NICOTINYL ALCOHOL ETHER DERIVATIVE, PREPARATION METHOD THEREFOR, AND PHARMACEUTICAL COMPOSITION AND USES THEREOF

Abstract

The present invention discloses a nicotinyl alcohol ether derivative, a preparation method therefor, and a pharmaceutical composition and uses thereof. Specifically, the invention relates to nicotinyl alcohol ether 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 nicotinyl alcohol ether derivative of Formula (I): ##STR00070## or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 is selected from ##STR00071## R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, 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, ##STR00072## X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, methoxy.

2. A nicotinyl alcohol ether derivative of claim 1, represented by formula (IA), or a pharmaceutically acceptable salt or a stereoisomer thereof; ##STR00073## wherein: R.sub.1 is selected from ##STR00074## R.sub.3 is selected from substituted C.sub.1-C.sub.8 saturated alkylamino, substituted C.sub.2-C.sub.6 unsaturated alkylamino, 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## X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

3. A nicotinyl alcohol ether derivative of claim 2, represented by formula (IA-1), 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, 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## X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

4. A nicotinyl alcohol ether derivative of claim 2, represented by formula (IA-2), 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, 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## X is selected from hydrogen, fluorine, chlorine, bromine, iodine, C.sub.1-C.sub.4 alkyl, ethenyl, trifluoromethyl, and methoxy.

5. A nicotinyl alcohol ether derivative of claim 1, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R.sub.3 is selected from: ##STR00080## ##STR00081## wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl; X is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethenyl, and trifluoromethyl.

6. A nicotinyl alcohol ether derivative of claim 1, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein the compound is selected from: Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride ##STR00082## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine ##STR00083## (S)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl)serinate ##STR00084## (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine ##STR00085## (S)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride ##STR00086## (S)-isopropyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride ##STR00087## (R)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate ##STR00088## (R)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine ##STR00089## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) glycine ##STR00090## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) valine ##STR00091## (E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) hut-2-enenitrile ##STR00092## N, N-bis(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine ##STR00093## N-(2-methanesulfonaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine ##STR00094## N-(2-acetylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine ##STR00095## (E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) but-2-enoic acid ##STR00096## 2-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) ethanesulfonic acid ##STR00097## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) leucine ##STR00098## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) tyrosine ##STR00099## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) isoleucine ##STR00100## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) asparagine ##STR00101## N-(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine ##STR00102## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) alanine ##STR00103## N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) proline ##STR00104## (S)-Sodium N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate ##STR00105## (S)-Calcium N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate ##STR00106## (S)-(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serinate ##STR00107##

7. A nicotinyl alcohol ether derivative 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 salt, 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.

8. A nicotinyl alcohol ether derivative of claim 7, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic, citric acid, maleic acid, tartaric acid, fumaric acid, citric acid or lactic acid; the alkali metal ion is selected from lithium ion, sodium ion, 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 or tris(2-hydroxyethyl) amine.

9. A process for the preparation of a nicotinyl alcohol ether derivative of claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00108## for the preparation of the compounds of the formula (I), according to its structure, the preparation method is divided into two steps: (a) 2-hydroxy-4-(2-bromo-3-R1 benzyloxy)-X-substituted benzaldehyde 1 as a starting material is reacted with pyridin-3-yl-methylene halide under basic conditions to obtain an aldehyde-containing intermediate compound 2; (b) the aldehyde-containing intermediate compound 2 as the starting material is condensed with an amino group- or an imino group-containing HR.sub.3 and the resultant product is reduced to obtain a target compound I; wherein R.sub.1, R.sub.3 and X each is defined as claim 1.

10. A pharmaceutical composition, characterized in that it comprises a nicotinyl alcohol ether derivative 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.

11. A method for preventing and/or treating a disease associated with the PD-1/PD-L1 signaling pathway in a subject in need of such treatment comprising administering to the subject an effective amount of a nicotinyl alcohol ether derivative of claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

12. The method of claim 11, wherein the disease associated with the PD-1/PD-L1 signaling pathway is selected from cancer, infectious disease, and autoimmune disease.

13. The method of claim 12, wherein the cancer is selected from skin cancer, lung cancer, urinary tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer; the infectious disease is selected from bacterial infection and viral infection; the autoimminue disease is selected from the organ specific autoimmune disease and the system autoimmune disease; wherein the organ specific autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes, severe myasthenia, ulcerative colitis, malignant anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome, primary biliary cirrhosis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis; the system autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, and autoimmune hemolytic anemia.

14. A pharmaceutical composition, characterized in that it comprises a nicotinyl alcohol ether derivative of claim 6, or a stereoisomer or a pharmaceutically acceptable salt thereof, as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

15. A method for preventing and/or treating a disease associated with the PD-1/PD-L1 signaling pathway in a subject in need of such treatment comprising administering to the subject an effective amount of the nicotinyl alcohol ether derivative of claim 6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

16. The method of claim 15, wherein the disease associated with the PD-1/PD-L1 signaling pathway is selected from cancer, infectious disease, and autoimmune disease.

17. The method of claim 16, wherein the cancer is selected from skin cancer, lung cancer, urinary tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer; the infectious disease is selected from bacterial infection and viral infection; the autoimminue disease is selected from the organ specific autoimmune disease and the system autoimmune disease; wherein the organ specific autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes, severe myasthenia, ulcerative colitis, malignant anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome, primary biliary cirrhosis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis; the system autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, and autoimmune hemolytic anemia.

Description

EXAMPLES

[0067] The invention is further illustrated by the following examples; however, the invention is not limited by the illustrative examples set herein below.

[0068] 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

Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride

[0069] ##STR00041##

2-Bromo-3-methyl-1, 1-biphenyl

[0070] To a 100 ml flask were added 2-bromo-3-iodotoluene (700 mg) and dioxane/water (volume ratio 5/1) with stirring. The solution was bubbled with argon for 10 min to remove dissolved oxygen. Then, phenylboronic acid (350 mg), cesium carbonate (1800 mg), and triphenylphosphine palladium (80 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 under reduced pressure to dryness to afford 2-bromo-3-methyl-1, 1-biphenyl as colorless oil (480 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). MS (FAB): 248 (M+1).

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

[0071] 2-Bromo-3-methyl-1,1-biphenyl (450 mg) as a starting material was weighed and was dissolved in 40 ml of CCl.sub.4 in a 100 ml flask. To this solution was added NBS (360 mg) while stirring. And the mixture was warmed to 80 C. and refluxed. Then benzoyl peroxide (8 mg) was added, and after 2 h, benzoyl peroxide (8 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 and water. The organic phase was washed with saturated brine, and dried over anhydrous sodium sulfate. The organic layer was filtered and evaporated under reduced pressure to dryness to afford 2-bromo-3-(bromomethyl)-1, 1-biphenyl as yellow oil (380 mg), which was used for the next step without further purification.

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

[0072] 5-chloro-2, 4-dihydroxybenzaldehyde (160 mg) was weighed and dissolved in 12 ml of anhydrous acetonitrile in a 50 ml flask, and sodium hydrogen carbonate (200 mg) was added. After stirring at room temperature for 40 min, 2-bromo-3-phenylbenzyl bromide (380 mg, dissolved in 16 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 under reduced pressure to dryness. The crude residue was purified by silica gel column chromatography to afford 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-hydroxy benzaldehyde (300 mg) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.99 (s, 1H, OH), 10.03 (s, 1H, CHO), 7.64 (d, 1H, ArH), 7.57 (d, 1H, ArH), 7.45 (m, 4H, ArH), 7.37 (d, 2H, ArH), 6.67 (d, 1H, ArH), 6.59 (s, 1H, ArH), 5.25 (s, 2H, CH.sub.2). MS (FAB): 418 (M+1).

4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzaldehyde

[0073] 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 3-bromomethylenepyridine (76.65 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 under reduced pressure to dryness. The crude residue was purified by silica gel column chromatography to afford a white solid (70 mg). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.26 (s, 1H, CHO), 8.00 (s, 1H, ArH), 7.83 (dd, 2H, ArH), 7.72 (d, 1H, ArH), 7.61 (t, 2H, ArH), 7.55-7.23 (m, 6H, ArH), 6.95 (s, 1H, ArH), 6.81 (d, 1H, ArH), 5.35 (s, 2H, CH.sub.2), 5.30 (s, 2H, CH.sub.2). MS (FAB): 510 (M+1).

Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride

[0074] 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde (80 mg) was dissolved in 5 ml of DMF, and then racemic ethyl ester of serine (59 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 under reduced pressure to dryness. The residue was dissolved in ethanol, heated to reflux until the reaction was complete. The mixture was evaporated to dryness. The crude residue was purified by silica gel column chromatography to afford ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate (60 mg) as yellow oil. The product was then reacted with a solution of hydrogen chloride in ethanol to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl )serinate dihydrochloride as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) 9.65 (s, 1H, HCl), 9.41 (s, 1H, HCl), 9.06 (s, 1H, ArH), 8.88-8.76 (m, 1H, ArH), 8.56 (d, J=7.9 Hz, 1H, ArH), 8.01-7.86 (m, 1H, ArH), 7.64 (d, J=13.4 Hz, 2H, ArH), 7.56-7.30 (m, 7H, ArH), 7.12 (s, 1H, ArH), 5.42 (s, 2H, CH.sub.2), 5.32 (s, 2H, CH.sub.2), 4.18 (s, 2H, CH.sub.2), 4.13-4.07 (m, 1H, CH), 4.04 (m, 2H, CH.sub.2), 3.94 (dd, 1H, CH.sub.2), 3.82 (dd, 1H, CH.sub.2), 1.15 (t, J=7.1 Hz, 3H, CH.sub.3). MS (FAB): 626 (M).

Example 2

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine

[0075] ##STR00042##

[0076] Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate as pale yellow oil (60 mg) was dissolved in methanol/H.sub.2O (4 ml/1 ml), and then 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 filtrated under reduced pressure to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine (45 mg) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.73 (s, 1H, ArH), 8.55 (d, 1H, ArH), 7.97 (d, 1H, ArH), 7.65 (d, 1H, ArH), 7.54-7.51 (m, 2H, ArH), 7.47 (d, 2H, ArH), 7.44 (dl H, ArH), 7.42-7.40 (m, 2H, ArH), 7.39-7.36 (m, 2H, ArH), 7.11 (s, 1H, ArH), 5.32 (s, 2H, CH.sub.2), 5.28 (m, 2H, CH.sub.2), 3.94 (s, 2H, CH.sub.2), 3.69 (dd, 1H, CH.sub.2), 3.62 (dd, 1H, CH.sub.2), 3.16 (t, 1H, CH). MS (FAB): 599 (M+1).

Example 3

(S)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate

[0077] ##STR00043##

[0078] The procedure was the same as in Example 1, except that ethyl ester of L-serine was used in place of racemic ethyl ester of serine to afford (S)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.68 (d, J=2.1 Hz, 1H, ArH), 8.55 (dd, J=4.8, 1.7 Hz, 1H, ArH), 7.88 (d, J=7.9 Hz, 1H, ArH), 7.65 (dd, J=7.6, 1.7 Hz, 1H, ArH), 7.54-7.45 (m, 3H, ArH), 7.45-7.35 (m, 6H, ArH), 7.06 (s, 1H, ArH), 5.30 (s, 2H, CH.sub.2), 5.24 (s, 2H, CH.sub.2), 4.81 (t, J=5.8 Hz, 1H, CH), 3.99 (q, J=7.1 Hz, 2H, CH.sub.2), 3.74-3.57 (m, 2H, CH.sub.2), 3.54 (t, J=5.6 Hz, 2H, CH.sub.2), 1.12 (t, J=7.1 Hz, 3H, CH.sub.3). MS (FAB): 626 (M).

Example 4

(S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine

[0079] ##STR00044##

[0080] The procedure was the same as in Example 2, except that (S)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate was used in place of racemic Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate to afford (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.73 (s, 1H, ArH), 8.55 (d, J=4.7 Hz, 1H, ArH), 7.97 (d, J=7.9 Hz, 1H, ArH), 7.65 (d, J=8.7 Hz, 1H, ArH), 7.54-7.51 (m, 2H, ArH), 7.47 (d, J=7.7 Hz, 2H, ArH), 7.44 (d, J=1.7 Hz, 1H, ArH), 7.42-7.40 (m, 2H, ArH), 7.39-7.36 (m, 2H, ArH), 7.11 (s, 1H, ArH), 5.32 (s, 2H, CH.sub.2), 5.28 (m, 2H, CH.sub.2), 3.94 (s, 2H, CH.sub.2), 3.69 (dd, J=11.1, 4.7 Hz, 1H, CH.sub.2), 3.62 (dd, J=11.1, 6.2 Hz, 1H, CH.sub.2), 3.16 (t, J=5.4 Hz, 1H, CH). MS (FAB): 599 (M+1). [].sup.27.sub.D=1.84 (C=0.434, DMSO).

Example 5

(S)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride

[0081] ##STR00045##

[0082] (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine (598 mg) and 60 ml of anhydrous ethanol were placed in a 100 ml round-bottom flask. To the mixture were added 6 ml of dichlorosulfoxide and two drops of DMF with stirring in an ice water bath. And after the mixture was stirred at room temperature for 2 h it was heated to reflux until the reaction was completed. The mixture was concentrated under reduced pressure to remove the solvent and afford (S)-ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) 9.65 (s, 1H, HCl), 9.41 (s, 1H, HCl), 9.06 (s, 1H, ArH), 8.88-8.76 (m, 1H, ArH), 8.56 (d, J=7.9 Hz, 1H, ArH), 8.01-7.86 (m, 1H, ArH), 7.64 (d, J=13.4 Hz, 2H, ArH), 7.56-7.30 (m, 7H, ArH), 7.12 (s, 1H, ArH), 5.42 (s, 2H, CH.sub.2), 5.32 (s, 2H, CH.sub.2), 4.18 (s, 2H, CH.sub.2), 4.13-4.07 (m, 1H, CH), 4.04 (m, 2H, CH.sub.2), 3.94 (dd, J=12.1, 2.9 Hz, 1H, CH.sub.2), 3.82 (dd, J=12.1, 3.8 Hz, 1H, CH.sub.2), 1.15 (t, J=7.1 Hz, 3H, CH.sub.3). MS (FAB): 626 (M). [].sup.27.sub.D=1.90 (C=0.422, ethanol).

Example 6

(S)-isopropyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride

[0083] ##STR00046##

[0084] (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine (598 mg) and 60 ml of anhydrous isopropanol were placed in a 100 ml round-bottom flask. To the mixture were added 6 ml of dichlorosulfoxide and two drops of DMF with stirring in an ice water bath. And after the mixture was stirred at room temperature for 2 h it was heated to reflux until the reaction was completed. The mixture was concentrated under reduced pressure to remove the solvent and afford (S)-isopropyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate dihydrochloride as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.62 (s, 1H, HCl), 9.40 (s, 1H, HCl), 9.10 (s, 1H, ArH), 8.86 (d, 1H, ArH), 8.59 (d, J=7.6 Hz, 1H, ArH), 8.01-7.91 (m, 1H, ArH), 7.73-7.64 (m, 2H, ArH), 7.57-7.46 (m, 3H, ArH), 7.46-7.37 (m, 4H, ArH), 7.16 (s, 1H, ArH), 5.47 (s, 2H, CH.sub.2), 5.36 (s, 2H, CH.sub.2), 4.92 (ml H, CH), 4.30-4.15 (m, 2H, CH.sub.2), 4.05 (s, 1H, CH), 3.97 (dd, J=12.0, 3.0 Hz, 1H, CH.sub.2), 3.84 (dd, J=12.0, 3.8 Hz, 1H, CH.sub.2), 1.20 (d, J=6.4 Hz, 3H, CH.sub.3), 1.18 (d, J=6.4 Hz, 3H, CH.sub.3). MS (FAB): 640 (M). [].sup.27.sub.D=5.33 (C=0.075, methanol).

Example 7

(R)-Ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate

[0085] ##STR00047##

[0086] The procedure was the same as in Example 1, except that ethyl ester of D-serine was used in place of racemic ethyl ester of serine to afford (R)-ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.68 (d, 1H, ArH), 8.55 (dd, 1H, ArH), 7.88 (d, 1H, ArH), 7.65 (dd, J=7.6, 1.7 Hz, 1H, ArH), 7.54-7.45 (m, 3H, ArH), 7.45-7.35 (m, 6H, ArH), 7.06 (s, 1H, ArH), 5.30 (s, 2H, CH.sub.2), 5.24 (s, 2H, CH.sub.2), 4.81 (t, J=5.8 Hz, 1H, CH), 3.99 (q, J=7.1 Hz, 2H, CH.sub.2), 3.74-3.57 (m, 2H, CH.sub.2), 3.54 (t, 2H, CH.sub.2), 1.12 (t, J=7.1 Hz, 3H, CH.sub.3). MS (FAB): 626 (M).

Example 8

(R)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine

[0087] ##STR00048##

[0088] The procedure was the same as in Example 2, except that (R)-ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate was used in place of racemic ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate to afford (R)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl)serine as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.73 (s, 1H, ArH), 8.55 (d, J=4.7 Hz, 1H, ArH), 7.97 (d, J=7.9 Hz, 1H, ArH), 7.65 (d, J=8.7 Hz, 1H, ArH), 7.54-7.51 (m, 2H, ArH), 7.47 (d, J=7.7 Hz, 2H, ArH), 7.44 (d, J=1.7 Hz, 1H, ArH), 7.42-7.40 (m, 2H, ArH), 7.39-7.36 (m, 2H, ArH), 7.11 (s, 1H, ArH), 5.32 (s, 2H, CH.sub.2), 5.28 (m, 2H, CH.sub.2), 3.94 (s, 2H, CH.sub.2), 3.69 (dd, J=11.1, 4.7 Hz, 1H, CH.sub.2), 3.62 (dd, J=11.1, 6.2 Hz, 1H, CH.sub.2), 3.16 (t, J=5.4 Hz, 1H, CH). MS (FAB): 599 (M+1). []

Example 9

(S)N-(4-(2-chloro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine

[0089] ##STR00049##

[0090] The procedure was the same as in Example 1, except that 4-(2-chloro-3-phenylbenzyloxy)-5-chloro-(pyridin-3-yl-methyleneoxy) benzaldehyde was used in place of 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzaldehyde, and ethyl ester of L-serine was used in place of racemic ethyl ester of serine to afford (S)-ethyl N-(4-(2-chloro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl)serinate.

[0091] The procedure was the same as in Example 2, except that (S)-ethyl N-(4-(2-chloro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate was used in place of ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate to afford (S)N-(4-(2-chloro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) 8.69 (s, 1H, ArH), 8.51 (d, J=4.6 Hz, 1H, ArH), 7.93 (d, J=7.6 Hz, 1H, ArH), 7.63 (d, J=7.4 Hz, 1H, ArH), 7.54-7.33 (m, 9H, ArH), 7.12 (s, 1H, ArH), 5.31 (s, 2H, CH.sub.2), 5.25 (m, 2H, CH.sub.2), 3.91 (s, 2H, CH.sub.2), 3.66 (dd, J=11.0, 4.0 Hz, 1H, CH.sub.2), 3.59 (dd, J=11.0, 6.0 Hz, 1H, CH.sub.2), 3.14 (t, J=4.8 Hz, 1H, CH). MS (FAB): 554 (M+1).

Example 10

(S)N-(4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine

[0092] ##STR00050##

[0093] The procedure was the same as in Example 1, except that 4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzaldehyde was used in place of 4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzaldehyde, and ethyl ester of L-serine was used in place of racemic ethyl ester of serine to afford (S)-ethyl N-(4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate.

[0094] The procedure was the same as in Example 2, except that (S)-ethyl N-(4-(2-fluoro-3-phenyl benzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate was used in place of ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate to afford (S)N-(4-(2-fluoro-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) 8.72 (dd, J=8.0, 1.9 Hz, 1H, ArH), 8.56 (dd, J=4.8, 1.5 Hz, 1H, ArH), 7.97 (d, J=7.9 Hz, 1H, ArH), 7.72-7.30 (m, 10H, ArH), 7.16 (d, J=15.1 Hz, 1H, ArH), 5.35 (d, J=6.7 Hz, 2H, CH.sub.2), 5.27 (dd, J=15.2, 3.2 Hz, 2H, CH.sub.2), 3.95 (s, 2H, CH.sub.2), 3.70 (dd, J=11.2, 4.5 Hz, 1H, CH.sub.2), 3.63 (dd, J=11.2, 6.2 Hz, 1H, CH.sub.2), 3.18 (t, J=5.3 Hz, 1H, CH). MS (FAB): 538 (M+1).

Example 11

(S)N-(4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine

[0095] ##STR00051##

[0096] The procedure was the same as in Example 1, except that 4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde was used in place of 4-(2-bromo-3-phenylbenzyl oxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzaldehyde, and ethyl ester of L-serine was used in place of racemic ethyl ester of serine to afford (S)-ethyl N-(4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate.

[0097] The procedure was the same as in Example 2, except that (S)-ethyl N-(4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate was used in place of ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate to afford (S)N-(4-(3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serine as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) 8.67 (s, 1H, ArH), 8.50 (s, 1H, ArH), 7.91 (d, J=5.6 Hz, 1H, ArH), 7.75 (s, 1H, ArH), 7.61 (d, J=13.2 Hz, 3H, ArH), 7.44 (d, J=15.2 Hz, 5H, ArH), 7.36 (d, J=8.4 Hz, 2H, ArH), 7.09 (s, 1H, ArH), 5.29 (s, 2H, CH.sub.2), 5.19 (m, 2H, CH.sub.2), 3.90 (s, 2H, CH.sub.2), 3.69-3.55 (m, 2H, CH.sub.2), 3.12 (m, 1H, CH). MS (FAB): 520 (M+1).

Example 12

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) glycine

[0098] ##STR00052##

(1) 2-bromo-3-phenyltoluene

[0099] 2-Bromo-3-iodotoluene (350 mg) was placed in 50 ml flask and dioxane/water was added with stirring. And the solution was bubbled with argon for 10 min to remove the dissolved oxygen. Then, phenylboronic acid (172.65 mg), cesium carbonate (961.2 mg) and triphenylphosphine palladium (40.91 mg) were added and the resulting mixture was stirred at 80-100 C. for 12 h under protection of argon. 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 by anhydrous Na.sub.2SO.sub.4. The solution was filtered and evaporated under reduced pressure to dryness. The residue was purified by silica gel column chromatography (petroleum ether) to give 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

[0100] 2-Bromo-3-phenyltoluene (234 mg) was weighed and placed in a 100 ml flask and 20 ml of CCl.sub.4 was added. After 2-Bromo-3-phenyltoluene was completely dissolved, NBS (178 mg) was added under stirring. And the resulting mixture was heated to 80 C. to reflux. Then benzoyl peroxide (4 mg) was added and another 4 mg of benzoyl peroxide was added after 2 h, and the reaction was continued for another 2 h. The reaction was stopped. The reaction was cooled to room temperature and a suitable amount of water was added and the mixture was extracted with dichloromethane. The organic phase was washed with saturated brine and dried by anhydrous Na.sub.2SO.sub.4. The solution was filtered and evaporated under reduced pressure to dryness to give yellow oil (192 mg). The material was used for the next step without further purification.

(3) 2-hydroxy-4-(2-bromo-3-phenylbenzoxy)-5-chlorobenzaldehyde

[0101] To anhydrous acetonitrile (6 ml) in 50 ml flask was added 2,4-dihydroxy-5-chlorobenzaldehyde (73.94 mg) and sodium bicarbonate (98.88 mg). After stirring 40 min at room temperature, the solution of 2-bromo-3-(bromomethyl)-1,1-biphenyl (192 mg) in 8 ml of DMF was slowly added dropwise to the reaction mixture via a constant pressure dropping funnel. The resulting mixture was refluxed until the reaction completed. After cooling to room temperature, the mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to dryness. The crude residue was purified by silica gel column chromatography to afford 2-hydroxy-4-(2-bromo-3-phenylbenzoxy)-5-chlorobenzaldehyde (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-phenylbenzoxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzaldehyde

[0102] 2-Hydroxy-4-(2-bromo-3-phenylbenzoxy)-5-chlorobenzaldehyde (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 3-bromomethyleneoxy pyridine (76.65 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 under reduced pressure 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-d.sub.6) 10.14 (s, 1H, CHO), 8.71 (d, J=1.6 Hz, 1H, ArH), 8.54 (d, J=4.8 Hz, 1H, ArH), 7.94 (d, J=7.8 Hz, 1H, ArH), 7.83 (s, 1H, ArH), 7.66 (d, J=7.6 Hz, 1H, ArH), 7.50 (t, J=7.6 Hz, 1H, ArH), 7.48-7.32 (m, 7H, ArH), 7.20 (s, 1H, ArH), 5.42 (s, 2H, CH.sub.2), 5.41 (s, 2H, CH.sub.2).

(5) N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) glycine

[0103] 4-(2-bromo-3-phenylbenzoxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzaldehyde (80 mg) was dissolved in 5 ml of DMF, and then ethyl glycinate (49 mg, 0.472 mmol) 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 under reduced pressure to dryness. The residue was dissolved in ethanol, heated to reflux until the reaction was complete. The mixture was evaporated to dryness. The crude residue was purified by silica gel column chromatography to afford ethyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) glycinate (70 mg) as yellow oil. The product was dissolved in methanol/H.sub.2O (4 ml/ml), and then 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 filtrated under reduced pressure to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) glycine (45 mg) as a white solid. .sup.1H NMR (400 MHz, DMSO) 8.73 (d, J=1.7 Hz, 1H, ArH), 8.55 (dd, J=4.8, 1.6 Hz, 1H, DMSO), 7.96 (dt, J=7.9, 1.9 Hz, 1H, DMSO), 7.65 (dd, J=7.6, 1.6 Hz, 1H, ArH), 7.55-7.35 (m, 9H, ArH), 7.12 (s, 1H, ArH), 5.33 (s, 2H, CH.sub.2), 5.29 (s, 2H, CH.sub.2), 3.91 (s, 2H, CH.sub.2), 3.11 (s, 2H, CH.sub.2). MS (FAB): 569 (M+1).

Example 13

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) valine

[0104] ##STR00053##

[0105] The procedure was the same as in Example 12, except that ethyl ester of valine was used in place of ethyl ester of glycine to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) valine as a white solid. .sup.1H NMR (400 MHz, DMSO) 8.69 (s, 1H, ArH), 8.56 (d, J=3.9 Hz, 1H, ArH), 7.88 (d, J=7.8 Hz, 1H, ArH), 7.66 (d, J=7.4 Hz, 1H, ArH), 7.55-7.33 (m, 9H, ArH), 7.06 (s, 1H, ArH), 5.31 (s, 2H, CH.sub.2), 5.22 (d, J=11.2 Hz, 2H, CH.sub.2), 3.52 (s, 2H, CH.sub.2), 2.91 (d, J=6.4 Hz, 1H, CH), 1.80 (dq, J=13.2, 6.5 Hz, 1H, CH), 0.86 (dd, J=18.9, 6.7 Hz, 6H, CH.sub.3). MS (FAB): 611 (M+1).

Example 14

(E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) but-2-enenitrile

[0106] ##STR00054##

[0107] The procedure was the same as in Example 12, except that (E)-3-aminobut-2-enenitrile was used in place of ethyl ester of glycine without hydrolysis to afford (E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) but-2-enenitrile as a white solid. .sup.1H NMR (400 MHz,) 8.70 (s, 1H, ArH), 8.55 (d, J=4.2 Hz, 1H, ArH), 7.99-7.81 (m, 1H, ArH), 7.65 (d, J=7.0 Hz, 1H, ArH), 7.58-7.31 (m, 9H, ArH), 7.11 (d, J=33.8 Hz, 1H), 6.30 (s, 1H, CH), 5.28 (d, J=14.1 Hz, 4H, CH.sub.2), 3.27 (s, 2H, CH.sub.2), 2.21-1.96 (m, 3H). MS (FAB): 576 (M+1).

Example 15

N, N-bis(2-hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine

[0108] ##STR00055##

[0109] The procedure was the same as in Example 12, except that bis(2-hydroxyethyl)amine was used in place of ethyl ester of glycine without hydrolysis to afford N, N-bis(2-hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.68 (s, 1H, ArH), 8.58-8.51 (m, 1H, ArH), 7.88 (d, J=7.2 Hz, 1H, ArH), 7.65 (d, J=7.6 Hz, 1H, ArH), 7.56-7.32 (m, 9H, ArH), 7.05 (d, J=2.4 Hz, 1H, ArH), 5.29 (s, 2H, OCH.sub.2), 5.24 (s, 2H, OCH.sub.2), 4.35 (s, 2H, OH), 3.58 (s, 2H, CH.sub.2), 3.41 (br s, 4H, CH.sub.2), 2.51 (m, 4H, CH.sub.2, overlapped in solvent peak). MS (FAB): 598 (M+1).

Example 16

N-(2-methanesulfonaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine

[0110] ##STR00056##

[0111] The procedure was the same as in Example 12, except that N-(2-aminoethyl)methanesulfonamide was used in place of ethyl ester of glycine without hydrolysis to afford N-(2-methanesulfonaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.69 (s, 1H, ArH), 8.55 (d, J=4.4 Hz, 1H, ArH), 7.89 (d, J=7.6 Hz, 1H, ArH), 7.65 (d, J=8.0 Hz, 1H, ArH), 7.56-7.33 (m, 9H, ArH), 7.07 (s, 1H, ArH), 6.95 (s, 1H, NH), 5.30 (s, 2H, OCH.sub.2), 5.25 (s, 2H, OCH.sub.2), 3.64 (s, 2H, CH.sub.2), 3.01 (m, 2H, CH.sub.2), 2.88 (s, 3H, CH.sub.3), 2.59 (t, J=6.4 Hz, 2H, CH.sub.2). MS (FAB): 631 (M).

Example 17

N-(2-acetylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamine

[0112] ##STR00057##

[0113] The procedure was the same as in Example 12, except that N-(2-aminoethyl)acetamide was used in place of ethyl ester of glycine without hydrolysis to afford N-(2-acetylaminoethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.69 (s, 1H, ArH), 8.55 (d, J=4.0 Hz, 1H, ArH), 7.88 (d, J=8.0 Hz, 1H, ArH), 7.80 (t, J=6.0 Hz, 1H, NH), 7.65 (d, J=6.0 Hz, 1H, ArH), 7.57-7.35 (m, 9H, ArH), 7.07 (s, 1H, ArH), 5.30 (s, 2H, OCH.sub.2), 5.25 (s, 2H, OCH.sub.2), 3.65 (s, 2H, CH.sub.2), 3.12 (q, J=6.0 Hz, 2H, CH.sub.2), 2.54 (t, J=6.4 Hz, 2H, CH.sub.2), 1.78 (s, 3H). MS (FAB): 595 (M).

Example 18

(E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino) but-2-enoic acid

[0114] ##STR00058##

[0115] The procedure was the same as in Example 12, except that (E)-3-aminobut-2-enoic acid was used in place of ethyl ester of glycine without hydrolysis to afford (E)-3-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamino) but-2-enoic acid as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.74 (s, 1H, ArH), 8.55 (d, J=4.4 Hz, 1H, ArH), 7.98 (d, J=8.0 Hz, 1H, ArH), 7.65 (dd, J.sub.1=7.6 Hz, J.sub.2=1.2 Hz, 1H, ArH), 7.55-7.34 (m, 9H, ArH), 7.13 (s, 1H, ArH), 5.33 (s, 2H, OCH.sub.2), 5.29 (s, 2H, OCH.sub.2), 3.85 (dd, J.sub.1=52.4 Hz, J.sub.2=13.2 Hz, 2H), 3.05 (s, 1H, CH), 1.12 (d, J=6.4 Hz, 3H). MS (FAB): 594 (M+1).

Example 19

2-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino)ethanesulfonic acid

[0116] ##STR00059##

[0117] The procedure was the same as in Example 12, except that 2-aminoethanesulfonic acid was used in place of ethyl ester of glycine without hydrolysis to afford 2-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzylamino)ethanesulfonic acid as a white solid. .sup.1H NMR (400 MHz, DMSO) 8.79 (s, 1H), 8.62 (d, J=4.8 Hz, 1H), 8.42 (d, J=7.2 Hz, 1H), 7.85 (t, J=4.8 Hz, 1H), 7.43 (m, 9H), 7.04 (s, 1H), 5.37 (s, 2H), 5.24 (s, 2H), 4.33 (s, 2H), 3.39 (m, 2H), 2.97 (m, 2H), 2.65 (br, 1H). MS (FAB): 618 (M+1).

Example 20

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) leucine

[0118] ##STR00060##

[0119] The procedure was the same as in Example 12, except that ethyl ester of leucine was used in place of ethyl ester of glycine to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) leucine as a white solid. .sup.1H NMR (400 MHz, DMSO) 8.69 (s, 1H), 8.52 (d, J=4.8 Hz, 1H), 8.42 (d, J=7.2 Hz, 1H), 7.85 (t, J=4.8 Hz, 1H), 7.43 (m, 9H), 7.04 (s, 1H), 5.37 (s, 2H), 5.24 (s, 2H), 3.79 (m, 2H), 3.18 (m, 1H), 1.86 (m, 1H), 1.41 (m, 2H), 0.74 (m, 6H). MS (FAB): 624 (M).

Example 21

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) tyrosine

[0120] ##STR00061##

[0121] The procedure was the same as in Example 12, except that ethyl ester of tyrosine was used in place of ethyl ester of glycine to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) tyrosine as a white solid. .sup.1H NMR (400 MHz, DMSO) 8.62 (s, 1H), 8.50 (d, J=3.2 Hz, 1H), 7.81 (t, J=7.6 Hz, 1H), 7.60 (t, J=9.2 Hz, 1H), 7.38 (m, 8H), 7.21 (s, 1H), 7.00 (s, 1H), 6.93 (d, J=8.0 Hz, 2H), 6.59 (d, J=8.0 Hz, 2H), 5.25 (s, 2H), 5.17 (s, 2H), 3.64 (m, 2H), 3.21 (t, J=6.8 Hz, 1H), 2.78 (m, 2H). MS (FAB): 674 (M).

Example 22

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) isoleucine

[0122] ##STR00062##

[0123] The procedure was the same as in Example 12, except that ethyl ester of isoleucine was used in place of ethyl ester of glycine to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) isoleucine as a white solid. .sup.1H NMR (400 MHz, DMSO) 8.64 (s, 1H), 8.50 (d, J=4.8 Hz, 1H), 8.38 (d, J=7.2 Hz, 1H), 7.82 (t, J=4.8 Hz, 1H), 7.43 (m, 9H), 7.00 (s, 1H), 5.25 (s, 2H), 5.18 (s, 2H), 3.63 (m, 2H), 3.48 (m, 1H), 2.28 (m, 1H), 1.50 (m, 2H), 1.06 (m, 3H), 0.73 (m, 3H). MS (FAB): 624 (M).

Example 23

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) asparagine

[0124] ##STR00063##

[0125] The procedure was the same as in Example 12, except that ethyl ester of asparagine was used in place of ethyl ester of glycine to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) asparagine as a white solid. .sup.1H NMR (400 MHz, DMSO) 11.96 (s, 1H), 8.72 (s, 1H), 8.52 (d, J=8.0 Hz, 1H), 8.16 (s, 1H), 7.68 (t, J=8.0 Hz, 1H), 7.44 (m, 10H), 7.01 (m, 2H), 5.26 (s, 2H), 5.21 (s, 2H), 3.65 (m, 2H), 3.35 (m, 1H), 2.65 (m, 2H). MS (FAB): 625 (M+1).

Example 24

N-(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine

[0126] ##STR00064##

[0127] The procedure was the same as in Example 12, except that 2-aminoethan-1-ol was used in place of ethyl ester of glycine without hydrolysis to afford N-(hydroxyethyl)-4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzylamine as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) 8.70 (s, 1H, ArH), 8.54 (d, J=15.4 Hz, 1H, ArH), 7.92 (s, 1H, ArH), 7.72-7.25 (m, 10H), 7.11 (s, 1H, ArH), 5.30 (s, 2H, CH.sub.2), 5.28 (s, 2H, CH.sub.2), 3.89 (m, 2H, CH.sub.2), 3.54 (s, 2H, CH.sub.2), 2.76 (s, 2H, CH.sub.2). MS (FAB): 554 (M+1).

Example 25

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) alanine

[0128] ##STR00065##

[0129] The procedure was the same as in Example 12, except that ethyl ester of alanine was used in place of ethyl ester of glycine to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) alanine as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) 8.69 (s, 1H, ArH), 8.53 (m, 1H, ArH), 7.93 (d, J=8.0 Hz, 1H, ArH), 7.61 (d, J=8.1 Hz, 1H, ArH), 7.53-7.31 (m, 9H, ArH), 7.08 (s, 1H, ArH), 5.29 (s, 2H, CH.sub.2), 5.27-5.23 (m, 2H, CH.sub.2), 3.96-3.77 (m, 2H, CH.sub.2), 3.12 (m, 1H, CH), 1.19 (d, J=7.0 Hz, 3H, CH.sub.3). MS (FAB): 582 (M+1).

Example 26

N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) proline

[0130] ##STR00066##

[0131] The procedure was the same as in Example 12, except that ethyl ester of proline was used in place of ethyl ester of glycine to afford N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) proline as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.72 (s, 1H, ArH), 8.56 (d, J=6.0 Hz, 1H, ArH), 7.95 (d, J=8.0 Hz, 1H, ArH), 7.66 (d, J=7.6 Hz, 1H, ArH), 7.5-7.34 (m, 9H, ArH), 7.12 (s, 1H, ArH), 5.35-5.27 (m, 4H, OCH.sub.2), 3.95 (dd, J.sub.1=51.6 Hz, J.sub.2=13.2 Hz, 2H, CH.sub.2), 3.12 (m, 1H, CH), 2.67 (m, 1H, CH.sub.2), 2.07 (m, 1H, CH.sub.2), 1.89 (m, 1H, CH.sub.2), 1.78 (m, 1H, CH.sub.2), 1.68 (m, 1H, CH.sub.2), 0.84 (m, 1H, CH.sub.2). MS (FAB): 608 (M).

Example 27

(S)-Sodium N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serinate

[0132] ##STR00067##

[0133] (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine (299 mg) was dissolved in 1 ml of 0.5 N sodium hydroxide aqueous solution. After being stirred for 30 min at room temperature, absolute ethanol was added to the solution until solids appeared. After being heated to dissolve and cooled to room temperature, the mixture was placed in the refrigerator for freezing. Then the mixture was filtered to afford the product (300 mg) as a white solid. .sup.1H NMR (400 MHz, Methanol-d4) 8.65 (s, 1H, ArH), 8.49 (d, 1H, ArH), 8.04 (d, 1H, ArH), 7.63 (d, J=8.0 Hz, 1H, ArH), 7.50-7.34 (m, 8H, ArH), 7.31 (d, J=7.5 Hz, 1H, ArH), 6.84 (s, 1H, ArH), 5.28 (s, 2H, CH.sub.2), 5.23 (s, 2H, CH.sub.2), 3.85-3.74 (m, 2H, CH.sub.2), 3.72 (d, J=4.6 Hz, 1H, CH.sub.2), 3.67 (m, 1H, CH.sub.2), 3.15 (t, J=5.6 Hz, 1H, CH). MS (FAB): 599 (M+1).

Example 28

(S)-Calcium N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serinate

[0134] ##STR00068##

[0135] (S)-Sodium N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate (243 mg) was dissolved in 5 ml of water. Aqueous solution of calcium dichloride (1%, 2.22 ml) was added dropwise while being stirred at room temperature. Then the mixture was stirred overnight, filtered, washed with water, and dried to afford the product (240 mg) as a white solid. MS (FAB): 599 (M+1).

Example 29

(S)-(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy)benzyl) serinate

[0136] ##STR00069##

[0137] (S)N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(pyridin-3-yl-methyleneoxy) benzyl) serine (299 mg) and 4-(chloromethyl)-5-methyl-1,3-dioxol-2-one (74 mg) were dissolved in 10 ml of DMF. After a catalytic amount of potassium iodide was added, the mixture was stirred at 30 C. until the reaction was completed. The mixture was poured into a stirred ice-cold saturated aqueous solution of sodium bicarbonate, then filtered, and the solid was washed with water and dried to afford the product (98 mg). MS (FAB):711 (M+1).

Pharmacological Experiments

[0138] 1. In vitro activity evaluation: Cisbio PD-1/PD-L1 binding assay kit was applied for the detection method of in vitro enzymology level.

[0139] 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.sup.+ 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.

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 1.48 10.sup.7 4 <10.sup.13 6 8.23 10.sup.8 8 4.29 10.sup.8 9 4.01 10.sup.8 10 1.34 10.sup.7 11 3.18 10.sup.7 12 4.11 10.sup.8 13 2.00 10.sup.7 14 2.69 10.sup.5 15 5.10 10.sup.8 16 1.99 10.sup.7 17 5.51 10.sup.7 18 2.33 10.sup.9 19 1.62 10.sup.5 20 6.10 10.sup.8 21 4.06 10.sup.7 22 4.99 10.sup.6 23 8.35 10.sup.7 24 5.38 10.sup.8 25 5.29 10.sup.9 26 10.sup.12~10.sup.13

[0140] Cisbio HTRF detection showed that the interaction of PD-1 and PD-L1 could be significantly inhibited by the Example 4 compound at the molecular level, with IC.sub.50<10.sup.13 mol/L.

2. The Example 4 compound's capacity of relieving the inhibition of IFN by ligand PD-L1:

[0141] 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.

[0142] 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 4 compound 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 4 compound (YPD29B) at 10 nM, and the level of IC.sub.50 was determined as the level of 1.810.sup.10 mol/L by testing different concentrations (FIG. 1).

3. The efficacy of the Example 4 compound in vivo

[0143] The methods of pharmacodynamics were as follows:

[0144] 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.

[0145] 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 C57BL/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.

[0146] 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 C57BL/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.

[0147] 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

[0148] For the high metastatic melanoma B16F10, the example compounds (45 mg/kg of Example 5 compound, 15 mg/kg of hydrochloride form of Example 4 compound and 15 mg/kg of sodium salt of Example 4 compound) can significantly inhibit the growth of the subcutaneous tumor, with the respect of tumor volume or weight (FIG. 2, FIG. 3 and Table 2) and the rate of inhibition of tumor weight can be 45.27%, 38.37% and 64.11% respectively.

TABLE-US-00002 TABLE 2 Inhibition of Bl6F10 subcutaneous xenograft tumors by Example compounds Number Body weight (g) (Begin/ Mean SD Tumor weight(g) Group Dose End) Begin End X SD T/C % (TGI %) Vehicle Control 6/6 20.3 1.0 23.7 1.7 2.58 1.56 NA Cyclophos- 80 mg/kg 5/5 20.3 0.8 23.3 1.4 1.55 0.59 60.00(40.00) phamide (CTX) Example 5 15 mg/kg 5/5 20.4 1.0 24.2 2.2 2.69 1.99 104.26(4.26) 45 mg/kg 5/5 20.3 1.0 22.6 1.4 1.41 0.60 54.73(45.27) Example 4 15 mg/kg 5/5 20.3 0.4 21.8 2.1 0.93 0.89 35.89(64.11)* sodium salt 45 mg/kg 5/5 20.4 0.9 23.8 1.4 2.57 0.85 99.77(0.23) Example 4 15 mg/kg 5/5 20.3 0.6 22.6 1.4 1.59 0.91 61.63(38.37) hydrochloride 45 mg/kg 5/5 20.3 0.7 23.1 1.7 2.19 0.92 84.81(15.19) form T/C: Relative tumor proliferation rate TGI: Tumor growth inhibition rate NA: Not applicable *P < 0.05 vs Vehicle control

[0149] From the analysis of mechanism, it can be seen that Example 5 compound, sodium salt of Example 4 compound and hydrochloride form of Example 4 compound can increase the proportion of tumor-infiltrating lymphocytes (FIG. 4, Table 3) and sodium salt of Example 4 compound can increase the proportion of lymphocytes in the spleen samples (FIG. 5, Table 4).

TABLE-US-00003 TABLE 3 Effects of Example 4 and Example 5 on tumor-infiltrating T lymphocytes Group CD3+ (%) CD4+ (%) CD8+ (%) Vehicle control 6.5 0.8 4.8 3.7 3.4 0.1 Cyclophosphamide (CTX) 3.6 1.5 1.7 0.4 1.4 0.3 Example 5 45 mg 10.1 4.5 9.0 4.7 5.2 2.8 Example 4 sodium salt 15 mg 13.3 6.9 7.2 3.4 3.9 1.4 Example 4 hydrochloride form 15.2 3.9 15.0 10.7 9.6 3.6 15 mg

TABLE-US-00004 TABLE 4 Effects of sodium salt of Example 4 compound on T lymphocytes in spleen Group CD3+ (%) CD4+ (%) CD8+ (%) Vehicle control 62.5 7.6 21.3 4.0 9.6 2.1 Cyclophosphamide (CTX) 78.6 2.5 24.6 2.6 15.2 3.1 Example 4 sodium salt 15 mg 74.3.3 3.5 27.0 1.8 13.6 1.8

(2) Lung Metastasis Model of High Metastatic Melanoma B16F10

[0150] For metastatic lung cancer models with high metastatic melanoma B16F10, the sodium salt of Example 4 compound can significantly inhibit the number of lung metastases at 15 mg/kg dose (FIG. 6, Table 5).

TABLE-US-00005 TABLE 5 Example compounds' inhibition effect on Pulmonary metastasis model of Bl6F10 Body weight(g) Tumor number Number Mean SD T/C % Group Dose (End/Begin) Begin End X SD (TGI %) Vehicle control 6/6 20.2 0.4 21.0 0.3 21 15 NA Cyclophosphamide 80 mg/kg 5/5 20.1 0.8 21.4 0.5 18 14 85.7(14.3) (CTX) Example 5 15 mg/kg 5/5 20.6 0.7 20.2 1.5 18 18 85.7(14.3) 45 mg/kg 5/5 20.5 0.6 21.2 0.7 16 7 76.2(23.8) Example 4 sodium salt 15 mg/kg 5/5 20.1 0.9 21.5 1.2 12 3 57.1(42.3) 45 mg/kg 5/5 20.3 0.6 21.4 0.3 19 15 90.5(9.5) Example 4 15 mg/kg 5/5 20.5 0.6 21.3 1.0 14 10 66.7(33.3) hydrochloride form 45 mg/kg 5/5 20.6 1.0 21.3 0.4 17 12 81.0(19.0) T/C: Relative tumor proliferation rate TGI: Tumor growth inhibition rate NA: Not applicable *P < 0.05 vs Vehicle control

[0151] From analysis of the mechanism, it can be seen Example 4 and Example 5 could increase the percentage of lymphocyte in mouse blood (FIG. 7, Table 6).

TABLE-US-00006 TABLE 6 Example compounds' effect on the percentage of T ymphocyte in mouse blood Group CD3+ CD4+ CD8+ Vehicle control 21.0 2.6 12.3 2.1 7.0 1.1 Cyclophosphamide (CTX) 22.4 5.5 13.0 2.4 7.5 2.4 Example 5 15 mg 22.7 4.8 14.4 3.3 7.4 1.9 Example 5 45 mg 25.8 3.0 15.7 2.5 7.6 1.8 Example 4 sodium salt 15 mg 29.0 3.7 17.8 2.4 9.6 0.8 Example 4 sodium salt 45 mg 23.2 3.6 14.7 2.5 7.6 1.3 Example 4 hydrochloride 15 mg 29.3 2.9 18.6 1.6 10.7 1.3 Example 4 hydrochloride 45 mg 26.8 4.1 17.4 2.0 8.5 2.3

(3) Subcutaneous Xenograft Model of Mouse Breast Cancer EMT6

[0152] For subcutaneous xenograft model of mouse breast cancer EMT6, sodium salt of Example 4 compound has some inhibition effect on mouse breast cancer EMT6. At the dose of 10 mg and 15 mg, sodium salt of Example 4 compound has 20% and 22% inhibition effect respectively (FIG. 8, Table 6). In addition, the combination of sodium salt of Example 4 compound and Cyclophosphamide can significantly increase the tumor growth inhibition rate of Cyclophosphamide from 85% to 95% (FIG. 8, Table 7).

TABLE-US-00007 TABLE 7 Example 4 compound's inhibition effect on mouse subcutaneous xenograft of EMT6 Number Body weight (g) (End/ Mean SD Tumorweight(g) Group Dose Begin) Begin End X SD T/C % (TGI %) Vehicle 6/6 18.4 0.3 20.3 0.3 1.72 0.22 NA control Cyclophosphamide 60 mg/kg 5/5 18.4 0.3 19.4 0.7* 0.25 0.17*** 14.1(85.9)*** Cyclophosphamide + 60 mg/kg + 5/5 17.8 1.1 18.9 0.4*** 0.08 0.04*** 4.8(95.2)*** Example 4 10 mg/kg sodium salt Example 4 1.5 mg/kg 6/6 18.4 0.9 22.0 1.6 1.5 0.61 86.9(13.1) sodium salt 5 mg/kg 6/6 18.4 0.4 21.0 1.6 1.37 0.23* 79.8(20.2)* 10 mg/kg 6/6 18.4 0.6 21.6 1.3 1.34 0.34* 77.7(22.3)* 15 mg/kg 6/6 18.5 0.4 22.0 1.1* 1.47 0.65 85.3(14.7) T/C: Relative tumor proliferation rate TGI: Tumor growth inhibition rate NA: Not applicable *P < 0.05 vs Vehicle control

(4) Mouse Lewis Lung Cancer Hydrothorax Model

[0153] Sodium salt of Example 4 compound has inhibition effect on mouse Lewis lung cancer hydrothorax model. The hydrothorax incidence rate in vehicle control group was 75%, whereas at the dose of 10 mg, sodium salt of Example 4 compound can reduce the rate to 33% (Table 8). The mean volume of the hydrothorax of mice was 0.3 ml in the vehicle control group and in the group administrated with sodium salt of Example 4 compound the mouse only had 0.2 ml of hydrothorax (FIG. 9, Table 9). Further, sodium salt of Example 4 compound can significantly increase thymus index (FIG. 10).

TABLE-US-00008 TABLE 8 Example 4's effect on hydrothorax incidence rate of Lewis lung cancer Vehicle control Example 4 sodium salt 10 mg 75% 33%

TABLE-US-00009 TABLE 9 Example 4's effect on hydrothorax volume of Lewis lung cancer Vehicle control Example 4 sodium salt 10 mg 0.3 ml 0.2 ml

(5) Subcutaneous Xenograft Model of Mouse Colon Cancer MC38

[0154] For subcutaneous xenograft model of mouse colon cancer MC38, sodium salt of Example 4 compound has significant inhibition effect. Furthermore, sodium salt of Example 4 compound has a synergistic antitumor effect on this cancer with Cyclophosphamide (CTX) (FIG. 11, Table 10).

TABLE-US-00010 TABLE 10 Example compounds' effect on subcutaneous xenograft model of mouse colon cancer MC38 Body weight (g) Number Mean SD Tumor weight(g) Group Dose (End/begin) Begin End Mean SD T/C % (TGI %) Vehicle 6/6 17.9 0.5 22.3 1.1 3.18 0.82 NA control Cyclophosphamide 60 mg/kg 6/6 17.9 0.6 19.1 0.8*** 0.17 0.05*** 5.4(94.6)*** Cyclophosphamide + 60 mg/kg + 6/6 18.2 0.6 18.8 0.8*** 0.06 0.04*** 1.9(98.1)*** Example 4 10 mg ## ## sodium salt Example 4 2.5 mg/kg 6/6 18.0 0.4 21.9 0.7 3.25 0.61 2.3(102.3) 5 mg/kg 6/6 18.2 0.3 22.8 0.6 2.78 0.44 87.3(12.7) 10 mg/kg 6/6 18.2 0.3 21.5 0.8 2.14 0.78* 67.3(32.7)* 20 mg/kg 6/6 18.2 0.4 20.9 1.1 1.87 0.90* 58.8(41.2)* T/C: Relative tumor proliferation rate TGI: Tumor growth inhibition rate NA: Not applicable *P < 0.05 vs Vehicle control; ***P < 0.001 vs Vehicle control; ##p < 0.01 vs Cyclophosphamide (CTX)
4. The interaction of Example 4 compound/PD-L1 antibody with PD-L1 protein was tested by Biacore

(1) Experimental Principle

[0155] 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

[0156] 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

[0157]

TABLE-US-00011 TABLE 11 The affinity of Example 4 compound and PD-L1 antibody to PD-L1 Ligand Analyte ka (1/Ms) kd (1/s) KD (M) PD-L1 protein PD-L1 antibody 2.016E+5 1.358E4 6.736E10 PD-L1protein Example 4 1.390E+6 2.815E5 2.025E11

[0158] It was preliminarily determined that the binding protein of the Example 4 is PD-L1 (FIG. 12). Further Biacore experiments confirmed that the combination of Example 4 has a strong ability of binding PD-L1 and the affinity kD value is 2.025E-11 which is even stronger than that of the antibody of PD-L1 (Table 11, FIG. 12-14).