HSD17B13 inhibitors

Abstract

The invention encompasses heteroaryl-substituted 2, 6-difluorophenol compounds of formula (I) wherein the groups A1 to A3 and Z have the meanings given in the claims and the description; the use of said compounds in pharmaceutical compositions containing these compounds and their use as medicaments, in particular for interfering with the progression of liver diseases from steatosis to the advanced stage of non-alcoholic steatohepatitis, fibrosis and cirrhosis. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to heteroaryl-substituted 2,6-difluorophenol compounds of formula (I),

[0002]

[0003] wherein A1 to A3 and Z have the meanings given in the claims and the description. In addition, their use as HSD17B13 inhibitors, pharmaceutical compositions containing said compounds and their use as medicaments (especially as medicaments for interfering with steatosis) are disclosed. Background Art

[0004] WO 2021 / 211974 and WO 2022 / 020714 disclose thiophene-carboxamide HSD17B13 inhibitors.

[0005] WO 2022 / 020730 discloses quinazolinone HSD17B13 inhibitors.

[0006] HSD17B13 is a member of the 17b-hydroxysteroid dehydrogenase family of oxidoreductases that act together on a range of lipid substrates.

[0007] In humans, HSD17B13 mRNA is expressed most abundantly in the liver, primarily in hepatocytes. Within the cells, HSD17B13 is associated with lipid droplets (Su et al., Proc National Acad Sci. 111: 11437-11442, 2014).

[0008] The physiological function of HSD17B13 is uncertain, and several substrates (including estradiol, retinol, and leukotriene B4) have been identified using an in vitro enzyme assay system in which NAD + (nicotinamide adenine dinucleotide, oxidized form) serves as a cosubstrate (Abdul-Husn et al., The New England Journal of Medicine. 378: 1096-1106, 2018).

[0009] Loss-of-function (LoF) genetic variants in humans provide evidence for a role for HSD17B13 activity in mediating the risk of certain liver diseases. The presence of the single nucleotide polymorphism (SNP) rs72613567, encoding a truncated, enzymatically inactive protein, is associated with liver diseases such as liver fibrosis or nonalcoholic steatohepatitis (NASH).

[0010] SNP rs72613567SNP also moderates the increased risk of liver disease.

[0011] The SNP rs72613567 was found to be less frequent in liver transplant recipients than in healthy controls.

[0012] SNP rs62305723, encoding the HSD17B13 LoF variant, is associated with reduced severity of NASH (Ma et al., Hepatology 69:1504-1519, 2018).

[0013] SNP rs80182459, encoding a possible LoF variant, has been found to be less frequent in certain patients with chronic liver disease (Kozlitina et al., The New England Journal of Medicine 379:1876-1877, 2018).

[0014] SNP rs6834314, which was highly correlated with SNP rs72613567, was also found to be associated with fatty liver disease.

[0015] Hepatocyte-directed small interfering RNA (siRNA) designed to deplete HSD17B13 in human livers was found to reduce serum alanine aminotransferase (ALT) activity, a biomarker of liver damage, in five patients with fatty liver disease.

[0016] In view of the above-mentioned data, it would be desirable to provide potent HSD17B13 inhibitors.

[0017] According to the present invention, "one or more HSD17B13 inhibitors" means compounds that inhibit HSD17B13 in the tests shown in Examples 4 and 6. DETAILED DESCRIPTION

[0018] It is not known that the compounds of the formula (I), in which the radicals A1 to A3 and Z have the meanings given below, act as HSD17B (17β-hydroxysteroid dehydrogenase) inhibitors selective for HSD17B13, as shown in Example 12 by comparative biochemical human IC against HSD17B11. 50 The data are shown. Therefore, the compounds according to the invention can be used, for example, to treat steatosis, such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0019] Therefore, the present invention relates to a compound of formula (I) or a salt thereof,

[0020]

[0021] a) Where Z-* is

[0022]

[0023]

[0024] and b) where the structure Selected from the group consisting of:

[0025]

[0026]

[0027]

[0028] The skilled artisan will appreciate that, for example, several heteroaryls may be described in the form of different tautomers (i.e., pyrazole, triazole, imidazole). Thus, the compounds of the invention may exist in the form of tautomers. For example, any compound of the invention containing a pyrazole moiety as a heteroaryl may exist in the form of a 1H tautomer, or a 2H tautomer, or even a mixture of two tautomers in any amount, or a triazole moiety may exist in the form of a 1H tautomer, a 2H tautomer, or a 4H tautomer, or even a mixture of said 1H, 2H, or 4H tautomers in any amount, i.e.:

[0029]

[0030] 1H-tautomer 2H-tautomer

[0031]

[0032] 1H-tautomer 2H-tautomer 4H-tautomer

[0033] The present invention includes all possible tautomers of the compounds of the present invention as single tautomers or as any mixture of said tautomers in any ratio.

[0034] In particular, the structure Can be

[0035]

[0036] In a group of compounds according to the invention, The structure is selected from the group consisting of:

[0037]

[0038] In another group of compounds of the present invention, The structure is selected from the group consisting of:

[0039]

[0040] In yet another group of compounds of the present invention, The structure is selected from the group consisting of:

[0041]

[0042] Z is preferably

[0043] The most preferred structure of Z is

[0044] The present invention relates to compounds of formula (I) or salts thereof that interfere with lipogenesis, wherein selective inhibition of HSD17B13 has therapeutic benefits, including but not limited to the treatment of non-alcoholic steatohepatitis. Therefore, in another aspect of the present invention, the compound of formula (I) or a pharmaceutically acceptable salt thereof is used as a medicament. The present invention also relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in a method for treating the human or animal body.

[0045] Particularly interesting is the use of one of the compounds of formula (I) or its salt in the treatment of metabolic disorders. Therefore, another aspect of the present invention is the use of a compound of formula (I) or its pharmaceutically acceptable salt for the preparation of a pharmaceutical composition, the pharmaceutical composition comprising at least one compound of formula (I) or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier for the treatment of one of the disorders. Particularly preferred are their uses in the preparation of a pharmaceutical composition to regulate metabolic disorders in humans or animals.

[0046] Also contemplated within the context of the present invention are methods of treating liver disease, metabolic disease, or cardiovascular disease using a compound disclosed herein, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, in combination with an additional therapeutic agent.

[0047] In some embodiments, the additional therapeutic agent is used to treat diabetes or a diabetes-related disorder or condition.

[0048] In some instances, the additional therapeutic agent comprises a statin, an insulin sensitizing drug, an insulin secretagogue, an α-glucosidase inhibitor, a GLP agonist, a THR β-agonist, a PDE inhibitor, a DPP-4 inhibitor (such as sitagliptin, vildagliptin, saxagliptin, linagliptin, anaelagliptin, teneligliptin, alogliptin, gemagliptin or dulagliptin), a catecholamine (such as epinephrine, norepinephrine or dopamine), a peroxisome proliferator-activated A peroxisome proliferator-activated receptor (PPAR)-γ agonist (e.g., a thiazolidinedione (TZD) [such as pioglitazone, rosiglitazone, rivoglitazone, or troglitazone]), aleglitazar, faraglitazar, muraglitazone, or tesaglitazar, a peroxisome proliferator-activated receptor (PPAR)-α agonist, a peroxisome proliferator-activated receptor (PPAR)-δ agonist, a farnesoid X receptor (FXR) agonist (e.g., obeticholic acid), or a combination thereof.

[0049] In some cases, statins are HMG-CoA reductase inhibitors.

[0050] In other cases, additional therapeutic agents include fish oil, fibrates, vitamins (such as nicotinic acid, retinoic acid (e.g., 9-cis retinoic acid), nicotinamide riboside or its analogs), or combinations thereof. In other cases, additional therapeutic agents include ACC inhibitors, FGF19 and FGF21 mimetics, CCR3 / CCR5 antagonists or combinations thereof.

[0051] In some embodiments, the additional therapeutic agent is vivitrol.

[0052] In some embodiments, the additional therapeutic agent is a statin, such as an HMG-CoA reductase inhibitor, fish oil, a fibrate, niacin, or a combination thereof. In other instances, the additional therapeutic agent is a dyslipidemia drug that prevents lipid absorption, such as orlistat.

[0053] In some embodiments, the additional therapeutic agent is a vitamin (such as retinoic acid or tocopherol acetate) used to treat diabetes and diabetes-related disorders or conditions (such as reducing weight gain due to food intake and / or reducing blood glucose increases due to food intake).

[0054] In some embodiments, the additional therapeutic agent is a glucose-lowering agent.

[0055] In some embodiments, the additional therapeutic agent is an anti-obesity agent.

[0056] In some embodiments, the additional therapeutic agent is selected from the group consisting of peroxisome proliferator-activated receptor (PPAR) agonists (gamma, dual or full), dipeptidyl peptidase (IV) inhibitors, glucagon-like peptide-1 (GLP-1) analogs, insulin or insulin analogs, insulin secretagogues, sodium glucose cotransporter 2 (SGLT2) inhibitors, Glucophages, human amylin analogs, biguanides, alpha-glucosidase inhibitors, meglitinides, thiazolidinediones, and sulfonylureas.

[0057] In some embodiments, the additional therapeutic agent is a lipid-lowering agent.

[0058] In some embodiments, the additional therapeutic agent is an antioxidant, a corticosteroid such as budesonide, an anti-tumor necrosis factor (TNF), or a combination thereof.

[0059] In some embodiments, the additional therapeutic agent is administered concurrently with the compounds disclosed herein.

[0060] In some embodiments, the additional therapeutic agent is administered less frequently than the compounds disclosed herein.

[0061] In some embodiments, the additional therapeutic agent is administered more frequently than the compounds disclosed herein.

[0062] In some embodiments, the additional therapeutic agent is administered prior to administration of a compound disclosed herein.

[0063] In some embodiments, an additional therapeutic agent is administered following administration of a compound disclosed herein.

[0064] In the treatment of mammals, the compounds according to the invention may be administered before, after or together with at least one other active substance or agent, such as a diuretic, antihypertensive, lipid-lowering agent or antidiabetic agent.

[0065] Preparations

[0066] Suitable formulations for administering the compounds of the invention will be clear to those of ordinary skill in the art and include, for example, tablets, pills, capsules, suppositories, lozenges, troches, solutions, elixirs, syrups, sachets, emulsions, inhalants, dispersible powders, etc. Preferred solutions are solutions for injection (sc, iv, im) or solutions for infusion (injectables).

[0067] The content of the pharmaceutically active compound needs to be an amount sufficient to achieve the dosage range specified below, for example in the range of 0.10 wt.-% to 90 wt.-%, preferably 0.5 wt.-% to 50 wt.-% of the composition as a whole. If necessary, the specified dose can be given several times a day.

[0068] Suitable tablets can be obtained, for example, by mixing one or more active substances of the present invention with known excipients, such as inert diluents, carriers, disintegrants, adjuvants, surfactants, binders and / or lubricants.

[0069] Coated tablets can be prepared accordingly by coating a core produced similarly to the tablets with a substance commonly used for tablet coatings, such as collidone or shellac, gum arabic, talc, titanium dioxide or sugar. To achieve delayed release or to prevent incompatibilities, the core can consist of multiple layers. Similarly, the tablet coating can consist of multiple layers to achieve delayed release, possibly using the excipients mentioned above for tablets.

[0070] Syrups or elixirs containing the active substances according to the invention or combinations thereof may additionally contain sweeteners (such as saccharin, cyclamates, glycerol or sugar) and flavour enhancers (e.g. flavouring agents such as vanillin or orange extract). They may also contain a suspending aid or thickener (such as sodium carboxymethylcellulose), a wetting agent (such as, for example, a condensation product of a fatty alcohol with ethylene oxide) or a preservative (such as a p-hydroxybenzoate).

[0071] Solutions for injection and infusion are prepared in a conventional manner, for example by adding isotonic agents, preservatives (such as p-hydroxybenzoic acid esters) or stabilizers (such as alkali metal salts of ethylenediaminetetraacetic acid), optionally using emulsifiers and / or dispersants, while if water is used as a diluent, for example, organic solvents may optionally be used as solvating agents or dissolution aids, and transferred to injection vials or ampoules or infusion bottles.

[0072] Capsules can be prepared, for example, by mixing the active substances with inert carriers, such as lactose or sorbitol, and packing them into gelatin capsules.

[0073] Suitable suppositories can be prepared, for example, by mixing with a carrier provided for this purpose, such as a neutral fat or polyethylene glycol or a derivative thereof.

[0074] Excipients that can be used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins (e.g., petroleum fractions), vegetable oils (e.g., peanut or sesame oil), monofunctional or polyfunctional alcohols (e.g., ethanol or glycerol), carriers such as natural mineral powders (e.g., kaolin, clay, talc, chalk), synthetic mineral powders (e.g., highly dispersed silicic acid and silicates), sugars (e.g., sucrose, lactose and glucose), emulsifiers (e.g., lignin, spent sulfite liquid, methylcellulose, starch and polyvinyl pyrrolidone) and lubricants (e.g., magnesium stearate, talc, stearic acid and sodium lauryl sulfate).

[0075] By conventional methods, preferably by oral or transdermal route, most preferably by oral route administration preparation.For oral administration, in addition to the carrier mentioned above, tablets can certainly contain additives, such as sodium citrate, calcium carbonate and dicalcium phosphate, together with various additives such as starch, preferably potato starch, gelatin.In addition, lubricants (such as magnesium stearate, sodium lauryl sulfate and talcum) can be used for tableting process simultaneously.In aqueous suspensions, in addition to the excipients mentioned above, active substances can also be combined with a variety of flavor enhancers or coloring agents.

[0076] For parenteral use, solutions of the active substance with suitable liquid carriers may be used.

[0077] The total amount of the active ingredient of formula (I) to be applied will generally be in the range of about 0.001 mg / kg body weight to about 200 mg / kg body weight per day, preferably in the range of about 0.01 mg / kg body weight to about 20 mg / kg body weight per day. Clinically useful dosage regimens will be in the range of one to three times daily administration to once every four weeks. In addition, "drug holiday" (wherein the patient is administered without drug in a certain time period) may be beneficial to the overall balance between pharmacological action and tolerance. The unit dose may contain about 0.5 mg to about 1500 mg of active ingredient, and may be applied once or more or less than once a day. The average daily dose applied by injection (including intravenous injection, intramuscular injection, subcutaneous injection and parenteral injection) and using infusion technology will preferably be 0.01 mg / kg to 200 mg / kg of total body weight. The average daily rectal dosage regimen will preferably be 0.01 mg / kg to 200 mg / kg of total body weight. The average daily vaginal dosage regimen will preferably be 0.01 mg / kg to 200 mg / kg of total body weight. The average daily topical dosage regimen will preferably be 0.1 mg to 200 mg, applied one to four times a day. The transdermal concentration will preferably be the concentration required to maintain a daily dose of 0.01 mg / kg to 200 mg / kg. The average daily inhalation dosage regimen will preferably be 0.01 mg / kg to 100 mg / kg of total body weight.

[0078] Sometimes it may be necessary to deviate from the specified amount, depending on the person's weight, age, route of administration, severity of the disease, individual response to the drug, the nature of the formulation, and the time or interval of administration of the drug (continuous or intermittent treatment of one or more doses per day). Therefore, in some cases, it may be sufficient to use a dose less than the minimum dose given above, while in other cases, the upper limit may have to be exceeded. When a higher dose is administered, it may be appropriate to divide the higher dose into a plurality of smaller doses spread throughout the day.

[0079] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which the parent compound is modified by preparing acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; etc.

[0080] For example, such salts include those derived from benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gentisic, hydrobromic, hydrochloric, maleic, malic, malonic, mandelic, methanesulfonic, 4-methyl-benzenesulfonic, phosphoric, salicylic, succinic, sulfuric, and tartaric acids.

[0081] Other pharmaceutically acceptable salts can be formed with cations derived from ammonia, L-arginine, calcium, 2,2'-iminobisethanol, L-lysine, magnesium, N-methyl-D-glucamine, potassium, sodium, and tris(hydroxymethyl)-aminomethane.

[0082] Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing alkaline or acidic moieties by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a sufficient amount of a suitable base or acid in water or in an organic diluent such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile or a mixture thereof.

[0083] Preparation of the compounds according to the invention

[0084] General

[0085] Unless otherwise stated, all reactions were performed in commercially available apparatus using methods commonly used in chemical laboratories. Starting materials sensitive to air and / or moisture were stored under protective gas, and the corresponding reactions and operations using them were performed under protective gas (nitrogen or argon).

[0086] The compounds according to the invention were named according to CAS conventions using the software MarvinSketch (Chemaxon).

[0087] Compounds according to the present 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 illustrate the present invention rather than to limit the scope of the compounds claimed for these embodiments and their subject matter. Where the preparation of the starting compounds is not described, they are commercially available or can be prepared similarly to known compounds or methods as described herein. The materials described in the literature are prepared according to the disclosed synthetic methods.

[0088] Overview

[0089] Synthesis of compounds:

[0090] The compounds of the present invention can be prepared as described in the following section. The schemes and procedures described below illustrate the general synthetic routes of the compounds of the general formula (I) of the present invention, and are not intended to be restrictive. It is clear to those skilled in the art that the transformation sequence illustrated in the scheme can be modified in a variety of ways. Therefore, the transformation sequence illustrated in these schemes is not intended to be restrictive. In addition, the interchange of any substituent can be achieved before and / or after the illustrated conversion. These modifications can be achieved in the following ways: introducing a protecting group, cutting a protecting group, exchanging, reducing or oxidizing a functional group, halogenating, metallizing, substituting or other reactions known to those skilled in the art. These conversions include those conversions that introduce functionality that allows substituents to be further interchanged. Suitable protecting groups and their introduction and cutting are well known to those skilled in the art (see, for example, PGM Wuts and TW Greene's " Protective Groups in Organic Synthesis ", 4th edition, Wiley 2006). Specific examples are described in the following paragraphs. Furthermore, it may be possible to carry out two or more consecutive steps without work-up between the steps, for example in a "one-pot" reaction, as is well known to those skilled in the art.

[0091] The synthesis of heteroaryl-substituted 2,6-difluorophenol compounds according to the present invention is preferably carried out according to the general synthetic sequence shown in Schemes 1-3.

[0092]

[0093] Scheme 1: Route to prepare compounds of the general formula 8 and 9, wherein A1, A2 and A3 have the same meanings as given above for formula (I), X has the meaning of Cl, Br, I, mesylate or triflate, and Y has the meaning of Cl, Br or I, and R has the meaning of alkyl.

[0094] Preparation of starting materials (Scheme 1):

[0095] Heterocyclic compounds of the general formula 1, 2, 3, 4, 5, 6 or 7 (Scheme 1) are commercially available or described in the literature.

[0096] Step 1-->2 (Solution 1)

[0097] Halogenation reaction

[0098] The conversion of compounds of general formula 1 to compounds of formula 2 is known to the skilled person. For Y = Br, the reaction can be carried out with reagents such as bromine, N-bromosuccinimide or copper (II) bromide. For Y = Cl, the reaction can be carried out with reagents such as N-chlorosuccinimide or chlorine. For Y = I, the reaction can be carried out with reagents such as iodine or N-iodosuccinimide.

[0099] Step 2-->8 (Solution 1)

[0100] Reduction of esters to alcohols

[0101] The conversion of compounds of general formula 2 to alcohols of formula 8 is known to the skilled person and can be carried out using reagents such as lithium aluminum hydride, sodium borohydride, calcium borohydride, diisobutylaluminum hydride.

[0102] Step 3-->8 (Solution 1)

[0103] Reduction of carboxylic acids to alcohols

[0104] The conversion of compounds of general formula 3 to alcohols of formula 8 is known to the skilled person and can be carried out using reagents such as lithium aluminum hydride.

[0105] Step 4-->8 (Solution 1)

[0106] Reduction of aldehydes to alcohols

[0107] The conversion of compounds of general formula 4 to alcohols of formula 8 is known to the skilled person and can be carried out using reagents such as sodium borohydride or lithium aluminum hydride.

[0108] Step 5-->6 (Solution 1)

[0109] Metal-catalyzed methylation reactions

[0110] The conversion of compounds of general formula 5 to compounds of general formula 6 is known to the skilled person and can be carried out, for example, under Suzuki conditions in an organic solvent (such as DMF or dioxane) using a reagent such as trimethylcyclotriboroxane, a palladium catalyst such as (1,1'-bis(diphenylphosphino)ferrocene)dichloropalladium(II) or tetrakis(triphenylphosphine)palladium(0) and a base such as potassium carbonate.

[0111] Step 7-->6 (Solution 1)

[0112] Halogenation reaction

[0113] The conversion of compounds of general formula 7 to compounds of general formula 6 is known to the skilled person. For Y = Br, the reaction can be carried out with reagents such as bromine, N-bromosuccinimide or copper (II) bromide. For Y = Cl, the reaction can be carried out with reagents such as N-chlorosuccinimide or chlorine. For Y = I, the reaction can be carried out with reagents such as iodine or N-iodosuccinimide.

[0114] Step 6-->8 (Solution 1)

[0115] Hydroxylation of methyl groups

[0116] The conversion of compounds of general formula 6 to alcohols of formula 8 is known to the skilled person and can be carried out using reagents such as selenium(IV) dioxide or tert-butyl hydroperoxide / manganese triacetate.

[0117] Step 8-->9 (Solution 1)

[0118] Convert hydroxyl groups to halogens (Br, Cl, I), mesylates or triflates

[0119] The conversion of alcohol 8 to a halogenated compound of formula 9 can be carried out, for example, using a chlorinating reagent such as thionyl chloride, methanesulfonyl chloride / triethylamine or triphenylphosphine / tetrachloromethane (for X=Cl). For X=Br, reagents such as phosphorus tribromide, trimethylsilyl bromide, hydrogen bromide, carbon tetrabromide / triphenylphosphine or boron tribromide / triphenylphosphine can be used. For X=I, reagents such as boron trifluoride etherate / potassium iodide, 1H-imidazole / iodine / triphenylphosphine can be used. For X=mesylate, reagents such as methanesulfonyl chloride and a base such as triethylamine can be used. For X=trifluoromethanesulfonate, reagents such as trifluoromethanesulfonic anhydride and a base such as pyridine can be used.

[0120] Step 7-->9 (Solution 1)

[0121] Free Radical Halogenation of Methyl

[0122] The conversion of compounds of general formula 7 to halides of formula 9 is known to the skilled person. Typically, a free radical initiator (such as dibenzoyl peroxide or 2,2'-azobis(isobutyronitrile)) and a halogenating agent are used. For X = Br, the reaction can be carried out with a halogenating agent such as N-bromosuccinimide. For X = Cl, the reaction can be carried out with a halogenating agent such as N-chloro-succinimide.

[0123]

[0124] Scheme 2: Route for the preparation of compounds of the general formula 11, 12 and 14, wherein R has the meaning of hydrogen or alkyl and R1 may be a protecting group known to those skilled in the art (see for example TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999).

[0125] Preparation of starting materials (Scheme 2):

[0126] Compounds of the general formula 10, 11, 12, 13 or 14 (Scheme 2) are commercially available or described in the literature. 2,6-Difluorophenol is commercially available.

[0127] Step 2, 6-Difluorophenol --> 10 (Scheme 2)

[0128] Introduction of phenol protecting group

[0129] The phenol group of 2,6-difluorophenol can be masked with a suitable protecting group R1 to generate compounds of general formula 10. The reaction conditions for introducing such suitable protecting groups R1 are known to the skilled person (see, for example, Green, Wuts, "Protective groups in organic synthesis" 1999, John Wiley & Sons and references therein). Preferably, benzyl, p-methoxybenzyl and 3,4-methoxybenzyl are used as protecting groups during the synthesis.

[0130] Step 10-->11 (Solution 2)

[0131] Introduction of boric acid or boric acid ester

[0132] The conversion of compounds of general formula 10 to boronic acid derivatives of formula 11 is known to the skilled person. For R = H, the reaction can be carried out with a reagent such as triisopropyl borate and a base such as n-butyl lithium, followed by an acidic work-up of the reaction. For RR = -C(CH3)2-C(CH3)2-, the reaction can be carried out with a reagent such as bis(pinacol)diborane and a catalyst such as Pt(N,N'-dicyclohexylimidazole-2-ylidene)(divinyltetramethylsiloxane).

[0133] Step 11-->12 (Solution 2)

[0134] Removal of phenol protecting group

[0135] Removal of the protecting group R1 from the compound of formula 11 generates the compound of formula 12. The reaction conditions for removing such suitable protecting groups R1 are known to the skilled person (see, for example, Green, Wuts, "Protective groups in organic synthesis" 1999, John Wiley & Sons and references therein). Preferably, benzyl, p-methoxybenzyl and 3,4-methoxybenzyl are used as protecting groups during the synthesis, which can be removed by hydrogenation.

[0136] Step 13-->14 (Solution 2)

[0137] Introduction of phenol protecting group

[0138] Compounds of general formula 13 can be masked with suitable protecting groups R1 to generate compounds of general formula 14. The reaction conditions for introducing such suitable protecting groups R1 are known to the skilled person (see, for example, Green, Wuts, "Protective groups in organic synthesis" 1999, John Wiley & Sons and references therein). Benzyl, p-methoxybenzyl and 3,4-methoxybenzyl are preferably used as protecting groups during the synthesis.

[0139] Step 14-->11 (Solution 2)

[0140] Introduction of borate esters

[0141] The conversion of compounds of general formula 14 to boronic acid derivatives of formula 11 is known to the skilled person. For RR = -C(CH3)2-C(CH3)2-, the reaction can be carried out with a reagent such as bis(pinacol)diborane, a catalyst such as [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and a base such as potassium acetate.

[0142] Step 13-->12 (Solution 2)

[0143] Introduction of borate esters

[0144] The conversion of compounds of general formula 13 to boronic acid derivatives of formula 12 is known to the skilled person. For RR = -C (CH3) 2-C (CH3) 2-, the reaction can be carried out with a reagent such as bis (pinacol) diborane, a catalyst such as [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II) and a base such as potassium acetate.

[0145]

[0146] Scheme 3: Routes for the preparation of compounds of general formula 18, wherein A1, A2, A3 and Z have the same meanings as given above for formula (I), X has the meanings of hydroxyl, Cl, Br, I, mesylate or triflate, Y has the meanings of Cl, Br or I, and R has the meaning of hydrogen or alkyl. In addition, R1 can be a protecting group known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis by TW Greene and PGM Wuts, 3rd edition, Wiley 1999).

[0147] Preparation of starting materials (Scheme 3):

[0148] Amide compounds of the general formula 15 (Scheme 3) are commercially available, described in the literature or can be prepared in analogy to literature procedures.

[0149] Step 8+15-->16 (Solution 3)

[0150] Mitsunobu reaction

[0151] The conversion of compounds of formula 8 and compounds of formula 15 to derivatives of formula 16 is known to the skilled person. The reaction can be carried out under Mitsunobu conditions using reagents such as diisopropyl azodicarboxylate / triphenylphosphine, di-tert-butyl azodicarboxylate / triphenylphosphine or diethyl azodicarboxylate / triphenylphosphine.

[0152] Step 9+15-->16 (Solution 3)

[0153] Alkylation of amides

[0154] The conversion of compounds of formula 9 and 15 to derivatives of formula 16 is known to the skilled person. For X = Cl, Br or I, the reaction can be carried out with a base such as potassium carbonate, cesium carbonate, sodium hydride or LDA in a solvent such as DMF, DMSO or acetonitrile.

[0155] Step 16+11-->17 (Scheme 3)

[0156] Palladium-catalyzed reactions with boronic acids

[0157] Heteroaryl halides of formula 16 can be reacted with boronic acid derivatives 11 to give compounds of formula 17. The boronic acid derivative can be boronic acid (R = H) or an ester of boronic acid, such as its isopropyl ester (R = -CH(CH3)2), preferably an ester derived from pinacol, wherein the boronic acid intermediate forms 2-aryl-4,4,5,5-tetramethyl-1,3,2,-dioxaborolane (RR = -C(CH3)2-C(CH3)2-).

[0158] The coupling reaction is catalyzed by a palladium catalyst, for example by a Pd(0) catalyst like tetrakis(triphenylphosphine)palladium(0)[Pd(PPh3)4], tris(dibenzylideneacetone)di-palladium(0)[Pd2(dba)3], or by a Pd(II) catalyst like dichlorobis(triphenylphosphine)-palladium(II)[Pd(PPh3)2Cl2], XPhos Pd G2, Pd-Peppsi 2Me-Ipent Cl, palladium(II) acetate and triphenylphosphine, or by [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.

[0159] The reaction is preferably carried out in a mixture of a solvent like 1,2-dimethoxymethane, dioxane, DMF, DME, THF, ethanol, or isopropanol with water and in the presence of a base like potassium carbonate, sodium bicarbonate, potassium acetate or potassium phosphate.

[0160] (Review: DH Gall, Boronic Acids, 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, ISBN 3-527-30991-8 and references therein).

[0161] The reaction is carried out at a temperature ranging from room temperature (i.e., about 20° C.) to the boiling point of the corresponding solvent. Further, the reaction can be carried out using a pressure tube and a microwave oven at a temperature above the boiling point. The reaction is preferably completed after a reaction time of 1 to 36 hours.

[0162] Step 17-->18 (Solution 3)

[0163] Removal of phenol protecting group

[0164] Removal of the protecting group R1 from the compound of formula 17 generates the compound of formula 18. The reaction conditions for removing such suitable protecting groups R1 are known to the skilled person (see, for example, Green, Wuts, "Protective groups in organic synthesis" 1999, John Wiley & Sons and references therein). Preferably, benzyl, p-methoxybenzyl and 3,4-methoxybenzyl are used as protecting groups during the synthesis, which can be removed by hydrogenation.

[0165] Step 16-->19 (Solution 3)

[0166] Introduction of borate esters

[0167] The conversion of compounds of general formula 16 to boronic acid derivatives of formula 19 is known to the skilled person. For RR = -C(CH3)2-C(CH3)2-, the reaction can be carried out with a reagent such as bis(pinacol)diborane, a catalyst such as [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and a base such as potassium acetate.

[0168] Step 19+13-->18 (Scheme 3)

[0169] Palladium-catalyzed reactions with boronic acids

[0170] Aryl halides of formula 13 can be reacted with boronic acid derivatives 19 to give compounds of formula 18. The boronic acid derivative can be boronic acid (R = H) or an ester of boronic acid, such as its isopropyl ester (R = -CH(CH3)2), preferably an ester derived from pinacol, wherein the boronic acid intermediate forms 2-aryl-4,4,5,5-tetramethyl-1,3,2,-dioxaborolane (RR = -C(CH3)2-C(CH3)2-).

[0171] The coupling reaction is catalyzed by a palladium catalyst, for example by a Pd(0) catalyst like tetrakis(triphenylphosphine)palladium(0)[Pd(PPh3)4], tris(dibenzylideneacetone)di-palladium(0)[Pd2(dba)3], or by a Pd(II) catalyst like dichlorobis(triphenylphosphine)-palladium(II)[Pd(PPh3)2Cl2], XPhos Pd G2, Pd-Peppsi 2Me-Ipent Cl, palladium(II) acetate and triphenylphosphine, or by [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.

[0172] The reaction is preferably carried out in a mixture of a solvent like 1,2-dimethoxymethane, dioxane, DMF, DME, THF, ethanol, or isopropanol with water and in the presence of a base like potassium carbonate, sodium bicarbonate, potassium acetate or potassium phosphate.

[0173] (Review: DH Gall, Boronic Acids, 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, ISBN 3-527-30991-8 and references therein).

[0174] The reaction is carried out at a temperature ranging from room temperature (i.e., about 20° C.) to the boiling point of the corresponding solvent. Further, the reaction can be carried out using a pressure tube and a microwave oven at a temperature above the boiling point. The reaction is preferably completed after a reaction time of 1 to 36 hours.

[0175] Step 16+12-->18 (Scheme 3)

[0176] Palladium-catalyzed reactions with boronic acids

[0177] Heteroaryl halides of formula 16 can be reacted with boronic acid derivatives 12 to give compounds of formula 18. The boronic acid derivative can be boronic acid (R = H) or an ester of boronic acid, such as its isopropyl ester (R = -CH(CH3)2), preferably an ester derived from pinacol, wherein the boronic acid intermediate forms 2-aryl-4,4,5,5-tetramethyl-1,3,2,-dioxaborolane (RR = -C(CH3)2-C(CH3)2-).

[0178] The coupling reaction is catalyzed by a palladium catalyst, for example by a Pd(0) catalyst like tetrakis(triphenylphosphine)palladium(0)[Pd(PPh3)4], tris(dibenzylideneacetone)di-palladium(0)[Pd2(dba)3], or by a Pd(II) catalyst like dichlorobis(triphenylphosphine)-palladium(II)[Pd(PPh3)2Cl2], XPhos Pd G2, Pd-Peppsi 2Me-Ipent Cl, palladium(II) acetate and triphenylphosphine, or by [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.

[0179] The reaction is preferably carried out in a mixture of a solvent like 1,2-dimethoxymethane, dioxane, DMF, DME, THF, ethanol, or isopropanol with water and in the presence of a base like potassium carbonate, sodium bicarbonate, potassium acetate or potassium phosphate.

[0180] (eg, reviewed in: DH Hall, Boronic Acids, 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, ISBN 3-527-30991-8 and references therein).

[0181] The reaction is carried out at a temperature ranging from room temperature (i.e., about 20° C.) to the boiling point of the corresponding solvent. Further, the reaction can be carried out using a pressure tube and a microwave oven at a temperature above the boiling point. The reaction is preferably completed after a reaction time of 1 to 36 hours.

[0182] Step 19+14-->17 (Scheme 3)

[0183] Palladium-catalyzed reactions with boronic acids

[0184] Aryl halides of formula 14 can be reacted with boronic acid derivatives 19 to give compounds of formula 17. The boronic acid derivative can be boronic acid (R = H) or an ester of boronic acid, such as its isopropyl ester (R = -CH(CH3)2), preferably an ester derived from pinacol, wherein the boronic acid intermediate forms 2-aryl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (RR = -C(CH3)2-C(CH3)2-).

[0185] The coupling reaction is catalyzed by a palladium catalyst, for example by a Pd(0) catalyst like tetrakis(triphenylphosphine)palladium(0)[Pd(PPh3)4], tris(dibenzylideneacetone)di-palladium(0)[Pd2(dba)3], or by a Pd(II) catalyst like dichlorobis(triphenylphosphine)-palladium(II)[Pd(PPh3)2Cl2], XPhos Pd G2, Pd-Peppsi 2Me-Ipent Cl, palladium(II) acetate and triphenylphosphine, or by [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.

[0186] The reaction is preferably carried out in a mixture of a solvent like 1,2-dimethoxymethane, dioxane, DMF, DME, THF, ethanol, or isopropanol with water and in the presence of a base like potassium carbonate, sodium bicarbonate, potassium acetate or potassium phosphate.

[0187] (Review: DH Gall, Boronic Acids, 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, ISBN 3-527-30991-8 and references therein).

[0188] The reaction is carried out at a temperature ranging from room temperature (i.e., about 20° C.) to the boiling point of the corresponding solvent. Further, the reaction can be carried out using a pressure tube and a microwave oven at a temperature above the boiling point. The reaction is preferably completed after a reaction time of 1 to 36 hours.

[0189] General preparation method of compounds of formula (I):

[0190] The present invention discloses the following method or process for preparing a compound of general formula (I) using compounds X1 and X2:

[0191]

[0192] Said process for obtaining compounds of general formula (I) is characterized in that:

[0193] In the compound of general formula X1, Z, A1, A2, A3 have the same meanings as defined for the compound of general formula (I),

[0194] · reacting a compound of formula X1 (wherein Y can be Br, I or Cl) with a compound of formula X2 using a palladium catalyst and a base; and

[0195] - The reaction is carried out in a solvent or solvent mixture at a temperature between ambient temperature and the boiling point of the solvent, preferably between 50°C and 120°C.

[0196] The preparation of the compounds of the general formula (I) can be carried out in an aprotic or protic solvent or solvent mixture, preferably in 1,4-dioxane, tetrahydrofuran or ethanol / water.

[0197] • Preferred bases that can be used to prepare compounds of general formula (I) are sodium carbonate or cesium carbonate.

[0198] The preferred palladium catalyst for preparing the compound of general formula (I) is

[0199] -chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (2nd generation XPhos precatalyst) or

[0200] -1,3-bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride.

[0201] Intermediate compounds:

[0202] The present invention also discloses intermediate compounds that can be used to prepare compounds of general formula (I). In particular, the present invention discloses compounds of general formula Ia, wherein Z is as defined above for compounds of general formula (I), and Y can be Br, I or Cl:

[0203]

[0204] The present invention discloses additional intermediate compounds that can be used to prepare compounds of formula (I). In particular, the present invention also discloses compounds of formula (Ib), wherein A1, A2 and A3 are as defined above for compounds of general formula (I):

[0205]

[0206] The following table lists compounds 01-30 prepared according to the present invention.

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218] Features and advantages of the present invention will become apparent from the following examples which are given by way of illustration of the invention without limiting its scope.

[0219] abbreviation

[0220]

[0221]

[0222] Example

[0223] Example 1: Analytical HPLC method

[0224] 1.1 Method A

[0225] Analytical column: Waters; Sunfire C18_3.0x30mm, 2.5μm; Column temperature: 60°C Apparatus: Agilent 1200 with DA detector and MS detector

[0226]

[0227] 1.2 Method B

[0228] Analytical column: Waters; XBridge C18_3.0x30mm, 2.5μm; Column temperature: 60°C Apparatus: Agilent 1200 with DA detector and MS detector

[0229]

[0230] 1.3 Method C

[0231] Analytical column: Waters; Sunfire C18_2.1x30mm, 2.5μm; Column temperature: 60°C Apparatus: Waters Acquity, with DA detector and MS detector

[0232]

[0233] 1.4 Method D

[0234] Analytical column: Sunfire C18_3.0x30mm, 2.5μm; Column temperature: 60℃ Device: Waters Acquity, QDa detector

[0235]

[0236] 1.5 Method E

[0237] Analytical column: Sunfire C18_3.0x30mm, 2.5μm; Column temperature: 60℃ Device: Waters Acquity, QDa detector

[0238]

[0239] Example 2: Preparation of intermediates

[0240] 2.1 Intermediate I

[0241] Intermediate I.1 (General Procedure)

[0242] 1-[(2-Bromo-1,3-thiazol-5-yl)methyl]-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0243]

[0244] 3-Methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (3.00 g; 23.79 mmol) and 3-bromothiazole-5-methanol (5.08 g; 26.17 mmol) were dissolved in THF (50 mL) and DMF (20 mL). TPP (polymer-bound, 3 mmol / g; 10.50 g; 30.92 mmol) was added and the reaction mixture was cooled in an ice bath. DTAD (7.12 g; 30.92 mmol) was added. After stirring overnight at RT, the reaction mixture was filtered and THF was evaporated. The residue was poured onto water and extracted several times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered out and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; DCM / MeOH, 100 / 0 until 95 / 5) to provide the product.

[0245] C9H8BrN3O2S: (M=302.2g / mol)

[0246] ESI-MS: 302[M+H] +

[0247] R t (HPLC): 0.67 min (Method A)

[0248] Yield: 4.03 g; 56%

[0249] 1 H NMR (400MHz, DMSO-d6) δppm: 3.16 (s, 3H), 5.10 (s, 2H), 5.76 (d, J = 7.86 Hz, 1H), 7.73 (s, 1H), 7.84 (d, J = 7.86 Hz, 1H).

[0250] Intermediate compounds 1.2 and 1.3 shown in the table below were prepared using procedures analogous to those described for intermediate 1.1 using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0251]

[0252]

[0253] Intermediate I.4

[0254] 1-[(5-Bromo-1,3,4-thiadiazol-2-yl)methyl]-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0255]

[0256] To a stirred mixture of (5-bromo-1,3,4-thiadiazol-2-yl)methanol (0.20 g; 1.03 mmol) and TEA (0.17 mL; 1.23 mmol) in DCM (5 mL) was added methanesulfonyl chloride (0.10 mL; 1.23 mmol) dropwise. After stirring for 1 h at RT, another portion of methanesulfonyl chloride (0.04 mL) was added and stirred for another hour. The reaction mixture was diluted with DCM and water. The organic layer was separated by a phase separator cartridge and evaporated. The residue was taken up in DMF (3 mL), 3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (0.10 g; 0.79 mmol) and K2CO3 (0.28 g; 2.03 mmol) were added. The reaction mixture was stirred at RT overnight, filtered and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the product.

[0257] C8H7BrN4O2S (M=303.1g / mol)

[0258] ESI-MS: 303 / 305 (Br) [M+H] +

[0259] R t (HPLC): 0.71 min (Method A)

[0260] Yield: 0.14 g; 58%

[0261] Intermediate I.5

[0262] 1-[(5-Bromo-1,2-thiazol-3-yl)methyl]-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0263]

[0264] 3-Methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (0.04 g; 0.32 mmol), 5-bromo-3-(bromomethyl)-1,2-thiazole (0.09 mL; 0.35 mmol) and K2CO3 (0.10 g; 0.70 mmol) in dry DMF (2 mL) were stirred at RT for 2 h. The reaction mixture was partitioned between EtOAc and water. The organic layer was washed with brine and water, dried over Na2SO4, filtered, and concentrated under reduced pressure.

[0265] C9H8BrN3O2S (M=302.2g / mol)

[0266] ESI-MS: 302[M+H] +

[0267] R t (HPLC): 0.71 min (Method A)

[0268] Yield: 0.09 g; 97%

[0269] 1 H NMR (400MHz, DMSO-d6) δppm: 3.15 (s, 3H), 5.07 (s, 2H), 5.76 (d, J = 7.86Hz, 1H), 7.56 (s, 1H), 7.77 (d, J = 7.86Hz, 1H).

[0270] 2.2 Intermediate II

[0271] [3-(Benzyloxy)-2,4-difluorophenyl]boronic acid

[0272]

[0273] 2-(Benzyloxy)-1,3-difluorobenzene (0.50 g; 2.27 mmol) in THF (10 mL) was cooled to -78 °C. n-BuLi (2.5 mol / L in THF; 1.36 mL; 3.63 mmol) was added dropwise and stirred at low temperature for 50 minutes. Triisopropyl borate (0.73 mL; 3.63 mmol) was added dropwise, stirred for another 10 min at -78 °C, and then stirred for another 30 minutes without cooling. The reaction was quenched with HCl (aqueous solution; 4 mol / L; 5 mL) and stirred for 10 minutes. The mixture was poured onto water and extracted several times with EtOAc. The combined organic layers were dried with MgSO4, filtered off, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; DCM / MeOH with a very small amount of AcOH, 100 / 0 until 96 / 4) to provide the product.

[0274] C 13 H 11 BF2O3 (M = 264.0 g / mol)

[0275] ESI-MS: 263[MH] -

[0276] R t (HPLC): 0.75 min (Method B)

[0277] Yield: 0.25 g; 42%

[0278] 1 H NMR (400MHz, DMSO-d6) δppm: 5.12 (s, 2H), 7.05 (ddd, J = 10.27, 8.62, 1.39Hz, 1H), 7.25 (dt, J = 8.40, 6.57Hz, 1H), 7.30-7.56 (m, 5H), 8.23 ​​(br s, 2H).

[0279] 2.3 Intermediate III

[0280] Intermediate III.1

[0281] (2,4-Difluoro-3-hydroxyphenyl)boronic acid

[0282]

[0283] Intermediate II.1 (0.10 g; 0.38 mmol) in a mixture of THF and MeOH (3 mL each) was hydrogenated in a parr apparatus using Pd / C 10% (0.02 g) at RT for 3 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to afford the product.

[0284] C6H5BF2O3 (M=173.9g / mol)

[0285] ESI-MS: 173[MH] -

[0286] R t (HPLC): 0.07 min (Method B)

[0287] Yield: 0.05 g; 77%

[0288] 1 H NMR (400MHz, DMSO-d6) δppm: 6.90-7.00 (m, 2H), 8.11 (br s, 2H), 9.81 (br s, 1H).

[0289] 2.4 Intermediate IV

[0290] Intermediate IV.1 (General Procedure)

[0291] 2,4-Difluoro-3-[(4-methoxyphenyl)methoxy]benzaldehyde

[0292]

[0293] 1-(Chloromethyl)-4-methoxybenzene (0.19 mL; 1.39 mmol) was added to a mixture of 2,4-difluoro-3-hydroxybenzaldehyde (0.20 g; 1.27 mmol) and K2CO3 (0.27 g; 1.94 mmol) in ACN (5 mL). After stirring at 60 °C for 3 h, the reaction mixture was filtered and diluted with DMF / water / TFA and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA). ACN was evaporated in the desired fractions and the product was partitioned between DCM and water. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure.

[0294] C 15 H 12 F2O3 (M = 278.3 g / mol)

[0295] ESI-MS: 277[MH] -

[0296] R t (HPLC): 1.12 min (Method A)

[0297] Yield: 0.22 g; 63%

[0298] 1 H NMR (400MHz, DMSO-d6) δppm: 3.75 (s, 3H), 5.14 (s, 2H), 6.93 (d, J = 8.74Hz, 2H), 7.26-7.39 (m, 3H), 7.59 (ddd, J = 8.81, 7.41, 6.08Hz, 1H), 10.11 (s, 1H).

[0299] Intermediate compounds IV.2 and IV.4 shown in the table below were prepared using procedures analogous to those described for intermediate IV.1 using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0300]

[0301]

[0302]

[0303] 2.5 Intermediate V

[0304] Intermediate V.1

[0305] ({2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}methylene)hydroxylamine

[0306]

[0307] Intermediate IV.1 (0.22 g; 0.79 mmol) and NaOAc (0.08 g; 1.03 mmol) in MeOH (8 mL) and water (3 mL) were stirred at RT. Hydroxylamine hydrochloride (0.04 mL; 1.03 mmol) was added and the reaction mixture was heated to 80 °C for 1.5 h. The reaction mixture was concentrated under reduced pressure and taken up in water / EtOAc. The aqueous layer was extracted several times with EtOAc. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure.

[0308] C 15 H 13 F2NO3 (M = 293.3 g / mol)

[0309] ESI-MS: 294 [M+H] +

[0310] R t (HPLC): 1.06 min (Method A)

[0311] Yield: 0.25 g; quantitative

[0312] 1 H NMR(400MHz,DMSO-d6)δppm:3.75(s,3H),5.10(s,2H),6.90-6.96(m,2H),7.10-7.19(m, 1H),7.31-7.37(m,2H),7.42(ddd,J=8.81,7.79,6.08Hz,1H),8.14(s,1H),11.59(s,1H).

[0313] 2.6 Intermediate VI

[0314] Intermediate VI.1

[0315] 1-[(3-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,2-oxazol-5-yl)methyl]-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0316]

[0317] Intermediate V.1 (0.10 g; 0.34 mmol), 3-methyl-1-(propyl-2-yn-1-yl)-1,2,3,4-tetrahydropyrimidine-2,4-dione (0.06 g; 0.34 mmol) and TEA (0.01 mL; 0.03 mmol) in t-BuOH (4 mL) and water (4 mL) were stirred at RT. NaOCl (0.45 mL; 0.58 mmol) was added. After stirring for 2.5 h at RT, EtOAc and water were added. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the product.

[0318] C 23 H 19 F2N3O5 (M = 455.4 g / mol)

[0319] ESI-MS: 456[M+H] +

[0320] R t (HPLC): 1.07 min (Method A)

[0321] Yield: 0.02 g; 12%

[0322] 1 H NMR (400MHz, DMSO-d6) δppm: 3.17 (s, 3H), 3.75 (s, 3H), 5.14 (s, 2H), 5.20 (s, 2H), 5.81 (d, J = 7.86Hz, 1H), 6.89 (d, J = 2.66Hz, 1H), 6.93 ( d,J=8.74Hz,2H),7.26(td,J=9.57,1.77Hz,1H),7.35(d,J=8.62Hz,2H),7.56(ddd,J=8.90,7.83,5.96Hz,1H),7.86(d,J=7.98Hz,1H).

[0323] Intermediate VI.2 (General Procedure)

[0324] 1-[(5-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,2-oxazol-3-yl)methyl]-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0325]

[0326] Intermediate IX.1 (0.10 g; 0.03 mmol), 3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (0.01 g; 0.05 mmol) and K2CO3 (0.01 g; 0.08 mmol) in DMF (2 mL) were stirred at RT for 1 h and at 50° C. for 30 min. The reaction mixture was filtered and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to afford the product.

[0327] C 23 H 19 F2N3O5 (M = 455.4 g / mol)

[0328] ESI-MS: 456[M+H] +

[0329] R t (HPLC): 1.07 min (Method A)

[0330] Yield: 0.01 g; 53%

[0331] The following intermediate compound VI.3, below, was prepared using a procedure analogous to that described for intermediate VI.2 using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0332]

[0333]

[0334] 2.7 Intermediate VII

[0335] Intermediate VII.1

[0336] Ethyl 5-(2,4-difluoro-3-hydroxyphenyl)-1,2-oxazole-3-carboxylate

[0337]

[0338] Ethyl 5-chloro-1,2-oxazole-3-carboxylate (0.15 g; 0.85 mmol), intermediate III.1 (0.22 g; 1.28 mmol) and K3PO4 (0.36 g; 1.71 mmol) were dissolved in dioxane (3 mL). The reaction mixture was flushed with nitrogen. Pd-Peppsi 2Me-Ipent Cl (0.04 g; 0.04 mmol) was added. After stirring at 65 °C for 20 min, the reaction mixture was diluted with DMF / water and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the product.

[0339] C 12 H9F2NO4 (M=269.2g / mol)

[0340] ESI-MS: 270 [M+H] +

[0341] R t (HPLC): 0.98 min (Method A)

[0342] Yield: 0.03 g; 11%

[0343] 1 H NMR (400MHz, DMSO-d6) δppm: 1.35 (t, J = 7.16Hz, 3H), 4.40 (q, J = 7.10Hz, 2H), 7.16 (d, J = 2. 91Hz,1H),7.25(td,J=9.57,1.77Hz,1H),7.43(ddd,J=8.84,7.64,5.70Hz,1H),10.77(br s,1H).

[0344] Intermediate VII.2

[0345] 2,6-Difluoro-3-[5-(hydroxymethyl)-1,3,4-thiadiazol-2-yl]phenol

[0346]

[0347] The reaction was carried out under a nitrogen atmosphere. (5-bromo-1,3,4-thiadiazol-2-yl)methanol (4.00 g; 20.51 mmol), intermediate III.1 (4.60 g; 24.61 mmol) and Na2CO3 (5.43 g; 51.27 mmol) were dissolved in EtOH (50 mL) and water (10 mL). Pd-Peppsi 2Me-Ipent Cl (0.86 g; 1.03 mmol) was added and the mixture was stirred at 80 ° C overnight. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was absorbed in water and the resulting precipitate was filtered out to provide the product.

[0348] C9H6F2N2O2S (M=244.2g / mol)

[0349] ESI-MS: 245 [M+H] +

[0350] R t (HPLC): 0.72 min (Method A)

[0351] Yield: 2.66 g; 53%

[0352] 1 H NMR (400MHz, DMSO-d6) δppm: 4.93 (d, J = 4.69Hz, 2H), 6.27 (br t, J = 5.39Hz, 1H), 7.19-7.34 (m, 1H), 7.65 (ddd, J = 8.84, 7.57, 5.89Hz, 1H), 10.75 (s, 1H).

[0353] 2.8 Intermediate VIII

[0354] Intermediate VIII.1

[0355] (5-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,2-oxazol-3-yl)methanol

[0356]

[0357] Intermediate IV.2 (0.03 g; 0.07 mmol) in THF (3 mL) was cooled to -20 °C. LiAlH4 (solution in THF; 1 mol / L; 0.05 mL; 0.05 mol / L) was added dropwise and stirred at low temperature for 30 min. Two drops of water and NaOH (aqueous solution; 4 mol / L) were added and stirred for another 20 min at RT. The reaction mixture was filtered, diluted with water, and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to afford the product.

[0358] C 18 H 15 F2NO4 (M = 347.3 g / mol)

[0359] ESI-MS: 348[M+H] +

[0360] R t (HPLC): 1.04min (Method A)

[0361] Yield: 0.02 g; 70%

[0362] 1 H NMR (400MHz, DMSO-d6) δppm: 3.75 (s, 3H), 4.57 (d, J = 6.08Hz, 2H), 5.16 (s, 2H), 5.55 (t, J = 6.02Hz, 1H), 6.82 (d, J = 3.55Hz, 1H) ,6.93(d,J=8.74Hz,2H),7.30(ddd,J=10.36,8.90,1.90Hz,1H),7.36(d,J=8.62Hz,2H),7.63(ddd,J=8.90,7.83,5.83Hz,1H).

[0363] 2.9 Intermediate IX

[0364] Intermediate IX.1

[0365] 3-(Chloromethyl)-5-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,2-oxazole

[0366]

[0367] Intermediate VIII.1 (0.02 g; 0.024 mmol), TPP (0.01 g; 0.04 mmol) and carbon tetrachloride (0.02 mL; 0.22 mmol) in ACN (2 mL) were stirred at 80° C. for 2.67 h. The reaction mixture was concentrated under reduced pressure. The residue was used as crude product in further reactions.

[0368] C 18 H 14 ClF2NO3 (M = 365.8 g / mol)

[0369] ESI-MS: 366[M+H] +

[0370] R t (HPLC): 1.20 min (Method A)

[0371] Yield: 0.01 g; 70% (determined by HPLC-MS)

[0372] Intermediate IX.2 (General Procedure)

[0373] 5-(Chloromethyl)-2-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,3-thiazole

[0374]

[0375] Intermediate XI.1 (0.99 g; 2.72 mmol) and TEA (0.60 mL; 4.33 mmol) in DCM (10 mL) were stirred in an ice bath. Methanesulfonyl chloride (0.32 mL; 4.13 mmol) was added dropwise. After stirring at low temperature for 5 min, the reaction mixture was stirred at RT overnight. The reaction mixture was diluted with additional DCM and washed with dilute citric acid. The organic layer was separated with a phase separator cartridge and evaporated.

[0376] C 18 H 14 ClF2NO2S (M = 381.8 g / mol)

[0377] ESI-MS: 382[M+H] +

[0378] R t (HPLC): 1.22 min (Method A)

[0379] Yield: 1.01 g; 97%

[0380] 1H NMR (400MHz, DMSO-d6) δppm: 3.75 (s, 3H), 5.16 (s, 2H), 5.18 (s, 2H), 6.93 (d, J=8.74Hz, 2H), 7.28 (td, J= 9.63, 1.90Hz, 1H), 7.36 (d, J = 8.62Hz, 2H), 7.89 (ddd, J = 9.00, 8.05, 6.02Hz, 1H), 8.03 (d, J = 2.28Hz, 1H).

[0381] The following intermediate compound IX.3 was prepared using a procedure analogous to that described for intermediate IX.2 using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0382]

[0383] 2.10 Intermediate X

[0384] Intermediate X.1

[0385] 2,4-Difluoro-N',3-dihydrobenzene-1-carboxamidine

[0386]

[0387] 2,4-difluoro-3-hydroxybenzonitrile (0.03 g; 0.20 mmol) in EtOH (0.5 mL) was stirred at RT. Hydroxylamine hydrochloride (0.03 g; 0.40 mmol) and TEA (0.06 mL; 0.04 mmol) in EtOH (1 mL) were added dropwise. After stirring at RT for 20 h, the reaction mixture was concentrated under reduced pressure and used further as a crude product.

[0388] C7H6F2N2O2 (M=188.1g / mol)

[0389] 2.11 Intermediate XI

[0390] Intermediate XI.1

[0391] (2-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,3-thiazol-5-yl)methanol

[0392]

[0393] Intermediate IV.3 (3.00 g; 9.11 mmol), bis(pinacolato)diboron (3.50 g; 13.78 mmol), KOAc (2.30 g; 23.44 mmol) and Pd(dppf)2x CHCl (0.75 g; 0.92 mmol) in dioxane (50 mL) were stirred at 90° C. overnight. Further bis(pinacolato)diboron (0.70 g; 2.76 mmol) was added and the reaction mixture was stirred at 100° C. for another 2 h. After cooling to RT, Pd(dppf)2x CHCl (0.38 g; 0.46 mmol), (2-bromo-1,3-thiazol-5-yl)methanol (1.80 g; 9.28 mmol) and NaCO (aqueous solution; 2 mol / L; 15.00 mL; 30.00 mmol) were added and the reaction mixture was stirred at reflux for 6 h. The mixture was cooled to RT, diluted with EtOAc and washed with The product was concentrated under reduced pressure. The residue was separated into EtOAc and water. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; cyclohexane / EtOAC 2:1 to 1:1). Et2O was added to the evaporated fraction, stirred and filtered out. The precipitate was dried in air.

[0394] C 18 H 15 F2NO3S (M = 363.4 g / mol)

[0395] ESI-MS: 364[M+H] +

[0396] R t (HPLC): 1.07 min (Method A)

[0397] Yield: 1.10 g; 30%

[0398] 1 H NMR (400MHz, DMSO-d6) δppm: 3.75 (s, 3H), 4.75 (d, J = 5.70Hz, 2H), 5.16 (s, 2H), 5.64 (t, J = 5.77Hz, 1H), 6.93 (d, J = 8.74H z, 2H), 7.26 (td, J = 9.63, 1.77Hz, 1H), 7.36 (d, J = 8.62Hz, 2H), 7.79-7.83 (m, 1H), 7.86 (ddd, J = 8.93, 8.11, 6.02Hz, 1H).

[0399] 2.12 Intermediate XII

[0400] Intermediate XII.1 (General Procedure)

[0401] 1-[(2-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,3-thiazol-5-yl)methyl]-3-ethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0402]

[0403] Intermediate VI.3 (0.03 g; 0.05 mmol), iodoethane (0.01 mL; 0.16 mmol) and K2CO3 (0.03 g; 0.16 mmol) in DMF (3 mL) were stirred at 50° C. for 2 h. The reaction mixture was diluted with water, filtered and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to afford the product.

[0404] C 24 H 21 F2N3O4S (M = 485.5 g / mol)

[0405] R t (HPLC): 1.12 min (Method A)

[0406] Yield: 0.02 g; 64%

[0407] The following intermediate compounds XII.2 to XII.7 below were prepared using procedures analogous to those described for intermediate compound XII.1 using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0408]

[0409]

[0410]

[0411] 2.13 Intermediate XIII

[0412] Intermediate XIII.1 (General Procedure)

[0413] 1-[(5-Bromo-1,3,4-thiadiazol-2-yl)methyl]-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0414]

[0415] The reaction was carried out under an argon atmosphere. 1,2,3,4-tetrahydropyrimidine-2,4-dione (0.50 g; 4.46 mmol) and (E)-trimethylsilyl N-(trimethylsilyl)acetimidate (2.75 mL; 11.25 mmol) in ACN (15 mL) were stirred at RT for 5 h. (5-Bromo-1,3,4-thiadiazol-2-yl)methyl methanesulfonate (1.40 g; 5.13 mmol) and tetrabutylazanium iodide (0.17 g; 0.46 mmol) were added. After stirring at 80 ° C for 6 hours and at RT overnight, 30 mL of water was slowly added. The resulting precipitate was filtered off and washed with water, ACN and Et2O.

[0416] C7H5BrN4O2S (M=289.1 g / mol)

[0417] Yield: 0.29 g; 23%

[0418] The following intermediate compounds XIII.2 to XIII.4, below, were prepared using procedures analogous to those described for intermediate compound XIII.1 using appropriate starting materials. As will be appreciated by one skilled in the art, analogous examples may involve variations in the general reaction conditions.

[0419]

[0420]

[0421]

[0422] 2.14 Intermediate XIV

[0423] Intermediate XIV.1

[0424] 1-[(5-Bromo-1,3,4-thiadiazol-2-yl)methyl]-6-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0425]

[0426] (5-Bromo-1,3,4-thiadiazol-2-yl)methyl methanesulfonate (0.20 g; 0.73 mmol) and 6-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (0.18 g; 1.46 mmol) were dissolved in DMF (5 mL) and K2CO3 (0.25 g; 1.83 mmol) was added. After stirring for 2 h at RT, iodoethane (0.15 mL; 1.83 mmol) was added and stirred for another hour at 50°C. The reaction mixture was purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the product.

[0427] C 10 H 11 BrN4O2S (M = 331.2 g / mol)

[0428] ESI-MS: 331[M+H] +

[0429] R t (HPLC): 0.83 min (Method A)

[0430] Yield: 0.03 g; 14%

[0431] 1 H NMR (400MHz, DMSO-d6) δppm: 1.08 (t, J = 7.03Hz, 3H), 2.31 (d, J = 0.63Hz, 3H), 3.82 (q, J = 6.97Hz, 2H), 5.42 (s, 2H), 5.71 (q, J = 0.63Hz, 1H).

[0432] 2.15 Intermediate XV

[0433] Intermediate XV.1

[0434] 1-[(2-Bromo-1,3-thiazol-5-yl)methyl]-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0435]

[0436] The reaction was carried out under a nitrogen atmosphere. 1,2,3,4-tetrahydropyrimidine-2,4-dione (1.35 g; 12.04 mmol), HMDS (2.76 mL; 13.25 mmol) and trimethylsilyl chloride (0.76 mL; 6.02 mmol) in dry ACN (10 mL) were stirred at 140 ° C for 5 h and then concentrated under reduced pressure. Half of the residue was taken up in dry ACN (22 mL) and 2-bromo-5-(bromomethyl)-1,3-thiazole (2.90 g; 11.29 mmol) in ACN (13 mL) was added dropwise at 0 ° C. After complete addition, the mixture was heated to 80 ° C and stirred overnight. After cooling to RT, the reaction mixture was diluted with NaHCO3 (9% aqueous solution) and extracted several times from EtOAc. The organic layer was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was treated with Et2O and filtered.

[0437] C8H6BrN3O2S (M=288.1g / mol)

[0438] ESI-MS: 288 / 290 [M+H] +

[0439] R t (HPLC): 0.67 min (Method A)

[0440] Yield: 2.66 g; 77%

[0441] 1 H NMR (400MHz, DMSO-d6) δppm: 5.04 (s, 2H), 5.62 (d, J = 7.86 Hz, 1H), 7.71 (s, 1H), 7.78 (d, J = 7.86 Hz, 1H), 11.41 (br s, 1H).

[0442] 2.16 Intermediate XVI

[0443] Intermediate XVI.1 (General Procedure)

[0444] 1-[(2-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,3-thiazol-5-yl)methyl]piperidin-2-one

[0445]

[0446] Piperidin-2-one (0.03 g; 0.26 mmol) and sodium hydride (0.01 g; 0.26 mmol) in DMF (2 mL) were stirred at RT for 10 min. Intermediate IX.2 (0.04 g; 0.10 mmol) was added. After stirring for 2 h, the mixture was diluted with water and MeOH and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to afford the desired product.

[0447] C 23 H 22 F2N2O3S (M = 444.5 g / mol)

[0448] ESI-MS: 445[M+H] +

[0449] R t (HPLC): 1.11 min (Method A)

[0450] Yield: 0.02 g; 52%

[0451] The following intermediate compounds XVI.2 to XVI.4 below were prepared using procedures analogous to those described for intermediate compound XVI.1 using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0452]

[0453]

[0454]

[0455] 2.17 Intermediate XVII

[0456] Intermediate XVII.1

[0457] tert-Butyl N-(3-{[(2-{2,4-difluoro-3-[(4-methoxyphenyl)methoxy]phenyl}-1,3-thiazol-5-yl)methyl]amino}propyl)-N-methylcarbamate

[0458]

[0459] Intermediate IX.2 (0.03 g; 0.07 mmol); tert-butyl N-(3-aminopropyl)-N-methylcarbamate (0.01 g; 0.07 mmol) and K2CO3 (0.02 g; 0.13 mmol) in ACN (3.84 mL) were stirred at 80° C. for 2 h. Another three equivalents of amine and base were added and stirred at 80° C. for another 1.4 h. The reaction mixture was diluted with water and MeOH and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to afford the desired product.

[0460] C 27 H 33 F2N3O4S (M = 533.6 g / mol)

[0461] ESI-MS: 534[M+H] +

[0462] R t (HPLC): 0.94 min (Method A)

[0463] Yield: 0.03 g; 86%

[0464] 2.18 Intermediate XVIII

[0465] Intermediate XVIII.1 (General Procedure)

[0466] 1-[(2-Bromo-1,3-thiazol-5-yl)methyl]-2,3-dihydro-1H-indol-2-one

[0467]

[0468] Methyl 2-(2-aminophenyl)acetate hydrochloride (0.10 g; 0.50 mmol); 2-bromo-5-(bromomethyl)-1,3-thiazole (0.14 g; 0.50 mmol) and DIPA (0.21 mL; 1.24 mmol) in DCM (5 mL) were stirred at RT. The reaction mixture was concentrated under reduced pressure. The residue was taken up in DMF and water and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the desired product.

[0469] C 12 H9BrN2OS(M=309.2 g / mol)

[0470] ESI-MS: 309[M+H] +

[0471] R t (HPLC): 0.96 min (Method A)

[0472] Yield: 0.02 g; 14%

[0473] 1 H NMR (400 MHz, DMSO-d6) δppm: 3.61 (s, 2 H), 5.09 (s, 2 H), 7.02 (td, J = 7.48, 1.01 Hz, 1 H), 7.16 (d, J = 7.73 Hz, 1 H), 7.23-7.30 (m, 2 H), 7.78 (s, 1 H).

[0474] 2.19 Intermediate XIX

[0475] Intermediate XIX.1 (General Procedure)

[0476] 1-Ethyl-7-methyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione

[0477]

[0478] Ethyl 4-amino-1-methyl-1H-imidazole-5-carboxylate (bromomethyl)-1,3-thiazole (0.20 g; 1.15 mmol) and ethyl isocyanate (0.16 mL; 1.95 mmol) in pyridine (1 mL) were stirred at 70 °C for 2 h. KOtBu (0.20 g; 1.72 mmol) was added. After stirring at 70 °C overnight, the reaction mixture was quenched with MeOH, concentrated under reduced pressure, taken up in DMF and water, and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the desired product.

[0479] 8H 10 N4O2(M=194.2 g / mol)

[0480] ESI-MS: 195[M+H] +

[0481] R t (HPLC): 0.28 min (Method C)

[0482] Yield: 0.10 g; 43%

[0483] 1 H NMR (400 MHz, DMSO-d6) δppm: 1.10 (t, J = 6.97 Hz, 3 H), 3.81-3.90 (m, 5 H), 7.90 (s, 1 H), 11.77 (s, 1 H).

[0484] The following intermediate compounds were prepared using procedures analogous to those described for Intermediate XIX.1 using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0485]

[0486]

[0487] 2.20 Intermediate XX

[0488] Intermediate XX.1 (General Procedure)

[0489] 3-[(5-Bromo-1,3,4-thiadiazol-2-yl)methyl]-1-ethyl-7-methyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione

[0490]

[0491] Intermediate XIX.1 (0.10 g; 0.51 mmol), (5-bromo-1,3,4-thiadiazol-2-yl)methyl methanesulfonate (0.27 g; 0.99 mmol) and KCO (0.21 g; 1.54 mmol) in DMF (6 mL) were stirred at 80 °C for 2.5 h. The reaction mixture was concentrated under reduced pressure, taken up in water and DCM. The organic layer was washed with water and brine, dried over NaSO, filtered, and concentrated under reduced pressure to give the desired product, which was used in the next step without further purification.

[0492] C 11 H 11 BrN6O2S (M = 371.2 g / mol)

[0493] ESI-MS: 371[M+H] +

[0494] R t (HPLC): 0.75 min (Method A)

[0495] Yield: 0.20 g; 89%

[0496] The following intermediate compounds were prepared using procedures analogous to those described for Intermediate XX.1 using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0497]

[0498] Example 3: Preparation of Example Compounds

[0499] 3.1 General Procedure

[0500] 1-{[2-(2,4-difluoro-3-hydroxyphenyl)-1,3-thiazol-5-yl]methyl}-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (Example Compound 1)

[0501]

[0502] Pd-Peppsi 2Me-Ipent Cl (0.06 g; 0.07 mmol) was added to a stirred mixture of intermediate I.1 (0.40 g; 1.32 mmol), intermediate III.1 (0.35 g; 2.01 mmol) and Cs2CO3 (1.10 g; 3.38 mmol) in water (1 mL) and EtOH (4 mL). After stirring at 100° C. for 1 h, the reaction mixture was stirred by Filter and wash with EtOH. The filtrate is concentrated under reduced pressure. The residue is taken up in DCN and water. The organic layer is evaporated and the resulting precipitate is filtered off and washed with water, MeOH and Et2O to obtain the desired product.

[0503] C 15 H 11 F2N3O3S (M = 351.3 g / mol)

[0504] ESI-MS: 352[M+H] +

[0505] R t (HPLC): 0.84 min (Method A)

[0506] Yield: 0.43 g; 92%

[0507] 1 H NMR (400MHz, DMSO-d6) δppm: 3.17 (s, 3H), 5.20 (s, 2H), 5.77 (d, J = 7.98Hz, 1H), 7.18 (td, J = 9.57, 1.90Hz, 1H) ,7.60(ddd,J=8.93,7.92,5.96Hz,1H),7.90(d,J=7.86Hz,1H),8.00(d,J=2.28Hz,1H),10.51-10.71(m,1H).

[0508] 3.2 Example Compounds (Examples) 2-14

[0509] The following compounds were prepared using procedures analogous to those described under 3.1 above using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0510]

[0511]

[0512]

[0513]

[0514] Analytical data of Example compounds 2-14 described in the above table

[0515]

[0516]

[0517]

[0518] 3.3 Example Compound 15

[0519] 1-{[5-(2,4-difluoro-3-hydroxyphenyl)-1,3-thiazol-2-yl]methyl}-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0520]

[0521] 3-Bromo-2,6-difluorophenol (0.04 g; 0.18 mmol), bis(pinacolato)diboron (0.05 g; 0.18 mmol), XPhos Pd G2 (0.01 g; 0.01 mmol), XPhos (0.01 g; 0.01 mmol) and KOAc (0.04 g; 0.36 mmol) in EtOH (2 mL) were stirred in a microwave oven at 120 ° C for 10 min. Intermediate I.2 (0.03 g; 0.12 mmol), Na2CO3 (aqueous solution; 2 mol / L; 0.18 mL; 0.36 mmol) and XPhos Pd G2 (0.01 g; 0.01 mmol) were added and stirred in a microwave oven at 120 ° C for another 10 min. The reaction mixture was diluted with DMF and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the desired product.

[0522] C 15 H 11 F2N3O3S (M = 351.3 g / mol)

[0523] ESI-MS: 352[M+H] +

[0524] R t (HPLC): 0.61 min (Method D)

[0525] Yield: 0.01 g; 31%

[0526] 1 H NMR (400MHz, DMSO-d6) δppm: 3.17 (s, 3H), 5.29 (s, 2H), 5.80 (d, J = 7.86Hz, 1H), 7.09-7.25 (m, 2H), 7.87 (d, J = 7.86Hz, 1H), 8.10 (s, 1H), 10.49 (s, 1H).

[0527] 3.4 Example Compound 16

[0528] 1-{[3-(2,4-difluoro-3-hydroxyphenyl)-1,2-oxazol-5-yl]methyl}-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0529]

[0530] Intermediate VI.1 (0.40 g; 1.32 mmol) in TFA (1.00 mL; 12.96 mmol) and DCM (3 mL) was stirred at RT for 20 min. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to afford the desired product.

[0531] C 15 H 11 F2N3O4 (M = 335.3 g / mol)

[0532] ESI-MS: 336[M+H] +

[0533] R t (HPLC): 0.83 min (Method A)

[0534] Yield: 0.01 g; 72%

[0535] 1H NMR (400MHz, DMSO-d6) δppm: 3.17 (s, 3H), 5.20 (s, 2H), 5.82 (d, J = 7.86Hz, 1H), 6.89 (d, J = 2.66Hz, 1 H),7.10-7.22(m,1H),7.28(ddd,J=8.71,7.57,5.89Hz,1H),7.86(d,J=7.86Hz,1H),10.57(s,1H).

[0536] The following examples, compounds 17 to 22, were prepared using procedures analogous to those described under 3.4 above using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0537]

[0538]

[0539] Analytical data of the compounds mentioned in the above table:

[0540]

[0541] 3.5 Example Compound 23

[0542] 1-{[3-(2,4-difluoro-3-hydroxyphenyl)-1,2,4-oxadiazol-5-yl]methyl}-3-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

[0543]

[0544] A mixture of 2-(3-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)acetic acid (0.04 g; 0.20 mmol), EDC hydrochloride (0.04 g; 0.20 mmol) and HOBT (0.03 g; 0.24 mmol) in DMF (1 mL) was stirred at RT for 15 min. The mixture was added to intermediate X.1 (0.04 g; 0.20 mmol) in DMF (1 mL). The resulting reaction mixture was stirred at 95 °C for 2 h and at RT overnight. The reaction mixture was diluted with DMF / water, filtered and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the desired product.

[0545] C 14 H 10 F2N2O4 (M = 336.3 g / mol)

[0546] ESI-MS: 337[M+H] +

[0547] R t (HPLC): 0.47 min (Method C)

[0548] Yield: 0.01 g; 6%

[0549] 1 H NMR (400MHz, DMSO-d6) δppm: 3.17 (s, 3H), 5.40 (s, 2H), 5.87 (d, J = 7.98Hz, 1H), 7.22 (td, J = 9. 54,1.71Hz,1H),7.40(ddd,J=8.87,7.35,5.96Hz,1H),7.88(d,J=7.98Hz,1H),10.67(s,1H).

[0550] 3.6 Example Compound 24

[0551] 1-{[2-(2,4-difluoro-3-hydroxyphenyl)-1,3-thiazol-5-yl]methyl}-3-methyl-1,3-diazin-2-one

[0552]

[0553] Intermediate XVII.1 (0.03 g; 0.06 mmol) and TFA (1.00 mL; 12.96 mmol) in DCM (3 mL) were stirred at RT for 25 min. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in THF (3 mL) and CDI (0.01 g; 0.07 mmol) was added. After stirring at 50 °C for 2 h, the reaction mixture was diluted with DMF and water and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the desired product.

[0554] C 15 H 15 F2N3O2S (M = 339.4 g / mol)

[0555] ESI-MS: 340 [M+H] +

[0556] R t (HPLC): 0.86 min (Method A)

[0557] Yield: 0.01 g; 52%

[0558] 1H NMR (400MHz, DMSO-d6) δppm: 1.86 (quin, J=5.92Hz, 2H), 2.82 (s, 3H), 3.22 (dt, J=20.40, 5.89Hz, 4H), 4 .63(s,2H),7.13-7.22(m,1H),7.60(ddd,J=8.78,8.02,6.02Hz,1H),7.83(d,J=2.28Hz,1H),10.55(br s,1H).

[0559] 3.7 Example Compound 25 (General Procedure)

[0560] 1-{[5-(2,4-difluoro-3-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]methyl}-3-hydroxy-3-methyl-2,3-dihydro-1H-indol-2-one

[0561]

[0562] Intermediate IX.3 (0.10 g; 0.26 mmol), 3-hydroxy-3-methyl-2,3-dihydro-1H-indol-2-one (0.04 g; 0.26 mmol) and K2CO3 (0.09 g; 0.65 mmol) in DMF (3 mL) were stirred at 70 ° C for 20 h. Water was added and the reaction mixture was extracted several times with DCM. The organic layer was separated, dried, and concentrated under reduced pressure. The residue was purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to give the intermediate. This intermediate was taken up in DCM (1.5 mL) and TFA (1.00 mL; 49.60 mmol) and stirred overnight at RT. After concentration under reduced pressure, the residue was taken up in ACN and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the desired product.

[0563] C 18 H 13 F2N3O3S (M = 389.4 g / mol)

[0564] ESI-MS: 390 [M+H] +

[0565] R t (HPLC): 0.79 min (Method A)

[0566] Yield: 0.01 g; 6%

[0567] 1H NMR(400MHz,DMSO-d6)δppm:1.44(s,3H),5.42(s,2H),6.09(br s,1H),7.09(td,J=7.45,0.82Hz,1H),7.13(d,J=7.86Hz,1H),7.24(td,J=9.60,1.84Hz,1H),7.27 -7.35(m,1H),7.39(dd,J=7.35,0.76Hz,1H),7.63(ddd,J=8.93,7.54,5.83Hz,1H),10.74(s,1H).

[0568] The following example compounds 26 to 30 were prepared using procedures analogous to those described under 3.7 above using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0569]

[0570] Analytical data of the above example compound 26

[0571]

[0572] 3.7 Example Compound 27

[0573] 1-{[2-(2,4-difluoro-3-hydroxyphenyl)-1,3-thiazol-5-yl]methyl}-2,3-dihydro-1H-1,3-benzothiazol-2-one

[0574]

[0575] 2,3-Dihydro-1H-1,3-benzodiazol-2-one (0.03 g; 0.08 mmol) and sodium hydride (55% dispersion in mineral oil; 0.01 g; 0.14 mmol) in THF (1 mL) were stirred at RT for 15 min. Intermediate IX.2 (0.02 g; 0.11 mmol) was added. After stirring at 60 °C for 6 h, the mixture was evaporated and taken up in water and DMF to be purified by column chromatography (reverse phase; XBridge C18; water / ACN / NH4OH). The residue was taken up in TFA (solution in DCM; 50%; 1 mL) and stirred at RT overnight. The reaction mixture was concentrated under reduced pressure, the residue was taken up in DMF and purified by column chromatography (reverse phase; SunFire C18; water / ACN / TFA).

[0576] C 17 H 11 F2N3O2S (M = 359.4 g / mol)

[0577] ESI-MS: 360 [M+H] +

[0578] R t (HPLC): 0.71 min (Method E)

[0579] Yield: 0.01 g; 10%

[0580] 1 H NMR(400MHz,DMSO-d6)δppm:5.30(s,2H),6.97-7.05(m,3H),7.12-7.21(m,1H),7.26 -7.31(m,1H),7.53-7.61(m,1H),8.05(d,J=2.28Hz,1H),10.56(s,1H),10.96(s,1H).

[0581] The following compounds 28 to 29 were prepared using procedures analogous to those described under 3.5 above using appropriate starting materials. As will be appreciated by those skilled in the art, these analogous examples may involve variations in the general reaction conditions.

[0582]

[0583] Analytical data of the above compounds 28 and 29

[0584]

[0585] 3.8 Example Compound 30

[0586] 1-{[5-(2,4-difluoro-3-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]methyl}-3-ethyl-1,2,3,4-tetrahydroquinazoline-2,4-dione

[0587]

[0588] Intermediate XIII.4 (0.07 g; 0.14 mmol), iodoethane (0.02 mL; 0.21 mmol) and K2CO3 (0.04 g; 0.28 mmol) in DMF (1 mL) were stirred at 80 °C for 2 h. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA). The residue was taken up in TFA (1.00 mL; 12-96 mmol) and DCM (2.5 mL). After stirring overnight at RT, the mixture was purified by column chromatography (reverse phase; Sunfire C18; water / ACN / TFA) to provide the desired compound.

[0589] C 19 H 14 F2N4O3S (M = 416.4 g / mol)

[0590] ESI-MS: 417[M+H] +

[0591] R t (HPLC): 0.90 min (Method A)

[0592] Yield: 0.01 g; 16%

[0593] 1 H NMR (400MHz, DMSO-d6) δppm: 1.20 (t, J = 7.03Hz, 3H), 4.04 (q, J = 7.05Hz, 2H), 5.84 (s, 2H), 7.24 (ddd, J = 10.30, 8.90, 1.84Hz, 1 H),7.31-7.37(m,1H),7.59-7.64(m,1H),7.65(d,J=8.24Hz,1H),7.76-7.81(m,1H),8.10(dd,J=7.86,1.52Hz,1H),10.75(br s,1H).

[0594] Example 4 : Biochemistry Human HSD17B13 - RapidFire MS / MS assay.

[0595] Estradiol (Sigma, catalog number E8875), NAD (Roche, catalog number 10621650001) and recombinant human HSD17B13 (full-length HSD17B13 with a C-terminal His tag (Uniprot ID Q7Z5P4-1), expressed in mammalian cells and purified to homogeneity) were diluted in assay buffer (100 mM Tris, Sigma, catalog number T2319; sodium chloride, Roth, catalog number 3957.2; 0.5 mM EDTA, Invitrogen, catalog number 15575020; 0.1% TCEP, Invitrogen, catalog number T2556; 0.05% BSA fraction V (protease and fatty acid free), Serva, catalog number 11945; 0.001% Tween20, Serva, catalog number 37470). Compounds were serially diluted in DMSO (Sigma, catalog number 5879) and spotted on 384-well microplates (PP, V-bottom) (Greiner, catalog number 781280) by LabcyteEcho 55x (1% DMSO in the assay). First, 6 μL / well of recombinant hHSD17B13 (final 1 nM) dilution was added, followed by incubation at RT for 15 min. Second, 6 μL / well of diluted estradiol (final 30 μM) and NAD (final 0.5 mM) were added, mixed, and incubated at RT for 4 h. 1 μL d4-estrone (final 50 nM; Sigma, catalog number 489204) was added, followed by 2.4 μL Girard's Reagent P (final 6.5 mM; TCI, catalog number G0030) dissolved in 90% methanol (Sigma, catalog number 34860) and 10% formic acid (Merck, catalog number 33015) to derivatize the analyte and stop the enzyme reaction. Incubate at RT for 12-24 h, then add 70 μL dH2O.

[0596] The analytical sample processing was performed by a RapidFire autosampler system (Agilent, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer (Triple Quad 6500, AB Sciex Germany GmbH, Darmstadt, Germany). The liquid sample was drawn into a 10 μL sample loop for 250 ms by a vacuum pump and then flushed for 3000 ms with an aqueous mobile phase (99.5% water, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.5 mL / min) onto a C18 column (Agilent, Waldbronn, Germany). The solid phase extraction step retained the analytes while removing interfering matrix (e.g., buffer components). The analyte was desorbed and eluted from the column in reverse direction for 3000 ms with an organic mobile phase (49.75% methanol, 49.75% acetonitrile, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.25 mL / min) and flushed into a mass spectrometer for MRM mode detection. The MRM transition of estrone was 404.1 < 157.1 Da (declustering voltage 27 V, collision energy 43 V), and the MRM transition of internal standard D4 estrone was 408.1 < 159.1 Da (declustering voltage 27 V, collision energy 43 V). The dwell time for each MRM transition was 25 ms, and the pause time between MRMs was 5 ms. The mass spectrometer was operated in positive ionization mode (curtain gas 35 Au, collision gas medium, ion spray voltage 4200 V, temperature 550° C., ion source gas 1 65 Au, ion source gas 2 80 Au). During the back flush into the mass spectrometer, the quantitative loop and related tubing were flushed with organic mobile phase to prevent carryover of analytes or matrix components in the next sample. The system equilibrium time was 500 ms. To minimize the carryover effect, the needle was washed with pure water (100%) and pure methanol (100%) between samples using the RapidFire system's wash station. The solvent delivery device of the RapidFire system consisted of two HPLC pumps (G1310A, Agilent, Waldbronn, Germany) running continuously and isocratically and a binary HPLC pump channel B (G4220A, Agilent, Waldbronn, Germany). MS data processing was performed at GMSU (Alpharetta, Georgia, USA), and the peak area ratios of analyte / internal standard were reported for IC50 calculations. IC50 values ​​were calculated using a 4-parameter nonlinear regression curve fitting model (developed in-house at Software Megalab). For data evaluation and calculations, the measurements of the bottom section (without HSD17B13 enzyme) were set as 0% control and the measurements of the top section (containing NAD, estrone and HSD17B13) were set as 100% control.IC50 values ​​were calculated using the following standard 4-parameter logistic regression formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50-X) x Slope + log((Top - Bottom) / (50 - Bottom) - 1))).

[0597] Example 5 :Biochemistry Mouse HSD17B13-RapidFire MS / MS Assay

[0598] Estradiol (Sigma, catalog number E8875), NAD (Roche, catalog number 10621650001) and recombinant mouse HSD17B13 (U-Protein Express BV, The Netherlands) were diluted in assay buffer (100 mM Tris, Sigma, catalog number T2319; sodium chloride, Roth, catalog number 3957.2; 0.5 mM EDTA, Invitrogen, catalog number 15575020; 0.1% TCEP, Invitrogen, catalog number T2556; 0.05% BSA fraction V (protease and fatty acid free), Serva, catalog number 11945; 0.001% Tween 20, Serva, catalog number 37470). The compounds were serially diluted in DMSO (Sigma, catalog number 5879) and spotted on 384-well microplates (PP, V-bottom) (Greiner, catalog number 781280) by Labcyte Echo 55x (1% DMSO in the assay). First, 6 μL / well of the dilution of recombinant mouse HSD17B13 (final 50 nM) was added, followed by incubation for 15 min at RT. Second, 6 μL / well of diluted estradiol (final 30 μM) and NAD (final 0.5 mM) were added, mixed, and incubated for 3 h at RT. 1 μL d4-estrone (final 50 nM; Sigma, catalog number 489204) was added, followed by 2.4 μL Giraert's reagent P (final 6.5 mM; TCI, catalog number G0030) dissolved in 90% methanol (Sigma, catalog number 34860) and 10% formic acid (Merck, catalog number 33015) to derivatize the analyte and stop the enzyme reaction. Incubate at RT for 12-24 h, then add 70 μL dH2O.

[0599] Sample processing for analysis was performed by a RapidFire autosampler system (Agilent, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer (Triple Quad 6500, AB Sciex Germany GmbH, Darmstadt, Germany). Liquid samples were drawn into a 10 μL loop by a vacuum pump for 250 ms and subsequently flushed for 3000 ms onto a C18 column (Agilent, Waldbronn, Germany) with an aqueous mobile phase (99.5% water, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.5 mL / min). The solid phase extraction step retained the analytes while removing interfering matrix (e.g., buffer components). The analytes were desorbed and back-eluted from the column with an organic mobile phase (49.75% methanol, 49.75% acetonitrile, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.25 mL / min) for 3000 ms and flushed into the mass spectrometer for detection in MRM mode. The MRM transition of estrone was 404.1 < 157.1 Da (declustering voltage 27 V, collision energy 43 V), and the MRM transition of internal standard D4 estrone was 408.1 < 159.1 Da (declustering voltage 27 V, collision energy 43 V). The dwell time for each MRM transition was 25 ms, and the pause time between MRMs was 5 ms. The mass spectrometer was operated in positive ionization mode (curtain gas 35 Au, collision gas medium, ion spray voltage 4200 V, temperature 550 ° C, ion source gas 1 65 Au, ion source gas 2 80 Au). During the back flush into the mass spectrometer, the quantitative loop and related pipes were flushed with organic mobile phase to prevent the carryover effect of analytes or matrix components in the next sample. The equilibration time of the system was 500 ms. In order to minimize the carryover effect, the needle was washed with pure water (100%) and pure methanol (100%) between samples using the RapidFire system's wash station. The solvent delivery device of the RapidFire system consists of two HPLC pumps (G1310A, Agilent, Waldbronn, Germany) running continuously and operating isocratically and one binary HPLC pump channel B (G4220A, Agilent, Waldbronn, Germany). MS data processing was performed at GMSU (Alpharetta, Georgia, USA) and the peak area ratios of analyte / internal standard were reported for IC50 calculations. IC50 values ​​were calculated using a 4-parameter nonlinear regression curve fitting model (developed in-house at Software Megalab). For data evaluation and calculations, the measured values ​​of the bottom (no HSD17B13 enzyme) were set to 0% control, and the measured values ​​of the top (containing NAD, estrone and HSD17B13) were set to 100% control.IC50 values ​​were calculated using the following standard 4-parameter logistic regression formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50-X) x Slope + log((Top - Bottom) / (50 - Bottom) - 1))).

[0600] Example 6: RapidFire MS / MS determination of human HSD17B13 in cells

[0601] Dilutions of estradiol (Sigma, catalog number E8875) and cells (-Myc / DDK-tagged cloned HEK293 cells stably overexpressing human HSD17B13, Lakepharma) were prepared in serum-free media (DMEM, Sigma, catalog number D5796; 10% heat-inactivated FBS, Gibco, catalog number 100500; 1x Glutamax, Gibco, catalog number 35050-087; 1x sodium pyruvate, Gibyo, catalog number 11360070).

[0602] 24 h before compound testing, 25 μL of 0.4*10^6 cells / mL dilution was seeded in a 384-well microplate (culture plate, Perkin Elmer, catalog number 6007680).

[0603] The compounds were serially diluted in DMSO (Sigma, catalog number 5879) and spotted on pre-seeded cell plates by Labcyte Echo55x (1% DMSO in the assay) and incubated for 30 min at 37° C. in a humidified incubator (rH=95%, CO2=5%). Afterwards, 25 μL of the 60 μM estradiol dilution was added to the plate and incubated for 3 h at 37° C. in a humidified incubator (rH=95%, CO2=5%).

[0604] Take 20 μL of supernatant and add 2.5 μL d4-estrone (final 50 nM; Sigma, catalog number 489204), followed by 5 μL Girardt's reagent P (final 6.5 mM; TCI, catalog number G0030) dissolved in 90% methanol (Sigma, catalog number 34860) and 10% formic acid (Merck, catalog number 33015) to derivatize the analyte. Incubate at RT for 12-24 h, then add 70 μL dH2O.

[0605] Sample processing for analysis was performed by a RapidFire autosampler system (Agilent, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer (Triple Quad 6500, AB Sciex Germany GmbH, Darmstadt, Germany). Liquid samples were drawn into a 10 μL loop by a vacuum pump for 250 ms and subsequently flushed for 3000 ms onto a C18 column (Agilent, Waldbronn, Germany) with an aqueous mobile phase (99.5% water, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.5 mL / min). The solid phase extraction step retained the analytes while removing interfering matrix (e.g., buffer components). The analytes were desorbed and back-eluted from the column with an organic mobile phase (49.75% methanol, 49.75% acetonitrile, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.25 mL / min) for 3000 ms and flushed into the mass spectrometer for detection in MRM mode. The MRM of estrone was changed to 404.1 < 157.1Da (declustering voltage 27V, collision energy 43V), and the MRM of internal standard D4 estrone was changed to 408.1 < 159.1Da (declustering voltage 27V, collision energy 43V). The residence time of each MRM transition was 25ms, and the pause time between MRMs was 5ms. The mass spectrometer was operated in positive ionization mode (curtain gas 35Au, collision gas medium, ion spray voltage 4200V, temperature 550°C, ion source gas 1 65Au, ion source gas 2 80Au). When backwashing into the mass spectrometer, the quantitative loop and related pipelines were rinsed with organic mobile phase to prevent the carryover effect.

[0606] MS data processing was performed at GMSU (Alpharetta, Georgia, USA), and the peak area ratio of the analyte / internal standard was reported for IC50 calculation. IC50 values ​​were calculated using a 4-parameter nonlinear regression curve fitting model (Software Megalab internal development). For data evaluation and calculation, the measured values ​​of the bottom (cells with estradiol and 10 μM of the internally identified HSD17B13 inhibitor) were set to 0% control, and the measured values ​​of the top (containing cells with estradiol) were set to 100% control. IC50 values ​​were calculated using the following standard 4-parameter logistic regression formula: Y=bottom+(top-bottom) / (1+10^((LogIC50-X)x slope+log((top-bottom) / (50-bottom)-1))).

[0607] Example 7: Determination of the viability of human HSD17B13 cells

[0608] Dilutions of estradiol (Sigma, catalog number E8875) and cells (-Myc / DDK-tagged cloned HEK293 cells stably overexpressing human HSD17B13, Lakepharma) were prepared in serum-free media (DMEM, Sigma, catalog number D5796; 10% heat-inactivated FBS, Gibco, catalog number 100500; 1x Glutamax, Gibco, catalog number 35050-087; 1x sodium pyruvate, Gibyo, catalog number 11360070).

[0609] 24 h before compound testing, 25 μL of 0.4*10^6 cells / mL dilution was seeded in a 384-well microplate (culture plate, Perkin Elmer, catalog number 6007680).

[0610] The compounds were serially diluted in DMSO (Sigma, catalog number 5879) and spotted on pre-seeded cell plates by Labcyte Echo55x (1% DMSO in the assay). Incubated at 37°C for 30 min in a humidified incubator (rH = 95%, CO2 = 5%). Afterwards, 25 μL of 60 μM estradiol dilution was added to the plate and incubated at 37°C for 3 h in a humidified incubator (rH = 95%, CO2 = 5%).

[0611] 5 μL of CellTiter Glo 2 (Promega, Cat. No. G9242) was added to the cell plate, incubated for 15 min at room temperature, and luminescence was measured on a PHERAstar FSX (BMG Labtech, Ortenberg, Germany).

[0612] IC50 values ​​were calculated using a 4-parameter nonlinear regression curve fitting model (Software Megalab internal development). For data evaluation and calculation, the measured value of the bottom (no cells, with estradiol) was set to 0% control, and the measured value of the top (containing cells and estradiol) was set to 100% control. IC50 values ​​were calculated using the following standard 4-parameter logistic regression formula: Y=bottom+(top-bottom) / (1+10^((LogIC50-X)*slope+log((top-bottom) / (50-bottom)-1))).

[0613] Example 8: Biochemistry Human HSD17B11-RapidFire MS / MS Assay.

[0614] Estradiol (Sigma, catalog number E8875), NAD (Roche, catalog number 10621650001) and recombinant hHSD17B11 (U-Protein Express BV, The Netherlands) were diluted in assay buffer (100 mM Tris, Sigma, catalog number T2319; sodium chloride, Roth, catalog number 3957.2; 0.5 mM EDTA, Invitrogen, catalog number 15575020; 0.1% TCEP, Invitrogen, catalog number T2556; 0.05% BSA fraction V (protease and fatty acid free), Serva, catalog number 11945; 0.001% Tween20, Serva, catalog number 37470). Compounds were serially diluted in DMSO (Sigma, catalog number 5879) and spotted on 384-well microplates (PP, V-bottom) (Greiner, catalog number 781280) by Labcyte Echo 55x (1% DMSO in the assay). First, 6 μL / well of the dilution of recombinant hHSD17B11 (final 35 nM) was added, followed by incubation for 15 min at RT. Second, 6 μL / well of diluted estradiol (final 30 μM) and NAD (final 0.5 mM) were added, mixed, and incubated for 4 h at RT. 1 μL d4-estrone (final 50 nM; Sigma, catalog number 489204) was added, followed by 2.4 μL Giraert's reagent P (final 6.5 mM; TCI, catalog number G0030) dissolved in 90% methanol (Sigma, catalog number 34860) and 10% formic acid (Merck, catalog number 33015) to derivatize the analyte and stop the enzyme reaction. Incubate at RT for 12-24 h, then add 70 μL dH2O.

[0615] Sample processing for analysis was performed by a RapidFire autosampler system (Agilent, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer (Triple Quad 6500, AB Sciex Germany GmbH, Darmstadt, Germany). Liquid samples were drawn into a 10 μL loop by a vacuum pump for 250 ms and subsequently flushed for 3000 ms onto a C18 column (Agilent, Waldbronn, Germany) with an aqueous mobile phase (99.5% water, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.5 mL / min). The solid phase extraction step retained the analytes while removing interfering matrix (e.g., buffer components). The analytes were desorbed and back-eluted from the column with an organic mobile phase (49.75% methanol, 49.75% acetonitrile, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.25 mL / min) for 3000 ms and flushed into the mass spectrometer for detection in MRM mode. The MRM transition of estrone was 404.1 < 157.1 Da (declustering voltage 27 V, collision energy 43 V), and the MRM transition of internal standard D4 estrone was 408.1 < 159.1 Da (declustering voltage 27 V, collision energy 43 V). The dwell time for each MRM transition was 25 ms, and the pause time between MRMs was 5 ms. The mass spectrometer was operated in positive ionization mode (curtain gas 35 Au, collision gas medium, ion spray voltage 4200 V, temperature 550 ° C, ion source gas 1 65 Au, ion source gas 2 80 Au). During the back flush into the mass spectrometer, the quantitative loop and related pipes were flushed with organic mobile phase to prevent the carryover effect of analytes or matrix components in the next sample. The equilibration time of the system was 500 ms. In order to minimize the carryover effect, the needle was washed with pure water (100%) and pure methanol (100%) between samples using the RapidFire system's wash station. The solvent delivery device of the RapidFire system consists of two HPLC pumps (G1310A, Agilent, Waldbronn, Germany) running continuously and operating isocratically and one binary HPLC pump channel B (G4220A, Agilent, Waldbronn, Germany). MS data processing was performed at GMSU (Alpharetta, Georgia, USA) and the peak area ratios of analyte / internal standard were reported for IC50 calculations. IC50 values ​​were calculated using a 4-parameter nonlinear regression curve fitting model (developed in-house at Software Megalab). For data evaluation and calculations, the measured values ​​of the bottom (no HSD17B13 enzyme) were set to 0% control, and the measured values ​​of the top (containing NAD, estrone and HSD17B13) were set to 100% control.IC50 values ​​were calculated using the following standard 4-parameter logistic regression formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50-X) x Slope + log((Top - Bottom) / (50 - Bottom) - 1))).

[0616] Example 9: Human HSD17B11-RapidFire MS / MS Assay

[0617] Estradiol (Sigma, catalog number E8875), NAD (Roche, catalog number 10621650001) and recombinant hHSD17B11 (U-Protein Express BV, The Netherlands) were diluted in assay buffer (100 mM Tris, Sigma, catalog number T2319; sodium chloride, Roth, catalog number 3957.2; 0.5 mM EDTA, Invitrogen, catalog number 15575020; 0.1% TCEP, Invitrogen, catalog number T2556; 0.05% BSA fraction V (protease and fatty acid free), Serva, catalog number 11945; 0.001% Tween20, Serva, catalog number 37470). Compounds were serially diluted in DMSO (Sigma, catalog number 5879) and spotted on 384-well microplates (PP, V-bottom) (Greiner, catalog number 781280) by Labcyte Echo 55x (1% DMSO in the assay). First, 6 μL / well of the dilution of recombinant hHSD17B11 (final 35 nM) was added, followed by incubation for 15 min at RT. Second, 6 μL / well of diluted estradiol (final 30 μM) and NAD (final 0.5 mM) were added, mixed, and incubated for 4 h at RT. 1 μL d4-estrone (final 50 nM; Sigma, catalog number 489204) was added, followed by 2.4 μL Giraert's reagent P (final 6.5 mM; TCI, catalog number G0030) dissolved in 90% methanol (Sigma, catalog number 34860) and 10% formic acid (Merck, catalog number 33015) to derivatize the analyte and stop the enzyme reaction. Incubate at RT for 12-24 h, then add 70 μL dH2O.

[0618] Sample processing for analysis was performed by a RapidFire autosampler system (Agilent, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer (Triple Quad 6500, AB Sciex Germany GmbH, Darmstadt, Germany). Liquid samples were drawn into a 10 μL loop by a vacuum pump for 250 ms and subsequently flushed for 3000 ms onto a C18 column (Agilent, Waldbronn, Germany) with an aqueous mobile phase (99.5% water, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.5 mL / min). The solid phase extraction step retained the analytes while removing interfering matrix (e.g., buffer components). The analytes were desorbed and back-eluted from the column with an organic mobile phase (49.75% methanol, 49.75% acetonitrile, 0.49% acetic acid, 0.01% trifluoroacetic acid, flow rate 1.25 mL / min) for 3000 ms and flushed into the mass spectrometer for detection in MRM mode. The MRM transition of estrone was 404.1 < 157.1 Da (declustering voltage 27 V, collision energy 43 V), and the MRM transition of internal standard D4 estrone was 408.1 < 159.1 Da (declustering voltage 27 V, collision energy 43 V). The dwell time for each MRM transition was 25 ms, and the pause time between MRMs was 5 ms. The mass spectrometer was operated in positive ionization mode (curtain gas 35 Au, collision gas medium, ion spray voltage 4200 V, temperature 550 ° C, ion source gas 1 65 Au, ion source gas 2 80 Au). During the back flush into the mass spectrometer, the quantitative loop and related pipes were flushed with organic mobile phase to prevent the carryover effect of analytes or matrix components in the next sample. The equilibration time of the system was 500 ms. In order to minimize the carryover effect, the needle was washed with pure water (100%) and pure methanol (100%) between samples using the RapidFire system's wash station. The solvent delivery device of the RapidFire system consists of two HPLC pumps (G1310A, Agilent, Waldbronn, Germany) running continuously and operating isocratically and one binary HPLC pump channel B (G4220A, Agilent, Waldbronn, Germany). MS data processing was performed at GMSU (Alpharetta, Georgia, USA) and the peak area ratios of analyte / internal standard were reported for IC50 calculations. IC50 values ​​were calculated using a 4-parameter nonlinear regression curve fitting model (developed in-house at Software Megalab). For data evaluation and calculations, the measured values ​​of the bottom (no HSD17B13 enzyme) were set to 0% control, and the measured values ​​of the top (containing NAD, estrone and HSD17B13) were set to 100% control.IC50 values ​​were calculated using the following standard 4-parameter logistic regression formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50-X) x Slope + log((Top - Bottom) / (50 - Bottom) - 1))).

[0619] Example 10: In vitro pharmacokinetic determination of metabolic stability in liver microsomes

[0620] Metabolic degradation of test compounds was determined using pooled liver microsomes at 37°C.

[0621] The final incubation volume of 60 μl for each time point contained TRIS buffer (pH 7.6, 0.1 M), magnesium chloride (5 mM), microsomal protein (0.5-2 mg / ml) and test compound at a final concentration of 1 μM at RT.

[0622] After a short pre-incubation period at 37°C, the reaction was started by adding the reduced form of β-nicotinamide adenine dinucleotide phosphate (NADPH, 1 mM) and terminated after different time points by transferring aliquots into solvent.

[0623] The quenched incubations were pelleted by centrifugation (10000 g, 5 min).

[0624] The amount of parent compound remaining in an aliquot of the supernatant was determined by LC-MS / MS.

[0625] The half-life (t1 / 2INVITRO) was determined by the slope of the semi-logarithmic plot of the concentration-time curve.

[0626] The intrinsic clearance (CL_INTRINSIC) was calculated by taking into account the amount of protein in the incubation:

[0627] CL_INTRINSIC [μl / min / mg protein] = (Ln 2 / (half-life [min] * protein content [mg / ml])) * 1000

[0628] Example 11: In vitro pharmacokinetic assay of metabolic stability in human hepatocytes (HHEP assay)

[0629] The assay was performed in human hepatocytes to assess the metabolic stability of the compounds. Metabolic degradation of the test compounds was determined in human hepatocyte suspensions. After recovery from cryopreservation, human hepatocytes were diluted in DMEM (supplemented with 3.5 μg glucagon / 500 ml, 2.5 mg insulin / 500 ml, 3.75 mg hydrocortisone / 500 ml, 5% or 50% human serum or in the absence of serum) to obtain 1.0×10 6 cells / ml or 4.0x106 The final cell density of 10 cells / ml depends on the metabolic turnover rate of the test compound.

[0630] After pre-incubation for 30 min in a cell culture incubator (37° C., 10% CO 2 ), the test compound solution was spiked into the hepatocyte suspension resulting in a final test compound concentration of 1 μM and a final DMSO concentration of 0.05%.

[0631] The cell suspension was incubated at 37°C (cell culture incubator, horizontal shaker), and samples were taken from the incubator after 0, 0.5, 1, 2, 4 and 6 hours. The samples were quenched with acetonitrile (containing internal standard) and precipitated by centrifugation. The supernatant was transferred to a 96-deep well plate and prepared for analysis of the decline of the parent compound by HPLC-MS / MS.

[0632] The percentage of test compound remaining was calculated using the peak area ratio (test compound / internal standard) at each incubation time point relative to the peak area ratio at time point 0. Log-transformed data were plotted against incubation time, and the absolute value of the slope obtained by linear regression analysis was used to estimate the in vitro half-life (T 1 / 2 ).

[0633] From in vitro T 1 / 2 Calculate the in vitro intrinsic clearance (Cl int ), and use 120x10 6 The hepatocyte composition of 10 cells / g liver, the liver weight of 25.7 g liver / kg human body weight and the in vitro incubation parameters were scaled up to the whole liver, using the following formula:

[0634] CL_INTRINSIC_INVIVO[ml / min / kg]=(CL_INTRINSIC[μl / min / 10 6 cells] x liver cells constitute [10 6 cells / g liver] x liver factor [g / kg body weight]) / 1000

[0635] Taking into account the average hepatic blood flow (QH) of 20.7 ml / min / kg, the hepatic in vivo blood clearance (CL) is predicted based on the well-stirred liver model:

[0636] CL [ml / min / kg] = CL_INTRINSIC_INVIVO [ml / min / kg] x liver blood flow [ml / min / kg] / (CL_INTRINSIC_INVIVO [ml / min / kg] + liver blood flow [ml / min / kg])

[0637] The results are expressed as a percentage of liver blood flow:

[0638] QH[%]=CL[ml / min / kg] / liver blood flow[ml / min / kg])

[0639] Example 12: Biological Data of Example Compounds 1-30

[0640]

[0641]

Claims

1. A compound having formula (I) or a salt thereof in a) Z——*Selected from And among them b) The structure is selected from the group consisting of:

2. The compound according to claim 1, wherein The structure is selected from the group consisting of:

3. The compound according to claim 1, wherein Z is 4. The compound or salt thereof according to claim 1, wherein the compound or salt thereof is selected from the group consisting of compounds of formula (I) 01-30.

5. A salt of a compound according to any one of claims 1 to 4 for use as a medicament.

6. A medicament prepared using the compound or salt thereof according to any one of claims 1 to 4.

7. A method for preparing a compound according to claim 1 having the general formula (I), the method comprising reacting compound X1 with compound X2 to produce compound (I), wherein Y may be Br, I or Cl, wherein in the compound of (I), Z, A1, A2 and A3 have the same meanings as defined in claim 1.

8. The process according to claim 7, which is carried out in an aprotic or protic solvent or solvent mixture at a temperature between ambient temperature and the boiling point of the solvent, preferably between 50°C and 120°C.

9. A pharmaceutical composition comprising at least one compound according to one or more of claims 1 to 4 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

10. A pharmaceutical composition according to claim 9, comprising a therapeutically effective amount of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, said therapeutically effective amount being in the range of 0.1 wt.-% to 90 wt.-% of the composition, preferably in the range of 0.5 wt.-% to 50 wt.-% of the composition.

11. A compound according to one or more of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 10, for use in the treatment or prevention of steatosis such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

12. The compound according to any one of claims 1 to 5 for use in the treatment and / or prevention of non-alcoholic steatohepatitis, wherein the compound is administered before, after or together with at least one other active substance.

13. A compound having formula (Ia) The compound can be used as an intermediate compound for preparing the compound of formula (I) according to claim 1, wherein Z is as defined in claim 1 and Y can be Br, I or Cl.

14. A compound having formula (Ib) The compound can be used as an intermediate compound for preparing the compound of formula (I) according to claim 1, wherein A1, A2 and A3 are as defined in claim 1.

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