N-acyl-(3-substituted)-(8-substituted)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazines as selective NK-3 receptor antagonists
09987274 ยท 2018-06-05
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61P25/18
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A61P13/02
HUMAN NECESSITIES
A61K31/4985
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61P25/14
HUMAN NECESSITIES
A61P15/08
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P15/00
HUMAN NECESSITIES
International classification
Abstract
A method for treating and/or preventing hot flashes, by administering, to a patient in need thereof, of a pharmaceutically effective amount of a compound of Formula I ##STR00001##
or a pharmaceutically acceptable solvate thereof.
Claims
1. A method for treating and/or preventing hot flashes in a patient, comprising administering to a patient in need thereof a pharmaceutically effective amount of a compound of Formula I: ##STR00094## wherein: R.sup.1 is H, F or methyl; R.sup.1 is H; R.sup.2 is H, F, Cl or methoxy; R.sup.2 is H or F; R.sup.3 is H, F, Cl, methyl, trifluoromethyl or cyano; R.sup.4 is methyl, ethyl, n-propyl, hydroxyethyl, methoxyethyl, trifluoromethyl, difluoromethyl or fluoromethyl; R.sup.5 is methyl, ethyl, methoxymethyl, trifluoromethyl, difluoromethyl or fluoromethyl; X.sup.1 is N and X.sup.2 is S or O; or X.sup.1 is S and X.sup.2 is N; represents a single or a double bond depending on X.sup.1 and X.sup.2; and
stands for the (R)-enantiomer or for the racemate of the compound of Formula I.
2. The method according to claim 1, wherein the hot flashes are related to peri-menopausal, menopausal and/or postmenopausal conditions.
3. The method according to claim 1, wherein the hot flashes are a consequence of hormone therapy intentionally lowering the level of sex hormones.
4. The method according to claim 3, wherein the hot flashes are therapy-induced hot flashes in breast, uterine or prostate cancer.
5. The method according to claim 1, wherein the compound is of Formula Ia: ##STR00095##
6. The method according to claim 1, wherein the compound is of Formula Ia-1: ##STR00096##
7. The method according to claim 1, wherein the compound is of Formula Ia-2: ##STR00097##
8. The method according to claim 1, wherein the compound is of Formula Ia-3: ##STR00098##
9. The method according to claim 1, wherein the compound is of Formula Ib: ##STR00099## wherein: R.sup.3 is F; and R.sup.5 is methyl, ethyl, trifluoromethyl, difluoromethyl or fluoromethyl.
10. The method according to claim 1, wherein the compound is of Formula Ic: ##STR00100## wherein R.sup.4 is methyl, ethyl, n-propyl or hydroxyethyl; and R.sup.5 is methyl, ethyl or trifluoromethyl.
11. The method according to claim 1, wherein the compound is selected from the group consisting of: (R)-(3,4-dichlorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(3-(3-ethyl-1,2,4-thiadiazol-5-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone; (R)-(4-chlorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(4-chloro-3-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(3-chloro-4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(3,4,5-trifluorophenyl)methanone; (R)-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(2,3,4-trifluorophenyl)methanone; (R)-(3,4-difluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(2,3,4,5-tetrafluorophenyl)methanone; (R)-(4-fluorophenyl)(8-(2-hydroxyethyl)-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (4-fluorophenyl)(8-(2-hydroxyethyl)-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(3-(3-ethyl-1,2,4-oxadiazol-5-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone; (4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(3-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(3-chlorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(3,5-difluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(2,4-difluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(p-tolyl)methanone; (R)-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(phenyl)methanone; (R)-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-(trifluoromethyl)phenyl)methanone; (R)-(8-ethyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone; (8-ethyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone; (R)-(4-fluorophenyl)(3-(3-methyl-1,2,4-thiadiazol-5-yl)-8-propyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(4-fluoro-3-methoxyphenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(o-tolyl)methanone; (R)-(3-methoxyphenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-oxadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-4-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzonitrile; (8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(2,3,4,5-tetrafluorophenyl)methanone; (3,4-difluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(2,3,4-trifluorophenyl)methanone; (8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(3,4,5-trifluorophenyl)methanone; (3-chloro-4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (4-chloro-3-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (4-chlorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (3,4-dichlorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (3-(3-ethyl-1,2,4-thiadiazol-5-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone; (3-(3-ethyl-1,2,4-oxadiazol-5-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone; (R)-(4-fluorophenyl)(8-methyl-3-(3-(trifluoromethyl)-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; and (R)-(3-(3-(difluoromethyl)-1,2,4-thiadiazol-5-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone.
12. The method according to claim 1, wherein the compound is selected from the group consisting of: (R)-(3-(3-ethyl-1,2,4-thiadiazol-5-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone; (R)-(4-chlorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; (4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; and (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-oxadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone.
13. The method according to claim 1, wherein the compound is (R)-(4-fluorophenyl)(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone.
14. A method for treating and/or preventing hot flashes in a patient, comprising administering to a patient in need thereof a pharmaceutically effective amount of a compound of Formula I: ##STR00101## wherein: R.sup.1 is H, F or methyl; R.sup.1 is H; R.sup.2 is H, F, Cl or methoxy; R.sup.2 is H or F; R.sup.3 is H, F, Cl, methyl, trifluoromethyl or cyano; R.sup.4 is methyl, ethyl, n-propyl, hydroxyethyl, methoxyethyl, trifluoromethyl, difluoromethyl or fluoromethyl; R.sup.5 is 1-fluoroethyl, 1,1-difluoroethyl or 2,2,2-trifluoroethyl; X.sup.1 is N and X.sup.2 is S or O; or X.sup.1 is S and X.sup.2 is N; represents a single or a double bond depending on X.sup.1 and X.sup.2; and
stands for the (R)-enantiomer or for the racemate of the compound of Formula I.
15. The method according to claim 14, wherein the compound is of Formula Ib: ##STR00102## wherein: R.sup.3 is F.
16. The method according to claim 14, wherein the compound is selected from the group consisting of: (R)-(3-(3-(1,1-difluoroethyl)-1,2,4-oxadiazol-5-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone; (R)-(4-fluorophenyl)(8-methyl-3-(3-(2,2,2-trifluoroethyl)-1,2,4-oxadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone; and ((8R)-3-(3-(1-fluoroethyl)-1,2,4-oxadiazol-5-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-fluorophenyl)methanone.
17. The method according to claim 14, wherein the hot flashes are related to peri-menopausal, menopausal and/or postmenopausal conditions.
18. The method according to claim 14, wherein the hot flashes are a consequence of hormone therapy intentionally lowering the level of sex hormones.
19. The method according to claim 18, wherein the hot flashes are therapy-induced hot flashes in breast, uterine or prostate cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
EXAMPLES
Chemistry Examples
(5) All reported temperatures are expressed in degrees Celsius (? C.); all reactions were carried out at room temperature (RT) unless otherwise stated.
(6) All reactions were followed by thin layer chromatography (TLC) analysis (TLC plates, silica gel 60 F.sub.254, Merck) was used to monitor reactions, establish silica-gel flash chromatography conditions. All other TLC developing agents/visualization techniques, experimental set-up or purification procedures that were used in this invention, when not described in specific details, are assumed to be known to those conversant in the art and are described in such standard reference manuals as: i) Gordon, A. J.; Ford, R. A. The Chemist's CompanionA Handbook of Practical Data, Techniques, and References, Wiley: New York, 1972; ii) Vogel's Textbook of Practical Organic Chemistry, Pearson Prentice Hall: London, 1989.
(7) HPLC-MS spectra were typically obtained on an Agilent LCMS using electrospray ionization (ESI). The Agilent instrument includes an autosampler 1100, a binary pump 1100, an ultraviolet multi-wavelength detector 1100 and a 6100 single-quad mass-spectrometer. The chromatography column used was Sunfire 3.5 ?m, C18, 3.0?50 mm in dimensions.
(8) Eluent typically used was a mixture of solution A (0.1% TFA in H.sub.2O) and solution B (0.1% TFA in MeCN).
(9) Gradient was applied at a flow rate of 1.3 mL per minute as follows: gradient A (for analysis of final compounds and intermediates): held the initial conditions of 5% solution B for 0.2 min, increased linearly to 95% solution B in 6 min, held at 95% during 1.75 min, returned to initial conditions in 0.25 min and maintained for 2.0 min; gradient B (for analysis of crude samples and reactions mixtures): held the initial conditions of 5% solution B for 0.2 min, increased linearly to 95% in 2.0 min, held at 95% during 1.75 min, returned to initial conditions in 0.25 min and maintained for 2 min.
(10) Determination of chiral purity was made using chiral HPLC that was performed on an Agilent 1100 (binary pump and a ultraviolet multi wavelength detector) with manual or automatic (Autosampler 1100) injection capabilities. Column used is CHIRALPAK IA 5 ?m, 4.6?250 mm 4.6?250 mm in isocratic mode. Choice of eluent was predicated on the specifics of each separation. Further details concerning the chiral HPLC methods used are provided below.
(11) Method A: column CHIRALPAK IA 5 ?m, 4.6?250 mm, eluent: EtOAc plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm; column at RT, eluent was used as sample solvent.
(12) Method B: column CHIRALPAK IA 5 ?m, 4.6?250 mm, eluent: EtOAc/hexane (50:50) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm; column at RT, eluent was used as sample solvent.
(13) Method C: column CHIRALPAK IA 5 ?m 4.6?250 mm, eluent: hexane/ethanol (80:20 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(14) Method D: column CHIRALPAK IA 5 ?m 4.6?250 mm, eluent: hexane/ethanol (50:50 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(15) Method E: column CHIRALPAK ID 5 ?m 4.6?250 mm, eluent: hexane/ethanol (80:20 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(16) Method F: column CHIRALPAK IA 5 ?m 4.6?250 mm, eluent: DCM/ethanol (98:2 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(17) Method G: column CHIRALPAK IA 5 ?m 4.6?250 mm, eluent: DCM/ethanol (98:2 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(18) Method H: column CHIRALPAK IB 5 ?m 4.6?250 mm, eluent: TBME plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(19) Method I: column CHIRALPAK IC 5 ?m 4.6?250 mm, eluent: TBME/ethanol (98:2 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(20) Method J: column CHIRALPAK ID 5 m 4.6?250 mm, eluent: EtOAc/DCM/IPAethanol (3:1:1 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(21) Method K: column CHIRALPAK IC 5 ?m 4.6?250 mm, eluent: TBME/methanol (98:2 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(22) Method L: column CHIRALPAK IB 5 ?m 4.6?250 mm, eluent: TBME/methanol (98:2 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 or 280 nm, column at RT, eluent was used as sample solvent.
(23) Preparative HPLC purifications were typically carried out on an Agilent 1200 instrument (preparative pump 1200 and ultraviolet multi wavelength detector 1200) with manual injection. The chromatography column used was Waters Sunfire 5 ?m, C18, 19?100 mm, or XBridge 5 ?m, C18, 19?100 mm depending on the type of eluent system employed, i.e. low pH or high pH conditions.
(24) For high-pH HPLC purifications, eluent typically consisted of a mixture of solution A (0.04 M ammonium bicarbonate in H.sub.2O plus 0.1% of conc. NH.sub.4OH) and solution B was MeCN. The gradient was adapted depending on the impurity profile in each sample purified, thereby allowing sufficient separation between the impurities and the desired compound.
(25) In rare cases when high-pH HPLC purification did not provide sufficient purity, low-pH HPLC was applied. For low-pH HPLC purifications, eluent typically consisted of a mixture of solution A (0.1% of TFA in H.sub.2O) and solution B was MeCN. The gradient was adapted depending on the impurity profile in each sample purified, thereby allowing sufficient separation between the impurities and the desired compound. TFA was removed from evaporated fractions by liquid-liquid extraction.
(26) Chiral preparative HPLC purifications were performed on an Agilent 1200 instrument (preparative pump 1200 and ultraviolet multi wavelength detector 1200) with manual injection. The chiral columns used are CHIRALPAK IA 5 ?m, 20?250 mm or CHIRALPAK IA 5 ?m, 10?250 mm. All chiral HPLC methods were employed in an isocratic mode. The eluent mixture was selected based on the analytical chiral HPLC experiment (see above) that provided the best chiral separation.
(27) .sup.1H (300 MHz), .sup.19F (282 MHz) and .sup.13C NMR (75 MHz) spectra were recorded on a Bruker Avance DRX 300 instrument. Chemical shifts are expressed in parts per million, (ppm, ? units). Coupling constants are expressed in Hertz (Hz). Abbreviations for multiplicities observed in NMR spectra are as follows: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), br (broad).
(28) Solvents, reagents and starting materials were purchased and used as received from commercial vendors unless otherwise specified.
(29) The following abbreviations are used:
(30) Boc: tert-Butoxycarbonyl,
(31) Cpd: Compound,
(32) DAST: (Diethylamino)sulfur trifluoride,
(33) DCM: Dichloromethane,
(34) DEA: Diethylamine,
(35) DMB: 2,4-Dimethoxybenzyl,
(36) DMB-CHO: 2,4-Dimethoxybenzaldehyde,
(37) DPP: Diphenylphosphiramide,
(38) ee: Enantiomeric excess,
(39) eq.: Equivalent(s),
(40) EtOAc: Ethyl acetate,
(41) EtOH: Ethanol,
(42) g: Gram(s),
(43) h: Hour(s),
(44) IPA: iso-Propylalcohol,
(45) L: Liter(s),
(46) MeOH: Methanol,
(47) ?L: Microliter(s),
(48) mg: Milligram(s),
(49) mL: Milliliter(s),
(50) mmol: Millimole(s),
(51) min: Minute(s),
(52) P: UV purity at 254 nm or 215 nm determined by HPLC-MS,
(53) PMB: 4-Methoxybenzyl,
(54) rt: Room temperature,
(55) SES: 2-Trimethylsilylethanesulfonyl,
(56) tBu: tert-Butyl,
(57) TBDPS: tert-Butyldiphenylsilyl,
(58) TBME: tert-Butyl methyl ether,
(59) TFA: Trifluoroacetic acid,
(60) TLC: Thin layer chromatography.
(61) The intermediates and compounds described below were named using ChemBioDraw? Ultra version 12.0 (PerkinElmer).
(62) I. Racemic Synthesis
(63) I.1. General Synthetic Scheme for Racemic Synthesis
(64) Compounds of the invention may be synthesized using the methodology described in Scheme 1, which represents the racemic product synthesis. The racemic products may then be subjected to chiral HPLC for chiral separation.
(65) ##STR00078##
(66) The general synthetic scheme comprises the following steps:
(67) Step 1: DMB-protected ketopiperazine 1.1 was converted to iminoether 1 by using the Meerwein reagent (Et.sub.3OBF.sub.4).
(68) Step 2: Ester 2.2 was subsequently converted to acyl hydrazide 2. Ester 2.2 may be obtained be esterification of acid 2.1.
(69) Step 3: Cyclodehydration between the acyl hydrazide 2 and the iminoether 1 furnished the protected triazolopiperazine 3.1. Thereafter, 3.1 was subjected to acidolytic deprotection to obtain 3. When applicable, R.sup.5 was introduced from R.sup.5 affording 3.
(70) Step 4: The thus obtained triazolopiperazine intermediate 3 (or 3) was acylated through reaction with the appropriate acid chloride 4.1 to obtain the racemic final target structure represented by the general Formula 4. Optionally, R.sup.4 may be transformed, for example by reduction of R.sup.4 when R.sup.4 contains a reducible group such as an ester group. The chiral compound 4 was subsequently obtained by purification using preparative chiral HPLC.
(71) I.2. Step 1: Protection and Conversion to Iminoether 1
(72) Method A: Conversion of DMB-Protected Ketopiperazine 1.1 to Iminoether 1
(73) Method A is the procedure used for the synthesis of the iminoether intermediates 1 with a DMB protecting group and is detailed below:
(74) ##STR00079##
(75) Method A is illustrated by the synthesis of intermediate 1a wherein R.sup.4 is Me.
Synthesis of 1-(2,4-dimethoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine 1a
(76) ##STR00080##
(77) Oven-dried (115? C.) sodium carbonate (18.6 g, 98 mmol, 2.25 eq.) was placed in a 500 mL round-bottom flask. The round-bottom flask was backfilled with Ar and then capped with a rubber septum. A solution of 4-(2,4-dimethoxybenzyl)-3-methylpiperazin-2-one 1.1a (20.6 g, 78 mmol, 1 eq.) in anhydrous DCM (250 mL) was added, followed by triethyloxonium tetrafluoroborate (18.6 g, 98 mmol, 1.25 eq.) in one portion. Thereafter, the reaction mixture was stirred further at RT for 1 h whereupon the reaction mixture was diluted with water (250 mL). The aqueous layer was extracted with DCM (3?150 mL). The organic layers were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude compound was then purified on silica gel (EtOAc) to afford the desired product 1a as orange oil. Yield: 13.2 g, 58%. LCMS: P=93%, retention time=1.8 min, (M+H+H.sub.2O).sup.+: 311; .sup.1H-NMR (CDCl.sub.3): ? 7.23 (d, J=8.8 Hz, 1H), 6.48 (d, J=8.8 Hz, 1H), 6.44 (s, 1H), 4.02 (m, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.86 (d, J.sub.AB=14.0 Hz, 1H), 3.46 (d, J.sub.AB=14.0 Hz, 1H), 3.44 (m, 2H), 3.10 (m, 1H), 2.79 (m, 1H), 2.32 (m, 1H), 1.35 (d, J=6.8 Hz, 3H), 1.24 (t, J=6.0 Hz, 3H).
(78) I.3. Step 2: Formation of Acyl Hydrazide 2
(79) Method B: Acyl Hydrazide2
(80) Method B is the procedure used for the synthesis of the acyl hydrazides 2 and is detailed below:
(81) ##STR00081##
(82) In a round-bottom flask equipped with a condenser, ester 2.2 (1 eq.) is dissolved in anhydrous EtOH and treated with hydrazine hydrate (1.2 to 20 eq., preferably 1.5 to 10 eq.) using a temperature range from RT to reflux. After allowing the reaction mixture to come to RT, the solution is concentrated under reduced pressure. Co-evaporations using a mixture of commercial DCM:MeOH (1:1) may be performed to remove residual water. The residue is then recrystallized and/or precipitated or purified on a pad of silica to afford 2.
(83) I.4. Step 3: Cyclodehydration Leading to Triazolopiperazine 3
(84) Method C: Cyclodehydration and Acydolysis
(85) Method C is the procedure used for the synthesis of the triazolopiperazine 3 and is detailed below:
(86) ##STR00082##
(87) Step 1: In a round-bottom flask equipped with a condenser, imino-ether 1 (1 eq.) is dissolved in anhydrous MeOH, to which is added 2 (1 eq.) in one portion. The resulting solution is stirred at reflux overnight. Thereafter, the reaction mixture is brought to RT and the volatiles are removed under reduced pressure. The crude compound is then purified using silica gel chromatography to afford the desired product 3.1.
(88) Step 2: In a round-bottom flask containing DCM is added 3.1 (1 eq.). Then, TFA (5 to 75 eq.), is added to the reaction mixture at RT. After 30 min stirring, the mixture is concentrated. Then DCM is added to the residue thus obtained, and washed with saturated NaHCO.sub.3. The aqueous layer is extracted twice with DCM, the organic layers are washed with brine, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to obtain crude 3. The crude 3 may be directly used in the next step without further purification.
(89) In one embodiment, alternative work-up equally used involves treatment of the dried residue obtained above with 4 M HCl/dioxane (20 eq.) at RT under stirring. After 5 min, Et.sub.2O is added to help precipitation. This precipitate is filtered off under vacuum, washed with Et.sub.2O and dried under high vacuum to furnish 3 as hydrochloride salt.
(90) In another embodiment, HCl could be used for Step 2: HCl 4M solution in 1,4-dioxane (3 to 20 eq.) is added in one portion to a solution of 3.1 (1 eq.) in commercial iso-propanol or ethanol. The reaction mixture is stirred at 60? C. After complete conversion monitored by HPLC-MS (1 to 10 h), the reaction mixture is allowed to cool to room temperature and then further cooled to 0? C. with an ice bath. Thereupon, Et.sub.2O is added. After 15-30 min stirring, the precipitate is filtered and dried in vacuo to afford 3 as hydrochloride salt.
(91) Remark:
(92) When R.sup.5?R.sup.5?H, introduction of groups such as trifluoro- or difluoromethyl through direct trifluoromethylation or direct difluoromethylation (Ji Y. et al., PNAS, 2011, 108(35), 14411-14415; Fujiwara Y. et al., JACS, 2012, 134, 1494-1497) may be performed.
(93) I.5. Step 4: Acylation Leading to Final Products
(94) Method D: Acylation and Chiral HPLC Purification
(95) Method D is the procedure used for the synthesis of the racemic product 4 and its purification to obtain (R)-enantiomer 4 compounds of general Formula I. Method D is detailed below:
(96) ##STR00083##
(97) To a solution of crude 3 (1 eq.) in anhydrous DCM are added, at RT, 4.1 (1.17 to 1.3 eq.), followed by N-methylmorpholine (1 eq. to 3.5 eq.) dropwise over 15 sec. The reaction mixture is stirred at RT for 1 to 30 minutes and the milky suspension is poured into 1 M HCl solution or directly diluted with DCM. The aqueous phase is extracted with DCM. The organic phases are combined, optionally washed with 1 M NaOH, water, brine, dried over MgSO.sub.4 and evaporated to dryness. The residue is solubilized in DCM and Et.sub.2O and is slowly added to induce precipitation. The solid was filtered off, washed with Et.sub.2O and dried under vacuum to afford 4. Alternatively, the residue is preliminary purified on silica gel before precipitation or purified on silica gel only.
(98) Substituent R.sup.4 may then be transformed, when applicable, into R.sup.4. One example of such transformation is illustrated by the synthesis of compound 12 wherein R.sup.4 is hydroxyethyl group, obtained by reduction of R.sup.4=CH.sub.2CO.sub.2Alkyl.
(99) To a solution of 4 (1 eq.) in anhydrous THF is added, at ?40? C., LAH (1 eq.), The reaction mixture is stirred at ?40? C. for 5 to 30 minutes and the mixture is quenched with 1 M NaOH solution. The resulting mixture is extracted with DCM twice. The organic phases are combined, dried over MgSO.sub.4 and evaporated to dryness. The residue 4 is then purified on silica gel.
(100) Compound 4 or 4 may be purified by chiral preparative HPLC according to the abovementioned method to yield the corresponding chiral(R)-compound 4. Compounds 4, 4 and 4 are compounds of Formula I of the invention.
(101) II. Chiral Synthesis
(102) II.1. General Synthetic Scheme for Chiral Synthesis
(103) Chiral compounds of the invention may be synthesized using the chiral process of the invention described in Scheme 7.
(104) ##STR00084##
(105) Chiral ketopiperazine B was protected with PG protecting group leading to PG-protected chiral ketopiperazine C. PG-protected chiral ketopiperazine C was converted to iminoether D by using the Meerwein reagent (Et.sub.3OBF.sub.4). Condensation reaction between the acyl hydrazide E and iminoether D was conducted under heating conditions in methanol to provide PG-protected piperazine F that was subsequently deprotected to yield compound of Formula G.
(106) In one embodiment, when the protecting group PG is DMB, the DMB group deprotection step (from F to G) is carried out using TFA in DCM at rt, followed by either TFA salt exchange with HCl or extraction at high pH recovering free piperazine G.
(107) When applicable, R.sup.5 was introduced from R.sup.5 of G, affording G.
(108) Acylation of G or G with the appropriate acid chloride H afforded the (R)-enantiomer of I typically in >90% enantiomeric excess (chiral HPLC). When applicable, R.sup.4 of I was then modified to afford R.sup.4, furnishing I.
(109) When applicable, R.sup.5 of I or I was then modified to afford R.sup.5, furnishing I or I respectively.
(110) II.2. Step 1: Protection of Ketopiperazine B
(111) II.2.1. Protection of Ketopiperazine B with an Allyl to Afford Protected Ketopiperazine C.sub.1
(112) ##STR00085##
(113) Allyl protection is illustrated by the synthesis of intermediate (R)-4-allyl-3-methylpiperazin-2-one (i.e. compound C.sub.1 wherein R.sup.4 is Me).
(114) To a solution of (R)-3-methylpiperazin-2-one (0.5 g, 4.38 mmol) in commercial anhydrous THF (44 mL) at rt was added K.sub.2CO.sub.3 (1.2 g, 8.76 mmol). 3-bromoprop-1-ene (0.41 mL, 4.82 mmol) was then added at once, and the reaction mixture was stirred under reflux for 14 h.
(115) The reaction mixture was allowed to cool to rt, concentrated and the residue was then solubilized with water (10 mL) and DCM (10 mL). The organic layer was separated, dried over MgSO.sub.4, filtered and concentrated to afford 460 mg of yellow oil. .sup.1H-NMR analysis shows that desired product was clearly the main product. Crude was used as-is in the following step.
(116) LCMS: P>90%, retention time=0.2 min, (M+H).sup.+: 155. .sup.1H-NMR (CDCl.sub.3): ? 6.2 (m, 1H), 5.8 (m, 1H), 5.3 (m, 2H), 3.4 (m, 3H), 3.3 (q, J=7.2 Hz, 1H), 3.1 (m, 2H), 2.6 (m, 1H).
(117) II.2.2. Protection of Ketopiperazine B with DPP to Afford Protected Ketopiperazine C.sub.2
(118) ##STR00086##
(119) DPP protection is illustrated by the synthesis of intermediate (R)-4-(diphenylphosphoryl)-3-methylpiperazin-2-one (i.e. compound C.sub.2 wherein R.sup.4 is Me).
(120) To a solution of (R)-3-methylpiperazin-2-one (0.5 g, 4.38 mmol) in commercial anhydrous DCM (9 mL) under Ar atmosphere at rt was added diphenylphosphinic chloride (0.84 mL, 4.38 mmol) in one portion, followed by N-methylmorpholine (1.2 mL, 8.76 mmol) dropwise. The reaction mixture was stirred under reflux for 72 h.
(121) The reaction mixture was concentrated and the crude compound was then purified on silica gel (DCM/MeOH 99/1) to afford the desired product as colorless oil. Yield: 0.54 g, 88%. LCMS: P=98%, retention time=2.0 min, (M+H).sup.+: 315; chiral HPLC retention time=26.7 min, ee=99.4%; .sup.1H-NMR (CDCl.sub.3): ? 7.9 (m, 4H), 7.5 (m, 6H), 6.1 (bs, 1H), 3.9 (m, 1H), 3.6 (m, 1H), 3.3 (m, 2H), 3.2 (m, 1H), 1.5 (m, 3H).
(122) II.2.3. Protection of Ketopiperazine B with Boc to Afford Protected Ketopiperazine C.sub.3
(123) ##STR00087##
(124) Boc protection is illustrated by the synthesis of intermediate (R)-tert-butyl 2-methyl-3-oxopiperazine-1-carboxylate (i.e. compound C.sub.3 wherein R.sup.4 is Me).
(125) To a solution of (R)-3-methylpiperazin-2-one (0.33 g, 2.87 mmol) in commercial anhydrous DCM (10 mL) at 0? C. was added Boc.sub.2O (0.77 mL, 3.30 mmol) in one portion. The reaction mixture was allowed to reach rt and stirred for 1 h.
(126) The reaction mixture was concentrated under reduced pressure and the residue was taken up in DCM (100 mL) and washed with HCl 0.5M (90 mL) and brine (120 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude compound was then purified on silica gel (DCM/MeOH 99/1) to afford the desired product as colorless oil. Yield: 0.45 g, 33%. LCMS: P=98%, retention time=1.9 min, (M+H).sup.+: 215; .sup.1H-NMR (CDCl.sub.3): ? 6.3 (bs, 1H), 4.6 (m, 1H), 4.1 (m, 1H), 3.5 (m, 1H), 3.3-3.1 (m, 2H), 1.5 (m, 3H), 1.4 (s, 9H).
(127) II.2.4. Protection of Ketopiperazine B with SES to Afford Protected Ketopiperazine C.sub.4
(128) ##STR00088##
(129) SES protection is illustrated by the synthesis of intermediate (R)-3-methyl-4-((2-(trimethylsilyl)ethyl)sulfonyl)piperazin-2-one (i.e. compound C.sub.4 wherein R.sup.4 is Me).
(130) To a solution of (R)-3-methylpiperazin-2-one (0.25 g, 2.19 mmol) in commercial anhydrous DCM (4.5 mL) under Ar atmosphere at rt was added 2-(trimethylsilyl)ethanesulfonyl chloride (0.44 mL, 2.30 mmol) in one portion, followed by N-methylmorpholine (0.45 mL g, 4.38 mmol) dropwise. The reaction mixture was stirred at rt for 16 h.
(131) The reaction mixture was diluted with water (10 mL) and DCM (10 mL). The organic layer was separated, dried over MgSO.sub.4, filtered and concentrated. The crude compound was then purified on silica gel (DCM/MeOH 99/1) to afford the desired product as colorless oil. Yield: 0.08 g, 13%. LCMS: P=95%, retention time=2.1 min, (M+H).sup.+: 279; chiral HPLC retention time=7.2 min, ee=99.6%; .sup.1H-NMR (CDCl.sub.3): ? 6.1 (bs, 1H), 4.5 (m, 1H), 3.8 (m, 1H), 3.6 (m, 1H), 3.4 (m, 1H), 3.3 (m, 1H), 2.9 (m, 2H), 1.6 (m, 3H), 1.0 (m, 2H), 0.1 (s, 9H).
(132) II.3. Step 2: Conversion to Iminoether D
(133) Method E: Conversion to Iminoether
(134) General Method E is the procedure used for the synthesis of intermediates D.
(135) ##STR00089##
(136) Method E is illustrated by the synthesis of intermediate (R)-1-(2,4-dimethoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine D.sub.5-1(i.e. compound D wherein PG is DMB and R.sup.4 is Me). The corresponding DMB-protected ketopiperazine C.sub.5 is commercially available.
(137) Oven dried (115? C.) sodium carbonate (2.48 g, 23.40 mmol, 2.25 eq.) was placed in a round-bottom flask. The round-bottom flask was backfilled with Ar and then capped with a rubber septum. A solution of (R)-4-(2,4-dimethoxybenzyl)-3-methylpiperazin-2-one C-1 (2.75 g, 10.40 mmol, 1 eq.) in anhydrous DCM (35 mL) was added, followed by freshly prepared triethyloxonium tetrafluoroborate (2.48 g, 13.05 mmol, 1.25 eq.) in one portion. Thereafter the reaction mixture was stirred further at rt for 45 min to 1 hour, whereupon the reaction mixture was diluted with saturated aqueous NaHCO.sub.3 (100 mL). The aqueous layer was extracted with DCM (3?200 mL). The organic layers were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford 3.1 g of yellow oil. The crude compound was then purified on silica gel (EtOAc/MeOH: 99/1) to afford the desired product D-1 as a pale yellow oil. Yield: 1.44 g, 48%. LCMS: P=95%, retention time=1.8 min, (M+H2O+H).sup.+: 311; chiral HPLC retention time=12.3 min, ee>97%. .sup.1H-NMR (CDCl.sub.3): ? 7.23 (d, J=8.8, 1H), 6.48 (d, J=8.8, 1H), 6.44 (s, 1H), 4.02 (m, 2H), 3.92 (s, 6H), 3.86 (d, J.sub.AB=14.0, 1H), 3.46 (d, J.sub.AB=14.0, 1H), 3.44 (m, 2H), 3.10 (m, 1H), 2.79 (m, 1H), 2.32 (m, 1H), 1.35 (d, J=6.8, 3H), 1.24 (t, J=6.0, 3H).
(138) The reaction mixture may alternatively be treated with brine. After stirring far about 20 min, additional water and DCM were added leading to phase separation. The organic layers were then dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude compound was then purified on silica gel.
(139) Method E is further illustrated by the synthesis of intermediate (R)-1-allyl-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine (i.e. compound D.sub.1 wherein PG is allyl and R.sup.4 is Me). To a solution of (R)-4-allyl-3-methylpiperazin-2-one (0.35 g, 2.27 mmol, 1 eq.) in DCM (7.6 mL) at 0? C. was added sodium carbonate (0.54 g, 5.11 mmol, 2.25 eq.) in one portion, followed by commercial triethyloxonium tetrafluoroborate (0.54 g, 2.84 mmol, 1.25 eq.) in one portion. Thereafter the reaction mixture was stirred further at rt for 45 min, whereupon the reaction mixture was diluted with DCM (10 mL) and brine (10 mL). The layers were separated and the aqueous layer was further extracted with DCM (2?5 mL). The organic layers were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude compound was then purified on silica gel (EtOAc) to afford the desired product as colorless oil. Yield: 0.19 g, 46%. LCMS: P=95%, retention time=1.5 min, (M+H).sup.+: 183; .sup.1H-NMR (CDCl.sub.3): ? 5.9 (m, 1H), 5.2 (m, 2H), 4.0 (m, 2H), 3.5 (m, 2H), 3.3 (m, 1H), 3.1-3.0 (m, 2H), 2.8 (m, 1H), 2.4 (m, 1H), 1.3 (m, 6H).
(140) The following intermediates were also prepared from the ad hoc reagents:
(141) (R)-(3-ethoxy-2-methyl-5,6-dihydropyrazin-1(2H)-yl)diphenylphosphine oxide in 44% yield. LCMS: P=98%, retention time=2.0 min, (M+H.sub.2O+H).sup.+: 361; chiral HPLC retention time=4.8 min, ee=99.4%; .sup.1H-NMR (CDCl.sub.3): ? 7.9 (m, 4H), 7.5 (m, 6H), 4.0 (m, 2H), 3.7 (m, 1H), 3.6 (m, 1H), 3.5 (m, 1H), 3.1 (m, 2H), 1.4 (m, 3H), 1.2 (m, 3H).
(142) (R)-tert-butyl 3-ethoxy-2-methyl-5,6-dihydropyrazine-1 (2H)-carboxylate in 68% yield. LCMS: P=98%, retention time=1.8 min, (M+H.sub.2O+H).sup.+: 261; .sup.1H-NMR (CDCl.sub.3): ? 4.3 (m, 1H), 4.1 (m, 2H), 3.9 (m, 1H), 3.5 (m, 2H), 2.9 (m, 1H), 1.5 (s, 9H), 1.3 (d, J=6.9 Hz, 3H), 1.2 (t, J=7.0 Hz, 3H).
(143) (R)-5-ethoxy-6-methyl-1-((2-(trimethylsilyl)ethyl)sulfonyl)-1,2,3,6-tetrahydropyrazine in 68% yield. LCMS: P=70%, retention time=2.0 min, (M+H.sub.2O+H).sup.+: 325; chiral HPLC retention time=4.8 min, ee=97.3%; .sup.1H-NMR (CDCl.sub.3): ? 4.3 (m, 1H), 4.1 (m, 2H), 3.6 (m, 3H), 3.2 (m, 1H), 2.9 (m, 2H), 1.5 (m, 3H), 1.3 (m, 3H), 1.0 (m, 2H), 0.0 (s, 9H).
(144) II.4. Step 3: Cyclodehydration Leading to F
(145) Method F: Cyclodehydration
(146) General Method F is the general procedure used for the synthesis of chiral triazolopiperazine intermediates F.
(147) ##STR00090##
(148) In a round-bottom flask equipped with a condenser, imino-ether D (1 eq.) was dissolved in anhydrous MeOH, to which was added E (1 eq.) in one portion. The resulting solution was stirred at a temperature ranging from 55? C. to 70? C. for a period of time ranging from 6 hours to 8 hours. Completion of the reaction was monitored by HPLC analysis. The reaction mixture was cooled down to rt and the solvent was removed under reduced pressure. The crude compound was then purified by silica gel chromatography to afford the desired product F.
(149) In an embodiment of the invention, the crude compound precipitates during cooling of the reaction mixture. In this case, the precipitate is stirred at rt in MeOH for about 5 hours before being filtered, washed with MeOH and oven dried.
(150) Cyclodehydration is illustrated by the synthesis of intermediate (R)-5-(7-allyl-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-3-methyl-1,2,4-thiadiazole (i.e. compound F.sub.1 wherein PG is allyl, R.sup.4 is Me, X.sup.1 is N, X.sup.2 is S and R.sup.5 is methyl).
(151) To (R)-1-allyl-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine (0.14 g, 0.77 mmol) at rt was added 3-methyl-1,2,4-thiadiazole-5-carbohydrazide (0.12 g, 0.77 mmol) at once. The mixture was diluted with commercially anhydrous MeOH (0.77 mL) to allow complete solubilization and the resulting mixture was heated to 60? C. for 16 h.
(152) The reaction mixture was then allowed to reach rt whereupon the solvent was removed under reduced pressure (1-2 mbar). The crude residue was then dissolved in DCM (10 mL), and thus-obtained organic phase washed with NaOH (1 M, 10 mL). The organic layer was then dried over MgSO.sub.4, filtered and concentrated under reduced pressure (1-2 mbar) the desired product as a yellow solid. Yield: 0.09 g, 42%. LCMS: P=95%, retention time=1.6 min, (M+H).sup.+: 277; chiral HPLC retention time=21.6 min, ee=98.9%; .sup.1H-NMR (CDCl.sub.3): ? 5.9 (m, 1H), 5.3 (m, 2H), 4.5 (m, 1H), 4.4 (m, 1H), 4.1 (m, 1H), 3.5 (m, 1H), 3.3 (m, 1H), 3.1 (m, 1H), 2.8 (m, 1H), 2.7 (s, 3H), 1.6 (m, 3H).
(153) The following intermediates were also prepared from the ad hoc reagents:
(154) (R)-(8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)diphenylphosphine oxide in 31% yield (reaction time: 48 h and silica gel purification (EtOAc)). LCMS: P=96%, retention time=2.2 min, (M+H).sup.+: 437; chiral HPLC retention time=7.5 min, ee=98.3%; .sup.1H-NMR (CDCl.sub.3): ? 7.9 (m, 4H), 7.5 (m, 6H), 4.9 (m, 1H), 4.8 (dd, J=3.1, 13.6 Hz, 1H), 4.3 (dt, J=4.9, 12.2 Hz, 1H), 3.6 (m, 1H), 3.5 (m, 1H), 2.7 (s, 3H), 1.6 (d, J=6.9 Hz, 3H).
(155) (R)-tert-butyl 8-methyl-3-(3-methyl-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]triazolo [4,3-a]pyrazine-7(8H)-carboxylate in 83% yield (reaction time: 48 h). LCMS: P=97%, retention time=2.3 min, (M+H).sup.+: 337; chiral HPLC retention time=19.4 min, ee=95.1%; .sup.1H-NMR (CDCl.sub.3): ? 5.7 (m, 1H), 4.9 (m, 1H), 4.5 (m, 1H), 4.2 (m, 1H), 3.3 (m, 1H), 2.7 (s, 3H), 1.6 (d, J=6.9 Hz, 3H), 1.5 (s, 9H).
(156) (R)-3-methyl-5-(8-methyl-7-((2-(trimethylsilyl)ethyl)sulfonyl)-5,6,7,8-tetrahydro-[1,2,4]triazol[4,3-a]pyrazin-3-yl)-1,2,4-thiadiazole in 28% yield (reaction time: 48 h). LCMS: P=40%, retention time=2.5 min, (M+H).sup.+: 401; chiral HPLC retention time=7.1 min, ee=92.4%; .sup.1H-NMR (CDCl.sub.3): ? 4.9 (m, 1H), 4.3 (m, 1H), 4.1 (m, 1H), 3.6 (m, 1H), 3.0 (m, 1H), 2.7 (s, 3H), 1.6 (m, 2H), 1.4 (m, 3H), 1.0 (m, 2H), 0.0 (s, 9H).
(157) II.5. Step 4: PG-Deprotection
(158) The methods of deprotection of above Protecting Groups (PGs) are known to those skill-in-the-art. As examples, one may refer to Greene's Protective Groups in Organic Synthesis: Allyl: p. 806 of fourth edition; DPP: p. 844 of fourth edition; Boc: p. 725 of fourth edition; SES: p. 854 in fourth edition.
(159) Method G: DMB DeprotectionTFA/DCM
(160) ##STR00091##
(161) Deprotection of DMB may be performed using TFA.
(162) When crude or precipitated F was used (in opposition to purified F on silica gel), pre-washing was performed before deprotection as follow: F was dissolved in DCM and optionally washed with 1M NaOH in order to remove remaining E. The DCM extracts were then dried over magnesium sulphate, filtered and the filter cake washed with DCM.
(163) F was diluted with DCM and TFA (7.6 eq.) was added to the DCM solution of F at RT. The mixture was stirred at rt for 2 h-2 h30. Completion of the deprotection was monitored by HPLC. Water was added, the mixture stirred for 30 minutes and filtered. The filter cake was washed with water and DCM. The filtrate layers were separated. The pH of the aqueous layer was adjusted to 12-13 by the addition of 4M NaOH. Sodium chloride was then added and the aqueous solution was extracted with DCM. The DCM extract comprising G was concentrated and was used in the next step without further purification.
(164) II.6. Optional Conversion of R.sup.5 to R.sup.5 in Triazolopiperazine G
(165) ##STR00092##
(166) Substituent R.sup.5 may then be introduced, when applicable, from R.sup.5 (especially when R.sup.5?H). One example of such transformation is illustrated by the synthesis of intermediate G wherein R.sup.5 is trifluoromethyl.
(167) To a solution of G (1 eq.) in DCM/water (3/1) are added, at rt, sodium trifluoromethansulfinate (3 eq.) and 2-hydroperoxy-2-methylpropane (5 eq.). The reaction mixture is not stirred and left at rt. Monitoring conversion by HPLC-MS, extra amount of each reagent can be added if required. The resulting mixture is diluted with DCM and quenched with 4 M NaOH saturated solution. Layers were separated and aqueous layer was extracted twice with EtOAc. The organic phases are combined, dried over MgSO.sub.4 and evaporated to dryness. The residue is purified on silica gel or used crude in next step.
(168) A further example of such transformation is illustrated by the synthesis of intermediate G wherein R.sup.5 is difluoromethyl:
(169) To a suspension of G (R.sup.5?H) (R)-5-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-1,2,4-thiadiazole (0.29 g, 1.13 mmol) and bis(difluoromethylsulfinyloxy)zinc (0.67 g, 2.26 mmol) in DCM (5 mL) and Water (2 mL), was added TFA (0.09 mL, 1.13 mmol), followed by slow addition of 2-hydroperoxy-2-methylpropane (0.77 mL, 5.64 mmol) with vigorous stirring.
(170) When conversion was not increasing any more (HPLC-MS monitoring) bis(difluoromethylsulfinyloxy)zinc and 2-hydroperoxy-2-methylpropane were added at rt still with vigorous stirring (3 additional times (1.001 g, 3.39 mmol) and (0.773 mL, 5.64 mmol) respectively).
(171) After 4 days in total, reaction mixture was diluted with EtOAc (50 mL) and carefully quenched with NaHCO.sub.3 sat. solution (30 mL) and then NaHCO.sub.3 solid until no bubbling was observed. Reaction mixture was filtered on Celite pad and phases of the filtrate were separated. Aqueous phase was filtered again on Celite pad then filtrate was extracted with EtOAc (2?50 mL). Organic phases were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude compound was then purified on silica gel (DCM/MeOH 99/1) to afford the desired product as colorless oil. Yield: 0.03 g, 10%. LCMS: P=97%, retention time=1.8 min, (M+H).sup.+: 273; .sup.1H-NMR (CDCl.sub.3): ? 6.8 (t, J.sub.H-F=53.5 Hz, 1H), 4.7 (m, 1H), 4.3 (m, 2H), 3.5 (m, 1H), 3.3 (m, 1H), 1.7 (d, J=6.7 Hz, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?113.5 (dd, J=3.2, 53.5 Hz, 2F).
(172) II.7. Step 5: Acylation Leading to Products I
(173) Method H: Acylation NMM/DCM
(174) General Method H is the general procedure used for the synthesis of (R)-enantiomer of Formula I of the invention.
(175) ##STR00093##
(176) To a solution of crude G or G (1 eq.) in anhydrous DCM were added at 0? C. H (1.3 eq.), followed by N-methylmorpholine (2.2 eq.) dropwise over 15 sec. The reaction mixture was stirred at rt for 10 minutes and, the milky suspension was poured into 1 M HCl. The aqueous phase was extracted with DCM. The organic phases were combined, washed with 1 M NaOH, brine, dried over MgSO.sub.4 and evaporated to dryness. The crude compound was purified by silica gel chromatography to afford the desired product (R)-I.
(177) Measurement of % ee confirmed that no detectable racemization occurs during the acidolytic deprotection and N-acylation steps.
(178) Method I: AcylationBiphasic Conditions
(179) Alternatively, the reaction may be performed under biphasic conditions.
(180) In this case, saturated sodium hydrogen carbonate solution was added to the DCM slurry of G or G (1 eq.) at rt. H (1 eq.) was added and the mixture stirred for a period of time ranging from about 20 minutes to overnight at rt. Completion of the reaction was monitored by HPLC. The layers were separated and the DCM phase washed with water. The DCM extracts were dried with magnesium sulphate and filtered, washing the filter cake with DCM. The DCM extracts were then concentrated. TBME was added and the resulting slurry stirred overnight at rt. The solid was collected by filtration, washed with TBME and pulled dry. The crude compound may be purified by silica gel chromatography or by crystallisation.
(181) Measurement of % ee confirmed that no detectable racemization occurs during the acidolytic deprotection and N-acylation steps.
(182) Substituent R.sup.4 may then be transformed, when applicable, into R.sup.4 (see racemic synthesis).
(183) II.8. Optional Further Transformation Leading to Products I/I from I/I
(184) Compound 45: From compound I/I wherein R.sup.5=1-((tert-butyldiphenylsilyl)oxy)ethyl, well known tert-butylamonium fluoride TBDPS deprotection of alkoxy was applied, followed by DAST fluorination of the latter alcohol, leading to racemic compound 45. Both diastereomers can be separated by purification on preparative HPLC to afford 45-1 and 45-2.
(185) Compound 43: From compound I/I wherein R.sup.5=1-((tert-butyldiphenylsilyl)oxy)ethyl, well known tert-butylamonium fluoride TBDPS deprotection of alkoxy was applied, followed by Dess-Martin oxidation, then followed by DAST fluorination of the latter ketone, leading to compound 43.
(186) III. Chemical Characterization
(187) Compound 1: HPLC-MS: t.sub.R=4.1 min, (M+H).sup.+=409; Chiral HPLC (Method C): % ee=99.0; .sup.1H-NMR (CDCl.sub.3): ? 7.6 (m, 2H), 7.3 (m, 1H), 5.8 (m, 1H), 4.9 (m, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 2.7 (s, 3H), 1.7 (d, 3H).
(188) Compound 2: HPLC-MS: t.sub.R=3.8 min, (M+H).sup.+=373; Chiral HPLC (Method A): % ee=98.0; .sup.1H-NMR (300 MHz, CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 3.1 (q, 2H), 1.8 (d, 3H), 1.4 (t, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?98.5.
(189) Compound 3: HPLC-MS: t.sub.R=3.8 min, (M+H).sup.+=375; Chiral HPLC (Method C): % ee>99.8; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 4H), 5.8 (m, 1H), 4.9 (m, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 2.7 (s, 3H), 1.7 (d, 3H).
(190) Compound 4: HPLC-MS: t.sub.R=3.9 min, (M+H).sup.+=393; Chiral HPLC (Method C): % ee=99.0; .sup.1H-NMR (CDCl.sub.3): ? 7.6 (m, 1H), 7.3 (s, 1H), 7.2 (m, 1H), 5.8 (m, 1H), 4.9 (m, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 2.7 (s, 3H), 1.7 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?98.4.
(191) Compound 5: HPLC-MS: t.sub.R=3.4 min, (M+H).sup.+=359; Chiral HPLC (Method C): % ee=99.0; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.3 (m, 2H), 5.8 (m, 1H), 4.9 (m, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.5 (m, 1H), 2.7 (s, 3H), 1.7 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?98.4.
(192) Compound 6: HPLC-MS: t.sub.R=3.8 min, (M+H).sup.+=393; Chiral HPLC (Method C): % ee=99.5; .sup.1H-NMR (CDCl.sub.3): ? 7.6 (m, 1H), 7.3 (m, 2H), 5.7 (m, 1H), 4.9 (m, 1H), 4.5 (m, 1H), 4.3 (m, 1H), 3.5 (m, 1H), 2.7 (s, 3H), 1.8 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?96.2.
(193) Compound 7: HPLC-MS: t.sub.R=3.8 min, (M+H).sup.+=395; Chiral HPLC (Method C): % ee=98.9; .sup.1H-NMR (CDCl.sub.3): ? 7.1 (m, 2H), 5.8 (m, 1H), 5.0 (m, 1H), 4.5 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 2.7 (s, 3H), 1.8 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?75.8.
(194) Compound 8: HPLC-MS: t.sub.R=3.7 min, (M+H).sup.+=395; Chiral HPLC (Method C): % ee=99.0; .sup.1H-NMR (CDCl.sub.3): ? 7.1 (m, 2H), 6.2 (m, 1H), 5.3-5.0 (m, 2H), 4.3-3.6 (m, 2H), 2.7 (s, 3H), 1.8 (m, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?49.4, ?72.0, ?77.4.
(195) Compound 9: HPLC-MS: t.sub.R=3.6 min, (M+H).sup.+=377; Chiral HPLC (Method C): % ee=99.4; .sup.1H-NMR (CDCl.sub.3): ? 7.3 (m, 3H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.7 (s, 3H), 1.7 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?72.1, ?74.4.
(196) Compound 10: HPLC-MS: t.sub.R=4.0 min, (M+H).sup.+=413; Chiral HPLC (Method C): % ee=99.0; .sup.1H-NMR (CDCl.sub.3): ? 7.2 (m, 1H), 6.2 (m, 1H), 5.2-5.0 (m, 2H), 4.3 (m, 1H), 3.9-3.4 (m, 2H), 2.7 (s, 3H), 1.8 (m, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?54.2, ?56.3, ?67.1, ?72.8.
(197) Compound 11: HPLC-MS: t.sub.R=3.2 min, (M+H).sup.+=389; Chiral HPLC (Method A): % ee>99.8; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 6.1 (m, 1H), 4.9 (dd, 1H), 4.3 (m, 2H), 3.9 (m, 2H), 3.6 (m, 1H), 2.7 (s, 3H), 2.4 (m, 1H), 2.2 (m, 1H); .sup.19F-NMR (CDCl.sub.3): ? ?97.9.
(198) Compound 12: is racemate of compound 11.
(199) Compound 13: HPLC-MS: t.sub.R=4.1 min, (M+H).sup.+=357; Chiral HPLC (Method B): % ee=98.7; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 5.8 (m, 1H), 4.8 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.9 (q, 2H), 1.8 (d, 3H), 1.4 (t, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?98.7.
(200) Compound 14: is racemate of compound 5.
(201) Compound 15: HPLC-MS: t.sub.R=3.4 min, (M+H).sup.+=359; Chiral HPLC (Method B): % ee>99.8; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 1H), 7.2 (m, 3H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.7 (s, 3H), 1.7 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?96.2.
(202) Compound 16: HPLC-MS: t.sub.R=3.7 min, (M+H).sup.+=375; .sup.1H-NMR (CDCl.sub.3): ? 7.5-7.3 (m, 4H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.7 (s, 3H), 1.7 (d, 3H).
(203) Compound 17: HPLC-MS: t.sub.R=3.6 min, (M+H).sup.+=377; .sup.1H-NMR (CDCl.sub.3): ? 7.3 (m, 1H), 7.0 (m, 2H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.8 (s, 3H), 1.8 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?101.2.
(204) Compound 18: HPLC-MS: t.sub.R=3.5 min, (M+H).sup.+=377; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 1H), 7.0-6.9 (m, 2H), 6.2 (m, 1H), 5.2-4.9 (m, 2H), 4.3 (m, 1H), 4.0-3.7 (m, 1H), 2.7 (s, 3H), 1.7 (m, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?95.7, ?102.5.
(205) Compound 19: HPLC-MS: t.sub.R=3.6 min, (M+H).sup.+=355; .sup.1H-NMR (CDCl.sub.3): ? 7.4 (m, 4H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.7 (s, 3H), 2.4 (s, 3H), 1.7 (d, 3H).
(206) Compound 20: HPLC-MS: t.sub.R=3.3 min, (M+H).sup.+=341; Chiral HPLC (Method C): % ee=96.8; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 5H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.7 (s, 3H), 1.7 (d, 3H).
(207) Compound 21: HPLC-MS: t.sub.R=4.0 min, (M+H).sup.+=409; .sup.1H-NMR (CDCl.sub.3): ? 7.7 (d, 2H), 7.6 (d, 1H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (m, 1H), 2.7 (s, 3H), 1.7 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?60.1.
(208) Compound 22: HPLC-MS: t.sub.R=3.7 min, (M+H).sup.+=373; Chiral HPLC (Method B): % ee>99.7; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.3 (m, 2H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 2.7 (s, 3H), 2.2-2.0 (m, 2H), 1.1 (m, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?98.4.
(209) Compound 23: is racemate of compound 22.
(210) Compound 24: HPLC-MS: t.sub.R=4.0 min, (M+H).sup.+=387; Chiral HPLC (Method D): % ee=95.5; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 5.8 (m, 1H), 4.9 (m, 1H), 4.6 (m, 1H), 4.2 (m, 1H), 3.6 (m, 1H), 2.7 (s, 3H), 2.1-2.0 (m, 2H), 1.6 (m, 2H), 1.0 (m, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?98.2.
(211) Compound 25: HPLC-MS: t.sub.R=3.5 min, (M+H).sup.+=389; .sup.1H-NMR (CDCl.sub.3): ? 7.2 (m, 2H), 7.0 (m, 2H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.9 (s, 3H), 3.5 (td, 1H), 2.7 (s, 3H), 1.8 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?76.3.
(212) Compound 26: HPLC-MS: t.sub.R=3.5 min, (M+H).sup.+=355; .sup.1H-NMR (CDCl.sub.3): ? 7.4-7.2 (m, 4H), 6.3 (m, 1H), 5.3-4.8 (m, 2H), 4.3-3.8 (m, 1H), 3.5-3.4 (m, 1H), 2.7 (2s, 3H), 2.3 (s, 3H). 1.7 (2s, 3H).
(213) Compound 27: HPLC-MS: t.sub.R=3.4 min, (M+H).sup.+=371; .sup.1H-NMR (CDCl.sub.3): ?7.4 (m, 1H), 7.0 (m, 3H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.8 (s, 3H), 3.5 (td, 1H), 2.7 (s, 3H), 1.7 (d, 3H).
(214) Compound 28: HPLC-MS: t.sub.R=3.1 min, (M+H).sup.+=343; Chiral HPLC (Method C): % ee=96.1; .sup.1H-NMR (CDCl.sub.3): ?7.5 (m, 2H), 7.2 (m, 2H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.5 (td, 1H), 2.5 (s, 3H), 1.7 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ??98.3.
(215) Compound 29: HPLC-MS: t.sub.R=3.2 min, (M+H).sup.+=366; Chiral HPLC (Method B): % ee=99.0; .sup.1H-NMR (CDCl.sub.3): ? 7.8 (d, 2H), 7.6 (d, 2H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.7 (s, 3H), 1.7 (d, 3H).
(216) Compound 30: HPLC-MS: t.sub.R=4.9 min, (M+H).sup.+=421; Chiral HPLC (Method A): % ee=98.2; .sup.1H-NMR (CDCl.sub.3): ?7.7 (d, 2H), 7.5 (d, 2H), 7.4 (m, 2H), 7.1 (m, 1H), 5.9 (m, 1H), 4.8 (dd, 1H), 4.7 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 2.9 (q, 2H), 1.8 (d, 3H), 1.4 (t, 3H).
(217) Compound 31: is racemate of compound 10.
(218) Compound 32: is racemate of compound 9.
(219) Compound 33: is racemate of compound 8.
(220) Compound 34: is racemate of compound 7.
(221) Compound 35: is racemate of compound 6.
(222) Compound 36: is racemate of compound 4.
(223) Compound 37: is racemate of compound 3.
(224) Compound 38: is racemate of compound 1.
(225) Compound 39: is racemate of compound 2.
(226) Compound 40: is racemate of compound 13.
(227) Compound 41: HPLC-MS: t.sub.R=4.8 min, (M+H).sup.+=413; Chiral HPLC (Method B): % ee=99.7; .sup.1H-NMR (CDCl.sub.3): ?7.5 (m, 2H), 7.2 (m, 2H), 5.8 (m, 1H), 4.9 (dd, 1H), 4.6 (m, 1H), 4.3 (td, 1H), 3.6 (td, 1H), 1.8 (d, 3H); .sup.19F-NMR (CDCl.sub.3): ??62.9, ?98.7.
(228) Compound 42: HPLC-MS: t.sub.R=4.3 min, (M+H).sup.+=395; Chiral HPLC (Method B): % ee=97.4; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.1 (m, 2H), 6.8 (t, J.sub.H-F=53.5 Hz, 1H), 5.8 (m, 1H), 4.9 (dd, J=3.1, 13.6 Hz, 1H), 4.6 (m, 1H), ), 4.3 (dt, J=4.6, 13.3 Hz, 1H), 3.6 (m, 1H), 1.8 (d, J=6.9 Hz, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?105.2 (s, 1F), ?113.4 (dd, J=9.6, 53.4 Hz, 2F).
(229) Compound 43: HPLC-MS: t.sub.R=4.4 min, (M+H).sup.+=393; Chiral HPLC (Method C): % ee=96.3; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 5.9 (m, 1H), 4.8 (dd, J=3.3, 13.5 Hz, 1H), 4.6 (m, 1H), 4.3 (dt, J=4.2, 12.7 Hz, 1H), 3.6 (m, 1H), 2.2 (t, J=8.6 Hz, 3H), 1.8 (d, J=6.9 Hz, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?88.3 (q, J=18.3 Hz, 2F), ?105.0 (s, 1F).
(230) Compound 44: HPLC-MS: t.sub.R=4.4 min, (M+H).sup.+=411; Chiral HPLC (Method C): % ee=98.6; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 5.8 (m, 1H), 4.8 (dd, J=3.5, 13.6 Hz, 1H), 4.6 (m, 1H), 4.3 (dt, J=4.0, 12.2 Hz, 1H), 3.7 (q, J=10.0 Hz, 2H), 3.6 (m, 1H), 1.8 (d, J=6.9 Hz, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?61.1 (t, J=9.6 Hz, 1F), ?105.0 (s, 1F).
(231) Compound 45: HPLC-MS: t.sub.R=4.4 min, (M+H).sup.+=375; Chiral HPLC (Method C): % ee=98.5; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 5.9 (m, 1H), 5.8 (m, 1H), 4.9 (m, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 1.9 (d, J=6.9 Hz, 3H), 1.8 (m, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?105.0 (s, 1F), ?175.0 (m, 1F).
(232) Compound 45-1: HPLC-MS: t.sub.R=4.4 min, (M+H).sup.+=375; Chiral HPLC (Method C): % ee=99.2; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 5.9 (m, 1H), 5.8 (m, 1H), 4.9 (m, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 1.9 (d, J=6.9 Hz, 3H), 1.8 (m, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?105.0 (s, 1F), ?175.0 (m, 1F).
(233) Compound 45-2: HPLC-MS: t.sub.R=4.4 min, (M+H).sup.+=375; Chiral HPLC (Method C): % ee=91.7; .sup.1H-NMR (CDCl.sub.3): ? 7.5 (m, 2H), 7.2 (m, 2H), 5.9 (m, 1H), 5.8 (m, 1H), 4.9 (m, 1H), 4.6 (m, 1H), 4.3 (m, 1H), 3.6 (m, 1H), 1.9 (d, J=6.9 Hz, 3H), 1.8 (m, 3H); .sup.19F-NMR (CDCl.sub.3): ? ?105.0 (s, 1F), ?175.0 (m, 1F).
Biology Examples
(234) Functional Assay
(235) Aequorin Assay with Human NK-3 Receptor
(236) Changes in intracellular calcium levels are a recognized indicator of G protein-coupled receptor activity. The efficacy of compounds of the invention to inhibit NKA-mediated NK-3 receptor activation was assessed by an in vitro Aequorin functional assay. Chinese Hamster Ovary recombinant cells expressing the human NK-3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. In the presence of the cofactor coelenterazine, apoaequorin emits a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium.
(237) Antagonist Testing
(238) The antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound (at various concentrations) with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC.sub.80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the concentration-dependent inhibition of the luminescence response to the addition of Neurokinin A.
(239) Inhibition curves are obtained for compounds of the invention and the concentrations of compounds which inhibit 50% of reference agonist response (IC.sub.50) were determined (see results in table 2 below). The IC.sub.50 values shown in table 2 indicate that compounds of the invention are potent NK-3 antagonist compounds.
(240) Competitive Binding Assays
(241) The affinity of compounds of the invention for the human NK-3 receptor was determined by measuring the ability of compounds of the invention to competitively and reversibly displace a well-characterized NK-3 radioligand in a concentration-dependent manner.
(242) .sup.3H-SB222200 Binding Competition Assay with Human NK-3 Receptor
(243) The ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist .sup.3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK-3 receptor. The membranes were incubated with 5 nM .sup.3H-SB222200 (ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl.sub.2 1 mM/MgCl.sub.2 5 mM/BSA 0.5%/Saponin 10 ?g/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of .sup.3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentration that displaced 50% of bound radioligand (IC.sub.50) were determined by linear regression analysis and then the apparent inhibition constant (K.sub.i) values were calculated by the following equation: K.sub.i=IC.sub.50/(1+[L]/K.sub.d) where [L] is the concentration of free radioligand and K.sub.d is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973) (see results in table 2 below).
(244) Table 2 shows biological results obtained using the .sup.3H-SB222200 binding competition assay with compounds of the invention. These results indicate that compounds of the invention display potent affinity for the human NK-3 receptor.
(245) TABLE-US-00002 TABLE 2 Functional assay: Aequorin assay with Competitive human NK-3 receptor binding assay with hNK-3 - AEQ(antagonist human NK-3 receptor Cpd n.sup.o IC.sub.50, nM) hNK-3 (K.sub.i, nM) 1 16 11 2 12 15 3 32 19 4 19 20 5 18 23 6 30 24 7 30 26 8 33 26 9 21 30 10 56 31 11 73 32 12 170 59 13 44 40 14 57 42 15 50 45 16 71 49 17 50 51 18 87 54 19 110 56 20 93 60 21 150 63 22 130 69 23 220 150 24 120 78 25 110 85 26 74 88 27 220 100 28 160 110 29 170 150 30 5 7 31 64 70 32 44 59 33 120 89 34 62 38 35 54 73 36 58 43 37 52 41 38 34 26 39 32 28 40 130 83 41 7 11 42 39 48 43 136 59 44 204 45 45 244 101 45-1 285 100 45-2 148 83
(246) Selectivity Assay
(247) Selectivity of the compounds of the invention was determined over the other human NK receptors, namely NK-1 and NK-2 receptors.
(248) Human NK-1
(249) The affinity of compounds of the invention for the NK-1 receptor was evaluated in CHO recombinant cells which express the human NK-1 receptor. Membrane suspensions were prepared from these cells. The following radioligand: [.sup.3H] substance P (PerkinElmer Cat#NET111520) was used in this assay. Binding assays were performed in a 50 mM Tris/5 mM MnCl2/150 mM NaCl/0.1% BSA at pH 7.4. Binding assays consisted of 25 ?l of membrane suspension (approximately 5 ?g of protein/well in a 96 well plate), 50 ?l of compound or reference ligand (Substance P) at increasing concentrations (diluted in assay buffer) and 2 nM [.sup.3H] substance P. The plate was incubated 60 min at 25? C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in 0.5% PEI for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC.sub.50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC.sub.50/(1+[L]/K.sub.D) where [L] is the concentration of free radioligand and K.sub.D is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).
(250) Human NK-2 The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [.sup.125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 ?g/ml saponin, at pH 7.4. Binding assays consisted of 25 ?l of membrane suspension (approximately 3.75 ?g of protein/well in a 96 well plate), 50 ?l of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [.sup.125]-Neurokinin A. The plate was incubated 60 min at 25? C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC.sub.50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC.sub.50/(1+[L]/K.sub.D) where [L] is the concentration of free radioligand and K.sub.D is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).
(251) The compounds of the invention, which were tested in the above NK-1 and NK-2 described assays, demonstrated a low affinity at the human NK-1 and human NK-2 receptors: more than 200 fold shift of the K.sub.i compared to the human NK-3 receptor (table 3). Thus, compounds according to the invention have been shown to be selective over NK-1 and NK-2 receptors.
(252) TABLE-US-00003 TABLE 3 Cpd n.sup.o hNK-3 (K.sub.i, nM) hNK-1 (K.sub.i, nM) hNK-2 (K.sub.i, nM) 1 11 10300 7500 2 15 23800 >30000 3 19 >30000 >30000 4 20 19900 23000 5 23 >30000 >30000 6 24 22100 25000 7 26 >30000 36000 8 26 >30000 >30000 9 30 >30000 >30000 10 31 >30000 49000 11 32 22000 >30000 12 59 NA NA 13 40 >30000 >30000 14 42 >30000 >30000 15 45 >30000 >30000 16 49 >30000 37000 17 51 >30000 >30000 18 54 >30000 >30000 19 56 >30000 >30000 20 60 >30000 >30000 21 63 >30000 >30000 22 69 >30000 >30000 23 150 NA NA 24 78 >30000 >30000 25 85 >30000 >30000 26 88 >30000 >30000 27 100 >30000 >30000 28 110 >30000 >30000 29 150 >30000 >30000 30 7 40000 32000 31 70 >30000 >30000 32 59 >30000 >30000 33 89 >30000 >30000 34 38 >30000 >30000 35 73 >30000 30000 36 43 >30000 36000 37 41 32000 >30000 38 26 21000 28000 39 28 >30000 >30000 40 83 >30000 >30000 41 11 NA NA 42 48 >30000 >30000 43 59 >30000 >30000 44 45 >30000 >30000 45 101 >30000 >30000 45-1 100 >30000 >30000 45-2 83 >30000 >30000 NA: not available
(253) hERG Inhibition Assay
(254) The human ether-a-go-go related gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (I.sub.Kr) which is involved in cardiac repolarisation. I.sub.Kr current inhibition has been shown to elongate the cardiac action potential, a phenomenon associated with increased risk of arrhythmia. I.sub.Kr current inhibition accounts for the vast majority of known cases of drug-induced QT-prolongation. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery.
(255) The hERG inhibition study aims at quantifying the in vitro effects of compounds of the invention on the potassium-selective IK.sub.r current generated in normoxic conditions in stably transfected HEK 293 cells with the human ether-a-go-go-related gene (hERG).
(256) Whole-cell currents (acquisition by manual patch-clamp) elicited during a voltage pulse were recorded in baseline conditions and following application of tested compounds (5 minutes of exposure). The concentrations of tested compounds (0.3 ?M; 3 ?M; 10 ?M; 30 ?M) reflect a range believed to exceed the concentrations at expected efficacy doses in preclinical models.
(257) The pulses protocol applied is described as follow: the holding potential (every 3 seconds) was stepped from ?80 mV to a maximum value of +40 mV, starting with ?40 mV, in eight increments of +10 mV, for a period of 1 second. The membrane potential was then returned to ?55 mV, after each of these incremented steps, for 1 second and finally repolarized to ?80 mV for 1 second.
(258) The current density recorded were normalized against the baseline conditions and corrected for solvent effect and time-dependent current run-down using experimental design in test compound free conditions.
(259) Inhibition curves were obtained for compounds and the concentrations which decreased 50% of the current density determined in the baseline conditions (IC.sub.50) were determined. All compounds for which the IC.sub.50 value is above 10 ?M are not considered to be potent inhibitors of the hERG channel whereas compounds with IC.sub.50 values below 1 ?M are considered potent hERG channel inhibitors.
(260) When tested in the hERG inhibition assay, compounds of the invention were determined to have IC.sub.50 values as shown in Table 4.
(261) Determination of Plasma Protein Binding
(262) The pharmacokinetic and pharmacodynamic properties of chemicals/drugs are largely a function of the reversible binding of chemicals to plasma or serum proteins. Generally, only the unbound or free fraction of a drug is available for diffusion or transport across cell membranes, and for interaction with a pharmacological/toxicological target. Consequently, the extent of the plasma protein binding (PPB) of a compound influences its action as well as its distribution and elimination.
(263) The determination of plasma protein binding (PPB) of a compound is enabled by equilibrium dialysis, an accepted and standard method for reliable estimation of the non-bound drug fraction in plasma. RED (Rapid Equilibrium Dialysis) device insert is made of two side-by-side chambers separated by an O-ring-sealed vertical cylinder of dialysis membrane (MWCO ?8,000). Plasma containing drug (at 5 ?M or blood concentrations otherwise corresponding to efficacious doses, if known) is added to one chamber while buffer is added to the second. After 4 hours incubation at 37? C. under shaking, an aliquot is removed from each chamber and analyzed by a LC-MS/MS procedure enables the determination of both free and bound drug.
(264) The percentages provided in Table 4 represent for the compounds of the invention the bound drug fraction to the plasma protein. The free fraction may be calculated as 100%-% rPPB (i.e. the complementary percentage of that disclosed in Table 4, corresponding to the drug concentration that is unbound and therefore available to engage biological target and elicit pharmacological activity).
(265) TABLE-US-00004 TABLE 4 Exposure CardioSafety Cpd n.sup.o (% rPPB) (hERG IC.sub.50, ?M) 1 67 42 2 47 32 3 42 66 4 40 70 5 22 70 6 53 45 7 26 70 8 29 70 9 22 70 10 30 70 11 24 50 12 20 NA 13 37 70 14 21 NA 15 20 70 16 36 70 17 24 46 18 23 70 19 51 NA 20 26 50 21 38 45 22 27 70 23 34 NA 24 48 61 25 19 NA 26 19 NA 27 24 70 28 12 NA 29 10 59 30 94 32 31 31 NA 32 25 NA 33 29 NA 34 24 NA 35 52 NA 36 60 NA 37 53 NA 38 76 NA 39 43 NA 40 24 NA 41 55 NA 42 16 NA 43 47 NA 44 31 NA 45 31 NA 45-1 27 NA 45-2 33 NA NA: not available
(266) In Vivo Assay to Assess Compound Activity in Rat (Oral Dosing)
(267) Castrated male rat model to assess the effect of compound of invention on circulating levels of luteinizing hormone (LH)
(268) The effect of compounds of the invention to inhibit luteinizing hormone (LH) secretion is determined by the following biological studies.
(269) In humans and rodents, castration is well-precedented to permit heightened, persistent GnRH signaling and consequent elevation of circulating LH. Thus, a castrated rat model is used to provide a broad index for measurement of LH inhibition as a marker of test compound inhibition of the GnRH signaling pathway.
(270) Castrated adult male Sprague-Dawley (SD) rats (150-175 g,) were purchased from Janvier (St Berthevin, France). All animals were housed 2 per cage in a temperature-controlled room (22?2? C.) and 50?5% relative humidity with a 12 hour/12 hour light/dark cycles (lights off at 6 h00 pm). The animals were allowed 3 weeks of postoperative recovery prior to study. Animals were handled on a daily basis. Standard diet and tap water were provided ad libitum. Animal cage litters were changed once a week. On the study day, animals were acclimated to the procedure room for a period of one hour prior to the initiation of the experiment.
(271) Compounds of the invention were formulated in 0.5% methyl cellulose.
(272) After basal sampling (T0) a single dose of compounds of the invention or vehicle was administrated orally to rats. Blood samples were then collected at several time points post dosing (45, 90, 150, 300 and 420 minutes). Blood samples were obtained via tail vein bleed, drawn into EDTA-containing tubes and centrifuged immediately. Plasma samples were collected and stored in a ?80? C. freezer until assayed. Serum LH levels were determined using radioimmunoassay kit from RIAZENRat LH, Zentech (Liege, Belgium). Baseline was defined as the initial basal blood sample.
(273) When tested in the castrated male rat model described above, compounds n.sup.o 1, 2, 4, 5, 8, 9, 11, 13, 20 and 30 of the invention significantly suppressed circulating LH levels (statistically significant, p<0.05) at a dose less than or equal to 30 mg/kg).
(274) Effect of Compounds of the Invention on Plasma Testosterone in Gonad Intact Male Rats
(275) The study was designed to evaluate the effect of compounds of the invention on testosterone circulating levels following oral administration at 3 mg/kg on SD gonad intact male rats.
(276) Briefly the experimental methods used for this study were as follows:
(277) Two groups of non-fasted rats (male, Sprague-Dawley, 200 to 225 g; n=4 rats/group) with jugular vein cannulation, were dosed via a single oral administration of compounds of the invention at 3 mg/kg. The control group was dosed with the vehicle. Compounds of the invention were prepared in a dose formulation of pyrogen-free water with 0.5% methylcellulose. Blood samples were collected via the catheter implanted in the jugular vein at pre-determined intervals using EDTA-3K as anti-coagulant. Samples were chilled and rapidly processed by centrifugation to obtain corresponding plasma samples. Testosterone hormone levels were determined by RIA performed on plasma samples collected for all the groups at 5 minutes before administration (basal time), and at 45, 90, 150, 300, 480 minutes and 24 hours after dosing.
(278) When tested in the gonad intact male rats, compound n.sup.o5 significantly suppressed plasma testosterone level over the test period as compared to the vehicle treated group (
(279) Effect of Compounds of the Invention on Prostate Weight Reduction in a Benign Prostatic Hyperplasia (BPH) Rat Model
(280) Briefly, adult male rats were injected daily for four weeks with testosterone to cause an enlargement of the prostate as per methods previously described in the literature (Scolnick et al., J. Andrology, 1994, 15(4), 287-297; Rick et al., J. Urol., 2012, 187, 1498-1504; see
(281) When tested in the Benign Prostatic Hyperplasia rat model, compound n.sup.o5, demonstrated a concentration-response to reduce prostate weight to normal levels (i.e. levels in rats not exposed to exogenous testosterone;
(282) Effect of Compounds of the Invention on Estradiol Circulating Level in Female Rats
(283) The aim of this study was to evaluate the effect of compounds of the invention on plasma estradiol levels following oral administration at 10 mg/kg (b.i.d.) for a period of 10 days in female rats.
(284) Briefly the experimental methods used for this study were as follows:
(285) Two groups of adult, female rats (Sprague-Dawley, ?320 g) were treated in-phase with their individual estrous cycles. Thus, treatment was started in the proestrus phase (coincident with peak estradiol levels, as shown on Day 1 in
(286) In vehicle-treated, adult female rats, estradiol peaks are observed every 4-5 days consistent with the anticipated duration of the rat estrous cycle. Treatment with compound n.sup.o5, significantly decreased estradiol levels over the time-course tracked over two consecutive estrous cycles. This finding is most apparent in the proestrus phase (i.e. for vehicle group, on Day 5 and Day 9) where estradiol levels rise coincident with ovulation.
(287) In Vivo Assay in OVX EwesActivity in Thermoregulation
(288) Experimental Methods: Evaluation of Thermoregulation in OVX Ewe
(289) Ten Corriedale ewes (body weight 56.6?3.4 kg) of 3-4 years of age were ovariectomized according to Standard Operating Procedures, as previously described (Barker-Gibb, Scott, Boublik, & Clarke, 1995). After 4 months recovery, animals were acclimatised to housing in single pens for a period of 7 days with ad libitum access to water and chaffed lucerne hay. One day prior to experimentation, the animals received a jugular vein cannula (Dwellcath, Tuta Laboratories, Lane Cove, Australia). The cannula were kept patent with heparinised saline. On the day of the experiment, compound n.sup.o5 of the present invention was formulated in physiological saline with 9% 2-hydroxypropyl-?-cyclodextrin at a concentration of 2 mg/mL. Compound n.sup.o5 (1 mg/kg, N=5) or vehicle (N=5) was administered at 11 h00 by intravenous bolus injection at a dose volume of 0.5 mL/kg through the jugular cannula and the injected material was flushed into the animal with 5 mL of heparinised saline. Animals were fed at 12 h00. Rectal temperatures were monitored with a probe at hourly intervals throughout the experiment, starting at 7 h40 and concluding at 15 h40.
(290) Results In response to feeding at 12 h00, vehicle-treated animals exhibited a transient, relative body temperature increase of ?0.7? C. measured at 12 h40, consistent with findings previously reported in the literature (Henry, Dunshea, Gould, & Clarke, 2008). However, this pyrogenic response to feeding in ovarectomized ewes was not observed in any animals treated with compound n.sup.o5. Thus, at the 12 h40 time point, the body temperature of the vehicle-treated group (39.7?0.3? C.) was significantly (p<0.05) higher than that of the compound n.sup.o5-treated group (38.9?0.2? C.), as presented in
(291) Conclusions: Ovariectomy in the ewe causes changes in core body temperature considered analogous to menopausal hot flashes (MacLeay, Lehmer, Enns, Mallinckrodt, Bryant, & Turner, 2003) to the extent that a transient, pyrogenic response to feeding is observed (Henry, Dunshea, Gould, & Clarke, 2008). It is herein demonstrated that the compounds of the invention protect against this hot flash induced by feeding.
(292) Therefore, the NK-3 receptor antagonists of the invention have therapeutic utility to protect against hot flashes in clinical situations where sex steroids (principally, estrogen in women and testosterone in men) are compromised, including such disorders as the induction of hot flashes due to menopause and the induction of hot flashes as a consequence of cancer therapy that lowers sex hormones (for example, therapy-induced hot flashes in breast, uterine and prostate cancer).
(293) Clinical Evaluation of Compound n.sup.o5 for the Treatment of Menopausal Hot Flashes
(294) Experimental Methods:
(295) Objectives: The study objective is to evaluate the effect of compound n.sup.o5 on the severity and frequency of hot flashes in postmenopausal women.
(296) Design and Setting: The Phase 2a clinical trial was conducted in 80 patients, divided over 2 treatment groups of 40 patients each. One group received placebo while the other group received 90 mg of compound n.sup.o5 twice daily (BID) for a period of 12 weeks. The Phase 2a trial was double blind and randomized. This Phase 2a trial was conducted on healthy menopausal women (40-65 y) experiencing at least 49 moderate or severe hot flashes or night sweats over a period of 7 consecutive days in the screening period (maximum of 4 weeks). The study was conducted on an ambulatory basis where patients were asked to visit the clinical site on the first day of dosing and at weeks 4, 8, 12 during the treatment period with a follow-up visit 2-3 weeks after conclusion of dosing.
(297) Hot flashes were to be reported daily in the morning and the evening using an electronic diary tool. These reported events were used to calculate the Hot Flash Frequency and the Hot Flash Score.
(298) The weekly number of hot flashes was calculated in two ways: cumulatively for all severity grades, and cumulatively but leaving out the number of mild hot flashes. Hot Flash Frequencies were calculated for weeks 4 and 12.
(299) The Hot Flash Score (HFS) is a composite score combining both frequency and severity. This score is an accepted method to assess the overall severity of the hot flash burden and is calculated weekly. The score is a general measure of the number and severity of all hot flashes occurred during a given time period. The Hot Flash Score (based on severity and frequency) is calculated as follows:
day-score=(number of mild hot flashes/day?1)+(number of moderate hot flashes/day?2)+(number of severe hot flashes/day?3) The weekly hot flash score is calculated: Mean HFS day-score over 1 week Scores were calculated for weeks 4 and 12
(300) Higher scores indicate worse symptoms. There is no maximum score since the number of hot flashes does not have an upper limit.
(301) Results: In response to treatment by compound n.sup.o5 (90 mg BID) both Score and Frequency of hot flashes in postmenopausal women were significantly reduced in comparison of placebo-controlled group as presented in Table 5 (Hot Flash Score) and in Table 6 (Hot Flash Frequencies). Data are presented as mean for placebo-treated versus compound n.sup.o5-treated patients (N=40/group). Statistical analyses performed by 2-way ANOVA.
(302) Table 5 is a table showing the calculated Hot Flashes Score for moderate and severe hot flashes expressed as absolute change from baseline after oral administration of compound n.sup.o5 (90 mg BID) or of a placebo.
(303) TABLE-US-00005 Mean Endpoint Visit Compound n.sup.o5 Placebo P value Hot Flash Baseline 29.08 25.92 n.s. Score Week 4 2.96 15.69 <0.0001 Week 12 1.70 13.68 <0.0001
(304) Table 6 is a table showing the calculated Hot Flashes Frequency for moderate and severe hot flashes expressed as absolute change from baseline after oral administration of compound n.sup.o5 (90 mg BID) or of a placebo.
(305) TABLE-US-00006 Mean Endpoint Visit Compound n.sup.o5 Placebo P value Frequency Baseline 11.84 10.72 n.s. Week 4 1.35 6.6 <0.0001 Week 12 0.79 5.6 <0.0001
CONCLUSIONS
(306) This well-controlled, Clinical trial study has been sufficiently powered to provide clear insight into the therapeutic value of compound n.sup.o5 for the treatment of menopausal hot flashes. It is herein demonstrated that the compounds of the invention can effectively prevent the occurrence of moderate and severe hot flashes in postmenopausal women.
(307) Therefore, the NK-3 receptor antagonists of the invention have therapeutic utility to protect against hot flashes in clinical situations where sex steroids (principally, estrogen in women and testosterone in men) are compromised, including such disorders as the induction of hot flashes due to menopause and the induction of hot flashes as a consequence of hormone therapy intentionally lowering the level of sex hormones (for example, therapy-induced hot flashes in breast, uterine and prostate cancer).