] The compounds according to the invention and one or more additional therapeutic agents may be present together in a formulation (e.g., tablets or capsules) or separately in two identical or different formulations (e.g., kits in so-called kit-of-part form).

[0250] Therefore, in another aspect, the present invention relates to a pharmaceutical composition comprising a compound according to the invention and one or more additional therapeutic agents as described in the context, optionally together with one or more inert carriers and / or diluents.

[0251] In another aspect, the present invention relates to the use of a compound according to the invention in a cough measuring device.

[0252] Other features and advantages of the invention will become apparent from the following more detailed embodiments, which illustrate the principles of the invention by way of example.

[0253] preparation

[0254] The compounds and intermediates of the present invention can be obtained using synthetic methods known to those skilled in the art and described in the literature on organic synthesis. Preferably, the compounds are obtained in a manner similar to the preparation methods explained more fully below, especially as described in the experimental section. In some cases, the order of the reaction steps may be varied. Variations of reaction methods known to those skilled in the art but not described in detail herein may also be used.

[0255] A general process for preparing the compounds according to the invention will become apparent to those skilled in the art who study the following procedures. Conventional protecting groups can be used to protect any functional groups in the starting material or intermediates. These protecting groups can be re-cleaved at appropriate stages within the reaction sequence using methods familiar to those skilled in the art.

[0256] The compounds according to the invention are prepared by the synthetic methods described below, wherein the substituents of the general formula have the meanings given above. These methods are intended to be illustrative of the invention but do not limit its subject matter or the scope of the compounds claimed in these examples. Where the preparation of the starting compound is not described, it may be commercially available or prepared in a manner similar to that of known compounds or methods described herein. Substances described in the literature are prepared according to the published synthetic methods. Abbreviations are as defined in the Examples section.

[0257] Compounds of formula (I) with E=O, represented by (Ia), can be prepared as shown in Scheme 1 below.

[0258] Option 1:

[0259]

[0260] In Scheme 1, chloromethyltetrazole is N-alkylated with a suitable ethyl ketone derivative carrying a leaving group “LG” (e.g., Cl or Br) adjacent to the carbonyl group in the presence of a base (e.g., K₂CO₃) to yield a mixture of two regiomeric isomers. Undesired regiomeric isomers (not illustrated) can be removed by chromatography using an appropriate gradient. The resulting ketone (A) can be enantioselectively reduced using a suitable catalytic system, combining a transition metal complex (e.g., Ru or Ir) with a chiral ligand (e.g., [(1S,2S)-2-amino-1,2-diphenylethyl](4-toluenesulfonyl)amide) and a hydrogen source (e.g., a triethylamine formate complex). The resulting alcohol (B) in the presence of a base can be used for direct alkylation (C) to yield a compound of general formula (Ia), or for alkylation (D) to produce an intermediate (E), which, upon methylation with a methylating agent (e.g., iodomethane) in the presence of a base, also yields a compound of general formula (Ia).

[0261] Compounds of formula (I) with E=O, represented by (Ia), can be prepared as shown in Scheme 2 below.

[0262] Option 2:

[0263]

[0264] In Scheme 2, compound (G) can be prepared by alkylating (D) with an acetonitrile derivative carrying a leaving group “LG” (e.g., Cl or Br) in the presence of a weak base such as DIPEA, followed by methylating intermediate (F) with a methylating agent (e.g., MeI) in the presence of a base (e.g., K₂CO₃). The formation of tetrazolium (H) can be achieved using typical reaction conditions for tetrazolium formation (e.g., using NaN₃ in the presence of a DMF solution of TEA / TEA hydrochloride). Alkylation of tetrazolium (H) with a suitable acetone derivative carrying a leaving group “LG” (e.g., Cl or Br) adjacent to the carbonyl group, in the presence of a base such as K₂CO₃, yields a mixture of two regiomeric isomers. Undesired regiomeric isomers (not illustrated) can be removed by chromatography using an appropriate gradient. Finally, the final compound (Ia) can be obtained by enantioselectively reducing the ketone group of (J) using a suitable catalytic system, combining a transition metal complex (e.g., Ru or Ir) with a chiral ligand (e.g., ([(1S,2S)-2-amino-1,2-diphenylethyl](4-toluenesulfonyl)amide) and a hydrogen source (e.g., a triethylamine formate complex). Alternatively, the final compound (Ia) can be prepared by alkylating the intermediate (H) with a suitable ethanol derivative (K) carrying a leaving group “LG” (e.g., Cl or Br) adjacent to a hydroxyl group in the presence of a base such as DIPEA, and subsequently isolating the desired regiomeric isomer.

[0265] Intermediates (C) and (D) of schemes 1 and 2 can be prepared as shown in scheme 3.

[0266] Option 3

[0267]

[0268] In scheme 3, intermediates (C) and (D) can be synthesized from 5-aminopyrimidine-4,6-diol as starting material. The 5-aminopyrimidine-4,6-diol is amidated with an activated carboxylic acid having a leaving group (LG) at the α-position (e.g., chloroacetyl chloride) to give (L), which can be further progressed to intermediate (D) in the presence of a base (e.g., DIPEA). Alternatively, intermediate (D) can be synthesized from 5-aminopyrimidine-4,6-diol via reaction under pure conditions with a carboxylic acid or carboxylic acid derivative carrying a leaving group (LG) at the α-position. N-alkylation of a pyrimidinone (D) having a protecting group such as p-methoxybenzyl can be achieved in the presence of a base (e.g., K₂CO₃) with a suitable reagent carrying a leaving group (e.g., p-methoxybenzyl chloride, PMB-Cl). This allows for subsequent methylation (e.g., with MeI) in the presence of a base (e.g., K₂CO₃) to give (N). Finally, the protecting group of (N) is cleaved under suitable conditions (e.g., for PMB: trifluoroacetic acid, 100 °C) to give intermediate (C).

[0269] Compounds of formula (I) with E=CH2, represented by (Ib), can be prepared as shown in Scheme 4 below.

[0270] Option 4

[0271]

[0272] In Scheme 4, sarcosine ethyl ester hydrochloride is amide-coupled with ethyl succinyl chloride in the presence of a base to give intermediate (XVI), which can be further reacted in a Claisen condensation in the presence of a base (e.g., NaOEt) to give intermediate (XVII). Subsequent condensation with a formamidinium salt (e.g., formamidinium acetate) gives intermediate (XVIII), which can be alkylated with an alcohol (B) in the presence of a base (e.g., K₂CO₃) to give (Ib).

[0273] Compounds of formula (I) having E=S, SO and SO2, as illustrated in scheme 5 below, can be prepared.

[0274] Option 5:

[0275]

[0276] In Scheme 5, intermediate (XIX) can be synthesized by methylating 4-chloro-5H,6H,7H-pyrimidine with [4,5-b][1,4]thiazin-6-one using a methylating agent (e.g., MeI) in the presence of a base (e.g., NaH). Subsequently, hydrolysis of (XIX) in the presence of an acid (e.g., formic acid) yields intermediate (XX), which can be N-alkylated with an alcohol (B) in the presence of a base (e.g., K₂CO₃) to give (Ic).

[0277] Example

[0278] preparation

[0279] The compounds and intermediates according to the invention can be obtained using synthetic methods known to those skilled in the art and described in the literature on organic synthesis, for example, using 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 are obtained by a method of preparation similar to that described more fully below, especially as described in the experimental section. In some cases, the order in which the reaction scheme is carried out may vary. Variations of these reactions known to those skilled in the art but not described in detail herein may also be used. Based on the study of the following schemes, the general process for preparing the compounds according to the invention will become apparent to those skilled in the art. The starting compounds are commercially available or can be prepared by methods described in the literature or herein, or can be prepared in a similar or analogous manner. Prior to the reaction, any corresponding functional groups in the starting compounds may be protected with conventional protecting groups. These protecting groups can be re-cleaved at appropriate stages within the reaction sequence using methods familiar to those skilled in the art and described in, for example, "Protecting Groups," 3rd edition, Philip J. Kocienski, Thieme, 2005 and "Protective Groups in Organic Synthesis," 4th edition, Peter GMWuts, Theodora W. Greene, John Wiley & Sons, 2006. The terms "ambient temperature" and "room temperature" are used interchangeably and represent approximately 20°C, for example, a temperature between 19 and 24°C.

[0280] abbreviation:

[0281]

[0282]

[0283] Preparation of intermediates Intermediate I

[0284] Intermediate I.1 (General Program)

[0285] 2-[5-(chloromethyl)-2H-1,2,3,4-tetrazol-2-yl]-1-(4-chlorophenyl)ethyl-1-one

[0286]

[0287] At RT, 1.63 g (11.8 mmol) of K₂CO₃ was added with stirring to 15 mL of a DMA solution containing 1.00 g (8.44 mmol) of 5-(chloromethyl)-2H-1,2,3,4-tetraazole and 2.17 g (9.28 mmol) of 4-chlorobenzoylmethyl bromide. The reaction mixture was stirred at RT for 30 min and then filtered. The filtrate was diluted with water and a saturated aqueous NaCl solution and extracted three times with EtOAc. The combined organic phases were washed with water, dried over Na₂SO₄, filtered through activated carbon, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel; CH / EtOAc, 80 / 20 to 50 / 50 gradient) to give the product.

[0288] C 10 H8Cl2N4O (M = 271.1 g / mol)

[0289] ESI-MS: 271 [M+H] +

[0290] R t (HPLC): 1.01 min (Method B)

[0291] The following compounds were prepared using a procedure similar to that described in Intermediate I.1, with appropriate starting materials. As those skilled in the art will appreciate, these similar examples may involve variations of general reaction conditions.

[0292]

[0293]

[0294]

[0295] * p-Methoxybenzoylmethyl bromide (1.04 eq.) was slowly added to a stirred solution of chloromethyltetrazole and K2CO3 (1.4 eq) in DMA at 18 °C; the mixture was stirred at RT for 1.5 h; and purified by reversed-phase HPLC (ACN / H2O gradient, 0.1% TFA).

[0296] Intermediate II

[0297] Intermediate II.1 (General Program)

[0298] (1R)-2-[5-(chloromethyl)-2H-1,2,3,4-tetrazol-2-yl-]-1-(4-chlorophenyl)ethyl-1-ol

[0299]

[0300] 1.30 g (4.80 mmol) of 1-(4-chlorophenyl)-2-[5-(chloromethyl)-2H-1,2,3,4-tetraazol-2-yl]ethyl-1-one (I.1) was dissolved in 20 mL of ACN under an inert atmosphere. 12 mg (0.02 mmol) of ruthenium chloride ([(1S,2S)-2-amino-1,2-diphenylethyl](4-toluenesulfonyl)amido(mesene)ruthenium(II) (CAS 174813-81-1) was added, followed by dropwise addition of 0.72 mL (1.73 mmol) of a triethylamine formate complex (5:2). After stirring at RT for 3 h, the solvent was removed under reduced pressure. Water was added to the remaining crude mixture, and the mixture was extracted with EtOAc. The combined organic layers were dried over Na2SO4, filtered, treated with activated carbon, filtered again, and the solvent was removed under reduced pressure to give intermediate II.1.

[0301] C 10 H 10 Cl2N4O (M = 273.1 g / mol)

[0302] ESI-MS: 273 [M+H] +

[0303] R t (HPLC): 0.96 min (Method B)

[0304] The following compounds were prepared using a procedure similar to that described in Intermediate II.1, with appropriate starting materials. As those skilled in the art will appreciate, these similar examples may involve variations of general reaction conditions.

[0305]

[0306]

[0307]

[0308] Intermediate III

[0309] Intermediate III.1 (General Program)

[0310] 1-(5,6-Difluoro-1-benzofuran-2-yl)ethyl-1-one

[0311]

[0312] A 50 mL acetone solution of 5.00 g (31.6 mmol) of 4,5-difluoro-2-hydroxybenzaldehyde was treated with 6.99 g (50.6 mmol) of potassium carbonate under argon atmosphere at 0 °C. After stirring at 0 °C for another 10 min, 3.78 mL (47.4 mmol) of chloroacetone was added dropwise, and the reaction mixture was stirred at 70 °C for 3 h. The reaction mixture was cooled to RT and concentrated. The crude product was extracted with EtOAc / water, and the organic phase was concentrated under reduced pressure to give intermediate III.1.

[0313] C 10 H6F2O2 (M = 196.2 g / mol)

[0314] 1 H NMR(300MHz,DMSO-d6)δppm:2.56(s,3H),7.89(m,1H),7.92(m,1H),8.01(m,1H)

[0315] The following compounds were prepared using a procedure similar to that described in Intermediate III.1, with appropriate starting materials. As those skilled in the art will appreciate, these similar examples may involve variations of general reaction conditions.

[0316]

[0317] Intermediate IV

[0318] Intermediate IV.1 (General Program)

[0319] 2-Bromo-1-(5,6-difluoro-1-benzofuran-2-yl)ethyl-1-one

[0320]

[0321] A solution of 1-(5,6-difluoro-1-benzofuran-2-yl)ethyl-1-one (III.1) in 6 mL of THF was treated dropwise with a solution of 1.23 g (2.55 mmol) tetrabutylammonium tribromide in 300 μL MeOH and 3 mL THF. The reaction mixture was stirred at RT for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was extracted with EtOAc / water. The organic phase was concentrated under reduced pressure and the crude product was purified by column chromatography (silica gel; hexane / EtOAc, 9 / 1 to 7 / 3 gradient).

[0322] C 10 H5BrF2O2 (M=275.0g / mol)

[0323] 1H NMR(300MHz,DMSO-d6)δppm:4.83(s,2H),7.98-8.12(m,3H)

[0324] The following compounds were prepared using a procedure similar to that described in Intermediate IV.1, with appropriate starting materials. As those skilled in the art will appreciate, these similar examples may involve variations of general reaction conditions.

[0325]

[0326]

[0327] **: The reaction was carried out at RT with bromine (13.6 eq) in dioxane / diethyl ether for 2 h, and quenched with sodium thiosulfate solution.

[0328] intermediate V

[0329] 3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione

[0330]

[0331] At 65 °C, 7.5 mL (94.41 mmol) of chloroacetyl chloride was slowly added to 300 mL of 10.0 g (127.10 mmol) of 5-aminopyrimidine-4,6-diol in DMF solution with stirring. After stirring at 65 °C for 1.5 h, 36.1 mL (129.94 mmol) of DIPEA was slowly added to the reaction mixture, and stirring was continued at 65 °C for 45 min. The reaction mixture was concentrated under reduced pressure, treated with water, and the precipitate was filtered, washed with a small amount of EtOH, and dried.

[0332] C6H5N3O3 (M=167.1g / mol)

[0333] ESI-MS: 168 [M+H] +

[0334] R t (HPLC): 0.20 min (Method M)

[0335] Intermediate VI

[0336] 3-[(4-methoxyphenyl)methyl]-5-methyl-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione

[0337]

[0338] 680 mg (4.07 mmol) of 3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione (intermediate V) in 15 mL of DMF was treated with 843 mg (6.10 mmol) of potassium carbonate and stirred at RT for 15 min. 0.58 mL (4.27 mmol) of 1-(chloromethyl)-4-methoxybenzene was added, and the reaction mixture was stirred for 20 h. 843 mg (6.10 mmol) of potassium carbonate and 0.30 mL (4.88 mmol) of iodomethane were added, and the reaction mixture was stirred at RT for 20 h. 0.26 mL (4.07 mmol) of iodomethane was added, and the reaction mixture was stirred at RT for 20 h. The mixture was filtered, concentrated under reduced pressure, and purified by reversed-phase HPLC (ACN / H₂O gradient, 0.1% TFA).

[0339] C 15 H 15 N3O4 (M=301.3g / mol)

[0340] ESI-MS: 302 [M+H] +

[0341] R t (HPLC): 0.95 min (Method B)

[0342] Intermediate VII

[0343] 5-Methyl-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione

[0344]

[0345] 5.30 g (14.07 mmol) of 3-[(4-methoxyphenyl)methyl]-5-methyl-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione (intermediate VI) was treated with 16 ml of TFA and stirred at 100 °C for 1.5 h. Subsequently, the reaction mixture was stirred in a microwave at 120 °C for 15 min, poured over ice water, and filtered. The lyophilized filtrate was used directly without further purification.

[0346] C7H7N3O3 (M=181.2g / mol)

[0347] ESI-MS: 182 [M+H] +

[0348] R t (HPLC): 0.37 min (Method B)

[0349] Intermediate VIII

[0350] 7-Methyl-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione

[0351]

[0352] A mixture of 200 mg (1.57 mmol) 5-aminopyrimidine-4,6-diol and 1.44 g (9.44 mmol) 2-bromopropionic acid was stirred at 100 °C under pure conditions for 22.5 h. The reaction mixture was diluted with DCM, filtered, and the concentrated filtrate was purified by column chromatography (silica gel; DCM / MeOH, 1 / 0 to 7 / 3 gradient).

[0353] C7H7N3O3 (M=181.2g / mol)

[0354] ESI-MS: 182 [M+H] +

[0355] R t (HPLC): 0.23 min (Method E)

[0356] Intermediate IX

[0357] Intermediate IX.1 (General Program)

[0358] 3-({2-[(2R)-2-(4-chlorophenyl)-2-hydroxyethyl]-2H-1,2,3,4-tetrazol-5-yl}methyl)-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione

[0359]

[0360] To 3 mL of a solution of 61 mg (0.37 mmol) 3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione (V) in DMF, 76 mg (0.55 mmol) of potassium carbonate and 100 mg (0.37 mmol) of (1R)-2-[5-(chloromethyl)-2H-1,2,3,4-tetraazol-2-yl]-1-(4-chlorophenyl)ethyl-1-ol (II.1) were added and the mixture was stirred overnight at RT. The reaction mixture was quenched with water / ACN / TFA, filtered, and purified by reversed-phase HPLC (ACN / H2O gradient, 0.1% TFA) to yield the desired product.

[0361] C 16 H 14 ClN7O4 (M = 403.8 g / mol)

[0362] ESI-MS: 404 [M+H] +

[0363] R t (HPLC): 0.77 min (Method H)

[0364] The following compounds were prepared using a procedure similar to that described in Intermediate IX.1, with appropriate starting materials. As those skilled in the art will appreciate, these similar embodiments may involve variations of general reaction conditions.

[0365]

[0366]

[0367] Intermediate X

[0368] 2-Bromo-2,2-difluoro-N-(4-hydroxy-6-oxo-1,6-dihydropyrimidin-5-yl)acetamide

[0369]

[0370] 200 mg (1.57 mmol) of 5-aminopyrimidine-4,6-diol was treated with 456 mg (2.36 mmol) of bromodifluoroacetyl chloride and stirred at 90 °C for 3.5 h under pure conditions. The reaction mixture was wet-milled with diethyl ether and filtered to give intermediate X.

[0371] C6H4BrF2N3O3 (M=284.0g / mol)

[0372] ESI-MS: 284 / 286 [M+H] +

[0373] R t (HPLC): 0.10 min (Method E)

[0374] Intermediate XI

[0375] 7,7-Difluoro-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione

[0376]

[0377] A 2.5 mL solution of 30 mg (0.11 mmol) of 2-bromo-2,2-difluoro-N-(4-hydroxy-6-oxo-1,6-dihydropyrimidin-5-yl)acetamide (X) was treated with 16 mg (0.37 mmol) sodium hydride and stirred at 50 °C for 18 h. The reaction mixture was purified by reversed-phase HPLC (ACN / H₂O gradient, 0.1% TFA) to yield the desired product.

[0378] C6H3F2N3O3 (M=203.1g / mol)

[0379] ESI-MS: 204 [M+H] +

[0380] R t (HPLC): 0.23 min (Method A)

[0381] Intermediate XII

[0382] Ethyl 6-acetyl-1-benzofuran-2-carboxylate

[0383]

[0384] Add 616 mg (4.46 mmol) of potassium carbonate to 1.00 g (3.72 mmol) of ethyl 6-bromo-1-benzofuran-2-carboxylate (III.4) in 12.5 mL of DMF. Purge the mixture with argon and add 92 mg (0.22 mmol) of 1,3-bis(diphenylphosphine)propane, 250 mg (0.11 mmol) of palladium acetate (II), and 670 mg (9.29 mmol) of ethyl vinyl ether. Stir the reaction mixture at 80 °C for 18 h, then cool to RT and adjust the pH to 1 by adding 1 M HCl aqueous solution. Extract the crude product with EtOAc, concentrate under reduced pressure, and purify by column chromatography (silica gel; hexane / EtOAc 7 / 3).

[0385] C 13 H 12 O4 (M = 232.2 g / mol)

[0386] ESI-MS: 233 [M+H] +

[0387] R t (HPLC): 1.38 min (Method Q)

[0388] Intermediate XIII

[0389] 6-Acetyl-1-benzofuran-2-carboxylic acid

[0390]

[0391] To a solution of 6.60 g (28.4 mmol) ethyl 6-acetyl-1-benzofuran-2-carboxylate (XII) in 66 mL THF and 33 mL THF, 3.3 mL ethanol and 1.43 g (34.1 mmol) LiOH monohydrate were added. The reaction mixture was stirred at room temperature for 1 hour and concentrated to dryness under reduced pressure to give an intermediate.

[0392] C 11 H8O4 (M = 204.2 g / mol)

[0393] 1 H NMR(300MHz,DMSO-d6)δppm:2.67(s,3H),7.73(m,1H),7.99-7.85(m,2H),8.32(m,1H),13.30-14.50(br s,1H)

[0394] Intermediate XIV

[0395] 6-Acetyl-1-benzofuran-2-carboxamide

[0396]

[0397] At 0 °C, 932 mg (7.34 mmol) oxaloyl chloride and 1 drop of DMF were added to 10 mL of a solution of 1.00 g (4.90 mmol) of 6-acetyl-1-benzofuran-2-carboxylic acid (XIII) in DCM. The reaction mixture was stirred at RT for 2 h and concentrated to dryness. The residue was dissolved in 10 mL of THF, cooled to 0 °C, and 15 mL of 25% ammonia was added. The reaction mixture was stirred at RT for 16 h and concentrated to dryness under reduced pressure to give an intermediate.

[0398] C 11 H9NO3 (M=203.2g / mol)

[0399] ESI-MS: 204 [M+H] +

[0400] R t (HPLC): 0.96 min (Method Q)

[0401] intermediate XV

[0402] 6-Acetyl-1-benzofuran-2-carboxynitrile

[0403]

[0404] 1.1 mL (7.77 mmol) of TFAA was added dropwise to 12 mL of THF solution containing 0.90 g (4.43 mmol) of 6-acetyl-1-benzofuran-2-carboxamide (XIV) and 1.4 mL (9.88 mmol) of TEA at 0 °C with stirring. The reaction mixture was stirred at 0 °C for 1 hour, quenched with water, and extracted three times with EtOAc. The combined organic layers were washed with saturated NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the intermediate.

[0405] C 11 H7NO2 (M = 185.2 g / mol)

[0406] 1 H NMR (300MHz, DMSO-d6) δppm: 2.68 (s, 3H), 7.92-8.05 (m, 2H), 8.21 (m, 1H), 8.37 (m, 1H).

[0407] Intermediate XVI

[0408] Ethyl 3-[(2-ethoxy-2-oxoethyl)(methyl)carbamoyl]propionate

[0409]

[0410] TEA (3.40 mL, 24.4 mmol) was added to 30 mL of DCM solution containing 1.50 g sarcosine ethyl ester hydrochloride (9.77 mmol, CAS: 5260-49-9) with stirring at 0 °C, followed by the addition of ethyl succinyl chloride (1.53 mL, 10.74 mmol, CAS: 14794-31-3). The mixture was slowly heated to room temperature and stirred at RT for 3 hours, followed by washing three times with water. The organic layer was concentrated under reduced pressure and purified by column chromatography (silica gel; CyH / EtOAc, gradient).

[0411] C 11 H 19 NO5 (M = 245.27 g / mol)

[0412] ESI-MS: 246 [M+H] +

[0413] R t (HPLC): 1.05 min (Method M)

[0414] Intermediate XVII

[0415] ethyl 1-methyl-3,6-dioxopiperidine-2-carboxylate

[0416]

[0417] Under stirring and at RT, 56 mg (2.45 mmol) of sodium was slowly added to 4.0 mL of anhydrous dioxane solution containing 0.40 g (1.63 mmol) of ethyl 3-[(2-ethoxy-2-oxoethyl)(methyl)carbamoyl]propionate (XVI), followed by the slow addition of 0.2 mL of anhydrous EtOH. The reaction mixture was stirred at 80 °C for 12 hours, cooled to RT, and the pH was adjusted to pH 7 by adding an aqueous HCl solution (4N). The reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the intermediate.

[0418] C9H 13 NO4 (M = 199.20 g / mol)

[0419] ESI-MS: 200 [M+H] +

[0420] R t (HPLC): 0.75 min (Method H)

[0421] Intermediate XVIII

[0422] 1-Methyl-1,2,3,4,7,8-hexahydro-1,7-naphthidine-2,8-dione

[0423]

[0424] At 0 °C, 0.60 mL of a 25 wt% sodium methoxide solution in MeOH was slowly added to a stirred mixture of 0.13 g (0.52 mmol) of 1-methyl-3,6-dioxopiperidine-2-carboxylate (XVII) and 0.27 g of formamidine acetate (2.61 mmol) in 5.0 mL of anhydrous MeOH. The reaction mixture was then refluxed for 4 hours, cooled to 0 °C, neutralized with acetic acid, and concentrated under reduced pressure. The desired product was obtained by purification by reversed-phase HPLC (ACN / H₂O gradient, 0.1% TFA).

[0425] C8H9N3O2 (M=179.18g / mol)

[0426] ESI-MS: 180 [M+H] +

[0427] R t (HPLC): 0.08 min (Method J)

[0428] intermediate XIX

[0429] 4-Chloro-5-methyl-5H,6H,7H-pyrimidino[4,5-b][1,4]thiazin-6-one

[0430]

[0431] Under argon atmosphere, 0.12 g (2.73 mmol) of sodium hydride was added to 3.0 mL of a solution of 0.50 g (2.48 mmol) of 4-chloro-5H,6H,7H-pyrimidino[4,5-b][1,4]thiazin-6-one (CAS: 20015-70-7) in DMF and stirred at room temperature for 20 minutes. Iodomethane (0.17 mL, 2.73 mmol) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with brine and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the intermediate.

[0432] C7H6ClN3OS (M=215.66g / mol)

[0433] ESI-MS: 215 [M+H] +

[0434] R t (HPLC): 0.80 min (Method B)

[0435] Intermediate XX

[0436] 5-Methyl-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]thiazin-6-one

[0437]

[0438] 0.34 g (1.58 mmol) of 4-chloro-5-methyl-5H,6H,7H-pyrimidinol[4,5-b][1,4]thiazin-6-one (XIX) was stirred in 4.0 mL of formic acid at 90 °C for 3 h and then stirred overnight at room temperature. The reaction mixture was then concentrated under reduced pressure and refluxed in EtOH with stirring for 30 min. After slow cooling to room temperature, the product was filtered off, washed with EtOH, and dried under reduced pressure.

[0439] C7H7N3O2S (M=197.22g / mol)

[0440] ESI-MS: 198 [M+H] +

[0441] R t (HPLC): 0.53 min (Method B)

[0442] Preparation of the final compound

[0443] Example 1 (General Program A)

[0444] 3-({2-[(2R)-2-(4-chlorophenyl)-2-hydroxyethyl]-2H-1,2,3,4-tetrazol-5-yl}methyl)-5-methyl-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione

[0445]

[0446] To 7 mL of 210 mg (0.77 mmol) of (1R)-2-[5-(chloromethyl)-2H-1,2,3,4-tetraazol-2-yl]-1-(4-chlorophenyl)ethyl-1-ol (II.1) in DMF, 159 mg (1.15 mmol) of K₂CO₃ and 185 mg (0.77 mmol) of 5-methyl-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione (VII) were added and the mixture was stirred overnight at room temperature. The mixture was purified by reversed-phase HPLC (ACN / H₂O gradient, 0.1% TFA) to yield the desired product.

[0447] C 17 H 16 ClN7O4 (M = 417.8 g / mol)

[0448] ESI-MS: 418 [M+H] +

[0449] R t (HPLC): 0.81 min (Method H)

[0450] 1 H NMR (400MHz, DMSO-d6) δppm: 3.33 (s, 3H), 4.74-4.84 (m, 4H), 5.10-5.16 (m, 1H), 5.45 (s, 2H), 5.92 (d, J = 4.8Hz, 1H), 7.36-7.43 (m, 4H), 8.46 (s, 1H)

[0451] The following compounds were prepared using a procedure similar to that described in Example 1, General Procedure A, with appropriate starting materials.

[0452] As those skilled in the art will understand, these similar embodiments may involve variations of general reaction conditions.

[0453]

[0454]

[0455]

[0456] The analytical data for the compounds are described in the table above:

[0457]

[0458]

[0459]

[0460] Example 1 (General Program B)

[0461] 3-({2-[(2R)-2-(4-chlorophenyl)-2-hydroxyethyl]-2H-1,2,3,4-tetrazol-5-yl}methyl)-5-methyl-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione

[0462]

[0463] To 15 mL of a solution of 0.74 g (1.83 mmol) of 3-({2-[(2R)-2-(4-chlorophenyl)-2-hydroxyethyl]-2H-1,2,3,4-tetrazol-5-yl}methyl)-3H,4H,5H,6H,7H-pyrimidino[4,5-b][1,4]oxazine-4,6-dione (IX.1) in DMF, 0.43 g (3.12 mmol) of potassium carbonate and 0.17 mL (2.75 mmol) of iodomethane were added. The reaction mixture was stirred overnight at room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with water and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under reduced pressure to give Example 1.

[0464] The following compounds were prepared using a procedure similar to that described in Example 1, General Procedure B, with appropriate starting materials.

[0465] As those skilled in the art will understand, these similar embodiments may involve variations of general reaction conditions.

[0466]

[0467] *The absolute configuration of the three-dimensional center in Example 23 is assigned via X-ray.

[0468] The analytical data for the compounds are described in the table above:

[0469]

[0470]

[0471] Analytical HPLC methods

[0472] Method A

[0473]

[0474] Analytical column: XBridge BEH C18 2.1×30mm, 1.7μm; column temperature: 60℃

[0475] Method B

[0476]

[0477] Analytical column: Stable Bond (Agilent) 1.8 μm; 3.0 × 30 mm; column temperature: 60℃

[0478] Method C

[0479]

[0480] Analytical column: Sunfire C18 (Waters) 2.5μm; 3.0×30mm; column temperature: 60℃

[0481] Method D

[0482]

[0483] Analytical column: XBridge C18 3.0×30mm 2.5μm (Waters); Column temperature: 60℃

[0484] Method E

[0485]

[0486] Analytical column: XSelect HSS PFP (Waters) 2.1×30mm 1.8μm; Column temperature: 60℃

[0487] Method F

[0488]

[0489] Analytical column: Sunfire (Waters) C18 3.0 × 30 mm 2.5 μm; Column temperature: 60℃

[0490] Method G

[0491]

[0492] Analytical column: Zorbax StableBond C18 (Agilent) 1.8 μm; 2.1 × 30 mm; column temperature: 60℃

[0493] Method H

[0494]

[0495] Analytical column: Sunfire (Waters) 2.5 μm; 3.0 × 30 mm; column temperature: 60℃

[0496] Method I

[0497]

[0498] Analytical column: Sunfire (Waters); C18 3.0 × 30 mm 2.5 μm; column temperature: 60℃

[0499] Method J

[0500]

[0501]

[0502] Analytical column: Xbridge (Waters); C18 3.0 × 30 mm 2.5 μm; column temperature: 60℃

[0503] Method K

[0504]

[0505] Analytical column: Sunfire (Waters); C18 3.0 × 30 mm 2.5 μm; column temperature: 60℃

[0506] Analytical column: Sunfire (Waters); C18 3.0 × 30 mm 2.5 μm; column temperature: 60℃

[0507] Method L

[0508]

[0509] Analytical column: Chiral Art Cellulose (YMC); SJ 4.6 × 250 mm 5 μm; Column temperature: 40 °C; Back pressure: 2175.0 psi

[0510] Method M

[0511]

[0512] Analytical column: Zorbax StableBond (Agilent) C18 3.0×30mm 1.8μm; Column temperature: 60℃

[0513] Method N

[0514]

[0515]

[0516] Analytical column: Kinetex XB; C18 4.6 × 50 mm 2.6 μm; column temperature: 25℃

[0517] Method O

[0518]

[0519] Analytical column: Acquity UPLC BEH; C8 2.1 × 150 mm 1.7 μm; column temperature: 55℃

[0520] Method P

[0521]

[0522] Analytical column: Xbridge (Waters); C18 3.0 × 30 mm 2.5 μm; column temperature: 60℃

[0523] Method Q

[0524] Time (min) water volume % ACN volume % Flow rate [mL / min] 0.0 80 20 0.5 0.1 80 20 0.5 1.1 0 100 0.5 2.5 80 20 0.5 3.0 80 20 0.5

[0525] Analytical column: Acquity UPLC BEH; C18 2.1×100mm 1.7μm; column temperature: 40℃

Claims

1. A compound according to formula (I) wherein A is selected from: E is selected from O, S and CH2; and R 1 and R 2 are independently selected from H, CH3, and F.

2. The compound of formula (I) according to claim 1, wherein E is O.

3. The compound of formula (I) according to claim 1, wherein R 1 and R 2 are H.

4. The compound of formula (I) according to claim 1, selected from:

5. The compound of formula (I) according to claim 1, selected from:

6. The compound of formula (I) according to claim 1, which is ###0002### 7. The compound of formula (I) according to claim 1, which is ###0002### 8. The compound of formula (I) according to claim 1, which is 9. The compound of formula (I) according to claim 1, which is ###0002### 10. The compound of formula (I) according to claim 1, which is ###0002### 11. The compound of formula (I) according to claim 1, which is ###00005### (I) 12. The compound of formula (I) according to claim 1, which is ###00006### (I) 13. The compound of formula (I) according to claim 1, which is ###0002### 14. The compound of formula (I) according to claim 1, which is ###00006### (I) 15. The compound of formula (I) according to claim 1, which is 16. A pharmaceutically acceptable salt of a compound according to any one of claims 1 to 15.

17. A pharmaceutical composition comprising at least one compound of formula (I) according to any one of claims 1 to 15 or a pharmaceutically acceptable salt according to claim 16, and one or more pharmaceutically acceptable excipients.

Images