AMINOTHIOLESTER COMPOUNDS AND USES THEREOF

Abstract

The present invention relates to novel aminoesters compounds or its pharmaceutically acceptable salts or optical isomers, racemates, diastereoisomers, enantiomers or tautomers. The present invention also relates to their process of preparation and to these compounds for use as a medicament, in particular for the treatment or the prevention of cancer. The present invention further relates to an antibody drug conjugate comprising such compounds.

Claims

1. A compound of formula (I): ##STR00039## in which: X is an atom chosen from O or S; R1 and R2 identical or different are independently chosen from: linear or branched (C.sub.1-C.sub.7)alkyl, linear or branched (C.sub.2-C.sub.7)alkenyl, aryl, heteroaryl, CHR.sub.5CHR.sub.6OR.sub.4 and (CHR.sub.5), OR.sub.4, said aryl and heteroaryl being optionally substituted by one or more substituents chosen from: linear or branched (C.sub.1-C.sub.7)alkyl, halogen, NO.sub.2 and CONH.sub.2; v is chosen from 2 to 4; R.sub.3 is chosen from linear or branched (C.sub.1-C.sub.7)alkyl, (C.sub.1-C.sub.7)alkyl —CO.sub.2Z and linear or branched (C.sub.1-C.sub.7)alkyl-NY.sub.1Y.sub.2; said linear or branched (C.sub.1-C.sub.7)alkyl-NY.sub.1Y.sub.2 being optionally substituted by (C.sub.1-C.sub.7)alkyl —CO.sub.2Z; R.sub.4 is chosen from: H, linear or branched (C.sub.2-C.sub.7)alkyl, linear or branched (C.sub.2-C.sub.7)alkenyl, —CONR.sub.7R.sub.8, aryl, heteroaryl, (C.sub.2-C.sub.7)cycloalkyl, linear or branched —(C.sub.1-C.sub.7)alkyl-aryl and linear or branched —(C.sub.1-C.sub.7)alkyl-heteroaryl; said aryl, (C.sub.2-C.sub.7)cycloalkyl, and heteroaryl being optionally substituted by one or more substituents chosen from: halogen, linear or branched (C.sub.1-C.sub.7)alkyl optionally substituted by one or more halogen atom, linear or branched (C.sub.1-C.sub.7)alkoxy optionally substituted by one or more halogen atom, —COOH, aryl, —NRR′, —NO.sub.2, or said aryl and heteroaryl being optionally fused to form an heterocycloalkyl; R.sub.5 and R.sub.6 identical or different are independently chosen from: H and linear or branched (C.sub.1-C.sub.7)alkyl, or R.sub.5 and R.sub.6 are linked together to form with the carbon atoms to which they are attached a cycloalkyl, aryl or heteroaryl, or R.sub.5 is H and R1 and R.sub.6 are linked together to form with the nitrogen atom linked to R1 an heterocycloalkyl or heteroaryl, or R.sub.6 is H and R1 and R.sub.5 are linked together to R1 to form with the nitrogen atom linked to R1 an heterocycloalkyl; R.sub.7 is —(C.sub.1-C.sub.3)alkyl; R8 is —(C.sub.1-C.sub.3)alkylNRR′; R and R′ identical or different, are independently chosen from H and linear or branched (C.sub.1-C.sub.7)alkyl, Y.sub.1 and Y.sub.2 identical or different are independently chosen from H and —CO—(C.sub.1-C.sub.7)alkyl; Z is chosen from H and linear or branched (C.sub.1-C.sub.7)alkyl; and in which, at least one of R1 and R2 is CHR.sub.5CHR.sub.6OR.sub.4 or (CHR.sub.5), OR.sub.4 when X is S and R3 is linear or branched (C.sub.1-C.sub.7)alkyl; or its pharmaceutically acceptable salts or optical isomers, racemates, diastereoisomers, enantiomers or tautomers.

2. A compound according to claim 1 in which X is S, R3 is linear or branched (C.sub.1-C.sub.7)alkyl, preferably methyl, R1 is linear or branched (C.sub.1-C.sub.7)alkyl, preferably methyl, R2 is CHR.sub.5CHR.sub.6OR.sub.4 or (CHR.sub.5).sub.vOR.sub.4 and R5 and R6 are: H, or R.sub.5 is H and R1 and R.sub.6 are linked together to form with the nitrogen atom linked to R1 an heterocycloalkyl, preferably pyrrolidinyl, or R.sub.6 is H and R1 and R.sub.5 are linked together to R1 to form with the nitrogen atom linked to R1 an heterocycloalkyl, preferably pyrrolidinyl.

3. A compound according to claim 1 in which X is S, R1 is linear or branched (C.sub.1-C.sub.7)alkyl, R3 is linear or branched (C.sub.1-C.sub.7)alkyl and R2 is CHR.sub.5CHR.sub.6OR.sub.4 or (CHR.sub.5).sub.vOR.sub.4.

4. A compound according to claim 2, in which R.sub.4 is chosen from linear or branched (C.sub.2-C.sub.7)alkyl, linear or branched (C.sub.2-C.sub.7)alkenyl, —CONR.sub.7R.sub.8, (C.sub.2-C.sub.7)cycloalkyl, linear or branched —(C.sub.1-C.sub.7)alkyl-heteroaryl, aryl, or benzyl; said (C.sub.2-C.sub.7) cycloalkyl being substituted by one or more substituents chosen from: linear or branched (C.sub.1-C.sub.7)alkyl, said benzyl being optionally substituted by one or more substituents chosen from: linear or branched (C.sub.1-C.sub.7)alkyl optionally substituted by one or more halogen atom, linear or branched (C.sub.1-C.sub.7)alkoxy optionally substituted by one or more halogen atom, halogen or said benzyl being optionally fused to form 1,3-benzodioxole.

5. A compound according to claim 2, in which R.sub.5 and R.sub.6 are H and R.sub.4 is chosen from linear or branched (C.sub.2-C.sub.7)alkyl, linear or branched (C.sub.2-C.sub.7)alkenyl, —CONR.sub.7R.sub.8, (C.sub.2-C.sub.7) cycloalkyl, linear or branched —(C.sub.1-C.sub.7)alkyl-heteroaryl, or benzyl; said (C.sub.2-C.sub.7) cycloalkyl being substituted by one or more substituents chosen from: linear or branched (C.sub.1-C.sub.7)alkyl, said benzyl being optionally substituted by one or more substituents chosen from: linear or branched (C.sub.1-C.sub.7)alkyl optionally substituted by one or more halogen atom, linear or branched (C.sub.1-C.sub.7)alkoxy optionally substituted by one or more halogen atom, halogen.

6. A compound according to claim 5, in which R1 is methyl and R.sub.4 is chosen from: —CONR.sub.7R.sub.8 with R.sub.7 being a methyl and R.sub.8 being NRR′ with R and R′ being methyl, ethyl, propenyl, benzyl, pyridyl, benzyloxybutyl, methyl-cyclohexenyl substituted by one or more methyl, and benzyl substituted by one of more fluorine, chlorine, methoxy or methyl.

7. A compound according to claim 1 in which X is S, R1 and R2 are linear or branched (C.sub.1-C.sub.7)alkyl and R3 is —(C.sub.1-C.sub.7)—CO.sub.2Z or linear or branched (C.sub.1-C.sub.7)alkyl-NY.sub.1Y.sub.2, said linear or branched (C.sub.1-C.sub.7)alkyl-NY.sub.1Y.sub.2 being optionally substituted by —(C.sub.1-C.sub.7)—CO.sub.2Z.

8. The compound according to claim 1, chosen in the group consisting of: S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate; S-methyl 4-[2-allyloxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate; S-methyl 4-[2-benzyloxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate; S-methyl 4-methyl-4-[methyl-[2-(m-tolylmethoxy)ethyl]amino]pent-2-ynethioate; S-methyl 4-[2-[(3,4-dimethylphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate; S-methyl 4-[2-[(4-methoxyphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate; S-methyl 4-[2-[(3,4-dimethoxyphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate; S-methyl 4-[2-[(3-chlorophenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate; S-methyl 4-[2-[(3-fluorophenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate; S-methyl 4-methyl-4-[methyl-[2-(2-pyridylmethoxy)ethyl]amino]pent-2-ynethioate; S-methyl 4-methyl-4-[methyl-[2-(3-pyridylmethoxy)ethyl]amino]pent-2-ynethioate; S-methyl 4-methyl-4-[methyl-[2-(4-pyridylmethoxy)ethyl]amino]pent-2-ynethioate; methyl 4-(dimethylamino)-4-methyl-pent-2-ynoate; ethyl 4-(dimethylamino)-4-methyl-pent-2-ynoate; tert-butyl 2-((4-(dimethylamino)-4-methylpent-2-ynoyl)thio)acetate; 2-((4-(dimethylamino)-4-methylpent-2-ynoyl)thio)acetic acid; S-methyl 4-((4-(benzyloxy)butyl)(methyl)amino)-4-methylpent-2-ynethioate; S-methyl 4-((2-hydroxyethyl)(methyl)amino)-4-methylpent-2-ynethioate; S-methyl 4-methyl-4-[methyl-[2-(2-naphthylmethoxy)ethyl]amino]pent-2-ynethioate; S-methyl 4-methyl-4-[methyl-[2-[(2,6,6-trimethylcyclohexen-1-yl)methoxy]ethyl]amino]pent-2-ynethioate; 2-[(1,1-dimethyl-4-methylsulfanyl-4-oxo-but-2-ynyl)-methylamino] ethyl-3,4-dimethoxybenzoate; 2[(1,1-dimethyl-4-methylsulfanyl-4-oxo-but-2-ynyl)-methylamino] ethyl acetate; S-methyl 2,5,10,11,11-pentamethyl-6-oxo-7-oxa-2,5,10-triazatetradec-12-yne-14-thioate; S-methyl 4-[2-(methoxymethyl)pyrrolidin-1-yl]-4-methylpent-2-ynethioate; S-methyl 4-(3-methoxypyrrolidin-1-yl)-4-methylpent-2-ynethioate; S-methyl 4-methyl-4-[methyl(2-phenoxycyclopentyl)amino]pent-2-ynethioate; (S)—S-methyl 4-(2-((benzyloxy)methyl)pyrrolidin-1-yl)-4-methylpent-2-ynethioate; S-methyl 4-[3(benzyloxy)-1pyrrolidinyl])-4-methylpent-2-ynethioate or its pharmaceutically acceptable salts or optical isomers, racemates, diastereoisomers, enantiomers or tautomers.

9. A compound according to claim 1, in which: X is S; R1 is linear or branched (C.sub.1-C.sub.7)alkyl; R2 is CHR.sub.5CHR.sub.6OR.sub.4 or (CHR.sub.5).sub.vOR.sub.4; R4 is chosen from H, aryl, heteroaryl, linear or branched —(C.sub.1-C.sub.7)alkyl-aryl and linear or branched —(C.sub.1-C.sub.7)alkyl-heteroaryl; said aryl and heteroaryl being optionally substituted by one or more substituents chosen from: —COOH, —NRR′ and —NO.sub.2; and R and R′ identical, are H.

10. A process for preparing a compound according to claim 1, comprising: a) reacting a compound of formula (II) with an organic or inorganic acid ##STR00040## b) reacting the compound obtained in step a) with a base; c) reacting the compound obtained in step b) with CO.sub.2; d) reacting the compound obtained in step c) with alkyl chloroformate, a reagent able of forming, with the compound obtained in step c), an acid halide or a reagent able of forming, with the compound obtained in step c), a mixed anhydride; e) reacting the compound obtained in step d) with an anion precursor compound SMe-; wherein R1 and R2 are defined as in claim 1.

11. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutical acceptable excipient.

12. (canceled)

13. A method for the prevention and/or treatment of cancer, comprising the administration to a subject in need thereof of an effective amount of a compound according to claim 1.

14. An antibody drug conjugate of formula: B-L-Ab, wherein: B is a compound of formula (I) as defined in claim 9; L is a linker; and Ab is an antibody.

15. An antibody drug conjugate according to claim 14, wherein the antibody is chosen from: rituximab, trastuzumab, alemtuzumab, ibritumomab, gemtuzumab, tiuxetan, tositumomab, brevacizumab, cetuximab, panitumumab, ofatumumab and obinutuzumab.

16. A method according to claim 12, for the prevention and/or treatment of leukemia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0257] FIG. 1: Mean viability of Raji cells in percentage comparing to the non-treated cells, after a treatment of 50 μg/ml of Rituximab (85.50%±2.268%) and Rituximab (102.9%±1.789%) coupled with compound 5. Difference between the two means was significantly using Unpaired t-test (P<0.01, **).

EXAMPLES

[0258] Representative compounds of the invention are summarized in the table 2 below:

TABLE-US-00002 TABLE 2 Example Structure Name 1 [00004] S-methyl 4-[2- ethoxyethyl)methyl)amino]-4-methyl- pent-2-ynethioate 2 [00005] S-methyl 4-[2- allyloxyethyl(methyl)amino]-4-methyl- pent-2-ynethioate 3 [00006] S-methyl 4-[2- benzyloxyethyl(methyl)amino]-4-methyl- pent-2-ynethioate 4 [00007] S-methyl 4-methyl-4-[methyl-[2-(m- tolylmethoxy)ethyl]amino]pent-2- ynethioate 5 [00008] S-methyl 4-[2-[(3,4- dimethylphenyl)methoxy]ethyl-methyl- amino]-4-methyl-pent-2-ynethioate 6 [00009] S-methyl 4-[2-[(4- methoxyphenyl)methoxy]ethyl-methyl- amino]-4-methyl-pent-2-ynethioate 7 [00010] S-methyl 4-[2-[(3,4- dimethoxyphenyl)methoxy]ethyl-methyl- amino]-4-methyl-pent-2-ynethioate 8 [00011] S-methyl 4-[2-[(3- chlorophenyl)methoxy]ethyl-methyl- amino]-4-methyl-pent-2-ynethioate 9 [00012] S-methyl 4-[2-[(3- fluorophenyl)methoxy]ethyl-methyl- amino]-4-methyl-pent-2-ynethioate 10 [00013] S-methyl 4-methyl-4-[methyl-[2-(2- pyridylmethoxy)ethyl]amino]pent-2- ynethioate 11 [00014] S-methyl 4-methyl-4-[methyl-[2-(3- pyridylmethoxy)ethyl]amino]pent-2- ynethioate 12 [00015] S-methyl 4-methyl-4-[methyl-[2-(4- pyridylmethoxy)ethyl]amino]pent-2- ynethioate 13 [00016] methyl 4-(dimethylamino)-4-methyl- pent-2-ynoate 14 [00017] ethyl 4-(dimethylamino)-4-methyl-pent- 2-ynoate 15 [00018] tert-butyl 2-((4-(dimethylamino)-4- methylpent-2-ynoyl)thio)acetate 16 [00019] 2-((4-(dimethylamino)-4-methylpent-2- ynoyl)thio)acetic acid 17 [00020] S-methyl 4-((4- (benzyloxy)butyl)(methyl)amino)-4- methylpent-2-ynethioate 18 [00021] S-methyl 4-((2- hydroxyethyl)(methyl)amino)-4- methylpent-2-ynethioate 19 [00022] S-methyl 4-methyl-4-[methyl-[2-(2- naphthylmethoxy)ethyl]amino]pent-2- ynethioate 20 [00023] S-methyl 4-methyl-4-[methyl-[2-[(2,6,6- trimethylcyclohexen-1- yl)methoxy]ethyl]amino]pent-2- ynethioate 21 [00024] 2-[(1,1-dimethyl-4-methylsulfanyl-4-oxo- but-2-ynyl)-methylamino] ethyl aetate 22 [00025] 2-[(1,1-dimethyl-4-methylsulfanyl-4-oxo- but-2-ynyl)-methylamino] ethyl-3,4-dimethoxybenzoate 23 [00026] S-methyl 2,5,10,11,11-pentamethyl-6- oxo-7-oxa-2,5,10-triazatetradec-12-yne- 14-thioate 24 [00027] S-methyl 4-[2- (methoxymethyl)pyrrolidin-1-yl]-4- methylpent-2-ynethioate 25 [00028] S-methyl 4-(3-methoxypyrrolidin-1-yl)-4- methylpent-2-ynethioate 26 [00029] S-methyl 4-methyl-4-[methyl(2- phenoxycyclopentyl)amino]pent-2- ynethioate 27 [00030] (S)-S-methyl 4-(2- ((benzyloxy)methyl)pyrrolidin-1-yl)-4- methylpent-2-ynethioate 28 [00031] S-methyl 4-[(3(benzyloxy)- 1pyrrolidinyl)-4-methylpent-2- ynethioate 29 [00032]

[0259] Representative compounds of the invention can be synthesized according to the following procedures.

General Analytical Procedures

[0260] The .sup.1H and .sup.13C NMR spectra were recorded on a Bruker Advance ALS300 and DRX400 MHz from Bruker. Chemical shifts are reported in ppm (δ) and were referenced to DMSO-d6 (.sup.1H, 2.50 ppm; .sup.13C, 39.52 ppm) or CDCl3 (7.26 ppm). The coupling constants (J were given in Hz.

[0261] The HRMS-ESI mass spectra were recorded in positive-ion mode on a hybrid quadrupole time-of-flight mass spectrometer (MicroTOFQ-II, Bruker Daltonics, Bremen) with an Electrospray Ionization (ESI) ion source. For the mass spectrometry of low resolution, LRMS-ESI mass spectra were recorded in a Thermo Finnigan MAT 95 XL spectrometer.

Part 1: Preparation of the Compounds According to the Invention

Example 1: S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate

[0262] Preparation of N-(2-ethoxyethyl)-N,2-dimethyl-but-3-yn-2-amine: To a solution of N-methyl-N-(2′hydroxyethyl)-3-amino-3methyl-1-butyne (Easton, Nelson R.; Hennion, George F. U.S. (1967), U.S. Pat. No. 3,337,625 19670822.) (1.0 g, 7.08 mmol) and iodoethane (0.98 mL, 7.6 mmol) in THF (12 mL) was added NaH (0.459 g, 11.5 mmol) at room temperature and the mixture was refluxed for 3 h. Mixture was then carefully hydrolyzed at room temperature by water and extracted by EtOAc (3×25 mL). Combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude by chromatography on silicagel (petroleum ether/EtOAc=70/30) gave pure N-(2-ethoxyethyl)-N,2-dimethyl-but-3-yn-2-amine (0.479 g, 40%).

[0263] .sup.1H NMR (300 MHz, DMSO) δ 3.45-3.36 (m, 4H), 3.11 (s, 1H), 2.51 (t, J=6.7 Hz. 2H), 2.20 (s, 3H), 1.27 (s, 6H), 1.09 (t, J=7.0 Hz, 3H).

[0264] ESI-LRMS 170.0 [M+H]+.

[0265] Preparation of S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate: To N-(2-ethoxyethyl)-N,2-dimethyl-but-3-yn-2-amine (0.367 g, 2.17 mmol) in THF (11 mL) was added dropwise a 2.28 M n-BuLi solution in hexane (1.14 mL, 2.60 mmol) at −70° C. After 5 min at −70° C. the reaction mixture was warmed to 0° C., maintained 10 min at this temperature then cooled at −70° C. before a 30 min bubbling with carbonyl sulfide (COS) through the solution. The yellow solution was warmed to 0° C., stirred for additional 10 min at this temperature before dropwise addition of iodomethane (0.162 mL, 2.60 mmol). The mixture was stirred for 2 h, carefully hydrolyzed at 0° C. by water and extracted with ether. Combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude by chromatography on silicagel (petroleum ether/EtOAc=90/10) gave pure S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate (0.369 g, 70%) as a near colorless oil.

[0266] .sup.1H NMR (300 MHz, DMSO) δ 3.42 (t, J=6.3 Hz, 2H), 3.42 (q, J=7.0 Hz, 2H), 2.56 (t, J=6.3 Hz, 2H), 2.39 (s, 3H), 2.25 (s, 3H), 1.36 (s, 6H), 1.10 (t, J=7.0 Hz, 3H).

[0267] ESI-HRMS calc for C.sub.12H.sub.22NO.sub.2S [M+H]+: 244.1366. found: 244.1362.

Example 2: S-methyl 4-[2-allyloxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate

[0268] Preparation of N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine: To N-methyl-N-(2′hydroxyethyl)-3-amino-3methyl-1-butyne (Easton, Nelson R.; Hennion, George F. U.S. (1967), U.S. Pat. No. 3,337,625 19670822.)) (1.0 g, 7.08 mmol) in THF (12 mL) was added NaH (0.340 g, 8.50 mmol) at 0° C. After 15 min at 0° C. and 15 min at room temperature, n-Bu.sub.4NI (0.026 g, 0.071 mmol) was added in one portion at 0° C. followed by dropwise addition of allyl bromide (0.735 mL, 8.50 mmol). Reaction mixture was allowed to reach room temperature, stirred overnight, then carefully hydrolyzed by water and extracted by ether (3×25 mL). Combined organic layers were washed with brine (25 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification by chromatography on silicagel (petroleum ether/ether=80/20 to 70/30) gave pure N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine (0.941 g, 73%) as an oil.

[0269] .sup.1H NMR (300 MHz, DMSO) δ 5.88 (ddt, J=17.3, 10.5, 5.3 Hz, 1H), 5.24 (ddd, J=17.3, 3.8, 1.7 Hz, 1H), 5.16-5.09 (m, 1H), 3.93 (dt, J=5.3, 1.6 Hz, 2H), 3.43 (t, J=6.4 Hz, 2H), 3.12 (s, 1H), 2.55 (t, J=6.4 Hz, 2H), 2.21 (s, 3H), 1.27 (s, 6H).

[0270] ESI-LRMS 182.0 [M+H]+.

[0271] Preparation of S-methyl 4-[2-allyloxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine. Scale: 2.2 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10 to 80/20). Yield: 65%. Near colorless oil.

[0272] .sup.1H NMR (300 MHz, DMSO) δ 5.88 (ddt, J=17.3, 10.5, 5.3 Hz, 1H), 5.24 (ddd, J=17.3, 3.8, 1.7 Hz, 1H), 5.17-5.10 (m, 1H), 3.94 (dt, J=5.3, 1.5 Hz, 2H), 3.45 (t, J=6.2 Hz, 2H), 2.58 (t, J=6.2 Hz, 2H), 2.39 (s, 3H), 2.26 (s, 3H), 1.36 (s, 6H).

[0273] ESI-HRMS calc for C.sub.13H.sub.22NO.sub.2S [M+H]+: 256.1366. found: 256.1364.

Example 3: S-methyl 4-[2-benzyloxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate

[0274] Preparation of N-(2-benzyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 1.015 eq of NaH and 1.01 eq. of benzyl bromide. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10). Yield: 81%. Colorless oil.

[0275] .sup.1H NMR (300 MHz, DMSO) δ 7.39-7.24 (m, 5H), 4.47 (s, 2H), 3.49 (t, J=6.3 Hz, 2H), 3.12 (s, 1H), 2.58 (t, J=6.3 Hz, 2H), 2.21 (s, 3H), 1.27 (s, 6H). ESI-LRMS 232.0 [M+H]+.

[0276] Preparation of S-methyl 4-[2-benzyloxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-(2-benzyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine. Scale: 2.2 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10). Yield: 79%. Colorless oil.

[0277] .sup.1H NMR (300 MHz, DMSO) δ 7.37-7.26 (m, 5H), 4.48 (s, 2H), 3.52 (t, J=6.1 Hz, 2H), 2.62 (t, J=6.1 Hz, 2H), 2.38 (s, 3H), 2.26 (s, 3H), 1.36 (s, 6H).

[0278] ESI-HRMS calc for C.sub.17H.sub.24NO.sub.2S [M+H]+: 306.1522. found: 306.1514.

[0279] Alternative protocol: To N-(2-benzyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine (0.650 g, 2.81 mmol) in THF (8 mL) was added dropwise a 2.28 M n-BuLi solution in hexane (1.36 mL, 3.09 mmol) at −70° C. After 5 min at −70° C. the reaction mixture was warmed to 0° C., maintained 30 min at this temperature and CO.sub.2 was bubbled through the solution for 30 min.

[0280] The mixture was warmed to room temperature within 5 min then re-cooled at 0° C. Isobutyl chloroformate (0.40 ml, 3.08 mmol) was added dropwise and the mixture stirred for 10 min before addition of sodium methoxide (0.236 g, 3.37 mmol) in one portion. The mixture was warmed to room temperature stirred for additional 15 min at this temperature then carefully hydrolyzed at 0° C. by water and extracted with ether. Combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude by chromatography on silicagel (petroleum ether/EtOAc=90/10) gave pure S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate (0.307 g, 36%).

Example 4: S-methyl 4-methyl-4-[methyl-[2-(m-tolylmethoxy)ethyl]amino]pent-2-ynethioate

[0281] Preparation of N-2-dimethyl-N-[2-(m-tolylmethoxy)ethyl]but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 3-Methylbenzyl bromide. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10). Scale: 4.5 mmol. Yield: 79%. Colorless oil.

[0282] .sup.1H NMR (300 MHz, DMSO) δ 7.26-7.19 (m, 1H), 7.16-7.05 (m, 3H), 4.43 (s, 2H), 3.48 (t, J=6.3 Hz, 2H), 3.12 (s, 1H), 2.57 (t, J=6.3 Hz, 2H), 2.30 (s, 3H), 2.21 (s, 3H), 1.27 (s, 6H).

[0283] ESI-LRMS 246.1 [M+H]+.

[0284] Preparation of S-methyl 4-methyl-4-[methyl-[2-(m-tolylmethoxy)ethyl]amino]pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-2-dimethyl-N-[2-(m-tolylmethoxy)ethyl]but-3-yn-2-amine. Scale: 1.3 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10). Yield: 77%. Colorless oil.

[0285] .sup.1H NMR (300 MHz, DMSO) δ 7.26-7.19 (m, 1H), 7.16-7.05 (m, 3H), 4.44 (s, 2H), 3.50 (t, J=6.1 Hz, 2H), 2.62 (t, J=6.1 Hz, 2H), 2.38 (s, 3H), 2.30 (s, 3H), 2.26 (s, 3H), 1.36 (s, 6H).

[0286] ESI-HRMS calc for C.sub.18H.sub.26NO.sub.2S [M+H]+: 320.1679. found: 320.1667.

Example 5: S-methyl 4-[2-[(3,4-dimethylphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate

[0287] Preparation of N-[2-[(3,4-dimethylphenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 3,4-Dimethylbenzyl bromide. Purification by chromatography on silicagel (petroleum ether/EtOAc=60/40). Scale: 3.8 mmol. Yield: 60%. Colorless oil.

[0288] .sup.1H NMR (300 MHz, DMSO) δ 7.12-7.06 (m, 2H), 7.04-6.99 (m, 1H), 4.39 (s, 2H), 3.45 (t, J=6.3 Hz, 2H), 3.12 (s, 1H), 2.56 (t, J=6.3 Hz, 2H), 2.20 (s, 6H), 2.19 (s, 3H), 1.27 (s, 6H).

[0289] ESI-LRMS 182.0 [M+H]+. ESI-LRMS 260.0 [M+H]+.

[0290] Preparation of S-methyl 4-[2-[(3,4-dimethylphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-[2-[(3,4-dimethylphenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine. Scale: 1.3 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10). Yield: 77%. Near colorless oil.

[0291] .sup.1H NMR (300 MHz, DMSO) δ 7.12-7.06 (m, 2H), 7.05-6.99 (m, 1H), 4.40 (s, 2H), 3.48 (t, J=6.2 Hz, 2H), 2.60 (t, J=6.2 Hz, 2H), 2.38 (s, 3H), 2.25 (s, 3H), 2.20 (s, 3H), 2.19 (s, 3H), 1.36 (s, 6H).

[0292] ESI-HRMS calc for C.sub.19H.sub.28NO.sub.2S [M+H]+: 334.1835. found: 334.1825.

Example 6: S-methyl 4-[2-[(4-methoxyphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate

[0293] Preparation of N-[2-[(4-methoxyphenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 1-(Bromomethyl)-4-methoxybenzene. Purification by chromatography on silicagel (DCM/MeOH=99/1 to 97.5/2.5). Scale: 4.0 mmol. Yield: 53%. Colorless oil.

[0294] 1H NMR (300 MHz, DMSO) δ 7.27-7.20 (m, 2H), 6.93-6.86 (m, 2H), 4.39 (s, 2H), 3.74 (s, 3H), 3.45 (t, J=6.3 Hz, 2H), 3.12 (s, 1H), 2.55 (t, J=6.4 Hz, 2H), 2.20 (s, 3H), 1.27 (s, 6H).

[0295] ESI-LRMS 261.9 [M+H]+.

[0296] Preparation of S-methyl 4-[2-[(4-methoxyphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-[2-[(4-methoxyphenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine. Scale: 1.3 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=80/20). Yield: 74%. Near colorless oil.

[0297] .sup.1H NMR (300 MHz, DMSO) δ 7.27-7.21 (m, 2H), 6.93-6.87 (m, 2H), 4.40 (s, 2H), 3.74 (s, 3H), 3.48 (t, J=6.2 Hz, 2H), 2.60 (t, J=6.2 Hz, 2H), 2.38 (s, 3H), 2.25 (s, 3H), 1.36 (s, 6H).

[0298] ESI-HRMS calc for C.sub.18H.sub.26NO.sub.3S [M+H]+: 336.1628. found: 336.1613.

Example 7: S-methyl 4-[2-[(3,4-dimethoxyphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate

[0299] Preparation of N-[2-[(3,4-dimethoxyphenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 4-(Bromomethyl)-1,2-dimethoxybenzene. Purification by chromatography on silicagel (DCM/MeOH=99/1 to 97.5/2.5). Scale: 4.0 mmol. Yield: 67%. Colorless oil.

[0300] .sup.1H NMR (300 MHz, DMSO) δ 6.94-6.88 (m, 2H), 6.87-6.80 (m, 1H), 4.39 (s, 2H), 3.74 (s, 3H), 3.73 (s, 3H), 3.46 (t, J=6.3 Hz, 2H), 3.12 (s, 1H), 2.56 (t, J=6.3 Hz, 2H), 2.21 (s, 3H), 1.27 (s, 6H).

[0301] ESI-LRMS 292.0 [M+H]+.

[0302] Preparation of S-methyl 4-[2-[(3,4-dimethoxyphenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-[2-[(3,4-dimethoxyphenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine. Scale: 1.0 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10). Yield: 67%. Near colorless oil.

[0303] .sup.1H NMR (300 MHz, DMSO) δ 6.94-6.87 (m, 1H), 6.87-6.81 (m, 1H), 4.40 (s, 2H), 3.74 (s, 3H), 3.73 (s, 3H), 3.48 (t, J=6.1 Hz, 2H), 2.61 (t, J=6.2 Hz, 2H), 2.38 (s, 3H), 2.26 (s, 3H), 1.36 (s, 6H).

[0304] ESI-HRMS calc for C.sub.19H.sub.28NO.sub.4S [M+H]+: 366.1734. found: 336.1720.

Example 8: S-methyl 4-[2-[(3-chlorophenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate

[0305] Preparation of N-[2-[(3-chlorophenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 3-Chlorobenzyl bromide. Purification by chromatography on silicagel (petroleum ether/EtOAc=70/30). Scale: 4.0 mmol. Yield: 71%. Colorless oil.

[0306] .sup.1H NMR (300 MHz, DMSO) δ 7.43-7.25 (m, 4H), 4.49 (s, 2H), 3.50 (t, J=6.2 Hz, 2H), 3.12 (s, 1H), 2.58 (t, J=6.2 Hz, 2H), 2.22 (s, 3H), 1.28 (s, 6H).

[0307] ESI-LRMS 266.0 [M+H]+.

[0308] Preparation of S-methyl 4-[2-[(3-chlorophenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-[2-[(3-chlorophenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine except that the reaction mixture was maintained at −70° C. after n-BuLi addition for 30 min before COS bubbling. Scale: 1.3 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=75/25). Yield: 63%. Near colorless oil.

[0309] .sup.1H NMR (300 MHz, DMSO) δ 7.44-7.24 (m, 4H), 4.50 (s, 2H), 3.52 (t, J=6.0 Hz, 2H), 2.63 (t, J=6.0 Hz, 2H), 2.38 (s, 3H), 2.27 (s, 3H), 1.37 (s, 6H).

[0310] ESI-HRMS calc for C.sub.17H.sub.23ClNO.sub.2S [M+H]+: 340.1133. found: 340.1120.

Example 9: S-methyl 4-[2-[(3-fluorophenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate

[0311] Preparation of N-[2-[(3-fluorophenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 3-fluorobenzyl bromide. Purification by chromatography on silicagel (petroleum ether/EtOAc=80/20). Scale: 4.0 mmol. Yield: 71%. Colorless oil.

[0312] Preparation of S-methyl 4-[2-[(3-fluorophenyl)methoxy]ethyl-methyl-amino]-4-methyl-pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-[2-[(3-fluorophenyl)methoxy]ethyl]-N,2-dimethyl-but-3-yn-2-amine except that the reaction mixture was maintained at −70° C. after n-BuLi addition for 30 min before COS bubbling. Scale: 1.3 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=70/30). Yield: 87%. Near colorless oil.

[0313] .sup.1H NMR (300 MHz, DMSO) δ 7.44-7.34 (m, 1H), 7.20-7.05 (m, 3H), 4.51 (s, 2H), 3.53 (t, J=6.1 Hz, 2H), 2.63 (t, J=6.1 Hz, 2H), 2.38 (s, 3H), 2.27 (s, 3H), 1.37 (s, 6H).

[0314] ESI-HRMS calc for C.sub.17H.sub.23FNO.sub.2S [M+H]+: 324.1428. found: 324.1415 Example 10: S-methyl 4-methyl-4-[methyl-[2-(2-pyridylmethoxy)ethyl]amino]pent-2-ynethioate

[0315] Preparation of N-2-dimethyl-N-[2-(2-pyridylmethoxy)ethyl]but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] starting from 2-(Bromomethyl)pyridine hydrobromide and using 4 eq of NaH. Purification by chromatography on silicagel (DCM/MeOH=99/1 to 95/5). Scale: 2.1 mmol. Yield: 71%. Yellow oil.

[0316] .sup.1H NMR (300 MHz, DMSO) δ 8.50 (ddd, J=4.8, 1.8, 0.9 Hz, 1H), 7.80 (td, J=7.7, 1.8 Hz, 1H), 7.44 (d, J=7.8 Hz, 1H), 7.32-7.24 (m, 1H), 4.55 (s, 2H), 3.56 (t, J=6.2 Hz, 2H), 3.13 (s, 1H), 2.61 (t, J=6.2 Hz, 2H), 2.23 (s, 3H), 1.28 (s, 6H).

[0317] ESI-LRMS 233.1 [M+H]+.

[0318] Preparation of S-methyl 4-methyl-4-[methyl-[2-(2-pyridylmethoxy)ethyl]amino]pent-2-ynethioate: The compound is obtained by using the same processes the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-2-dimethyl-N-[2-(2-pyridylmethoxy)ethyl]but-3-yn-2-amine and using 1.5 eq of n-BuLi, 1.5 eq of Mel and DCM extractions. Scale: 0.9 mmol. Purification by chromatography on silicagel (DCM/MeOH=99/1 to 90/10). Yield: 18%. Yellow oil.

[0319] .sup.1H NMR (300 MHz, DMSO) δ 8.54-8.48 (m, 1H), 7.80 (td, J=7.7, 1.8 Hz, 1H), 7.44 (d, J=7.8 Hz, 1H), 7.28 (dd, J=6.7, 5.1 Hz, 1H), 4.56 (s, 2H), 3.59 (t, J=6.1 Hz, 2H), 2.65 (t, J=6.1 Hz, 2H), 2.38 (s, 3H), 2.28 (s, 3H), 1.37 (s, 6H).).

[0320] ESI-HRMS calc for C.sub.16H.sub.23N.sub.2O.sub.2S [M+H]+: 3071475. found: 307.1471.

Example 11: S-methyl 4-methyl-4-[methyl-[2-(3-pyridylmethoxy)ethyl]amino]pent-2-ynethioate

[0321] Preparation of N-2-dimethyl-N-[2-(3-pyridylmethoxy)ethyl]but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] starting from 3-(Bromomethyl)pyridine hydrobromide and using 4 eq of NaH. Purification by chromatography on silicagel (DCM/MeOH=99/1 to 95/5). Scale: 2.1 mmol. Yield: 67%. Yellow oil.

[0322] .sup.1H NMR (300 MHz, DMSO) δ 8.56-8.52 (m, 1H), 8.49 (dd, J=4.8, 1.7 Hz, 1H), 7.78-7.70 (m, 1H), 7.38 (ddd, J=7.8, 4.8, 0.8 Hz, 1H), 4.52 (s, 2H), 3.52 (t, J=6.2 Hz, 2H), 3.12 (s, 1H), 2.58 (t, J=6.2 Hz, 2H), 2.21 (s, 3H), 1.27 (s, 6H).

[0323] ESI-LRMS 233.1 [M+H]+.

[0324] Preparation of S-methyl 4-methyl-4-[methyl-[2-(3-pyridylmethoxy)ethyl]amino]pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-2-dimethyl-N-[2-(3-pyridylmethoxy)ethyl]but-3-yn-2-amine and using 1.5 eq of n-BuLi, 1.5 eq of Mel and DCM extractions. Scale: 0.4 mmol. Purification by chromatography on silicagel (DCM/MeOH=99/1 to 95/5). Yield: 15%. Yellow oil.

[0325] .sup.1H NMR (300 MHz, DMSO) δ 8.54 (d, J=1.5 Hz, 1H), 8.49 (dd, J=4.8, 1.7 Hz, 1H), 7.78-7.70 (m, 1H), 7.38 (ddd, J=7.8, 4.8, 0.8 Hz, 1H), 4.53 (s, 2H), 3.54 (t, J=6.1 Hz, 2H), 2.63 (t, J=6.1 Hz, 2H), 2.38 (s, 3H), 2.26 (s, 3H), 1.36 (s, 6H)).

[0326] ESI-HRMS calc for C.sub.16H.sub.23N.sub.2O.sub.2S [M+H]+: 3071475. found: 307.1474.

Example 12: S-methyl 4-methyl-4-[methyl-[2-(4-pyridylmethoxy)ethyl]amino]pent-2-ynethioate

[0327] Preparation of N-2-dimethyl-N-[2-(4-pyridylmethoxy)ethyl]but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] starting from 4-(Bromomethyl)pyridine hydrobromide and using 4 eq of NaH. Purification by chromatography on silicagel (DCM/MeOH=99/1 to 95/5). Scale: 2.1 mmol. Yield: 95%. Yellow oil.

[0328] .sup.1H NMR (300 MHz, DMSO) δ 8.56-8.49 (m, 2H), 7.38-7.27 (m, 2H), 4.54 (s, 2H), 3.53 (t, J=6.2 Hz, 2H), 3.13 (s, 1H), 2.61 (t, J=6.2 Hz, 2H), 2.23 (s, 3H), 1.28 (s, 6H).

[0329] ESI-LRMS 233.1 [M+H]+.

[0330] Preparation of S-methyl 4-methyl-4-[methyl-[2-(4-pyridylmethoxy)ethyl]amino]pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-2-dimethyl-N-[2-(4-pyridylmethoxy)ethyl]but-3-yn-2-amine and using 1.5 eq of n-BuLi, 1.5 eq of Mel and DCM extractions. Scale: 1.0 mmol. Purification by chromatography on silicagel (DCM/MeOH=99/1 to 95/15). Yield: 24%. Yellow oil.

[0331] .sup.1H NMR (300 MHz, DMSO) δ 8.60-8.45 (m, 2H), 7.37-7.26 (m, 2H), 4.55 (s, 2H), 3.56 (t, J=6.0 Hz, 2H), 2.65 (t, J=6.0 Hz, 2H), 2.38 (s, 3H), 2.28 (s, 3H), 1.37 (s, 6H).

[0332] ESI-HRMS calc for C.sub.16H.sub.23N.sub.2O.sub.2S [M+H]+: 3071475. found: 307.1470.

Example 13: methyl 4-(dimethylamino)-4-methyl-pent-2-ynoate

[0333] Preparation of 4-(dimethylamino)-4-methyl-pent-2-ynoic acid chlorhydrate: To N,N,2-trimethylbut-3-yn-2-amine (0.928 g, 8.35 mmol) in THF (42 mL) was added dropwise a 2.35 M n-BuLi solution in hexane (3.73 mL, 8.76 mmol) at −70° C. After 5 min at −70° C. the reaction mixture was warmed to 0° C., maintained 10 min at this temperature then cooled at −70° C. before a 45 min bubbling with carbon dioxide. The mixture was warmed to 0° C. within 2 h, then carefully hydrolyzed at 0° C. by water and washed (2×25 mL) with ether. Aqueous layers were acidified (PH1-2) with 6N HCl then concentrated in vacuo. The solid obtained was triturated and washed twice with MeOH. The crude 4-(dimethylamino)-4-methyl-pent-2-ynoic acid chlorhydrate (0.721 g, 45%) obtained as a white solid was used in the next step without purification.

[0334] .sup.1H NMR (300 MHz, D.sub.2O) δ 2.94 (s, 6H), 1.70 (s, 6H). .sup.13C NMR (75 MHz, D.sub.2O) δ 158.97 (C), 83.64 (C), 76.05 (C), 60.35 (C), 38.46 (2CH.sub.3), 23.66 (2CH.sub.3).

[0335] Preparation of Methyl 4-(dimethylamino)-4-methyl-pent-2-ynoate: 4-(dimethylamino)-4-methyl-pent-2-ynoic acid chlorhydrate (0.500 g, 2.61 mmol) in MeOH (10 mL) was treated with conc.H.sub.2SO.sub.4 (0.15 mL) at 0° C. then stirred overnight at room temperature. After concentration in vacuo the residue was diluted in AcOEt. Organic layer was washed with NaHCO.sub.3 aq.sat. and brine, dried over Na.sub.2SO.sub.4 and solvent evaporated in vacuo to give (yield <10%, not optimized) the methyl ester as a near colorless oil.

[0336] .sup.1H NMR (300 MHz, DMSO) δ 3.71 (s, 3H), 2.19 (s, 6H), 1.35 (s, 6H).

[0337] ESI-LRMS [M+H]+ calc for C.sub.9H.sub.15NO.sub.2 [M+H]+: 170.11. found: 170.1.

Example 14: ethyl 4-(dimethylamino)-4-methyl-pent-2-ynoate

[0338] The compound is obtained by using the same process as the one described in example 13 using EtOH in the esterification step. Scale 5.22 mmol. Yield: 73%. Colorless oil.

[0339] .sup.1H NMR (300 MHz, DMSO) δ 4.17 (q, J=7.1 Hz, 2H), 2.19 (s, 6H), 1.34 (s, 6H), 1.22 (t, J=7.1 Hz, 3H).

[0340] ESI-HRMS calc for C.sub.10H.sub.18NO.sub.2 [M+H]+: 184.1332. found: 184.1326.

Example 15: tert-butyl 2-((4-(dimethylamino)-4-methylpent-2-ynoyl)thio)acetate

[0341] The compound was obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N,N,2-trimethylbut-3-yn-2-amine and using tert-butyl iodoacetate instead of iodomethane. Purification by chromatography on silicagel (cyclohexane/EtOAc=70/30). Scale 3 mmol Yield: 57%. Red oil.

[0342] .sup.1H NMR (300 MHz, DMSO) δ 3.82 (s, 2H), 2.21 (s, 6H), 1.41 (s, 9H), 1.37 (s, 6H).

[0343] ESI-HRMS: calc for C.sub.14H.sub.24NO.sub.3S [M+H]+ 286.1471. found 286.1472.

Example 16: 2-((4-(dimethylamino)-4-methylpent-2-ynoyl)thio)acetic acid

[0344] To a solution of tert-butyl 2-((4-(dimethylamino)-4-methylpent-2-ynoyl)thio)acetate (200 mg, 0.7 mmol) in dichloromethane (3.6 mL) is added trifluoroacetic acid (0.36 mL). The mixture is stirred overnight in the dark. After evaporation under reduced pressure the crude was triturated and washed with Et.sub.2O. The TFA salt was obtained as an amorphous solid. Yield: 84%.

[0345] 1H NMR (300 MHz, acetone) δ 12.14 (s, 1H), 3.95 (s, 2H), 2.99 (s, 6H), 1.87 (s, 6H).

[0346] ESI-HRMS: calc for C.sub.10H.sub.16NO.sub.3S [M+H]+ 230.0845. found 230.0847 Example 17: S-methyl 4-((4-(benzyloxy)butyl)(methyl)amino)-4-methylpent-2-ynethioate

[0347] Preparation of 4-(methyl(2-methylbut-3-yn-2-yl)amino)butan-1-ol: This compound was prepared by standard protocols previously described for the synthesis of N-methyl-N-(2′hydroxyethyl)-3-amino-3methyl-1-butyne (Easton, Nelson R.; Hennion, George F. U.S. (1967), U.S. Pat. No. 3,337,625 19670822.) starting from commercially available 4-(methylamino)butan-1-ol. 4-(methyl(2-methylbut-3-yn-2-yl)amino)butan-1-ol was obtained as a bright yellow oil. Scale 3 mmol. Yield: 99%.

[0348] .sup.1H NMR (300 MHz, DMSO) δ 4.41 (t, J=5.2 Hz, 1H), 3.42-3.33 (m, 2H), 3.09 (s, 1H), 2.38-2.29 (m, 2H), 2.14 (s, 3H), 1.45-1.36 (m, 4H), 1.27 (s, 6H).

[0349] ESI-LRMS: 170.1 [M+H]+

[0350] Preparation of N-(4-(benzyloxy)butyl)-N,2-dimethylbut-3-yn-2-amine: The compound was obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] starting from 4-(methyl(2-methylbut-3-yn-2-yl)amino)butan-1-ol and using 1.015 eq of NaH and 1.01 eq. of benzyl bromide. Purification by chromatography on silicagel (cyclohexane/EtOAc=80/20). Scale 2.4 mmol. Yield: 55%. Yellow oil.

[0351] .sup.1H NMR (300 MHz, DMSO) δ 7.39-7.22 (m, 5H), 4.44 (s, 2H), 3.42 (t, J=6.3 Hz, 2H), 3.08 (s, 1H), 2.34 (t, J=6.9 Hz, 2H), 2.13 (s, 3H), 1.60-1.37 (m, 4H), 1.26 (s, 6H).

[0352] ESI-LRMS: 260.2 [M+H]+

[0353] Preparation of S-methyl 4-((4-(benzyloxy)butyl)(methyl)amino)-4-methylpent-2-ynethioate: The compound was obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N-(4-benzyloxybutyl)-N,2-dimethyl-but-3-yn-2-amine. Purification by chromatography on silicagel (cyclohexane/EtOAc=90/10). Scale 0.77 mmol. Yield: 80%. Yellow oil.

[0354] 1H NMR (300 MHz, CDCl.sub.3) δ 7.38-7.19 (m, 5H), 4.48 (s, 2H), 3.47 (t, J=6.1 Hz, 2H), 2.45 (t, J=6.9 Hz, 2H), 2.35 (s, 3H), 2.26 (s, 3H), 1.73-1.48 (m, 4H), 1.39 (s, 6H).

[0355] ESI-HRMS: calc for C.sub.19H.sub.28NO.sub.2S [M+H]+ 334.1835. found 334.1840.

Example 18: S-methyl 4-((2-hydroxyethyl)(methyl)amino)-4-methylpent-2-ynethioate

[0356] ##STR00033##

[0357] Preparation of Compound 3: N,2-dimethyl-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)but-3-yn-2-amine: To 2-(methyl(2-methylbut-3-yn-2-yl)amino)ethanol 2 (3.00 g, 21.2 mmol) and 3,4-Dihydro-2H-pyran (5.0 eq) in anhydrous DCM (135 mL) was added p-toluenesulfonic acid (0.1 eq) at room temperature. The reaction mixture was stirred overnight, washed with aqueous saturated NaHCO.sub.3 (30 mL) then brine (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was first purified by short-path distillation using Kugelrohr apparatus (10-12 Torrs, oven 155° C.) then by flash chromatography on silica gel (petroleum ether/ethyl acetate 95/5 to 60/40) to give compound 3 as an oil (yield 70%).

[0358] .sup.1H NMR (300 MHz, DMSO) δ 4.60-4.50 (m, 1H), 3.82-3.70 (m, 1H), 3.59-3.56 (m, 1H), 3.49-3.35 (m, 2H), 3.12 (s, 1H), 2.55 (t, J=6.5 Hz, 2H), 2.21 (s, 3H), 1.77-1.35 (m, 6H), 1.27 (s, 6H).

[0359] Preparation of Compound 4: S-methyl 4-methyl-4-(methyl(2-((tetrahydro-2H-pyran-2-yl)oxy) ethyl) amino) pent-2-ynethioate: To the acetylenic amine 3 (1.00 g, 4.44 mmol) in anhydrous THF (22 mL) was added n-Butyllithium solution (2.2 M in hexanes, 1.5 eq) dropwise. The mixture was allowed to reach to 0° C. within 10 minutes then re-cooled to −70° C. before carbonyl sulfide bubbling. After 30 minutes the bright yellow solution was carefully warmed to 0° C., stirred 30 minutes at this temperature and methyl iodide (1.2 eq) was added dropwise. The reaction mixture was stirred for 2 hours at 0° C. before hydrolysis by water. Extractive work-up by DCM (washing with brine, drying with sodium sulfate and concentration under reduced pressure) gave a crude which was purified by chromatography on silica gel (petroleum ether/ethyl acetate 90/10 to 60/40) to give compound 4 as an oil (yield 59%).

[0360] .sup.1H NMR (300 MHz, DMSO) δ 4.58 (t, J=3.2 Hz, 1H), 3.75 (ddd, J=11.4, 7.9, 3.3 Hz, 1H), 3.70-3.60 (m, 1H), 3.49-3.38 (m, 2H), 2.59 (t, J=6.3 Hz, 2H), 2.39 (s, 3H), 2.27 (s, 3H), 1.77-1.39 (m, 6H), 1.36 (s, 6H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 176.62 (C═O), 98.94 (CH), 96.46 (C), 80.97 (C), 66.55 (CH.sub.2), 62.37 (CH.sub.2), 55.19 (C), 52.43 (CH.sub.2), 37.92 (CH.sub.3), 30.75 (CH.sub.2), 28.01 (2×CH.sub.3), 25.59 (CH.sub.2), 19.63 (CH.sub.2), 12.61 (CH.sub.3).

[0361] Preparation of Compound 5: S-methyl 4-((2-hydroxyethyl)(methyl)amino)-4-methylpent-2-ynethioate: To the aminothiolester 4 (1.00 g, 3.34 mmol) in methanol (15 mL) was added p-toluenesulfonic acid (1.1 eq) at room temperature. The reaction mixture was stirred overnight, washed with aqueous saturated NaHCO.sub.3 (30 mL) then brine (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate 80/20 to 20/80) to give compound 5 as an oil (yield 90%).

[0362] .sup.1H NMR (300 MHz, DMSO) δ 4.42 (t, J=5.6 Hz, 1H), 3.44 (td, J=6.7, 5.6 Hz, 2H), 2.46 (, J=6.7 Hz, 2H), 2.39 (s, 3H), 2.24 (s, 3H), 1.36 (s, 6H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 176.46 (C═O), 95.56 (C), 80.89 (C), 58.92 (CH.sub.2), 54.99 (C), 53.52 (CH.sub.2), 36.12 (CH.sub.3), 27.88 (2×CH.sub.3), 12.53 (CH.sub.3). ESI-HRMS: Calc. for C.sub.10H.sub.18NO.sub.2S [M+H]+ 216.1053 found 216.1043.

Example 19. S-methyl 4-methyl-4-[methyl-[2-(2-naphthylmethoxy)ethyl]amino]pent-2-ynethioate

[0363] Preparation of N,2-dimethyl-N-[2-(2-naphthylmethoxy)ethyl]but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 1.015 eq of NaH and 1.01 eq. of 2-naphtyl bromide. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10). Yield: 61%. orange oil.

[0364] 1H NMR (300 MHz, DMSO) δ 7.91-7.88 (m, 4H), 7.50-7.48 (m, 3H), 4.65 (s, 2H), 3.55 (t, 6.1 Hz, 2H), 3.13 (s, 1H), 2.62 (t, 6.2 Hz, 2H), 2.23 (s, 3H), 1.28 (s, 6H).

[0365] Preparation of S-methyl 4-methyl-4-[methyl-[2-(2-naphthylmethoxy)ethyl]amino]pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from N,2-dimethyl-N-[2-(2-naphthylmethoxy)ethyl]but-3-yn-2-amine. Scale: 0.65 mmol. Purification by chromatography on silicagel (petroleum ether/EtOAc=85/15). Yield: 28%. Yellow oil.

[0366] 1H NMR (300 MHz, DMSO) δ7.95-7.82 (m, 4H), 7.55-7.41 (m, 3H), 4.66 (s, 2H), 3.57 (t, J=6.2 Hz, 2H), 2.66 (t, J=6.1 Hz, 2H), 2.38 (s, 3H), 2.28 (s, 3H), 1.37 (s, 6H).

[0367] ESI-HRMS: calc. for C.sub.21H.sub.26NO.sub.2S 356.1683. found 356.1679 [M+H]+

Example 20: S-methyl 4-methyl-4-[methyl-[2-[(2,6,6-trimethylcyclohexen-1-yl)methoxy]ethyl]amino]pent-2-ynethioate

[0368] Preparation of N,2-dimethyl-N-[2-[(2,6,6-trimethylcyclohexen-1-yl)methoxy]ethyl]but-3-yn-2-amine: The compound is obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 1.015 eq of NaH and 1.01 eq. of 2-(Bromomethyl)-1,3,3-trimethyl-1-cyclohexene (prepared from p-Cyclocitral by known protocols (WO 2015048363)). Purification by chromatography on silicagel (petroleum ether/EtOAc=95/05). Yield: 67%. Pale yellow oil.

[0369] 3.86 (s, 2H), 3.41 (t, J=6.5 Hz, 2H), 3.12 (s, 1H), 2.53 (t, J=6.4 Hz, 2H), 2.20 (s, 3H), 1.90 (t, J=5.9 Hz, 2H), 1.62 (s, 3H), 1.57-1.49 (m, 2H), 1.41-1.33 (s, 2H), 1.27 (s, 6H), 0.97 (s, 6H).

[0370] Preparation of S S-methyl 4-methyl-4-[methyl-[2-[(2,6,6-trimethylcyclohexen-1-yl)methoxy]ethyl]amino]pent-2-ynethioate: The compound is obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate (example 19) starting from N,2-dimethyl-N-[2-[(2,6,6-trimethylcyclohexen-1-yl)methoxy]ethyl]but-3-yn-2-amine. Purification by chromatography on silicagel (petroleum ether/EtOAc=90/10). Yield: 63%. Yellow oil.

[0371] H NMR (300 MHz, DMSO) δ 3.87 (s, 2H), 3.44 (t, J=6.3 Hz, 2H), 2.57 (t, J=6.3 Hz, 2H), 2.38 (s, 3H), 2.26 (s, 3H), 1.91 (t, J=6.2 Hz, 2H), 1.62 (s, 3H), 1.57-1.49 (m, 2H), 1.36 (s, 6H), 1.39-1.34 (m, 2H), 0.97 (s, 6H).

[0372] ESI-HRMS calc for C20H34NO2S [M+H]+: 352.2305. found: 352.2289.

Example 21: 2-[(1,1-dimethyl-4-methylsulfanyl-4-oxo-but-2-ynyl)-methylamino]ethyl acetate

[0373] To S-methyl 4-[2-hydroxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate (100 mg, 0.46 mmol) and di-isopropylethylamine (1.2 eq) in dichloromethane (2.3 mL) was added dropwise acetyl chloride (1.2 eq) at 0° C. After 10 min at 0° C. the reaction mixture was warmed up to rt and stirred until complete conversion (TLC checking). The mixture is then diluted in dichloromethane (30 mL), washed with brine (40 mL), dried over sodium sulfate and the solvent evaporated under reduced pressure. The crude was purified by chromatography on silica gel (petroleum ether/ethyl acetate 75/25) to give product 21 as a yellow oil. Yield 58%.

[0374] 1H NMR (300 MHz, DMSO) δ 4.05 (t, J=6.1 Hz, 2H), 2.62 (t, J=6.1 Hz, 2H), 2.39 (s, 3H), 2.26 (s, 3H), 2.01 (s, 3H), 1.36 (s, 6H).

[0375] ESI-HRMS calc for C12H19NO3S [M+H]+: 258.1086. found: 258.1150.

Example 22: 2-[(1,1-dimethyl-4-methylsulfanyl-4-oxo-but-2-ynyl)-methylamino]ethyl-3,4-dimethoxybenzoate

[0376] The compound is obtained by using the same process as the one described for 2-[(1,1-dimethyl-4-methylsulfanyl-4-oxo-but-2-ynyl)-methylamino]ethyl acetate [example 21] starting from 3,4-dimethoxybenzoyl chloride. Scale: 0.46 mmol. Purification by chromatography on silica gel (petroleum ether/EtOAc=60/40). Yield: <10%. yellow oil.

[0377] 1H NMR (300 MHz, DMSO) δ 7.59 (dd, J=8.4, 2.0 Hz, 1H), 7.45 (d, J=2.0 Hz, 1H), 7.08 (d, J=8.5 Hz, 1H), 4.28 (t, J=7.1 Hz, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 2.72 (t, J=7.1 Hz, 2H), 2.38 (s, 3H), 2.33 (s, 3H), 1.39 (s, 6H).

Example 23: S-methyl 2,5,10,11,11-pentamethyl-6-oxo-7-oxa-2,5,10-triazatetradec-12-yne-14-thioate

[0378] The compound is obtained by using the same process as the one described for S-methyl 4-methyl-4-[methyl-[2-[methyl-[2-(methylamino)ethyl]carbamoyl]oxyethyl]amino]pent-2-ynethioate [compound 7/Example 29] starting from N,N,N′-Trimethylethylenediamine. Purification by chromatography on silica gel DCM/MeOH (85/15). Yield: 34%. Yellow oil.

[0379] 1H NMR (300 MHz, DMSO) δ 4.01 (t, J=5.9 Hz, 2H), 3.32-3.25 (m, 2H), 2.83 (large s, 3H), 2.62 (t, J=5.8 Hz, 2H), 2.6-2.5 (m partially hide by solvent peak, 2H), 2.39 (s, 3H), 2.27 (s, 3H), 2.17 (large s, 6H), 1.36 (s, 6H).

[0380] ESI-HRMS calc for C16H30N3O3S [M+H]+: 344.1992. found: 344.1993.

Example 24: (S)—S-methyl 4-(2-(methoxymethyl)pyrrolidin-1-yl)-4-methylpent-2-ynethioate

[0381] Preparation of (S)-2-(methoxymethyl)-1-(2-methylbut-3-yn-2-yl)pyrrolidine: The compound was obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] using 1 eq of (S)-pyrrolidin-2-ylmethanol, 1.015 eq of NaH and 1.01 eq. of iodomethane. Purification by chromatography on silicagel (dichloromethane/methanol=90/10). Scale 3.0 mmol. Yield: 61%. Orange oil.

[0382] .sup.1H NMR (300 MHz, DMSO) δ 3.23 (s, 3H), 3.16-3.06 (m, 2H), 3.05 (s, 1H), 3.03-2.91 (m, 1H), 2.91-2.80 (m, 1H), 2.67-2.54 (m, 1H), 1.77-1.49 (m, 4H), 1.30 (s, 3H), 1.24 (s, 3H).

[0383] ESI-LRMS: 182.2 [M+H]+.

[0384] Preparation of (S)—S-methyl 4-(2-(methoxymethyl)pyrrolidin-1-yl)-4-methylpent-2-ynethioate: The compound was obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from (S)-2-(methoxymethyl)-1-(2-methylbut-3-yn-2-yl)pyrrolidine. Purification by chromatography on silicagel (petroleum ether/EtOAc=80/20). Scale 0.8 mmol. Yield: 57%.

[0385] Yellow oil.

[0386] .sup.1H NMR (300 MHz, DMSO) δ 3.24 (s, 3H), 3.19-2.89 (m, 4H), 2.67-2.53 (m, 1H), 2.38 (s, 3H), 1.83-1.50 (m, 4H), 1.39 (s, 3H), 1.33 (s, 3H).

[0387] ESI-HRMS calc for C.sub.13H.sub.22NO.sub.2S [M+H]+: 256.1366. found: 256.1363.

Example 25: S-methyl 4-[(3R)-3-methoxypyrrolidin-1-yl]-4-methyl-pent-2-ynethioate

[0388] Preparation of (R)-3-methoxy-1-(2-methylbut-3-yn-2-yl)pyrrolidine: The compound was obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] starting from (R)-1-(2-methylbut-3-yn-2-yl)pyrrolidin-3-ol using 1.015 eq of NaH and 1.01 eq. of iodomethane. Purification by chromatography on silicagel (dichloromethane/methanol=90/10). Scale 3.3 mmol. Yield 30%. Yellow oil.

[0389] .sup.1H NMR (300 MHz, DMSO) δ 3.85 (ddd, J=10.8, 7.2, 3.6 Hz, 1H), 3.17 (s, 3H), 3.13 (s, 1H) 2.82 (dd, J=9.8, 6.5 Hz, 1H), 2.66-2.60 (m, 1H), 2.56-2.50 (m, 2H), 2.01-1.87 (m, 1H), 1.73-1.55 (m, 1H), 1.28 (s, 6H).

[0390] ESI-LRMS: 168.0 [M+H].sup.+.

[0391] Preparation of (R)—S-methyl 4-(3-methoxypyrrolidin-1-yl)-4-methylpent-2-ynethioate:

[0392] The compound was obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from (R)-3-methoxy-1-(2-methylbut-3-yn-2-yl)pyrrolidine. Purification by chromatography on silicagel (petroleum ether/EtOAc=60/40). Scale 0.8 mmol. Yield: 55%. Yellow oil.

[0393] .sup.1H NMR (300 MHz, DMSO) δ 3.87 (ddd, J=10.5, 6.9, 3.4 Hz, 1H), 3.18 (s, 3H), 2.90-2.76 (m, 1H), 2.77-2.65 (m, 1H), 2.65-2.54 (m, 2H), 2.38 (s, 3H), 2.07-1.86 (m, 1H), 1.76-1.60 (m, 1H), 1.36 (s, 6H).

Example 26: S-methyl 4-(((1R,2R)-2-(benzyloxy)cyclopentyl)(methyl)amino)-4-methylpent-2-ynethioate

[0394] Preparation of (1R,2R)-2-benzyloxy-N-(1,1-dimethylprop-2-ynyl)cyclopentanamine:

[0395] To a solution of commercially available (1R,2R)-2-(benzyloxy)cyclopentanamine (0.93 g, 4.86 mmol), 3-chloro-3-methylbut-1-yne (1.3 eq) and triethylamine (1.3 eq) in THF (20 mL) was added CuI (8 mol %) at room temperature. The mixture was left to stir overnight. The solvent was evaporated under reduced pressure and the crude was then diluted in aqueous saturated NaHCO.sub.3 solution, extracted with ethyl acetate. Combined organic layers were washed with 2% NH.sub.4OH aqueous solution then brine, dried over Na.sub.2SO.sub.4 and the solvent evaporated under reduced pressure. (1R,2R)-2-benzyloxy-N-(1,1-dimethylprop-2-ynyl)cyclopentanamine was obtained as a brown oil. Yield: 99%.

[0396] .sup.1H NMR (300 MHz, DMSO) δ 7.37-7.16 (m, 5H), 4.56-4.36 (m, 2H), 3.72-3.58 (m, 1H), 3.28-3.19 (m, 1H), 3.08 (s, 1H), 2.00-1.88 (m, 1H), 1.86-1.67 (m, 2H), 1.65-1.49 (m, 3H), 1.40-1.28 (m, 1H), 1.25 (s, 3H), 1.25 (s, 3H).

[0397] Preparation of (1R,2R)-2-(benzyloxy)-N-methyl-N-(2-methylbut-3-yn-2-yl)cyclopentanamine: To (1R,2R)-2-(benzyloxy)-N-(2-methylbut-3-yn-2-yl)cyclopentanamine (0.25 g, 0.97 mmol) were added 5 eq of formic acid and 1.5 eq of formaldehyde (37% in water). The mixture was refluxed overnight then 2N HCl was added until pH 1 was reached and washed with ether. The aqueous layer was basified with 1N NaOH, and extracted with DCM. The organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and the solvents evaporated under reduced pressure. The crude is then purified by chromatography on silica gel (petroleum ether/EtOAc=80/20), giving a yellow oil. Yield 68%.

[0398] .sup.1H NMR (300 MHz, DMSO) δ 7.43-7.17 (m, 5H), 4.52 (m, 2H), 3.94-3.78 (m, 1H), 3.62-3.49 (m, 1H), 3.11 (s, 1H), 2.20 (s, 3H), 1.78-1.45 (m, 6H), 1.37 (s, 3H), 1.34 (s, 3H).

[0399] ESI-LRMS: 272.1 [M+H]+.

[0400] Preparation of S-methyl 4-(((1R,2R)-2-(benzyloxy)cyclopentyl)(methyl)amino)-4-methylpent-2-ynethioate: The compound was obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from (1R,2R)-2-(benzyloxy)-N-methyl-N-(2-methylbut-3-yn-2-yl)cyclopentanamine. Purification by chromatography on silicagel (petroleum ether/EtOAc=80/20 then DCM=100). Scale 0.64 mmol. Yield: 47%. Yellow oil.

[0401] .sup.1H NMR (300 MHz, DMSO) δ 7.35-7.20 (m, 5H), 4.51 (s, 2H), 3.92-3.80 (m, 1H), 3.59-3.45 (m, 1H), 2.36 (s, 3H), 2.24 (s, 3H), 1.78-1.49 (m, 6H), 1.45 (s, 3H), 1.42 (s, 3H).

[0402] ESI-HRMS calc for C.sub.20H.sub.28NO.sub.2S [M+H]+: 346.1835. found: 346.1824.

Example 27: (S)—S-methyl 4-(2-((benzyloxy)methyl)pyrrolidin-1-yl)-4-methylpent-2-ynethioate

[0403] Preparation of (S)-2-((benzyloxy)methyl)-1-(2-methylbut-3-yn-2-yl)pyrrolidine: The compound was obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] starting from (S)-(1-(2-methylbut-3-yn-2-yl)pyrrolidin-2-yl)methanol 1.015 eq of NaH and 1.01 eq. of benzyl bromide. Purification by chromatography on silicagel (dichloromethane/methanol=95/5). Scale 3.0 mmol. Yield 60%. Orange oil.

[0404] .sup.1H NMR (300 MHz, DMSO) δ 7.41-7.19 (m, 5H), 4.46 (s, 2H), 3.24 (dd, J=8.3, 2.7 Hz, 1H), 3.14 (dd, J=7.6, 3.2 Hz, 1H), 3.11-3.02 (m, 2H), 2.86 (dd, J=8.4, 6.0 Hz, 1H), 2.65-2.54 (m, 1H), 1.81-1.51 (m, 4H), 1.25 (s, 3H), 1.23 (s, 3H).

[0405] ESI-LRMS: 258.1 [M+H]+.

[0406] Preparation of (S)—S-methyl 4-(2-((benzyloxy)methyl)pyrrolidin-1-yl)-4-methylpent-2-ynethioate: The compound was obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from (S)-2-((benzyloxy)methyl)-1-(2-methylbut-3-yn-2-yl)pyrrolidine. Purification by chromatography on silicagel (petroleum ether/EtOAc=80/20). Scale 1.2 mmol. Yield: 50%. Orange oil.

[0407] .sup.1H NMR (300 MHz, DMSO) δ 7.46-7.18 (m, 5H), 4.47 (s, 2H), 3.29-3.23 (m, 1H), 3.17-3.03 (m, 2H), 2.94 (dd, J=8.5, 5.7 Hz, 1H), 2.62-2.53 (m, 1H), 2.38 (s, 3H), 1.85-1.58 (m, 4H), 1.34 (s, 3H), 1.32 (s, 3H).

Example 28: (R)—S-methyl 4-(3-(benzyloxy)pyrrolidin-1-yl)-4-methylpent-2-ynethioate

[0408] Preparation of (R)-3-(benzyloxy)-1-(2-methylbut-3-yn-2-yl)pyrrolidine: The compound was obtained by using the same process as the one described for N-(2-allyloxyethyl)-N,2-dimethyl-but-3-yn-2-amine [example 2] starting from (R)-1-(2-methylbut-3-yn-2-yl)pyrrolidin-3-ol using 1.01 eq of NaH and 1.01 eq. of benzyl bromide. Purification by chromatography on silicagel (dichloromethane/methanol=98/2). Scale 3.3 mmol. Yield 69%. Orange oil.

[0409] .sup.1H NMR (300 MHz, DMSO) δ 7.40-7.17 (m, 5H), 4.42 (s, 2H), 4.07 (tt, J=7.3, 3.7 Hz, 1H) 3.13 (s, 1H), 2.86 (dd, J=9.6, 6.7 Hz, 1H), 2.75-2.65 (m, 2H), 2.60-2.53 (m, 1H), 2.06-1.95 (m, 1H), 1.75-1.65 (m, 1H), 1.29 (s, 6H).

[0410] ESI-LRMS 244.1 [M+H]+.

[0411] Preparation of (R)—S-methyl 4-(3-(benzyloxy)pyrrolidin-1-yl)-4-methylpent-2-ynethioate: The compound was obtained by using the same process as the one described for S-methyl 4-[2-ethoxyethyl(methyl)amino]-4-methyl-pent-2-ynethioate [example 1] starting from (R)-3-(benzyloxy)-1-(2-methylbut-3-yn-2-yl)pyrrolidine. Purification by chromatography on silicagel (dichloromethane/methanol=98/2). Scale 1.2 mmol. Yield: 69%. Orange oil.

[0412] .sup.1H NMR (300 MHz, DMSO) δ 7.32 (m, 5H), 4.44 (s, 2H), 4.09 (dq, J=10.2, 3.5 Hz, 1H), 2.97-2.83 (m, 1H), 2.80-2.65 (m, 2H), 2.65-2.55 (m, 1H), 2.37 (s, 3H), 2.11-1.90 (m, 1H), 1.83-1.67 (m, 1H), 1.37 (s, 6H).

[0413] ESI-HRMS calc for C.sub.18H.sub.24NO.sub.2S [M+H]+: 318.1522. found: 318.1518.

Example 29: ADC Compound (without Antibody)

[0414] Preparation of a linker (Mc-Val-Cit-PAB-PNP): The selected linker for the conjugate preparation was designed on known platform already used in Rituximab and others, comprising the maleimide for attachment to the Antibody, the Cathepsin cleavable group and the p-amino benzyl system for the 1,6-elimination: Mc-Val-Cit-PAB-PNP, CAS 159857-81-5.

[0415] It was prepared following standard protocols starting from Fmoc-Val-OSu [CAS 3392-12-9] or may be purchased from commercial suppliers (ex. creative biolabs, ALB technology, Carbosynth etc.).

[0416] The general formula of the linker is indicated below:

##STR00034##

[0417] Preparation of the Compound According to the Invention (Example 18) Coupled to Mc-Val-Cit-PAB-PNP

##STR00035##

Compound 6:

[0418] To p-nitrochloroformate (140 mg, 0.70 mmol, 1.5 eq) in of DCM (2.5 mL) was added dropwise a solution of compound 5 (100 mg, 0.46 mmol) and TEA (1.5 eq) in DCM (1.5 mL) at 0° C. After 10 minutes at 0° C. the reaction mixture was warmed up to room temperature and stirred until complete conversion (TLC checking, 1 h). The mixture is then diluted in DCM (30 mL), washed with brine (40 mL), dried over Na.sub.2SO.sub.4 and the solvent evaporated under reduced pressure. The crude was purified by chromatography on silica gel (petroleum ether/ethyl acetate 100/ to 70/30) to give compound 6 as an amorphous solid (yield 82%). .sup.1H NMR (300 MHz, DMSO): δ(ppm) 8.35-8.30 (m, 2H), 7.59-7.53 (m, 2H), 4.30 (t, J=5.7 Hz, 2H), 2.75 (t, J=5.7 Hz, 2H), 2.39 (s, 3H), 2.30 (s, 3H), 1.39 (s, 6H).

Compound 7:

[0419] To compound 6 (200 mg, 0.53 mmol) in DCM (3 mL) is added TEA (1.2 eq) at room temperature. Then a solution of tert-butyl N-methyl-N-[2-(methylamino)ethyl]carbamate (1.2 eq) in DCM (2.3 mL) is added at 0° C. The bright yellow reaction mixture obtained was warmed up to room temperature and stirred overnight. The mixture was diluted in 30 mL of DCM, washed by 40 mL of brine, dried over Na.sub.2SO.sub.4 and the solvent evaporated under reduced pressure. The crude was purified by chromatography on silica gel (petroleum ether/ethyl acetate 75/25 to 20/80) to give compound 7 as a visquous oil (yield 70%).

[0420] .sup.1H NMR (300 MHz, DMSO): δ(ppm) 3.99 (t, J=5.7 Hz, 2H), 3.30 (s, 3H), 2.85-2.70 (m, 7H), 2.66-2.58 (m, 2H), 2.38 (s, 3H), 2.26 (s, 3H), 1.37 (s, 9H), 1.36 (s, 6H). ESI-LRMS: Calc. for C.sub.20H.sub.36N.sub.3O.sub.5S [M+H]+: 430.2. found: 430.2.

Compound 8:

[0421] To compound 7 (44.5 mg, 0.104 mmol) in 4.5 mL of DCM (4.5 mL) was added TFA (0.5 mL) at 0° C. The mixture was then allowed to warm up to room temperature and stirred overnight. After concentration in vacuo (bath T° C.<45° C.) The oily residue was triturated and sonicated in Et.sub.2O. The resulting sticky solid (bis trifluoroacetate salt) was washed with Et.sub.2O and dried. Yield: 67%.

[0422] .sup.1H NMR (300 MHz, D.sub.2O) δ 4.57-4.44 (m, 2H), 3.73-3.69 (m, 2H), 3.62 (t, J=5.7 Hz, 2H), 3.30-3.19 (t, J=5.8 Hz, 2H), 3.05-2.99 (m, 3H), 2.99-2.90 (m, 3H), 2.73 (s, 3H), 2.45 (s, 3H), 1.79 (s, 6H). ESI-LRMS: Calc. for C.sub.15H.sub.28N.sub.3O.sub.3S [M+H].sup.+: 330.2. found: 330.1.

##STR00036##

Compound 9:

[0423] To MC-Val-Cit-PABC-PNP (52 mg, 0.07 mmol) and compound 8 (1.06 eq) in DMF (1.4 mL) were sequentially added at 0° C.: HOBt (1 eq) in one portion then DIPEA (3 eq) dropwise. After 5 minutes at 0° C., the mixture was warmed up to room temperature and stirred overnight. The mixture is then concentrated under vacuum (¾ evaporation off, bath T° C.<45° C.) and flocculated with Et.sub.2O. A white crude solid was obtained after washings/triturations (×3) in Et2O. A final purification by chromatography on silica (MeOH/DCM 95/5 to 90/10%) gave the final product (compound 9) as a white solid. Yield: 75%.

[0424] .sup.1H NMR (300 MHz, DMSO) δ 9.99 (s, 1H), 8.08 (d, J=7.4 Hz, 1H), 7.80 (d, J=8.6 Hz, 1H), 7.59 (d, J=8.5 Hz, 2H), 7.35-7.22 (m, 2H), 7.01 (s, 2H), 5.97 (t, J=5.8 Hz, 1H), 5.41 (s, 2H), 4.98 (s, 2H), 4.44-4.32 (m, 1H), 4.25-4.14 (m, 1H), 3.99 (s, 2H), 3.45-3.33 (m, 4H), 3.09-2.89 (m, 2H), 2.89-2.70 (m, 6H), 2.65-2.56 (m, 2H), 2.38 (s, 3H), 2.30-2.11 (m, 5H), 2.10-1.90 (m, 1H), 1.77-1.09 (m, 18H), 0.84 (dd, J=9.5, 6.8 Hz, 6H).

[0425] LC: Zorbax, ACN/H.sub.2O 0.1% TFA, 254 nm, 96%.

[0426] ESI-HRMS: Calc. for C.sub.44H.sub.66N.sub.9O.sub.11S [M+H].sup.+ 928.4597 found 928.4586.

Part 2: Use of a Compound According to the Invention

Example 1: Activity of the Compounds According to the Invention

Material and Methods

[0427] Cell lines. Leukemic cell line, HL-60 (derived from a 36-year-old female with AML-M2), was used for determination of drug efficacy. Cells were obtained from the European Collection of Cell Cultures (ECACC). All cells were cultivated in appropriate media according to supplier recommendations.

[0428] Cell Viability assay, 96-well format. Cells were seeded into 96-well cell culture plates at concentrations required to ensure approximately 80% confluence in control (untreated cells) at the end of experiment (0.5×10.sup.4-5×10.sup.4 cell/well).

[0429] The sensitivity towards compounds according to the invention was determined using different concentrations of each compound ranging from 0.5 to 100 μM (0.5, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 100 μM). Following 48 hours of incubation at 37° C. in a humidified atmosphere containing 5% CO.sub.2, the growth-inhibitory effect of compounds was analyzed using Resazurin, according to manufactures instructions.

[0430] To ensure good data quality and to minimize impact of pipetting errors, each compounds concentration was assessed based on mean fluorescence intensity from 8 separate wells. Compounds response were quantified by the half maximal inhibitory concentration (IC.sub.50) for each particular cell line, and determined by non-linear regression analysis of log-dose/response curves.

[0431] Statistical Analysis. Values were expressed as mean±SD or frequencies and proportions. Cell viability curves were determined using four parameter regression line. Differences between groups were determined by unpaired t test, Chi-square, Fisher's exact test or ANOVA, where appropriate. P<0.05 was considered statistically significant. Analysis was performed using GraphPad prism version 5.0 (GraphPad software, San Diego Calif. USA).

Results

[0432] The IC.sub.50 obtained by testing the cytotoxicity activity of the compounds according to the invention in HL60 cells are mentioned in table 3 below.

TABLE-US-00003 TABLE 3 Example Name IC.sub.50 at 48 h 1 S-methyl 4-[2- 4.977 μM ethoxyethyl(methyl)amino]-4- methyl-pent-2-ynethioate 2 S-methyl 4-[2- 6.5 μM allyloxyethyl(methyl)amino]-4- methyl-pent-2-ynethioate 3 S-methyl 4-[2- 1.169 μM benzyloxyethyl(methyl)amino]-4- methyl-pent-2-ynethioate 4 S-methyl 4-methyl-4-[methyl-[2-(m- 5.622 μM tolylmethoxy)ethyl]amino]pent-2- ynethioate 5 S-methyl 4-[2-[(3,4- 4.722 μM dimethylphenyl)methoxy]ethyl- methyl-amino]-4-methyl-pent-2- ynethioate 6 S-methyl 4-[2-[(4- 6.478 μM methoxyphenyl)methoxy]ethyl- methyl-amino]-4-methyl-pent-2- ynethioate 7 S-methyl 4-[2-[(3,4- 6.085 μM dimethoxyphenyl)methoxy]ethyl- methyl-amino]-4-methyl-pent-2- ynethioate 8 S-methyl 4-[2-[(3- 7.069 μM chlorophenyl)methoxy]ethyl-methyl- amino]-4-methyl-pent-2-ynethioate 9 S-methyl 4-[2-[(3- 6.647 μM fluorophenyl)methoxy]ethyl-methyl- amino]-4-methyl-pent-2-ynethioate 10 S-methyl 4-methyl-4-[methyl-[2-(2- 5.622 μM pyridylmethoxy)ethyl]amino]pent-2- ynethioate 11 S-methyl 4-methyl-4-[methyl-[2-(3- 6.560 μM pyridylmethoxy)ethyl]amino]pent-2- ynethioate 12 S-methyl 4-methyl-4-[methyl-[2-(4- 5.684 μM pyridylmethoxy)ethyl]amino]pent-2- ynethioate 13 methyl 4-(dimethylamino)-4-methyl- 42.66 μM pent-2-ynoate 14 ethyl 4-(dimethylamino)-4-methyl- 40.00 μM pent-2-ynoate 15 tert-butyl 2-((4-(dimethylamino)-4- 25 μM methylpent-2-ynoyl)thio)acetate 16 2-((4-(dimethylamino)-4- 22.48 μM methylpent-2-ynoyl)thio)acetic acid 17 S-methyl 4-((4- 11.31 μM (benzyloxy)butyl)(methyl)amino)-4- methylpent-2-ynethioate 18 S-methyl 4-((2- 6.75 μM hydroxyethyl)(methyl)amino)-4- methylpent-2-ynethioate 19 S-methyl 4-methyl-4-[methyl-[2-(2- 11.31 μM naphthylmethoxy)ethyl]amino]pent- 2-ynethioate 20 S-methyl 4-methyl-4-[methyl-[2- 13.62 μM [(2,6,6-trimethylcyclohexen-1- yl)methoxy]ethyl]amino]pent-2- ynethioate 21 2-[(1,1-dimethyl-4-methylsulfanyl-4- 12.72 μM oxo-but-2-ynyl)-methylamino] ethyl acetate 22 2-[(1,1-dimethyl-4-methylsulfanyl-4- 15.32 μM oxo-but-2-ynyl)-methylamino] ethyl-3,4-dimethoxybenzoate 23 S-methyl 2,5,10,11,11-pentamethyl- 10.61 μM 6-oxo-7-oxa-2,5,10-triazatetradec- 12-yne-14-thioate 24 S-methyl 4-[2- 20.5 μM (methoxymethyl)pyrrolidin-1-yl]-4- methylpent-2-ynethioate 25 S-methyl 4-(3-methoxypyrrolidin-1- 16.5 μM yl)-4-methylpent-2-ynethioate 26 S-methyl 4-methyl-4-[methyl(2- 17.5 μM phenoxycyclopentyl)amino]pent-2- ynethioate 27 (S)-S-methyl 4-(2- 10.38 μM ((benzyloxy)methyl)pyrrolidin-1-yl)- 4-methylpent-2-ynethioate 28 S-methyl 4-[3(benzyloxy)- 11.83 μM 1pyrrolidinyl])-4-methylpent-2- ynethioate

Example 2: Activity of Compounds 3 and 5 in Enzymatic Assays with Human Recombinant ALDH1A1, ALDH1A2, ALDH1A3, ALDH2 and ALDH3A1

Material and Methods

[0433] Human recombinant ALDH1A1, ALDH1 A2, ALDH1A3, ALDH2 (Creative BioMart, NY, USA) were prepared at 1 mg/mL. The enzymatic reactions were performed using saturating concentrations of substrate. To test the enzymatic activity of the enzymes, 10 μL of enzyme was added into a reaction buffer containing 50 mM HEPES pH 7.2, 30 mM MgCl.sub.2, plus 20 mM NAD.sup.+ cofactor and 2 mM Hexanal (Sigma-Aldrich, St. Louis, Mo., USA) in the presence or absence of the different tested compounds. Internal standards were prepared with Nicotinamide adenine dinucleotide reduce form (NADH, 500 μM, Sigma-Aldrich) in Reaction Buffer (50 mM HEPES pH 7.2, 30 mM MgCl.sub.2).

[0434] For ALDH3A1 (1 mg/mL), 10 μL of enzyme was added into a reaction buffer containing 50 mM Tris, 5 mM DTT, pH 8, plus 40 mM Nicotinamide adenine dinucleotide phosphate (oxidized form, NADP.sup.+) and 4-Nitrobenzaldehyde (4-NBA) (Sigma-.Aldrich). Internal standards were prepared with Nicotinamide adenine dinucleotide phosphate reduced form (NADPH, 5 μM, Sigma-Aldrich) in Reaction Buffer (50 mM Tris, 5 mM DTT, pH 8).

[0435] Time-dependent inhibition assays were performed for 0-2-5-10-15-20-30-45-60 min at 37° C. in 1 mL-quartz cuvette. The formation of NADH was monitored, reading samples at excitation wavelength=340 nm/emission wavelength=460 nm (fluorescence) for at least 600 sec using Cary Eclipse Varian fluorimeter.

[0436] Negative control consisted in the same reactions except that enzyme was not added (enzyme blank). To determine the slope for enzyme blank and calculate product concentration (Unit of fluorescence) the following formulae was used:

[00001]$v=\frac{\mathrm{dF}}{\mathrm{dt}}\times \frac{\mathrm{Cst}}{\mathrm{Fst}}$

where Cst is the standard NADH concentration, Fst is the standard fluorescence and dF/dt is the slope of the time dependent fluorescence (S. Sotobodowska et al, 2012).
The specific activity of the enzymes (μmol/min.Math.mg-U/mg) in the absence of presence of inhibitors was calculated as follows:

[00002]$\text{Specific Activity}=\frac{\begin{array}{c}\mathrm{Adjusted}\mathrm{slope}\left(\mu \mathrm{mols}\mathrm{NADH}/\sec \right)\times \mathrm{time}\left(60\sec /\min \right)\times \\ \mathrm{dilution}\mathrm{factor}\end{array}}{\begin{array}{c}\mathrm{Final}\mathrm{volume}\mathrm{assay}\left(1000\mathrm{\mu L}\right)\times \mathrm{enzyme}\mathrm{volume}\left(0.01\mathrm{\mu L}\right)\times \\ \mathrm{enzyme}\mathrm{concentration}\left(\mathrm{mg}/\mathrm{ml}\right)\end{array}}\text{Specific Activity}=\frac{\begin{array}{c}\mathrm{Adjusted}\mathrm{slope}\left(\mu \mathrm{mols}\mathrm{NADH}/\sec \right)\times \mathrm{time}\left(60\sec /\min \right)\times \\ \mathrm{dilution}\mathrm{factor}\end{array}}{\begin{array}{c}\mathrm{Final}\mathrm{volume}\mathrm{assay}\left(1000\mathrm{\mu L}\right)\times \mathrm{enzyme}\mathrm{volume}\left(0.01\mathrm{\mu L}\right)\times \\ \mathrm{enzyme}\mathrm{concentration}\left(\mathrm{mg}/\mathrm{ml}\right)\end{array}}$

[0437] In said reactions, activity of compounds 3 and 5 according to the invention was compared to the one of DIMATE (S-methyl 4-(dimethylamino)-4-methylpent-2-ynethioate, described in EP 1296946).

Results

[0438] The IC.sub.50 obtained are mentioned in table 4 below.

TABLE-US-00004 TABLE 4 ALDH1A1 ALDH1A2 ALDH1A3 ALDH3A1 ALDH2 COMPOUND IC.sub.50 (μM) IC.sub.50 (μM) IC.sub.50 (μM) IC.sub.50 (μM) IC.sub.50 (μM) DIMATE 37 ± 5  18 ± 4 20 ± 2  303 ± 46 72 ± 9 (not part of the invention) Compound 3 3.8 ± 1.1 0.568 ± 0.09 3.4 ± 0.1 242 ± 11  3.1 ± 0.8 Compound 5 4.3 ± 0.4  1.3 ± 0.2 4.8 ± 0.1 143 ± 2  12 ± 3

[0439] Compounds 3 and 5 showed higher inhibition of the ALDH class 1 enzymes than that of DIMATE.

Example 3: Kinetic Parameters for Compounds 3 and 5 in Reactions with Human Recombinant ALDH1A1, ALDH1A2, ALDH1A3, ALDH2 or ALDH3A1

Material and Methods

[0440] The kinetic data are expressed as the mean±standard error from three independent determination. Kinact/Ki was determined from Kobs versus concentration of the inhibitor [I] plots. Kobs was determined from product concentration vs time incubation plot of the enzymes with compounds 3 and 5 or DIMATE at different concentration (i.e. 300 PM, 200 μM, 100 μM, 50 μM, 20 μM, and 10 μM of inhibitors). The Kobs were obtained from negative exponential fit using non-linear regression program GraFit 5.0 (Erithacus software).

Results

[0441] The kinetic parameters obtained are mentioned in table 5 below.

TABLE-US-00005 TABLE 5 ALDH1A1 ALDH1A2 ALDH1A3 ALDH3A1 ALDH2 Kinact/Ki Kinact/Ki Kinact/Ki Kinact/Ki Kinact/Ki COMPOUND (M.sup.−1 .Math. min.sup.−1) (M.sup.−1 .Math. min.sup.−1) (M.sup.−1 .Math. min.sup.−1) (M.sup.−1 .Math. min.sup.−1) (M.sup.−1 .Math. min.sup.−1) DIMATE 900  5 100  1 700 Ki = 253 ± 48 μM 425 (not part of the invention) Compound 3 500 100 000  38 500 Ki = 233 ± 16 μM 540 Compound 5 3 700 21 100 11 800 300 2450

[0442] The inhibitory potency of compounds 3 and 5 is between 4 and 20-fold higher than that of DIMATE for ALDH class 1 recombinant enzymes, in particular ALDH1A2 and ALDH1A3.

Example 4: Inhibition Type Following Full Enzymatic and Biochemical Characterization of compounds 3 and 5

Material and Methods

[0443] To determine the inhibition mechanisms of the tested compounds for each isoenzyme, the corresponding Kobs were calculated as mentioned above, plotting product concentration vs time incubation of the enzymes with the test compounds at 300 μM, 200 μM, 100 μM, 50 μM, 20 μM and 10 μM. The k.sub.obs were obtained for each concentration of inhibitor tested, from the negative exponential fit using non-linear regression program GraFit 5.0 (Erithacus software). Finally, the kinetic parameter of k.sub.inact/K.sub.I was determined from the plot of k.sub.obs versus the corresponding concentration of inhibitor. The slope of the linear fit of data indicated the rate constant in μM.sup.−1.Math.min.sup.−1. Based on the different plots obtained, the irreversible inhibition was characterized as Specific or Non-Specific Affinity Labelling.

[0444] In case of Specific Affinity Labelling the plot exhibits a saturated kobs versus concentration of inhibitor diagram (similar to a reversible inhibition plot), achieving a plateau at high concentrations of inhibitor. In Non-Specific Affinity Labelling, the dependence of k.sub.obs on inhibitor concentration appears as non-saturating.

[0445] In said experiment, compounds 3 and 5 were compared to Dimate.

Results

[0446] The results obtained are mentioned in table 6 below.

TABLE-US-00006 TABLE 6 ALDH1A1 ALDH1A2 ALDH1A3 ALDH3A1 ALDH2 Inhibition Inhibition Inhibition Inhibition Inhibition COMPOUND Type Type Type Type Type DIMATE specific Non-specific Non-specific Non- Non-specific affinity label affinity label competitive affinity label Compound 3 Non-specific Non-specific specific Non- Non-specific affinity label affinity label competitive affinity label Compound 5 Non-specific Non-specific specific Non-specific Non-specific affinity label affinity label affinity label affinity label

[0447] Although all the characterized compounds showed irreversible inhibition, the type of inactivation observed for the different isoenzymes varied between specific and non-specific affinity label. Notably, compounds 3 and 5 interact with higher specificity with ALDH1A3 while for DIMATE that inhibition takes place by a single-step mechanism of inactivation as described for non-specific affinity label, irreversible inhibitors.

Part 3: Preparation of an Antibody Drug Conjugate (ADC) According to the Invention

[0448] ADC is a three-components system including a cytotoxic agent linked via a biodegradable linker to an antibody. The antibody binds to specific markers (antigens or receptors) at the surface if the cancer cell. The whole antibody-drug conjugate is then internalized within the cancer cell, where the linker is degraded and the active drug released.

[0449] In the context of the present invention, cytotoxic agent, a compound according to the invention, is coupled to antibody via an attachment group (maleimide, succinimidyl ester, specific peptidic sequence substrate of enzyme, etc. . . . ), linked to a cleavable linker (protease site, hydrazine, disulfide) or non-cleavable and with or not a self-imolative spacer.

Synthesis of the Compound According to the Invention

[0450] Reference is made to part 1 example 29.

Conjugation with the Antibody Rituximab

[0451] Antibody rituximab (Roche@ was mixed with DTT at 37° C. for 30 minutes and then diafiltered against PBS containing 1 mmol/L EDTA using Amicon Ultra-15, MWCO 30 kDa, Merck-Millipore. The thiol concentration was quantified by Ellman's reagent, 5,5′-dithio-bis(2-nitrobenzoic acid) [DTNB]. A 50-fold molar excess of the final compound 9 obtained in the precedent paragraph dissolve in DMF, was added to the reduced antibody at 4° C. for 1 hour. Antibody-Drug conjugate was diafiltered in PBSx1 using Amicon Ultra-15, MWCO 30 kDa, Merck-Millipore. For the determination of Drug Antibody Ratio (DAR), the thiol concentration of modified antibody after coupling was quantified by Ellman's reagent.

[0452] The mechanism of release of the compound according to the invention followed by the cathepsin cleavage group is shown in the scheme below:

##STR00037##

[0453] Released Fragments:

##STR00038##

Part 4: Use of an ADC According to the Invention

Drugs/Antibody Ratio (DAR) Determination.

[0454] DAR of Antibody-Drug Conjugate mentioned in part 3 was controlled by the difference between the thiol quantification using Ellman's Reagent, after the mild thiolation of rituximab by dithiothreitol (DTT) and the quenching of these free thiol by the coupling of the maleimide group. After DTT thiolation, 10 new free thiol group were produced by Rituximab molecule. After coupling of the final product 9, the totality of these new free thiol group was quenched resulting a coupling of 10 compounds 9 per Rituximab molecule.

Cell Cytoxicity by Rituximab-Compound 9.

[0455] 50 000 viable Raji cells were plated in triplicate. Then, serial 1:2 dilutions of Rituximab-compound 9 or a control Rituximab were added to yield the final concentrations (starting concentrations 50 μg/mL). The cells were incubated for 48 h at which time 20 μl of Alamar Blue (Thermo Fisher Scientific) was added to each well. The plates were incubated for an additional four hours and the fluorescence intensity read on a plate reader using excitation wavelength of 540 nm and an emission wavelength of 620 nm.

[0456] The results show that Raji cells viability was significantly (FIG. 1; p-value <0.01; **) less using a 500 μg/ml treatment of Rituximab-compound 9 for 48 hours than Rituximab per se.