WO2012047156A1 - Isoxazol-3(2h)-one analogs as plasminogen inhibitors and their use in the treatment of fibrinolysis related diseases - Google Patents

Abstract

A compound of formula (I): wherein R1 is hydrogen; R2 is C₁-C₁₀ alkyl, which C₁-C₁₀ alkyl comprises 0, 1, 2, 3 or 4 heteroatoms, selected from O and N, or 0, 1 or 2 acid, ester or amide functionalities, and said C₁-C₁₀ alkyl is substituted by 0, 1, 2, 3 or 4 CH₃, halogen or CF₃, or -XR20, wherein X is a bond or _-CH₂-, and R20 is a 4 to 10 membered, monocyclic or bicyclic heterocyclic aromatic or non-aromatic ring containing 1, 2, 3 or 4 heteroatoms selected from O and N, and R20 is substituted by 0, 1, 2, 3 or 4 CH3, halogen or CF3, pharmaceutical compositions, medical uses and treatment or prophylaxis of a disease or condition in which modulation of fibrinolysisis is beneficial.

Description

ISOXAZOL-3(2H)-ONE ANALOGS AS PLASMINOGEN INHIBITORS AND THEIR USE IN THE TREATMENT OF FIBRINOLYSIS RELATED DISEASES

Field of invention

The present invention relates to compounds of formula I, to a pharmaceutical composition comprising them, and to their use in treating fibrinolysis related diseases or conditions, for example inherited bleeding disorders, stroke,

menorrhagia and liver diseases and for the treatment of hereditary

angioedema.

Background

Bleeding is a common clinical problem in trauma, surgery, bleeding disorders, stroke, menorrhagia and liver diseases. Treatment of bleeding includes agents in primary and secondary haemostasis as well as fibrinolysis inhibition.

The fibrinolytic system comprises an inactive zymogen, plasminogen (PLG), that can be activated to the active enzyme plasmin (PLN). PLN degrades insoluble fibrin into soluble fibrin fragments. The result of this activity is the dissolution of the fibrin clot. The activation of plasminogen to plasmin occurs on the clot surface after binding to fibrin. Mediators of the activation are urokinase plasminogen activator (u-PA) or tissue-type plasminogen activator (t-PA).

Activation of the fibrinolytic process can be used to treat thrombotic conditions. Conversely, inhibition of fibrinolysis can be, and is, used for treatment of bleeding conditions. There are several possible targets for the inhibition of fibrinolysis. Activation of plasminogen activator inhibitor 1 (PAI-1 ), inhibition of u-PA and/or t- PA activity, inhibition of PLN activity and activation of antiplasmin are examples. Specific inhibition of proteolytic sites in tPA, uPA and PLN is difficult. However, bleeding control via inhibition of plasmin(ogen) fibrin binding by lysine analogues has been proven in humans as a safe and effective mechanism of action.

Inhibition of fibrinolysis via a lysine mimetic is a validated concept for reducing blood-loss, without increased risk for thrombotic complications, for instance following surgery, in menorrhagia, haemophilia and von Willebrands disease. Potential uses of the compounds are to block plasmin-induced proteolysis as a universal pathomechanism propagating cancer, and cardiovascular, inflammatory, and many other diseases.

Antifibrinolytics have also been successfully used to treat hereditary angioedema. In this disease the skin or mucosa around the mouth, throat and tongue rapidly swell up. Swelling can occur at other places like limbs or genitals. For reasons that are not well-understood antifibrinolytics can be used as a prophylaxis or acute treatment of hereditary angioedema.

Tranexamic acid, currently the compound on the market to treat menorrhagia, requires very high and multiple doses and has gastrointestinal side effects. Its use has been described in "Tranexamic acid. A review of its use in surgery and other indications"; Dunn, C.J.; Goa K.L.; Drugs 1999, June 57(6): 1005-1032.

Object of the invention

An object of the invention is to provide for a pharmaceutical compound to prevent or treat excess bleeding, the treatment or prevention therapy having one or more improvements such as enhanced efficacy, selectivity, permeability, duration, less side effects and improved bio availability.

Description of the invention

The present invention provides for a compound of formula I:

wherein R1 is hydrogen; R2 is

C-i-C-io alkyl, which C1-C10 alkyl comprises 0, 1 , 2, 3 or 4 heteroatoms, selected from O and N, or 0, 1 or 2 acid, ester or amide functionalities, and said C-I-C-IO alkyl is substituted by 0, 1 , 2, 3 or 4 CH_3, halogen or CF₃, or

-XR20, wherein X is a bond or -CH₂- , and

R20 is a 4 to 10 membered, monocyclic or bicyclic heterocyclic aromatic or non-aromatic ring containing 1 , 2, 3 or 4 heteroatoms selected from O and N, and R20 is substituted by 0, 1 , 2, 3 or 4 CH_3, halogen or CF₃, or a pharmaceutically acceptable salt thereof.

A further embodiment of the invention discloses a compound of formula I as described herein, wherein R2 is -XR20 and X is -CH₂- , and

R20 is a 4 to 10 membered, monocyclic or bicyclic heterocyclic non-aromatic ring containing 1 , 2, 3 or 4 heteroatoms selected from O and N, and R20 is substituted by 0, 1 , 2, 3 or 4 CH₃, halogen or CF₃.

Still a further embodiment of the invention discloses a compound of formula I as described herein, wherein R2 is -XR20 and at least one heteroatom is N and/or halogen (if present) is fluoro.

The compounds of formula I may exist in stereoisomeric and/or tautomeric forms. It is to be understood that all enantiomers, diastereomers, racemates, tautomers and mixtures thereof are included within the scope of the invention.

Different isomers may have different biological activity. It may be the case that different compounds of the general formula I may show the highest biological activity with different configuration. For instance for one compound the (2R,4S) configuration may have the highest biological activity, but for another compound the (2S,4S) may have the highest activity.

According to one aspect of the invention it is provided for a compound of formula II with the following configuration:

II

According to one aspect of the invention it is provided for a compound of formula III with the following configuration:

III

According to one aspect of the invention it is provided for a compound of formula IV with the following configuration:

IV According to one aspect of the invention it is provided for a compound of formula V with the following configuration:

In a further embodiment of the invention, as described herein, a compound of formula I is selected from:

5-((2R,4S)-2-(Morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-(Trans-2-(morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-((3-(Trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2S,4R)-2-((3-(Trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2S,4S)-2-((3-(Trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2R,4R)-2-((3-(Trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-(((R)-2-(Trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidin- 4-yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-(((S)-2-(Trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidin- 4-yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-((3,3-Difluoropyrrolidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one;

5-((2S,4S)-2-((3,3-Difluoropyrrolidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one; 5-((2R,4S)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one;

5-((2S,4S)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one;

5-(Cis-2-(isoindolin-2-ylmethyl)piperidin-4-yl)isoxazol-3(2H)-one; and 5-((2S,4S)-2-(lsoindolin-2-ylmethyl)piperidin-4-yl)isoxazol-3(2H)-one.

According to another aspect of the invention it is provided for a pharmaceutical composition comprising at least one compound and a pharmaceutically acceptable carrier or diluent.

According to another aspect of the invention it is provided for a method for

treatment or prophylaxis of a disease or condition, such as heavy bleeding, inherited or acquired bleeding disorders, stroke, menorrhagia and liver diseases, in which modulation of fibrinolysis is beneficial, and for the treatment of hereditary angioedema, comprising administering to a warm-blooded animal in need of such treatment a therapeutically effective amount of at least one compound of formula I. Other areas of use for the compound according to formula I would be in wound healing, reduce blood loss in connection with surgery including dental surgery and for treating patients who are treated with anti-coagulation, for instance in preparation for surgery. The compound may also be used to reduce the blood transfusion needed in different situations. The compound may additionally be used in prophylactic treatment of von

Willebrand's disease, haemophilia or in the case of anti-coagulating patients.

According to another aspect of the invention it is provided for a method wherein said disease or condition is selected from inherited bleeding disorders, stroke, menorrhagia and liver diseases.

According to another aspect of the invention it is provided for the treatment or prophylaxis of a disease or condition in which modulation of fibrinolysis is beneficial. According to another aspect of the invention it is provided for wherein said disease or condition is selected from inherited bleeding disorders, stroke, menorrhagia and liver diseases.

According to another aspect of the invention it is provided for the use of a

compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis of a disease or condition in which modulation of fibrinolysis is beneficial.

According to another aspect of the invention it is provided for the use of a

compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis of a disease wherein said disease is selected from inherited bleeding disorders, stroke, menorrhagia and liver diseases.

Compounds of formula l-V may be prepared by the following route:

TSE2011/051172

NOTE: Stereochemical assignments marked with asterisks shall be taken to be definitive of the relative configurations of the carbon atoms marked, but not their absolute configurations, unless expressly stated otherwise

Assay Method

The assay method used for evaluating biological activity of the compounds according to the invention was Clot-lysis buffer assay.

Measurement of inhibition of plasminogen were performed in a 200 μΙ_ reaction mixture containing 15 mM HEPES, pH 7.4, 100 mM NaCI, 0.008 % Tween-80, 13 g/mL human Glu-plasminogen (Chromogenix, Italy), 1 .7 mg/mL human fibrinogen (Aniara, USA), 0.02 nM human single-chain tissue type plasminogen activator (Biopool, Sweden), 1 % DMSO and 0.05 NIH U/mL human thrombin (Sigma, USA). Tested substance were added to the reaction mixture in 10% DMSO. Reaction was started by the addition of thrombin.

Fibrin formation and degradation were followed in a spectrophotometer at 405 nm for 15 hrs at 37° C.

Biological activity

Measured according to the Clot-lysis buffer assay described above.

Example 1 showed an IC₅₀ value of 2.4 μΜ. Example 2 showed an IC₅₀ value of 5.4 μΜ. Example 3 showed an IC50 value of 0.62 μΜ. Example 4 showed an IC50 value of 45.6 μΜ. Example 5 showed an IC50 value of 4.84 μΜ. Example 6 showed an IC₅₀ value of 55.7 μΜ. Example 7 showed an IC₅₀ value of 2.78 μΜ. Example 8 showed an IC₅₀ value of 1 .88 μΜ. Example 9 showed an IC₅₀ value of 1 .48 μΜ. Example 10 showed an IC50 value of 9.1 μΜ. Example 1 1 showed an IC50 value of 3.3 μΜ. Example 12 showed an IC50 value of 2.5 μΜ. Example 13 showed an IC₅₀ value of 14.4 μΜ. Example 14 showed an IC₅₀ value of 1 .1 μΜ.

Preparation of Reference Compounds Reference compound 1

1 -(Methoxycarbonyl)-2-(morpholinomethyl)pipehdine-4-carboxylic acid Step 1 : Methyl 2-(hvdroxymethyl)isonicotinate

The reaction was performed in 2 separate 1000 ml_ round -bottomed flasks. To a solution of methyl isonicotinate (70 g, 510.44 mmol) and sulfuric acid (2.340 ml_, 43.90 mmol) in MeOH (700 ml_) under reflux, was added a solution of ammonium peroxydisulfate (210 g, 918.80 mmol) in water (350 ml_) over 20 min. The reaction was refluxed for 20 min and was then allowed to cool to room temperature. The solid was filtered off and washed with MeOH. MeOH was removed from the filtrate under reduced pressure and then was neutralised by cautious stepwise addition of solid Na₂CO₃ under ice-cooling. The aqueous solution was extracted with ethyl acetate and the combined organic layers were dried with Na₂SO₄ and evaporated. The dark-brown residue was treated with cyclohexane (3 x 300 ml_) and the cyclohexane phase was decanted. The remaining dark-brown residue was purified by automated flash chromatography on 2 Biotage® KP-SIL 340g columns. A gradient from 25 % to 100 % of EtOAc in heptane over 10 CV was used as mobile phase. Methyl 2-(hydroxymethyl)isonicotinate (27.5 g, 32 %) was isolated. ¹H NMR (400 MHz, cdcls) δ 3.90 (s, 3H), 4.78 (s, 2H), 7.57 - 7.90 (m, 2H), 8.64 (s, 1 H). MS m/z 168 (M+H)⁺

Step 2: Methyl 2-formyl isonicotinate

Methyl 2-(hydroxymethyl)isonicotinate (8.36 g, 50 mmol) was dissolved in dichloromethane (150 ml_). Dess-Martin periodinane (25 g, 58.94 mmol) was added and the mixture stirred at room temperature for 2 h 30 min. Sodium sulfothioate (59.3 g, 375.00 mmol) was dissolved in satd NaHCO₃ and added to the reaction mixture. The suspension was vigorously stirred at room temperature for 15 min, DCM was added and the phases were separated. The aqueous phase was extracted with DCM twice and the combined organic layers were dried over MgSO and evaporated. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 340g column. Gradient heptanes / EtOAc 80:20 to 65:35 over 5 CV was used as mobile phase. Methyl 2-formylisonicotinate (7 g, 85 %) was isolated as an off-white solid. ¹H NMR (400 MHz, cdcl₃) δ 4.00 (s, 3H), 8.09 (dd, 1 H), 8.49 (s, 1 H), 8.95 (d, 1 H), 10.15 (s, 1 H). MS m/z 165 (M+H)+

Step 3: Methyl 2-(morpholinomethyl)isonicotinate

Methyl 2-formylisonicotinate (1 .48 g, 8.96 mmol) was dissolved in THF (35 mL) to give a yellow solution. Morpholine (1 .951 mL, 22.40 mmol) was added to the solution. Acetic acid (1 .539 mL, 26.89 mmol) and sodium triacetoxyhydroborate (2.279 g, 10.75 mmol) were added and the mixture was stirred at room temperature for 4 h. Satd NaHCO₃ was added and the mixture was stirred at room temperature for 15 min. DCM was added and the phases were separated. The aqueous phase was extracted with DCM and the combined organic phases were filtered through a phase separator and evaporated. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100 g column. Mobile phase: 100 % ethyl acetate over 5 CV, then DCM / MeOH 20:1 over 5 CV. Methyl 2-(morpholinomethyl)isonicotinate (1 .89 g, 89 %) was isolated. ¹H NMR (400 MHz, cdcls) δ 2.47 - 2.57 (m, 4H), 3.72 (s, 2H), 3.73 - 3.80 (m, 4H), 3.96 (s, 3H), 7.72 (dd, 1 H), 7.96 (s, 1 H), 8.72 (d, 1 H). MS m/z 237 (M+H)+

Step 4: Methyl 2-(morpholinomethyl)pipehdine-4-carboxylate

Methyl 2-(morpholinomethyl)isonicotinate (1 .89 g, 8.00 mmol) was dissolved in acetic acid (40 mL) to give a colorless solution. Platinum(IV) oxide (0.182 g, 0.80 mmol) was added and the mixture hydrogenated at 5 bar and room temperature for 6 h 30 min. The catalyst was removed by filtration and the solvent evaporated. The residue was taken up in DCM and the organic phase was washed with satd NaHCO₃. The aqueous phase was extracted with DCM and the combined organic phases were filtered through a phase separator and evaporated. Crude methyl 2- (morpholinomethyl)piperidine-4-carboxylate (1 .5 g, 77 %) was isolated. MS m/z 243 (M+H)+ Step 5: Dimethyl 2-(morpholinomethyl)piperidine-1 ,4-dicarboxylate

Methyl 2-(morpholinomethyl)piperidine-4-carboxylate (1 .5 g, 6.19 mmol) was dissolved in dichloromethane (50 mL) to give a colorless solution. A/-ethyl-/V- isopropylpropan-2-amine (1 .510 mL, 8.67 mmol) and methyl carbonochloridate (0.585 mL, 7.43 mmol) were added and the mixture was stirred at room temperature for 4 h. Satd NH₄CI was added and the aqueous phase extracted with DCM. The combined organic layers were filtered through a phase separator and evaporated. Crude dimethyl 2-(morpholinomethyl)piperidine-1 ,4-dicarboxylate (2.1 g) was isolated. MS m/z 301 (M+H)+

Step 6: 1 -(Methoxycarbonyl)-2-(morpholinomethyl)piperidine-4-carboxylic acid

Dimethyl 2-(morpholinomethyl)piperidine-1 ,4-dicarboxylate (2.1 1 g, 7.03 mmol) was dissolved in THF (30 mL) and lithium hydroxide (9.13 mL, 9.13 mmol) (1 M in water) was added. The mixture was stirred at room temperature for 3 h and hydrogen chloride (1 M) (9.13 mL, 9.13 mmol) was added. The solvents were removed in vacuo, the residue was dissolved in water and purified by preparative HPLC on a Kromasil C8 column (10 μιτι 250x50 ID mm) using a gradient of 0-15 % Acetonitrile in H₂O/ACN/FA 95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. 1 -(Methoxycarbonyl)-2-(morpholinomethyl)-piperidine-4-carboxylic acid (1 .32 g, 66 %) was isolated. MS m/z 287 (M+H)+

Reference compound 2

1 -(Methoxycarbonyl)-2-((3-(trifluoromethyl)-5,6-dihvdro-n ,2,41triazolor4,3- alpyrazin-7(8H)-yl)methyl)piperidine-4-carboxylic acid

Step 1 : Methyl 2-((3-(trifluoromethyl)-5,6-dihvdro-ri ,2,41triazolor4,3-alpyrazin- 7(8H)-yl)methyl)isonicotinate

methyl 2-formylisonicotinate (1 .23 g, 7.45 mmol) (from reference compound 1 , step 2) was dissolved in THF (50 mL) to give a yellow solution. 3-(Trifluoromethyl)- 5,6,7, 8-tetrahydro-[1 ,2,4]triazolo[4,3-a]pyrazine hydrochloride (2.55 g, 1 1 .17 mmol) was added to give a suspension. Acetic acid (0.639 mL, 1 1 .17 mmol) and sodium triacetoxyhydroborate (1 .894 g, 8.94 mmol) were added and the mixture was stirred at room temperature for 4 h. Saturated NaHCO₃ was added and the mixture was stirred at room temperature for 15 minutes. DCM was added and the phases were separated. The aqueous phase was extracted with DCM and the combined organic phases were filtered through a phase separator and evaporated. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100g column. Mobile phase: 100 % ethyl acetate over 8 CV, then DCM/MeOH 20:1 over additional 8 CV. Methyl 2-((3-(trifluoromethyl)-5,6- dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)isonicotinate (1 .85 g, 73 %) was isolated. ¹H NMR (400 MHz, cdcl₃) δ 3.06 (t, 2H), 3.93 - 4.05 (m, 7H), 4.19 (t, 2H), 7.81 (d, 1 H), 7.96 (s, 1 H), 8.76 (d, 1 H). MS m/z 342 (M+H)⁺

Step 2: Methyl 2-((3-(trifluoromethyl)-5,6-dihvdro-ri ,2,41triazolor4,3-alpyrazin- 7(8H)-yl)methyl)piperidine-4-carboxylate

Methyl 2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)- yl)methyl)isonicotinate (1 .85 g, 5.42 mmol) was dissolved in acetic acid (40 mL) to give a colorless solution. Platinum(IV) oxide (0.098 g, 0.43 mmol) was added and the mixture was hydrogenated at 5 bar and room temperature for 3.5 h in a Buchi hydrogenator. Conversion of the starting material was about 50%. The catalyst was removed by filtration and a new portion catalyst platinum(IV) oxide (0.095 g, 0.42 mmol) was added. Hydrogenation was continued at 5 bar and room temperature for 4.5 h. The catalyst was removed by filtration and the solvent evaporated. The residue was taken up in DCM and the organic phase was washed with satd NaHCO3, filtered through a phase separator and evaporated. Crude methyl 2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)- yl)methyl)piperidine-4-carboxylate (1 .51 g, 80 %) was isolated. MS m/z 348 (M+H)⁺

Step 3: Dimethyl 2-((3-(trifluoromethyl)-5,6-dihvdro-ri ,2,41triazolor4,3-alpyrazin- 7(8H)-yl)methyl)piperidine-1 ,4-dicarboxylate

Methyl 2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)- yl)methyl)piperidine-4-carboxylate (1 .51 g, 4.35 mmol) was dissolved in dichloromethane (50 ml_) to give a colorless solution. A/-ethyl-/V-isopropylpropan-2- amine (1 .060 ml_, 6.09 mmol) and methyl carbonochloridate (0.41 1 ml_, 5.22 mmol) were added and the mixture was stirred at room temperature for 4 h. Saturated NH CI was added and the aqueous layer was extracted with DCM. The combined organic layers were filtered through a phase separator and evaporated. Crude dimethyl 2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin- 7(8H)-yl)methyl)piperidine-1 ,4-dicarboxylate (1 .75 g, 99 %) was isolated. MS m/z 406 (M+H)⁺

Step 4: 1 -(Methoxycarbonyl)-2-((3-(trifluoromethyl)-5.6 lihvclro-ri .2.41triazolor4.3- alpyrazin-7(8H)-yl)methyl)piperidine-4-carboxylic acid

Dimethyl 2-((3-(trifluorOmethyl)-5_>6-dihydro-[1 _>2_>4]triazolo[4_>3-a]pyrazin-7(8H)- yl)methyl)piperidine-1 ,4-dicarboxylate (1 .75 g, 4.32 mmol) was dissolved in THF (30 ml_) and lithium hydroxide (5.61 ml_, 5.61 mmol) (1 M in water) was added. The mixture was stirred at room temperature for 5 h and then acidified with 1 M hydrogen chloride to pH 4-5. Water and ethyl acetate were added and the phases were separated. The aqueous phase was extracted with ethyl acetate and the combined organic layers were dried over Na₂SO and evaporated. Crude 1 - (methoxycarbonyl)-2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin- 7(8H)-yl)methyl)piperidine-4-carboxylic acid (1 .64 g, 97 %) was isolated. MS m/z 392 (M+H)⁺

Reference compound 3

1 -(Methoxycarbonyl)-2-(((R)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-4- carboxylic acid

Step 1 : (R)-Methyl 2-((2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)isonicotinate

Methyl 2-formylisonicotinate (4.76 g, 28.82 mmol) (from reference compound 1 , step 2) was dissolved in THF (150 ml_) to give a yellow solution. (R)-2- (Trifluoromethyl)pyrrolidine (4.01 g, 28.82 mmol) was added to the solution. Acetic acid (4.95 ml_, 86.47 mmol) and sodium triacetoxyhydroborate (7.33 g, 34.59 mmol) were added and the mixture was stirred at room temperature for 1 h. Saturated NaHCO₃ was added and the mixture was stirred at room temperature for 15 min. Diethyl ether was added and the phases were separated. The organic phase was dried over Na₂SO₄, filtered through celite and evaporated. Crude (R)- methyl 2-((2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)isonicotinate (7.8 g, 94 %) was isolated. MS m/z 289 (M+H)⁺

Step 2: Methyl 2-(((R)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-4- carboxylate

(R)-Methyl 2-((2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)isonicotinate (8 g, 27.75 mmol) was dissolved in acetic acid (150 mL). Platinum(IV) oxide (315 mg, 1 .39 mmol) was added and the resulting mixture was hydrogenated at 8 bar and room temperature overnight. The catalyst was filtered off and the solvent was removed by evaporation. The crude was dissolved between dichloromethane and saturated Na₂CO₃. The organic layer was isolated, dried over Na₂SO , filtered through celite and the solvent evaporated. Crude methyl 2-(((R)-2-(trifluoromethyl)pyrrolidin-1 - yl)methyl)piperidine-4-carboxylate (6 g, 73.5 %) was isolated. MS m/z 295 (M+H)⁺

Step 3: Dimethyl 2-(((R)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 ,4- dicarboxylate

Methyl 2-(((R)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-4-carboxylate (6 g, 20.39 mmol) and A/-ethyl-/V-isopropylpropan-2-amine (5.33 mL, 30.58 mmol) were dissolved in dichloromethane (100 mL). Methyl carbonochloridate (1 .93 mL, 24.5 mmol) was added to the reaction mixture dropwise at room temperature. After 4 h the reaction mixture was washed with satd NaHCO3. The organic layer was isolated, dried over Na₂SO₄, filtered through celite and the solvent evaporated. Crude dimethyl 2-(((R)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 ,4- dicarboxylate (7.3 g) was isolated. MS m/z 353 (M+H)⁺

Step 4: 1 -(Methoxycarbonyl)-2-(((R)-2-(trifluoromethyl)pyrrolidin-1 - yl)methyl)piperidine-4-carboxylic acid

Dimethyl 2-(((R)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 ,4- dicarboxylate (7.3 g, 20.72 mmol) was dissolved in acetonitrile (50 mL) and water (1 ml_), then lithium bromide (14.39 g, 165.74 mmol) was added in one portion. Triethylamine (1 1 .49 ml_, 82.87 mmol) was added in one portion and the resulting suspension was heated under reflux for 3 h. Water and MTBE were added. The organic phase was extracted twice with water. The pooled aqueous layer was acidified to pH 3 with 3.8 M hydrogen chloride and then extracted twice with MTBE. The combined organic layer was dried over Na₂SO , filtered and evaporated. Crude amount 3.9 g. The water layer was extracted twice with dichloromethane. The organic layer was dried over Na₂SO₄, filtered through celite and combined with the crude earlier isolated. The solvent was evaporated and crude 1 -(methoxycarbonyl)-2-(((R)-2-(trifluoromethyl)pyrrolidin-1 - yl)methyl)piperidine-4-carboxylic acid (5.9 g, 84 %) was isolated. MS m/z 339 (M+H)⁺

Reference compound 4

1 -(Methoxycarbonyl)-2-(((S)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-4- carboxylic acid

Step 1 : (S)-Methyl 2-((2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)isonicotinate

Methyl 2-formylisonicotinate (1 .667 g, 10.09 mmol) (from reference compound 1 , step 2) dissolved in THF (35 ml_) to give a yellow solution. (S)-2- (Trifluoromethyl)pyrrolidine (4.21 g, 30.28 mmol) was added to the solution. Acetic acid (1 .733 ml_, 30.28 mmol) and sodium triacetoxyhydroborate (2.57 g, 12.1 1 mmol) were added and the mixture was stirred at room temperature for 1 h. Saturated NaHCO3 was added and the mixture was stirred at room temperature for 15 min. DCM was added and the phases were separated. The aqueous phase was extracted with DCM and the combined organic phases were filtered through a phase separator and evaporated. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100g column. A gradient from 2:1 to 1 :1 of EtOAc in heptane over 10 CV was used as mobile phase. (S)-Methyl 2-((2- (trifluoromethyl)pyrrolidin-1 -yl)methyl)isonicotinate (2.310 g, 79 %) was yielded as yellow oil. ¹H NMR (400 MHz, cdcl₃) δ 1 .75 - 2.14 (m, 4H), 2.56 (td, 1 H), 3.06 (t, 1 H), 3.38 - 3.53 (m, 1 H), 3.97 (s, 3H), 3.98 - 4.07 (m, 1 H), 4.30 (d, 1 H), 7.74 (d, 1 H), 8.00 (s, 1 H), 8.67 (d, 1 H), MS m/z 289 (M+H)⁺

Step 2: Methyl 2-(((S)-2-(tnfluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-4- carboxylate hydrochloride

(S)-Methyl 2-((2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)isonicotinate (2.31 g, 8.01 mmol) was disssolved in methanol and hydrogen chloride (1 .25 M in methanol, 9.62 ml_, 12.02 mmol) was added. The solvent was evaporated and the residue redissolved in methanol (50 ml_) and the solution was transferred to a Buchi hydrogenation vessel. Platinum(IV) oxide (0.182 g, 0.80 mmol) was added, the vessel sealed and the mixture was hydrogenated at 5 bar and room temperature for 7 h. The conversion was not complete and more platinum(IV) oxide (1 10 mg, 0.48 mmol) was added and hydrogenation was continued for 14 h at room temperature and 5 bar. The conversion was still not complete, the catalyst was removed by filtration, the solvent evaporated and the residue redissolved in methanol (50 ml_). Platinum(IV) oxide (1 15 mg, 0.51 mmol) was added and hydrogenation was continued for 4 h in a Buchi hydrogenation vessel at 5 bar and room temperature. The conversion was now complete and the catalyst was filtered off and the solvent was evaporated. Crude methyl 2-(((S)-2- (trifluoromethyl)pyrrolidin-l -yl)methyl)piperidine-4-carboxylate hydrochloride (2.39 g, 90 %) was obtained as a colorless solid. MS m/z 295 (M+H)⁺

Step 3: Dimethyl 2-(((S)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 ,4- dicarboxylate

Methyl 2-(((S)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-4-carboxylate hydrochloride (2.39 g, 7.23 mmol) was dissolved in dichloromethane (50 ml_) to give a colorless solution. A/-ethyl-/V-isopropylpropan-2-amine (3.15 ml_, 18.06 mmol) and methyl carbonochloridate (0.683 ml_, 8.67 mmol) were added and the mixture was stirred at room temperature for 3 h. Satd NH₄CI was added and the aqueous phase was extracted with DCM. The combined organic layers were filtered through a phase separator and evaporated. Crude dimethyl 2-(((S)-2- (trifluoromethyl)pyrrolidin-l -yl)methyl)piperidine-1 ,4-dicarboxylate (2.76 g) was obtained as a yellow oil. MS m/z 353 (M+H)⁺

Step 4: 1 -(Methoxycarbonyl)-2-(((S)-2-(trifluoronnethyl)pyrrolidin-1 - yl)methyl)pipehdine-4-carboxylic acid

Dimethyl 2-(((S)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 ,4- dicarboxylate (2.76 g, 7.83 mmol) was dissolved in acetonitnle (50 mL) and water (1 .000 mL), then lithium bromide (5.44 g, 62.67 mmol) was added in one portion. Triethylamine (4.34 mL, 31 .33 mmol) was added in one portion and the resulting suspension was heated under reflux for 7 h. MTBE (170 mL) and water (90 mL) were added, the phases separated and the organic layer was extracted with water. The combined aqueous layers were neutralised (pH 7-6) with 3.8 M hydrogenchloride and extracted with EtOAc. The combined organic layers were dried over Na₂SO₄ and evaporated. Crude 1 -(methoxycarbonyl)-2-(((S)-2- (trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-4-carboxylic acid (2.350 g, 89 %) was yielded as a colorless solid. MS m/z 339 (M+H)⁺

Reference compound 5

2-((3,3-Difluoropyrrolidin-1 -yl)methyl)-1 -(methoxycarbonyl)piperidine-4-carboxylic acid

Step 1 : Methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)isonicotinate

To a solution of methyl 2-formylisonicotinate (3.02 g, 18.28 mmol) (from reference compound 1 , step 2) in DCM (80 mL) was added acetic acid (1 .495 mL, 26.12 mmol) and 3,3-difluoropyrrolidine hydrochloride (2.5 g, 17.41 mmol). To the resulting suspension was added sodium triacetoxyhydroborate (5.54 g, 26.12 mmol) in one portion. The reaction was stirred at room temperature for 1 h, satd NaHCO₃ (100 mL) was added and stirring continued for 30 min. The phases were separated and the aqueous phase extracted with DCM (100 mL). The combined organic phases were dried with a phase separator and evaporated in vacuo. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100 g column. A gradient from 20 - 50 % of EtOAc in heptane over 1 1 CV was used as mobile phase. The product fractions were evaporated in vacuo to yield methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)isonicotinate (4.12 g, 92 %) as a pale oil. ¹H NMR (600 MHz, CDCI3) δ 2.25 - 2.39 (m, 2H), 2.81 - 2.92 (m, 2H), 3.01 (t, 2H), 3.88 (s, 2H), 3.95 (s, 3H), 7.74 (d, 1 H), 7.94 (s, 1 H), 8.70 (d, 1 H). MS m/z 257.2 (M+H)⁺.

Step 2: Methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)piperidine-4-carboxylate dihydrochloride

Methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)isonicotinate (5.847 g, 22.82 mmol) was dissolved in 1 .25 M hydrogen chloride in MeOH (54.8 mL, 68.45 mmol) and stirred at room temperature for 30 min. The solvent was evaporated in vacuo. The remaining salt was redissolved in MeOH (40 mL), platinum(IV) oxide (0.155 g, 0.68 mmol) added and the reaction mixture hydrogenated in a Buchi hydrogenator at 8 bar and room temperature for 3 h. The catalyst was filtered off, washed with MeOH and the filtrate evaporated in vacuo to yield crude methyl 2-((3,3- difluoropyrrolidin-1 -yl)methyl)piperidine-4-carboxylate dihydrochloride (7.50 g, 98 %) as a white solid. MS m/z 263.2 (M+H)⁺

Step 3: Dimethyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)piperidine-1 ,4-dicarboxylate Methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)piperidine-4-carboxylate dihydrochloride (8.202 g, 24.47 mmol) and DIPEA (14.96 mL, 85.64 mmol) were dissolved in DCM (40 mL). Methyl chloroformate (2.274 mL, 29.36 mmol) was added and the reaction stirred at room temperature for 3 h. More methyl chloroformate (0.569 mL, 7.34 mmol) was added and stirring continued for 1 h. DCM (170 mL) and satd NaHCO₃ (200 mL) were added, shaken and the phases separated. The aqueous phase was extracted with DCM (200 mL). The combined organic phases were dried with a phase separator and evaporated in vacuo to yield crude dimethyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)piperidine-1 ,4- dicarboxylate (8.09 g) as a pale oil. ¹H NMR (600 MHz, CDCI₃) δ 1 .08 (d, 1 H), 1 .49 - 1 .84 (m, 1 H), 1 .86 - 2.14 (m, 3H), 2.14 - 2.41 (m, 2H), 2.41 - 2.64 (m, 2H), 2.66 - 2.77 (m, 1 H), 2.78 - 3.03 (m, 1 H), 3.06 - 3.21 (m, 1 H), 3.49 - 3.77 (m, 8H), 3.82 - 3.96 (m, 1 H), 4.10 - 4.27 (m, 1 H). MS m/z 321 .2 (M+H)⁺.

Step 4: 2-((3,3-Difluoropyrrolidin-1 -yl)methyl)-1 -(^

carboxylic acid

Dimethyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)piperidine-1 ,4-dicarboxylate (8.087 g, 25.25 mmol) was dissolved in THF (40 mL). 1 M LiOH (32.8 mL, 32.82 mmol) was added and the reaction was stirred at room temperature for 2 h. The solvent was then concentrated in vacuo. 3 M hydrogen chloride (10.94 mL, 32.82 mmol) was added until pH 5-6. Water (150 mL) was added and the aqueous phase was extracted with MTBE (4x150 mL). The combined organic phases were dried over Na₂SO , filtered and evaporated in vacuo to yield 2-((3,3-difluoropyrrolidin-1 - yl)methyl)-1 -(methoxycarbonyl)piperidine-4-carboxylic acid (4.61 g, 59.6 %) as a colorless oil. MS m/z 307.2 (M+H)⁺, 305.2 (M-H)⁺.

Reference compound 6

1 -(Methoxycarbonyl)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidine-4- carboxylic acid

Step 1 : Methyl 2-(bromomethyl)isonicotinate

Methyl 2-(hydroxymethyl)isonicotinate (5 g, 29.91 mmol) (from reference compound 1 , step 1 ) and triphenylphosphine (9.41 g, 35.89 mmol) were dissolved in DCM (100 mL) and cooled with an ice-bath. Perbromomethane (1 1 .90 g, 35.89 mmol) was added in small portions and the reaction mixture was stirred at 0°C for 2 h and then at room temperature for 3 h. The solvent was concentrated in vacuo. The residue was purified by automated flash chromatography on 2 Biotage® KP- SIL 100g columns. A gradient from 20 - 50 % of EtOAc in heptane over 10 CV was used as mobile phase. Methyl 2-(bromomethyl)isonicotinate (5.84 g, 85 %) was isolated as a dark blue oil. ¹H NMR (600 MHz, CDCI3) δ 3.96 (s, 3H), 4.59 (s, 2H), 7.76 (dd, 1 H), 7.98 (s, 1 H), 8.72 (d, 1 H). Step 2: Methyl 2-((2,2,6,6-tetramethylpipendin-1 -yl)methyl)isonicotinate

Two 20 ml_ microwave reaction vials were charged with methyl 2- (bromomethyl)isonicotinate (5 g, 21 .73 mmol) and 2,2,6,6-tetramethylpiperidine (18.34 ml_, 108.67 mmol). DMF (10 ml_) was added to each vial and the vials were sealed and heated in a microwave reactor at 130^°C for 15 min. The contents of the vessels were partioned between water and DCM and the aqueous layer was extracted with DCM. The combined organic layers were filtered through a phase evaporator and evaporated. Remaining DMF was removed by repetitive co- evaporation with touene. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100 g column. A gradient from 4:1 to 1 :1 of heptane:ethyl acetate over 10 CV was used as mobile phase. Methyl 2-((2, 2,6,6- tetramethylpiperidin-1 -yl)methyl)isonicotinate (2.73 g, 43 %) was isolated. ¹H NMR (400 MHz, cdcls) δ 1 .00 (s, 12H), 1 .46 - 1 .76 (m, 6H), 3.93-3.98 (m, 5H), 7.60 (d, 1 H), 8.29 (s, 1 H), 8.60 (d, 1 H). MS m/z 291 (M+H)⁺

Step 3: Methyl 2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidine-4-carboxylate hydrochloride

Methyl 2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)isonicotinate (3.9 g, 13.43 mmol) was disssolved in methanol and hydrogen chloride (1 .25 M in methanol, 23.64 ml_, 29.55 mmol) was added. The solvent was evaporated and the residue redissolved in methanol (80 ml_) and the solution was transferred to a Buchi hydrogenation vessel. Platinum(IV) oxide (0.305 g, 1 .34 mmol) was added, the vessel sealed and the mixture was hydrogenated at 5 bar and room temperature for 25 h. Only partial conversion. The catalyst was removed by filtration and a new catalyst portion platinum(IV) oxide (0.305 g, 1 .34 mmol) was added. The hydrogenation was continued at 5 bar for another 24 h. Still only partial conversion. More catalyst platinum(IV) oxide (0.305 g, 1 .34 mmol) was added, and the reaction was continued for another 24 h. A last portion of catalyst platinum(IV) oxide (0.305 g, 1 .34 mmol) was added and the hydrogenation was completed over night at 8 bar and room temperature. The catalyst was removed by filtration and the solvent was evaporated. Crude methyl 2-((2,2,6,6-tetramethylpiperidin-1 - yl)methyl)piperidine-4-carboxylate hydrochloride (4.46 g, 100 %) was isolated. MS m/z 297 (M+H)⁺

Step 4: Dimethyl 2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidine-1 ,4- dicarboxylate

Methyl 2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidine-4-carboxylate hydrochloride (4.46 g, 13.4 mmol) was dissolved in dichloromethane (100 mL) to give a colorless solution. A/-ethyl-/V-isopropylpropan-2-amine (5.84 mL, 33.50 mmol) and methyl carbonochloridate (1 .266 mL, 16.08 mmol) were added and the mixture was stirred at room temperature for 3 h. Satd NH₄CI was added and the aqueous solution was extracted with DCM. The combined organic layers were filtered through a phase separator and evaporated. Crude dimethyl 2-((2,2,6,6- tetramethylpiperidin-1 -yl)methyl)piperidine-1 ,4-dicarboxylate (4.75 g, 100 %) was obtained as yellow oil. MS m/z 355 (M+H)⁺

Step 5: 1 -(Methoxycarbonyl)-2-((2,2,6,6-tetramethylpipehdin-1 - yl)methyl)piperidine-4-carboxylic acid

Dimethyl 2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidine-1 ,4-dicarboxylate (4.75 g, 13.4 mmol) was dissolved in THF (50 mL) and lithium hydroxide (17.42 mL, 17.42 mmol) (1 M in water) was added. The mixture was stirred at room temperature for 5 h and acidified with 1 M hydrogen chloride to pH 5-4. Water and ethyl acetate were added and the phases were separated. The product was not found in the organic phase but completely in the aq. phase. The aq. phase was freeze dried and the residue was treated with a mixture of DCM (200 mL) and methanol (20 mL). The suspension was filtered and the filtrate was evaporated to yield crude 1 -(methoxycarbonyl)-2-((2,2,6,6-tetramethylpiperidin-1 - yl)methyl)piperidine-4-carboxylic acid (1 .800 g, 39.5 %) as light yellow oil. MS m/z 341 (M+H)⁺

Reference compound 7

2-(lsoindolin-2-ylmethyl)-1 -(methoxycarbonyl)piperidine-4-carboxylic acid Step 1 : Methyl 2-(isoindolin-2-ylnnethyl)isonicotinate

Methyl 2-(bromomethyl)isonicotinate (5.837 g, 25.37 mmol) (from reference compound 6, step 1 ) and isoindoline (12.09 g, 101 .49 mmol) were dissolved in acetonitrile (25 ml_), divided up in two microwave vials and heated at 1 10°C for 15 min. The solvent was then evaporated in vacuo. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 340g column. A gradient from 10 - 70 % of EtOAc/MeOH/TEA 100:10:1 in heptane over 1 1 CV was used as mobile phase. The product fractions were evaporated in vacuo to yield methyl 2- (isoindolin-2-ylmethyl)isonicotinate (5.18 g, 76 %) as a brown solid. ¹H NMR (600 MHz, CDCI3) δ 3.94 (s, 3H), 4.1 1 (s, 4H), 4.18 (s, 2H), 7.15 - 7.24 (m, 4H), 7.75 (d, 1 H), 8.07 (s, 1 H), 8.73 (d, 1 H). MS m/z 269.2 (M+H)⁺.

Step 2: Methyl 2-(isoindolin-2-ylmethyl)piperidine-4-carboxylate dihydrochloride Methyl 2-(isoindolin-2-ylmethyl)isonicotinate (5.175 g, 19.29 mmol) was dissolved in 1 .25 M hydrogen chloride in MeOH (46.3 ml_, 57.86 mmol) and stirred at room temperature for 30 min. The solvent was evaporated in vacuo. The remaining HCI salt was redissolved in MeOH (80 ml_), platinum(IV) oxide (0.131 g, 0.58 mmol) added and the reaction mixture hydrogenated in a Buchi hydrogenator at 5 bar and room temperature for 2 h. The catalyst was filtered off, washed with MeOH and the filtrate evaporated in vacuo to yield crude methyl 2-(isoindolin-2- ylmethyl)piperidine-4-carboxylate dihydrochloride (quant.) as a light brown solid. MS m/z 275.3 (M+H)⁺.

Step 3: Dimethyl 2-(isoindolin-2-ylmethyl)piperidine-1 ,4-dicarboxylate

Methyl 2-(isoindolin-2-ylmethyl)piperidine-4-carboxylate dihydrochloride (8.191 g, 23.59 mmol) and DIPEA (14.42 ml_, 82.55 mmol) were dissolved in DCM (40 ml_). Methyl chloroformate (2.192 ml_, 28.30 mmol) was added and the reaction stirred at room temperature for 1 .5 h. DCM (170 ml_) was added. The organic phase was extracted with 0.1 M hydrogen chloride (2x200 ml_). The combined aqueous phases were basified to pH 8 with 3.8 M HCI (aq) and extracted with DCM (2x250 ml_). The combined organic phases were dried with a phase separator and evaporated in vacuo to yield crude dimethyl 2-(isoindolin-2-ylmethyl)piperidine-1 ,4- dicarboxylate (5.58 g, 71 .2 %) as a dark oil. MS m/z 333.3 (M+H)⁺.

Step 4: 2-(lsoindolin-2-ylmethyl)-1 -(methoxycarbonyl)piperidine-4-carboxylic acid Dimethyl 2-(isoindolin-2-ylmethyl)piperidine-1 ,4-dicarboxylate (5.58 g, 16.79 mmol) was dissolved in THF (40 mL). 1 M LiOH (21 .82 mL, 21 .82 mmol) was added and the reaction was stirred at room temperature for 2 h. The solvent was then concentrated in vacuo. 3 M HCI (7.27 mL, 21 .82 mmol) was added to pH 5-6. Water (150 mL) was added. The aqueous phase was extracted with EtOAc. The product remained in the aqueous phase. pH was adjusted several times between 4-6 but the product remained in the aqueous phase during extraction with EtOAc. The aqueous phase was concentrated in vacuo to 15 mL and the product purified by preparative HPLC on a XBridge C18 column (10 μιτι 250x50 ID mm) using a gradient of 5-45% Acetonithle in H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. The product fractions were freeze dried to yield 2- (isoindolin-2-ylmethyl)-1 -(methoxycarbonyl)piperidine-4-carboxylic acid (3.96 g, 74.1 %) as a white solid. ¹H NMR (600 MHz, CDCI₃) δ 1 .72 - 1 .91 (m, 1 H), 2.05 -

2.17 (m, 2H), 2.21 - 2.30 (m, 1 H), 2.64 - 2.77 (m, 1 H), 2.94 - 3.08 (m, 1 H), 3.09 - 3.32 (m, 2H), 3.36 - 3.52 (m, 1 H), 3.65 - 3.78 (m, 3H), 3.91 (d, 1 H), 4.57 (s, 4H),

7.18 - 7.37 (m, 4H). MS m/z 319.3 (M+H)⁺.

Preparation of Compound Examples. Formation of 5-isoxazol-3-ones Example 1

5-((2R,4S)-2-(Morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one

Step 1 : Trans-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-

(morpholinomethyl)pipe dine-l -carboxylate and cis-methyl 4-(3-ethoxy-3- oxopropanoyl)-2-(morpholinomethyl)pipehdine-1 -carboxylate

1 -(Methoxycarbonyl)-2-(morpholinomethyl)piperidine-4-carboxylic acid (1 .32 g, 4.61 mmol) (reference compound 1 ) was dissolved in methyl THF (30 ml_) and di(1 H-imidazol-1 -yl)methanone (1 .12 g, 6.92 mmol) was added. The suspension was stirred at room temperature for 3 h under nitrogen (flask 1 ). In a separate flask potassium 3-ethoxy-3-oxopropanoate (1 .41 g, 8.3 mmol) was suspended in methyl THF (30 ml_) and magnesium chloride (0.79 g, 8.3 mmol) added. The suspension was stirred at 50°C for 22 h under nitrogen (flask 2). The suspension in flask 1 was then added to the suspension in flask 2. The resulting suspension was stirred at room temperature for 20 h. Conversion was not complete and in a separate flask was prepared a new portion of the magnesium salt mixture: potassium 3-ethoxy- 3-oxopropanoate (0.71 g, 4.17 mmol) was suspended in methyl THF (15 ml_) under nitrogen atmosphere and magnesium chloride (0.41 g, 4.31 mmol) was added. The suspension was stirred vigorously at 50°C for 6 h. The suspension was added to the reaction mixture and stirring was continued at room temperature for 17 h. Saturated NH CI and DCM were added and the phases were separated. The aqueous phase was extracted with DCM and the combined organic layers were filtered through a phase separator and evaporated. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100 g column. 100% ethyl acetate over 8 CV was used as mobile phase to elute cis-methyl 4-(3-ethoxy- 3-oxopropanoyl)-2-(morpholinomethyl)piperidine-1 -carboxylate (317 mg, 19 %). DCM/MeOH 30:1 over another 8 CV was used to elute trans-methyl 4-(3-ethoxy-3- oxopropanoyl)-2-(morpholinomethyl)piperidine-1 -carboxylate (226 mg, 14 %). Cis- isomer: ¹H NMR (400 MHz, cdcl₃) δ 1 .22 - 1 .32 (m, 3H), 1 .59 - 2.56 (m, 9H), 2.71 - 2.80 (m, 1 H), 3.04 - 3.17 (m, 1 H), 3.32 (s, 2H), 3.61 - 3.76 (m, 7H), 3.77 - 3.95 (m, 2H), 4.20 (q, 2H), 4.25 - 4.35 (m, 1 H). MS m/z 357 (M+H)+. Trans-isomer: ¹H NMR (400 MHz, cdcl₃) δ 1 .24 - 1 .31 (m, 3H), 1 .42 - 2.55 (m, 10H), 2.69 - 2.89 (m, 2H), 3.48 (s, 2H), 3.62 - 3.72 (m, 7H), 3.79 - 4.64 (m, 2H), 4.19 (q, 2H). MS m/z 357 (M+H)+

Step 2: Cis-methyl 2-(morpholinomethyl)-4-(3-oxo-2,3-dihvdroisoxazol-5- yl)piperidine-1 -carboxylate

Cis-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(morpholinomethyl)piperidine-1 - carboxylate (317 mg, 0.89 mmol) was dissolved in MeOH (4 mL) and cooled to -40°C under nitrogen. Sodium hydroxide (35.6 mg, 0.89 mmol) dissolved in water (0.400 ml_) was added and the mixture was stirred at -40°C for 15 min. Hydroxylamine (50 % by weight in water, 0.055 mL, 0.89 mmol) was added. The resulting solution was stirred at -40°C for 1 h. The mixture was then transfered into a pre-warmed (80°C) solution of 6 M hydrogen chloride (4.60 mL, 27.57 mmol) and the mixture was stirred at 80°C for 20 min. Water and DCM were added and the phases separated. The aqueous phase was extracted with DCM and the combined organic layers were filtered through a phase separator and evaporated. The residue was purified by preparative HPLC on a Kromasil C8 column (10 μιτι 250x50 ID mm) using a gradient of 0-20% Acetonitrile in H2O/ACN/FA 95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. Cis- methyl 2-(morpholinomethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 - carboxylate (163 mg, 56 %) was isolated. ¹H NMR (400 MHz, cdcl₃) δ 1 .86 - 2.58 (m, 10H), 2.98 - 3.16 (m, 2H), 3.61 - 3.75 (m, 7H), 3.90 - 4.00 (m, 1 H), 4.20 - 4.29 (m, 1 H), 5.75 (s, 1 H). MS m/z 326 (M+H)+

Step 3: (2R,4S)-Methyl 2-(morpholinomethyl)-4-(3-oxo-2,3-dihvdroisoxazol-5- yl)piperidine-1 -carboxylate

Cis-methyl 2-(morpholinomethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 - carboxylate (160 mg, 0.49 mmol) was subjected to chiral preparative HPLC (Column: Chiralcel OJ (250x20), 5 μιτι particle size, mobile phase: Heptane/EtOH 70/30, flow rate 18 mL/min) to yield (2R,4S)-methyl 2-(morpholinomethyl)-4-(3- oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 -carboxylate (62.7 mg, 39 %). Chiral purity 99.9 % ee. Optical rotation [af° = +38.4 (acetonitrile, c=1 ). ¹ H NMR (400

MHz, cdcls) δ 1 .83 - 2.10 (m, 4H), 2.21 - 2.54 (m, 6H), 2.98 (p, 1 H), 3.05 - 3.16 (m, 1 H), 3.60 - 3.71 (m, 7H), 3.84 - 3.94 (m, 1 H), 4.19 - 4.30 (m, 1 H), 5.70 (s, 1 H).

Step 4: 5-((2R,4S)-2-(Morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one

(2R,4S)-Methyl 2-(morpholinomethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine- 1 -carboxylate (62 mg, 0.19 mmol) was dissolved in hydrogen bromide (33 % in acetic acid (1502 μΙ, 8.58 mmol) and stirred at room temperature for 16 h. The solvent was removed in vacuo and the compound was purified by preparative HPLC on a XBridge C18 column (10 μηη 250x19 ID mm) using a gradient of 0-10% Acetonitrile in H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minutes with a flow of 19 mL/min. 5-((2R,4S)-2-(morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one (46 mg, 90 %) was isolated. ¹H NMR (400 MHz, cd₃od) δ 1 .44 - 1 .59 (m, 1 H), 1 .71 - 1 .87 (m, 1 H), 2.20 - 2.32 (m, 2H), 2.35 - 2.47 (m, 2H), 2.51 - 2.56 (m, 2H), 2.59 - 2.69 (m, 2H), 3.02 - 3.20 (m, 2H), 3.41 - 3.55 (m, 2H), 3.68 - 3.77 (m, 4H), 5.73 (s, 1 H). HRMS Calcd for [Ci₃H2iN₃O3+H]+: 268.1661 . Found: 268.1662.

Example 2

5-(Trans-2-(morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one

Step 1 : Trans-methyl 2-(morpholinomethyl)-4-(3-oxo-2,3-dihvdroisoxazol-5- yl)piperidine-1 -carboxylate

Trans-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(morpholinomethyl)piperidine-1 - carboxylate (225 mg, 0.63 mmol) (from example 1 , step 1 ) was dissolved in MeOH (3 ml_) and cooled to -40°C under nitrogen. Sodium hydroxide (25.2 mg, 0.63 mmol) dissolved in water (0.300 ml_) was added and the mixture was stirred at - 40°C for 15 min. Hydroxylamine (50 % by weight in water, 0.039 ml_, 0.63 mmol) was added. The resulting solution was stirred at -40°C for 1 h. The mixture was then transferred into a pre-warmed (80°C) solution of 6 M hydrogen chloride (3.26 ml_, 19.57 mmol) and the mixture was stirred at 80°C for 20 min. The compound was purified by preparative HPLC on a Kromasil C8 column (10 μιτι 250x20 ID mm) using a gradient of 0-20% Acetonitrile in H₂O/ACN/FA 95/5/0.2 buffer over 20 minutes with a flow of 19 mL/min. Trans-methyl 2- (morpholinomethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 -carboxylate (160 mg, 78 %) was isolated. ¹H NMR (400 MHz, cdcl₃) δ 1 .41 - 2.23 (m, 4H), 2.57 - 3.61 (m, 8H), 3.74 (s, 3H), 3.80 - 4.94 (m, 6H), 5.64 (s, 1 H), 8.06 (s, 1 H). MS m/z 326 (M+H)+

Step 2: 5-(Trans-2-(morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one

Trans-methyl 2-(morpholinomethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 - carboxylate (160 mg, 0.49 mmol) was dissolved in hydrogen bromide (33 % in acetic acid, 3876 μΙ, 22.13 mmol) and stirred at room temperature for 16 h. The solvent was removed in vacuo and the residue was redissolved in water. The compound was purified by preparative HPLC on a XBridge C18 column (10 μιτι 250x19 ID mm) using a gradient of 0-15% Acetonitrile in H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minutes with a flow of 19 mL/min. 5-(Trans-2- (morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one (90 mg, 68 %) was isolated. ¹H NMR (400 MHz, cd₃od) δ 1 .76 - 1 .94 (m, 2H), 2.00 - 2.24 (m, 3H), 2.36 - 2.51 (m, 3H), 2.56 - 2.67 (m, 3H), 3.06 - 3.15 (m, 1 H), 3.22 - 3.28 (m, 2H), 3.35 - 3.45 (m, 1 H), 3.66 - 3.74 (m, 4H), 5.62 (s, 1 H). HRMS Calcd for [C₁₃H2i N₃O3+H]+: 268.1661 . Found: 268.1652.

Example 3

5-((2R,4S)-2-((3-(Trifluoromethyl)-5.6-dihvdro-ri ,2,41triazolor4,3-alpyrazin-7(8H)- yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one

Step 1 : Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-((3-(trifluoromethyl)-5,6-dihvdro- [1 ,2,41triazolo[4,3-alpyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate 1 -(Methoxycarbonyl)-2-((3-(tnfluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidine-4-carboxylic acid (1 .64 g, 4.19 mmol) (reference compound 2) was dissolved in methyl THF (30 mL) under nitrogen atmosphere and di(1 H-imidazol-1 -yl)methanone (1 .019 g, 6.29 mmol) added. The suspension was stirred at room temperature for 6 h (flask 1 ). In a separate flask was potassium 3-ethoxy-3-oxopropanoate (1 .284 g, 7.54 mmol) suspended in methyl THF (30.0 mL) and magnesium chloride (0.718 g, 7.54 mmol) was added. The suspension was stirred at 50^°C under nitrogen for 22 h using an oversized stirring bar (flask 2). To flask 2 was added the contents of flask 1 and the resulting white suspension was stirred at room temperature for 64 h. Satd NH CI and DCM were added and the phases were separated. The aqueous phase was extracted with DCM and the combined organic layers were filtered through a phase separator and evaporated. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100g column. Mobile phase: DCM/MeOH 50:1 over 10 CV. Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-((3- (trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)- yl)methyl)piperidine-1 -carboxylate (1 .04 g, 54 %) was isolated. MS m/z 462 (M+H)⁺

Step 2: Methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6- dihydro-H ,2,41triazolo[4,3-alpyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate

Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-((3-(trifluoromethyl)-5,6-dihydro- [1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate (1 .04 g, 2.25 mmol) was dissolved in MeOH (10 mL) and cooled to -40^°C under nitrogen. Sodium hydroxide (0.090 g, 2.25 mmol) dissolved in water (1 .000 mL) was added and the mixture was stirred at -40^°C for 15 min. Hydroxylamine (50 % by weight in water, 0.138 mL, 2.25 mmol) was added. The resulting solution was stirred at - 40^°C for 2 h. The mixture was then transferred into a pre-warmed (80 C) solution of 6 M hydrogen chloride (1 1 .64 mL, 69.87 mmol) and the mixture was stirred at 80^°C for 30 min. Water and DCM were added and the phases were separated. The aqueous phase was extracted with DCM, the combined organic layers filtered through a phase separator and evaporated. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100g column. DCM/MeOH/HCOOH 50:1 :0.1 over 10 CV was used as mobile phase. Methyl 4-(3- oxo-2,3-dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate (710 mg, 73 %) was isolated. MS m/z 431 (M+H)⁺

Step 3: (2R,4S)-Methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-((3-(trifluoromethyl)- 5,6-dihydro-H ,2,41triazolo[4,3-alpyrazin-7(8H)-yl)methyl)pipehdine-1 -carboxylate,

(2S,4R)-methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6- dihydro-H ,2,41triazolo[4,3-alpyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate,

(2S,4S)-methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6- dihydro-H ,2,41triazolo[4,3-alpyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate and

(2R,4R)-methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6- dihydro-[1 ,2,41triazolo[4,3-alpyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate

Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6-dihydro- [1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate (710 mg, 1 .65 mmol) was subjected to chiral preparative HPLC in two steps (first using Column: Chiralpak AD (250x20), 5 μιτι particle size, mobile phase: Heptane/EtOH 60/40, flow rate 18 mL/min and then using Column: Chiralpak IC (250x20), 5 μιτι particle size, mobile phase: Heptane/EtOH 40/60, flow rate 18 mL/min or using Column: Chiralpak AD (250x20), 5 μιτι particle size, mobile phase: Heptane/lPA 80/20, flow rate 18 mL/min) to yield (2R,4S)-methyl 4-(3-oxo-2,3-dihydroisoxazol- 5-yl)-2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)- yl)methyl)piperidine-1 -carboxylate (150 mg, 21 %), Chiral purity 99.9 %ee, Optical rotation [af° = +6.6 (acetonitrile, c=1 ), (2S,4R)-methyl 4-(3-oxo-2,3- dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate, (167 mg, 24 %), Chiral purity

99.8 %ee, Optical rotation = -5.3 (acetonitrile, c=1 ), (2S,4S)-methyl 4-(3-oxo- 2,3-dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate (43 mg, 6 %), Chiral purity 99.8 %ee, Optical rotation [a £° = +24.1 (acetonitrile, c=1 ) and (2R,4R)-methyl 4-(3-oxo- 2,3-dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate (40 mg, 6 %), Chiral purity 98.6

%ee, Optical rotation [a = -31.9 (acetonitrile, c=1 ).

Step 4: 5-((2R,4S)-2-((3-(Trifluoromethyl)-5,6-dihvdro-ri ,2,41triazoloi4,3-alpyrazin- 7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one

(2R,4S)-Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6- dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)nnethyl)piperidine-1 -carboxylate (150 mg, 0.35 mmol) was dissolved in hydrogen bromide (33 % in acetic acid, 2747 μΙ, 15.68 mmol) and stirred at room temperature for 16 h. The solvent was evaporated and the residue purified by preparative HPLC (Instrument: FractionLynx III, Mobilphase: gradient 5-95% MeCN in 0.2% NH₃, pH 10, Column: Xbridge Prep C18 5μηη OBD 19^*150 mm) to yield 5-((2R,4S)-2-((3- (trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidin-

4- yl)isoxazol-3(2H)-one (69 mg, 53 %). ¹H NMR (400 MHz, cd₃od) δ 1 .40 (q, 1 H), 1 .63 - 1 .78 (m, 1 H), 2.15 (d, br., 2H), 2.71 - 2.83 (m, 2H), 2.87 - 3.07 (m, 3H), 3.1 1 - 3.22 (m, 1 H), 3.26 - 3.40 (m, 2H), 3.90 (d, 1 H), 4.08 (d, 1 H), 4.27 (t, 2H), 5.53 (s, 1 H). HRMS Calculated for [Ci₅H₁₉F3N6O2+H]⁺: 373.1600. Found: 373.1585

Example 4

5- ((2S.4R)-2-((3-(Trifluoromethyl)-5.6-dihvdro-ri ,2,41triazolor4,3-alpyrazin-7(8H)- yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one

(2S,4R)-Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6- dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate (169 mg, 0.39 mmol) (from example 3, step 3) was dissolved in hydrogen bromide (33 % in acetic acid, 3058 μΙ, 17.46 mmol) and stirred at room temperature for 16 h. The solvent was evaporated and the residue purified by preparative HPLC (Instrument: FractionLynx III, Mobilphase: gradient 5-95% MeCN in 0.2% NH₃, pH 10, Column: Xbridge Prep C18 5μηη OBD 19^*150 mm) to yield 5-((2S,4R)-2-((3- (trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidin-

4- yl)isoxazol-3(2H)-one (100 mg, 69 %). ¹H NMR (400 MHz, cd₃od) δ 1 .40 (dd, 1 H), 1 .64 - 1 .79 (m, 1 H), 2.15 (d, 2H), 2.65 (s, 2H), 2.73 - 2.82 (m, 2H), 2.89 - 3.06 (m, 3H), 3.1 1 - 3.23 (m, 1 H), 3.36 - 3.40 (m, 1 H), 3.90 (d, 1 H), 4.09 (d, 1 H), 4.27 (t, 2H), 5.53 (s, 1 H). HRMS Calculated for [Ci₅H₁₉F3N6O2+H]⁺: 373.1600. Found: 373.1606

Example 5

5- ((2S.4S)-2-((3-(Trifluoromethyl)-5.6-dihvdro-ri ,2,41triazolor4,3-alpyrazin-7(8H)- yl )methyl )piperid in-4-yl )isoxazol-3(2H )-one

(2S,4S)-Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6- dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate (43 mg, 0.1 mmol) (from example 3, step 3) was dissolved in hydrogen bromide (33 % in acetic acid, 787 μΙ, 4.50 mmol) and stirred at room temperature for 16 h. The solvent was evaporated and the residue purified by preparative HPLC (Instrument: FractionLynx III, Mobilphase: gradient 5-95% MeCN in 0.2% NH₃, pH 10, Column: Xbridge Prep C18 5μηη OBD 19^*150 mm) to yield 5-((2S,4S)-2-((3- (trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidin-

4- yl)isoxazol-3(2H)-one (17 mg, 45 %). ¹H NMR (400 MHz, cd₃od) δ 1 .75 - 1 .86 (m, 1 H), 1 .97 - 2.26 (m, 3H), 2.75 - 3.50 (m, 8H), 3.99 (dd, 2H), 4.25 (t, 2H), 5.63 (s, 1 H). HRMS Calculated for [Ci₅H₁₉F3N6O2+H]⁺: 373.1600. Found: 373.1606

Example 6

5- ((2R,4R)-2-((3-(Trifluoromethyl)-5.6-dihvdro-ri ,2,41triazolor4,3-alpyrazin-7(8H)- yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one

(2R,4R)-Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((3-(trifluoromethyl)-5,6- dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidine-1 -carboxylate (40 mg, 0.09 mmol) (from example 3, step 3) was dissolved in hydrogen bromide (33 % in acetic acid, 732 μΙ, 4.18 mmol) and stirred at room temperature for 16 h. The solvent was evaporated and the residue purified by preparative HPLC (Instrument: FractionLynx III, Mobilphase: gradient 5-95% MeCN in 0.2% NH₃, pH 10, Column: Xbridge Prep C18 5μηη OBD 19^*150 mm) to yield 5-((2R,4R)-2-((3- (trifluoromethyl)-5,6-dihydro-[1 ,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methyl)piperidin-

4- yl)isoxazol-3(2H)-one (18 mg, 52 %). ¹H NMR (400 MHz, cd₃od) δ 1 .77 - 1 .87 (m, 1 H), 2.04 - 2.09 (m, 1 H), 2.12 - 2.25 (m, 2H), 2.65 (s, 2H), 2.78 - 2.89 (m, 2H), 2.94 - 3.12 (m, 2H), 3.17 - 3.26 (m, 2H), 3.38 - 3.49 (m, 1 H), 3.99 (dd, 2H), 4.25 (t, 2H), 5.63 (s, 1 H). HRMS Calculated for [C₁₅H₁₉F₃N₆O2+H]⁺: 373.1600. Found: 373.1570

Example 7

5- ((2R,4S)-2-(((R)-2-(Trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidin-4-yl)isoxazol- 3(2H)-one

Step 1 : Cis-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(((R)-2-

(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate

1 -(Methoxycarbonyl)-2-(4-(trifluoromethyl)phenyl)piperidine-4-carboxylic acid (5.9 g, 17.44 mmol) (reference compound 3) was dissolved in THF (75 mL) under nitrogen atmosphere and di(1 H-imidazol-1 -yl)methanone (4.24 g, 26.16 mmol) was added. The suspension was stirred at room temperature for 3 h (flask 1 ). In a separate flask was potassium 3-ethoxy-3-oxopropanoate (2.99 g, 17.44 mmol) suspended in THF (75 mL) and magnesium chloride (2.99 g, 31 .39 mmol) was added. The suspension was stirred at 50°C under nitrogen for 3.5 h using an oversized stirring bar (flask 2). The contents of flask 1 was added to flask 2 and the resulting white suspension was stirred at room temperature overnight. Saturated NH CI and DCM were added and the phases separated. The aqueous phase was extracted with DCM, the combined organic layers dried over Na₂SO , filtered through celite and evaporated. The residue was purified via automated flash chromatography (Mobile phase heptane/EtOAc 2:1 , Biotage® KP-SIL 340g column, 10 CV). Cis-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(((R)-2- (trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate (3.32 g, 46.6 %) was isolated. MS m/z 409 (M+H)⁺

Step 2: Cis-methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-(((R)-2-

(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate

Cis-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(((R)-2-(trifluoromethyl)pyrrolidin-1 - yl)methyl)piperidine-1 -carboxylate (3.32 g, 8.13 mmol) was dissolved in MeOH (50 ml_) and cooled to -40°C. Sodium hydroxide (0.341 g, 8.54 mmol) dissolved in water (5.00 ml_) was added and the mixture was stirred at -40°C for 15 min. Hydroxylamine (50 % by weight in water, 0.523 ml_, 8.54 mmol) was added. The resulting solution was stirred at -40°C for 1 h. The mixture was then transferred into a pre-warmed (80°C) solution of 6 M hydrogen chloride (42.0 ml_, 252.00 mmol) and the mixture was stirred at 80°C for 20 min. Satd Na₂CO₃(aq) and DCM were added and the phases separated. The aqueous phase was extracted with DCM and the combined organic layers were isolaled, dried over Na2SO₄, filtered through celite and evaporated. Crude cis-methyl 4-(3-oxo-2,3-dihydroisoxazol-5- yl)-2-(((R)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate (3.23 g) was isolated as a light yellow oil. MS m/z 378 (M+H)⁺

Step 3: (2R,4S)-Methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-(((R)-2- (trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate

Cis-methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-(((R)-2-(trifluoromethyl)pyrrolidin- 1 -yl)methyl)piperidine-1 -carboxylate (3.23 g, 8.56 mmol) was subjected to preparative HPLC (Column: Kromasil-C18 (250x50), 10 μιτι particle size, mobile phase: ACN/H₂O/FA 45/55/0.1 , flow rate 120 mL/min) to yield (2R,4S)-methyl 4-(3- oxo-2,3-dihydroisoxazol-5-yl)-2-(((R)-2-(trifluoromethyl)pyrrolidin-1 - yl)methyl)piperidine-1 -carboxylate (334 mg, 10 %). Chiral purity 99.6 %ee, Optical rotation = +67.7 (acetonitrile, c=1 ), ¹H NMR (600 MHz, cdcl₃) δ 1 .65 - 1 .95 (m, 6H), 2.07 - 2.19 (m, 1 H), 2.20 - 2.31 (m, 1 H), 2.46 (dd, 1 H), 2.74 - 2.94 (m, 3H), 3.02 - 3.24 (m, 3H), 3.70 (s, 3H), 3.81 - 3.91 (m, 1 H), 3.96 - 4.12 (m, 1 H), 5.67 (s, 1 H). Step 4: 5-((2R^S)-2-(((R)-2-(Trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidin^^ yl )isoxazol-3(2H )-one

(2R,4S)-Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-(((R)-2- (trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate (334 mg, 0.89 mmol) was dissolved in HBr (33 % in acetic acid, 4.32 ml_, 17.7 mmol) and stirred overnight at room temperature. The solvent was evaporated and the residue purified by preparative HPLC (Instrument: FractionLynx III, Mobilphase: gradient 5- 95 % MeCN in 0.2 % NH₃, pH 10, Column: Xbridge Prep C18 5pm OBD 19^*150 mm) to yield 5-((2R,4S)-2-(((R)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one (103 mg, 36 %). ¹H NMR (600 MHz, dmso) δ 0.91 (dd, 1 H), 1 .37 (qd, 1 H), 1 .62 - 1 .70 (m, 1 H), 1 .71 - 1 .82 (m, 3H), 1 .91 - 2.05 (m, 2H), 2.33 - 2.44 (m, 2H), 2.50 - 2.56 (m, 6H), 2.56 - 2.64 (m, 3H), 2.68 (t, 1 H), 2.99 (d, 1 H), 3.06 (t, 1 H), HRMS Calculated for [C₁₄H₂oF3N₃O2+H]⁺: 320.1586. Found: 320.1578

Example 8

5-((2R,4S)-2-(((S)-2-(Trifluoromethyl)pyrrolidin-1 -yl)methyl)pipehdin-4-yl)isoxazol- 3(2H)-one

Step 1 : Cis-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(((S)-2-

(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate

1 -(Methoxycarbonyl)-2-(((S)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-4- carboxylic acid (2.35 g, 6.95 mmol) (reference compound 4) was dissolved in methyl THF (50 mL) under nitrogen atmosphere and di(1 H-imidazol-1 - yl)methanone (1 .689 g, 10.42 mmol) was added. The suspension was stirred at room temperature for 3 h (flask 1 ). In a separate flask was potassium 3-ethoxy-3- oxopropanoate (2.128 g, 12.50 mmol) suspended in methyl THF (25 mL) and magnesium chloride (1 .190 g, 12.50 mmol) was added. The suspension was stirred at 50^°C under nitrogen for 3 days using an oversized stirring bar (flask 2). The contents of flask 1 was added to flask 2 and the resulting white suspension was stirred at room temperature for 20 h. Conversion was not complete and in a separate flask was potassium 3-ethoxy-3-oxopropanoate (1 .064 g, 6.25 mmol) suspended in methyl THF (25 mL) and magnesium chloride (0.595 g, 6.25 mmol) was added. The suspension was stirred at 50^°C for 5 h and then added to the reaction mixture. The mixture was stirred at room temperature for 15 h. Saturated NH CI and DCM were added and the phases separated. The aqueous phase was extracted with DCM, the combined organic layers filtered through a phase separator and evaporated. The residue was purified via automated flash chromatography (2:1 heptane:EtOAc, Biotage® KP-SIL 340g column, 10 CV). Cis- methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(((S)-2-(trifluoromethyl)pyrrolidin-1 - yl)methyl)piperidine-1 -carboxylate (1 .917 g, 67.6 %) was yielded as colorless oil. MS m/z 409 (M+H)⁺

Step 2: Cis-methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-(((S)-2- (trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate

Cis-methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(((S)-2-(trifluoromethyl)pyrrolidin-1 - yl)methyl)piperidine-1 -carboxylate (1 .915 g, 4.69 mmol) was dissolved in MeOH (20 mL) and cooled to -40^°C under nitrogen. Sodium hydroxide (0.197 g, 4.92 mmol) dissolved in water (2.000 mL) was added and the mixture was stirred at - 40^°C for 15 min. Hydroxylamine (50 % by weight in water, 0.302 mL, 4.92 mmol) was added. The resulting solution was stirred at -40 C for 1 h. The mixture was then transfered into a pre-warmed (80^°C) solution of 6 M hydrogen chloride (24.23 mL, 145.36 mmol) and the mixture was stirred at 80^°C for 20 min. Water and DCM were added and the phases separated. The aqueous phase was extracted with DCM and the combined organic layers were filtered through a phase separator and evaporated. The residue contained only very little amount of the product, the major part remained in the aqueous phase. Sodium hydroxide (0.197 g, 4.92 mmol) was added to the aqueous phase, and the pH was basic after addition. The product could still not be extracted with DCM from the aqueous phase. The aqueous phase was freeze-dried and the resulting white powder was triturated with DCM containing acetic acid (0.268 mL, 4.69 mmol). The salt was filtered off and the solvent was evaporated to yield crude cis-methyl 4-(3-oxo-2,3- dihydroisoxazol-5-yl)-2-(((S)-2-(trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 - carboxylate (0.588 g, 33.2 %) as a light yellow oil. MS m/z 378 (M+H)⁺ Step 3: (2R,4S)-methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-(((S)-2- (trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate

Cis-methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-(((S)-2-(trifluoromethyl)pyrrolidin- 1 -yl)methyl)piperidine-1 -carboxylate (0.58 g, 1 .54 mmol) was subjected to preparative HPLC (Colunnn: Kromasil-C18 (250x50), 10 μηη particle size, mobile phase: ACN/H₂O/FA 50/50/0.1 , flow rate 100 mL/min) to yield (2R,4S)-methyl 4-(3- oxo-2,3-dihydroisoxazol-5-yl)-2-(((S)-2-(trifluoromethyl)pyrrolidin-1 - yl)methyl)piperidine-1 -carboxylate (177 mg, 31 %). Chiral purity 99.5 %ee, Optical rotation [a^ = +12.6 (acetonitrile, c=1 )

Step 4: 5-((2R,4S)-2-(((S)-2-(Trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one

(2R,4S)-Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-(((S)-2- (trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidine-1 -carboxylate (177 mg, 0.47 mmol) was dissolved in hydrogen bromide (33 % in acetic acid, 3697 μΙ, 21 .1 1 mmol) and stirred at room temperature for 16 h. The solvent was removed in vacuo and the residue purified by preparative HPLC (Instrument: FractionLynx III, Mobilphase: gradient 5-95 % MeCN in 0.2 % NH₃, pH 10, Column: Xbridge Prep C18 5μηη OBD 19*150 mm) to yield 5-((2R,4S)-2-(((S)-2-(trifluoromethyl)pyrrolidin- 1 -yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one (130 mg, 87 %). ¹ H NMR (600 MHz, dmso) δ 1 .01 - 1 .12 (m, 1 H), 1 .38 - 1 .48 (m, 1 H), 1 .64 - 1 .74 (m, 1 H), 1 .74 - 1 .85 (m, 4H), 1 .93 - 2.03 (m, 1 H), 2.25 - 2.39 (m, 2H), 2.52 - 2.57 (m, 1 H), 2.59 - 2.77 (m, 4H), 3.05 (d, 1 H), 3.10 (t, 1 H), 5.72 (s, 1 H). HRMS Calculated for [C₁₄H₂oF3N₃O2+H]⁺: 320.1586. Found: 320.1591

Example 9

5-((2R,4S)-2-((3,3-Difluoropyrrolidin-1 -yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one Step 1 : Methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)-4-(3-ethoxy-3- oxopropanoyl)piperidine-1 -carboxylate 2-((3,3-Difluoropyrrolidin-1 -yl)methyl)-1 -(me^^

acid (4.608 g, 15.04 mmol) was dissolved in anhydrous THF (50 ml_) and di(1 H- imidazol-1 -yl)methanone (3.66 g, 22.57 mmol) added. The suspension was stirred at room temperature under nitrogen atmosphere for 5 h (flask 1 ). In a separate flask 3-ethoxy-3-oxopropanoic acid, potassium salt (5.15 g, 30.09 mmol) and magnesium chloride (2.86 g, 30.09 mmol) were suspended in anhydrous THF (50 ml_) and stirred with an oversized stirring bar at 50°C under a nitrogen atmosphere for 5 h (flask 2). The grey suspension in flask 2 was then added to flask 1 . The thick suspension was stirred at room temperature for 18 h. The reaction mixture was acidified by addition of 3 M hydrogen chloride to pH 1 . The solvent was evaporated in vacuo. EtOAc (250 ml_) and water (250 ml_) were added, shaken and the phases separated. The aqueous phase was washed with EtOAc (250 ml_), basified to pH 10 with 3.8 M NaOH (aq) and extracted with EtOAc (3x250 ml_). The combined extracts were dried with Na₂SO (s), filtered and the filtrate evaporated in vacuo to yield crude methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)-4- (3-ethoxy-3-oxopropanoyl)piperidine-1 -carboxylate (3.08 g, 54.4 %) as a pale solid. MS m/z 377.3 (M+H)+, 375.2 (M-H)\

Step 2: Methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)-4-(3-oxo-2,3-dihvdroisoxazol- 5-yl)piperidine-1 -carboxylate

Methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)-4-(3-ethoxy-3- oxopropanoyl)piperidine-1 -carboxylate (3.078 g, 8.18 mmol) was dissolved in MeOH (25 ml_) and cooled to - 40°C. 3.8 M NaOH (2.152 ml_, 8.18 mmol) dissolved in water (2.500 ml_) was added and the reaction stirred at - 40°C for 20 min. Hydroxylamine (50 % by weight in water, 0.501 ml_, 8.18 mmol) was added and stirring continued for 3.5 h at - 40°C. The reaction mixture was then added to a pre warmed 80°C solution of 6 M HCI (aq) (42.3 ml_, 253.50 mmol) and stirred for 20 min. The reaction was cooled to room temperature and the solvent concentrated in vacuo. Water (10 ml_) was added and the solution neutralized to pH 5 by addition of 3.8 M NaOH. The solution was then purified by preparative HPLC on a XBridge C18 column (10 μηη 250x50 ID mm) using a gradient of 0-40% acetonitrile in H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. The product fractions were collected and freeze dried to yield methyl 2- ((3,3-difluoropyrrolidin-1-yl)methyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1- carboxylate (1.010 g, 35.8 %) as a white solid. MS m/z 346.2 (M+H)⁺ 344.1 (M-H)\

Step 3: (2R,4S)-Methyl 2-((3,3-difluoropyrrolidin-1-yl)methyl)-4-(3-oxo-2,3- dihvdroisoxazol-5-yl)piperidine-1-carboxylate and (2S,4S)-methyl 2-((3,3- difluoropyrrolidin-1-yl)methyl)-4-(3-oxo-2,3-dihvdroisoxazol-5-yl)pipehdine-1- carboxylate

Methyl 2-((3,3-difluoropyrrolidin-1-yl)methyl)-4-(3-oxo-2,3-dihydroisoxazol-5- yl)piperidine-1-carboxylate (1.010 g, 2.92 mmol) was subjected to chiral preparative HPLC (Column: Chiralcel OJ (250x20), 5 μιτι particle size, mobile phase: heptane/I PA/FA 80/20/0.1, flow rate 18 mL/min) to yield (2R,4S)-methyl 2- ((3,3-difluoropyrrolidin-1-yl)methyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1- carboxylate (210 mg, 21 %), Chiral purity: > 99 % ee. Optical rotation [af° =+30.0

(acetonitrile, c=1).¹H NMR (600 MHz, CDCI₃) δ 1.83 - 1.93 (m, 1H), 1.92 - 2.08 (m, 3H), 2.14 - 2.27 (m, 2H), 2.43 (dd, 1H), 2.51 (dd, 1H), 2.73 - 2.82 (m, 2H), 2.90-3.03 (m, 3H), 3.03-3.13 (m, 1H), 3.66 (s, 3H), 3.83-3.92 (m, 1H), 4.12- 4.23 (m, 1H), 5.72 (s, 1H), 10.12 (bs, 1H) and (2S,4S)-methyl 2-((3,3- difluoropyrrolidin-1-yl)methyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1- carboxylate (279 mg, 28 %), Chiral purity: > 99 % ee. Optical rotation [a]^² = ND

(acetonitrile, c=1).¹H NMR (600 MHz, CDCI₃) δ 1.34 - 1.51 (m, 1H), 1.54 - 1.68 (m, 1H), 1.74 - 2.11 (m, 2H), 2.11 - 2.28 (m, 2H), 2.54 - 3.11 (m, 8H), 3.62 (s, 3H), 3.91 -4.21 (m, 1H), 4.41 (d, 1H), 5.53 (s, 1H), 9.94 (bs, 1H).

Step 4: 5-((2R,4S)-2-((3,3-Difluoropyrrolidin-1-yl)methyl)pipehdin-4-yl)isoxazol- 3(2H)-one

(2R,4S)-Methyl 2-((3,3-difluoropyrrolidin-1-yl)methyl)-4-(3-oxo-2,3-dihydroisoxazol- 5-yl)piperidine-1-carboxylate (210 mg, 0.61 mmol) was dissolved in HBr (33 % in AcOH, 1789 μΙ, 25.54 mmol) and stirred at room temperature for 20 h. The solvent was then evaporated in vacuo. The residue was dissolved in DMSO/water and purified by preparative HPLC on a XBridge C18 column (5 μιτι OBD 19^*150 mm) using a gradient of 5-95% Acetonitrile in 0.2 % NH₃, pH 10, to yield 5-((2R,4S)-2- ((3,3-difluoropyrrolidin-1 -yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one (94 mg, 53.6 %). ¹H NMR (600 MHz, DMSO-c/6) δ 1 .02 (dd, 1 H), 1 .31 - 1 .42 (m, 1 H), 1 .77 - 1 .89 (m, 2H), 2.14 - 2.25 (m, 2H), 2.25 - 2.33 (m, 1 H), 2.33 - 2.43 (m, 2H), 2.54 - 2.75 (m, 5H), 2.77 - 2.94 (m, 2H), 2.96 - 3.04 (m, 1 H), 5.70 (s, 1 H). HRMS Calcd for [C₁₃H₁₉F₂N3O2+H]⁺: 288.1523. Found: 288.1498.

Example 10

5-((2S,4S)-2-((3,3-Difluoropyrrolidin-1 -yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one (2S,4S)-Methyl 2-((3,3-difluoropyrrolidin-1 -yl)methyl)-4-(3-oxo-2,3-dihydroisoxazol- 5-yl)piperidine-1 -carboxylate (273 mg, 0.79 mmol) (from example 9, step 3) was dissolved in HBr (33 % in AcOH, 4 mL, 57.10 mmol) and stirred at room temperature for 20 h. The solvent was then evaporated in vacuo. The residue was dissolved in DMSO/water and purified by preparative HPLC on a XBridge C18 column (5 μιτι OBD 19^*150 mm) using a gradient of 5-95% Acetonitrile in 0.2 % NH₃, pH 10, to yield 5-((2S,4S)2-((3,3-difluoropyrrolidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one (194 mg, 85 %). HRMS Calcd for [C₁₃H₁₉F₂N3O2+H]⁺: 288.1523. Found: 288.1513.

Example 1 1

5-((2R,4S)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidin-4-yl)isoxazol- 3(2H)-one

Step 1 : Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-((2,2,6,6-tetramethylpiperidin-1 - yl )methyl )piperid ine-1 -carboxylate

1 -(Methoxycarbonyl)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidine-4- carboxylic acid (1 .8 g, 5.29 mmol) (reference compound 6) was dissolved in THF (50 mL) and di(1 H-imidazol-1 -yl)methanone (1 .29 g, 7.93 mmol) was added. A precipitate formed. Dichloromethane (25 mL) was added followed by di(1 H- imidazol-1 -yl)methanone (1 .29 g, 7.93 mmol) and the resulting mixture stirred for 2 h (flask 1 ). In a separate flask 3-ethoxy-3-oxopropanoic acid, potassium salt (1 .63 g, 9.52 mmol) and magnesium chloride (0.91 g, 9.52 mmol) were suspended in anhydrous THF (25 mL) and stirred at 50°C for 5 h (flask 2). The contents of flask 1 was added to flask 2 and the resulting mixture stirred overnight at room temperature. The salts were filtered off and the solvent evaporated. The residue was purified by automated flash chromatography (gradient 0 to 100% Hex EtOAc; Biotage® KP-SIL 100g column). Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-((2,2,6,6- tetramethylpiperidin-1 -yl)methyl)piperidine-1 -carboxylate (0.64 g, 29 %) was isolated. MS m/z 41 1 (M+H)⁺

Step 2: Methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-((2,2,6,6-tetramethylpiperidin- 1 -yl)methyl)piperidine-1 -carboxylate

Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-((2,2,6,6-tetramethylpiperidin-1 - yl)methyl)piperidine-1 -carboxylate (0.64 g, 1 .56 mmol) was dissolved in MeOH (10 ml_) and cooled to -40°C. Sodium hydroxide (0.079 g, 1 .97 mmol) dissolved in water (1 .000 ml_) was added and the mixture was stirred at -40°C for 15 min. Hydroxylamine (50 % by weight in water, 0.124 g, 1 .88 mmol) was added. The resulting solution was stirred at -40°C for 1 h. The mixture was then transferred into a pre-warmed (80°C) solution of 6 M hydrogen chloride (9.40 mL, 56.39 mmol) and the mixture was stirred at 80°C for 20 min. Water and DCM were added and the phases separated. The aqueous phase was extracted with DCM and the combined organic layers were isolaled, dried (Na₂SO ), filtered through celite and evaporated. Crude methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((2,2,6,6- tetramethylpiperidin-1 -yl)methyl)piperidine-1 -carboxylate (0.46 g, 64 %) was isolated. MS m/z 380 (M+H)⁺

Step 3: (2R,4S)-Methyl 4-(3-oxo-2,3-dihvdroisoxazol-5-yl)-2-((2,2,6,6- tetramethylpiperidin-1 -yl)methyl)piperidine-1 -carboxylate and (2S,4S)-methyl 4-(3- oxo-2,3-dihvdroisoxazol-5-yl)-2-((2,2,6,6-tetramethylpiperidin-1 - yl)methyl)pipehdine-1 -carboxylate

Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((2,2,6,6-tetramethylpiperidin-1 - yl)methyl)piperidine-1 -carboxvlate (0.46 g, 1 .21 mmol) was subjected to chiral preparative HPLC (Column: Chiralpak IC (250x20), 5 μιτι particle size, mobile phase: heptane/EtOH/DEA 75/25/0.1 , flow rate 18 mL/min) to yield (2R,4S)-methyl

4- (3-oxo-2,3-dihydroisoxazol-5-yl)-2-((2,2,6,6-tetramethylpiperidin-1 - yl)methyl)piperidine-1 -carboxylate (151 mg, 33 %), Chiral purity: 99.9 % ee and (2S,4S)-methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((2,2,6,6-tetramethylpiperidin- 1 -yl)methyl)piperidine-1 -carboxylate (52 mg, 1 1 %), Chiral purity: 99 % ee.

Step 4: 5-((2R,4S)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidin-4- yl )isoxazol-3(2H )-one

(2R,4S)-Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((2,2,6,6-tetramethylpiperidin- 1 -yl)methyl)piperidine-1 -carboxylate (0.151 g, 0.40 mmol) was dissolved in hydrogen bromide (33 % in acetic acid, 5 mL, 28.50 mmol) and stirred overnight at room temperature. The acetic acid and HBr were evaporated. The crude was dissolved in methanol/water 50/50 and purified by preparative HPLC on a XBridge C18 column (5 μιτι OBD 19^*150 mm) using a gradient of 5-95% Acetonitrile in 0.2 % NH₃, pH 10, to yield 5-((2R,4S)-2-((2,2,6,6-tetramethylpiperidin-1 - yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one (29 mg, 23 %). ¹H NMR (600 MHz, cd3od) d 1 .06 (s, 6H), 1 .10 (s, 6H), 1 .49 (d, 6H), 1 .66 - 1 .76 (m, 1 H), 2.06 (tt, 1 H), 2.20 (d, 1 H), 2.33 (d, 1 H), 2.69 - 2.81 (m, 2H), 2.99 (td, 1 H), 3.14 - 3.24 (m, 1 H), 3.29 (dt, 2H), 5.55 (s, 1 H).

Example 12

5- ((2S,4S)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)piperidin-4-yl)isoxazol- 3(2H)-one

(2S,4S)-Methyl 4-(3-oxo-2,3-dihydroisoxazol-5-yl)-2-((2,2,6,6-tetramethylpiperidin- 1 -yl)methyl)piperidine-1 -carboxylate (0.052 g, 0.14 mmol) (from example 1 1 , step 3) was dissolved in hydrogen bromide 33 % in acetic acid, 5 mL, 28.50 mmol) and stirred overnight at room temperature. The acetic acid and HBr were evaporated. The residue was dissolved in methanol and purified by preparative HPLC on a XBridge C18 column (5 μηη OBD 19^*150 mm) using a gradient of 5-95% Acetonitrile in 0.2 % NH₃, pH 10, to yield 5-((2S,4S)-2-((2,2,6,6- tetramethylpiperidin-1 -yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one (0.44 mg, 1 %). Calcd for [C₁₈H3i N₃O2+H]⁺: 322.2495. Found: 322.2506.

Example 13

5-(Cis-2-(isoindolin-2-ylmethyl)piperidin-4-yl)isoxazol-3(2H)-one

Step 1 : Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(isoindolin-2-ylmethyl)piperidine-1 - carboxylate

2-(lsoindolin-2-ylmethyl)-1 -(methoxycarbonyl)piperidine-4-carboxylic acid (3.958 g, 12.43 mmol) (reference compound 7) was dissolved in THF (60 ml_) and di(1 H- imidazol-1 -yl)methanone (3.02 g, 18.65 mmol) added. The suspension was stirred at room temperature under N₂ atmosphere for 5 h (flask 1 ). In a separate flask 3- ethoxy-3-oxopropanoic acid, potassium salt (4.26 g, 24.86 mmol) and magnesium chloride (2.367 g, 24.86 mmol) were suspended in THF (60 ml_) and stirred using an oversized stirring bar at 50°C under an N₂ atmosphere for 5 h (flask 2). The grey suspension in flask 2 was then added to flask 1 . The thick suspension was stirred at room temperature for 18 h. The reaction mixture was acidified by addition of 3 M hydrogen chloride to pH 1 . The solvent was evaporated in vacuo. EtOAc (250 ml_) and water (250 ml_) were added, shaken and the phases separated. The aqueous phase was washed with EtOAc (250 ml_). The aqueous phase was then basified to pH 10 with 3.8 M NaOH (aq) and extracted with EtOAc (3x250 ml_). The combined organic phases were dried with Na₂SO (s), filtered and the filtrate evaporated in vacuo. The residue was purified by automated flash chromatography on a Biotage® KP-SIL 100g column. A gradient from 10 - 40 % of EtOAc/MeOH/TEA 100:10:1 in heptane over 9 CV was used as mobile phase. The product fractions were evaporated in vacuo to yield methyl 4-(3-ethoxy-3- oxopropanoyl)-2-(isoindolin-2-ylmethyl)piperidine-1 -carboxylate (2.92 g, 60.5 %). MS m/z 389.3 (M+H)⁺.

Step 2: Methyl 2-(isoindolin-2-ylmethyl)-4-(3-oxo-2,3-dihvdroisoxazol-5- yl)piperidine-1 -carboxylate Methyl 4-(3-ethoxy-3-oxopropanoyl)-2-(isoindolin-2-ylmethyl)piperidine-1 - carboxylate (2.923 g, 7.52 mmol) was dissolved in MeOH (25 ml_) and cooled to - 40°C. 3.8 M NaOH (1 .980 ml_, 7.52 mmol) dissolved in water (2.500 ml_) was added and the reaction stirred at - 40°C for 20 min. Hydroxylamine (50 % in water, 0.461 ml_, 7.52 mmol) was added and stirring continued for 3.5 h at - 40 °C. The reaction mixture was then added to a prewarmed 80°C solution of 6 M HCI (38.9 ml_, 233.26 mmol) and stirred for 20 min. The solvent was concentrated in vacuo. DCM (200 ml_) and water (150 ml_) were added, shaken and the phases separated. The aqueous phase was neutralized with 3.8 M NaOH to pH 5-6. The aqueous phase was extracted with DCM (4x150 ml_). The combined organic phase were dried with a phase separator and evaporated in vacuo. The compound was purified by preparative HPLC on a XBridge C18 column (10 μιτι 250x50 ID mm) using a gradient of 5 - 45% acetonitrile in H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. The product fractions were collected, concentrated in vacuo and freeze dried to yield 756 mg as a light purple-grey solid. LCMS analysis of the aqueous phase showed product remaining. The aqueous phase was concentrated in vacuo, solids filtered off and the filtrate purified by preparative HPLC on a XBridge C18 column (10 μιτι 250x50 ID mm) using a gradient of 5 - 45% acetonitrile in H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. The product fractions were collected, concentrated in vacuo and freeze dried to yield 192 mg of a pale solid. The solid from the filtration was redissolved in acetonitrile/water and freeze dried to yield 648 mg of a pale solid. The three differently isolated solids were combined (1 .596 g, 4.47 mmol), dissolved in MeOH and repurified by preparative HPLC on a XBridge C18 column (10 μηη 250x50 ID mm) using a gradient of 5 - 45% acetonitrile in H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. The product fractions were collected, concentrated in vacuo and freeze dried to yield methyl 2- (isoindolin-2-ylmethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 -carboxylate (1 .174 g, 43.6 %) as a solid. MS m/z 358.3 (M+H)⁺ 356.2 (M-H)\ Step 3: Cis-methyl 2-(isoindolin-2-ylmethyl)-4-(3-oxo-2,3-dihvdroisoxazol-5- yl)piperidine-1 -carboxylate and (2S,4S)-methyl 2-(isoindolin-2-ylnnethyl)-4-(3-oxo- 2,3-dihvdroisoxazol-5-yl)piperidine-1 -carboxylate

Methyl 2-(isoindolin-2-ylmethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 - carboxylate (1 .174 g, 3.28 mmol) was subjected to chiral preparative HPLC (Column: Chiralcel AD (250x20), 5 μιτι particle size, mobile phase: heptane/EtOH/TEA 70/30/0.1 , flow rate 18 mL/min) to yield cis-methyl 2- (isoindolin-2-ylmethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 -carboxylate (600 mg, 51 %), ¹H NMR (400 MHz, CDCI₃) δ 1 .20 - 1 .46 (m, 3H), 1 .58 - 1 .72 (m, 1 H), 1 .94 - 2.1 1 (m, 2H), 2.17 - 2.29 (m, 1 H), 2.62 - 2.82 (m, 1 H), 2.87 - 3.13 (m, 3H), 3.14 - 3.33 (m, 1 H), 3.34 - 3.57 (m, 2H), 3.62 - 3.80 (m, 4H), 3.99 - 4.33 (m, 6H), 4.42 - 4.58 (m, 1 H), 4.60 - 4.73 (m, 1 H), 5.35 (s, 1 H), 7.16 - 7.24 (m, 4H) and (2S,4S)-methyl 2-(isoindolin-2-ylmethyl)-4-(3-oxo-2,3-dihydroisoxazol-5- yl)piperidine-1 -carboxylate (37 mg, 3 %), Chiral purity: 98.1 % ee. Optical rotation [a f° = +5.2 (acetonitrile, c=0.5).

Step 4: 5-(Cis-2-(isoindolin-2-ylmethyl)piperidin-4-yl)isoxazol-3(2H)-one

Cis-methyl 2-(isoindolin-2-ylmethyl)-4-(3-oxo-2,3-dihydroisoxazol-5-yl)piperidine-1 - carboxylate (479 mg, 1 .34 mmol) was dissolved in HBr (33 % in AcOH, 5 ml_, 71 .37 mmol) and stirred at room temperature for 24 h. The reaction mixture turned dark brown. The reaction mixture was then evaporated in vacuo. The residue was dissolved in DMSO/water and purified twice by preparative HPLC on a XBridge C18 column (5 μιτι OBD 19^*150 mm) using a gradient of 5-95% Acetonitrile in 0.2 % NH₃, pH 10, to yield 5-(Cis-2-(isoindolin-2-ylmethyl)piperidin-4-yl)isoxazol- 3(2H)-one (62.0 mg, 15.45 %). ¹H NMR (600 MHz, DMSO-c/6) δ 1 .48 - 1 .55 (m, 1 H), 1 .71 - 1 .84 (m, 2H), 1 .87 - 1 .94 (m, 1 H), 2.34 - 2.39 (m, 1 H), 2.54 - 2.61 (m, 1 H), 2.61 - 2.73 (m, 2H), 2.75 - 2.86 (m, 2H), 3.07 - 3.14 (m, 2H), 3.78 (d, 2H), 3.86 (d, 2H), 5.81 (s, 1 H), 7.13 - 7.17 (m, 2H), 7.17 - 7.22 (m, 2H). HRMS Calcd for [C₁₇H₂iN₃O₂+H]⁺: 300.1712. Found: 300.1715.

Example 14 5-((2S^S)-2-(lsoindolin-2-ylmethyl)pipendin-4-yl)isoxazol-3(2H)-one (2S,4S)-Methyl 2-(isoindolin-2-ylmethyl)-4-(3-oxo-2,3-dihydroisoxazol-5- yl)piperidine-1 -carboxylate (37 mg, 0.10 mmol) was dissolved in HBr (33 % in AcOH, 1 mL, 14.27 mmol) and stirred at room temperature for 24 h. The reaction mixture was then evaporated in vacuo. The residue was dissolved in DMSO/water and purified twice by preparative HPLC on a XBridge C18 column (5 μιτι OBD 19^*150 mm) using a gradient of 5-95% Acetonitrile in 0.2 % NH₃, pH 10, to yield 5- ((2S,4S)-2-(isoindolin-2-ylmethyl)piperidin-4-yl)isoxazol-3(2H)-one (1 .5 mg, 4.84 %). HRMS Calcd for [C₁₇H2i N₃O2+H]⁺: 300.1712. Found: 300.1708.

Claims

Claims

1 . A compound of formula I:

wherein R1 is hydrogen; R2 is

C-i-C-io alkyl, which C-I-C-IO alkyl comprises 0, 1 , 2, 3 or 4 heteroatoms, selected from O and N, or 0, 1 or 2 acid, ester or amide functionalities, and said C-I-C-IO alkyl is substituted by 0, 1 , 2, 3 or 4 CH_3, halogen or CF₃, or

-XR20, wherein X is a bond or -CH₂- , and

R20 is a 4 to 10 membered, monocyclic or bicyclic heterocyclic aromatic or non-aromatic ring containing 1 , 2, 3 or 4 heteroatoms selected from O and N, and R20 is substituted by 0, 1 , 2, 3 or 4 CH_3, halogen or CF₃, or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 wherein R2 is -XR20 and X is -CH₂- , and R20 is a 4 to 10 membered, monocyclic or bicyclic heterocyclic non- aromatic ring containing 1 , 2, 3 or 4 heteroatoms selected from O and N, and R20 is substituted by 0, 1 , 2, 3 or 4 CH₃, halogen or CF₃.

3. A compound according to claim 1 or 2 selected from one or more of the following compounds: 5-((2R,4S)-2-(Morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-(Trans-2-(morpholinomethyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-((3-(Trifluoromethyl)-5,6-dihydro-[1 ,2,4]tnazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2S,4R)-2-((3-(Trifluoromethyl)-5,6-dihydro-[1 ,2,4]tnazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2S,4S)-2-((3-(Trifluoromethyl)-5,6-dihydro-[1 ,2,4]tnazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2R,4R)-2-((3-(Trifluoromethyl)-5,6-dihydro-[1 ,2,4]tnazolo[4,3- a]pyrazin-7(8H)-yl)methyl)piperidin-4-yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-(((R)-2-(Trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidin^ 4-yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-(((S)-2-(Trifluoromethyl)pyrrolidin-1 -yl)methyl)piperidin-

4- yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-((3,3-Difluoropyrrolidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one;

5-((2S,4S)-2-((3,3-Difluoropyrrolidin-1 -yl)methyl)piperidin-4- yl)isoxazol-3(2H)-one;

5-((2R,4S)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)pipendin-4- yl)isoxazol-3(2H)-one;

5- ((2S,4S)-2-((2,2,6,6-tetramethylpiperidin-1 -yl)methyl)pipendin-4- yl)isoxazol-3(2H)-one;

5-(Cis-2-(isoindolin-2-ylmethyl)piperidin-4-yl)isoxazol-3(2H)-one; and 5-((2S,4S)-2-(lsoindolin-2-ylmethyl)piperidin-4-yl)isoxazol-3(2H)-one.

4. A pharmaceutical composition comprising at least one compound according any of to claims 1 -3 and a pharmaceutically acceptable carrier or diluent.

5. A method for treatment or prophylaxis of a disease or condition in which modulation of fibrinolysisis is beneficial comprising administering to a warm- blooded animal in need of such treatment a therapeutically effective amount of at least one compound in accordance with any of claims 1 -3.

6. The method according to claim 5, wherein said disease or condition is selected from inherited bleeding disorders, stroke, menorrhagia and liver diseases.

7. The use of a compound according to any of claims 1 -3 for the treatment or prophylaxis of a disease or condition in which modulation of fibrinolysis is beneficial.

8. The use according to claim 7, wherein said disease or condition is selected from inherited bleeding disorders, stroke, menorrhagia and liver diseases.

9. The use of a compound according to any of claims 1 -3 in the

manufacture of a medicament for the treatment or prophylaxis of a disease or condition in which modulation of fibrinolysis is beneficial.

10. The use according to claim 9, wherein said disease is selected from inherited bleeding disorders, stroke, menorrhagia and liver diseases.