Phenylate derivative, preparation method therefor, and pharmaceutical composition and uses thereof

Abstract

The present invention discloses a phenylate derivative, a preparation method therefor, and a pharmaceutical composition and uses thereof. Specifically, the invention relates to phenylate derivatives represented by formula (I), a pharmaceutically-acceptable salt thereof, a stereoisomer thereof, a preparation method therefor, a pharmaceutical composition containing the one or more compounds, and uses of the compounds in treating diseases related to PD-1/PD-L1 signal channels, such as cancers, infectious diseases and autoimmune diseases. ##STR00001##

Claims

1. A phenylate compound of Formula (I), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00065## wherein: R.sub.1 is selected from ##STR00066## R.sub.2 is unsubstituted or substituted C.sub.1-C.sub.8 aliphatic hydrocarbonyl, and when substituted, the substituent is selected from fluorine, chlorine, bromine, iodine, cyano, trifluoromethyl, hydroxy, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino (CH.sub.3CONH), methanesulfonyl (SO.sub.2CH.sub.3), hydroxyformyl (COOH), carbamoyl (CONH.sub.2), and hydroxycarbamoyl (CONHOH); R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00067## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

2. A phenylate compound of claim 1, represented by formula (IA), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00068## wherein: R.sub.2 is unsubstituted or substituted C.sub.1-C.sub.8 aliphatic hydrocarbonyl, and when substituted, the substituent is selected from halogen, cyano, trifluoromethyl, hydroxy, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, methanesulfonyl (SO.sub.2CH.sub.3), hydroxyformyl (COOH), carbamoyl (CONH.sub.2), and hydroxycarbamoyl (CONHOH); R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00069## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

3. A phenylate compound of claim 2, represented by formula (IA-1), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00070## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00071## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

4. A phenylate compound of claim 2, represented by formula (IA-2), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00072## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00073## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

5. A phenylate compound of claim 2, represented by formula (IA-3), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00074## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00075## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

6. A phenylate compound of claim 2, represented by formula (IA-4), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00076## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00077## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

7. A phenylate compound of claim 2, represented by formula (IA-5), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00078## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00079## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

8. A phenylate compound of claim 1, represented by formula (TB), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00080## wherein: R.sub.2 is unsubstituted or substituted C.sub.1-C.sub.8 aliphatic hydrocarbonyl, and when substituted, the substituent is selected from halogen, cyano, trifluoromethyl, hydroxy, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, methanesulfonyl (SO.sub.2CH.sub.3), hydroxyformyl (COOH), and hydroxycarbamoyl (CONHOH); R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00081## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

9. A phenylate compound of claim 8, represented by formula (IB-1), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00082## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00083## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

10. A phenylate compound of claim 8, represented by formula (IB-2), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00084## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00085## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

11. A phenylate compound of claim 8, represented by formula (IB-3), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00086## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00087## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

12. A phenylate compound of claim 8, represented by formula (IB-4), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00088## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00089## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

13. A phenylate compound of claim 8, represented by formula (IB-5), or a pharmaceutically acceptable salt or a stereoisomer thereof: ##STR00090## wherein: R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, and substituted N-containing C.sub.2-C.sub.6 heterocycle-1-yl, wherein each is mono-, di-, tri-, or tetra-substituted with substituent(s) selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkoxy, amino, C.sub.1-C.sub.6 alkylamino, acetylamino, cyano, ureido (NH(CO)NH.sub.2), guanidino (NH(CNH)NH.sub.2), ureido amino (NHNH(CO)NH.sub.2), guanidino amino (NHNH(CNH)NH.sub.2), sulfonylamino (NHSO.sub.3H), sulfamoyl (SO.sub.2NH.sub.2), methanesulfonylamino (NHSO.sub.2CH.sub.3), hydroxyformyl (COOH), C.sub.1-C.sub.8 alkoxyl carbonyl, sulfydryl, imidazolyl, thiazolyl, oxazolyl, tetrazolyl, ##STR00091## and X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

14. A phenylate compound of claim 1, or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein R.sub.3 is of one of the following formulae: ##STR00092## ##STR00093## wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.

15. A phenylate compound of claim 1, or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the compound is selected from: (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(carbamoylmethoxy) benzyl) serine ##STR00094## N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-methoxy benzylamine hydrochloride ##STR00095## N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-allyloxy benzylamine ##STR00096## N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(prop-2-ynyloxy) benzylamine ##STR00097## N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropyl methoxy) benzylamine ##STR00098## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(N, N-dimethylcarbamoylmethoxy) benzyl) serine ##STR00099## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-methylbut-2-enyloxy) benzyl) serine ##STR00100## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzyl) threonine ##STR00101## 2-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzylamino)-3-hydroxypropanamide ##STR00102## N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropyl methoxy) benzylamine ##STR00103## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzyl) Citrulline ##STR00104## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(N-methoxy-N-methylcarbamoyl methoxy) benzyl) serine ##STR00105## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(2-morpholino-2-oxoethoxy) benzyl) serine ##STR00106## (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzyl) serine ##STR00107## (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(N-hydroxycarbamoylmethoxy) benzyl) Pipecolinic acid ##STR00108## (S,S)N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(carbamoylmethoxy) benzyl)-4-hydroxyproline ##STR00109## and (S,S)N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(cyclopropylmethoxy) benzyl)-4-hydroxyproline ##STR00110##

16. A phenylate compound of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt comprises a salt formed with an inorganic acid, a salt formed with an organic acid, an alkali metal ion salt, alkaline earth metal ion salt or a salt formed with organic base which provides a physiologically acceptable cation, and an ammonium salt.

17. A phenylate compound of claim 16, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, and sulfuric acid; the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic, citric acid, maleic acid, tartaric acid, fumaric acid, citric acid, and lactic acid; the alkali metal ion is selected from lithium ion, sodium ion, and potassium ion; the alkaline earth metal ion is selected from calcium ion and magnesium ion; and the organic base which provides a physiologically acceptable cations is selected form methylamine, dimethylamine, trimethylamine, piperidine, morpholine, and tris(2-hydroxyethyl) amine.

18. A phenylate compound of claim 2, or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein R.sub.3 is of one of the following formulae: ##STR00111## ##STR00112## wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.

19. A phenylate compound of claim 3, or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein R.sub.3 is of one of the following formulae: ##STR00113## ##STR00114## wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, and octyl; and X is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.

20. A process for the preparation of the phenylate compound of claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, comprising the following five steps: ##STR00115## (a) 2-bromo-3-iodotoluene 1 and benzene boronic acid or substituted benzene boronic acid or boric acid ester of benzene or substituted benzene as starting materials are reacted via a Suzuki coupling reaction to obtain intermediate compound 2; (b) intermediate 2 as a starting material is subjected to bromination of the methyl group by a bromination reagent to give the bromo intermediate 3; (c) intermediate 3 as a starting material is reacted with substituted 2,4-dihydroxy-X-substituted benzaldehyde under basic conditions to obtain benzyl aryl ether intermediate 4; (d) intermediate 4 as a starting material is reacted with a halide under basic conditions to give intermediate compound 5; and (e) an aldehyde group-containing intermediate compound 5 as a starting material is condensed with an amino group- or an imino group-containing HR.sub.3 and the resultant product is reduced to obtain the target compound I; wherein each of R.sub.1, R.sub.2, R.sub.3 and X are as defined in claim 1.

21. A pharmaceutical composition comprising the phenylate compound of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

22. A pharmaceutical composition comprising the phenylate compound of claim 15, or a stereoisomer or a pharmaceutically acceptable salt thereof, as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

Description

EXAMPLES

(1) The invention is further illustrated by the following examples; however, the invention is not limited by the illustrative examples set herein below.

(2) Measuring instrument: Nuclear magnetic resonance spectroscopy was carried out by using a Vaariaan Mercury 300 nuclear magnetic resonance apparatus. Mass spectrometry was performed by using ZAD-2F mass spectrometer and VG300 mass spectrometer.

Example 1

(S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(carbamoylmethoxy)benzyl)serine

(3) ##STR00048##

(1) 2-Bromo-3-phenyltoluene

(4) To a 50 ml flask were added 2-bromo-3-iodotoluene (350 mg) and dioxane/water with stirring. The solution was bubbled with argon for 10 min to remove dissolved oxygen. Then, phenylboronic acid (172.65 mg), cesium carbonate (961.2 mg), and triphenylphosphine palladium (40.91 mg) were sequentially added. The mixture was stirred for 12 h at 80-100 C. under argon protection. The reaction was stopped. After cooling to room temperature, the mixture was filtered with diatomaceous earth. The filtrate was concentrated under reduced pressure and extracted with water and ethyl acetate for three times. The organic phase was combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The organic layer was filtered and evaporated in vacuo to dryness. The crude residue was purified by silica gel column chromatography (petroleum ether) to afford colorless oil (221 mg). .sup.1H NMR (400 MHz, DMSO-d.sub.6), 7.49-7.29 (m, 7H, ArH), 7.14 (d, 1H, ArH), 2.42 (s, 3H, ArCH.sub.3).

(2) 2-Bromo-3-(bromomethyl)-1,1-biphenyl

(5) 2-Bromo-3-phenyltoluene (234 mg) was weighed and was dissolved in 20 ml of CCl.sub.4 in a 100 ml flask. To this solution was added NBS (178 mg) while stirring. And the mixture was warmed to 80 C. and refluxed. Then benzoyl peroxide (4 mg) was added, and after 2 h, benzoyl peroxide (4 mg) was added again, and the reaction was continued for another 2 h. The reaction was stopped. After cooling to room temperature, the mixture was quenched with water, extracted with dichloromethane. The organic phase was washed with saturated brine, and dried over anhydrous sodium sulfate. The organic layer was filtered and evaporated in vacuo to dryness to afford yellow oil (192 mg), which was used for the next step without further purification.

(3) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde

(6) 2,4-dihydroxy-5-chlorobenzaldehyde (73.94 mg) was weighed and dissolved in 6 ml of anhydrous acetonitrile in a 50 ml flask, and then sodium hydrogen carbonate (98.88 mg) was added. After stirring at room temperature for 40 min, 2-bromo-3-phenylbenzyl bromide (192 mg, dissolved in 8 ml of DMF) was slowly added dropwise to the reaction mixture via a constant pressure dropping funnel, and heated to reflux until the reaction was completed. After cooling to room temperature, the mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine, and dried over anhydrous sodium sulfate, then filtrated and evaporated in vacuo to dryness. The crude residue was purified by silica gel column chromatography to afford 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde (152 mg) as a white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) 11.18 (s, 1H, OH), 10.09 (s, 1H, CHO), 7.74 (s, 1H, ArH), 7.66 (d, 1H, ArH), 7.57 (t, 1H, ArH), 7.51 (m, 2H, ArH), 7.46 (d, 1H, ArH), 7.42 (d, 3H, ArH), 6.85 (s, 1H, ArH), 5.37 (s, 2H, CH.sub.2).

(4) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(carbamoylmethoxy)benzaldehyde

(7) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde (100 mg) was dissolved in 6 ml of DMF in a 50 ml flask, and then cesium carbonate (127.53 mg) was added. After stirring at room temperature for 15 min, a solution of 2-bromoacetamide (68.25 mg) in DMF (4 ml) was added dropwise. After the mixture was stirred at 80 C. for 2 h, the reaction was stopped. After cooling to room temperature, the mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine, and dried over anhydrous sodium sulfate, then filtrated and evaporated in vacuo to dryness. The crude residue was purified by silica gel column chromatography to afford a white solid (60 mg). .sup.1H NMR (400 MHz, DMSO-6d) 10.28 (s, 1H, CHO), 7.74 (s, 1H, ArH), 7.72-7.63 (m, 2H, ArH), 7.58-7.36 (m, 8H, ArH, CONH.sub.2), 7.11 (s, 1H, ArH), 5.44 (s, 2H, CH.sub.2), 4.75 (s, 2H, CH.sub.2). MS (FAB): 476 (M+1).

(5) (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(carbamoylmethoxy)benzyl)serine

(8) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(carbamoylmethoxy)benzaldehyde (80 mg) was dissolved in 5 ml of DMF, and then ethyl ester of serine (49 mg) and acetic acid glacial (57 mg) were added. After stirring at room temperature for 20 min, sodium cyanoborohydride (25 mg) was added and the mixture was stirred at 25 C. for 14 h. The reaction was stopped. The mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine, and dried over anhydrous sodium sulfate, then filtrated and evaporated in vacuo to dryness. The residue was dissolved in ethanol, heated to reflux until the reaction was complete. The mixture was evaporated in vacuo to dryness. The crude residue was purified by silica gel column chromatography to afford ethyl (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(carbamoylmethoxy)benzyl)serinate (70 mg) as pale yellow oil. Then it was dissolved in methanol/H.sub.2O (4 ml/1 ml), and lithium hydroxide monohydrate (20 mg) was added. After stirring at room temperature for 2 h, a few drops of acetic acid were added to the mixture in an ice bath to adjust the pH to acidity. The mixture was evaporated in vacuo to afford (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(carbamoylmethoxy)benzyl)serine (45 mg) as a white solid. .sup.1H NMR (400 MHz, DMSO) 8.03 (s, 1H, ArH), 7.62 (d, J=6.7 Hz, 1H, ArH), 7.56-7.35 (m, 9H, ArH, CONH.sub.2), 7.00 (s, 1H, ArH), 5.32 (s, 2H, CH.sub.2), 4.60 (m, 2H, CH.sub.2), 4.03 (m, 2H, CH.sub.2), 3.78-3.56 (m, 3H, CH.sub.2, CH). MS (FAB): 565 (M+1).

Example 2

N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-methoxy benzylamine hydrochloride

(9) ##STR00049##

(1) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-methoxybenzaldehyde

(10) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxybenzaldehyde (100 mg, 0.24 mmol) was weighed and dissolved in 5 ml of anhydrous THF, and then cesium carbonate (93.4 mg, 0.29 mmol) was added. After the mixture was stirred for 15 min in an ice bath under argon protection, iodomethane (110 ul, 1.8 mmol) was added. The reaction was transferred to room temperature and continued for 3 h, and was extracted with ethyl acetate and water for three times. The organic phase was combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude residue was purified by silica gel column chromatography to afford a white solid (78.2 mg). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.13 (s, 1H, CHO), 7.66 (s, 1H, ArH), 7.51 (s, 1H, ArH), 7.44 (q, 3H, ArH), 7.39 (s, 1H, ArH), 7.36 (s, 2H, ArH), 7.34 (s, 1H, ArH), 7.06 (s, 1H, ArH), 5.44 (s, 2H, CH.sub.2), 3.97 (s, 3H, OCH.sub.3).

(2) N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-methoxy benzylamine hydrochloride

(11) 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-methoxybenzaldehyde (50 mg, 0.116 mmol) was dissolved in 5 ml of DMF, and then N-(2-aminoethyl) acetamide (35.54 mg, 0.348 mmol) was added and acetic acid glacial (42.11 mg, 0.696 mmol) was added dropwise. After stirring at room temperature for 20 min, sodium cyanoborohydride (18 mg, 0.29 mmol) was added and the mixture was stirred at 25 C. for 14 h. The reaction was stopped. The mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine, and dried over anhydrous sodium sulfate, then filtrated and evaporated in vacuo to dryness. The crude residue was purified by silica gel column chromatography to afford a viscous product. 10 ml of saturated HCl methanol solution was added, stirred at room temperature overnight. The mixture was evaporated in vacuo to dryness, and washed with diethyl ether to afford pale yellow solid powder (28 mg). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.31 (s, 1H, HCl), 9.04 (s, 1H, NH), 8.33 (s, 1H, CONH), 7.61 (m, 2H, ArH), 7.48 (s, 1H, ArH), 7.42 (m, 2H, ArH), 7.37 (m, 1H, ArH), 7.33 (m, 3H, ArH), 6.95 (s, 1H, ArH), 5.32 (s, 2H, CH.sub.2), 3.98 (s, 2H, CH.sub.2), 3.85 (s, 3H, OCH.sub.3), 3.16 (m, 2H, CH.sub.2), 2.86 (m, 2H, CH.sub.2), 1.78 (s, 3H, COCH.sub.3). MS (FAB): 517 (M).

Example 3

N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-allyloxy benzylamine

(12) ##STR00050##

(13) The procedure was the same as in Example 1, except that 3-bromoprop-1-ene was used in place of 2-bromoacetamide, N-(2-aminoethyl)acetamide was used in place of ethyl ester of serine to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.10 (s, 1H, CONH), 7.61 (d, 1H, ArH), 7.54 (d, 1H, ArH), 7.51-7.46 (m, 1H, ArH), 7.43 (d, 2H, ArH), 7.39 (d, 1H, ArH), 7.35 (s, 2H, ArH), 7.33 (s, 1H, ArH), 6.95 (s, 1H, ArH), 6.04 (m, 1H, CH), 5.39 (d, 1H, CH.sub.2), 5.31 (s, 2H, CH.sub.2), 5.24 (d, 1H, CH.sub.2), 4.68 (d, 2H, CH.sub.2), 3.98 (s, 2H, CH.sub.2), 2.85 (t, 2H, CH.sub.2), 1.79 (s, 3H, COCH.sub.3). MS (FAB): 545 (M+1).

Example 4

N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(prop-2-ynyloxy)benzylamine

(14) ##STR00051##

(15) The procedure was the same as in Example 1, except that 3-bromoprop-1-yne was used in place of 2-bromoacetamide, N-(2-aminoethyl)acetamide was used in place of ethyl ester of serine to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.50 (s, 1H, NH), 8.08 (t, 1H, CONH), 7.63 (dd, 1H, ArH), 7.57 (s, 1H, ArH), 7.49 (t, 1H, ArH), 7.44 (m, 1H, ArH), 7.42 (s, 1H, ArH), 7.41-7.37 (m, 1H, ArH), 7.36 (m, 2H, ArH), 7.34 (q, 1H, ArH), 7.09 (s, 1H, ArH), 5.31 (s, 2H, CH.sub.2), 4.93 (d, 2H, CH.sub.2), 3.95 (s, 2H, CH.sub.2), 3.59 (t, 1H, CH), 3.27-3.28 (m, 2H, CH.sub.2), 2.83 (m, 2H, CH.sub.2), 1.79 (s, 3H, COCH.sub.3). MS (FAB): 543 (M+1).

Example 5

N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropyl methoxy)benzylamine

(16) ##STR00052##

(17) The procedure was the same as in Example 1, except that (bromomethyl)cyclopropane was used in place of 2-bromoacetamide, N-(2-aminoethyl)acetamide was used in place of ethyl ester of serine to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.06 (s, 1H, CONH), 7.59 (m, 1H, ArH), 7.51-7.46 (m, 2H, ArH), 7.43 (m, 2H, ArH), 7.40 (s, 1H, ArH), 7.34 (m, 3H, ArH), 6.91 (d, 1H, ArH), 5.36-5.26 (m, 2H, CH.sub.2), 3.94-3.88 (m, 2H, CH.sub.2), 3.29-3.19 (m, 4H, CH.sub.2), 2.76 (m, 2H, CH.sub.2), 2.34 (m, 1H, CH.sub.2), 2.02 (m, 1H, CH.sub.2), 1.78 (s, 3H, COCH.sub.3), 1.27-1.15 (m, 1H, CH), 0.53 (m, 1H, CH.sub.2), 0.31 (m, 1H, CH.sub.2). MS (FAB): 559 (M+1).

Example 6

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(N,N-dimethylcarbamoylmethylcarbamoylmethoxy)benzyl)serine

(18) ##STR00053##

(19) The procedure was the same as in Example 1, except that N,N-dimethyl-2-bromoacetamide was used in place of 2-bromoacetamide to afford a white solid. .sup.1H NMR (400 MHz, DMSO) 7.65 (d, J=7.1 Hz, 1H, ArH), 7.45 (ddd, J=23.8, 18.3, 6.8 Hz, 8H, ArH), 7.03 (s, 1H, ArH), 5.25 (d, J=42.2 Hz, 2H, CH.sub.2), 5.05 (s, 2H, CH.sub.2), 4.05 (dd, J=32.7, 13.2 Hz, 2H, CH.sub.2), 3.78-3.56 (m, 3H, CH.sub.2, CH), 2.99 (s, 3H, CH.sub.3), 2.83 (s, 3H, CH.sub.3). MS (FAB): 593 (M+1).

Example 7

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(3-methylbut-2-enyloxy)benzyl)serine

(20) ##STR00054##

(21) The procedure was the same as in Example 1, except that 1-bromo-3-methylbut-2-ene was used in place of 2-bromoacetamide to afford a white solid. .sup.1H NMR (400 MHz, DMSO) 7.64 (dd, J=7.7, 1.7 Hz, 1H, ArH), 7.55-7.49 (m, 1H, ArH), 7.49-7.45 (m, 3H, ArH), 7.43 (dt, J=5.6, 2.3 Hz, 1H, ArH), 7.40-7.36 (m, 3H, ArH), 6.92 (s, 1H, ArH), 5.46-5.40 (m, 1H, CH), 5.33 (s, 2H, CH.sub.2), 4.65 (d, J=6.5 Hz, 2H, CH.sub.2), 3.88 (d, J=2.5 Hz, 2H, CH.sub.2), 3.64 (ddd, J=17.3, 11.0, 6.2 Hz, 2H, CH.sub.2), 3.15-3.09 (m, 1H, CH), 1.72 (d, J=1.0 Hz, 6H, CH.sub.3). MS (FAB): 576 (M+1).

Example 8

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzyl)threonine

(22) ##STR00055##

(23) The procedure was the same as in Example 1, except that (bromomethyl)cyclopropane was used in place of 2-bromoacetamide, and ethyl ester of threonine was used in place of ethyl ester of serine to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 7.65-6.86 (m, 10H, ArH), 5.27 (s, 2H, CH.sub.2), 3.90-3.81 (m, 5H, CH.sub.22, CH), 2.93 (d, J=4.0 Hz, 1H, CH), 1.21 (m, 1H, CH), 1.11 (d, J=4.0 Hz, 3H, CH.sub.3), 0.52-0.32 (m, 4H, CH.sub.22). MS (FAB): 576 (M+1).

Example 9

2-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzyl amino)-3-hydroxypropanamide

(24) ##STR00056##

(25) The procedure was the same as in Example 1, except that (bromomethyl) cyclopropane was used in place of 2-bromoacetamide, and 2-amino-3-hydroxypropanamide was used in place of ethyl ester of serine to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 7.63-6.82 (m, 12H, ArH, CONH.sub.2), 5.26 (s, 2H, CH.sub.2), 3.85 (q, 2H, J=4.0 Hz, CH.sub.2), 3.66-3.40 (m, 4H, CH.sub.22), 2.97 (s, 1H, CH), 1.17 (m, 1H, CH), 0.55-0.26 (m, 4H, CH.sub.22). MS (FAB): 561 (M+1).

Example 10

N-acetylaminoethyl-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropyl methoxy)benzylamine

(26) ##STR00057##

(27) The procedure was the same as in Example 1, except that (bromomethyl)cyclopropane was used in place of 2-bromoacetamide, N-(2-aminoethyl)acetamide was used in place of ethyl ester of serine to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 7.78 (m, 1H, CONH), 7.62-6.80 (m, 10H, ArH), 5.26 (s, 2H, CH2), 3.86 (d, J=8.0 Hz, 2H, CH2), 3.61 (s, 2H, CH2), 3.10 (q, J=4.0 Hz, 2H, CH2), 2.52 (t, J=4.0 Hz, 2H, CH2), 1.75 (s, 3H, CH3), 1.17 (m, 1H, CH), 0.55-0.26 (m, 4H, CH.sub.22). MS (FAB): 559 (M+1).

Example 11

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzyl)Citrulline

(28) ##STR00058##

(29) The procedure was the same as in Example 1, except that (bromomethyl)cyclopropane was used in place of 2-bromoacetamide, ethyl ester of Citrulline was used in place of ethyl ester of serine to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 7.58-6.73 (m, 10H, ArH), 5.29 (s, 1H, CONH), 5.20 (s, 2H, CH.sub.2), 3.82 (d, J=4.0 Hz, 2H, CH.sub.2), 3.68 (q, J=16.0 Hz, 2H, CH.sub.2), 2.86 (q, J=8.0 Hz, 1H, CH), 1.68-1.36 (m, 4H, CH.sub.22), 0.81 (m, 2H, CH.sub.2), 1.19 (m, 1H, CH), 0.52-0.27 (m, 4H, CH.sub.22). MS (FAB): 632 (M+1).

Example 12

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(N-methoxy-N-methylcarbamoyl methoxy)benzyl)serine

(30) ##STR00059##

(31) The procedure was the same as in Example 1, except that 2-bromo-N-methoxy-N-methylacetamide was used in place of 2-bromoacetamide to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 7.66 (d, J=7.3 Hz, 1H, ArH), 7.52 (d, J=7.7 Hz, 1H, ArH), 7.49 (s, 1H, ArH), 7.47 (s, 1H, ArH), 7.45 (s, 1H, ArH), 7.43 (s, 1H, ArH), 7.38 (d, J=6.7 Hz, 3H, ArH), 7.14 (s, 1H, ArH), 5.75 (d, J=0.9 Hz, 1H), 5.28 (s, 3H, OCH.sub.3), 4.69-4.52 (m, 3H, NCH.sub.3), 4.12 (d, J=12.5 Hz, 2H, CH.sub.2), 4.00 (d, J=15.7 Hz, 2H, CH.sub.2), 3.79 (d, J=12.4 Hz, 2H, CH.sub.2), 3.75 (d, J=5.5 Hz, 2H, CH.sub.2). MS (FAB): 609 (M+1).

Example 13

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(2-morpholino-2-oxoethoxy)benzyl)serine

(32) ##STR00060##

(33) The procedure was the same as in Example 1, except that 2-bromo-1-morpholinoethanone was used in place of 2-bromoacetamide to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 7.65 (dd, J=7.7, 1.7 Hz, 1H, ArH), 7.55-7.51 (m, 1H), 7.50 (s, 1H, ArH), 7.48 (t, J=1.8 Hz, 1H, ArH), 7.46 (q, J=1.3 Hz, 1H, ArH), 7.45-7.41 (m, 1H, ArH), 7.40 (d, J=1.7 Hz, 2H, ArH), 7.38 (dq, J=4.3, 1.9 Hz, 2H, ArH), 7.00 (s, 1H), 5.30 (s, 2H, CH.sub.2), 5.05 (s, 2H, CH.sub.2), 4.19-4.02 (m, 2H, CH.sub.2), 4.02-3.91 (m, 2H, CH.sub.2), 3.74 (dd, J=11.2, 4.5 Hz, 2H, CH.sub.2), 3.67 (q, J=6.7, 5.0 Hz, 2H, CH.sub.2), 3.62-3.58 (m, 2H, CH.sub.2), 3.55-3.52 (m, 2H, CH.sub.2), 3.25-3.21 (m, 1H, CH). MS (FAB): 635 (M+1).

Example 14

(S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzyl)serine

(34) ##STR00061##

(35) The procedure was the same as in Example 1, except that (bromomethyl)cyclopropane was used in place of 2-bromoacetamide to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 7.68-7.58 (m, 1H, ArH), 7.55-7.47 (m, 3H, ArH), 7.47-7.41 (m, 2H, ArH), 7.38 (m, 3H, ArH), 6.93 (m, 1H, ArH), 5.34-5.20 (m, 2H, CH.sub.2), 4.20-4.02 (m, 1H, CH.sub.2), 3.94 (m, 2H, CH.sub.2), 3.93-3.91 (m, 1H, CH.sub.2), 3.76-3.62 (m, 2H, CH.sub.2), 3.25-3.17 (m, 1H, CH.sub.2), 1.26 (m, 1H, CH) 0.56 (m, 1H, CH.sub.2), 0.42-0.28 (m, 1H, CH.sub.2). MS (FAB): 562 (M+1).

Example 15

(S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(N-hydroxycarbamoylmethoxy)benzyl)Pipecolinic Acid

(36) ##STR00062##

(37) The procedure was the same as in Example 1, except that 2-bromo-N-hydroxyacetamide was used in place of 2-bromoacetamide, and (S)-ethyl piperidine-2-carboxylate was used in place of ethyl ester of serine to afford a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.21 (s, 1H, ArH), 7.81-7.32 (m, 9H, ArH), 5.43-5.19 (s, 2H, CH.sub.2), 4.18-4.00 (m, 2H, CH.sub.2), 3.78-3.56 (m, 2H, CH.sub.2), 3.16 (d, J=11.0 Hz, 1H, CH), 2.94-2.86 (m, 1H, CH.sub.2), 2.28-2.22 (m, 1H, CH.sub.2), 1.91-1.85 (m, 2H, CH.sub.2), 1.56-1.40 (m, 3H, CH.sub.2), 1.36-1.30 (m, 1H, CH.sub.2). MS (FAB): 605 (M+1).

Example 16

(S,S)N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(carbamoylmethoxy)benzyl)-4-hydroxyproline

(38) ##STR00063##

(1) 2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)toluene

(39) The procedure was the same as in Example 1, except that 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was used in place of phenylboronic acid, and [1,1-bis (diphenylphosphino)ferrocene]dichloropalladium (II) was used in place of triphenylphosphine palladium, and potassium carbonate was used in place of cesium carbonate to afford 2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)toluene as pale yellow oil. .sup.1H NMR (400 MHz, Chloroform-d) : 7.21 (d, 2H, ArH), 7.11 (m, 1H, ArH), 6.90 (d, 2H, ArH), 6.86 (d, 1H, ArH), 4.30 (m, 4H, OCH.sub.2CH.sub.2O), 2.48 (s, 3H, CH.sub.3).

(2) (S,S)N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(carbamoyl methoxy) benzyl)-4-hydroxyproline

(40) The procedure was the same as in Example 1, except that 2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)toluene in place of 2-bromo-3-methyl-1,1-biphenyl, and (S,S)-ethyl 4-hydroxyprolinate was used in place of ethyl ester of serine to afford (S,S)N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(carbamoylmethoxy)benzyl)-4-hydroxyproline as a white solid. MS (FAB): 633 (M+1).

Example 17

(S,S)N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(cyclopropylmethoxy)benzyl)-4-hydroxyproline

(41) ##STR00064##

(1) 2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)toluene

(42) The procedure was the same as in Example 1, except that 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was used in place of phenylboronic acid, [1,1-bis (diphenylphosphino)ferrocene]dichloropalladium (II) was used in place of triphenylphosphine palladium, and potassium carbonate was used in place of cesium carbonate to afford 2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)toluene as pale yellow oil. .sup.1H NMR (400 MHz, Chloroform-d) : 7.21 (d, 2H, ArH), 7.11 (m, 1H, ArH), 6.90 (d, 2H, ArH), 6.86 (d, 1H, ArH), 4.30 (m, 4H, OCH.sub.2CH.sub.2O), 2.48 (s, 3H, CH.sub.3).

(2) (S,S)N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(cyclopropyl methoxy)benzyl)-4-hydroxyproline

(43) The procedure was the same as in Example 1, except that 2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)toluene in place of 2-(bromo-3-methyl-1,1-biphenyl, (bromomethyl)cyclopropane was used in place of 2-bromoacetamide, and (S,S)-ethyl 4-hydroxyprolinate was used in place of ethyl ester of serine to afford (S,S)N-(4-(2-bromo-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzyloxy)-5-chloro-2-(cyclopropyl methoxy) benzyl)-4-hydroxyproline as a white solid. MS (FAB): 646 (M+1).

(44) Pharmacological Experiments

(45) 1. In vitro activity evaluation: Cisbio PD-1/PD-L1 binding assay kit was applied for the detection method of in vitro enzymology level.
Screening Principles and Methods of PD-1/PD-L1 Small Molecule Inhibitors 1) Principle: PD-1 protein is with HIS tag, and PD-1 ligand PD-L1 is with hFc tag. Eu labeled anti-hFc antibody and XL665 labeled anti-HIS antibody are combined with the above two label proteins respectively. After laser excitation, energy can be transferred from donor Eu to receptor XL665, allowing XL665 to glow. After adding inhibitors (compounds or antibodies), blocking the binding of PD-1 and PD-L1 makes the distance between Eu and XL665 far away, the energy can not be transferred, and XL665 does not glow. 2) Experimental method: The specific method can be referred to Cisbio's PD-1/PD-L1 Kit (item 64CUS000C-2). Reagents should be dispensed in the following order. For 384-well white ELISA plate, 2 l of diluent or target compound diluted with diluent was added to each well, and then 4 l of PD-1 protein and 4 l of PD-L1 protein were added per well, incubated for 15 min at room temperature; and 10 l of a mixture of anti-Tag1-Eu3+ and anti-Tag2-XL665 was added per well and incubated for 1 h to 4 h at room temperature and the fluorescence signals at 665 nm and 620 nm were measured with an Envison instrument. HTRF rate=(665 nm/620 nm)*10.sup.4. 8-10 concentrations were detected for each compound and IC.sub.50 was calculated by Graphpad software. 3) The results of the screening were shown in Table 1.

(46) TABLE-US-00001 TABLE 1 Evaluation of the inhibitory activity of the example compounds at molecular level on the interaction between PD-1 and PD-L1: Example IC.sub.50(M) 1 3.12 10.sup.7 2 7.32 10.sup.7 3 1.26 10.sup.7 4 1.80 10.sup.6 5 1.15 10.sup.6 6 4.25 10.sup.8 7 1.82 10.sup.8 8 1.02 10.sup.8 9 8.16 10.sup.8 10 1.00 10.sup.6 11 1.41 10.sup.6 12 1.91 10.sup.7 13 4.13 10.sup.8 14 4.17 10.sup.8 15 1.83 10.sup.7 16 1.82 10.sup.7 17 4.05 10.sup.8 2. The Example Compounds' Capacity of Relieving the Inhibition of IFN by Ligand PD-L1:

(47) The expression level of IFN can reflect the proliferative activity of T lymphocytes. Using the extracted human PBMC (peripheral blood mononuclear cell), on the basis that T lymphocyte could be activated by the anti-CD3/anti-CD28 antibody, the ligand PD-L1 was added to the inhibit T lymphocyte, the example compounds' capacity of relieving the inhibition by the PD-L1 was investigated.

(48) The specific procedure is as follows. DAKEWE human lymphocyte separation solution (DKW-KLSH-0100) was used to extract PBMC from human whole blood, and PBMC was inoculated into 96 well plate, with 310.sup.5 cells per well. Human PD-L1 protein (final concentration 5 g/ml), anti-CD3/anti-CD28 antibody (final concentration 1 g/ml) and proportional dilution of the example compounds were added respectively. After 72 h, the expression level of IFN in the supernatant was detected by Cisbio IFN test kit. The experimental results showed that the inhibition of PD-L1 to expression level of IFN could be partially relieved by the example compounds at 10 nM. 3. The Efficacy of the Example Compounds In Vivo

(49) The methods of pharmacodynamics were as follows:

(50) The method in subcutaneous xenograft tumor was as follows. The cultured specific tumor cells were digested and collected by centrifugation, and washed with sterile physiological saline for two times and then counted. The cell concentration was adjusted to 510.sup.6/ml by physiological saline, and 0.2 ml of cell suspension was inoculated to the right armpit of C57BL/6 or Bablc mice. After inoculation, the animals were randomly divided into two groups in next day. Each group had 6-7 mice. After weighing, the animals were dosed once each day to monitor tumor size. When the tumor size reached to a certain size, the mice was weighed and blood was collected from mice orbit and then the mice were killed by removing the neck. The tumor tissue, thymus tissue and spleen tissue were collected and weighed respectively. Finally, the tumor growth inhibition rate was calculated, and the tumor growth inhibition rate was used to evaluate the level of anti-tumor effect.

(51) The method in B16F10 lung metastasis model was as follows. The cultured B16F10 tumor cells were digested and centrifuged and washed for two times with sterile physiological saline and then counted. And the cell concentration was adjusted to 2.510.sup.6/ml by physiological saline. 0.2 ml of cells were injected into the C.sub.57BL/6 mice through the tail vein, and the tumor cells will gather in the lung of the mice. After inoculation, the animals were randomly divided into two groups in next day. Each group had 6-7 mice. After weighing, the animals were dosed once each day. After 3 weeks, the mice were weighed and killed, the lung tissue was collected and weighed, and the number of lung tumors was counted after being fixed by the Bouin's Fluid. Finally, the tumor growth inhibition rate was calculated, and the tumor growth inhibition rate was used to evaluate the level of anti-tumor effect.

(52) The method in Lewis lung cancer hydrothorax model was as follows: The subcutaneous xenograft tumor of Lewis lung cancer was homogenized and washed for two times with sterile physiological saline, and the cell concentration was adjusted to 2.510.sup.5/ml by physiological saline. 0.2 ml of cells were injected into the thoracic cavity of C.sub.57BL/6 mice. After inoculation, the animals were randomly divided into two groups in next day. Each group had 6-7 mice. After weighing, the animals were dosed once each day. Animals were sacrificed when the weight of the animals in the control group suddenly dropped. The liquid in thoracic cavity was extracted with syringe and the volume of fluid was recorded.

(53) In the study of the mechanism of the above models, the method of flow cytometry was adopted in measuring the total cell proportion of T cells of various types. The specific steps were as follows. The samples were treated at first. For blood tissue, the orbital blood was taken. The red cell lysate was used to remove the red blood cells, and then the PBS buffer was used for wash. After being washed, the cells were collected. For the tumor and spleen, the tissues were grinded with a homogenizer, and then diluted with PBS buffer, then filtered by 300 meshes of screen. After the number of cells was counted for each sample, 110.sup.6 cells were added into EP tube and stained for flow antibody. After incubation for 1 h on ice, each sample was washed 2 times with PBS buffer. The cell population was analyzed by VERSE flow instrument of BD Company. The total number of cells in tumor tissue was 110.sup.5 and the total number of cells in blood and spleen tissues was 110.sup.4. The ratio of T cells to total number of cells was analyzed after flow cytometry. (1) Subcutaneous Xenograft Model of high Metastatic Melanoma B16F10

(54) For the high metastatic melanoma B16F10, the example compounds can significantly inhibit the growth of the subcutaneous tumor, with the respect of tumor volume or weight.

(55) From the analysis of mechanism, the example compounds can increase the proportion of tumor-infiltrating lymphocytes and the proportion of lymphocytes in the spleen. (2) Lung Metastasis Model of High Metastatic Melanoma B16F10

(56) For metastatic lung cancer models with high metastatic melanoma B16F10, the example compounds can significantly inhibit the number of lung metastases.

(57) From analysis of the mechanism, the example compounds can increase the percentage of lymphocyte in mouse blood. (3) Subcutaneous Xenograft Model of Mouse Breast Cancer EMT6

(58) For subcutaneous xenograft model of mouse breast cancer EMT6, the example compounds have some inhibition effect on mouse breast cancer EMT6, and the combination of the example compounds and CTX can significantly increase the tumor growth inhibition rate of CTX. (4) Mouse Lewis Lung Cancer Hydrothorax Model

(59) The example compounds have significant inhibition effect on mouse Lewis lung cancer hydrothorax model, and can reduce the hydrothorax incidence rate. (5) Subcutaneous Xenograft Model of Mouse Colon Cancer MC38

(60) For subcutaneous xenograft model of mouse colon cancer MC38, the example compounds have significant inhibition effect on mouse colon cancer MC38, and have a synergistic antitumor effect on this cancer in combination with CTX. 4. The Interaction of Example Compound/PD-L1 Antibody with PD-L1 Protein was Tested by Biacore (1) Experimental Principle

(61) Surface plasmon is a kind of electromagnetic wave on the surface of metal, produced by the interaction of photon and electron in free vibration. Surface plasmon resonance (SPR) is an optical phenomenon that occurs on the surface of two kinds of media, which can be induced by photon or electron. The phenomenon of total reflection of light from light dense medium into light scattering medium will form evanescent wave into light scattering medium. When the total reflected evanescent wave meets the plasma wave on the metal surface, the resonance may occur, and the energy of reflected light decreases and the resonance peak appears on the reflected light energy spectrum. This resonance is called the surface plasmon resonance. The incident angle of the surface plasmon resonance is called the SPR angle. The SPR biosensor provides a sensitive, real-time, non-label detection technique for monitoring the interaction of molecules. The sensor detects the change of the SPR angle, and SPR is also related to the refractive index of the metal surface. When an analyte is bond on the surface of the chip, it leads to the change of the refractive index of the chip surface, which leads to the change of the SPR angle. This is the basic principle of the real-time detection of intermolecular interaction by the SPR biosensor. In the interaction analysis, the change of SPR angle is recorded on the sensor map in real time. (2) Experimental Methods

(62) The PD-L1 protein was captured on the Fc4 channel of NTA chip by capture method, and the buffer system was PBS-P+, pH7.4, 0.01% DMSO. A series of concentration of compounds and PD-L1 antibodies were prepared and flowed through the surface of the chip for the determination of interaction. (3) Experimental Results

(63) It was preliminarily determined that the binding protein of the example compounds was PD-L1. Further Biacore experiments confirmed that the example compounds had a strong ability of binding PD-L1.