OXIMES AND THEIR USE IN TREATMENT OF GBA-RELATED DISEASES

Abstract

The present invention relates to oximes, their synthesis, and their use for increasing GBA activity and/or levels as well as treatment of GBA-related diseases, such as Parkinson's disease.

Claims

1. A compound of formula (I), ##STR00124## or a pharmaceutically acceptable salt thereof, wherein R.sup.1 is selected from the group consisting of: H, C.sub.1-6 alkyl, and halogen; Y is selected from the group consisting of: OH, and C.sub.1-6 alkoxy; X is selected from the group consisting of: chlorine and bromine; J is an aliphatic cycle comprising at least one nitrogen atom, wherein the aliphatic cycle is optionally substituted; and wherein A is selected from the group consisting of: ##STR00125## wherein each k is 1, 2, 3, or 4; R.sup.2 and R.sup.3 are independently of each other selected from the group consisting of: C.sub.1-6 alkyl, C.sub.1-6 acyl, and CO.sub.2C.sub.1-6 alkyl; n1, n2, u1, and u2 are independently of each other selected from the group consisting of: 1, 2, or 3; each R.sup.4 is independently selected from the group consisting of: H, halogen, and C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; each R.sup.5 is independently selected from the group consisting of: H, halogen, and C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; each R.sup.6 is independently selected from the group consisting of: H, halogen, and C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; each R.sup.7 is independently selected from the group consisting of: H, halogen, and C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; R.sup.8 is selected from the group consisting of: H, and C.sub.1-4 alkyl; G is selected from the group consisting of: CH.sub.2, CH(R.sup.9), C(R.sup.9).sub.2, NH, and N(R.sup.9); wherein each R.sup.9 is independently selected from the group consisting of hydrogen, halogen, C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; and wherein HetAr is a heteroaryl optionally substituted by one or more R.sup.10, wherein each R.sup.10 is independently selected from the group consisting of: hydrogen, C.sub.1-6 alkyl, halogen, hydroxy, C.sub.1-6 alkoxy, amino, amido, and C.sub.1-6 acyl; and wherein T is selected from the group consisting of: ##STR00126## or any tautomer thereof, wherein a is 0, 1, 2, or 3; X.sup.1, X.sup.2, X.sup.3, X.sup.4, and X.sup.5 independently are selected from the group consisting of: C, CH, and N; and each one, two, or three Subst. is independently selected from the group consisting of: hydrogen, alkyl, halogen, hydroxy, alkoxy, amino, amido, acyl, cycloalkyl, and heterocycloalkyl, wherein each methylene group of the alkyl is optionally replaced by O; with the proviso that when X is chlorine, A is not ##STR00127##

2. The compound according to claim 1, wherein J is of formula (J1): ##STR00128## wherein n3 and n4 are independently 1, 2, or 3; each R.sup.11 is independently selected from the group consisting of: H, halogen, and C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; each R.sup.12 is independently selected from the group consisting of: H, halogen, and C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; each R.sup.13 is independently selected from the group consisting of: H, halogen, and C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; each R.sup.14 is independently selected from the group consisting of: H, halogen, and C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; Q is selected from the group consisting of: a bond, CH.sub.2, CH(R.sup.15), C(R.sup.15).sub.2, NH, and N(R.sup.15); and wherein each R.sup.15 is independently selected from the group consisting of hydrogen, halogen, C.sub.1-4 alkyl, wherein each methylene group optionally is replaced by O; and wherein a pair of R.sup.11 and R.sup.13 are optionally linked together to form a ring.

3. The compound according to claim 2, wherein Q is selected from the group consisting of: a bond, CH.sub.2, CHF, N(Me)-, and NH.

4. The compound according to any one of the preceding claims, wherein J is selected from the group consisting of: ##STR00129##

5. The compound according to any one of the preceding claims, wherein Y is OH.

6. The compound according to any one of the preceding claims, wherein A is of formula (Ia); ##STR00130## k is 2, R.sup.3 is C.sub.1-3 alkyl, and R.sup.2 is C.sub.1-3 alkyl or CO.sub.2tBu.

7. The compound according to any one of the preceding claims, wherein A is of formula (Ib); ##STR00131## wherein n1 and n2 are each 2; R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each hydrogen, R.sup.8 is hydrogen or C.sub.1-3 alkyl; and T is of formula (T3); ##STR00132## wherein a is 0 or 1; wherein X.sup.1, X.sup.2, and/or X.sup.3 is N and the remainder of X.sup.1X.sup.5 are independently C or CH; and wherein each one, two, or three Subst. is independently selected from the group consisting of: hydrogen, C.sub.1-4 alkyl, halogen, hydroxy, C.sub.1-4 alkoxy, and C.sub.1-4 acyl.

8. The compound according to any one of the preceding claims, wherein only one Subst. is present and is: a) methyl; or b) chlorine.

9. The compound according to any one claims 1-5, wherein A is of formula (Ib); ##STR00133## wherein n1 and n2 are each 2; R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each hydrogen; R.sup.8 is hydrogen or C.sub.1-3 alkyl; and T is of formula (T1); ##STR00134## wherein a is 0 and Subst is C.sub.1-6 alkyl, wherein each methylene group is optionally replaced by O.

10. The compound according to any one claims 1-5, wherein A is of formula (Ic); ##STR00135## wherein k is 1, 2, or 3; u1 and u2 are each 1 or 2; R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each hydrogen; and G is selected from the group consisting of: a bond, CH.sub.2, NH, and N(C.sub.1-3 alkyl)-.

11. The compound according to any one of claims 1-5, wherein A is of formula (Id); ##STR00136## wherein HetAr is a C.sub.5-13 heteroaryl comprising one or more ring heteroatoms selected from nitrogen, oxygen and sulfur, and which is monocyclic, bicyclic, or tricyclic.

12. The compound according to any one of the preceding claims, wherein A is selected from the group consisting of: ##STR00137## ##STR00138##

13. The compound according to claim 1, wherein the compound is selected from the group consisting of: ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144##

14. The compound according to any one of the preceding claims, wherein the compound is a GBA inducer and increases glucocerebrosidase (GBA) enzyme levels and/or GBA enzyme activity.

15. A compound as defined in any one of claims 1-14 for use in the treatment of Parkinson's disease (PD).

Description

EXAMPLES

Example 1: Synthesis of Oximes

Materials and Abbreviations

[0167] The abbreviations used:

TABLE-US-00001 DCM Dichloromethane DCE Dichloroethane TFA Trifluoroacetic acid CDI Carbonyldiimidazole MeCN Acetonitrile HOAc Acetic acid MeOH Methanol H.sub.2O Water NaBH.sub.3CN Sodium cyanoborohydride NBS 1-Bromo-2,5-pyrrolidinedione K.sub.2CO.sub.3 Potassium carbonate DIPEA N,N-Diisopropylethylamine TEA N,N-Diethylethanamine HATU 1-[Bis(dimethylamino)methylene]- 1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate DMF Dimethylformamide NaOH Sodium hydroxide THF Tetrahydrofuran LiHMDS Lithium bis(trimethylsilyl)amide DIAD Diisopropyl azodicarboxylate PPh.sub.3 Triphenylphosphine TBAF Tetra-n-butylammonium fluoride NaBH(OAc).sub.3 Sodium triacetoxyboranuide MeNH.sub.2 Methanamine Boc.sub.2O Di-tert-butyl dicarbonate Paraform Paraformaldehyde t-BuONO tert-Butyl nitrite CuCl.sub.2 Copper (II) chloride IPA Propan-2-ol NH.sub.2OH*HCl Hydroxylamine hydrochloride Rac Racemic (Some compounds may contain both known and unknown stereochemistry) r.t. Room temperature

[0168] A straight line towards a chiral center in the schemes and structures below indicate the material is a racemate. If nothing else is noted, the structures are racemates.

[0169] The salt stoichiometry are assumptions based on normal acid base reaction considerations. The exact salt content has not been absolutely determined.

[0170] Analytical and preparative instruments used. One or more of the following instruments were used in the process of analysing composition of isolated material:

LC/MS

Instrument Specifications:

[0171] Agilent 1100 Series LC/MSD system with DAD\ELSD Alltech 2000ES and Agilent LC\MSD VL (G1956B), SL (G1956B) mass-spectrometer.

[0172] Agilent 1200 Series LC/MSD system with DAD\ELSD Alltech 3300 and Agilent LC\MSD G6130A, G6120B mass-spectrometer.

[0173] Agilent Technologies 1260 Infinity LC/MSD system with DAD\ELSD Alltech 3300 and Agilent LC\MSD G6120B mass-spectrometer.

[0174] Agilent Technologies 1260 Infinity II LC/MSD system with DAD\ELSD G7102A 1290 Infinity II and Agilent LC\MSD G6120B mass-spectrometer.

[0175] Agilent 1260 Series LC/MSD system with DAD\ELSD and Agilent LC\MSD (G6120B) mass-spectrometer.

[0176] UHPLC Agilent 1290 Series LC/MSD system with DAD\ELSD and Agilent LC\MSD (G6125B) mass-spectrometer.

[0177] All the LC/MS data were obtained using positive/negative mode switching.

H-NMR

[0178] Bruker AVANCE III 400 [0179] Varian UNITY INOVA 400

[0180] For chiral analysis or separation the following instruments were used:

Analytical Separation:

[0181] Column: Chiralpak IA (250*4.6 mm, 5 mkm) [0182] HPLC instrument: [0183] Agilent Technologies HPLC Systems 1200 Series with DAD Detector (G1315B).

Preparative Separation:

[0184] Column: Chiralpak IA (250*20 mm, 5 mkm)

HPLC Instrument:

[0185] Agilent Technologies HPLC Preparative Systems 1260 Infinity II Series with DAD Detector (G7115B).

General Synthesis of Building Blocks

Synthesis of 2-methyl-4-azoniaspiro[3.5]nonan-2-ol chloride

[0186] ##STR00047##

[0187] To a solution 8.5 g of piperidine in 200 ml of methanol was added 10.64 g of 2-(chloromethyl)-2-methyl-oxirane. The mixture was stirred at 25? C. 24 h and then evaporated to dryness. The residue was triturated with ether three times, and then dried in vacuo to give 16.5g crude title compound which was used as such.

1. General Synthesis Using Quaternary Salt

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride dihydrochloride, ID 336

General Scheme

[0188] ##STR00048##

1.1 Synthesis of tert-butyl 4-(N-hydroxycarbamimidoyl)-4-methylpiperidine-1-carboxylate

##STR00049##

[0189] tert-Butyl 4-cyano-4-methyl-piperidine-1-carboxylate (29.0 g, 122.83 mmol) was dissolved in IPA (200 ml), after that hydroxylamine hydrochloride (12.80 g, 184.24 mmol, 1.5 eq) was added to the resulting solution, followed by the addition of sodium hydrogen carbonate (15.48 g, 184.24 mmol, 1.5 eq). The reaction mixture was then left while stirring at 60? C. overnight. After 24 hours the reaction mixture was diluted with water (500 ml). The formed solid was collected by filtration, washed with water (100 ml) and airdried to afford the title product (25 g, 75%) as white solid. LCMS [M?t-Bu H].sup.+ 202.0. The product obtained was used as such without additional purification.

1.2 Synthesis of tert-butyl 4-(N-(2-hydroxy-3-(piperidin-1-yl)propoxy)carbamimidoyl)-4-methylpiperidine-1-carboxylate

[0190] ##STR00050##

[0191] tert-Butyl 4-(N-hydroxycarbamimidoyl)-4-methylpiperidine-1-carboxylate (1.5 g, 5.54 mmol, 1 eq) was dissolved in IPA (50 ml), followed by sodium hydroxide (0.222 g, 5.54 mmol, 1 eq) and 4-azoniaspiro[3.5]nonan-2-ol chloride (0.98 g, 5.54 mmol, 1 eq). The reaction mixture was then stirred for 24 hours at 50? C., after which the inorganic precipitate was removed by filtration and the filtrate collected was concentrated under reduced pressure to afford the title product (3 g, 95.15%) as a yellow oil. LCMS [M+1].sup.+ 399.2.

1.3 Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride dihydrochloride

[0192] ##STR00051##

[0193] tert-Butyl 4-(N-(2-hydroxy-3-(piperidin-1-yl)propoxy)carbamimidoyl)-4-methylpiperidine-1-carboxylate, obtained in the previous experiment (3.0 g, 5.27 mmol, 1 eq) was dissolved in a mixture of acetic acid (5 ml) and aqueous hydrochloric acid (4N, 5 ml) at 0? C. Sodium nitrite (727 mg, 10.54 mmol, 2 eq) was added portion wise to the resulting solution while cooling, maintaining the temperature interval 0-5? C. After the addition was completed aqueous hydrochloric acid (4N, 5 ml) was added at 0? C. to the reaction mixture, which was left while stirring at 0? C. for 1 hour. The cooling bath was removed and the reaction mixture was allowed to warm up to room temperature and then left while stirring overnight. After 12 hours the mixture was* concentrated under reduced pressure to afford crude semi-solid product (3.1 g), which was subjected for prep HPLC purification with HCl addition to result in 386 mg (17.8%) of the title N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride dihydrochloride as a yellow oil (METHOD A). *In an alternative work up and purification procedure, the mixture was concentrated under reduced pressure to afford crude semi-solid residue, which was diluted with 30% aqueous solution of potassium carbonate to adjust pH 10 and then extracted with DCM (3?5 ml). The organic layers were combined, dried over anhydrous sodium sulfate and filtered. The filtrate collected was concentrated under reduced pressure to afford crude oil, which was subjected for prep HPLC with trifluoroacetate addition (METHOD B LCMS [M?.Math.Cl.sup.?].sup.+ 282.2. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 4.32-4.23 (m, 1H), 4.19-4.03 (m, 2H), 3.53-3.35 (m, 2H), 3.24-3.12 (m, 3H), 3.10-2.88 (m, 4H), 2.82 (t, J=12.1, 12.1 Hz, 1H), 2.23 (d, J=14.2 Hz, 2H), 1.88-1.74 (m, 2H), 1.75-1.57 (m, 5H), 1.44-1.30 (m, 1H), 1.17 (s, 3H).

Example 2

Synthesis of 4-ethyl-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)piperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate, NME200409

[0194] ##STR00052##

[0195] In a generally similar manner with non-critical variations was made 4-ethyl-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)piperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate (254 mg, 29.6%) as a yellow oil from the commercially available tert-butyl 4-cyano-4-ethylpiperidine-1-carboxylate in line with the synthesis described in 1.1 to 1.3. .sup.1H NMR (Deuterium Oxide, 500 MHz): ? (ppm) 4.34-4.25 (m, 1H), 4.23-4.09 (m, 2H), 3.46 (dd, J=31.0, 11.7 Hz, 2H), 3.27-3.06 (m, 4H), 3.05-2.92 (m, 3H), 2.83 (t, J=12.1, 12.1 Hz, 1H), 2.27 (d, J=14.6 Hz, 2H), 1.88-1.77 (m, 2H), 1.72-1.61 (m, 5H), 1.60-1.49 (m, 2H), 1.46-1.34 (m, 1H), 0.67 (t, J=7.4, 7.4 Hz, 3H).

Example 3

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(pyridin-2-yl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate, NME200415

[0196] ##STR00053##

[0197] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(pyridin-2-yl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate (115 mg, 12.46%) as a yellow oil from the commercially available tert-butyl 4-cyano-4-(pyridin-2-yl)piperidine-1-carboxylate in line with the synthesis described in 1.1 to 1.3. LCMS [M?.Math.Cl.sup.?].sup.+ 345.0. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 8.59 (d, J=4.9 Hz, 1H), 8.38 (t, J=8.4, 8.4 Hz, 1H), 7.95 (d, J=8.2 Hz, 1H), 7.81 (t, 1H), 4.35-4.24 (m, 2H), 4.16 (dd, 1H), 3.55-3.14 (m, 7H), 3.15-3.01 (m, 2H), 2.93-2.86 (m, 1H), 2.82-2.76 (m, 2H), 2.42-2.31 (m, 2H), 1.85-1.71 (m, 2H), 1.70-1.51 (m, 3H), 1.44-1.27 (m, 1H).

Example 4

General Scheme Preparation of Nitrile Precursors

[0198] ##STR00054##

1.4 synthesis of tert-butyl 4-cyano-4-(3-pyridylmethyl)piperidine-1-carboxylate

[0199] ##STR00055##

[0200] To a solution of tert-butyl 4-cyanopiperidine-1-carboxylate (1.5 g. 7.13 mmol) under nitrogen atmosphere in a round-bottomed flask was slowly added at ?76? C. lithium bis(trimethylsilyl)azanide (1.0 M in THF/Ethylbenzol, 21.4 ml, 21.4 mmol). After the mixture was stirred for 2 hours at ?76? C., 3-(bromomethyl)pyridine hydrobromide (1.98 g, 10.46 mmol) was added into the system. The reaction mixture was stirred for further 30 minutes and then warmed to room temperature and stirred overnight. The mixture was quenched with 50 ml saturated aqueous NH.sub.4Cl.sub.2 further diluted with water and extracted with EtOAc. The organic layers were washed with water and brine then dried over sodium sulfate, filtered and concentrated to afford target compound (2.6 g, 33%), which was used as such LCMS: [M+H].sup.+ 302

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(pyridin-3-ylmethyl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate, NME200419

[0201] ##STR00056##

[0202] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(pyridin-3-ylmethyl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate (720 mg, 33.19%) as a yellow oil using tert-butyl 4-cyano-4-(3-pyridylmethyl)piperidine-1-carboxylate instead of tert-butyl 4-cyanopiperidine-1-carboxylate in line with the synthesis described in 1.1 to 1.3, and 1.4 respectively LCMS [M+1].sup.+ 396.2. .sup.1H NMR (Chloroform-d, 400 MHz): ? (ppm) 8.48 (d, J=4.5 Hz, 1H), 8.37 (s, 1H), 7.44 (d, 1H), 7.23-7.17 (m, 1H), 4.46-4.31 (m, 1H), 4.05 (d, J=13.2 Hz, 1H), 3.68 (dd, J=11.6, 5.7 Hz, 1H), 3.38-3.22 (m, 2H), 3.16-2.98 (m, 2H), 2.88-2.74 (m, 2H), 2.66-2.24 (m, 6H), 2.27-2.15 (m, 2H), 1.91 (t, J=14.4, 14.4 Hz, 2H), 1.64-1.51 (m, 4H), 1.46-1.34 (m, 2H).

Example 5

Synthesis of 3-(dimethylamino)-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)propanimidoyl chloride, NME200446

[0203] ##STR00057##

[0204] In a generally similar manner with non-critical variations was made 3-(dimethylamino)-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)propanimidoyl chloride (445.2 mg, 41.56%) as a yellow oil from the commercially available 3-(dimethylamino)propanenitrile in line with the synthesis described in 1.1 to 1.3. LCMS [M+1].sup.+ 292.2. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): ? (ppm) 4.67-4.58 (m, 1H), 4.08-4.00 (m, 1H), 3.99-3.92 (m, 1H), 3.88-3.75 (m, 1H), 2.64-2.56 (m, 2H), 2.49-2.45 (m, 2H), 2.42-2.31 (m, 4H), 2.29-2.21 (m, 2H), 2.13 (s, 6H), 1.52-1.42 (m, 4H), 1.39-1.28 (m, 2H).

Example 6

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(2-methoxyethyl)piperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate, NME200448

[0205] ##STR00058##

[0206] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(2-methoxyethyl)piperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate (26.5 mg, 6.63%) as a yellow oil from the commercially available tert-butyl 4-cyano-4-(2-methoxyethyl)piperidine-1-carboxylate in line with the synthesis described in 1.1 to 1.3. LCMS [M?.Math.Cl.sup.?].sup.+ 326.2. .sup.1H NMR (Methanol-d.sub.4, 400 MHz): ? (ppm) 4.38-4.28 (m, 1H), 4.22 (d, J=4.9 Hz, 2H), 3.64-3.51 (m, 2H), 3.40 (t, J=6.1, 6.1 Hz, 2H), 3.34-3.21 (m, 8H), 3.19-2.96 (m, 4H), 2.39 (d, J=14.5 Hz, 2H), 2.00-1.67 (m, 8H), 1.62-1.47 (m, 1H).

Example 7

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)pyridazine-4-carbimidoyl bromide, NME200452

[0207] ##STR00059##

[0208] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)pyridazine-4-carbimidoyl bromide (159.9 mg, 12.36%) as a brown oil from the commercially available pyridazine-4-carbonitrile in line with the synthesis described in 1.1 to 1.3 but using hydrobromic acid instead of hydrochloric acid in experimental procedure 1.3. LCMS [M+1].sup.+ 345.2. .sup.1H NMR (Chloroform-d, 400 MHz): ? (ppm) 9.57 (s, 1H), 9.24 (d, J=5.3 Hz, 1H), 7.78 (d, J=7.7 Hz, 1H), 4.39 (d, J=5.0 Hz, 2H), 4.12-4.02 (m, 1H), 3.53 (s, 1H), 2.65-2.55 (m, 2H), 2.46-2.40 (m, 2H), 2.38-2.28 (m, 2H), 1.63-1.50 (m, 4H), 1.47-1.38 (m, 2H).

Example 8

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-2-methylthiazole-5-carbimidoyl chloride, NME200453

[0209] ##STR00060##

[0210] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-2-methylthiazole-5-carbimidoyl chloride (136 mg, 21.28%) as a yellow oil from the commercially available 2-methyl-1,3-thiazole-5-carbonitrile in line with the synthesis described in 1.1 to 1.3. LCMS [M+1].sup.+ 318.2. .sup.1H NMR (Chloroform-d, 400 MHz): ? (ppm) 7.93 (s, 1H), 7.24 (s, 1H), 4.29-4.16 (m, 2H), 4.07-3.99 (m, 1H), 2.68 (s, 3H), 2.62-2.51 (m, 2H), 2.42-2.27 (m, 4H), 1.63-1.51 (m, 4H), 1.47-1.38 (m, 2H).

Example 9

Synthesis of 6-chloro-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)nicotinimidoyl chloride, NME200466

[0211] ##STR00061##

[0212] In a generally similar manner with non-critical variations was made 6-chloro-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)nicotinimidoyl chloride (239 mg, 23.82%) as a yellow oil from the commercially available 6-chloronicotinonitrile in line with the synthesis described in 1.1 to 1.3. LCMS [M+1].sup.+ 332.2. .sup.1H NMR (Chloroform-d, 400 MHz): ? (ppm) 8.80 (d, J=2.3 Hz, 1H), 8.05 (dd, J=8.4, 2.4 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 4.31-4.25 (m, 2H), 4.08-4.01 (m, 1H), 3.47 (s, 1H), 2.66-2.57 (m, 2H), 2.40-2.29 (m, 4H), 1.60-1.53 (m, 4H), 1.44-1.39 (m, 2H).

Example 10

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-3-methylisoxazole-5-carbimidoyl chloride, NME200475

[0213] ##STR00062##

[0214] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-3-methylisoxazole-5-carbimidoyl chloride (360 mg, 24.46%) as a yellow oil from the commercially available 3-methylisoxazole-5-carbonitrile in line with the synthesis described in 1.1 to 1.3. LCMS [M+1].sup.+ 302.2. .sup.1H NMR (Chloroform-d, 400 MHz): ? (ppm) 6.50 (s, 1H), 4.31 (d, J=5.0 Hz, 2H), 4.09-4.01 (m, 1H), 3.76 (s, 1H), 2.65-2.54 (m, 2H), 2.42-2.25 (m, 7H), 1.64-1.48 (m, 4H), 1.47-1.36 (m, 2H).

Example 11

Synthesis of 6-chloro-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylnicotinimidoyl chloride, NME200501

[0215] ##STR00063##

[0216] In a generally similar manner with non-critical variations was made 6-chloro-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylnicotinimidoyl chloride (66.3 mg, 2.48%) as a yellow oil from the commercially available 6-chloro-4-methylnicotinonitrile in line with the synthesis described in 1.1 to 1.3. LCMS [M+1].sup.+ 346.2. .sup.1H NMR (Chloroform-d, 400 MHz): ? (ppm) 8.44 (s, 1H), 7.20 (s, 1H), 4.26 (d, J=5.6 Hz, 2H), 4.08-4.00 (m, 1H), 3.76 (s, 1H), 2.62-2.55 (m, 2H), 2.44 (s, 3H), 2.41-2.29 (m, 4H), 1.62-1.52 (m, 4H), 1.46-1.38 (m, 2H).

Example 12

Synthesis of 6-chloro-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)nicotinimidoyl bromide, NME200504

[0217] ##STR00064##

[0218] In a generally similar manner with non-critical variations was made 6-chloro-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)nicotinimidoyl bromide (421.8 mg, 18.49%) as a yellow oil from the commercially available 6-chloronicotinonitrile in line with the synthesis described in 1.1 to 1.3 but using hydrobromic acid instead of hydrochloric acid in experimental procedure 1.3. LCMS [M+1].sup.+ 376.2. .sup.1H NMR (Chloroform-d, 500 MHz): ? (ppm) 8.82 (s, 1H), 8.07 (d, J=10.6 Hz, 1H), 7.36 (d, J=8.4 Hz, 1H), 4.48-4.27 (m, 2H), 4.13-4.04 (m, 1H), 2.74-2.54 (m, 2H), 2.51-2.24 (m, 4H), 1.64-1.52 (m, 4H), 1.49-1.36 (m, 2H).

Example 13

Synthesis of 3-(dimethylamino)-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)propanimidoyl bromide, NME200532

[0219] ##STR00065##

[0220] In a generally similar manner with non-critical variations was made 3-(dimethylamino)-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)propanimidoyl bromide (123.6 mg, 16.69%) as a yellow oil from the commercially available 3-(dimethylamino)propanenitrile in line with the synthesis described in 1.1 to 1.3 but using hydrobromic acid instead of hydrochloric acid in experimental procedure 1.3. LCMS [M+1].sup.+ 337.8. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): ? (ppm) 4.64 (s, 1H), 4.15-4.04 (m, 1H), 4.03-3.93 (m, 1H), 3.89-3.79 (m, 1H), 2.69 (t, J=6.7, 6.7 Hz, 2H), 2.49-2.43 (m, 2H), 2.40-2.20 (m, 6H), 2.14 (s, 6H), 1.54-1.42 (m, 4H), 1.40-1.30 (m, 2H).

Example 14

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(pyridin-3-yl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate, NME200492

[0221] ##STR00066##

[0222] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(pyridin-3-yl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate (116.4 mg, 17.22%) as a yellow oil from the commercially available tert-butyl 4-cyano-4-(pyridin-3-yl)piperidine-1-carboxylate in line with the synthesis described in 1.1 to 1.3. LCMS [M?.Math.Cl.sup.?].sup.+ 345.4. .sup.1H NMR (Methanol-d.sub.4, 400 MHz): ? (ppm) 8.82 (s, 1H), 8.72 (d, J=4.2 Hz, 1H), 8.32 (d, J=8.1 Hz, 1H), 7.82 (dd, J=8.0, 5.3 Hz, 1H), 4.40-4.28 (m, 2H), 3.64-3.52 (m, 2H), 3.43-3.26 (m, 8H), 3.25-3.13 (m, 2H), 3.07-2.93 (m, 2H), 2.85 (d, J=14.9 Hz, 2H), 2.42 (t, J=12.8, 12.8 Hz, 2H), 1.96-1.87 (m, 2H), 1.85-1.75 (m, 2H), 1.59-1.47 (m, 1H).

Example 15

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-((6-methylpyridin-3-yl)methyl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate, NME200493

[0223] ##STR00067##

[0224] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-((6-methylpyridin-3-yl)methyl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate (108.2 mg, 27.64%) as a pale brown oil in line with the synthesis described in 3.1-3.3, but using 5-(bromomethyl)-2-methylpyridine hydrobromide instead of 3-(bromomethyl)-pyridine hydrobromide in experimental procedure 1.4. LCMS [M+1].sup.+ 410.2. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 8.26 (s, 1H), 8.06 (d, J=9.6 Hz, 1H), 7.68 (d, J=8.3 Hz, 1H), 4.18-4.06 (m, 1H), 4.02-3.86 (m, 2H), 3.40 (t, J=11.0, 11.0 Hz, 2H), 3.24 (d, J=13.2 Hz, 2H), 3.10-2.83 (m, 7H), 2.79 (t, J=12.0, 12.0 Hz, 1H), 2.60 (s, 3H), 2.22 (d, J=14.5 Hz, 2H), 1.91-1.70 (m, 4H), 1.73-1.58 (m, 3H), 1.41-1.29 (m, 1H).

Example 16

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-((6-methoxy-2-methylpyridin-3-yl)methyl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate, NME200524

[0225] ##STR00068##

[0226] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-((6-methoxy-2-methylpyridin-3-yl)methyl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate (102.5 mg, 18.89%) as a pale brown oil in line with the synthesis described in 1.1-1.3, but using 3-(chloromethyl)-6-methoxy-2-methylpyridine instead of 3-(bromomethyl)-pyridine hydrobromide in experimental procedure 1.4. LCMS [M?.Math.Cl.sup.?].sup.+ 403.2. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 7.95 (d, J=9.0 Hz, 1H), 7.13 (d, J=9.0 Hz, 1H), 4.20-4.12 (m, 1H), 4.07-3.90 (m, 5H), 3.40 (t, J=11.3, 11.3 Hz, 2H), 3.23 (d, J=13.0 Hz, 2H), 3.10-2.98 (m, 2H), 2.96-2.74 (m, 6H), 2.42 (s, 3H), 2.27 (d, J=14.7 Hz, 2H), 1.85-1.73 (m, 4H), 1.70-1.58 (m, 3H), 1.39-1.26 (m, 1H).

Example 17

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-((6-methoxy-4-methylpyridin-3-yl)methyl)piperidine-4-carbimidoyl chloride, NME200525

[0227] ##STR00069##

[0228] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-((6-methoxy-4-methylpyridin-3-yl)methyl)piperidine-4-carbimidoyl chloride (62.6 mg, 11.42%) as a yellow oil in line with the synthesis described in 1.1-1.3, but using 5-(chloromethyl)-2-methoxy-4-methylpyridine instead of 3-(bromomethyl)-pyridine hydrobromide in experimental procedure 1.4. LCMS [M+1].sup.+ 439.2. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 7.77 (s, 1H), 7.22 (s, 1H), 4.23-3.92 (m, 7H), 3.41 (t, J=12.8, 12.8 Hz, 2H), 3.27-3.20 (m, 2H), 3.10-3.01 (m, 2H), 2.94-2.86 (m, 4H), 2.83-2.76 (m, 1H), 2.38 (s, 3H), 2.29 (d, J=14.4 Hz, 2H), 1.89-1.75 (m, 4H), 1.71-1.56 (m, 3H), 1.42-1.28 (m, 1H).

Example 18

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(pyridin-2-yl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate, NME200534

[0229] ##STR00070##

[0230] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-(pyridin-2-yl)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate (115 mg, 12.46%) as a yellow oil from the commercially available tert-butyl 4-cyano-4-(pyridin-2-yl)piperidine-1-carboxylate in line with the synthesis described in 1.1 to 1.3. LCMS [M?.Math.Cl.sup.?].sup.+ 345.2. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 8.59 (d, J=4.9 Hz, 1H), 8.38 (t, J=8.4, 8.4 Hz, 1H), 7.95 (d, J=8.2 Hz, 1H), 7.86-7.78 (m, 1H), 4.34-4.22 (m, 2H), 4.19-4.11 (m, 1H), 3.47-3.21 (m, 6H), 3.15-2.98 (m, 2H), 2.96-2.86 (m, 1H), 2.85-2.75 (m, 3H), 2.36 (t, J=14.8, 14.8 Hz, 2H), 1.86-1.71 (m, 2H), 1.69-1.54 (m, 3H), 1.42-1.29 (m, 1H).

Example 19

Synthesis of 4-(3-chloropyridin-4-yl)-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate, NME200535

[0231] ##STR00071##

[0232] In a generally similar manner with non-critical variations was made 4-(3-chloropyridin-4-yl)-N-(2-hydroxy-3-(piperidin-1-yl)propoxy)piperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate (65 mg, 13.72%) as a yellow oil from the starting material tert-butyl 4-(3-chloropyridin-4-yl)-4-cyanopiperidine-1-carboxylate in line with the synthesis described in 1.1 to 1.3. The synthesis of the starting material described above. LCMS [M+1].sup.+ 416.0. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 8.59 (s, 1H), 8.52 (d, J=5.3 Hz, 1H), 7.72 (d, 1H), 4.33-4.19 (m, 3H), 3.51-3.26 (m, 6H), 3.20-3.02 (m, 2H), 2.95-2.76 (m, 4H), 2.41-2.25 (m, 2H), 1.87-1.75 (m, 2H), 1.72-1.56 (m, 3H), 1.46-1.30 (m, 1H).

Example 20

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-3-(pyrrolidin-1-yl)propanimidoyl chloride, NME200562

[0233] ##STR00072##

[0234] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-3-(pyrrolidin-1-yl)propanimidoyl chloride (168.1 mg, 31.57%) as a yellow oil from the commercially available 3-(pyrrolidin-1-yl)propanenitrile in line with the synthesis described in 1.1 to 1.3. LCMS [M+1].sup.+ 318.2. .sup.1H NMR (Methanol-d.sub.4, 400 MHz): ? (ppm) 4.07-4.01 (m, 2H), 3.32-3.25 (m, 3H), 2.84-2.65 (m, 4H), 2.61-2.56 (m, 3H), 2.53-2.41 (m, 5H), 1.92-1.71 (m, 4H), 1.64-1.55 (m, 4H), 1.50-1.41 (m, 2H).

Example 21

Synthesis of N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-3-(piperidin-1-yl)propanimidoyl chloride, NME200563

[0235] ##STR00073##

[0236] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-3-(piperidin-1-yl)propanimidoyl chloride (119.3 mg, 23.71%) as a yellow oil from the commercially available 3-(piperidin-1-yl)propanenitrile in line with the synthesis described in 1.1 to 1.3. LCMS [M+1].sup.+ 332.2. .sup.1H NMR (Methanol-da, 400 MHz): ? (ppm) 4.14-3.95 (m, 3H), 3.30 (s, 2H), 2.75-2.60 (m, 4H), 2.51-2.37 (m, 8H), 1.68-1.52 (m, 8H), 1.50-1.36 (m, 4H).

Example 22

Synthesis of 6-chloro-N-(2-hydroxy-2-methyl-3-(piperidin-1-yl)propoxy)nicotinimidoyl chloride, NME200502

[0237] ##STR00074##

[0238] In a generally similar manner with non-critical variations was made 6-chloro-N-(2-hydroxy-2-methyl-3-(piperidin-1-yl)propoxy)nicotinimidoyl chloride (433.3 mg, 38.85%) as a yellow oil from the commercially available 6-chloronicotinonitrile in line with the synthesis described in 1.1 to 1.3 but using 2-hydroxy-2-methyl-4-azaspiro[3.5]nonan-4-ium chloride described above instead of 2-hydroxy-4-azaspiro[3.5]nonan-4-ium chloride in experimental procedure 1.2. LCMS [M+1].sup.+ 346.2. .sup.1H NMR (Chloroform-d, 400 MHz): ? (ppm) 8.80 (s, 1H), 8.05 (d, 1H), 7.33 (d, J=8.4 Hz, 1H), 4.20-4.11 (m, 2H), 3.75 (s, 1H), 2.61-2.47 (m, 5H), 2.24 (d, J=13.9 Hz, 1H), 1.58-1.50 (m, 4H), 1.45-1.35 (m, 2H), 1.20 (s, 3H).

Example 23

Synthesis of tert-butyl (3-chloro-3-((2-hydroxy-3-(piperidin-1-yl)propoxy)imino)propyl)(methyl)carbamate, NME200599 General scheme

[0239] ##STR00075##

[0240] 3.4 Synthesis of tert-butyl (3-chloro-3-((2-hydroxy-3-(piperidin-1-yl)propoxy)imino)propyl)(methyl)carbamate

##STR00076##

[0241] tert-Butyl N-[3-amino-3-[2-hydroxy-3-(1-piperidyl)propoxy]imino-propyl]-N-methyl-carbamate, obtained in a similar manner with non-critical variations from commercially available tert-butyl (2-cyanoethyl)(methyl)carbamate in line with the synthesis described in 1.1 to 1.2 (2 g, 3.12 mmol, 1 eq) was dissolved in acetonitrile (50 ml), after that tert-butyl nitrite (0.97 g, 9.37 mmol, 3 eq) was added to the resulting solution, followed by the addition of CuCl.sub.2 (1.26 g, 9.37 mmol, 3 eq). The reaction mixture was left while stirring at room temperature for 2 days in the dark. After 48 hours the reaction mixture was concentrated under reduced pressure, diluted with 4.0 M aqueous solution of sodium carbonate (50 ml), and extracted with ethyl acetate (2?30 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate collected was concentrated under reduced pressure to afford crude yellow oil (1 g), which was subjected to prep HPLC purification to give the title product (95 mg, 7.6%) as a pink oil. LCMS [M+1].sup.+ 378.4. .sup.1H NMR (Chloroform-d, 400 MHz): ? (ppm) 4.20-4.02 (m, 2H), 4.01-3.88 (m, 1H), 3.58-3.31 (m, 2H), 2.84 (s, 3H), 2.74-2.47 (m, 4H), 2.43-2.27 (m, 4H), 1.71-1.45 (m, 6H), 1.43 (s, 9H).

1. Approach Through Usage of Nosyl Epoxide

Example 1

Synthesis of N-(3-(4-fluoropiperidin-1-yl)-2-hydroxypropoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate, NME200399 General Scheme

[0242] ##STR00077##

1.1 Synthesis of tert-butyl 4-(N-(3-(4-fluoropiperidin-1-yl)-2-hydroxypropoxy)carbamimidoyl)-4-methylpiperidine-1-carboxylate

[0243] ##STR00078##

[0244] A suspension of tert-Butyl 4-(N-hydroxycarbamimidoyl)-4-methylpiperidine-1-carboxylate (2 g, 7.77 mmol, 1 eq) in dry DMF (5 ml) was cooled down to 0? C. Sodium hydride in mineral oil, 60% (0.311 g, 7.77 mmol, 1 eq) was added, and the the reaction mixture was stirred at 0? C. for 30 minutes. Then oxiran-2-ylmethyl 3-nitrobenzenesulfonate (2.015 g, 7.77 mmol, 1 eq) dissolved in dry DMF (5 ml) was added the mixture was allowed to warm up to room temperature and stirred for additional 2 hours. 4-Fluoropiperidine hydrochloride (1.085 g, 7.77 mmol, 1 eq) and N,N-diethylethanamine (0.787 g, 7.77 mmol, 1 eq) in dry DMF (5 ml) was added to the reaction mixture in a drop-wise manner. The reaction mass was heated up to 60? C. and left while stirring for 48 hours after which the it was concentrated under reduced pressure, diluted with distilled water (20 ml) and extracted with DCM (3?15 ml). The organic layers were combined and washed saturated solution of sodium hydrocarbonate (aq, 2?15 ml), dried over anhydrous sodium sulfate, filtered. The collected was concentrated under reduced pressure to afford brown oil (3.5 g). The material obtained was subjected for flash chromatography purification (Companion combiflash, 80 g SiO.sub.2, acetonitrile/methanol with methanol from 0-25%, flow rate=60 mL/min) to afford the title product (1.515 g, 44.5%) as pale brown oil of satisfactorily. LCMS [M+1].sup.+ 417.2.

1.2 Synthesis of N-(3-(4-fluoropiperidin-1-yl)-2-hydroxypropoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate

[0245] ##STR00079##

[0246] tert-Butyl 4-[N-[3-(4-fluoro-1-piperidyl)-2-hydroxy-propoxy]carbamimidoyl]-4-methyl-piperidine-1-carboxylate (500 mg, 1.14 mmol, 1 eq) was dissolved in distilled water (2 ml) and acetic acid (1 ml). The resulting solution was cooled down to 0? C. and aqueous hydrochloric acid, 30% (0.603 ml, 692.97 mg, 5.7 mmol, 5 eq) was added drop-wise to the reaction mixture, followed by a slow addition a solution of sodium nitrite (157.37 mg, 2.28 mmol, 2 eq) in distilled water (1 ml). The reaction mixture was left while stirring at 0? C. for 2 hours, and then allowed to warm up to room temperature. Aqueous hydrochloric acid, 30% (0.603 ml, 692.97 mg, 5.7 mmol, 5 eq) was added to the reaction mixture after 6 hours and then left while stirring over night at room temperature and reduced in volume and subjected for prep HPLC to afford the title product (290.7 mg, 42.95%) as a yellow oil. LCMS [M?.Math.Cl.sup.?].sup.+ 301.0. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): ? (ppm) 9.54 (s, 1H), 8.75 (s, 1H), 8.61 (s, 1H), 5.13-4.68 (m, 1H), 4.31-4.17 (m, 1H), 4.16-4.04 (m, 2H), 3.61-3.34 (m, 2H), 3.34-3.01 (m, 7H), 3.01-2.87 (m, 2H), 2.22-1.90 (m, 6H), 1.75 (t, J=13.3, 13.3 Hz, 2H), 1.24 (s, 3H).

Example 2

Synthesis of N-(2-hydroxy-3-(4-methylpiperazin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate, NME200402

[0247] ##STR00080##

[0248] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(4-methylpiperazin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride tri-2,2,2-trifluoroacetate (420 mg, 51.47%) as a yellow oil from the commercially available tert-butyl 4-(N-hydroxycarbamimidoyl)-4-methylpiperidine-1-carboxylate in line with the synthesis described in 4.1 to 4.2 but using 1-methylpiperazine instead of 4-fluoropiperidine hydrochloride in experimental procedure 4.1. LCMS [M?.Math.Cl.sup.?].sup.+ 298.0. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): ? (ppm) 8.80 (s, 1H), 8.67 (s, 1H), 4.25-4.01 (m, 3H), 3.69-3.34 (m, 5H), 3.29-3.12 (m, 4H), 3.13-3.06 (m, 1H), 3.06-2.84 (m, 4H), 2.55-2.49 (m, 6H), 2.22-2.07 (m, 2H), 1.83-1.62 (m, 2H), 1.24 (s, 3H).

Example 3

Synthesis of N-(3-(3-azabicyclo[3.1.0]hexan-3-yl)-2-hydroxypropoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate, NME200403

[0249] ##STR00081##

[0250] In a generally similar manner with non-critical variations was made N-(3-(3-azabicyclo[3.1.0]hexan-3-yl)-2-hydroxypropoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate (89.5 mg, 15.50%) as a yellow oil from the commercially available tert-butyl 4-(N-hydroxycarbamimidoyl)-4-methylpiperidine-1-carboxylate in line with the synthesis described in 2.1 to 2.2 but using 3-azabicyclo[3.1.0]hexane hydrochloride instead of 4-fluoropiperidine hydrochloride in experimental procedure 2.1. LCMS [M?.Math.Cl.sup.?].sup.+ 280.0. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): ? (ppm) 9.59 (s, 1H), 8.76 (s, 1H), 8.64 (s, 1H), 4.19-3.84 (m, 4H), 3.65-3.55 (m, 2H), 3.44-3.32 (m, 2H), 3.27-3.21 (m, 1H), 3.05-2.84 (m, 3H), 2.15-2.02 (m, 2H), 1.90-1.67 (m, 5H), 1.24 (s, 4H), 0.88-0.77 (m, 1H), 0.72-0.59 (m, 1H).

Example 4

Synthesis of N-(3-(2-azabicyclo[2.2.1]heptan-2-yl)-2-hydroxypropoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate, NME200410

[0251] ##STR00082##

[0252] In a generally similar manner with non-critical variations was made N-(3-(2-azabicyclo[2.2.1]heptan-2-yl)-2-hydroxypropoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate (104 mg, 19.97%) as a pale brown oil from the commercially available tert-butyl 4-(N-hydroxycarbamimidoyl)-4-methylpiperidine-1-carboxylate in line with the synthesis described in 2.1 to 2.2 but using 2-azabicyclo[2.2.1]heptane hydrochloride instead of 4-fluoropiperidine hydrochloride in experimental procedure 2.1. LCMS [M?.Math.Cl.sup.?].sup.+ 294.4. .sup.1H NMR (Methanol-d.sub.4, 400 MHz): ? (ppm) 4.32-4.09 (m, 4H), 3.59-3.23 (m, 7H), 3.21-2.97 (m, 4H), 2.88-2.72 (m, 1H), 2.34 (d, J=14.4 Hz, 2H), 2.05-1.97 (m, 1H), 1.86-1.68 (m, 5H), 1.62-1.49 (m, 1H), 1.31 (s, 3H).

Example 5

Synthesis of (S)N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2-trifluoroacetate, NME200384

[0253] ##STR00083##

[0254] In a generally similar manner with non-critical variations was made (S)N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate (93 mg, 20.57%) as a yellow oil from the commercially available tert-butyl 4-(N-hydroxycarbamimidoyl)-4-methylpiperidine-1-carboxylate in line with the synthesis described in 2.1 to 2.2 but using piperidine instead of 4-fluoropiperidine hydrochloride and using [(2S)-oxiran-2-yl]methyl 3-nitrobenzenesulfonate instead of oxiran-2-ylmethyl 3-nitrobenzenesulfonate in experimental procedure 4.1. LCMS [M?.Math.Cl.sup.?].sup.+ 282.0. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 4.32-4.23 (m, 1H), 4.17-4.03 (m, 2H), 3.51-3.37 (m, 2H), 3.22-3.11 (m, 3H), 3.09-2.97 (m, 3H), 2.95-2.86 (m, 1H), 2.82 (t, J=12.7, 12.7 Hz, 1H), 2.23 (d, J=15.2 Hz, 2H), 1.86-1.74 (m, 2H), 1.74-1.55 (m, 5H), 1.43-1.28 (m, 1H), 1.18 (s, 3H)

Example 6

Synthesis of (R)N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate, NME200387

[0255] ##STR00084##

[0256] In a generally similar manner with non-critical variations was made (R)N-(2-hydroxy-3-(piperidin-1-yl)propoxy)-4-methylpiperidine-4-carbimidoyl chloride di-2,2,2-trifluoroacetate (450 mg, 82.13%) as a yellow oil from the commercially available tert-butyl 4-(N-hydroxycarbamimidoyl)-4-methylpiperidine-1-carboxylate in line with the synthesis described in 2.1 to 2.2 but using piperidine instead of 4-fluoropiperidine hydrochloride and using [(2R)-oxiran-2-yl]methyl 3-nitrobenzenesulfonate instead of oxiran-2-ylmethyl 3-nitrobenzenesulfonate in experimental procedure 2.1. LCMS [M?.Math.Cl.sup.?].sup.+ 282.0. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): ? (ppm) 9.35 (s, 1H), 8.86 (s, 1H), 8.68 (s, 1H), 4.26-4.19 (m, 1H), 4.16-4.05 (m, 2H), 3.47-3.38 (m, 2H), 3.26-2.73 (m, 9H), 2.11 (d, J=15.0 Hz, 2H), 1.80-1.58 (m, 7H), 1.43-1.34 (m, 1H), 1.23 (s, 3H)

Example 7

Synthesis of N-(2-hydroxy-3-(4-methylpiperazin-1-yl)propoxy)-4-(pyridin-3-ylmethyl)piperidine-4-carbimidoyl chloride tetra-2,2,2-trifluoroacetate, NME200513

[0257] ##STR00085##

[0258] In a generally similar manner with non-critical variations was made N-(2-hydroxy-3-(4-methylpiperazin-1-yl)propoxy)-4-(pyridin-3-ylmethyl)piperidine-4-carbimidoyl chloride tetra-2,2,2-trifluoroacetate (344 mg, 23.14%) as a yellow oil from the commercially available tert-butyl 4-cyanopiperidine-1-carboxylate in line with the synthesis described in 1.4, 1.1, 2.1-2.2, but using 3-(bromomethyl)pyridine hydrobromide instead of 3-(bromomethyl)-5-fluoro-pyridine hydrobromide in experimental procedure 1.4 and using 1-methylpiperazine instead of 4-fluoropiperidine hydrochloride in experimental procedure 2.1. LCMS [M+1].sup.+ 410.2. .sup.1H NMR (Deuterium Oxide, 400 MHz): ? (ppm) 8.57 (d, J=5.5 Hz, 1H), 8.45 (s, 1H), 8.23 (d, J=8.0 Hz, 1H), 7.87 (t, 1H), 4.23-4.13 (m, 1H), 3.99-3.93 (m, 1H), 3.92-3.87 (m, 1H), 3.83-3.57 (m, 4H), 3.54-3.29 (m, 4H), 3.26-3.18 (m, 4H), 3.08 (s, 2H), 2.95-2.86 (m, 5H), 2.22 (d, J=14.2 Hz, 2H), 1.81 (t, J=13.7, 13.7 Hz, 2H).

Example 2: Determination of Potencies and Efficacies of Oximes Using GCase Assay

Materials

[0259] Human fibroblast cell line GM10915 harboring the L444P GBA mutation was obtained from Coriell Biorepositories. All chemicals (Glacial acetic acid, Glycine, 4-Methylumbelliferyl b-D-glucopyranoside (4-MUG), Sodium acetate trihydrate, Sodium hydroxide, Crystal violet, SDS, Ammonium hydroxide) were obtained from Sigma-Aldrich (Denmark). Compounds tested for GCase activity were dissolved in H.sub.2O or DMSO.

Methods

[0260] The GM10915 cell line was cultured under standard cell culture conditions (37? C. and 5% CO.sub.2) in complete DMEM medium supplemented with nonessential amino acids (NEAA), 1% Pen-Strep and 12% FCS. Cells were seeded at a density of 10.sup.4 cells/well in 100 ?L complete medium in one black 96-well plate for glucosylceramidase (GCase) activity measurement and in one clear 96-well plate for crystal violet staining to correct for cell density. Crystal violet staining is performed to obtain quantitative information about the relative density of cells adhering to multi-wells plates.

Assay of GCase Activity

[0261] The assay was adapted from Sawkar et al (2002) and briefly described in the following. The day after seeding of cells, the medium was replaced with fresh medium containing the compounds to be tested. Compounds were tested in duplicate and in an 8-point diluted dose range to obtain a dose response. Cells were exposed with compounds for five days. Fresh compound was added every 2-3 days. PBS was included to define the basal level of GCase activity.

[0262] Cells were washed three times with 200 ?L PBS per well and 50 ?L of 2.5 mM 4-MUG buffer (4-MUG dissolved in 0.2 M acetate buffer pH 4.0) was added and the cells were incubated at 37? C., 5% CO.sub.2 for 23 hours. The reaction was stopped by adding 150 ?L 0.2 M glycine buffer (pH 10.8). Fluorescence was measured with a Varioskan? Flash reader (Thermo Scientific) at an excitation/emission setting of 365/445 nm.

Crystal Violet Staining

[0263] Cells were treated with compounds in a parallel setup identical to the setup to test for GCase activity. At the end of compound treatment, cells were washed once with 200 ?L PBS per well and 50 ?L 0.1% w/v crystal violet (in H.sub.2O) was added. Following 10 min. of incubation, the crystal violet solution was removed, and the cells were washed three times with 200 ?L PBS and 100 ?L 1% SDS was added to solubilize the stain. The plate was agitated on an orbital shaker for 10-30 min. Absorbance (A) is measured at 570 nM using a Varioskan? Flash reader (Thermo Scientific).

Calculations

[0264] The fluorescence signal (F) derived from the GCase measurement is normalized to the absorbance signal (A) derived from the crystal violet staining. The percent GCase activity resulting from compound treatment is calculated relative to the basal activity obtained from untreated cells.

[00001]$\mathrm{Percent}\mathrm{GCase}\mathrm{activity}=100*\frac{{\left(F/A\right)}_{\mathrm{compound}}}{{\left(F/A\right)}_{\mathrm{untreated}}}$

[0265] The potency, EC.sub.1.5, is determined based on the dose response effects of the compounds as the concentration where Percent GCase activity=150% corresponding to at 1.5-fold induction of GCase activity. Maximal effect of compounds (Emax) is determined from the dose response effects as the maximum Percent GCase activity achieved in the dose range tested.

Results

[0266] The GBA potencies and Emax were determined as described above in the present example and the results are shown in Table 1 below.

TABLE-US-00002 ID Structure GBA EC.sub.1.5 (?M) GBA Emax (%) Ambroxol reference [00086] >20* *The 1.5-fold induction criteria was not reached within the tested concentration range 127 LTI-291 reference [00087] 112 232 524 (II) [00088] 1 149 525 (II) [00089] 3 168 563 (I) [00090] 3 211 562 (I) [00091] 3 239 446 (I) [00092] 6 176 534 (II) [00093] 19 226 504 (III) [00094] 20 310 419 (II) [00095] 12 387 452 (III) [00096] 18 572 466 (III) [00097] 18 221 493 (II) [00098] 18 279 501 (III) [00099] 19 128 415 (II) [00100] 23 235 502 (III) [00101] 23 131 475 (III) [00102] 24 354 409 (II) [00103] 25 255 550 (II) [00104] 27 159 532 (I) [00105] 30 239 384 (II) [00106] 32 352 448 (II) [00107] 34 468 399 (II) [00108] 34 314 257 (III) [00109] 35 307 387 (II) [00110] 35 379 535 (II) [00111] 36 186 336 (II) [00112] 37 320 453 (III) [00113] 39 202 492 (II) [00114] 42 383 410 (II) [00115] 43 344 352 (III) [00116] 45 474 513 (II) [00117] 35 174 403 (II) [00118] 48 264 402 (II) [00119] 50 408 503 (III) [00120] 50 142 512 (II) [00121] 50 147 593 (I) [00122] 50 136 599 (I) [00123] 48 136

CONCLUSION

[0267] This example demonstrates that the oximes of the present disclosure are highly potent and efficacious in comparison with state-of-the-art GBA inducers like Ambroxol and LTI-291. These effects render the oximes of the present disclosure promising candidates for treatment of GBA-mediated disorders.