Deuterated compounds as rock inhibitors

Abstract

Provided are compounds of Formula (I), or pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising these compounds thereof, and use of these compounds in preparing drugs for inhibiting ROCK. ##STR00001##

Claims

1. A compound of formula (I): ##STR00061## or a pharmaceutically acceptable salt thereof, wherein R.sup.1 is selected from CH.sub.3, CH.sub.2D, CHD.sub.2 and CD.sub.3; R.sup.2 is selected from CH.sub.3, CH.sub.2D, CHD.sub.2 and CD.sub.3; Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, Y.sup.6, Y.sup.7, Y.sup.9, Y.sup.10, Y.sup.11, Y.sup.12, Y.sup.14, Y.sup.15 and Y.sup.16 are each independently selected from hydrogen and deuterium; each Y.sup.8 is independently selected from hydrogen and deuterium, provided that both Y.sup.8 are the same; each Y.sup.13 is independently selected from hydrogen and deuterium, provided that both Y.sup.13 are the same; provided that the compound of formula (I) contains at least one deuterium atom.

2. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein Y.sup.1 is deuterium.

3. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein Y.sup.2 is deuterium.

4. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein Y.sup.3 is deuterium.

5. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein Y.sup.7 is deuterium.

6. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein both Y.sup.8 are deuterium.

7. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein both Y.sup.13 are deuterium.

8. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein R.sup.1 is selected from CH.sub.3 and CD.sub.3.

9. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein R.sup.2 is selected from CH.sub.3 and CD.sub.3.

10. The compound of claim 1, selected from ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066## and pharmaceutically acceptable salts thereof.

11. The compound of claim 10 or the pharmaceutically acceptable salt thereof, selected from ##STR00067## ##STR00068## and pharmaceutically acceptable salts thereof.

12. The compound of claim 11 or the pharmaceutically acceptable salt thereof, selected from ##STR00069## and pharmaceutically acceptable salts thereof.

13. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt thereof is hydrochloride.

14. The compound of claim 13 or the pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt thereof is dihydrochloride.

15. A pharmaceutical composition, comprising the compound of claim 1 or the pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

16. A method of treating, ameliorating or preventing a condition, which responds to inhibition of ROCK, comprising administering to a subject in need of such treatment an effective amount of the compound of claim 1, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, and optionally in combination with a second therapeutic agent.

17. A method of treating elevate intraocular pressure, comprising administering to a subject in need of such treatment an effective amount of the compound of claim 1 or the pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition, comprising the compound of claim 10 or the pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

19. A method of treating, ameliorating or preventing a condition, which responds to inhibition of ROCK, comprising administering to a subject in need of such treatment an effective amount of the compound of claim 10, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, and optionally in combination with a second therapeutic agent.

20. A method of treating elevated intraocular pressure, comprising administering to a subject in need of such treatment an effective amount of the compound of claim 10 or the pharmaceutically acceptable salt thereof.

Description

EXAMPLES

(1) Various methods may be developed for synthesizing a compound of formula (I) or a pharmaceutically acceptable salt thereof. Representative methods for synthesizing a compound of formula (I) or a pharmaceutically acceptable salt thereof are provided in the Examples. It is noted, however, that a compound of formula (I) or a pharmaceutically acceptable salt thereof may also be synthesized by other synthetic routes that others may devise.

(2) It will be readily recognized that certain compounds of formula (I) have atoms with linkages to other atoms that confer a particular stereochemistry to the compound (e.g., chiral centers). It is recognized that synthesis of a compound of formula (I) or a pharmaceutically acceptable salt thereof may result in the creation of mixtures of different stereoisomers (enantiomers, diastereomers). Unless a particular stereochemistry is specified, recitation of a compound is intended to encompass all of the different possible stereoisomers.

(3) A compound of formula (I) can also be prepared as a pharmaceutically acceptable acid addition salt by, for example, reacting the free base form of the at least one compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of the at least one compound of formula (I) can be prepared by, for example, reacting the free acid form of the at least one compound with a pharmaceutically acceptable inorganic or organic base. Inorganic and organic acids and bases suitable for the preparation of the pharmaceutically acceptable salts of compounds of formula (I) are set forth in the definitions section of this Application. Alternatively, the salt forms of the compounds of formula (I) can be prepared using salts of the starting materials or intermediates.

(4) The free acid or free base forms of the compounds of formula (I) can be prepared from the corresponding base addition salt or acid addition salt form. For example, a compound of formula (I) in an acid addition salt form can be converted to the corresponding free base thereof by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of formula (I) in a base addition salt form can be converted to the corresponding free acid thereof by, for example, treating with a suitable acid (e.g., hydrochloric acid, etc).

(5) The N-oxides of the a compound of formula (I) or a pharmaceutically acceptable salt thereof can be prepared by methods known to those of ordinary skill in the art. For example, N-oxides can be prepared by treating an unoxidized form of the compound of formula (I) with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta-chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at approximately 0 to 80° C. Alternatively, the N-oxides of the compounds of formula (I) can be prepared from the N-oxide of an appropriate starting material.

(6) Compounds of formula (I) in an unoxidized form can be prepared from N-oxides of compounds of formula (I) by, for example, treating with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, and the like) in an suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, and the like) at 0 to 80° C.

(7) Protected derivatives of the compounds of formula (I) can be made by methods known to those of ordinary skill in the art. A detailed description of the techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, Protecting Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999.

(8) As used herein the symbols and conventions used in these processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Standard single-letter or three-letter abbreviations are generally used to designate amino acid residues, which are assumed to be in the L-configuration unless otherwise noted. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification. For example, the following abbreviations may be used in the examples and throughout the specification: g (grams); mg (milligrams); L (liters); mL (milliliters); μL (microliters); psi (pounds per square inch); M (molar); mM (millimolar); i.v. (intravenous); Hz (Hertz); MHz (megahertz); mol (moles); mmol (millimoles); RT (room temperature); min (minutes); h (hours); mp (melting point); TLC (thin layer chromatography); Rt (retention time); RP (reverse phase); MeOH (methanol); i-PrOH (isopropanol); TEA (triethylamine); TFA (trifluoroacetic acid); TFAA (trifluoroacetic anhydride); THF (tetrahydrofuran); DMSO (dimethyl sulfoxide); EtOAc (ethyl acetate); DME (1,2-dimethoxyethane); DCM (dichloromethane); DCE (dichloroethane); DMF (N,N-dimethylformamide); DMPU (N,N′-dimethylpropyleneurea); CDI (1,1-carbonyldiimidazole); IBCF (isobutyl chloroformate); HOAc (acetic acid); HOSu (N-hydroxysuccinimide); HOBT (1-hydroxybenzotriazole); Et.sub.2O (diethyl ether); EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); BOC (tert-butyloxycarbonyl); FMOC (9-fluorenylmethoxycarbonyl); DCC (dicyclohexylcarbodiimide); CBZ (benzyloxycarbonyl); Ac (acetyl); atm (atmosphere); TMSE (2-(trimethylsilyl)ethyl); TMS (trimethylsilyl); TIPS (triisopropylsilyl); TBS (t-butyldimethylsilyl); DMAP (4-dimethylaminopyridine); Me (methyl); OMe (methoxy); Et (ethyl); tBu (tert-butyl); HPLC (high pressure liquid chomatography); BOP (bis(2-oxo-3-oxazolidinyl)phosphinic chloride); TBAF (tetra-n-butylammonium fluoride); m-CPBA (meta-chloroperbenzoic acid).

(9) References to ether or Et.sub.2O are to diethyl ether; brine refers to a saturated aqueous solution of NaCl. Unless otherwise indicated, all temperatures are expressed in ° C. (degrees Centigrade). All reactions were conducted under an inert atmosphere at RT unless otherwise noted.

(10) .sup.1H NMR spectra were recorded on a Varian Mercury Plus 400. Chemical shifts are expressed in parts per million (ppm). Coupling constants are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad).

(11) Low-resolution mass spectra (MS) and compound purity data were acquired on a Shimadzu LC/MS single quadrapole system equipped with electrospray ionization (ESI) source, UV detector (220 and 254 nm), and evaporative light scattering detector (ELSD). Thin-layer chromatography was performed on 0.25 mm Superchemgroup silica gel plates (60F-254), visualized with UV light, 5% ethanolic phosphomolybdic acid, ninhydrin, or p-anisaldehyde solution. Flash column chromatography was performed on silica gel (200-300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd).

(12) Synthetic Schemes

(13) A compound of formula (I) or a pharmaceutically acceptable salt thereof may be synthesized according to a variety of reaction schemes. Some illustrative schemes are provided below and in the examples. Other reaction schemes could be readily devised by those skilled in the art in view of the present disclosure.

(14) In the reactions described herein after it may be necessary to protect reactive functional groups, for example hydroxyl, amino, imino, thio or carboxyl groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups may be used in accordance with standard practice, for examples see T. W. Greene and P. G. M. Wuts in “Protective Groups in Organic Chemistry” John Wiley and Sons, 1991.

(15) Synthetic methods for preparing the compounds of the present invention are illustrated in the following Schemes and Examples. Starting materials are commercially available or may be made according to procedures known in the art or as illustrated herein.

(16) The intermediates shown in the following schemes are either known in the literature or may be prepared by a variety of methods familiar to those skilled in the art.

(17) As an illustration, one synthesis of compounds of formula I of the present disclosure is outlined in Scheme 1. As shown in the Scheme, the compounds of formula I can be disassembled into the intermediates II and III, the preparation of which are either known in the literature or may be prepared by a variety of methods familiar to those skilled in the art. In the route, the intermediates II can be obtained via a three-step sequence including protection of free carboxyl group of IV, coupling with V and cleavage of the protecting groups. Finally, coupling of carboxylic acid II with amino-arenes such as those of formula III via a condensation reaction leads to compounds of formula I.

(18) ##STR00022##

(19) As an illustration of the preparation of intermediates of formula IV, one synthetic route of Intermediate IVa is shown in Scheme 2. Starting from the commercially available IVa-A. IVa-C can be prepared by carbonyl insertion reaction and TBS protection of free hydroxyl group of IVa-B. Reduction of ester IVa-C with LiAlD.sub.4 leads to IVa-D, which can be further converted to IVa-E through a condensation with benzoic acid VI. Finally, Intermediate of formula IVa can be obtained from IVa-E through a sequence of cleavage of the protecting groups on the oxygen and oxidation of the resulting alcohol.

(20) ##STR00023##

(21) As a further illustration of the preparation of intermediates of formula IV, one synthetic route of intermediate IVb is provided in Scheme 3 The commercially available dimethyl terephthalate IVb-A is selected as starting material. Reduction of IVb-A with LiAlD.sub.4 leads to IVb-B, which can be further converted to IVb-D through selective TBDPS protection of the hydroxyl group and iodination The nucleophilic displacement of the iodide of IVb-D with TMSCN yields intermediate IVb-E. Conversion of the cyano group in IVb-E into methyl ester group results in intermediate of formula IVb-G. Finally, Compounds of formula IVb can be prepared by condensation with benzoic acid VI followed by hydrolysis of IVb-H.

(22) ##STR00024## ##STR00025##

(23) Another illustration of the preparation of intermediates of formula IV is shown is Scheme 4 which demonstrates preparation of compound of formula IVc. Starting from commercially available 2,4-dimethylbenzoic acid IVc-A. The intermediate 2,4-bis(methyl-d.sub.3)benzoic acid IVc-B can be prepared by treating IVc-A with t-BuOK and DMSO-d.sub.6. Coupling of 2,4-bis(methyl-d.sub.3)benzoic acid IVc-B with the intermediates of formula IVc-C via a condensation reaction leads to compounds of formula IVc-D. Finally, hydrolysis of IVc-D results in compounds of formula IVc.

(24) ##STR00026##

(25) As an illustration of the preparation of intermediates of formula III, one synthetic route of compound IIIa is shown in Scheme 5. Starting from 1,4-dibromobenzene IIIa-A, which is commercially available, aldehyde IIIa-B can be prepared by treating IIIa-A with n-BuLi and DMF-d.sub.7. The intermediates IIIa-E can be obtained via reductive amination with 2,2-dimethoxyethan-1-amine and NaBD.sub.4, followed by protecting of amino group of IIIa-D. Cyclization of IIIa-E, promoted by Lewis acid such as AlCl.sub.3, leads to isoquinoline of formula IIIa-F which can be further converted to IIIa-G through the Palladium-catalyzed coupling with benzophenone imine. Finally, deprotecting of IIIa-G leads to compounds of formula IIIa.

(26) ##STR00027##

(27) As a further illustration of the preparation of intermediates of formula III, one synthetic route of compound Mb is shown in Scheme 6. Starting from commercially available 2-oxoacetic acid IIIb-A. The intermediate IIIb-B can be prepared by treating IIIb-A with trimethoxymethane and p-TsOH. Ammonolysis of IIIb-B gives compounds of formula IIIb-C which can be further transformed into compounds of formula IIIb-D via reduction of amide with LiAlD.sub.4. The intermediate IIIb-G can be obtained from the intermediate IIIb-D via reductive amination with 4-bromobenzaldehyde and NaBH.sub.4, followed by protecting of amino group of IIIb-F. Cyclization of IIIb-G, promoted by Lewis acid such as AlCl.sub.3, leads to isoquinoline of formula IIIb-H which can be further converted to IIIb-I through the Palladium-catalyzed coupling with benzophenone imine. Finally, deprotecting of IIIb-I leads to compounds of formula IIIb.

(28) ##STR00028## ##STR00029##

(29) As an illustration of the preparation of intermediates of formula V, one synthetic route of compound Va is shown in Scheme 7. Starting from commercially available 1H-benzo[d][1,2,3]triazole Va-A. The intermediates Va-B can be prepared by treating Va-A with formaldehyde-d.sub.2. Then compounds of formula Va can be obtained via a conversion of the hydroxyl group in Va-B into the Boc-protected amino group with tert-butyl carbamate and 4-methylbenzenesulfonic acid.

(30) ##STR00030##

(31) In some cases, the order of carrying out the foregoing reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products. The following examples are provided so that the invention might be more fully understood. These examples are illustrative only and should not be construed as limiting the invention in any way.

Intermediate A

Isoquinolin-1-d-6-amine (A)

(32) ##STR00031##

(4-Bromophenyl)-deuterio-methanone (A1)

(33) To a solution of 1,4-dibromobenzene (2.50 g, 10.6 mmol) in THF (5 mL) at −78° C. was added n-BuLi (1.6 M, 7 mL, 11.7 mmol) dropwise, and the resulting mixture was stirred at −78° C. for 0.5 h. Then DMF-d.sub.7 (1.08 mL, 12.7 mmol) was added dropwise to the mixture at −78° C. and the resulting mixture was stirred at −78° C.˜−30° C. for 1.5 h. The reaction was quenched with saturated aqueous NH.sub.4Cl (2 mL) at −30° C. The mixture was diluted with water and extracted with EtOAc (3×50 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to give the crude product of title compound (4-bromophenyl)-deuterio-methanone (A1), which was used without further purification for next step.

N-((4-bromophenyl)methyl-d.SUB.2.)-2,2-dimethoxyethan-1-amine (A2)

(34) A mixture of (4-bromophenyl)-deuterio-methanone (A1) (2.00 g, 10.8 mmol), p-TsOH.H.sub.2O (0.210 g, 1.08 mmol) and 2,2-dimethoxyethan-1-amine (1.05 g, 10.0 mmol) in toluene (30 mL) was stirred at 138° C. for 3.5 h. The mixture was poured into saturated NaHCO.sub.3 aqueous solution (8 mL), washed sequentially with aqueous NaHCO.sub.3 solution (2×100 mL) and water (1×100 mL), and the aqueous phase was extracted with toluene (2×100 mL). The combined organic phase was dried over Na.sub.2SO.sub.4 and concentrated. The residue was dissolved in EtOH (20 mL), then NaBD.sub.4 (220 mg, 5.50 mmol) was added to the mixture at RT and the resulting mixture was stirred at RT for 2 h. Then HOAc (0.2 mL) was added to the mixture. The reaction mixture was diluted with water and extracted with EtOAc. The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to give the crude product of title compound N-((4-bromophenyl)methyl-d.sub.2)-2,2-dimethoxyethan-1-amine (A2). MS-ESI (m/z): 276 [M+1].sup.+.

N-((4-bromophenyl)methyl-d.SUB.2.)-N-(2,2-dimethoxyethyl)-4-methylbenzenesulfonamide (A3)

(35) To a solution of N-((4-bromophenyl)methyl-d.sub.2)-2,2-dimethoxyethan-1-amine (A2) (2.5 g, 9.1 mmol) in DCM (16 mL) and pyridine (2 mL) was added TsCl (3.10 g, 16.3 mmol). The mixture was stirred at RT for 2 h. The mixture was washed with 1 N HCl (3×50 mL), dried and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (20:1˜5:1) to give the title compound N-((4-bromophenyl)methyl-d.sub.2)-N-(2,2-dimethoxyethyl)-4-methylbenzenesulfonamide (A3). MS-ESI (m/z): 430 [M+1].sup.+.

6-Bromoisoquinoline-1-d (A4)

(36) To a mixture of AlCl.sub.3 (2.95 g, 22.0 mmol) in DCM (10 mL) was added a solution of N-((4-bromophenyl)methyl-d.sub.2)-N-(2,2-dimethoxyethyl)-4-methylbenzenesulfonamide (A3) (1.9 g, 4.4 mmol) in DCM (10 mL) dropwise. After being stirred at RT for 4 h. the mixture was poured into ice (50 g), and extracted with DCM (3×100 mL). The organic phase was washed sequentially with 1 N NaOH (2×100 mL), aqueous NaHCO.sub.3 solution (1×100 mL) and brine (100 mL), dried and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (5:1) to give the title compound 6-bromoisoquinoline-1-d (A4). MS-ESI (m/z): 209 [M+1].sup.+.

Isoquinolin-1-d-6-amine (A)

(37) To a mixture of 6-bromoisoquinoline-1-d A4) (455 mg, 2.19 mmol), diphenylmethanimine (441 μl, 2.62 mmol) and t-BuONa (631 mg, 6.57 mmol) in toluene (10 mL) was added BINAP (954 mg, 1.53 mmol) and Pd.sub.2(dba).sub.3 (401 mg, 0.438 mmol) at RT. The mixture was stirred at 100° C. for 3 h. The mixture was concentrated. The residue was dissolved in MeOH (20 mL), then NH.sub.2OH.HCl (241 mg, 3.50 mmol) and NaOAc (312 mg, 3.80 mmol) was added to the mixture at RT and the resulting mixture was stirred at RT for 1.5 h. The mixture was concentrated and the residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (1:1) to give the title compound isoquinolin-1-d-6-amine (A). MS-ESI (m/z): 146 [M+1].sup.+.

Intermediate B

isoquinolin-3-d-6-amine (B-a) and isoquinolin-3,4-d2-6-amine (B-b)

(38) ##STR00032##

2,2-dimethoxyethan-1,1-d.SUB.2.-1-amine (B1)

(39) To a solution of 2,2-dimethoxyacetamide (0.10 g, 0.84 mmol) in THF (3 mL) was added LiAlD.sub.4 (42 mg, 1.0 mmol) at RT, and the mixture was stirred at 65° C. for 1.5 h. Then NaSO.sub.4.10H.sub.2O was added to the mixture under ice water bath. After being stirred for 20 min, the mixture was filtered. The filtered cake was washed with THF and the solution was evaporated to give the crude product of 2,2-dimethoxyethan-1,1-d.sub.2-1-amine (B), which was used for next step without further purification. MS-ESI (m/z): 108 [M+1].sup.+.

N-(4-bromobenzyl)-2,2-dimethoxyethan-1,1-d.SUB.2.-1-amine (B2)

(40) A mixture of 2,2-dimethoxyethan-1,1-d.sub.2-1-amine (B1) (100 mg, 0.920 mmol), p-TsOH.H.sub.2O (17.5 mg, 0.0920 mmol) and 4-bromobenzaldehyde (170 mg, 0.920 mmol) in toluene (50 mL) was stirred at 135° C. for 4.5 h. After being concentrated, the residue was dissolved in EtOH (30 mL), then NaBH.sub.4 (300 mg, 7.90 mmol) was added to the mixture at RT. The resulting mixture was stirred at RT for 1 h. Then HOAc (0.2 mL) was added to the mixture. Solvent was concentrated, the residue was diluted with water and extracted with EtOAc. The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to give the crude product of title compound N-(4-bromobenzyl)-2,2-dimethoxyethan-1,1-d.sub.2-1-amine (B2). MS-ESI (m/z): 276 [M+1].sup.+.

isoquinolin-3-d-6-amine (B-a) and isoquinolin-3,4-d.SUB.2.-6-amine (B-b)

(41) The title compound isoquinolin-3-d-6-amine (B-a) (30%) and isoquinolin-3,4-d.sub.2-6-amine (B-b) (70%) was prepared according to the synthetic method of A by replacing N-((4-bromophenyl)methyl-d.sub.2)-2,2-dimethoxyethan-1-amine (A2) with N-(4-bromobenzyl)-2,2-dimethoxyethan-1,1-d.sub.2-1-amine (B2). MS-ESI (m/z) B-a: 146 [M+1].sup.+ and B-b: 147 [M+1].sup.+

Intermediate C

2,4-Bis(methyl-d.SUB.3.)benzoic acid (C)

(42) ##STR00033##

2,4-Bis(methyl-d.SUB.3.)benzoic acid (C)

(43) To a solution of 2,4-dimethylbenzoic acid (1.0 g, 6.1 mmol) in DMSO-d.sub.6 (10 mL) was added t-BuOK (3.40 g, 30.5 mmol). The mixture was stirred at 120° C. under N.sub.2 for 3 h. After cooled to RT, the mixture was diluted with water. The pH was adjusted to 2˜3 and extracted with EtOAc (3×). The extracts were washed with water, brine, dried over Na.sub.2SO.sub.4 and concentrated to give a solid. This solid was treated for 2 times according to the above process to give the title compound 2,4-bis(methyl-d.sub.3)benzoic acid (C).

Intermediate D

(4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)methan-d.SUB.2.-ol (D)

(44) ##STR00034##

Ethyl 4-(2-hydroxyethyl)benzoate (D1)

(45) A mixture of 2-(4-bromophenyl)ethan-1-ol (1.0 g, 5.0 mmol), TEA (0.83 ml, 6.0 mmol) and Pd(dppf)Cl.sub.2 (365 mg, 0.500 mmol) in EtOH (35 mL) and DMSO (22 mL) was stirred at 80° C. under CO atmosphere for 3.5 h. The reaction mixture was cooled to RT, quenched with saturated aqueous NH.sub.4Cl solution and extracted with EtOAc. The extracts were washed with brine (30 mL), dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (5:1˜4:1) to give the title compound ethyl 4-(2-hydroxyethyl)benzoate (D1). MS-ESI (m/z): 195 [M+1].sup.+.

Ethyl 4-(2-((tert-butyldimethylsilyl)oxy)ethyl)benzoate (D2)

(46) To a solution of ethyl 4-(2-hydroxyethyl)benzoate (D) (0.96 g, 4.9 mmol) in DMF (20 mL) was added imidazole (0.84 g, 12 mmol). The mixture was cooled to 0° C. and then TBSCl (0.894 g, 5.91 mmol) was added. The mixture was stirred at RT for 30 min, diluted with water and extracted with EtOAc. The extracts were washed with brine (30 mL), dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (50:1) to give the title compound ethyl 4-(2-((tert-butyldimethylsilyl)oxy)ethyl) benzoate (D2). MS-ESI (m/z): 309 [M+1].sup.+.

(4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)methan-d.SUB.2.-ol (D)

(47) The title compound (4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)methan-d.sub.2-ol (D) was prepared according to the synthetic method of 2,2-dimethoxyethan-1,1-d.sub.2-1-amine (B1) by replacing 2,2-dimethoxyacetamide with ethyl 4-(2-((tert-butyldimethylsilyl)oxy)ethyl)-benzoate (D2). MS-ESI (m/z): 269 [M+1].sup.+.

Intermediate E

Methyl 2-(4-(hydroxymethyl-d.SUB.2.)phenyl)acetate-d.SUB.2 .(E)

(48) ##STR00035##

1,4-Phenylenebis(methan-d.SUB.2.-ol) (E1)

(49) To a solution of dimethyl terephthalate (5.9 g, 30 mmol) in THF (100 mL) at 0° C., was added LiAlD.sub.4 (3.5 g, 59 mmol) with several portions. The mixture was stirred at 65° C. for 1 h. After being cooled to 0° C., the mixture was quenched with Na.sub.2SO.sub.410H.sub.2O carefully and filtered. The filtrate was concentrated to give the title compound 1,4-phenylenebis(methan-d.sub.2-ol) (E1).

(4-(((Tert-butyldimethylsilyl)oxy)methyl-d.SUB.2.)phenyl)methan-d.SUB.2.-ol (E2)

(50) To a solution of 1,4-phenylenebis(methan-d.sub.2-ol) (E1) (4.3 g, 30 mmol) and 1H-imidazole (2.2 g, 33 mmol) in DMF (50 mL) was added tert-butylchlorodimethylsilane (8.3 g, 30 mmol) dropwise. After being stirred at RT for 18 h, the mixture was poured into H.sub.2O, and extracted with EtOAc (3×). The extracts were washed with H.sub.2O, brine, dried over Na.sub.2SO.sub.4 and concentrated to give the title compound (4-(((tert-butyldimethylsilyl)oxy)methyl-d.sub.2)phenyl)methan-d.sub.2-ol (E2).

Tert-butyl((4-(iodomethyl-d.SUB.2.)phenyl)methoxy-d.SUB.2.)dimethylsilane (E3)

(51) A mixture of (4-(((tert-butyldimethylsilyl)oxy)methyl-d.sub.2)phenyl)methan-d.sub.2-ol (E2) (3.8 g, 10 mmol), 1H-imidazole (1.05 g, 15.0 mmol), iodine (3.3 g, 13 mmol) and triphenylphosphane (3.9 g, 15 mmol) in DCM (60 mL) was stirred at RT under N.sub.2 atmosphere for 5 h. The mixture was diluted with DCM, washed with Na.sub.2S.sub.2O.sub.3 aqueous solution and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (10:1) to give the title compound tert-butyl((4-(iodomethyl-d.sub.2)phenyl)methoxy-d.sub.2)dimethylsilane (E3).

2-(4-(((Tert-butyldimethylsilyl)oxy)methyl-d.SUB.2.)phenyl)acetonitrile-d.SUB.2 .(E4)

(52) To a mixture of tert-butyl((4-(iodomethyl-d.sub.2)phenyl)methoxy-d.sub.2)dimethylsilane (E4) (3.6 g, 7.3 mmol) and K.sub.2CO.sub.3 (1.8 g, 13 mmol) in acetonitrile (40 mL) was added TMSCN (1.6 mL, 13 mmol). After being refluxed for 20 h, the mixture was cooled to RT and filtered. The filtrate was concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (10:1) to give the title compound 2-(4-(((tert-butyldimethylsilyl)oxy)methyl-d.sub.2)phenyl)acetonitrile-d.sub.2 (E5).

2-(4-(Hydroxymethyl-d.SUB.2.)phenyl)acetic-2,2-d.SUB.2 .acid (E6)

(53) To a solution of NaH (5.00 g, 120 mmol) in D.sub.2O (20 ml) was added 2-(4-(((tert-butyldimethylsilyl)oxy)methyl-d.sub.2)phenyl)acetonitrile-d.sub.2 (E5) (2.5 g, 6.0 mmol) in EtOD (20 mL). The mixture was stirred at 80° C. for 15 h. After cooling to RT, the mixture was diluted with D.sub.2O (30 ml), adjusted pH to 2˜3 and extracted with EtOAc (3×). The extracts were washed with brine and dried over Na.sub.2SO.sub.4. The solvents were concentrated to give the title compound 2-(4-(hydroxymethyl-d.sub.2)phenyl)acetic-2,2-d.sub.2 acid (E6). MS-ESI (m/z): 169 [M−1].sup.−.

Methyl 2-(4-(hydroxymethyl-d.SUB.2.)phenyl)acetate-d.SUB.2 .(E)

(54) To a solution of 2-(4-(hydroxymethyl-d.sub.2)phenyl)acetic-2,2-d.sub.2 acid (E6) (1.35 g, 7.94 mmol) in MeOH (13 mL) was added Con.H.sub.2SO.sub.4 (1.3 mL) dropwise. The mixture was stirred at RT for overnight. The mixture was poured into water, and extracted with EtOAc (3×). The extracts were washed with sat. NaHCO.sub.3 (aqueous solution) and brine, dried over Na.sub.2SO.sub.4, and concentrated to give the title compound methyl 2-(4-(hydroxymethyl-d.sub.2)phenyl)acetate-d.sub.2 (E). MS-ESI (m/z): 185 [M+1].sup.+.

Intermediate F

Tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl-d2)carbamate (F)

(55) ##STR00036##

(1H-benzo[d][1,2,3]triazol-1-yl)methan-d.SUB.2.-ol (F1)

(56) To a solution of 1H-benzo[d][1,2,3]triazole (625 mg, 5.25 mmol) in H.sub.2O (40 mL) was added formaldehyde-d.sub.2 (20% in D.sub.2O) (840 mg, 5.25 mmol). The mixture was stirred at RT for 3 h. The solid was collected by filtration, and dried to give the title compound (1H-benzo[d][1,2,3]triazol-1-yl)methan-d.sub.2-ol (F1). MS-ESI (m/z): 152 [M+1].sup.+.

Tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl-d.SUB.2.)carbamate (F)

(57) A mixture of (1H-benzo[d][1,2,3]triazol-1-yl)methan-d.sub.2-ol (F1) (650 mg, 4.28 mmol), tert-butyl carbamate (500 mg, 4.28 mmol) and 4-methylbenzenesulfonic acid (26.7 mg, 0.155 mmol) in toluene (50 mL) was refluxed using Dean-Stark trap for 1 h. After being cooled to RT, the mixture was concentrated to ¼ volume. Then, the solid was collected by filtration, and dried to give the title compound tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl-d.sub.2)carbamate (F). MS-ESI (m/z): 251 [M+1].sup.+.

Example 1

(S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1)

(58) ##STR00037##

(S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)oxy)methyl)phenyl)-propanoic acid (1a)

(59) (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)oxy)methyl)-phenyl)propanoic acid (1a) was prepared according to the method described in WO2017/086941.

(S)-4-(3-((tert-butoxycarbonyl)amino)-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1b)

(60) To a mixture of (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)-oxy)methyl)phenyl)propanoic acid (1a) (56 mg, 0.13 mmol), isoquinolin-1-d-6-amine (A) (25 mg, 0.17 mmol) and 2,4-lutidine (18 mg, 0.17 mmol) in DMF (1 mL) was added 1,1,1-trichloro-2-methylpropan-2-yl carbonochloridate (41 mg, 0.17 mmol) and stirred at RT for 3 h. The mixture was poured into water (20 mL), extracted with EtOAc (3×50 mL), washed with brine (15 mL), dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by preparative TLC, eluting with DCM/MeOH (15:1) to give (S)-4-(3-((tert-butoxycarbonyl)amino)-1-((isoquinolin-6-yl-1-d) amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1b). MS-ESI (m/z): 555 [M+1].sup.+.

(S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1)

(61) To a mixture of (S)-4-(3-((tert-butoxycarbonyl)amino)-1-((isoquinolin-6-yl-1-d)-amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1b) (25 mg, 0.050 mmol) in DCM (5 mL) was added TFA (1 mL) dropwise. The mixture was stirred at RT for 1 h. The mixture was poured into saturated NaHCO.sub.3 and extracted with DCM (3×30 mL). The organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by preparative TLC eluting with DCM/NH.sub.3 in MeOH (20:1) to give (S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1). MS-ESI (m/z): 455 [M+1].sup.+.

Example 2

(S)-4-(3-amino-1-((isoquinolin-6-yl-3-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (2-A) and (S)-4-(3-amino-1-((isoquinolin-6-yl-3,4-d.SUB.2.)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (2-B)

(62) ##STR00038##

(S)-4-(3-amino-1-((isoquinolin-6-yl-3-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (2)

(63) The title compound (S)-4-(3-amino-1-((isoquinolin-6-yl-3-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (2-A) (30%) and (S)-4-(3-amino-1-((isoquinolin-6-yl-3,4-d.sub.2)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (2-B) (70%) was prepared according to the synthetic method of (S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethyl-benzoate (1) by replacing isoquinolin-1-d-6-amine (A) with isoquinolin-3-d-6-amine (B-a) and isoquinolin-3,4-d.sub.2-6-amine (B-b). MS-ESI (m/z): 2-A: 455 [M+1].sup.+ and 2-B: 456 [M+1].sup.+

Example 3

(S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3)

(64) ##STR00039##

(4-(2-((tert-Butyldimethylsilyl)oxy)ethyl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3a)

(65) To a solution of 2,4-dimethylbenzoic acid (31.0 mg, 0.204 mmol) in DCM (1 mL) was added EDCI (65.0 mg, 0.335 mmol). The mixture was stirred at RT for 1 h, and then, a solution of (4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)methan-d.sub.2-ol (D) (50.0 mg, 0.186 mmol) in DCM and DMAP (5.0 mg, 0.0372 mmol) were added. The mixture was stirred at RT for overnight. The mixture was diluted with water and extracted with DCM (3×25 mL). The extracts were washed with brine (15 mL), dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (50:1) to give the title compound (4-(2-((tert-butyldimethylsilyl)oxy)ethyl)-phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3a). MS-ESI (m/z): 401 [M+1].sup.+.

(4-(2-Hydroxyethyl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3b)

(66) To a solution of (4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3a) (4.100 g, 10.25 mmol) in MeOH (20 mL) was added 1 N HCl (2 mL). The mixture was stirred at RT for 1 h. The mixture was diluted with water and extracted with DCM (3×25 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to give the title compound (4-(2-hydroxyethyl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3b). MS-ESI (m/z): 287 [M+1].sup.+.

(4-(2-Oxoethyl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3c)

(67) To a solution of (4-(2-hydroxyethyl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3b) (2.90 g, 10.3 mmol) in DCM (100 mL) was added DMP (5.20 g, 12.3 mmol). The mixture was stirred at RT for 30 min. The reaction was quenched with saturated aqueous NaHCO.sub.3 solution (100 mL) and extracted with DCM. The organic phase was washed with brine (20 mL), dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (10:1) to give the title compound (4-(2-oxoethyl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3c). MS-ESI (m/z): 285 [M+1].sup.+.

2-(4-(((2,4-Dimethylbenzoyl)oxy)methyl-d.SUB.2.)phenyl)acetic acid (3d)

(68) To a solution of (4-(2-oxoethyl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3c) (50 mg, 0.18 mmol) in DMF (1 mL) was added Oxone (36 mg, 0.21 mmol). The mixture was stirred at RT for 4 h. The mixture was poured into water (20 mL). The precipitated solid was collected by filtration, washed with water (30 mL) and dried to give the crude product of 2-(4-(((2,4-dimethylbenzoyl)oxy)-methyl-d.sub.2)phenyl)acetic acid (3d), which was used for next step without further purification. MS-ESI (m/z): 301 [M+1].sup.+.

(4-(2-Chloro-2-oxoethyl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3e)

(69) To a solution of 2-(4-(((2,4-dimethylbenzoyl)oxy)-methyl-d.sub.2)phenyl)acetic acid (3d) (1.9 g, 6.2 mmol) in DCM (40 mL) was added oxalyl chloride (0.79 ml, 7.4 mmol). The mixture was stirred at RT for overnight. The mixture was concentrated to give the crude product of (4-(2-chloro-2-oxoethyl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3e), which was used for next step without further purification.

(R)-(4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3f)

(70) To a solution of (R)-4-benzyloxazolidin-2-one (1.1 g, 6.2 mmol) in THF (40 mL) at −70° C. was added n-BuLi (1.6 M, 4.3 mL, 6.8 mmol) dropwise, then a solution of (4-(2-chloro-2-oxoethyl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3e) (2.0 g, 6.2 mmol) in THF was added dropwise and the resulting mixture was stirred at −70° C. for 1 h. The reaction was quenched with saturated aqueous NH.sub.4Cl solution and extracted with EtOAc (2×50 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (10:1˜5:1) to give the title compound (R)-(4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)phenyl)methyl-d.sub.2 2,4-dimethyl-benzoate (3f). MS-ESI (m/z): 460 [M+1].sup.+.

(4-((S)-1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3 g)

(71) To a solution of (R)-(4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)phenyl)-methyl-d.sub.2 2,4-dimethylbenzoate (3f) (1.0 g, 2.2 mmol) in THF (20 mL) at −70° C. was added KHMDS (1.0 M, 2.6 mL, 2.61 mmol) dropwise. The mixture was stirred at −70° C. for 30 min, and then, a solution of tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl)carbamate (0.65 g, 2.6 mmol) (WO2017/086941) in THF (3 mL) was added dropwise at −70° C. The mixture was stirred at 0° C. for 1 h. The reaction was quenched with saturated aqueous NH.sub.4Cl solution and extracted with EtOAc (3×50 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (10:1˜5:1) to give the title compound (4-((S)-1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3 g). MS-ESI (m/z): 589 [M+1].sup.+.

(S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)oxy)methyl-d.SUB.2.)phenyl) propanoic acid (3h)

(72) To a solution of (4-((S)-1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3 g) (1.1 g, 1.9 mmol) in THF/H.sub.2O (22 ml/7 mL) at 0° C. was added H.sub.2O.sub.2 (30%, 0.85 g, 7.5 mmol) and LiOH.H.sub.2O (78 mg, 1.9 mmol) sequentially, The mixture was stirred at 0° C. for 10 min. The reaction was quenched with 10% Na.sub.2SO.sub.3 aqueous solution. The mixture was extracted with EtOAc (3×50 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with DCM/MeOH (100:1˜20:1) to give the title compound (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)oxy)methyl-d.sub.2)phenyl)propanoic acid (3h). MS-ESI (m/z): 430 [M+1].sup.+.

(S)-(4-(3-((tert-butoxycarbonyl)amino)-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3i)

(73) To a solution of isoquinolin-6-amine (23 mg, 0.16 mmol) and (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)oxy)methyl-d.sub.2)phenyl)propanoic acid (3h) (43 mg, 0.10 mmol) in DF (1 mL) was added EDCI (39 mg, 0.21 mmol), TEA (33 mg, 0.30 mmol) and HOBT (27 mg, 0.21 mmol). The mixture was stirred at RT for overnight. The mixture was diluted with water and extracted with EtOAc (3×5 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with DCM/MeOH (15:1) to give the title compound (S)-(4-(3-((tert-butoxycarbonyl)amino)-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3i). MS-ESI (m/z): 556 [M+1].sup.+.

(S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)phenyl)methyl-d.SUB.2 .2,4-dimethylbenzoate (3)

(74) The title compound (S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)-phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3) was prepared according to the synthetic method of (S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1) by replacing (S)-4-(3-((tert-butoxycarbonyl)amino)-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1b) with (S)-(4-(3-((tert-butoxycarbonyl)amino)-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3i). MS-ESI (m/z): 456 [M+1].sup.+.

Example 4

(S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl-3,3-d.SUB.2.)benzyl 2,4-dimethylbenzoate dihydrochloride (4)

(75) ##STR00040##

(R)-4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)benzyl 2,4-dimethylbenzoate (4a)

(76) The title compound (R)-4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)benzyl 2,4-dimethylbenzoate (4a) was prepared according to the method described in WO2017/086941. MS-ESI (m/z): 458 [M+1].sup.+.

4-((S)-1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl-3,3-d2)benzyl 2,4-dimethylbenzoate (4b)

(77) To a solution of the title compound (R)-4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)benzyl 2,4-dimethylbenzoate (4a) (1.0 g, 2.2 mmol) in THF (20 mL) at −70° C. was added KHMDS (1.0 M, 2.6 mL, 2.61 mmol) dropwise. The mixture was stirred at −70° C. for 30 min, and then, a solution of tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl-d.sub.2)carbamate (F) (0.65 g, 2.6 mmol) in THF (3 mL) was added dropwise at −70° C. The mixture was stirred at 0° C. for 1 h. The reaction was quenched with saturated aqueous NH.sub.4Cl solution and extracted with EtOAc (3×50 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (10:1˜5:1) to give the title compound 4-((S)-1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl-3,3-d.sub.2)benzyl 2,4-dimethylbenzoate (4b). MS-ESI (m/z): 589 [M+1].sup.+.

(S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)oxy)methyl)phenyl)propanoic-3,3-d2 acid (4c)

(78) The title compound (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethyl-benzoyl)oxy)methyl)phenyl)propanoic-3,3-d.sub.2 acid (4c) was prepared according to the synthetic method of (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)oxy)methyl-d.sub.2)-phenyl)propanoic acid (3h) by replacing (4-((S)-1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate (3 g) with 4-((S)-1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl-3,3-d.sub.2)benzyl 2,4-dimethylbenzoate (4b). MS-ESI (m/z): 430 [M+1].sup.+

(S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl-3,3-d2)benzyl 2,4-dimethylbenzoate (4d)

(79) The title compound (S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl-3,3-d.sub.2)benzyl 2,4-dimethylbenzoate (4d) was prepared according to the synthetic method of (S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate (1) by replacing (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethyl-benzoyl)oxy)methyl) phenyl)propanoic acid (1a) with (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl) oxy)methyl)phenyl)propanoic-3,3-d.sub.2 acid (4c). MS-ESI (m/z): 457 [M+1].sup.+.

(S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl-3,3-d)benzyl 2,4-dimethylbenzoate dihydrochloride (4)

(80) To a solution of (S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl-3,3-d.sub.2)benzyl 2,4-dimethylbenzoate (4d) (22 mg, 0.048 mmol) in DCM (1 mL) and MeOH (0.1 mL) was added HCl (30% EtOH solution, 0.108 mmol). After being stirred for 10 min, the mixture was concentrated to give the title compound (S)-4-(3-amino-1-((isoquinolin-6-yl-1-d)amino)-1-oxopropan-2-yl-3,3-d.sub.2)benzyl 2,4-dimethylbenzoate dihydrochloride (4). MS-ESI (m/z): 457 [M+1].sup.+.

Example 5

(S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2,3,3-d)phenyl)-methyl-d.SUB.2 .2,4-bis(methyl-d.SUB.3.)benzoate dihydrochloride (5)

(81) ##STR00041##

(4-(2-Methoxy-2-oxoethyl-1,1-d2)phenyl)methyl-d.SUB.2.2,4-bis(methyl-d.SUB.3.)benzoate (5a)

(82) A mixture of methyl 2-(4-(hydroxymethyl-d.sub.2)phenyl)acetate-d.sub.2 (E) (610 mg, 3.30 mmol), 2,4-bis(methyl-d.sub.3)benzoic acid (C) (517 mg, 3.30 mmol), EDCI (1.2 g, 6.0 mmol) and DMAP (81 mg, 0.66 mmol) in DCM (20 mL) was stirred at RT for overnight. The mixture was diluted with DCM and washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel, eluting with PE/EtOAc (10:1) to give the title compound (4-(2-methoxy-2-oxoethyl-1,1-d.sub.2)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3) benzoate (5a). MS-ESI (m/z): 323 [M+1].sup.+.

2-(4-(((2,4-Bis(methyl-d.SUB.3.)benzoyl)oxy)methyl-d.SUB.2.)phenyl)acetic-2,2-d.SUB.2 .acid (5b)

(83) To a solution of (4-(2-methoxy-2-oxoethyl-1,1-d.sub.2)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate (5a) (752 mg, 2.33 mmol) in D.sub.2O (15 mL) and THF (10 mL) was added LiOH.H.sub.2O (108 mg, 2.6 mmol). The mixture was stirred at RT for 2 h. The mixture was acidified to pH=2˜3, diluted with H.sub.2O and extracted with EtOAc (3×). The combined EtOAc phase was washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4 and concentrated to give the title compound 2-(4-(((2,4-bis(methyl-d.sub.3)benzoyl)oxy)methyl-d.sub.2)phenyl)acetic-2,2-d.sub.2 acid (5b). MS-ESI (m/z): 307 [M−1].sup.−.

(R)-(4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl-1,1-d.SUB.2.)phenyl)methyl-d.SUB.2 .2,4-bis(methyl-d.SUB.3.)benzoate (5c)

(84) The title compound (R)-(4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl-1,1-d.sub.2)-phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate (5c) was prepared according to the synthetic method of 3f by replacing 2-(4-(((2,4-dimethylbenzoyl)oxy)-methyl-d.sub.2)phenyl)acetic acid (3d) with 2-(4-(((2,4-bis(methyl-d.sub.3)benzoyl)oxy)methyl-d.sub.2)phenyl)acetic-2,2-d.sub.2 acid (5b). MS-ESI (m/z): 468 [M+1].sup.+.

(S)-(4-(1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl-2,3,3-d.SUB.3.)phenyl)methyl-d.SUB.2 .2,4-bis(methyl-d.SUB.3.)benzoate (5d)

(85) The title compound (S)-(4-(1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl-2,3,3-d.sub.3)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate (5d) was prepared according to the synthetic method of 3 g by replacing (R)-(4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)phenyl)-methyl-d.sub.2 2,4-dimethylbenzoate (3f) and tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl)carbamate with (R)-(4-(2-(4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl-1,1-d.sub.2)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate (5c) and tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl-d.sub.2)carbamate (F). MS-ESI (m/z): 598 [M+1].sup.+.

(S)-2-(4-(((2,4-bis(methyl-d.SUB.3.)benzoyl)oxy)methyl-d2)phenyl)-3-((tert-butoxycarbonyl)amino)propanoic-2,3,3-d.SUB.3 .acid (5e)

(86) To a solution of (S)-(4-(1-((R)-4-benzyl-2-oxooxazolidin-3-yl)-3-((tert-butoxycarbonyl)amino)-1-oxopropan-2-yl-2,3,3-d.sub.3)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate (5d) (538 mg, 0.904 mmol) in D.sub.2O (5 mL) and THF (10 mL) at 0° C. was added H.sub.2O.sub.2 (0.41 ml, 3.61 mmol) followed by LiOH.H.sub.2O (42 mg, 0.99 mmol). The mixture was stirred at 0° C. for 0.5 h, then quenched with NaHSO.sub.3 (10% aqueous solution). The mixture was acidified to pH=2˜3 and extracted with EtOAc (3×50 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by column chromatography on silica gel, eluting with DCM/MeOH (100:1˜10:1) to give the title compound (S)-2-(4-(((2,4-bis(methyl-d.sub.3)benzoyl)oxy)-methyl-d.sub.2)phenyl)-3-((tert-butoxycarbonyl)amino)propanoic-2,3,3-d.sub.3 acid (5e). MS-ESI (m/z): 439 [M−1].sup.−.

(S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2,3,3-d.SUB.3.)phenyl)methyl-d.SUB.2 .2,4-bis(methyl-d.SUB.3.)benzoate (5f)

(87) The title compound (S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2,3,3-d.sub.3)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate (5f) was prepared according to the synthetic method of 3 by replacing (S)-3-((tert-butoxycarbonyl)amino)-2-(4-(((2,4-dimethylbenzoyl)oxy)methyl-d.sub.2)phenyl) propanoic acid (3h) with (S)-2-(4-(((2,4-bis(methyl-d.sub.3)benzoyl)oxy)methyl-d.sub.2)phenyl)-3-((tert-butoxycarbonyl)amino)propanoic-2,3,3-d.sub.3 acid (5e). MS-ESI (m/z): 465 [M+1].sup.+.

(S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2,3,3-d3)phenyl)methyl-d.SUB.2 .2,4-bis(methyl-d.SUB.3.)benzoate dihydrochloride (5)

(88) To a solution of (S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2,3,3-d)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate (5f) (20 mg, 0.043 mmol) in DCM (1 mL) and MeOH (0.1 mL) was added HCl (30% EtOH solution, 0.108 mmol). After being stirred for 10 min, the mixture was concentrated to give the title compound (S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2,3,3-d.sub.3)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate dihydrochloride (5). MS-ESI (m/z): 465 [M+1].sup.+.

Example 6

(S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2-d)phenyl)methyl-d.SUB.2 .2,4-bis(methyl-d.SUB.3.)benzoate dihydrochloride (6)

(89) ##STR00042##

(90) The title compound (S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2-d)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate dihydrochloride (6) was prepared according to the synthetic method of 5 by replacing tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl-d.sub.2)carbamate (F) with tert-butyl ((1H-benzo[d][1,2,3]triazol-1-yl)methyl)carbamate. MS-ESI (m/z): 463 [M+1].sup.+.

Reference Compound 1

(S)-4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate dihydrochloride (Reference compound 1)

(91) (S)-4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate was disclosed and prepared following essentially the same procedures outlined on pages 21 of WO2017/086941. Reference compound 1 was prepared according to the synthetic method of (5) by replacing(S)-(4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl-2,3,3-d.sub.3)phenyl)methyl-d.sub.2 2,4-bis(methyl-d.sub.3)benzoate (5f) with (S)-4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate.

(92) Following essentially the same procedures described for Examples 1-6, Examples 7-24 listed in Table 1 were prepared from the appropriate starting materials which are commercially available or known in the literature. The structures and names of Examples 7-24 are given in Table 1.

(93) TABLE-US-00001 TABLE 1 EXAMPLE STRUCTURE NAME DATA 7 (S)-(4-(3-amino-1- ((isoquinolin-6-yl-1- d)amino)-1-oxopropan- 2-yl-2,3,3-d.sub.3) phenyl)methyl-d.sub.2 2,4-dimethylbenzoate dihydrochloride MS-ESI (m/z): 460 [M + 1].sup.+ 8 (S)-(4-(3-amino-1- (isoquinolin-6- ylamino)-1-oxopropan- 2-yl-3,3-d.sub.2) phenyl)methyl-d.sub.2 2,4- dimethylbenzoate dihydrochloride MS-ESI (m/z): 458 [M + 1].sup.+ 9 (S)-(4-(3-amino-1- (isoquinolin-6-ylamino)- 1-oxopropan-2-yl-2-d) phenyl)methyl-d.sub.2 2,4- dimethylbenzoate dihydrochloride MS-ESI (m/z): 457 [M + 1].sup.+ 10 (S)-(4-(3-amino-1- (isoquinolin-6-ylamino)- 1-oxopropan-2-yl- 2,3,3-d.sub.3)phenyl)methyl-d.sub.2 2,4-dimethylbenzoate dihydrochloride MS-ESI (m/z): 459 [M + 1].sup.+ 11 (S)-4-(3-amino-1- (isoquinolin-6- ylamino)-1-oxopropan- 2-yl-3,3-d.sub.2) benzyl 2,4- dimethylbenzoate dihydrochloride MS-ESI (m/z): 456 [M + 1].sup.+ 12 (S)-(4-(3-amino-1- ((isoquinolin-6-yl-1- d)amino)-1-oxopropan- 2-yl-3,3-d.sub.2) phenyl)methyl-d.sub.2 2,4- dimethylbenzoate dihydrochloride MS-ESI (m/z): 459 [M + 1].sup.+ 13 (S)-(4-(3-amino-1- ((isoquinolin-6-yl-1- d)amino)-1-oxopropan- 2-yl-2-d)phenyl) methyl-d.sub.2 2,4- dimethylbenzoate dihydrochloride MS-ESI (m/z): 458 [M + 1].sup.+ 14 0 (S)-4-(3-amino-1- (isoquinolin-6- ylamino)-1-oxopropan- 2-yl)benzyl 2,4- bis(methyl-d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 460 [M + 1].sup.+ 15 (S)-(4-(3-amino-1- (isoquinolin-6- ylamino)-1-oxopropan- 2-yl)phenyl)methyl- d.sub.2 2,4-bis(methyl- d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 462 [M + 1].sup.+ 16 (S)-4-(3-amino-1- (isoquinolin-6-ylamino)- 1-oxopropan-2-yl- 3,3-d.sub.2)benzyl 2,4- bis(methyl-d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 462 [M + 1].sup.+ 17 (S)-(4-(3-amino-1- (isoquinolin-6-ylamino)- 1-oxopropan-2-yl- 3,3-d.sub.2)phenyl)methyl-d.sub.2 2,4- bis(methyl-d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 464 [M + 1].sup.+ 18 (S)-(4-(3-amino-1- ((isoquinolin-6-yl-1- d)amino)-1-oxopropan- 2-yl)phenyl) methyl-d.sub.2 2,4- dimethylbenzoate MS-ESI (m/z): 457 [M + 1].sup.+ 19 (S)-4-(3-amino-1- ((isoquinolin-6-yl-1- d)amino)-1- oxopropan-2-yl)benzyl 2,4- bis(methyl-d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 461 [M + 1].sup.+ 20 (S)-(4-(3-amino-1- ((isoquinolin-6-yl-1- d)amino)-1-oxopropan- 2-yl)phenyl) methyl-d.sub.2 2,4-bis (methyl-d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 463 [M + 1].sup.+ 21 (S)-4-(3-amino-1- ((isoquinolin-6-yl-1- d)amino)-1-oxopropan- 2-yl-3,3-d.sub.2) benzyl 2,4-bis(methyl- d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 463 [M + 1].sup.+ 22 (S)-(4-(3-amino-1- ((isoquinolin-6-yl-1-d) amino)-1-oxopropan- 2-yl-3,3-d.sub.2)phenyl) methyl-d.sub.2 2,4-bis (methyl-d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 465 [M + 1].sup.+ 23 (S)-(4-(3-amino-1- ((isoquinolin-6-yl-1-d) amino)-1-oxopropan- 2-yl-2-d)phenyl) methyl-d.sub.2 2,4-bis (methyl-d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 464 [M + 1].sup.+ 24 0 (S)-(4-(3-amino-1- ((isoquinolin-6-yl-1-d) amino)-1-oxopropan- 2-yl-2,3,3-d.sub.3) phenyl)methyl-d.sub.2 2,4-bis(methyl- d.sub.3)benzoate dihydrochloride MS-ESI (m/z): 466 [M + 1].sup.+
Cell Proliferation Assays
ROCK-I(h) In Vitro Inhibition Activity-Method A

(94) ROCK-I(h) kinase assays were conducted using the KinaseProfiler™ service of Eurofins Pharma Discovery Services UK Limited. ROCK-I(h) was incubated with the test compound in assay buffer containing 8 mM MOPS pH 7.0 (VWR cat #441644J) 0.2 mM EDTA (Sigma cat #E5134), 30 μM KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK (Pepceuticals), 10 mM magnesium acetate (Merck Millipore cat #105819) and [γ-33P-ATP] (Perkin Elmer cat #NEG602K). Test compound in 100% DMSO (Calbiochem cat #317275) was spotted into a 96-well assay plate prior using a Mosquito X1 (TTP Labtech) prior to addition of the reaction mix. The reaction was initiated by the addition of the Mg/ATP mix using a Matrix Wellmate. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of a 3% phosphoric acid solution (Fisher Scientific cat #O/0500/PB17) using a Matrix Wellmate. An aliquot of the reaction was then spotted onto a filtermat and washed in phosphoric acid followed by a rinse in methanol prior to drying and scintillation counting in a Trilux Wallac Microbeta detector (Perkin Elmer). Results were expressed in relation to controls containing DMSO only in place of test compound. Where applicable, IC50 curve analysis was performed using XLFit version 5.3 (ID Business Solutions). Sigmoidal dose-response (variable slope) curves are fitted using non-linear regression analysis.

(95) ROCK-II(h) kinase assays were conducted using the KinaseProfiler™ service of Eurofins Pharma Discovery Services UK Limited. ROCK-II(h) was incubated with the test compound in assay buffer containing 50 mM Tris pH 7.5 (VWR cat #103157P), 0.1 mM EGTA (VWR cat #20308), 30 μM KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK (Pepceuticals), 10 mM magnesium acetate and [γ-33P-ATP] (Perkin Elmer cat #NEG602K). Test compound in 100% DMSO (Calbiochem cat #317275) was spotted into a 96-well assay plate prior using a Mosquito X1 (TTP Labtech) prior to addition of the reaction mix. The reaction was initiated by the addition of the Mg/ATP mix using a Matrix Wellmate. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of a 3% phosphoric acid solution (Fisher Scientific cat #O/0500/PB17) using a Matrix Wellmate. An aliquot of the reaction was then spotted onto a filtermat and washed in phosphoric acid followed by a rinse in methanol prior to drying and scintillation counting in a Trilux Wallac Microbeta detector (Perkin Elmer). Results were expressed in relation to controls containing DMSO only in place of test compound. Where applicable, IC.sub.50 curve analysis was performed using XLFit version 5.3 (ID Business Solutions). Sigmoidal dose-response (variable slope) curves are fitted using non-linear regression analysis.

(96) Select compounds prepared as described above were assayed according to the biological procedures described in Method A. The results are given in the table 2.

(97) TABLE-US-00002 TABLE 2 ROCK-I (h) ROCK-II (h) Example IC.sub.50 (nM) IC.sub.50 (nM)  1 44 15  3 64 18 18 26 8
ROCK-I(h) In Vitro Inhibition Activity-Method B

(98) ROCK-I (Invitrogen, PV3691) kinase assays were conducted using the HTRF® KinEASE-STK kit service of CISBIO. Transfer 10 nl compound dilutions into each well of assay plates (784075, Greiner) using Echo 550; Centrifuge compound plates at 1000 g for 1 min. Seal the assay plate. Prepare 5×ROCK-I (Invitrogen, PV3691) in 1× kinase buffer (1 volume of enzymatic buffer 5× with 4 volumes of distilled water; 5 mM MgCl.sub.2; 1 mM DTT). Add 2 μl of 5× ROCK-I into 384-well assay plate (784075, Greiner). Add 4 μl× kinase buffer into each well of the assay plate and centrifuge plates at 1000 g for 30 s, RT for 10 min. Prepare 5× TK-substrate-biotin (5 μM) in kinase buffer and 5×ATP (100 μM) in kinase buffer. Start the reaction by adding 2 μl STK-substrate-biotin and 2 μl ATP. Centrifuge plates at 1000 g for 30 s. Seal the assay plate, RT for 20 min. Prepare 4× Sa-XL 665 (250 nM) and TK-antibody-Cryptate in HTRF detection buffer. Add 5 μl Sa-XL 665 and 5 μl TK-antibody-Cryptate into each well of the assay plate. Centrifuge plate at 1000 g for 30 s, RT for 1 h. Read fluorescence signal at 615 nm (Cryptate) and 665 nm (XL665) on Envision 2104 plate reader (Perkin Elmer). A Ratio (665/615 nm) is calculated for each well. Calculate IC.sub.50 by fitting % Inhibition values and log of compound concentrations to nonlinear regression (dose response−variable slope) with GraphPad 6.0.

(99) Select compounds prepared as described above were assayed according to the biological procedures described in Method B. The results are given in the table 3.

(100) TABLE-US-00003 TABLE 3 ROCK-I (h) Example IC.sub.50 (nM)  4 14  5 39  6 21  7 27  8 23  9 21 10 17 11 21 12 15 13 17 14 19 15 27 16 19 17 21 19 26 20 19 21 20 22 33 23 18 Reference 18 compound 1
ROCK In Vivo Inhibition Activity-Method C

(101) Oxybuprocaine (Santen Co. JP lot: B2031); Carbonate restrainer (harvard apparatus); TONOVET Plus (Finland icare); Chinchilla rabbits: 2˜8 months of age and 2.23˜4.41 kg of body weight (Dongfang Breeding Co., Ltd., Pizhou, Licence: SCXK (su) 2014-0005, Certificate No.: 201814493, 201826314).

(102) Eighteen male rabbits were randomly assigned to 3 groups (6 animals/group). Vehicle control, Rhopressa® (0.02% netarsudil, equivalent to 0.2 mg/ml netarsudil), or 0.2 mg/mL Example 11 were topically administered to right eye (30 μL/eye/dose) once daily for 3 days, with the fellow left eye treated with saline as the internal control. Clinical observations and ophthalmic examinations were performed once daily. IOP was measured by TONOVET Plus before first dosing, and at 0.5, 2, 4, 6 and 24 h after each first dosing on day 1 and day 3.

(103) The change of IOP % was calculated using the following formula:
The change of IOP %(ΔIOP %)=[IOP(time)−IOP(control)]/IOP(control)*100).

(104) Select compounds prepared as described above were assayed according to the biological procedures described in Method C. The results of dosing on day 3 at 24 h are given in the table 4.

(105) TABLE-US-00004 TABLE 4 Example ΔIOP %  1 −1.5  3 −5.02  4 −15.63  5 1.19  6 −16.08  7 0.48  8 −7.52  9 −3.06 10 −0.78 11 −9.57 12 −5.23 13 −8.48 14 −5.81 15 −4.25 16 −1.44 18 −1.1 19 −3.3 20 −4.89 21 −5.06 22 0.08 23 −0.87 Reference −4.35 compound 1